Discovery of novel 5,6,7,8-tetrahydro[1,2,4]triazolo[4,3-a]pyridine derivatives as γ-secretase modulators (Part 2)

Article abstract of DOI:10.1016/j.bmc.2016.05.040

γ-Secretase modulators (GSMs), which lower pathogenic amyloid beta (Aβ) without affecting the production of total Aβ or Notch signal, have emerged as a potential therapeutic agent for Alzheimer's disease (AD). A novel series of 5,6,7,8-tetrahydro[1,2,4]tr

Full text of DOI:10.1016/j.bmc.2016.05.040

Bioorganic & Medicinal Chemistry 24 (2016) 3192–3206
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Discovery of novel 5,6,7,8-tetrahydro[1,2,4]triazolo[4,3-a]pyridine
derivatives as c-secretase modulators (Part 2)
⇑
Takafumi Takai , Tatsuki Koike, Minoru Nakamura, Yuichi Kajita, Toshiro Yamashita, Naohiro Taya,
Tetsuya Tsukamoto, Tomomichi Watanabe, Koji Murakami, Tomoko Igari, Makoto Kamata
Pharmaceutical Research Division, Takeda Pharmaceutical Company Ltd, 26-1, Muraokahigashi 2-chome, Fujisawa, Kanagawa 251-8555, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
c
-Secretase modulators (GSMs), which lower pathogenic amyloid beta (Ab) without affecting the
Received 13 April 2016
Revised 18 May 2016
Accepted 20 May 2016
Available online 20 May 2016
production of total Ab or Notch signal, have emerged as a potential therapeutic agent for Alzheimer’s
disease (AD). A novel series of 5,6,7,8-tetrahydro[1,2,4]triazolo[4,3-a]pyridine derivatives was discovered
and characterized as GSMs. Optimization of substituents at the 8-position of the core scaffold using
ligand-lipophilicity efficiency (LLE) as a drug-likeness guideline led to identification of various types of
high-LLE GSMs. Phenoxy compound (R)-17 exhibited especially high LLE as well as potent in vivo
Ab42-lowering effect by single administration. Furthermore, multiple oral administration of (R)-17
significantly reduced soluble and insoluble brain Ab42, and ameliorated cognitive deficit in novel object
recognition test (NORT) using Tg2576 mice as an AD model.
Keywords:
Alzheimer’s disease
Amyloid beta
c
c
-Secretase
-Secretase modulator
Ó 2016 Elsevier Ltd. All rights reserved.
1. Introduction
lipophilicity of the distal phenyl moiety helped in identification
of 2 as a potent GSM (Fig. 1). Although oral administration of 2 sig-
Alzheimer’s disease (AD) is characterized by impaired memory
and loss of other cognitive functions, and is the most common form
of dementia, accounting for 50–75% of all dementias.1 A character-
istic neuropathology of AD is the formation of amyloid plaques,
which are composed of insoluble deposits of amyloid beta (Ab).
nificantly decreased brain Ab42 in mice, further reduction of the
drug exposure level in both brain and plasma (2.332 g/g in cortex
and 6.450 g/mL in plasma at 3 h after oral dosing) at the pharma-
l
l
cologically effective dose remains desirable.7 Furthermore, increas-
ing lipophilicity for 2 to enhance the Ab42-lowering effect could
induce a promiscuous effect, which could induce off-target risks.
Through LLE-based optimization, we accordingly pursued more
potent compounds without increasing lipophilicity to discover a
safer GSM that could reduce Ab42 at a lower drug exposure. The
synthetic intermediate 3 in the course of previous modification
maintained the equipotent GSM activity compared to 1, as shown
in Figure 1. Encouraged by the tolerance for introduction of an
additional polar group at the 8-position of 1, we explored the
optimal functional group based on LLE. Herein we describe
identification of (R)-17 by the optimization study, and also report
the Ab42-lowering effect and the behavioral pharmacological
evaluation in Tg2576 mice.
b-Secretase and
through a stepwise cleavage of amyloid precursor protein (APP).2
Since -secretase catalyzes the degradation of many other sub-
strates besides APP, such as Notch,3 the inhibition of
-secretase
led to induction of mechanism-based toxicities in clinical trials.4
As -secretase modulators (GSMs) lower pathogenic Ab through
c-secretase are responsible for producing Ab
c
c
c
cleavage shift without affecting Notch signal, GSMs have emerged
as a potential therapeutic agent for AD.5 A recent analysis of GSMs
indicated that their increased lipophilicity could enhance their
potency, but might reduce their drug-likeness.6 We therefore used
ligand-lipophilicity efficiency (LLE) as a drug-likeness guideline in
a lead generation study of GSM, and discovered 5,6,7,8-tetrahydro
[1,2,4]triazolo[4,3-a]pyridine derivative 1. Finally, increasing
2. Chemistry
Abbreviations: GSM, c-secretase modulator; Ab, amyloid beta; AD, Alzheimer’s
Various functional groups were introduced into the 8-position
of 1, as shown in Scheme 1. Oxidation of the 8-position of 1 under
basic conditions and subsequent methylation gave 8-methoxy ana-
log 4. Hydroxymethylation to the 8-position was achieved by
disease; LLE, ligand-lipophilicity efficiency; NORT, novel object recognition test;
APP, amyloid precursor protein; MLM, mice liver microsomes; CNS, central nervous
system; WT, wild type.
⇑
Corresponding author. Tel.: +81 466 32 1133; fax: +81 466 29 4454.
E-mail address: takafumi.takai@takeda.com (T. Takai).
http://dx.doi.org/10.1016/j.bmc.2016.05.040
0968-0896/Ó 2016 Elsevier Ltd. All rights reserved.

T. Takai et al. / Bioorg. Med. Chem. 24 (2016) 3192–3206
3193
Figure 1. 5,6,7,8-Tetrahydro[1,2,4]triazolo[4,3-a]pyridines.
Scheme 1. Synthesis of the 8-position-substituted derivatives. Reagents and conditions: (a) NaH, air, then NaH, MeI, DMF, 31%; (b) (i) NaH, paraformaldehyde, DMF, 13%; (ii)
TsCl, Py, 69%; (c) (i) NaN3, DMSO; (ii) PPh3, H2O, THF, 49% (2 steps); (d) AcCl, Et3N, THF, 72%; (e) NaH, diethyl carbonate, THF, 90–100%; (f) NaOEt, acrylonitrile, t-BuOH; (g) H2,
Raney cobalt, NH3, EtOH, 41–73%; (h) Lawesson’s reagent, toluene, 79–90%; (i) MeI, MeCN, then 30, EtOH, 59–68%; (j) MeMgBr, THF, 36–41%.
nucleophilic alkylation with paraformaldehyde, and the following
tosylation afforded 24. The tosyl group was converted to the amino
group in 5 through azidation and subsequent reduction. Acetyla-
tion of 5 smoothly proceeded to give amide derivative 6. Tertiary
alcohol analog 7 was then synthesized through a straightforward
synthesis. Ethyl carboxylation of the
a-position of 25a followed

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T. Takai et al. / Bioorg. Med. Chem. 24 (2016) 3192–3206
by the Michael reaction with acrylonitrile gave 27a. Hydrogenation
of cyano group of 27a and subsequent intracyclization were
achieved as a one-pot reaction to yield oxopiperidine 28a, which
was then converted to thioxo analog 29a by use of Lawesson’s
reagent. The 5,6,7,8-tetrahydro[1,2,4]triazolo[4,3-a]pyridine core
of 31a was constructed by activation of 29a by iodomethane and
the following cyclization reaction with hydrazide 30.7 Finally, the
ester moiety of 31a was converted to 2-hydroxypropan-2-yl group
by treatment with a Grignard reagent to give 7. Difluorophenyl
analog 8 was prepared by a similar manner.
