Discovery and in vitro and in vivo profiles of N-ethyl-N-[2-[3-(5-fluoro-2-pyridinyl)-1H-pyrazol-1-yl]ethyl]-2-(2H-1,2,3-triazol-2-yl)-benzamide as a novel class of dual orexin receptor antagonist

Article abstract of DOI:10.1016/j.bmc.2015.01.044

Orexins play an important role in sleep/wake regulation, and orexin receptor antagonists are a focus of novel therapy for the treatment of insomnia. We identified 27e (TASP0428980) as a potent dual orexin receptor antagonist through the systematic modification of our original designed lead A. We demonstrated the potent sleep-promoting effects of 27e at ip dose of 3 mg/kg in a rat polysomnogram study. 27e exhibited relatively short half-life profiles in rats and dogs. Furthermore, accumulating evidence regarding ADME profiles indicates that the predicted human half-life of 27e should be 1.2-1.4 h. These data indicated that 27e has a short-acting hypnotic property, suggesting that 27e might be useful for treating primary insomnia while exhibiting a low risk of next-day residual somnolence. Thus, 27e and its related compounds should be further evaluated to enable advancement to clinical trials.

Full text of DOI:10.1016/j.bmc.2015.01.044

Bioorganic & Medicinal Chemistry 23 (2015) 1260–1275
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Discovery and in vitro and in vivo profiles of N-ethyl-N-[2-[3-(5-
fluoro-2-pyridinyl)-1H-pyrazol-1-yl]ethyl]-2-(2H-1,2,3-triazol-2-yl)-
benzamide as a novel class of dual orexin receptor antagonist
Ryo Suzuki a, Dai Nozawa a, Aya Futamura a, Rie Nishikawa-Shimono a, Masahito Abe a, Nobutaka Hattori a,
Hiroshi Ohta a, Yuko Araki a, Daiji Kambe b, Mari Ohmichi c, Seiken Tokura a, Takeshi Aoki b,
Norikazu Ohtake a, Hiroshi Kawamoto a,
⇑
a Chemistry Laboratories, Taisho Pharmaceutical Co., Ltd, 1-403 Yoshino-cho, Kita-ku, Saitama 331-9530, Japan
b Pharmacology Laboratories, Taisho Pharmaceutical Co., Ltd, 1-403 Yoshino-cho, Kita-ku, Saitama 331-9530, Japan
c Drug Safety and Pharmacokinetics Laboratories, Taisho Pharmaceutical Co., Ltd, 1-403 Yoshino-cho, Kita-ku, Saitama 331-9530, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Orexins play an important role in sleep/wake regulation, and orexin receptor antagonists are a focus of
novel therapy for the treatment of insomnia. We identified 27e (TASP0428980) as a potent dual orexin
receptor antagonist through the systematic modification of our original designed lead A. We demon-
strated the potent sleep-promoting effects of 27e at ip dose of 3 mg/kg in a rat polysomnogram study.
27e exhibited relatively short half-life profiles in rats and dogs. Furthermore, accumulating evidence
regarding ADME profiles indicates that the predicted human half-life of 27e should be 1.2–1.4 h. These
data indicated that 27e has a short-acting hypnotic property, suggesting that 27e might be useful for
treating primary insomnia while exhibiting a low risk of next-day residual somnolence. Thus, 27e and
its related compounds should be further evaluated to enable advancement to clinical trials.
Ó 2015 Elsevier Ltd. All rights reserved.
Received 19 November 2014
Revised 23 January 2015
Accepted 23 January 2015
Available online 10 February 2015
Keywords:
Dual orexin receptor antagonist
Insomnia
Short half-life
1. Introduction
ute to the activation of phospholipase C, leading to the elevation of
intracellular Ca2+ concentrations.2 The OX2 receptor can also be
The orexin system is composed of two widely expressed G-pro-
tein-coupled receptors, orexin 1 (OX1) receptor and orexin 2 (OX2)
receptor, and the two peptide agonists orexin A and orexin B (also
known as hypocretin 1 and hypocretin 2), which are selectively
expressed in a small cluster of neurons in the lateral hypothala-
mus.1,2 The OX1 receptor has a high affinity for orexin A, with its
affinity for orexin B being 1 to 2 orders of magnitude lower,
whereas the OX2 receptor exhibits an equally high affinity for both
peptides.2 Both receptors are coupled to Gq/11 proteins and contrib-
coupled to the Gs and Gi pathways.3 The orexin neurons and recep-
tors are widely distributed in the brain, including the basal fore-
brain, limbic structures, and brainstem regions. The patterns of
receptor expression partially overlap, but some regions express
only one of the receptors. This pattern of expression suggests that
the two types of receptors may have different physiological roles.4
The orexin system also has been implicated in numerous phys-
iological functions, including feeding, addiction, depression, anxi-
ety, panic, and lung/respiratory function, as suggested from
animal studies.4,5 A key role of the orexin system in the regulation
of the sleep/wake cycle has been suggested by observations that
mice lacking orexin peptides or both receptors display a strong
narcoleptic-like phenotype with cataplexy4 and that familial
canine narcolepsy is caused by defective OX2 receptor signaling.6
Furthermore, the orexin levels in cerebrospinal fluid (CSF) vary
during the circadian cycle in rodents, monkeys, dogs, and humans,
with levels being highest at the end of the wake-active period and
lowest toward the end of the sleep period.7
Abbreviations: OX1, orexin 1; OX2, orexin 2; CSF, cerebrospinal fluid; POC, proof-
of-concept; DORA, dual orexin receptor antagonist; REM, rapid eye movement;
NREM, non-REM; HATU, N-[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridin-1-
ylmethylene]-N-methylmethanaminium hexafluorophosphate N-oxide; LBDD,
ligand-based drug design; PSG, polysomnogram; ip, intraperitoneal; EEG, electro-
encephalogram; EMG, electromyogram; WSCÁHCl, 1-(3-dimethylaminopropyl)-3-
ethylcarbodiimide hydrochloride; HOBtÁH2O, 1-hydroxy-1H-benzotriazole hydrate;
TEA, triethylamine; DIPEA, N,N-diisopropylethylamine; T3P, propylphosphonic acid
anhydride.
Positive allosteric modulators of GABAA receptors, such as zolpi-
dem, are responsible for mediating the mechanism of action
⇑
Corresponding author. Tel.: +81 48 669 3029; fax: +81 48 652 7254.
E-mail address: h-kawamoto@so.taisho.co.jp (H. Kawamoto).
http://dx.doi.org/10.1016/j.bmc.2015.01.044
0968-0896/Ó 2015 Elsevier Ltd. All rights reserved.

R. Suzuki et al. / Bioorg. Med. Chem. 23 (2015) 1260–1275
1261
involved in the current standard of care for insomnia. This mecha-
nism is widely known to promote sleep through the suppression of
CNS activity, which can be associated with serious adverse effects
including muscular relaxation, memory disturbance, rebound
insomnia, tolerance, and dependence. These concerns have encour-
aged further efforts to discover and develop treatments for insom-
nia with mechanisms of action that differ from those of current
therapies. Novel treatments for insomnia have therefore targeted
the orexin system, and the discovery of dual OX1/OX2 antagonist
has been reported.8,9 Positive clinical proof-of-concept (POC) stud-
ies have demonstrated sleep induction by four dual orexin receptor
antagonists (DORAs): almorexant, SB-649868, suvorexant, and fil-
orexant (MK-6096).9 Importantly, these compounds have been
shown to induce a physiological sleep architecture characterized
by increases in the time spent in rapid eye movement (REM) and
non-REM (NREM) sleep in animals and humans, differentiating
them from GABAA modulators that reduce the time spent in these
sleep stages, which are believed to be important for the consolida-
tion of memory and the restorative functions of sleep. However, in
Phase 2 and Phase 3 clinical trials, the most common adverse event
associated with suvorexant, the most advanced DORA, was somno-
lence, with a dose-related increase in events observed across treat-
ment groups.10 The half-life of suvorexant in humans is 9–13 h,
which is much longer than that of SB-649868 when analyzed clin-
ically.11 SB-649868 has been reported to have a half-life in the
range of 3.4–3.9 h in healthy volunteers.12 Furthermore, patients
with primary insomnia reported that SB-649868 significantly
improved the quality of their sleep, while objectively increasing
the total sleep time, reducing sleep latency, and suppressing night-
time awakenings.14 The assessment of next-day residual effects
indicated no significant differences between SB-649868 and
placebo.13,14 Thus, SB-649868 showed positive results in terms of
efficacy, despite its short half-life profile, without inducing residual
next-day effects.12–14
Therefore, we defined the discovery of a potent and short-acting
DORA, compared with suvorexant, as a clear research goal. Here,
we describe the characterization of N-ethyl-N-[2-[3-(5-fluoro-2-
pyridinyl)-1H-pyrazol-1-yl]ethyl]-2-(2H-1,2,3-triazol-2-yl)-benz-
amide (27e, TASP0428980), a potent and structurally distinct
DORA that is currently being evaluated in preclinical studies for
primary insomnia (Fig. 1).
2. Chemistry
The routes of synthesis are summarized in Schemes 1–7. The
pyrazole derivatives 4a–4f were synthesized as described in
Scheme 1. Commercially available 1-(tetrahydro-2H-pyran-2-yl)-
5-(4,4,5,5-tetramethyl-1,3-dioxaborolan-2-yl)-1H-pyrazole (1) was
transformed into 2a–2f via Suzuki–Miyaura coupling with appropri-
ate aryl bromides and the removal of the THP group in acceptable
yields. N-Alkylation of 2a–2f with 3-ethyl-1,2,3-oxathiazolidine
2,2-dioxide using sodium hydroxide as a base yielded 3a–3f. Finally,
Figure 1. Representative dual OX1 and OX2 receptor antagonists.
Scheme 1. Synthesis of pyrazole derivatives 4a–4f. Reagents and conditions: (a) ArBr, Pd(PPh3)4, Na2CO3, H2O, EtOH, toluene, 90 °C; (b) 4N HCl–AcOEt, MeOH, rt; (c) (1) 3-
ethyl-1,2,3-oxathiazolidine 2,2-dioxide, NaOH, CH3CN, 70 °C; (2) 25% H2SO4, 70 °C; (d) 5-methyl-2-(2H-1,2,3-triazol-2-yl)benzoic acid, T3P or WSCÁHCl or HATU.
Scheme 2. Synthesis of oxadiazole derivative 9. Reagents and conditions: (a) (1) SOCl2, CHCl3, reflux; (2) 6, TEA, CHCl3, rt; (b) (1) MsCl, TEA, CHCl3, rt; (2) NaCN, DMF, 80 °C;
(c) 50% NH2OH, EtOH, 80 °C; (d) (1) 5-fluoropicolinic acid, CDI, CH3CN, DMF, rt; (2) DBU, 70 °C.