Benzyl derivatives 9–14 and phenoxy derivatives 15–17 were
synthesized as illustrated in Scheme 2. Activation of oxopiperidine
32 by trimethyloxonium tetrafluoroborate and subsequent cycliza-
tion reaction with hydrazide 30 gave bicyclic compound 33. Alky-
lation of the 8-position of 33 with benzyl bromide afforded 34,
which was reacted with the Grignard reagent to give 2-hydrox-
ypropan-2-yl derivative 9 and ketone 11 as a by-product. Reduc-
tion of ester 34 with LAH gave the primary alcohol 10. Direct
conversion of the ester group of 34 to carboxamide group was
achieved by treatment with formamide under basic conditions to
afford 12. Trifluoroethyl amide derivative 13 was obtained through
hydrolysis of 34 and subsequent condensation with 2,2,2-trifluo-
roethylamine, and methylation of 13 gave tertiary amide 14. The
a-position of ester 33 was chlorinated with NCS to give 35. Nucle-
ophilic substitution at the quaternary carbon atom of 35 with phe-
nols afforded 36–38. Finally, tertiary alcohols 15–17 were prepared
from the corresponding ester using the Grignard reagent.
Synthetic routes for spiro compounds 18–23 are illustrated in
Scheme 3. Introduction of 2-nitrobenzyl moiety into 33 was
accomplished by nucleophilic substitution with 2-nitrobenzyl bro-
mide to afford 39. Nitrophenyl derivative 40 was also obtained
using 1-fluoro-2-nitrobenzene under similar conditions. After
reduction of the nitro group, cyclized compounds 44 and 45 were
smoothly obtained from the corresponding ester. The amide moi-
ety was then alkylated with 2,2,2-trifluoroethyltriflate to give 18
and 19, respectively. The 8-position of 33 was easily oxidized
under basic conditions, and the introduced hydroxyl group was
trapped by 2-nitrobenzyl bromide to give benzyloxy analog 41.
Scheme 2. Synthesis of benzyl derivatives and phenoxy derivatives. Reagents and conditions: (a) (i) Me3OBF4, MeCN; (ii) 30, MeCN, 38% (2 steps); (b) NaH, 3,4-
difluorobenzylbromide, DMF, 87%; (c) MeMgBr, THF, 48% for 9, 8% for 11; (d) LAH, THF, 73%; (e) HCONH2, NaOMe, MeOH, DMF, 85%; (f) (i) 1 M NaOH aq, MeOH, THF; (ii) 2,2,2-
trifluoroethylamine, HATU, Et3N, DMF, 31% (2 steps); (g) MeI, NaH, DMF, 25%; (h) NCS, NaH, DMF, 69%; (i) various phenols, K2CO3, DMF, 43–62%; (j) MeMgBr, THF, 58–66%.

T. Takai et al. / Bioorg. Med. Chem. 24 (2016) 3192–3206
3195
Scheme 3. Synthesis of spiro compounds. Reagents and conditions: (a) NaH, 2-nitrobenzyl bromide, DMF for 39, 60%; NaH, 1-fluoro-2-nitrobenzene, DMF for 40, 46%; NaH,
air, DMF, then NaH, 2-nitrobenzyl bromide analogs for 41–43, 35–64%; (b) (i) H2, Pd/C, MeOH, EtOH; (ii) EtOH for 44, 43% (2 steps); Fe, AcOH for 45, 39%; (i) H2, PtO2, MeOH;
(ii) NaOH aq, THF; (iii) HATU, Et3N, DMF for 46, 57% (3 steps); (i) NH2NH2ꢁH2O, FeCl3ꢁ6H2O, activated charcoal, THE, MeOH; (ii) NaOH aq, THF, MeOH; (iii) HATU, Et3N, DMF for
47 and 48, 45–69%; (c) 2,2,2-trifluoroethyltriflate, NaH or Cs2CO3, DMF, 40–73%; (d) NaH, paraformaldehyde, DMF, then H2O; (e) 2-benzyloxyaniline, HATU, DMF, 55% (2
steps); (f) (i) H2, Pd/C, MeOH, THF; (ii) PPh3, DEAD, THF, then PBu3, ADDP; (g) 2,2,2-trifluoroethyltriflate, NaH, DMF, 22% (3 steps).
Table 1
For benzoxazepine derivative 46, HATU was used for intracycliza-
tion after preparing the aminobenzoic acid derived from 41. The
amide moiety of 46 was then alkylated with 2,2,2-trifluoroethyltri-
flate to give 20. Benzoxazepine derivatives 22 and 23 were synthe-
sized in a similar manner with that of 20. Synthesis of the
regioisomeric benzoxazepine analog 51 was achieved by the
Mitsunobu reaction of the phenol derived by deprotection of 50,
which was prepared from 33 by hydroxymethylation of the 8-posi-
tion and subsequent benzamide formation. Finally, 2,2,2-trifluo-
roethylation of amide moiety of 51 was conducted to give 21.
Ab42-lowering effects, logD values, and LLE values of 1 and 3–9
R1
H
R2
Ab42 IC50 (nM)
LogDb
LLEc
3.5
a
Compound
1
240
3.2
3
4
5
OH
290
120
280
2.9
3.7
3.1
3.6
3.2
3.5
3. Results and discussion
OMe
All compounds were evaluated for inhibitory activity against
Ab42 production in rat primary neuronal cells, lipophilicity (logD
at pH 7.4),8 and LLE (= pIC50 ꢀ logD). We initially introduced vari-
ous substituents to the 8-position of 1 as shown in Table 1. Methy-
lation of the hydroxyl group of 3 slightly enhanced potency.
However, the increased lipophilicity of 4 resulted in the decreased
LLE value. While replacement of hydroxyl group of 3 with an ami-
nomethyl group (5) or an acetamidomethyl group (6) did not offer
positive effect on the potency or LLE, 2-hydroxypropan-2-yl
derivative 7 showed an 8-fold enhancement of potency as well as
a significant improvement of LLE. High LLE was also observed with
3,4-difluoro phenyl analog 8 as we reported in the previous work.7
Furthermore, insertion of methylene linker (9) into the distal phe-
nyl group of 8 greatly enhanced the LLE value, indicating that the
LLE is controlled by not only the polar group at the 8-position,
but also the position of the distal phenyl group. Since benzyl
derivative 9 showed high clearance in mouse liver microsomes
6
300
3.2
3.3
7
8
9
35
3.7
2.9
3.0
3.8
3.9
4.3
160
48
a
b
c
IC50 values are means of triplicate measurements.
Measured at pH 7.4.
LLE = pIC50 ꢀ logD.
(93 lL/min/mg), we next modified both the 2-hydroxypropan-2-
yl group and the benzyl moiety of 9, which were suspected to be
metabolically labile, in order to improve the microsomal stability,
as shown in Figure 2.
We modified the 2-hydroxypropan-2-yl group of 9, and
assessed the effect on metabolic stability and LLE by the modifica-
tion (Table 2). Although neither removing geminal dimethyl moi-
eties from the 2-hydroxypropan-2-yl group (10) nor converting
the hydroxyl group to a carbonyl group (11) affected the microso-
mal stability, carboxamide derivative 12 showed a significantly
improved metabolic stability with slight decrease of LLE. In addi-
tion, more lipophilic trifluoroethyl amide analog 13 also showed
low clearance in mouse liver microsomes, with higher LLE than
Figure 2. Approaches to improve metabolic stability of 9.

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T. Takai et al. / Bioorg. Med. Chem. 24 (2016) 3192–3206
Table 2
substituents, the benzyl moiety and the methyl group on the amide
moiety, were connected to fix the high-LLE conformation while
improving microsomal stability, as depicted in Figure 3. In
addition, undesirable flexibility of 14 as a drug for use in central
nervous system (CNS) could be reduced by this modification.9
As shown in Table 4, the six-membered spiro derivative 18
showed both high LLE and low metabolic clearance. The spiro ring
size of 18 was enlarged or reduced to seek a favorable conforma-
tion for LLE. While ring contraction resulted in slight decrease of
the LLE (19), great enhancement of potency and LLE were observed
with benzoxazepine derivative 20, with moderate metabolic stabil-
ity. Unfortunately, shifting the oxygen atom in the benzoxazepine
ring of 20 to its benzyl position had a negative effect on the activity
and LLE (21) in spite of improved metabolic stability, which sug-
gests that the benzyl position of 20 is a main metabolized part.
We accordingly introduced bulky substituents into the 6-position
of benzoxazepine ring of 20 to metabolically shield its benzyl posi-
tion by the steric hindrance. However, the metabolic stability could
not be improved in either the 6-chloro (22) or the 6-trifluo-
romethyl (23) analog. As further reduction of metabolic clearance
would be difficult while retaining high LLE, 22 and 23, with potent
Ab42-lowering effect and very high LLE, were selected from spiro
series for in vivo Ab42-lowering test.
We next evaluated in vivo Ab42-lowering effect of the selected
compounds in wild-type mice (C57BL/6J), and the results are
shown in Table 5. Trifluoroethyl amide analog 13, with moderate
in vitro Ab42-lowering effect, was dosed at 30 mg/kg. On the basis
of metabolic stabilities in mouse liver microsomes, 2-hydrox-
ypropan-2-yl derivatives 16 and 17, and spiro compounds 22 and
23 were orally administered at doses of 10 mg/kg and 30 mg/kg,
respectively. Tested compounds except 13 (30 mg/kg) afforded
positive Ab42-lowering effects in mouse brain, and especially 16
(10 mg/kg) and 17 (10 mg/kg) exhibited the same or more potent
activity than 22 (30 mg/kg) and 23 (30 mg/kg), most likely due to
the much lower microsomal clearances of 16 and 17. Consequently
we selected 3,4,5-trifluorophenyl analog 17, which exhibited the
most potent Ab42-lowering effect in mouse brain, and optically
resolved 17 into compounds (R)-17 and (S)-17.10 The chiral config-
uration greatly affected the potency, and the eutomer (R)-17 exhib-
ited more than 460 times the Ab42 inhibitory activity compared to
the distomer (S)-17, as shown in Table 6. We also reported that
compound (R)-17 reduced both Ab42 and Ab40, but increased
Ab37, without affecting total Ab level in cellular assay using Neu-
Ab42-lowering effects, logD values, LLE values, and in vitro metabolic clearance of 10–
14 in mouse liver microsomes
LogDb
LLEc
MLMd
(lL/min/mg)
a
Compound
R
Ab42 IC50
(nM)
10
11
250
290
2.5
2.6
4.1
3.9
144
148
12
13
870
75
2.3
3.0
3.8
4.1
22
3
14
50
2.9
4.4
101
a
b
c
IC50 values are means of triplicate measurements.
Measured at pH 7.4.
LLE = pIC50 ꢀ logD.
d
In vitro metabolic clearance in mice liver microsomes.
that of 12. Introducing a methyl group into the amide moiety of 13
further enhanced Ab42-lowering effect and LLE (14), though this
modification significantly deteriorated the metabolic stability.
We accordingly selected 13 for in vivo Ab42-lowering test, and next
turned our attention to modifying the benzyl moiety of 9.
As shown in Table 3, we replaced the methylene at the benzyl
position of 9, a potential metabolic soft spot, with an oxygen atom
(15). This modification significantly improved the microsomal sta-
bility while maintaining the high LLE value. Enhancement of Ab42
-
lowering effect was then achieved by increasing minimal
lipophilicity of the distal phenyl ring while retaining LLE (16 and
17). Since 16 and 17 maintained the good microsomal stability,
we selected 16 and 17 for further in vivo examination.
We next pursued further improvement of LLE. Starting from tri-
fluoroethyl amide analog 14, which showed the highest LLE value,
we designed spiro compounds in which the 8-position geminal
ro2A cells, indicating that (R)-17 would modulate
activity by shifting the APP cleavage site.11 In addition, (R)-17
exhibited no activity against Notch signal (IC50 >10 M). Potent
c-secretase
Table 3
l
Ab42-lowering effects, logD values, LLE values, and in vitro metabolic clearance of
15–17 in mouse liver microsomes
in vivo Ab42-lowering effect in the brain of (R)-17 was confirmed
at a dose of 6 mg/kg in normal mice (Table 6) with lower drug
exposure level (0.677 lg/g in cortex and 3.307 lg/mL in plasma
at 3 h after oral dosing) than those of 2. Repeated oral treatment
of (R)-17 (3, 6 mg/kg) to APP-transgenic Tg2576 mice was then
performed to further investigate the pharmacological profiles.
We first confirmed that concentration of (R)-17 in cortex 18 h after
single oral administration to Tg2576 mice was below the measur-
able limit, and the multiple administration had little effect on the
compound concentration in plasma or cortex (data not shown).
Compound
R
Ab42
IC50 (nM)
LogDb
LLEc
MLMd
(lL/min/mg)
a
15
16
88
38
2.8
3.2
4.3
4.2
ꢀ1
ꢀ7
17
41
3.2
4.2
ꢀ3
a
b
c
IC50 values are means of triplicate measurements.
Measured at pH 7.4.
LLE = pIC50 ꢀ logD.
d
In vitro metabolic clearance in mice liver microsomes.
Figure 3. Design of spiro compounds from trifluoroethyl amide analog 14.

T. Takai et al. / Bioorg. Med. Chem. 24 (2016) 3192–3206
3197
Table 4
Introduction of the 2-hydroxypropan-2-yl group to the 8-position
of the core scaffold and insertion of methylene linker into the distal
phenyl group at the 8-position effectively enhanced LLE to give 9.
Improvement of the insufficient microsomal stability of 9 in mouse
liver microsomes was achieved by converting the 2-hydrox-
ypropan-2-yl group to trifluoroethyl amide, or replacing the benzyl
moiety with a phenoxy group, while retaining the high LLE. In
addition, spiro compounds that showed extremely high LLE were
discovered by the design from the trifluoroethyl amide analog to
reduce the flexibility and fix its active conformation. Through
in vivo Ab42-lowering effect screening, we identified highly potent
GSM (R)-17. Chronic treatment with (R)-17 significantly reduced
brain Ab42 and then successfully ameliorated cognitive impairment
in Tg2576 mice as an AD model. These results suggest that GSM
(R)-17 should be a promising candidate for AD therapy. Further
pharmacological profiling of (R)-17 is in progress and will be
reported in due course.