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R. Suzuki et al. / Bioorg. Med. Chem. 23 (2015) 1260–1275
Scheme 3. Synthesis of pyrazole derivative 12. Reagents and conditions: (a) 2-bromo-5-fluoropyridine, Pd(PPh3)4, Na2CO3, H2O, EtOH, toluene, 90 °C; (b) (1) MsCl, TEA, CHCl3,
rt; (2) 11, Cs2CO3, DMF, 80 °C.
Scheme 4. Synthesis of oxazole derivative 16. Reagents and conditions: (a) 5-methyl-2-(2H-1,2,3-triazol-2-yl)benzoic acid, HATU, DIPEA, DMF, rt; (b) (1) 1.2 M HClaq, THF,
rt; (2) NH2OHÁHCl, AcONa, EtOH, H2O, 0 °C; (3) NCS, DMF, rt; (4) 2-ethynyl-5-fluoropyridine, TEA, THF, rt; (c) EtI, NaH, DMF, rt.
the hydroxyl group in intermediate 7 followed by the N-alkylation
of pyrazole 11 using cesium carbonate yielded the desired com-
pound 12.
The steps for synthesizing isoxazole derivative 16 are depicted
in Scheme 4. The amide condensation of 5-methyl-2-(2H-1,2,3-
triazol-2-yl)benzoic acid with amine 13 in the presence of HATU
yielded 14. The removal of the acetal group of 14 followed by con-
densation with hydroxylamine, the chlorination of oxime, and
isoxazole cyclization provided the desired compound 16.
The condensation of commercially available 5-fluoropicolinal-
dehyde 17 and hydroxylamine followed by the chlorination of oxime
Scheme 5. Synthesis of isoxazole derivative 21. Reagents and conditions: (a) (1)
NH2OHÁHCl, AcONa, EtOH, H2O, rt; (2) NCS, DMF, rt; (3) N-(3-butynyl)phthalimide,
and isoxazole cyclization yielded 18 (Scheme 5). The removal of the
phthalimide group of 18 yielded 19. The resulting amine 19 was
reacted with 5-methyl-2-(2H-1,2,3-triazol-2-yl)benzoic acid to
yield the intermediate 20. N-Ethylation of intermediate 20 provided
the final target 21.
TEA, THF, rt; (b) H2NNH2ÁH2O, EtOH, 90 °C; (c) 5-methyl-2-(2H-1,2,3-triazol-2-
yl)benzoic acid, HATU, DIPEA, DMF, rt; (d) EtI, NaH, DMF, rt.
amide condensation of 5-methyl-2-(2H-1,2,3-triazol-2-yl)benzoic
acid with 3a–3f provided the pyrazole derivatives 4a–4f.
The N-alkylated amides (26a, 26b) were prepared by the
condensation of amine 24 with 5-methyl-2-(2H-1,2,3-triazol-
2-yl)benzoic acid, followed by N-alkylation of the amide of
intermediate 25. Intermediate 24 was obtained in 2 steps from
tert-butyl(2-bromoethyl)carbamate: first the N-alkylation of pyra-
zole 2a, followed by removal of the Boc-group of 23 (Scheme 6).
To obtain the pyrazole derivatives (27a–27e, Scheme 7), amine
3a was coupled with the appropriate benzoic acids. Intermediate
3a was synthesized as described in Scheme 1.
The 1,2,4-oxadiazole derivative 9 was synthesized as described
in Scheme 2. The amide condensation of 5-methyl-2-(2H-1,2,3-
triazol-2-yl)benzoic acid 5 with 2-(ethylamino)ethanol 6 in the
presence of N-[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridin-
1-ylmethylene]-N-methylmethanaminium hexafluorophosphate
N-oxide (HATU) provided alcohol 7. The mesylation of the hydroxyl
group followed by cyanation yielded 8. Finally, the addition reac-
tion of 8 with hydroxylamine followed by O-acylation of amidox-
ime and cyclodehydration provided 9.
Compound 12 was prepared as shown in Scheme 3.
Commercially available tert-butyl 4-(4,4,5,5-tetramethyl-1,
3-dioxaborolan-2-yl)-1H-pyrazole-1-carboxylate 10 was converted
to the pyrazole 11 using the Suzuki–Miyaura coupling reaction.
The Boc-group was removed in this reaction. The mesylation of
3. Results and discussion
We have been pursuing potent dual OX1 and OX2 receptor
antagonists in terms of their sleep-promoting effects with or with-
Scheme 6. Synthesis of N-alkyl derivatives 26a and 26b. Reagents and conditions: (a) 2a, Cs2CO3, DMF, 80 °C; (b) 4 N HCl–EtOAc, EtOAc, rt; (c) 5-methyl-2-(2H-1,2,3-triazol-
2-yl)benzoic acid, HATU, DIPEA, DMF, rt; (d) RI, NaH, DMF, rt.

R. Suzuki et al. / Bioorg. Med. Chem. 23 (2015) 1260–1275
1263
resulted in a maintained or slightly improved antagonistic potency
for the OX1 and OX2 receptors, compared with open compound 29b
(Fig. 2).15
We attempted to superimpose representative OX1 and OX2
receptor antagonists (suvorexant, filorexant and SB-674042) onto
the quinazoline analogue 28 with the U-shaped conformation
using the flexible alignment method of the Molecular Operating
Environment program16 with the default parameters and found
that these molecules overlapped quite closely, as expected
(Fig. 3). Based on this information, we created a novel pharmaco-
phore model based on the superposition of these molecules, as
shown in Figure 4. The pharmacophore model suggested that the
cyclic amide linkages utilized coincidentally in the representative
antagonists could be replaced with a linear amide linkage. As a
result of the design of novel pharmacophore model and their fitting
trials using the Ligand Pharmacophore Mapping protocol of the
Discovery Studio program17 with the default parameters, a pyra-
zoylethylbenzamide designed lead A was created as a linear linker.
We decided to use designed lead A as a starting point for chemical
modifications.
Scheme 7. Synthesis of biarylamide derivative 27a–27e. Reagents: (a) ArCO2H,
WSCÁHCl or T3P.
out total sleep maintenance. Our strategy for designing lead com-
pounds was to use a ligand-based drug design (LBDD) method,
since several research groups have already reported various OX1
and OX2 receptor ligands. In addition, we adopted a hypothesis
that a U-shaped conformation of N,N-disubstituted-1,4-diazepane
28 would elicit bioactivity through OX1 and OX2 receptors, since
enforcing the U-shaped conformation by macrocyclization (29a)
Our goals were to investigate the structure-activity relationship
(SAR) of our novel lead class and to identify a potent dual OX1 and
OX2 receptor antagonist in which the potency and pharmacoki-
netic profiles were appropriately balanced.
Figure 2. N,N-Disubstituted-1,4-diazepane derivatives 28 and 29.
Figure 3. Superposition of compound 28 (gray), suvorexant (deep blue), filorexant (red) and SB-674042 (yellow).

1264
R. Suzuki et al. / Bioorg. Med. Chem. 23 (2015) 1260–1275
Table 2
SAR of 5-membered heteroaromatics
Compd
Ar
Kb SEa (nM)
OX2R
ClogPb
OX1R
4a
9
0.43 0.04
8.09 1.64
3.81 0.62
15.24 4.32
0.89 0.24
22.99 1.24
7.22 1.76
19.78 2.03
3.88
2.46
3.67
3.18
12
16
Figure 4. The pharmacophore model and the pyrazoylethylbenzamide designed
lead A (Ar = phenyl). The pharmacophore model consists of two hydrogen bond
acceptors (green arrows and spheres), one hydrophobic site (blue sphere), and three
aromatic rings (orange arrows and spheres).
21
45.49 2.93
32.19 3.52
3.18
We initiated chemical modification to clarify the SAR in order
from the aryl moiety at the right end of designed lead A. Table 1
highlights the SAR for the Ar part. First, the introduction of 5-flu-
oro-2-pyridine, which is used as a typical right-end component
of known ligands such as filorexant, resulted in strong dual OX1
and OX2 receptor antagonistic activities. Various kinds of Ar
replacements were then introduced with the exception of 2- and
4-fluoropyridine derivatives, since these analogues have a possibil-
ity of drug-protein covalent binding because of the potential
electrophilicity of such fluoropyridine structures.18 Among them,
2-trifluoromethyl-6-pyridine (4d), 6-methyl-2-pyridine (4e), and
4-fluorobenzene (4f) analogues showed almost the same antago-
nistic potency as that of 4a. Unfortunately, these analogues had
relatively high lipophilic natures. The logD/ClogP values tended
to have limits between the values of 3 and 4.19 Therefore, 4a
a
The Kb value is the mean of multiple results (at least three independent
determinations performed in duplicate) with the standard error of the means.
The ClogP value was calculated using software from Daylight Chemical Infor-
mation Systems, Inc.
b
showing relatively low ClogP value was used as a template for
the following modifications. The ClogP value was calculated using
software from Daylight Chemical Information Systems, Inc.
Next, we attempted to find a novel and potent 5-membered
heteroaromatic component. The replacement of the pyrazole with
several kinds of ring systems, which could reduce their lipophilic-
ity, was conducted as shown in Table 2. Oxadiazole analogue 9,
1,4-pyrazole analogue 12, and isoxazole analogues 16 and 21
showed moderate antagonistic activities for the OX1 and OX2
receptors. Only pyrazole analogues 4a and 12 had potent dual
OX1 and OX2 receptor antagonistic activities.
Table 1
The effects of substituents at the amide nitrogen atom on intrin-
sic potency were investigated (Table 3). An amide NH proton ana-
logue 25 dramatically decreased both the OX1 and OX2 receptor
antagonistic activities. This effect might have been caused by the
difficulty in forming a U-shaped conformation. The introduction
of a methyl group (26a) at the amide nitrogen atom showed a sin-
gle digit nano-molar potency against both OX1 and OX2 receptors.
A more bulky substitution, such as an isopropyl group in 26b, led
SAR of right-end Ar substituents
Compd
Ar
Kb SEa (nM)
ClogPb
OX1R
OX2R
Table 3
SAR of N-alkyl substituents
4a
4b
0.43 0.04
0.89 0.24
3.88
4.68
48.57 3.05
902.41 144.99
4c
28.01 3.58
3.19 0.71
66.43 10.10
3.92 2.04
4.68
4.68
4d
Compd
R
Kb SEa (nM)
OX2R
0.89 0.24
OX1R
0.43 0.04
4e
4f
0.41 0.04
0.32 0.04
1.96 0.17
1.25 0.36
4.17
5.02
4a
Et
25
26a
26b
H
Me
iPr
151.88 35.05
2.27 0.10
38.46 6.31
376.10 83.80
8.58 1.32
37.85 3.91
a
The Kb value is the mean of multiple results (at least three independent
determinations performed in duplicate) with the standard error of the means.
The ClogP value was calculated using software from Daylight Chemical Infor-
mation Systems, Inc.
a
b
The Kb value is the mean of multiple results (at least three independent
determinations performed in duplicate) with the standard error of the means.

R. Suzuki et al. / Bioorg. Med. Chem. 23 (2015) 1260–1275
1265
Table 4
SAR of ethylpyrazole derivatives
Compd
Ar
Kb SEa (nM)
ClogPb
hMSc (%)
OX1R
OX2R
4a
0.43 0.04
0.89 0.24
3.88
35.0
27a
27b
27c
6.67 0.44
1.48 0.33
11.42 0.97
2.82 0.19
3.58
4.15
3.40
35.3
46.9
NT
119.30 41.58
638.10 89.48
27d
27e
2.52 0.22
5.00 1.95
4.01 0.45
3.08
3.38
19.7
24.1
13.06 5.58
a
b
c
The Kb value is the mean of multiple results (at least three independent determinations performed in duplicate) with the standard error of the means.
The ClogP value was calculated using software from Daylight Chemical Information Systems, Inc.
Metabolized percentage after 15 min of incubation in human liver microsomes (1 mg protein/mL, 5 lM).
Table 5
Pharmacokinetic profiles of 27d and 27e in rats and dogs
Compd
27d
27e
Rat
Dog
Rat
Dog
Vd (mL/kg)
1790
3910
0.318
0.667
4.62
0.13
0.03
97.3
1290
786
1.13
0.500
100
—
1710
3960
0.299
0.833
14.9
0.17
0.06
79.9
745
598
0.962
0.667
80.2
—
CL (mL/h/kg)
*
T1/2 (h)
Tmax (h)
F (%)
Brain/Plasma ratio
CSF/Plasma ratio
MSa (%)
—
10.1
—
7.3
27d: For rats, intravenous administration at 2 mg/kg, oral administration (BA), or intraperitoneal administration (Brain/Plasma ratio and Cerebrospinal fluid/Plasma ratio) at
30 mg/kg to male rats. For dogs, intravenous administration at 0.5 mg/kg, and oral administration at 3 mg/kg to male dogs.
27e: For rats, intravenous administration at 2 mg/kg, and oral administration at 10 mg/kg to male rats. For dogs, intravenous administration at 0.5 mg/kg, and oral
administration at 3 mg/kg to male dogs.
Vd, CL and T1/2 were determined by intravenous administration. Tmax was determined by oral administration. F (%) was comparison between intravenous administration and
oral administration.
a
MS (%): Metabolized percentage after 15 min of incubation in liver microsomes of rats or dogs (1 mg protein/mL, 5 lM).
T1/2: ln 2 * Vd/CL.
*
to a moderate potency. These results suggested that relatively
small N-alkyl substituents were acceptable and that the ethyl
group showing more potent intrinsic potency than the methyl
group was an optimal N-alkyl substituent in terms of the OX1
and OX2 receptor antagonistic activities.
We optimized the three parts in 4a as described above, and
finally we undertook the optimization of the left end part, taking
into account the bioavailability of the molecules in humans by
assessing the in vitro stability in human microsomes. The replace-
ment of the methyl group with fluoro, chloro, and methoxy groups
was conducted. Among them, the fluoro and chloro analogues 27a
and 27b exhibited maintained orexin receptor antagonistic activi-
ties but did not show any improvements in metabolic stability.
These results might have been due to their relatively high lipo-
philic natures. The methoxy group in 27c played an important role
in reducing the lipophilicity, while 27c was not tolerated in terms
of its OX1 and OX2 receptor antagonistic activities. On the other
hand, alternative approaches to reducing lipophilicity worked to
some extent. These approaches, including 1) the replacement of
the triazole moiety in 4a with a pyrimidine group (leading to