Ab42-lowering effects, logD values, LLE values, and in vitro metabolic clearance of 18–
23 in mouse liver microsomes
Compound
R
Ab42
IC50 (nM)
LogDb LLEc MLMd
L/min/mg)
a
(l
18
19
240
520
2.4
2.2
4.2
4
29
ꢀ10
20
21
140
420
2.2
2.5
4.6
3.9
62
11
5. Experimental procedure
Melting points were determined on a Yanaco micro melting
point apparatus or a Büchi melting point apparatus B-545 and
were uncorrected. Proton nuclear magnetic resonance (1H NMR)
spectra were recorded on a Varian Mercury-300 (300 MHz), a Bru-
ker AVANCE III (300 MHz) or a Bruker Advance III plus (400 MHz)
spectrometer. Chemical shifts are given in parts per million (ppm)
with tetramethylsilane as an internal standard. Abbreviations are
used as follows: s = singlet, d = doublet, t = triplet, q = quartet,
quin = quintet, m = multiplet, dd = doublet of doublets, dt = dou-
blet of triplets, td = triplet of doublets, qd = quartet of doublets,
br s = broad singlet. Coupling constants (J values) are given in hertz
(Hz). Elemental analyses were carried out by Takeda Analytical
Laboratories Ltd, and were within 0.4% of the theoretical values
unless otherwise noted. LC–MS analysis was performed on a
Waters Liquid Chromatography–Mass Spectrometer System (LC–
MS) or a Shimadzu LC–MS, operating in APCI (+ or ꢀ) or ESI (+ or ꢀ)
ionization mode. Analytes were eluted using a linear gradient of
0.05% TFA containing water/acetonitrile or 5 mM ammonium
acetate containing water/acetonitrile mobile phase and detected
at 220 nm. Reagents and solvents were obtained from commercial
sources and used without further purification. Chromatographic
purification was carried out on silica gel columns [(Merck Kieselgel
60, 70–230 mesh or 230–400 mesh, Merck) or (Chromatorex
NH-DM 1020, 100–200 mesh, Fuji Silysia Chemical, Ltd)] or on
Purif-Pack (Si or NH, Moritex). Our method employed in determining
purity utilized a Shimadzu UFLC instrument with an L-column 2
22
23
30
31
2.8
2.9
4.8
4.6
107
83
a
b
c
IC50 values are means of triplicate measurements.
Measured at pH 7.4.
LLE = pIC50 ꢀ logD.
d
In vitro metabolic clearance in mice liver microsomes.
Table 5
In vivo Ab42-lowering effects in C57BL/6J mice
a
a
Brain Ab42 (%)
Plasma Ab42 (%)
13 (30 mg/kg)
16 (10 mg/kg)
17 (10 mg/kg)
22 (30 mg/kg)
23 (30 mg/kg)
96
71
66
77
75
80
42
35
49
39
a
Expressed as % vehicle at 3 h after p.o. administration, n = 4–6.
ODS 2
l
m 2.1 ꢂ 30 mm. All tested compounds analyzed were
After 58-day oral administration of (R)-17 at both doses, significant
and dose-dependent reduction of insoluble brain Ab42 as well as
soluble brain Ab42 was observed, as shown in Figure 4A and B.
We also confirmed decreased level of Ab40 without effect on total
Ab (Fig. 4C and D). We used the novel object recognition test
(NORT) to assess the effect of (R)-17 on cognitive function on the
45th day of the course of the multiple administration. The prefer-
ence ratio was significantly improved to the normal range com-
pared to the vehicle-treated group (Fig. 5). Furthermore,
continuous cognitive improvement was observed even after with-
drawal of (R)-17 administration for 7 days, as we had reported.11
These studies indicate the chronic treatment (R)-17 ameliorated
cognitive deficit of Tg2576 mice by lowering pathogenic Ab, and
(R)-17 might have a disease-modifying effect.
>95% pure unless otherwise indicated.
5.1. 8-(3,4-Dichlorophenyl)-8-methoxy-3-[3-methoxy-4-(2-meth
yl-1,3-oxazol-5-yl)phenyl]-5,6,7,8-tetrahydro[1,2,4]triazolo[4,3-
a]pyridine (4)
NaH (60% in oil, 0.014 g, 0.35 mmol) was added to a mixture of
1 (0.16 g, 0.35 mmol) in DMF (2 mL) at 0 °C. After the mixture was
stirred at 0 °C under air for 30 min, NaH (60% in oil, 0.014 g,
0.35 mmol) was added to the reaction mixture. After the mixture
was stirred at 0 °C under N2 for 30 min, MeI (22 lL, 0.35 mmol)
was added to the reaction mixture. After being stirred at room
temperature for 1 h, the reaction mixture was diluted with EtOAc
and brine, and extracted with EtOAc. The extract was washed
with brine, dried over MgSO4 and concentrated in vacuo. The
residue was purified by preparative HPLC (L-Column 2 ODS,
eluted with H2O in acetonitrile containing 0.1% TFA) to give a
solid, which was recrystallized from EtOAc/hexane to give 4
4. Conclusion
In conclusion, we optimized 5,6,7,8-tetrahydro[1,2,4]triazolo
[4,3-a]pyridine derivatives using LLE as a drug-likeness guideline.

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T. Takai et al. / Bioorg. Med. Chem. 24 (2016) 3192–3206
Table 6
Ab42-lowering effects, logD values, LLE values, and in vitro metabolic clearance in mouse liver microsomes, and in vivo Ab42-lowering effects of (R)-17 and (S)-17 in C57BL/6J mice
LogDb
LLEc
4.4
MLMd
(lL/min/mg)
In vivo Ab42 (%)
Brain Plasma
a
e
Compound
R
Ab42 IC50 (nM)
(R)-17
(S)-17
26
3.2
3.2
ꢀ7
60
36
>10000
ꢀ20
N.T.f
N.T.f
a
b
c
d
e
f
IC50 values are means of triplicate measurements.
Measured at pH 7.4.
LLE = pIC50 ꢀ logD.
In vitro metabolic clearance in mice liver microsomes.
Expressed as % vehicle at 3 h after p.o. administration, 6 mg/kg, n = 6.
Not tested.
Figure 4. Effect of (R)-17 on insoluble Ab42 (A), soluble Ab42 (B), soluble Ab40 (C), and soluble total Ab (D) in Tg2576 mouse cortex. (R)-17 was administrated for 58 days and at
3 h prior to decapitation, n = 10–15. The bars represent means SEM. +++p <0.001, Aspin–Welch test versus wild-type (WT) mice treated with vehicle. $p <0.025, Shirley–
Williams test versus Tg mice treated with vehicle. #p <0.025, Williams test versus Tg mice treated with vehicle.
(0.053 g, 31%) as a colorless solid, mp 166–169 °C. 1H NMR (CDCl3)
d: 1.89–2.07 (2H, m), 2.34–2.52 (2H, m), 2.57 (3H, s), 3.31 (3H, s),
3.98–4.11 (4H, m), 4.24–4.33 (1H, m), 7.29–7.36 (1H, m),
7.44–7.49 (1H, m), 7.50–7.53 (2H, m), 7.59 (1H, d, J = 1.9 Hz),
7.86 (1H, d, J = 8.0 Hz). MS m/z: 485.1 (M+H)+. Anal. Calcd for
5.2. {8-(3,4-Dichlorophenyl)-3-[3-methoxy-4-(2-methyl-1,3-oxa
zol-5-yl)phenyl]-5,6,7,8-tetrahydro[1,2,4]triazolo[4,3-a]pyridin
-8-yl}methyl 4-methylbenzenesulfonate (24)
NaH (60% in oil, 0.032 g, 0.79 mmol) was added to a mixture of
1 (0.30 g, 0.66 mmol) in DMF (5 mL) at 0 °C. After the mixture was
stirred at room temperature under N2 for 1 h under, paraformaldehyde
C24H22Cl2N4O3: C, 59.39; H, 4.57; N, 11.54. Found: C, 59.33; H,
4.56; N, 11.51.

T. Takai et al. / Bioorg. Med. Chem. 24 (2016) 3192–3206
3199
4.22 (1H, m), 7.14 (1H, dd, J = 8.4, 2.3 Hz), 7.29 (1H, dd, J = 8.2,
1.4 Hz), 7.36–7.44 (2H, m), 7.50 (2H, s), 7.83 (1H, d, J = 8.0 Hz).
MS m/z: 484.3 (M+H)+.