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R. Suzuki et al. / Bioorg. Med. Chem. 23 (2015) 1260–1275
Figure 5. Effects of compound 27e on sleep parameters in a rat polysomnography study. The sleep-promoting effects on the latency until the sleep stage (A), the percentage
of time spent in total sleep (B), the time spent in non-REM (NREM) sleep (C), and the time spent in REM sleep (D) for the 2-h period after the intraperitoneal administration of
the vehicle or compound 27e are shown. Values are shown as the mean and SEM (n = 8–10 animals). ⁄⁄P <0.01, ⁄⁄⁄P <0.001 versus vehicle-treated group, based on the Bartlett
test followed by the Dunnett multiple comparison post-hoc test.
compounds were not relatively potent. As expected, 27d and 27e
exhibited relatively short half-life profiles, with a rapid Tmax in rats
and dogs. These compounds had poor bioavailability in rats (27d:
4.62%, 27e: 14.9%) and good bioavailability in dogs (27d: 100%,
27e: 80.2%). Compound 27e was then evaluated in greater detail
as a representative of N-pyrazolylethylbenzamide derivatives,
since 27e had a better CSF penetrability than 27d. A major metab-
olite of 27e was estimated to be the same in rat, dog, and human
liver microsomes. Furthermore, the major elimination pathway
was thought to be hepatic metabolism in rats, based on the low
excretion rates of the parent compound 27e in urine (0.4%) and bile
(0.2%). The gastrointestinal absorption of 27e seemed to be excel-
lent, considering that it has moderate lipophilicity and acceptable
aqueous solubility (30.2 lg/mL in pH6.8 phosphate buffer). There-
fore, hepatic microsomal stability might be a good indicator of bio-
availability. In fact, the hepatic microsomal stabilities data
(%metabolized: rat: 79.9%, dog: 7.3%) agreed well with in vivo data
demonstrating that 27e has a low bioavailability in rats and a high
bioavailability in dogs. Assuming that hepatic metabolism is the
primary mode of elimination in vivo, 27e was predicted to have
good bioavailability in humans. Therefore, further in vivo evalua-
tion of 27e was conducted as a representative of N-pyrazolyleth-
ylbenzamide derivatives (Table 5).
The sleep-promoting effect of the most balanced compound,
27e, was evaluated using a rat polysomnogram (PSG) study at an
intraperitoneal (ip) dose of 1–30 mg/kg in consideration of its
low oral bioavailability in rats. Freely moving rats with chronically
implanted electrodes were well habituated to the experiment
boxes and had access to food and water ad libitum. The compound
27e or vehicle was administered prior to lights off (start of the
active phase) and the start of recording. In the present study, 27e
showed a fast onset of action after dosing. The effects of 27e at
Figure 6. Effects of compound 27e on rotarod performance test in rats. The
compounds were administered 30 min prior to the test. ⁄⁄P <0.01 versus vehicle-
treated group (Steel test).
27d), and 2) the des-methylation of the tolyl moiety in 4a (leading
to 27e), led to slight improvements not only in lipophilicity but
also in microsomal stability while their OX1 and OX2 receptor
antagonistic activities were tolerated.
All the compounds shown in Table 4, except for 27c, had no sig-
nificant difference. Accordingly, we thought that the lower lipo-
philic compounds would show smaller Vd values and, by
extension, that these compounds might have relatively short
half-life profiles. To confirm whether our strategy was correct,
we selected the lowest lipophilic compounds 27d and 27e, and
used them in rat and dog PK studies, even though these

R. Suzuki et al. / Bioorg. Med. Chem. 23 (2015) 1260–1275
1267
Table 6
Profiles of 27e
a The Kb value is the mean of multiple results (at least three independent determinations performed in duplicate) with the standard error of the means.
b Brain/plasma and CSF/plasma of rats were collected at 0.5 h after drug administration (10 mg/kg, po) and the drug concentrations were measured (n = 3
rats/group).
c The pharmacokinetic study was conducted in fasted Sprague–Dawley rats (n = 3) and dogs dosed at 2 mg/kg and 0.5 mg/kg for intravenous dosing and
dosed at 10 mg/kg and 3 mg/kg for oral dosing. Vd, CL and T1/2 were determined by intravenous administration. Tmax was determined by oral adminis-
tration. F (%) was comparison between intravenous administration and oral administration. T1/2 was calculated from ln 2 * Vd/CL equation.
d All the assays were performed at Cerep, France.
3–30 mg/kg showed a significant reduction in the latency until
total sleep in a dose-dependent manner (Cmax plasma levels:
728 nM at 3 mg/kg, ip, 0.33 h). Furthermore, the duration of NREM
sleep was significantly increased at doses of 10 or 30 mg/kg, ip. The
duration of REM sleep also tended to increase, but the difference
was not significant during the 2-h period after administration.
The current standards of care for insomnia, such as zolpidem (a
GABAA receptor modulator), are well known to attenuate sleep
latency and to promote NREM sleep, but they also suppress the
REM components of normal sleep. Meanwhile, the sleep architec-
ture induced by 27e, a dual OX1 and OX2 receptor antagonist, is
characterized by increases in both NREM and REM sleep (Fig. 5).
Furthermore, GABAA receptor modulator-induced sleep is also
associated with a significant impairment of motor coordination.
In the present study, compound 27e at doses of 3, 10, and 30 mg/
kg, ip, did not affect the rotarod performance of rats, while zolpi-
dem at an oral dose of 10 mg/kg significantly impaired motor coor-
dination (Fig. 6). These results suggest that compound 27e is
unlikely to impair motor coordination even when administered
at a dose 10-fold above its effective dose in the PSG study. These
results indicate that 27e has potent sleep-promoting effects with-
out inducing motor impairment.
Finally, we identified 27e as a representative of N-pyrazolyleth-
ylbenzamide derivatives for further development. The detailed
profiles of 27e are summarized in Table 6. Compound 27e showed
potent OX1 and OX2 receptor dual antagonistic activities, and no
species differences were observed between humans and rats
(hOX1: 5.00 nM, rOX1: 5.08 nM, hOX2: 13.06 nM, rOX2: 6.06 nM).
Compound 27e was highly selective for orexin receptors when
tested in 92 receptor/transporter binding or enzyme activity
assays, including benzodiazepine, GABAA, adenosine, melatonin
and histamine receptors; it also exhibited acceptable solubility in
aqueous media. We demonstrated potent sleep-promoting effects
in a rat PSG study when 27e was administered at 3 mg/kg, ip, with-
out inducing motor impairment on the rat rotarod performance
test when administered at doses of up to 30 mg/kg, ip. Further-
more, 27e showed acceptable DMPK profiles, good oral bioavail-
ability in dogs (F = 80%) and moderate bioavailability in rats
(F = 15%), good brain and CSF penetrability, and short half-life pro-
files with a rapid Tmax in rats and dogs. The predicted human half-
life (single-species scaling) was calculated to be 1.2–1.4 h.20
Finally, 27e was also predicted to have a rapid onset of action in
humans. These data suggest that 27e could be a useful drug for
insomnia with a low risk of next-day drowsiness.
4. Conclusion
Our SAR characterization of a novel series of dual orexin
receptor antagonist, represented by N-pyrazolylethylbenzamide
derivatives, has led to the identification of 27e as a potent, short
half-life, brain-penetrating agent for the treatment of insomnia.
The design of designed lead A was used as an LBDD method
based on published DORAs.
A reduced lipophilicity through
systematic modification led to 27d and 27e, which exhibited
acceptable liver microsomal stability and moderate lipophilicity
profiles to obtain
a
relatively low Vd value compound.
Finally, we identified N-ethyl-N-[2-[3-(5-fluoro-2-pyridinyl)-1H-
pyrazol-1-yl]ethyl]-2-(2H-1,2,3-triazol-2-yl)-benzamide 27e as
an advanced compound. Compound 27e had an excellent phar-
macokinetic profile with a rapid Tmax and a short half-life as well
as good dual OX1 and OX2 receptor antagonistic activities. The
compound also demonstrated potent sleep-promoting effects in
a rat PSG study at a dose of 3 mg/kg, ip. Furthermore, the pre-
dicted human half-life (single-species allometric scaling) was
calculated to be 1.2–1.4 h.20 Compound 27e fits the profile that
was set as our initial goal: a short-acting DORA. These data sug-
gest that 27e might be useful as an insomnia drug without the
risk of next-day drowsiness. In the near future, we plan to select
a clinical candidate with the best profile from among these
derivatives.

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R. Suzuki et al. / Bioorg. Med. Chem. 23 (2015) 1260–1275
socketed into an electrode pedestal (MS363; PlasticsOne) and fixed
to the skull with dental resin and superglue. The rats were allowed
to recover for 7 days or more before the PSG recording.
5. Experimental section
5.1. Biology
5.1.1. Intracellular Ca2+ mobilization assay
5.1.4. Polysomnogram recordings
Rats were weighed and transferred to the measurement cage
(width, 220 mm  depth, 300 mm  height, 380 mm; lighting per-
iod: 7:00–19:00; free access to food and water) 8–11 h before drug
administration. Their electrode pedestal was tethered to a lead
wire with a slip ring, and the animals were acclimated to the mea-
surement environment under a freely moving and unrestrained
condition. The EEG/EMG recordings were started at 16:00 on the
administration day. The test compound and vehicle were adminis-
tered between 18:50 and 19:00. In addition, the measurements
were continued for up to 13 h after administration. The signals
from the EEG and EMG electrodes were amplified and passed
through bandpass filters (EEG, 0.16–30 Hz; EMG, 5.3–1000 Hz)
using a biophysical amplifier (Dia Medical System, Tokyo, Japan).
The signals were digitized at a sampling rate of 512 Hz with an
analog-to-digital converter (Contec, Osaka, Japan) and were
recorded using the data acquisition program SleepSignÒ ver. 2.0
(Kissei Comtec, Nagano, Japan).
Chinese hamster ovary (CHO) cells stably expressing the recom-
binant human OX1 receptor (hOX1/CHO) or human OX2 receptor
(hOX2/CHO) were used in the present study. The cells were seeded
into a 96-well black/clear bottom plate (Thermo Fisher Scientific,
Rochester, NY) at 2.4 Â 104 cells/well and cultured for 18 h. The
cells were then incubated with a loading buffer Ca2+-sensitive fluo-
rescent dye, Fluo-4 AM (0.5 lM final concentration; Molecular
Probes, Eugene, OR) in an assay buffer containing Hank’s balanced
salt solution (Life Technologies, Carlsbad, CA), 20 mM HEPES, 0.1%
BSA, 2.5 mM probenecid and 0.2 mg/mL amaranth at pH7.4 (load-
ing buffer) at 37 °C for 1 h. The loading buffer was removed, and
the cells were washed once with the assay buffer to remove extra-
cellular Fluo-4 AM. Then, the cells were incubated with various
concentrations of test compounds for 30 min at room temperature.
Intracellular Ca2+ mobilization was measured using a Functional
Drug Screening System FDSS/lCELL (Hamamatsu Photonics, Shi-
zuoka, Japan).
Responses were measured as the peak increase in fluorescence
minus the basal level, normalized to the maximal stimulatory
effect induced by the modified human orexin peptide ligand
[Ala6,12]orexin-A (Peptide Institute, Osaka, Japan). The concentra-
tion-response curves were fitted, and the agonist EC50 values and
the antagonist IC50 values were calculated using GraphPad Prism
5.0 (GraphPad Software, San Diego, CA). Then, the Kb values were
calculated according to the following equation: Kb = IC50/(1+[L]/
EC50), where L is the concentration of agonist added to the assay
and the IC50 and EC50 values were derived from the antagonist inhi-
bition and agonist response curves, respectively. The concentra-
tion-response curve data shown were the mean with SEM of
three individual experiments carried out in duplicate.
5.1.5. Sleep-wake state determination and data analysis
The sleep-wake states were classified into 8-s epochs as wake-
fulness, NREM sleep, and REM sleep based on the EEG and EMG
patterns, followed by visual verification according to the standard
criteria. Each state was characterized as follows: wakefulness, high
EMG amplitude and low EEG amplitude; NREM sleep, low EMG
amplitude and high EEG amplitude with a high-power density in
the delta band (0.5–4.0 Hz); and REM sleep, very low EMG ampli-
tude and low EEG amplitude with high values in the theta band
(4.0–8.0 Hz). The cumulative times of wakefulness, NREM sleep,
REM sleep, and total sleep (NREM plus REM sleep) during a 2-h
period were calculated and were expressed as a percentage of all
the states. The latency of sleep was defined as the time from drug
administration until the first 15 consecutive 8-s epochs of total
sleep.
5.1.2. Animals
Male Wistar rats (9 weeks old, Charles River Laboratories, Yoko-
hama, Japan) were used to perform the polysomnogram (PSG)
study. Male Sprague–Dawley rats (6 weeks old, Charles River Lab-
oratories) were used for the rotarod test. The animals were housed
four-to-five per cage under controlled temperature (23 3 °C) and
humidity (50 20%) conditions and a 12-h light-dark cycle (lights
on 07:00–19:00). Electrode-implanted rats were singly housed
before the performance of the PSG study at an age of 11–12 weeks.
Food and water were available ad libitum.
All the experimental procedures were reviewed and approved
by the Taisho Pharmaceutical Co., Ltd. Animal Care Committee
and met the Japanese Experimental Animal Research Association
standards, as defined in the Guidelines for Animal Experiments
(1987).
5.1.6. Rotarod performance
The rotarod (ENV-577; Med Associates, St. Albans, VT), con-
sisted of a gritted plastic roller (3.0 cm in diameter, 8.9 cm long)
flanked by two large round plates to prevent the animals from
escaping and was run at 10 rpm. All the animals were given control
trials prior to the test. A rat was placed on the roller, and the length
of time it remained there was measured. A maximum of 2 min was
allowed for each animal. The test compound 27e (3–30 mg/kg, ip)
and the positive control zolpidem (10 mg/kg, po) were adminis-
tered 30 min prior to the test.
5.1.7. Statistical analysis
Data from the PSG study (sleep latency and accumulated time
of each sleep stage) and rotarod performance (duration time on
gritted plastic roller) were expressed as the mean SEM. The
statistical analysis was performed using SAS System Version
8.2 (SAS Institute Japan, Tokyo, Japan). In the case of the PSG
test, the effect of the test compound was analyzed using the
Bartlett test (for homogeneity of variance) within the vehicle-
treated group and at each dosage of the test compound in the
test compound-treated groups. If the variance was homogeneous,
then the data were analyzed using the Dunnett test. If the vari-
ance was not homogeneous, then the data were analyzed using
the Steel test. For the rotarod performance test, the data were
statistically evaluated using the Steel test. Values of P <0.05 were
regarded as significant.
5.1.3. Surgical procedures for electrode implantation
Ten-week-old rats were anaesthetized with pentobarbital
sodium (50 mg/kg, ip) and fixed on a stereotaxic apparatus. After
drilling holes in the skull, stainless steel screw electrodes (E363/
20; PlasticsOne, Roanoke, VA) were placed on the cerebral dura
mater of the bilateral frontal cortex (as the recording electrode:
2.0 mm anterior, 1.5 mm bilateral from the bregma) and the pari-
etal cortex (as the reference electrode: 6.0 mm posterior, 2.0 mm
lateral from the bregma) for the electroencephalogram (EEG)
recording, according to a rat brain atlas. To record the electromyo-
gram (EMG), two stainless steel subcutaneous electrodes (E363/76;
PlasticsOne) were used, with needles that were bipolarly
implanted into the dorsal neck region. These electrodes were

R. Suzuki et al. / Bioorg. Med. Chem. 23 (2015) 1260–1275
1269
5.2. Chemistry
J = 8.75, 4.29 Hz, 1H), 8.48 (d, J = 2.74 Hz, 1H); 13C NMR
(500 MHz, CDCl3, 25 °C) d ppm 103.50, 121.22, 121.26, 123.95,
124.10, 136.71, 137.76, 137.95, 146.38, 158.11, 160.16.
5.2.1. General methods
All the solvents and reagents were obtained from commercial
suppliers and were used without further purification or were pre-
pared according to published procedures. The 1H and 13C NMR
spectra were recorded using a JOEL 500 MHz NMR spectrometer
and all the chemical shifts were reported in parts per million
(ppm) relative to tetramethylsilane (TMS) as an internal standard.
High resolution mass spectral data were acquired using a Shima-
dzu LCMS-IT-TOF equipped with an ESI/APCI dual ion source. The
final compounds exhibited a P95% purity, as determined using
HPLC and LC–MS on an Agilent instrument using electrospray ion-
ization. The HPLC conditions were as follows: Shimadzu LC-20A;
5.2.3. 2-(1H-Pyrazol-3-yl)-5-(trifluoromethyl)pyridine
hydrochloride (2b)
The title compound was synthesized according to the procedure
described for compound 2a from 2-bromo-5-trifluoromethylpyri-
dine (69% over
2
steps). HRMS calcd for C9H6F3N3 [M+H]+
214.0587, found 214.0577. LC–MS t = 0.73 min, [M+H]+ = 214.
1H NMR (500 MHz, DMSO-d6, 25 °C) d ppm 6.91 (d, J = 2.06 Hz,
1H), 7.81 (d, J = 2.40 Hz, 1H), 8.10–8.13 (m, 1H), 8.20 (dd, J = 8.58,
2.06 Hz, 1H), 8.89–8.93 (m, 1H); 13C NMR (500 MHz, DMSO-d6,
25 °C) d ppm 104.77, 119.85, 123.36, 123.80, 124.06, 125.53,
134.92, 146.58, 148.73, 155.70.
column Shim-Pack XR-ODS 2.2
lm, 3.0 mm  75 mm; eluent A,
water + 0.1% phosphoric acid; eluent B, acetonitrile; (1) 10% B for
4.0 min, 10–90% B for 5.0 min, 90% B for 2.0 min, (2) 20% B
for 4.0 min, 20–90% B for 5.0 min, 90% B for 2.0 min or (3) 40% B
for 4.0 min, 40–90% B for 5.0 min, 90% B for 2.0 min; flow rate
0.8 mL/min; UV detection, k = 210 nm. The LC–MS conditions were
as follows: Agilent 1290 infinity and Agilent 6150; column Waters
5.2.4. 2-(1H-Pyrazol-3-yl)-4-(trifluoromethyl)pyridine
hydrochloride (2c)
The title compound was synthesized according to the procedure
described for compound 2a from 2-bromo-4-trifluoromethylpyri-
dine (70% over
2
steps). HRMS calcd for C9H6F3N3 [M+H]+
Acquity CSH C18, 1.7
l
m, 2.1 mm  50 mm; eluent A, water + 0.1%
214.0587, found 214.0581. LC–MS t = 0.71 min, [M+H]+ = 214.
1H NMR (500 MHz, DMSO-d6, 25 °C) d ppm 6.94 (d, J = 2.41 Hz,
1H), 7.65 (dt, J = 5.15, 0.86 Hz, 1H), 7.81 (d, J = 2.40 Hz, 1H), 8.17
(s, 1H), 8.83 (d, J = 5.15 Hz, 1H); 13C NMR (500 MHz, DMSO-d6,
25 °C) d ppm 104.47, 115.10, 115.13, 118.31, 118.34, 122.40,
124.57, 132.86, 137.70, 137.97, 138.24, 138.51, 148.38, 151.35,
153.30.
formic acid; eluent B, acetonitrile + 0.1% formic acid; 20–99% B for
1.2 min, 99% B for 0.2 min; flow rate 0.8 mL/min; UV detection,
k = 254 nm.
The N-alkylated benzamide orexin receptor antagonists
described in this paper exist in two conformations as a result of
amide rotamers that are slow on the NMR timescale. The 1H and
13C NMR spectra of compounds 4a–4f, 7, 8, 9, 12, 16, 21, 26a,
26b and 27a–27e consist of broad, overlapping multiplets preclud-
ing a detailed coupling constant analysis. Thus, the NMR reso-
nances of these compounds are not listed in numerical format.
Instead, pictures of the 1H and 13C NMR spectra of these com-
pounds at 25 °C are included in the Supplementary data. For com-
pound 27e only, the 1H and 13C chemical shifts were assigned
based on an analysis of the COSY, HSQC, and HMBC spectra
acquired for a sample dissolved in acetone-d6 at 25 °C.
5.2.5. 2-(1H-Pyrazol-3-yl)-6-(trifluoromethyl)pyridine (2d)
The title compound was synthesized according to the procedure
described for compound 2a from 2-bromo-6-trifluoromethylpyri-
dine (48% over
2
steps). HRMS calcd for C9H6F3N3 [M+H]+
214.0587, found 214.0575. LC–MS t = 0.90 min, [M+H]+ = 214.
1H NMR (500 MHz, CDCl3, 25 °C) d ppm 6.92 (d, J = 1.71 Hz, 1H),
7.59 (d, J = 8.58 Hz, 1H), 7.68 (d, J = 2.06 Hz, 1H), 7.87–7.93 (m, 1H),
7.94–7.99 (m, 1H); 13C NMR (500 MHz, CDCl3, 25 °C) d ppm 104.44,
118.20, 119.21, 120.38, 122.55, 124.75, 136.31, 138.29, 147.67,
147.95, 148.23, 148.51, 150.39.
5.2.2. 5-Fluoro-2-(1H-pyrazol-3-yl)pyridine (2a)
Pd(PPh3)4 (9.23 g, 7.99 mmol) and a 2 mol/L aqueous Na2CO3
solution (2.4 L) were added to an EtOH (1.6 L)/ toluene (1.6 L)
mixed solution of 1 (500 g, 1.8 mol) and 2-bromo-5-fluoropyridine
(281.2 g, 1.60 mol), and the mixture was heated to 90 °C and stir-
red for 2 h. After standing to cool at 0 °C, water was added to the
reaction mixture, followed by extraction with EtOAc. The extracted
organic layer was distilled off under reduced pressure. The
obtained residue was stirred in EtOAc (1.2 L), and NH silica gel
was added thereto. The mixture was stirred at room temperature
for 1 h. Then, the silica gel was filtered off, and the solvent was dis-
tilled off under reduced pressure to obtain 5-fluoro-2-(1-(tetrahy-
dro-2H-pyran-2-yl)-1H-pyrazol-5-yl)pyridine as a brown oil.
A 4 mol/L HCl–EtOAc solution (1.2 L) was added to a solution of
5-fluoro-2-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-5-yl)pyridine
in MeOH (1.0 L), and the mixture was stirred at room temperature
for 5 h. The deposited solid was then collected by filtration. The
obtained solid was stirred in water (2.0 L), and an 8 mol/L aqueous
NaOH solution (0.3 L) was added thereto under ice cooling. The
mixture was extracted with EtOAc, and the solvent was distilled
off under reduced pressure. The obtained residue was stirred for
1 h in Et2O. Then, the deposited solid was collected by filtration
and dried by heating under reduced pressure to obtain the title
compound 2a as a colorless powder (185 g, 71% over 2 steps).
HRMS calcd for C8H6FN3 [M+H]+ 164.0619, found 164.0610.
LC–MS t = 0.50 min, [M+H]+ = 164.
5.2.6. 2-Methyl-6-(1H-pyrazol-3-yl)pyridine (2e)
The title compound was synthesized according to the procedure
described for compound 2a from 2-bromo-6-trifluoromethylpyri-
dine (35% over 2 steps). HRMS calcd for C9H9N3 [M+H]+ 160.0869,
found 160.0878. LC–MS t = 0.24 min, [M+H]+ = 160.
1H NMR (500 MHz, CDCl3, 25 °C) d ppm 2.57 (s, 3H), 6.74 (d,
J = 1.72 Hz, 1H), 7.07 (d, J = 7.89 Hz, 1H), 7.49 (d, J = 7.89 Hz, 1H),
7.57–7.62 (m, 1H), 7.63 (d, J = 2.06 Hz, 1H); 13C NMR (500 MHz,
CDCl3, 25 °C)
d ppm 24.51, 103.16, 105.12, 117.16, 122.49,
137.16, 138.31, 148.39, 158.44.
5.2.7. N-Ethyl-2-[3-(5-fluoropyridin-2-yl)-1H-pyrazol-1-yl]etha-
namine dihydrochloride (3a)
A solution of 3-ethyl-1,2,3-oxathiazolidine-2,2-dioxide (2.22 g,
14.7 mmol) in CH3CN (5.0 mL) was added dropwise to a mixture
of 5-fluoro-2-(1H-pyrazol-3-yl)pyridine (2.00 g, 12.3 mmol), NaOH
(0.74 g, 18.4 mmol), and CH3CN (15 mL) at 70 °C, and the mixture
was stirred at the same temperature as above for 1 h. After ice
cooling, 25% H2SO4 (12 mL) was added thereto at 25 °C. The reac-
tion mixture was heated and stirred at 70 °C for 4 h. After ice cool-
ing, an 8 mol/L aqueous NaOH solution was added thereto, and the
mixture was then separated into aqueous and organic layers. The
solvent in the organic layer was distilled off under reduced pres-
sure. The residue was stirred in EtOAc (20 mL), and a 4 mol/L
HCl–EtOAc solution (4.6 mL) was added thereto under ice cooling.
The mixture was stirred at room temperature for 1 h, and the
1H NMR (500 MHz, CDCl3, 25 °C) d ppm 6.77 (d, J = 2.06 Hz, 1H),
7.46 (td, J = 8.40, 3.09 Hz, 1H), 7.64 (d, J = 2.06 Hz, 1H), 7.78 (dd,