5.4. N-({8-(3,4-Dichlorophenyl)-3-[3-methoxy-4-(2-methyl-1,3-
oxazol-5-yl)phenyl]-5,6,7,8-tetrahydro[1,2,4]triazolo[4,3-a]pyri
din-8-yl}methyl)acetamide (6)
To a mixture of 5 (0.077 g, 0.16 mmol) and triethylamine
(26
(16
l
L, 0.19 mmol) in THF (1 mL) was added acetic anhydride
lL, 0.17 mmol) at 0 °C. After being stirred at room temperature
for 2 h, the reaction mixture was diluted with water, and the pre-
cipitate was collected by filtration. The solid was purified by silica
gel column chromatography (MeOH/EtOAc) to give 6 (0.060 g, 72%)
as a colorless amorphous. 1H NMR (CDCl3) d: 1.74–1.86 (1H, m),
1.89 (3H, s), 1.97–2.12 (2H, m), 2.33–2.44 (1H, m), 2.56 (3H, s),
3.58 (1H, dd, J = 13.6, 3.7 Hz), 3.94–4.10 (4H, m), 4.16–4.27 (1H,
m), 4.32 (1H, dd, J = 13.6, 8.4 Hz), 7.03–7.13 (2H, m), 7.21 (1H, d,
J = 2.5 Hz), 7.31 (1H, dd, J = 8.0, 1.4 Hz), 7.40 (1H, d, J = 8.2 Hz),
7.52 (2H, s), 7.85 (1H, d, J = 8.0 Hz). MS m/z: 526.3 (M+H)+.
Figure 5. Effect of (R)-17 on novel object recognition test in Tg2576 mouse on the
45th day of 58-day oral administration, n = 10–15. The bars represent means SEM.
***p <0.001, t-test versus WT mice treated with vehicle. $p <0.025, Shirley–Williams
test versus Tg mice treated with vehicle.
(0.040 g) was added to the reaction mixture. After being stirred
for 30 min, the reaction mixture was diluted water and
extracted with EtOAc. The extract was washed with water and
brine, dried over MgSO4 and concentrated in vacuo. The residue
was purified by silica gel column chromatography (MeOH/EtOAc)
to give {8-(3,4-Dichlorophenyl)-3-[3-methoxy-4-(2-methyl-1,3-
oxazol-5-yl)phenyl]-5,6,7,8-tetrahydro[1,2,4]triazolo[4,3-a]pyr-
idin-8-yl}methanol (0.043 g, 13%) as a colorless solid. 1H NMR
(CDCl3) d: 1.76–1.93 (1H, m), 2.03–2.20 (2H, m), 2.30–2.40 (1H,
m), 2.56 (3H, s), 3.83–3.89 (1H, m), 3.95–4.03 (2H, m), 4.05 (3H,
s), 4.13–4.26 (2H, m), 7.09 (1H, dd, J = 8.5, 2.2 Hz), 7.29 (1H, dd,
J = 8.1, 1.5 Hz), 7.33 (1H, d, J = 2.2 Hz), 7.41 (1H, d, J = 8.5 Hz),
7.50 (1H, d, J = 1.6 Hz), 7.51 (1H, s), 7.83 (1H, d, J = 8.0 Hz). MS
m/z: 485.2 (M+H)+. Anal. Calcd for C24H22Cl2N4O3: C, 59.39; H,
4.57; N, 11.54. Found: C, 59.33; H, 4.64; N, 11.77.
5.5. Diethyl(3,4-dichlorophenyl)propanedioate (26a)
To a solution of ethyl 2-(3,4-dichlorophenyl)acetate (5.0 g,
21 mmol) in THF (60 mL) were added NaH (60% in oil, 1.7 g,
43 mmol) and diethyl carbonate (13 mL, 110 mmol) at room tem-
perature. After being refluxed for 2 h, the reaction mixture was
quenched with satd NH4Cl aq and extracted with EtOAc. The
extract was washed with brine, dried over Na2SO4 and concen-
trated in vacuo. The residue was purified by silica gel column chro-
matography (EtOAc/hexane) to give 26a (6.6 g, 100%) as a pale
yellow oil. 1H NMR (CDCl3) d: 1.25–1.33 (6H, m), 4.11–4.28 (4H,
m), 4.54 (1H, s), 7.24–7.27 (1H, m), 7.43 (1H, d, J = 8.1 Hz), 7.51
(1H, d, J = 2.4 Hz).
To a mixture of the methanol derivative (0.18 g, 0.37 mmol) in
pyridine (3 mL) was added 4-methylbenzenesulfonyl chloride
(0.084 g, 0.44 mmol) at 0 °C. After the mixture was stirred at room
temperature for 4 h, 4-methylbenzenesulfonyl chloride (0.084 g,
0.44 mmol) was added to reaction the mixture. After being stirred
at room temperature for 2 h, and at 50 °C overnight, the reaction
mixture was diluted with water, and the precipitate was collected
by filtration. The solid was purified by silica gel column chro-
matography (MeOH/EtOAc) to give 24 (0.16 g, 69%) as a colorless
amorphous. 1H NMR (CDCl3) d: 1.73–1.94 (1H, m), 2.08–2.21 (1H,
m), 2.26–2.39 (1H, m), 2.44 (3H, s), 2.55 (3H, s), 2.64–2.76 (1H,
m), 3.94–4.15 (5H, m), 4.43–4.49 (1H, m), 4.51–4.57 (1H, m),
7.21–7.26 (2H, m), 7.26–7.30 (2H, m), 7.31–7.35 (1H, m), 7.43
(1H, d, J = 1.6 Hz), 7.47 (1H, d, J = 2.2 Hz), 7.50 (1H, s), 7.57–7.64
(2H, m), 7.83 (1H, d, J = 8.2 Hz). MS m/z: 638.9 (M+H)+.
5.6. Diethyl(3,4-difluorophenyl)propanedioate (26b)
Compound 26b was prepared from 25b by a manner similar to
that described for 26a in 90% yield as a pale yellow oil. 1H NMR
(CDCl3) d: 1.27 (6H, t, J = 7.1 Hz), 4.16–4.29 (4H, m), 4.55 (1H, s),
7.08–7.20 (2H, m), 7.26–7.35 (1H, m).
5.7. Ethyl 3-(3,4-dichlorophenyl)-2-oxopiperidine-3-carboxylate
(28a)
NaOEt (20% in ethanol, 0.84 mL) was added to a solution of 26a
(6.6 g, 21 mmol) in t-BuOH (20 mL) under N2 at room temperature.
After the mixture was stirred for 30 min, acrylonitrile (1.4 mL,
21 mmol) was added to the reaction mixture at room temperature.