1270
R. Suzuki et al. / Bioorg. Med. Chem. 23 (2015) 1260–1275
deposited solid was then collected by filtration. The obtained solid
was dried by heating under reduced pressure to obtain the title
compound 3a as a colorless powder (2.7 g, 72%). HRMS calcd for
1H NMR (500 MHz, DMSO-d6, 25 °C) d ppm 1.21 (t, J = 7.20 Hz,
3H), 2.75 (s, 3H), 2.87–3.04 (m, 2H), 3.41 (quin, J = 6.17 Hz, 2H),
4.62 (t, J = 6.17 Hz, 2H), 7.27 (br s, 1H), 7.54–7.68 (m, 1H), 8.04
(d, J = 2.40 Hz, 1H), 8.11 (d, J = 7.20 Hz, 1H), 8.25 (d, J = 0.69 Hz,
1H); 13C NMR (500 MHz, DMSO-d6, 25 °C) d ppm 11.33, 21.32,
42.81, 45.94, 48.35, 106.99, 120.58, 125.65, 134.20, 144.11,
146.84, 155.90.
C
12H15FN4 [M+H]+ 235.1354, found 235.1347. LC–MS t = 0.26 min,
[M+H]+ = 235.
1H NMR (500 MHz, DMSO-d6, 25 °C) d ppm 1.17 (t, J = 7.38 Hz,
3H), 2.87–2.97 (m, 2H), 3.38 (quin, J = 6.26 Hz, 2H), 4.53 (t,
J = 6.52 Hz, 2H), 6.81 (d, J = 2.06 Hz, 1H), 7.77 (td, J = 8.75,
2.74 Hz, 1H), 7.90 (d, J = 2.40 Hz, 1H), 7.98 (dd, J = 8.75, 4.63 Hz,
1H), 8.55 (d, J = 3.09 Hz, 1H); 13C NMR (500 MHz, DMSO-d6,
25 °C) d ppm 11.37, 42.65, 46.08, 48.02, 104.82, 121.47, 121.50,
124.74, 124.90, 133.41, 137.35, 137.54, 148.70, 150.97, 158.00,
160.02.
5.2.12. N-Ethyl-2-[3-(4-fluorophenyl)-1H-pyrazol-1-yl]ethana-
mine dihydrochloride (3f)
The title compound was synthesized according to the procedure
described for compound 3a from 3-(4-fluorophenyl)-1H-pyrazole
2f (59%). HRMS calcd for C13H16FN3 [M+H]+ 234.1401, found
234.1383. LC–MS t = 0.39 min, [M+H]+ = 234.
1H NMR (500 MHz, DMSO-d6, 25 °C) d ppm 1.16 (t, J = 7.20 Hz,
3H), 2.87–2.97 (m, 2H), 3.36 (quin, J = 6.26 Hz, 2H), 4.48 (t,
J = 6.35 Hz, 2H), 6.73 (d, J = 2.40 Hz, 1H), 7.20 (t, J = 8.92 Hz, 2H),
7.79–7.87 (m, 3H); 13C NMR (500 MHz, DMSO-d6, 25 °C) d ppm
11.40, 42.65, 46.13, 47.84, 103.47, 115.93, 116.10, 127.65,127.71,
130.26, 130.29, 133.22, 150.48, 161.29, 163.23.
5.2.8. N-Ethyl-2-{3-[5-(trifluoromethyl)pyridin-2-yl]-1H-pyrazol-
1-yl}ethanamine dihydrochloride (3b)
The title compound was synthesized according to the procedure
described for compound 3a from 2-(1H-pyrazol-3-yl)-5-(trifluoro-
methyl)pyridine hydrochloride 2b (45%). HRMS calcd for C13H15
F3N4 [M+H]+ 285.1322, found 285.1307. LC–MS t = 0.41 min,
[M+H]+ = 285.
1H NMR (500 MHz, DMSO-d6, 25 °C) d ppm 1.01–1.29 (m, 3H),
2.80–3.08 (m, 2H), 3.42 (quin, J = 6.17 Hz, 2H), 4.54 (t, J = 6.35 Hz,
2H), 6.94 (d, J = 2.40 Hz, 1H), 7.95 (d, J = 2.40 Hz, 1H), 8.11 (d,
J = 8.23 Hz, 1H), 8.22 (dd, J = 8.40, 2.23 Hz, 1H), 8.93 (dd, J = 1.37,
0.69 Hz, 2H); 13C NMR (500 MHz, DMSO-d6, 25 °C) d ppm 11.40,
42.74, 46.11, 48.30, 105.82, 119.93, 123.33, 124.02, 124.28,
125.51, 133.75, 134.88, 134.90, 146.71, 146.74, 150.80, 155.70.
5.2.13. N-Ethyl-N-{2-[3-(5-fluoropyridin-2-yl)-1H-pyrazol-1-yl]
ethyl}-2-(2H-1,2,3-triazol-2-yl)benzamide (27e)
1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochlo-
ride (WSCÁHCl) (37.4 g, 0.20 mol) was added to a solution of N-
ethyl-2[3-(5-fluoro-pyridin-2-yl)-1H-pyrazol-1-yl]
ethanamine
dihydrochloride (50 g, 0.16 mol), 5-methyl-2-(2H-1,2,3-triazol-2-
yl)benzoic acid (33.9 g, 0.18 mol), 1-hydroxy-1H-benzotriazole
hydrate (HOBtÁH2O) (26.4 g, 0.20 mol) and triethylamine (TEA)
(39.5 g, 0.39 mol) in THF (500 mL) at 0 °C, followed by stirring at
room temperature for 20.5 h. The solvent of the reaction solution
was distilled off under reduced pressure. An aqueous NaHCO3 solu-
tion and EtOAc were added to the resulting residue, followed by
extraction with EtOAc. The organic layer was washed with water,
and the solvent was distilled off under reduced pressure to yield
the crude product (72 g). An additional 196.7 g of the crude prod-
uct was synthesized using the same method from N-ethyl-2[3-(5-
fluoro-pyridin-2-yl)-1H-pyrazol-1-yl] ethanamine dihydrochloride
(140 g, 0.46 mol) and 5-methyl-2-(2H-1,2,3-triazol-2-yl)benzoic
acid (94.8 g, 0.50 mol). The obtained crude product (268.7 g) was
recrystallized with heptane and EtOAc. The deposited solid was
then collected by filtration. The obtained solid was dried by heat-
ing under reduced pressure to yield the title compound 27e as a
colorless powder (217.3 g). HRMS calcd for C21H20FN7O [M+H]+
406.1786, found 406.1805. LC–MS t = 0.95 min, [M+H]+ = 406.
Compound 27e exists as a mixture of two rotameric compo-
nents in chemical exchange in acetone-d6 at room temperature
(1:0.57). The 1H and 13C chemical shifts of the two rotamers were
assigned based on an analysis of the COSY, HSQC, and HMBC spec-
tra acquired for a sample dissolved in acetone-d6 at 25 °C.
Rotamer 1 (64% of total): 1H NMR (500 MHz, acetone-d6, 25 °C)
d ppm 0.90 (3H), 3.02–3.14 (2H), 3.87–3.97 (2H), 4.39–4.51 (1H),
4.57–4.67 (1H), 6.87 (1H), 7.47–7.53 (2H), 7.58–7.67 (2H), 7.84
(1H), 8.00 (1H), 8.01 (2H), 8.11 (1H), 8.48 (1H).
5.2.9. N-Ethyl-2-{3-[4-(trifluoromethyl)pyridin-2-yl]-1H-pyrazol-
1-yl}ethanamine dihydrochloride (3c)
The title compound was synthesized according to the procedure
described for compound 3a from 2-(1H-pyrazol-3-yl)-4-(trifluoro-
methyl)pyridine hydrochloride 2c (85%). HRMS calcd for
C
13H15F3N4 [M+H]+ 285.1322, found 285.1311. LC–MS t = 0.28
min, [M+H]+ = 285.
1H NMR (500 MHz, DMSO-d6, 25 °C) d ppm 1.17 (t, J = 7.20 Hz,
3H), 2.85–3.00 (m, 2H), 3.41 (quin, J = 6.17 Hz, 2H), 4.55 (t,
J = 6.35 Hz, 2H), 6.93 (d, J = 2.06 Hz, 1H), 7.67 (d, J = 4.12 Hz, 1H),
7.96 (d, J = 2.40 Hz, 1H), 8.14 (s, 1H), 8.84 (d, J = 5.15 Hz, 1H); 13C
NMR (500 MHz, DMSO-d6, 25 °C) d ppm 11.37, 42.68, 46.07,
48.20, 105.36, 114.98, 115.01, 118.48, 118.51, 120.25, 122.43,
124.60, 126.78, 133.80, 137.50, 137.77, 138.04, 138.31, 150.75,
151.62, 153.52.
5.2.10. N-Ethyl-2-{3-[6-(trifluoromethyl)pyridin-2-yl]-1H-pyra-
zol-1-yl}ethanamine hydrochloride (3d)
The title compound was synthesized according to the procedure
described for compound 3a from 2-(1H-pyrazol-3-yl)-6-(trifluoro-
methyl)pyridine 2d (44%). HRMS calcd for C13H15F3N4 [M+H]+
285.1322, found 285.1314. LC–MS t = 0.45 min, [M+H]+ = 285.
1H NMR (500 MHz, DMSO-d6, 25 °C) d ppm 0.97–1.34 (m, 3H),
2.93 (q, J = 7.32 Hz, 2H), 3.40 (t, J = 6.35 Hz, 2H), 4.54 (t,
J = 6.35 Hz, 2H), 6.88 (d, J = 2.40 Hz, 1H), 7.79 (dd, J = 7.55,
0.69 Hz, 1H), 7.94 (d, J = 2.40 Hz, 1H), 8.07–8.14 (m, 1H), 8.15–
8.22 (m, 1H); 13C NMR (500 MHz, DMSO-d6, 25 °C) d ppm 11.41,
42.71, 46.14, 48.23, 105.40, 119.90, 121.04, 123.22, 123.51,
125.41, 133.66, 139.75, 146.42, 146.69, 146.96, 147.22, 150.63,
152.80.
13C NMR (500 MHz, acetone-d6, 25 °C) d ppm 13.33, 44.92,
45.76, 49.95, 104.77, 104.80, 121.35, 123.03, 124.22, 128.82,
128.94, 130.49, 131.05, 131.09, 132.82, 132.87, 136.95, 137.13,
137.80, 150.16, 151.92, 160.72, 169.54.
Rotamer 2 (36% of total): 1H NMR (500 MHz, acetone-d6, 25 °C)
d ppm 1.19 (3H), 3.16–3.25 (1H), 3.49–3.57 (1H), 3.65–3.75 (1H),
3.79–3.87 (1H), 4.22–4.28 (1H), 4.39–4.51 (1H), 6.83 (1H), 6.91
(1H), 7.17 (1H), 7.47–7.53 (1H), 7.58–7.67 (1H), 7.64 (1H), 7.89
(1H), 7.92 (1H), 7.96 (2H), 8.48 (1H).
5.2.11. N-Ethyl-2-[3-(6-methylpyridin-2-yl)-1H-pyrazol-1-yl]
ethanamine dihydrochloride (3e)
The title compound was synthesized according to the procedure
described for compound 3a from 2-methyl-6-(1H-pyrazol-3-
yl)pyridine 2e (44%). HRMS calcd for C13H18N4 [M+H]+ 231.1604,
found 231.1593. LC–MS t = 0.25 min, [M+H]+ = 231.
13C NMR (500 MHz, acetone-d6, 25 °C) d ppm 11.45, 39.56,
48.72, 50.70, 104.77, 104.80, 121.46, 122.80, 124.08, 128.82,