After being stirred at room temperature overnight, the reaction
mixture was quenched with satd NH4Cl aq and extracted with
EtOAc. The extract was washed with brine, dried over Na2SO4
and concentrated in vacuo. The residue was purified by silica gel
column chromatography (EtOAc/hexane) to give 27a as crude. A
mixture of the crude compound 27a and Raney cobalt (30 g) in
2 M ammonia ethanol solution (60 mL) was hydrogenated under
balloon pressure at room temperature overnight. The catalyst
was removed by filtration through Celite and the filtrate was con-
centrated in vacuo. The residue was purified by silica gel column
chromatography (EtOAc/hexane) to give 28a (2.8 g, 41% from
5.3. 1-{8-(3,4-Dichlorophenyl)-3-[3-methoxy-4-(2-methyl-1,3-
oxazol-5-yl)phenyl]-5,6,7,8-tetrahydro[1,2,4]triazolo[4,3-a]pyri
din-8-yl}methanamine (5)
A mixture of 25 (0.15 g, 0.24 mmol) and sodium azide (0.046 g,
0.71 mmol) in DMSO (2 mL) was stirred at 100 °C for 2 days. Water
was added to the reaction mixture, and the precipitate was col-
lected by filtration. After the solid was dissolved in THF (2 mL)/
water (2 mL), diphenylphosphino-polystyrene (1.99 mmol/g,
0.24 g) was added to the mixture. After being stirred at 60 °C over-
night, the reaction mixture was filtered, and the filtrate was and
concentrated in vacuo. The residue was recrystallized from
EtOAc/hexane to give 5 (0.056 g, 49%) as a colorless solid. 1H
NMR (CDCl3) d: 1.77–1.91 (1H, m), 2.01–2.13 (1H, m), 2.23 (1H,
td, J = 13.2, 2.6 Hz), 2.30–2.40 (1H, m), 2.56 (3H, s), 3.19 (1H, d,
J = 13.2 Hz), 3.42 (1H, d, J = 13.4 Hz), 3.96–4.08 (4H, m), 4.12–
26a) as
a
colorless solid. 1H NMR (CDCl3) d: 1.27 (3H, t,
J = 7.0 Hz), 1.56–1.91 (2H, m), 2.23–2.34 (1H, m), 2.61–2.74 (1H,
m), 3.28–3.51 (2H, m), 4.20–4.30 (2H, m), 6.41 (1H, br s), 7.21
(1H, dd, J = 8.5, 2.5 Hz), 7.41 (1H, d, J = 8.5 Hz), 7.46 (1H, d,
J = 2.5 Hz).

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T. Takai et al. / Bioorg. Med. Chem. 24 (2016) 3192–3206
5.8. Ethyl 3-(3,4-difluorophenyl)-2-oxopiperidine-3-carboxylate
(28b)
5.13. 2-{8-(3,4-Dichlorophenyl)-3-[3-methoxy-4-(2-methyl-1,3-
oxazol-5-yl)phenyl]-5,6,7,8-tetrahydro[1,2,4]triazolo[4,3-a]
pyridin-8-yl}propan-2-ol (7)
Compound 28b was prepared from 26b by a manner similar to
that described for 28a in 73% yield as an off-white solid. 1H NMR
(CDCl3) d: 1.26 (3H, t, J = 7.2 Hz), 1.58–1.90 (2H, m), 2.23–2.34
(1H, m), 2.61–2.72 (1H, m), 3.28–3.50 (2H, m), 4.25 (2H, q,
J = 7.2 Hz), 6.47 (1H, br s), 7.06–7.30 (3H, m). MS m/z: 284.3 (M
+H)+.
Methylmagnesium bromide (1 M in THF, 2.4 mL, 2.4 mmol) was
added to a solution of 31a (0.25 g, 0.47 mmol) in THF (2.5 mL)
under Ar at room temperature. After being stirred under Ar at
0 °C for 1 h, and at room temperature overnight, the reaction mix-
ture was quenched with satd NH4Cl aq and extracted with EtOAc.
The extract was washed with satd NaHCO3 aq and brine, dried over
Na2SO4 and concentrated in vacuo. The residue was purified by NH
silica gel column chromatography (EtOAc/hexane) and recrystal-
lization from EtOAc/hexane to give 7 (0.099 g, 41%) as a colorless
solid, mp 186–188 °C. 1H NMR (CDCl3) d: 1.15 (3H, s), 1.35 (3H,
s), 1.75–1.92 (1H, m), 2.01–2.20 (2H, m), 2.55 (3H, s), 2.68–2.79
(1H, m), 3.94–4.15 (5H, m), 5.25 (1H, br s), 7.23–7.29 (1H, m),
7.36–7.41 (2H, m), 7.46–7.51 (2H, m), 7.60–7.63 (1H, m), 7.82
(1H, d, J = 8.2 Hz). MS m/z: 513.2 (M+H)+.
5.9. Ethyl 3-(3,4-dichlorophenyl)-2-thioxopiperidine-3-carboxylate
(29a)
Lawesson’s reagent (0.90 g, 2.2 mmol) was added to a suspen-
sion of 28a (1.0 g, 3.2 mmol) in toluene (20 mL) under Ar at room
temperature. After being stirred at 110 °C for 2 h, the reaction mix-
ture was cooled to room temperature and concentrated in vacuo.
The residue was purified by NH silica gel column chromatography
(EtOAc/hexane) to give 29a (0.83 g, 79%) as a colorless solid. 1H
NMR (CDCl3) d: 1.29 (3H, t, J = 7.1 Hz), 1.46–1.64 (1H, m), 1.77–
1.90 (1H, m), 2.26–2.36 (1H, m), 2.63–2.75 (1H, m), 3.28–3.54
(2H, m), 4.20–4.33 (2H, m), 7.22–7.28 (1H, m), 7.41 (1H, d,
J = 8.5 Hz), 7.50 (1H, d, J = 2.5 Hz), 8.56 (1H, br s). MS m/z: 332.1
(M+H)+.
5.14. 2-{8-(3,4-Difluorophenyl)-3-[3-methoxy-4-(2-methyl-1,3-
oxazol-5-yl)phenyl]-5,6,7,8-tetrahydro[1,2,4]triazolo[4,3-a]
pyridin-8-yl}propan-2-ol (8)
Compound 8 was prepared from 31b by a manner similar to that
described for 7 in 36% yield as a colorless solid. 1H NMR (CDCl3) d:
1.15 (3H, s), 1.35 (3H, s), 1.77–1.93 (1H, m), 2.00–2.23 (2H, m),
2.55 (3H, s), 2.67–2.81 (1H, m), 3.92–4.18 (5H, m), 5.26 (1H, br s),
7.03–7.18 (1H, m), 7.22–7.32 (2H, m), 7.35–7.44 (1H, m), 7.47–
7.53 (2H, m), 7.82 (1H, d, J = 8.0 Hz). MS m/z: 481.1 (M+H)+.
5.10. Ethyl 3-(3,4-difluorophenyl)-2-thioxopiperidine-3-carbo
xylate (29b)
Compound 29b was prepared from 28b by a manner similar to
that described for 29a in 90% yield as a colorless solid. 1H NMR
(CDCl3) d: 1.29 (3H, t, J = 7.1 Hz), 1.45–1.62 (1H, m), 1.77–1.91
(1H, m), 2.26–2.35 (1H, m), 2.63–2.75 (1H, m), 3.28–3.53 (2H,
m), 4.26 (2H, q, J = 7.1 Hz), 7.07–7.19 (2H, m), 7.23–7.32 (1H, m),
8.57 (1H, br s).
5.15. Ethyl 3-[3-methoxy-4-(2-methyl-1,3-oxazol-5-yl)phenyl]-
5,6,7,8-tetrahydro[1,2,4]triazolo[4,3-a]pyridine-8-carboxylate
(33)
A mixture of 32 (35 g, 200 mmol), trimethyloxonium tetrafluo-
roborate (95%, 32 g, 200 mmol) in MeCN (600 mL) was stirred at
room temperature for 16 h. To the reaction mixture was added
30 (50 g, 200 mmol). After being refluxed for 24 h, the reaction
mixture was concentrated in vacuo. After satd NaHCO3 aq was
added the residue, the insoluble material was removed by filtra-
tion, and the filtrate was extracted with EtOAc. The extract was
washed with brine, and dried over Na2SO4 and concentrated in
vacuo. The residue was crystallized from MeOH/EtOAc to give 33
(29 g, 38%) as a brown solid. 1H NMR (CDCl3) d: 1.32 (3H, t,
J = 7.1 Hz), 1.94–2.42 (4H, m), 2.55 (3H, s), 3.95–4.35 (8H, m),
7.23 (1H, dd, J = 8.0, 1.6 Hz), 7.44 (1H, d, J = 1.6 Hz), 7.49 (1H, s),
7.82 (1H, d, J = 8.0 Hz). MS m/z: 383.3 (M+H)+.
5.11. Ethyl 8-(3,4-dichlorophenyl)-3-[3-methoxy-4-(2-methyl-
1,3-oxazol-5-yl)phenyl]-5,6,7,8-tetrahydro[1,2,4]triazolo[4,3-a]
pyridine-8-carboxylate (31a)
MeI (0.47 ml, 7.5 mmol) was added to a solution of 29a (0.83 g,
2.5 mmol) in MeCN (10 mL) at room temperature. After being stir-
red at 50 °C for 4 h, the reaction mixture was concentrated in vacuo
and diluted with EtOH (10 mL). To the mixture was added 30
(0.62 g, 2.5 mmol) at room temperature. After being stirred at
90 °C overnight, the reaction mixture was quenched with satd
NaHCO3 aq and extracted with EtOAc. The extract was washed
with brine, dried over Na2SO4 and concentrated in vacuo. The resi-
due was purified by silica gel column chromatography (MeOH/
EtOAc) to give 31a (0.78 g, 59%) as an off-white amorphous. 1H
NMR (CDCl3) d: 1.25 (3H, t, J = 7.1 Hz), 1.89–2.20 (2H, m),
2.32–2.44 (1H, m), 2.55 (3H, s), 2.80–2.91 (1H, m), 4.04 (3H, s),
4.08–4.36 (4H, m), 7.26–7.30 (2H, m), 7.42 (1H, d, J = 8.5 Hz),
7.48–7.51 (3H, m), 7.83 (1H, d, J = 8.0 Hz). MS m/z: 527.1 (M+H)+.
5.16. Ethyl 8-(3,4-difluorobenzyl)-3-[3-methoxy-4-(2-methyl-
1,3-oxazol-5-yl)phenyl]-5,6,7,8-tetrahydro[1,2,4]triazolo[4,3-a]
pyridine-8-carboxylate (34)
To a suspension of NaH (60% in oil, 0.34 g, 8.5 mmol) in DMF
(5 mL) was added 33 (3.0 g, 7.8 mmol) at 0 °C. After the mixture
was stirred for 30 min at 0 °C, 3,4-difluorobenzyl bromide (1.5 g,
9.4 mmol) was added at 0 °C. After being stirred for 1 h at 0 °C,
the reaction mixture was diluted with EtOAc, washed with satd
NH4Cl aq and brine, dried over Na2SO4, and concentrated in vacuo.
The residue was purified by silica gel column chromatography
(MeOH/EtOAc) to give 34 (3.5 g, 87%) as a colorless amorphous.
1H NMR (CDCl3) d: 1.37 (3H, t, J = 7.0 Hz), 2.16–2.30 (1H, m),
2.34–2.60 (5H, m), 2.70–2.83 (1H, m), 3.97–4.16 (4H, m), 4.19–
4.31 (1H, m), 4.39 (2H, q, J = 7.0 Hz), 7.21–7.28 (1H, m), 7.46 (1H,
d, J = 1.4 Hz), 7.50 (1H, s), 7.83 (1H, d, J = 8.2 Hz). MS m/z: 509.2
(M+H)+.
5.12. Ethyl 8-(3,4-difluorophenyl)-3-[3-methoxy-4-(2-methyl-
1,3-oxazol-5-yl)phenyl]-5,6,7,8-tetrahydro[1,2,4]triazolo[4,3-a]
pyridine-8-carboxylate (31b)
Compound 31b was prepared from 29b by a manner similar to
that described for 31a in 68% yield as a light brown amorphous. 1H
NMR (CDCl3) d: 1.25 (3H, t, J = 7.2 Hz), 1.89–2.21 (2H, m), 2.31–
2.45 (1H, m), 2.55 (3H, s), 2.79–2.92 (1H, m), 4.00–4.06 (3H, m),
4.08–4.36 (4H, m), 7.08–7.18 (2H, m), 7.24–7.37 (2H, m), 7.51
(2H, s), 7.84 (1H, d, J = 7.9 Hz). MS m/z: 495.2 (M+H)+.

T. Takai et al. / Bioorg. Med. Chem. 24 (2016) 3192–3206
3201
5.17. 2-{8-(3,4-Difluorobenzyl)-3-[3-methoxy-4-(2-methyl-1,3-
oxazol-5-yl)phenyl]-5,6,7,8-tetrahydro[1,2,4]triazolo[4,3-a]pyri
din-8-yl}propan-2-ol (9) and 1-{8-(3,4-difluorobenzyl)-3-[3-
methoxy-4-(2-methyl-1,3-oxazol-5-yl)phenyl]-5,6,7,8-tetrahyd
ro[1,2,4]triazolo[4,3-a]pyridin-8-yl}ethanone (11)
5.20.
8-(3,4-Difluorobenzyl)-3-[3-methoxy-4-(2-methyl-1,3-
oxazol-5-yl)phenyl]-N-(2,2,2-trifluoroethyl)-5,6,7,8-tetrahydro
[1,2,4]triazolo[4,3-a]pyridine-8-carboxamide (13)
To a mixture of 34 (0.20 g, 0.39 mmol) in THF (1 mL) / MeOH
(1 mL) was added 1 M NaOH aq (0.39 mL, 0.39 mmol) at room tem-
perature, and the mixture was stirred at room temperature for 3 h.
The reaction mixture was concentrated in vacuo. Et3N (0.066 mL,
0.47 mmol) and 2,2,2-trifluoroethylamine (0.037 mL, 0.47 mmol)
in DMF (2 mL) were added to the residue. After HATU (0.18 g,
0.47 mmol) was added to the mixture at 0 °C, the mixture was stir-
red at 0 °C for 1 h. The reaction mixture was diluted with EtOAc,
washed with brine and dried over dried over MgSO4 and concen-
trated in vacuo. The residue was purified by silica gel column chro-
matography (EtOAc/hexane) and recrystallization from EtOAc/MeOH
to give 13 (0.069 g, 31%) as a colorless solid, mp 247–249 °C. 1H
NMR (CDCl3) d: 1.65–1.86 (2H, m), 1.94–2.06 (1H, m), 2.57 (3H,
s), 2.68–2.79 (1H, m), 3.30 (1H, d, J = 13.5 Hz), 3.60 (1H, d,
J = 13.5 Hz), 3.71–3.92 (2H, m), 3.99–4.13 (5H, m), 6.78–6.86 (1H,
m), 6.86–6.96 (1H, m), 6.97–7.09 (1H, m), 7.22 (1H, d, J = 8.0 Hz),
7.47 (1H, s), 7.52 (1H, s), 7.84 (1H, d, J = 8.0 Hz), 8.27 (1H, t,
J = 6.3 Hz). MS m/z: 562.4 (M+H)+. Anal. Calcd for C27H24F5N5O3:
C, 57.74; H, 4.49; N, 12.50. Found: C, 57.75; H, 4.31; N, 15.47.
Methylmagnesium bromide (1 M in THF, 13 mL, 13 mmol) was
added to a solution of 34 (1.6 g, 3.2 mmol) in THF (16 mL) at 0 °C.