R. Suzuki et al. / Bioorg. Med. Chem. 23 (2015) 1260–1275
1271
129.06, 130.14, 131.05, 131.09, 132.82, 132.87, 136.83, 136.90,
137.61, 150.00, 152.10, 158.71, 169.24.
2-yl)-1H-pyrazol-1-yl]ethanamine dihydrochloride 3e and 5-
methyl-2-(2H-1,2,3-triazol-2-yl)benzoic acid (47%). HRMS calcd
for
C
23H25N7O [M+H]+ 416.2193, found 416.2185. LC–MS
5.2.14. N-Ethyl-N-{2-[3-(5-fluoropyridin-2-yl)-1H-pyrazol-1-yl]
ethyl}-5-methyl-2-(2H-1,2,3-triazol-2-yl)benzamide (4a)
TEA (1.20 mL, 8.63 mmol) was added to a solution of N-ethyl-
2[3-(5-fluoro-pyridin-2-yl)-1H-pyrazol-l-yl] ethanamine dihydro-
chloride (0.50 g, 1.63 mmol) in CHC13 (5 mL) at room temperature.
5-methyl-2-(2H-1,2,3-triazol-2-yl)benzoic acid (0.36 g, 1.79 mmol)
and propylphosphonic acid anhydride (cyclic trimer) (50% solution
in DMF [approximately 1.6 mol/L], 1.32 mL, 2.12 mmol) were
added to the reaction solution under cooling in ice water. The
resultant mixture was stirred at 50 °C for 5 h. After standing until
cooled to room temperature, water was added thereto, followed
by extraction with CHCl3. The organic layer was washed with brine.
Then, the organic layer was dried over MgSO4 and the desiccant
was filtered off. Then, the solvent was distilled off under reduced
pressure. The obtained residue was purified by column chromatog-
raphy (20–80% EtOAc in hexanes) and washed with Et2O to yield
the title compound 4a as a colorless powder (0.51 g, 75%). HRMS
calcd for C22H22FN7O [M+H]+ 420.1943, found 420.1936. LC–MS
t = 1.00 min, [M+H]+ = 420. Please see the Supplementary data for
pictures of 500 MHz 1H and 500 MHz 13C NMR spectra in
acetone-d6 at 25 °C.
t = 0.52–0.64 min, [M+H]+ = 416. Please see the Supplementary
data for pictures of 500 MHz 1H and 500 MHz 13C NMR spectra in
acetone-d6 at 25 °C.
5.2.19. N-Ethyl-N-{2-[3-(4-fluorophenyl)-1H-pyrazol-1-yl]
ethyl}-5-methyl-2-(2H-1,2,3-triazol-2-yl)benzamide (4f)
The title compound was synthesized according to the procedure
described for compound 4a from N-ethyl-2-[3-(4-fluorophenyl)-
1H-pyrazol-1-yl]ethanamine dihydrochloride 3f and 5-methyl-
2-(2H-1,2,3-triazol-2-yl)benzoic acid (88%). HRMS calcd for
C
23H23FN6O [M+H]+ 419.1990, found 419.1980. LC–MS
t = 1.16 min, [M+H]+ = 419. Please see the Supplementary data for
pictures of 500 MHz 1H and 500 MHz 13C NMR spectra in
acetone-d6 at 25 °C.
5.2.20. N-Ethyl-5-fluoro-N-{2-[3-(5-fluoropyridin-2-yl)-1H-
pyrazol-1-yl]ethyl}-2-(2H-1,2,3-triazol-2-yl)benzamide (27a)
The title compound was synthesized according to the procedure
described for compound 27e from 5-fluoro-2-(2H-1,2,3-triazol-2-
yl)benzoic acid (60%). HRMS calcd for
C
21H19F2N7O [M+H]+
424.1692, found 424.1687. LC–MS t = 0.93 min, [M+H]+ = 424.
Please see the Supplementary data for pictures of 500 MHz 1H
and 500 MHz 13C NMR spectra in acetone-d6 at 25 °C.
5.2.15. N-Ethyl-5-methyl-2-(2H-1,2,3-triazol-2-yl)-N-(2-{3-[5-
(trifluoromethyl)pyridin-2-yl]-1H-pyrazol-1-yl}ethyl)benzamide
(4b)
5.2.21. 5-Chloro-N-ethyl-N-{2-[3-(5-fluoropyridin-2-yl)-1H-
pyrazol-1-yl]ethyl}-2-(2H-1,2,3-triazol-2-yl)benzamide (27b)
The title compound was synthesized according to the procedure
described for compound 4a from 5-chloro-2-(2H-1,2,3-triazol-2-
The title compound was synthesized according to the procedure
described for compound 27e from N-ethyl-2-{3-[5-(trifluoro-
methyl)pyridin-2-yl]-1H-pyrazol-1-yl}ethanamine dihydrochlo-
ride 3b and 5-methyl-2-(2H-1,2,3-triazol-2-yl)benzoic acid (52%).
HRMS calcd for C23H22F3N7O [M+H]+ 470.1911, found 470.1901.
LC–MS t = 1.07 min, [M+H]+ = 470. Please see the Supplementary
data for pictures of 500 MHz 1H and 500 MHz 13C NMR spectra in
acetone-d6 at 25 °C.
yl)benzoic acid (42%). HRMS calcd for
C
21H19ClFN7O [M+H]+
440.1396, found 440.1390. LC–MS t = 0.93–1.05 min, [M+H]+ =
440. Please see the Supplementary data for pictures of 500 MHz
1H and 500 MHz 13C NMR spectra in acetone-d6 at 25 °C.
5.2.22. N-Ethyl-N-{2-[3-(5-fluoropyridin-2-yl)-1H-pyrazol-1-yl]
ethyl}-5-methoxy-2-(2H-1,2,3-triazol-2-yl)benzamide (27c)
The title compound was synthesized according to the procedure
described for compound 4a from 5-methoxy-2-(2H-1,2,3-triazol-2-
5.2.16. N-Ethyl-5-methyl-2-(2H-1,2,3-triazol-2-yl)-N-(2-{3-[4-
(trifluoromethyl)pyridin-2-yl]-1H-pyrazol-1-yl}ethyl)benzamide
(4c)
The title compound was synthesized according to the procedure
described for compound 27e from N-ethyl-2-{3-[4-(trifluoro-
methyl)pyridin-2-yl]-1H-pyrazol-1-yl}ethanamine dihydrochlo-
ride 3c, 5-methyl-2-(2H-1,2,3-triazol-2-yl)benzoic acid and HATU
which was used as condensation agent instead of WSCÁHCl (62%).
HRMS calcd for C23H22F3N7O [M+H]+ 470.1911, found 470.1900.
LC–MS t = 1.06 min, [M+H]+ = 470. Please see the Supplementary
data for pictures of 500 MHz 1H and 500 MHz 13C NMR spectra in
acetone-d6 at 25 °C.
yl)benzoic acid (44%). HRMS calcd for C
22H22FN7O2 [M+H]+
436.1892, found 436.1878. LC–MS t = 0.98 min, [M+H]+ = 436.
Please see the Supplementary data for pictures of 500 MHz 1H
and 500 MHz 13C NMR spectra in acetone-d6 at 25 °C.
5.2.23. N-Ethyl-N-{2-[3-(5-fluoropyridin-2-yl)-1H-pyrazol-1-yl]
ethyl}-5-methyl-2-(pyrimidin-2-yl)benzamide (27d)
The title compound was synthesized according to the procedure
described for compound 4a from 5-methyl-2-(pyrimidin-2-yl)ben-
zoic acid (36%). HRMS calcd for C24H23FN6O [M+H]+ 431.1990,
found 431.2001. LC–MS t = 0.98 min, [M+H]+ = 431. Please see the
Supplementary data for pictures of 500 MHz 1H and 500 MHz 13C
NMR spectra in acetone-d6 at 25 °C.
5.2.17. N-Ethyl-5-methyl-2-(2H-1,2,3-triazol-2-yl)-N-(2-{3-[6-
(trifluoromethyl)pyridin-2-yl]-1H-pyrazol-1-yl}ethyl)benzamide
(4d)
The title compound was synthesized according to the procedure
described for compound 4a from N-ethyl-2-{3-[6-(trifluoro-
methyl)pyridin-2-yl]-1H-pyrazol-1-yl}ethanamine hydrochloride
3d and 5-methyl-2-(2H-1,2,3-triazol-2-yl)benzoic acid (78%).
HRMS calcd for C23H22F3N7O [M+H]+ 470.1911, found 470.1896.
LC–MS t = 1.19 min, [M+H]+ = 470. Please see the Supplementary
data for pictures of 500 MHz 1H and 500 MHz 13C NMR spectra in
acetone-d6 at 25 °C.
5.2.24. N-Ethyl-N-(2-hydroxyethyl)-5-methyl-2-(2H-1,2,3-
triazol-2-yl)benzamide (7)
To a suspension of 5-methyl-2-(2H-1,2,3-triazol-2-yl)benzoic
acid (2.0 g, 9.84 mmol) in CHCl3 (20 mL) was added SOCl2
(1.1 mL, 14.8 mmol) at 0 °C. The reaction mixture was heated to
reflux for 1 h. After standing until cooled to room temperature,
the solvent in the reaction mixture was distilled off under reduced
pressure. TEA (2.7 mL, 19.7 mmol) and 2-(ethylamino)ethanol
(1.2 mL, 10.8 mmol) were then added to a solution of the residue
obtained in the previous step in CHCl3 (20 mL) at 0 °C. The solution
was stirred for 16 h at room temperature. An aqueous NaHCO3
solution and water were added to the reaction mixture, followed
5.2.18. N-Ethyl-5-methyl-N-{2-[3-(6-methylpyridin-2-yl)-1H-
pyrazol-1-yl]ethyl}-2-(2H-1,2,3-triazol-2-yl)benzamide (4e)
The title compound was synthesized according to the procedure
described for compound 4a from N-ethyl-2-[3-(6-methylpyridin-

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R. Suzuki et al. / Bioorg. Med. Chem. 23 (2015) 1260–1275
by extraction with CHCl3. The organic layer was washed with brine
and was dried over MgSO4. The desiccant was then removed by fil-
tration, and the solvent was distilled off under reduced pressure.
The obtained residue was purified by column chromatography
(20–80% EtOAc in hexanes) to yield the title compound 7 as a col-
5.2.27. 5-Fluoro-2-(1H-pyrazol-4-yl)pyridine (11)
A 2 mol/L aqueous Na2CO3 solution (45.2 g, 426.2 mmol) and
Pd(PPh3)4 (4.93 g, 4.26 mmol) were added to a solution of tert-butyl
4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole-1-
carboxylate (46.0 g, 156.3 mmol) and 2-bromo-5-fluoropyridine
(25.0 g, 142.1 mmol) in 1,4-dioxane (284 mL). The mixture was
then stirred in an oil bath with a temperature of 90 °C for 4 h. Next,
the mixture was stirred at room temperature for 2 days. Brine was
added to the reaction mixture, followed by extraction with EtOAc.
The organic layer was dried over Na2SO4, and the desiccant was fil-
tered off. Then, the solvent was distilled off under reduced pressure.
EtOAc was added to the obtained residue, and the deposited solid
was collected by filtration to obtain the title compound 11 as a col-
orless powder (13.0 g, 56%). HRMS calcd for C8H6FN3 [M+H]+
164.0619, found 164.0620. LC–MS t = 0.52 min, [M+H]+ = 164.
1H NMR (500 MHz, CDCl3, 25 °C) d ppm 7.36–7.44 (m, 1H), 7.49
orless oil (2.4 g, 89%). HRMS calcd for
C
14H18N4O2 [M+H]+
275.1503, found 275.1503. LC–MS t = 0.62–0.73 min, [M+H]+ =
275. Please see the Supplementary data for pictures of 500 MHz
1H and 500 MHz 13C NMR spectra in CDCl3 at 25 °C.
5.2.25. N-(2-Cyanoethyl)-N-ethyl-5-methyl-2-(2H-1,2,3-triazol-
2-yl)benzamide (8)
Methanesulfonyl chloride (MsCl) (0.30 mL, 3.83 mmol) was
added to a solution of compound 7 (1.0 g, 3.65 mmol) and TEA
(0.76 mL, 5.47 mmol) in CHC13 (10 mL) under cooling in ice
water, and the mixture was heated to room temperature and stir-
red for 1 h. Water was then added under cooling in ice water, fol-
lowed by extraction with CHC13. Next, the organic layer was
washed with brine and allowed to pass through an ISOLUTEÒ
Phase Separator. Then, the filtrate was concentrated under
reduced pressure. NaCN (0.27 g, 5.47 mmol) was added to a solu-
tion of the obtained residue in DMF (10 mL), and the mixture was
stirred in an oil bath with a temperature of 80 °C for 3.5 h. After
standing until cooled to room temperature, water was added
thereto, followed by extraction with EtOAc. The organic layer
was washed with brine and was dried with MgSO4. The desiccant
was then removed by filtration, and the solvent was distilled off
under reduced pressure. The obtained residue was purified by
column chromatography (20–80% EtOAc in hexanes) to yield the
title compound 8 as a colorless powder (0.96 g, 93% over 2 steps).
HRMS calcd for C15H17N5O [M+H]+ 284.1506, found 284.1512.
LC–MS t = 0.86 min, [M+H]+ = 284. Please see the Supplementary
data for pictures of 500 MHz 1H and 500 MHz 13C NMR spectra
in CDCl3 at 25 °C.
(dd, J = 8.75, 4.29 Hz, 1H), 8.06 (s, 2H), 8.43 (d, J = 2.74 Hz, 1H); 13
NMR (500 MHz, CDCl3, 25 °C) d ppm 120.51, 120.55, 122.59,
123.57, 123.72, 132.37, 137.69, 137.88, 148.25, 148.28, 157.10,
159.13.
C
5.2.28. N-Ethyl-N-{2-[4-(5-fluoropyridin-2-yl)-1H-pyrazol-1-yl]
ethyl}-5-methyl-2-(2H-1,2,3-triazol-2-yl)benzamide (12)
MsCl (0.15 mL, 1.91 mmol) was added to a solution of com-
pound 7 (0.50 g, 1.82 mmol) and TEA (0.38 mL, 2.73 mmol) in
CHC13 (2 mL) under cooling in ice water, and the mixture was
warmed to room temperature and stirred for 1 h. Water was then
added under cooling in ice water, followed by extraction with
CHC13. Next, the organic layer was washed with brine and allowed
to pass through an ISOLUTEÒPhase Separator. Then, the filtrate was
concentrated under reduced pressure. Cs2CO3 (1.31 g, 4.01 mmol)
was added to a solution of the obtained residue and compound
11 (0.33 g, 2.00 mmol) in DMF (2 mL), and the mixture was stirred
in an oil bath with a temperature of 80 °C for 4 h. After standing
until cooled to room temperature, water was added thereto, fol-
lowed by extraction with EtOAc. The organic layer was washed
with brine and was dried over MgSO4. The desiccant was then
removed by filtration, and the solvent was distilled off under
reduced pressure. The obtained residue was purified by column
chromatography (20–80% EtOAc in hexanes) and washed with
Et2O to yield the title compound 12 as a colorless powder
(0.30 g, 39% over 2 steps). HRMS calcd for C22H22FN7O [M+H]+
420.1943, found 420.1939. LC–MS t = 0.98 min, [M+H]+ = 420.
Please see the Supplementary data for pictures of 500 MHz 1H
and 13C NMR spectra in acetone-d6 at 25 °C.
5.2.26. N-Ethyl-N-{2-[5-(5-fluoropyridin-2-yl)-1,2,4-oxadiazol-
3-yl]ethyl}-5-methyl-2-(2H-1,2,3-triazol-2-yl)benzamide (9)
An aqueous 50% hydroxylamine solution (0.31 mL, 5.29 mmol)
was added to a solution of N-(2-cyanoethyl)-N-ethyl-5-methyl-2-
(2H-1,2,3-triazol-2-yl)benzamide 8 (0.50 g, 1.76 mmol) in EtOH
(10 mL), followed by stirring at 80 °C for 4 h. After cooling at room
temperature, the reaction solution was concentrated under
reduced pressure. Water was added to the obtained residue, fol-
lowed by extraction with EtOAc. The organic layer was washed
with brine and was dried over MgSO4. The desiccant was then
removed by filtration, and the solvent was distilled off under
reduced pressure. A solution of the resulting residue in CH3CN
(5 mL) and DMF (0.5 mL) was added to a solution of 5-fluoropyri-
din-2-carboxylic acid (0.27 g, 1.95 mmol) and carbonyldiimidazole
(0.34 g, 2.12 mmol) in CH3CN (5 mL) and DMF (0.5 mL) that was
then stirred in advance at room temperature for 3 h. The reaction
solution was stirred for 2 h at room temperature. Next, 1,8-diaza-
bicyclo[5.4.0]undec-7-ene (DBU) (0.26 mL, 1.77 mmol) was added
to the reaction mixture, followed by stirring at 70 °C for 1 h. After
cooling at room temperature, water was added to the reaction mix-
ture, followed by extraction with EtOAc. The organic layer was
washed with a 1.2 mol/L aqueous HCl solution and brine and was
dried over MgSO4. The desiccant was then removed by filtration,
and the solvent was distilled off under reduced pressure. The
resulting residue was purified by column chromatography
(20–80% EtOAc in hexanes) and washed with Et2O to give the
title compound 9 as a colorless powder (0.47 g, 63% over 2 steps).
HRMS calcd for C21H20FN7O2 [M+H]+ 422.1735, found 422.1739.
LC–MS t = 0.98 min, [M+H]+ = 422. Please see the Supplementary
data for pictures of 500 MHz 1H and 13C NMR spectra in acetone-
d6 at 25 °C.
5.2.29. N-{2-[5-(5-Fluoropyridin-2-yl)-1,2-oxazol-3-yl]ethyl}-5-
methyl-2-(2H-1,2,3-triazol-2-yl)benzamide (15)
HATU (1.94 g, 5.09 mmol) was added to a solution of 3,3-dieth-
oxypropan-1-amine (0.50 g, 3.40 mmol), 5-methyl-2-(2H-1,2,
3-triazol-2-yl)benzoic acid (0.83 g, 4.08 mmol) and N,N-diisopropyl-
ethylamine (DIPEA) (2.97 mL, 17.0 mmol) in DMF (5 mL), followed
by stirring at room temperature for 15 h. An aqueous NaHCO3
solution was added to the reaction solution, followed by extraction
with EtOAc. The organic layer was washed with brine and was
dried with MgSO4. The desiccant was then removed by filtration,
and the solvent was distilled off under reduced pressure.
The resulting residue was purified by column chromatography
(20–80% EtOAc in hexanes) to yield N-(3,3-diethoxy-propyl)-5-
methyl-2-(2H-1,2,3-triazol-2-yl)benzamide 14 as
(1.13 g, 100%).
a brown oil
An aqueous 1.2 mol/L HCl solution was then added to a solution
of compound 14 (0.82 g, 2.47 mmol) in THF (10 mL), followed by
stirring at room temperature for 18 h. An aqueous NaHCO3
solution was added to the reaction solution to adjust the pH to a
neutral level, followed by extraction with EtOAc. The organic layer

R. Suzuki et al. / Bioorg. Med. Chem. 23 (2015) 1260–1275
1273
was washed with brine and was dried over MgSO4. The desiccant
was then removed by filtration, and the solvent was distilled off
under reduced pressure. Sodium acetate (0.28 g, 3.45 mmol) and
hydroxylamine hydrochloride (0.24 g, 3.45 mmol) were added to
an aqueous solution of the residue in 95% EtOH (10 mL), followed
by stirring in an ice bath for 3 h. An aqueous NaHCO3 solution
was added to the reaction solution, followed by extraction with
EtOAc. The organic layer was washed with brine and was dried
over MgSO4. The desiccant was then removed by filtration, and
the solvent was distilled off under reduced pressure. The resulting
solid was washed with Et2O and was then collected by filtration to
give N-[3-(hydroxyimino)propyl]-5-methyl-2-(2H-1,2,3-triazol-2-
yl)benzamide as a colorless powder (0.23 g).
N-Chlorosuccinimide (0.081 g, 0.60 mmol) was added to a solu-
tion of N-[3-(hydroxylimino)propyl]-5-methyl-2-(2H-1,2,3-triazol-
2-yl)benzamide (0.15 g, 0.55 mmol) in DMF (2 mL) in an ice bath.
The mixture was warmed to room temperature, followed by stirring
for 16 h. Water was added to the reaction solution, followed by
extraction with EtOAc. The organic layer was washed with brine
and was dried over MgSO4. The desiccant was then removed by fil-
tration, and the solvent was distilled off under reduced pressure.
The residue was dissolved in THF (4 mL), and a solution of 2-ethy-
nyl-5-fluoropyridine (0.10 g, 0.83 mmol) in THF (1 mL) was drop-
wise added to the solution in an ice bath. TEA (0.084 mL,
0.83 mmol) was further dropwise added thereto. The mixture was
warmed to room temperature, followed by stirring for 16 h. Water
was added to the reaction solution, followed by extraction with
EtOAc. The organic layer was washed with brine and was dried over
MgSO4. The desiccant was then removed by filtration, and the sol-
vent was distilled off under reduced pressure. The resulting residue
was purified by column chromatography (20–80% EtOAc in hex-
anes) to give the title compound 15 as a colorless powder
(0.050 g, 8% over 4 steps). HRMS calcd for C20H17FN6O2 [M+H]+
393.1470, found 393.1466. LC–MS t = 0.85 min, [M+H]+ = 393.
1H NMR (500 MHz, CDCl3, 25 °C) d ppm 2.37–2.44 (m, 3H), 2.96
(t, J = 6.52 Hz, 2H), 3.74 (q, J = 6.17 Hz, 2H), 6.24 (t, J = 5.32 Hz, 1H),
6.73 (s, 1H), 7.28–7.34 (m, 1H), 7.41 (d, J = 1.72 Hz, 1H), 7.51 (td,
J = 8.32, 2.92 Hz, 1H), 7.60 (d, J = 8.23 Hz, 1H), 7.72 (s, 2H), 7.86
(dd, J = 8.92, 4.46 Hz, 1H); 13C NMR (500 MHz, CDCl3, 25 °C) d
ppm 21.06, 26.35, 37.89, 102.17, 122.08, 122.12, 123.74, 123.89,
124.28, 129.68, 131.03, 131.33, 134.78, 135.64, 138.72, 138.91,
139.07, 142.92, 142.96, 158.55, 160.62, 162.63, 167.81, 168.49.
5-fluoropyridine-2-carbaldehyde (0.35 g, 2.80 mmol) in EtOH
(10 mL) and H2O (1 mL) under ice cooling. The mixture was
warmed to room temperature, followed by stirring for 15 h. An
aqueous NaHCO3 solution was added to the reaction solution, fol-
lowed by extraction with EtOAc. The organic layer was washed
with brine and was dried over MgSO4. The desiccant was then
removed by filtration, and the solvent was distilled off under
reduced pressure to give 1-(5-fluoropyridin-2-yl)-N-hydroxymeth-
animine as a pale yellow powder (0.27 g, 69%).
N-Chlorosuccinimide (0.28 g, 2.12 mmol) was added to a solu-
tion of 1-(5-fluoropyridin-2-yl)-N-hydroxymethanimine (0.27 g,
1.93 mmol) in DMF (5 mL) in an ice bath. The mixture was warmed
to room temperature, followed by stirring for 16 h. Water was
added to the reaction solution, followed by extraction with EtOAc.
The organic layer was washed with brine and was dried with
MgSO4. The desiccant was then removed by filtration, and the
solvent was distilled off under reduced pressure. The residue was
dissolved in THF (5 mL), and a solution of 2-(but-3-yn-1-yl)-1H-iso-
indole-1,3(2H)-dione (0.58 g, 2.92 mmol) in THF (5 mL) was
dropwise added to the solution in an ice bath. TEA (0.30 mL,
2.92 mmol) was further dropwise added thereto. The mixture was
warmed to room temperature, followed by stirring for 3 days.
Water was added to the reaction solution, followed by extraction
with EtOAc. The organic layer was washed with brine and was dried
with MgSO4. The desiccant was then removed by filtration, and the
solvent was distilled off under reduced pressure. The resulting res-
idue was purified by column chromatography (5–50% EtOAc in hex-
anes) to give the title compound 18 as a pale yellow powder (0.12 g,
18% over 2 steps). HRMS calcd for C18H12FN3O3 [M+H]+ 338.0935,
found 338.0932. LC–MS t = 1.02 min, [M+H]+ = 338.
1H NMR (500 MHz, CDCl3, 25 °C) d ppm 3.25 (t, J = 7.03 Hz, 2H),
4.09 (t, J = 7.20 Hz, 2H), 6.72 (s, 1H), 7.44–7.51 (m, 1H), 7.66–7.75
(m, 2H), 7.85 (dd, J = 5.49, 3.09 Hz, 2H), 8.05 (dd, J = 8.58, 4.46 Hz,
1H), 8.49 (d, J = 2.74 Hz, 1H); 13C NMR (500 MHz, CDCl3, 25 °C) d
ppm 26.07, 35.84, 101.02, 122.83, 122.86, 123.55, 123.67, 123.82,
132.01, 134.22, 137.97, 138.16, 144.89, 144.92, 158.85, 160.91,
162.49, 168.01, 170.36
5.2.32. 2-[3-(5-Fluoropyridin-2-yl)-1,2-oxazol-5-yl]ethanamine
(19)
Hydrazine monohydrate (0.035 mL, 0.71 mmol) was dropwise
added to a solution of 2-{2-[3-(5-fluoropyridin-2-yl)-1,2-oxazol-
5-yl]ethyl}-1H-isoindole-1,3(2H)-dione (0.12 g, 0.36 mmol) in
EtOH (5 mL), followed by stirring at 90 °C for 2 h. After cooling at
room temperature, the precipitated solid was removed by filtra-
tion. The filtrate was concentrated under reduced pressure. The
resulting residue was purified by column chromatography (20–
100% EtOAc in hexanes) to give the title compound 19 as a pale yel-
low powder (0.053 g, 72%). HRMS calcd for C10H10FN3O [M+H]+
208.0881, found 208.0892. LC–MS t = 0.25 min, [M+H]+ = 208.
1H NMR (500 MHz, CDCl3, 25 °C) d ppm 2.93–3.01 (m, 2H),
3.09–3.14 (m, 2H), 6.67 (s, 1H), 7.49 (td, J = 8.32, 2.92 Hz, 1H),
8.07 (dd, J = 8.92, 4.46 Hz, 1H), 8.51 (d, J = 2.74 Hz, 1H); 13C NMR
(500 MHz, CDCl3, 25 °C) d ppm 31.26, 40.29, 100.56, 122.83,
123.76, 123.92, 137.98, 138.18, 145.04, 145.08, 158.85, 160.91,
162.48, 172.46.
5.2.30. N-Ethyl-N-{2-[5-(5-fluoropyridin-2-yl)-1,2-oxazol-3-yl]
ethyl}-5-methyl-2-(2H-1,2,3-triazol-2-yl)benzamide (16)
60% NaH (0.011 g, 0.24 mmol) was added to a solution of com-
pound 15 (0.080 g, 0.20 mmol) in DMF (0.68 mL), followed by stir-
ring at room temperature for 1 h. EtI (0.018 mL, 0.22 mmol) was
dropwise added to the reaction solution, followed by stirring at
room temperature for 3 h. Water was added to the reaction solu-
tion, followed by extraction with EtOAc. The organic layer was
washed with water and brine and was dried with Na2SO4. The des-
iccant was then removed by filtration, and the solvent was distilled
off under reduced pressure. The resulting residue was purified by
column chromatography (15–100% EtOAc in hexanes). The result-
ing solid was washed with hexane/EtOAc with stirring, followed
by filtration to give the title compound 16 as a colorless powder
(0.060 g, 70%). HRMS calcd for C22H21FN6O2 [M+H]+ 421.1783,
found 421.1777. LC–MS t = 1.05 min, [M+H]+ = 421. Please see the
Supplementary data for pictures of 500 MHz 1H and 13C NMR spec-
tra in acetone-d6 at 25 °C.
5.2.33. N-{2-[3-(5-Fluoropyridin-2-yl)-1,2-oxazol-5-yl] ethyl}-5-
methyl-2-(2H-1,2,3-triazol-2-yl)benzamide (20)
HATU (0.090 g, 0.24 mmol) was added to a solution of 2-[3-
(5-fluoropyridin-2-yl)-1,2-oxazol-5-yl]ethanamine (0.053 g, 0.16
mmol), 5-methyl-2-(2H-1,2,3-triazol-2-yl)benzoic acid (0.038 g,
0.19 mmol) and DIPEA (0.082 mL, 0.47 mmol) in DMF (5 mL),
followed by stirring at room temperature for 16 h. An aqueous
NaHCO3 solution was added to the reaction solution, followed
by extraction with EtOAc. The organic layer was washed with
5.2.31. 2-{2-[3-(5-Fluoropyridin-2-yl)-1,2-oxazol-5-yl]ethyl}-
1H-isoindole-1,3(2H)-dione (18)
Sodium acetate (0.32 g, 3.92 mmol) and hydroxylamine
hydrochloride (0.27 g, 3.92 mmol) were added to a solution of

1274
R. Suzuki et al. / Bioorg. Med. Chem. 23 (2015) 1260–1275
brine and was dried over MgSO4. The desiccant was then removed
by filtration, and the solvent was distilled off under reduced pres-
sure. The resulting residue was purified by column chromatogra-
phy (20–80% EtOAc in hexanes) to give the title compound 20 as
a colorless powder (0.060 g, 97%). HRMS calcd for C20H17FN6O2
[M+H]+ 393.1470, found 393.1485. LC–MS t = 0.90 min,
[M+H]+ = 393.
ppm 39.21, 49.14, 104.76, 121.55, 121.59, 124.73, 124.88, 133.39,
137.30, 137.49, 148.75, 150.94, 158.00, 160.02.
5.2.37. N-{2-[4-(5-Fluoropyridin-2-yl)-1H-pyrazol-1-yl]ethyl}-5-
methyl-2-(2H-1,2,3-triazol-2-yl)benzamide (25)
HATU (0.061 g, 0.16 mmol) was added to a solution of 2-[3-(5-
fluoropyridin-2-yl)-1H-pyrazol-l-yl]ethanamine dihydrochloride
(0.030 g, 0.11 mmol), 5-methyl-2-(2H-1,2,3-triazol-2-yl)benzoic
acid (0.026 g, 0.13 mmol) and DIPEA (0.094 mL, 0.54 mmol) in
DMF (5 mL), followed by stirring at room temperature
for 3 h. An aqueous NaHCO3 solution was added to the reaction
solution, followed by extraction with EtOAc. The organic layer
was washed with brine and was dried over MgSO4. The desiccant
was then removed by filtration, and the solvent was distilled off
under reduced pressure. The resulting residue was purified by col-
umn chromatography (20–80% EtOAc in hexanes). The resulting
solid was washed with Et2O with stirring, followed by filtration
to give the title compound 25 as a colorless powder (0.021 g,
1H NMR (500 MHz, CDCl3, 25 °C) d ppm 2.41 (s, 3H), 3.09 (t,
J = 6.52 Hz, 2H), 3.73 (q, J = 6.40 Hz, 2H), 6.11 (br s, 1H), 6.65 (s,
1H), 7.33 (dd, J = 8.23, 2.06 Hz, 1H), 7.42 (d, J = 1.72 Hz, 1H), 7.49
(td, J = 8.40, 3.09 Hz, 1H), 7.61 (d, J = 8.23 Hz, 1H), 7.76 (s, 2H),
8.05 (dd, J = 8.58, 4.46 Hz, 1H), 8.50 (d, J = 2.74 Hz, 1H); 13C NMR
(500 MHz, CDCl3, 25 °C)
d ppm 21.06, 26.88, 37.85, 100.83,
122.78, 122.82, 123.77, 123.92, 124.43, 129.72, 130.80, 131.49,
134.77, 135.75, 138.06, 138.25, 139.18, 144.88, 158.87, 160.94,
162.53, 167.89, 171.53.
5.2.34. N-Ethyl-N-{2-[3-(5-fluoropyridin-2-yl)-1,2-oxazol-5-yl]
ethyl}-5-methyl-2-(2H-1,2,3-triazol-2-yl)benzamide (21)
The title compound 21 was prepared according to the procedure
described for compound 16 from N-{2-[3-(5-Fluoropyridin-2-yl)-
1,2-oxazol-5-yl]ethyl}-5-methyl-2-(2H-1,2,3-triazol-2-yl)benzam-
ide as a colorless powder (0.032 g, 75%). HRMS calcd for C22H21FN6O2
[M+H]+ 421.1783, found 421.1773. LC–MS t = 1.06 min,
[M+H]+ = 421. Please see the Supplementary data for pictures of
500 MHz 1H and 13C NMR spectra in acetone-d6 at 25 °C.
50%). HRMS calcd for
C
20H18FN7O [M+H]+ 392.1630, found
392.1612. LC–MS t = 0.82 min, [M+H]+ = 392.
1H NMR (500 MHz, acetone-d6, 25 °C) d ppm 2.35 (s, 4H), 3.74
(q, J = 6.17 Hz, 3H), 4.37 (t, J = 6.00 Hz, 4H), 6.77 (d, J = 2.40 Hz,
2H), 7.23–7.45 (m, 3H), 7.60 (td, J = 8.66, 2.92 Hz, 1H), 7.64 (d,
J = 8.23 Hz, 1H), 7.78 (d, J = 2.40 Hz, 1H), 7.88 (s, 2H), 8.01–8.05
(m, 1H); 13C NMR (500 MHz, acetone-d6, 25 °C) d ppm 20.01,
40.07, 50.97, 103.81, 120.57, 123.23, 123.38, 123.71, 129.31,
130.70, 131.80, 131.99, 135.30, 135.61, 136.77, 136.96, 138.39,
149.45, 151.08, 157.88, 159.90, 167.32.
5.2.35. tert-Butyl {2-[3-(5-fluoropyridin-2-yl)-1H-pyrazol-1-yl]
ethyl}carbamate (23)
5.2.38. N-{2-[3-(5-Fluoropyridin-2-yl)-1H-pyrazol-1-yl]ethyl}-
N,5-dimethyl-2-(2H-1,2,3-triazol-2-yl)benzamide (26a)
tert-Butyl N-(2-bromoethyl)carbamate (1.00 g, 4.49 mmol) and
Cs2CO3 (1.83 g, 5.61 mmol) were added to a solution of 5-fluoro-
2-(1H-pyrazol-3-yl)pyridine (0.61 g, 3.74 mmol) in DMF (10 mL),
and the mixture was stirred at 80 °C for 4 h. After standing until
cooled to room temperature, the reaction mixture was added to
water, followed by extraction with EtOAc. The extracted organic
layer was washed with brine and was dried over MgSO4. Then,
the desiccant was filtered off, and the solvent was distilled off
under reduced pressure. The obtained residue was purified by col-
umn chromatography (20–80% EtOAc in hexanes) to obtain the
title compound 23 as a colorless powder (0.55 g, 48%). HRMS calcd
The title compound 26a was prepared according to the
procedure described for compound 16 from N-{2-[4-(5-
fluoropyridin-2-yl)-1H-pyrazol-1-yl]ethyl}-5-methyl-2-(2H-1,2,3-
triazol-2-yl)benzamide and MeI which was used instead of EtI as
colorless powder (0.34 g, 100%). HRMS calcd for C21H20FN7O
[M+H]+ 406.1786, found 406.1775. LC–MS t = 0.93 min,
[M+H]+ = 406. Please see the Supplementary data for pictures of
500 MHz 1H and 13C NMR spectra in acetone-d6 at 25 °C.
5.2.39. N-{2-[4-(5-Fluoropyridin-2-yl)-1H-pyrazol-1-yl]ethyl}-5-
methyl-N-(propan-2-yl)-2-(2H-1,2,3-triazol-2-yl)benzamide
(26b)
for
C
15H19FN4 O2 [M+H]+ 307.1565, found 307.1568. LC–MS
t = 0.92 min, [M+H]+ = 307.
1H NMR (500 MHz, CDCl3, 25 °C) d ppm 1.42 (s, 9H), 3.61 (q,
J = 5.49 Hz, 2H), 4.28 (t, J = 5.49 Hz, 2H), 6.81 (d, J = 2.06 Hz, 1H),
7.39–7.46 (m, 2H), 7.91 (dd, J = 8.58, 4.46 Hz, 1H), 8.46 (d,
J = 2.74 Hz, 1H); 13C NMR (500 MHz, CDCl3, 25 °C) d ppm 28.42,
40.87, 51.91, 79.81, 104.30, 121.05, 123.48, 123.63, 131.80,
137.38, 137.58, 148.70, 148.73, 151.52, 155.96, 157.88, 159.92.
The title compound 26b was prepared according to the
procedure described for compound 16 from N-{2-[4-(5-fluoro-
pyridin-2-yl)-1H-pyrazol-1-yl]ethyl}-5-methyl-2-(2H-1,2,3-tria-
zol-2-yl)benzamide and iPrI which was used instead of EtI as
colorless powder (0.052 g, 12%). HRMS calcd for C23H24FN7O
[M+H]+ 434.2099, found 434.2099. LC–MS t = 1.10 min,
[M+H]+ = 434. Please see the Supplementary data for pictures of
500 MHz 1H and 13C NMR spectra in acetone-d6 at 25 °C.
5.2.36. 2-[3-(5-Fluoropyridin-2-yl)-1H-pyrazol-l-yl]ethanamine
dihydrochloride (24)
A 4 mol/L HCl-EtOAc solution (5 mL) was added to a solution
of tert-butyl {2-[3-(5-fluoropyridin-2-yl)-1H-pyrazol-1-yl]ethyl}
carbamate (0.30 g, 0.98 mmol) in EtOAc (5 mL), and the mixture
was stirred at room temperature for 15 h. The deposited solid
was then collected by filtration. The obtained solid was dried
by heating under reduced pressure to obtain the title compound
Acknowledgments
The authors would like to thank Dr. Junichi Yamaguchi and Mr.
Takeshi Tani for helpful discussion: Dr. Atsushi Okada, Dr. Hideki
Shinonaga, Mr. Naoto Ohsaki and Mr. Akira Higuchi for analytical
and spectrum studies: and Dr. Hirohiko Hikichi for in vivo pharma-
cological experiments.
24 as
a colorless powder (0.27 g, 100%). HRMS calcd for
C
10H11FN4 [M+H]+ 207.1041, found 207.1034. LC–MS t = 0.25
min, [M+H]+ = 207.
Supplementary data
1H NMR (500 MHz, CDCl3, 25 °C) d ppm 3.01–3.48 (m, 2H), 4.44
(t, J = 6.17 Hz, 2H), 6.80 (d, J = 2.06 Hz, 1H), 7.77 (td, J = 8.75,
3.09 Hz, 1H), 7.88 (d, J = 2.40 Hz, 1H), 8.02 (dd, J = 8.75, 4.63 Hz,
1H), 8.55 (d, J = 2.74 Hz, 1H); 13C NMR (500 MHz, CDCl3, 25 °C) d
Supplementary data (reproduction of the 1H and 13C NMR spec-
tra for key compounds) associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2015.01.044.

R. Suzuki et al. / Bioorg. Med. Chem. 23 (2015) 1260–1275
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