After being stirred at 0 °C for 1 h, the reaction mixture was
quenched with satd NH4Cl aq and extracted with EtOAc. The
extract was washed with brine, dried over Na2SO4 and concen-
trated in vacuo. The residue was purified by NH silica gel column
chromatography (EtOAc/hexane) to give 9 (0.74 g, 48%) as a color-
less amorphous, and 11 (0.12 g, 8%) as a colorless solid. 9: 1H NMR
(CDCl3) d: 1.09 (3H, s), 1.42 (3H, s), 1.61–1.73 (2H, m), 1.91–2.04
(2H, m), 2.55 (3H, s), 2.84 (1H, d, J = 13.5 Hz), 3.43–3.55 (1H, m),
3.66–3.78 (1H, m), 3.94 (1H, d, J = 13.5 Hz), 4.04 (3H, s), 5.15 (1H,
br s), 6.54–6.75 (2H, m), 6.87–6.98 (1H, m), 7.06 (1H, dd, J = 8.0,
1.4 Hz), 7.38 (1H, d, J = 1.4 Hz), 7.50 (1H, s), 7.80 (1H, d,
J = 8.0 Hz). MS m/z: 495.1 (M+H)+. 11: 1H NMR (CDCl3) d: 1.51–
1.60 (1H, m), 1.76–1.94 (2H, m), 2.41–2.52 (4H, m), 2.55 (3H, s),
3.21 (1H, d, J = 13.5 Hz), 3.70–3.83 (2H, m), 4.01–4.10 (4H, m),
6.80–6.88 (1H, m), 6.89–7.06 (2H, m), 7.20 (1H, dd, J = 8.0,
1.6 Hz), 7.47 (1H, d, J = 1.6 Hz), 7.50 (1H, s), 7.81 (1H, d,
J = 8.0 Hz). MS m/z: 479.3 (M+H)+.
5.21. 8-(3,4-Difluorobenzyl)-3-[3-methoxy-4-(2-methyl-1,3-oxa
zol-5-yl)phenyl]-N-methyl-N-(2,2,2-trifluoroethyl)-5,6,7,8-tetra
hydro[1,2,4]triazolo[4,3-a]pyridine-8-carboxamide (14)
5.18. {8-(3,4-Difluorobenzyl)-3-[3-methoxy-4-(2-methyl-1,3-ox
azol-5-yl)phenyl]-5,6,7,8-tetrahydro[1,2,4]triazolo[4,3-a]pyrid
in-8-yl}methanol (10)
To a mixture of 13 (0.10 g, 0.18 mmol) in DMF (2 mL) was added
NaH (60% in oil, 7.8 mg) at 0 °C, and the mixture was stirred at
room temperature for 1 h. After methyl iodide (0.012 mL,
0.20 mmol) was added to the reaction mixture at 0 °C, the mixture
was stirred at room temperature for 3 h. The reaction mixture was
diluted with EtOAc, washed with brine and dried over dried over
MgSO4 and concentrated in vacuo. The residue was purified by sil-
ica gel column chromatography (EtOAc/hexane) and recrystalliza-
tion from EtOAc/MeOH to give 14 (0.025 g, 25%) as a colorless
solid, mp 113–116 °C. 1H NMR (CDCl3) d: 1.63–1.76 (1H, m),
1.79–1.94 (1H, m), 1.98–2.11 (1H, m), 2.43–2.61 (4H, m), 2.92
(3H, s), 3.58–3.73 (2H, m), 3.84–4.02 (2H, m), 4.03–4.09 (4H, m),
4.32–4.51 (1H, m), 6.82–7.09 (3H, m), 7.18 (1H, d, J = 8.0 Hz),
7.44 (1H, s), 7.52 (1H, s), 7.84 (1H, d, J = 8.0 Hz). MS m/z: 576.4
(M+H)+. Anal. Calcd for C28H26F5N5O3: C, 58.43; H, 4.55; N, 12.17.
Found: C, 58.26; H, 4.79; N, 11.87.
A solution of 34 (0.18 g, 0.35 mmol) in THF (2 mL) was added to
a suspension of LAH (0.054 g, 1.4 mmol) in THF (2 mL) at 0 °C, and
the reaction mixture was stirred for 10 min at 0 °C. After sodium
sulfate decahydrate (0.50 g) was added to the mixture at 0 °C,
the mixture was filtered and concentrated in vacuo. The residue
was purified by NH silica gel column chromatography (EtOAc/hex-
ane) to give 10 (0.12 g, 73%) as a colorless amorphous. 1H NMR
(CDCl3) d: 1.51–1.74 (1H, m), 177–2.04 (3H, m), 2.55 (3H, s), 3.16
(1H, t, J = 13.7 Hz), 3.27 (1H, d, J = 13.7 Hz), 3.58–3.74 (2H, m),
3.95 (2H, t, J = 5.9 Hz), 4.03 (3H, s), 4.07–4.20 (1H, m), 6.85–6.94
(1H, m), 6.96–7.12 (2H, m), 7.17 (1H, dd, J = 8.1, 1.5 Hz), 7.41
(1H, d, J = 1.5 Hz), 7.49 (1H, s), 7.80 (1H, d, J = 8.1 Hz). MS m/z:
467.0 (M+H)+.
5.19. 8-(3,4-Difluorobenzyl)-3-[3-methoxy-4-(2-methyl-1,3-oxa
zol-5-yl)phenyl]-5,6,7,8-tetrahydro[1,2,4]triazolo[4,3-a]pyridi
ne-8-carboxamide (12)
5.22. Ethyl 8-chloro-3-[3-methoxy-4-(2-methyl-1,3-oxazol-5-yl)
phenyl]-5,6,7,8-tetrahydro[1,2,4]triazolo[4,3-a]pyridine-8-carb
oxylate (35)
A
mixture of 34 (0.10 g, 0.20 mmol), formamide (79 lL,
2.0 mmol) and NaOMe (28% in MeOH, 0.2 mL) in DMF (1 mL) was
stirred for 30 min at 70 °C. After being cooled to room temperature,
satd NH4Cl aq was added to the mixture. The precipitate was col-
lected by filtration, washed with water and IPE to give 12
(0.080 g, 85%) as an off-white solid, mp 208–210 °C. 1H NMR
(CDCl3) d: 1.55–1.70 (1H, m), 1.74–2.08 (2H, m), 2.55 (3H, s),
2.64–2.75 (1H, m), 3.34 (1H, d, J = 13.5 Hz), 3.63 (1H, d,
J = 13.5 Hz), 3.76–3.91 (1H, m), 4.00–4.14 (4H, m), 5.54 (1H, d,
J = 3.0 Hz), 6.82–6.91 (1H, m), 6.92–7.08 (2H, m), 7.21 (1H, dd,
J = 8.1, 1.5 Hz), 7.44 (1H, d, J = 1.5 Hz), 7.50 (1H, s), 7.61 (1H, d,
J = 3.0 Hz), 7.82 (1H, d, J = 8.1 Hz). MS m/z: 480.4 (M+H)+. Anal.
Calcd for C25H23F2N5O3: C, 62.62; H, 4.83; N, 14.61. Found: C,
62.43; H, 4.89; N, 14.39. Purity: 90%.
To a suspension of NaH (60% in oil, 0.12 g, 2.9 mmol) in DMF
(10 mL) was added 33 (1.0 g, 2.6 mmol) under Ar at 0 °C. After
the reaction mixture was stirred for 20 min at 0 °C, NCS (0.38 g,
2.9 mmol) was added to the mixture at 0 °C. After being stirred
for 20 min at 0 °C, the reaction mixture was diluted with EtOAc
and satd NH4Cl aq, and extracted with EtOAc. The extract was
washed with brine, dried over Na2SO4 and concentrated in vacuo.
The residue was purified by NH silica gel column chromatography
(MeOH/EtOAc) to give 35 (0.75 g, 69%) as an off-white solid. 1H
NMR (CDCl3) d: 1.37 (3H, t, J = 7.1 Hz), 2.16–2.61 (6H, m), 2.70–
2.85 (1H, m), 3.96–4.17 (4H, m), 4.19–4.30 (1H, m), 4.34–4.46
(2H, m), 7.20–7.29 (1H, m), 7.46 (1H, d, J = 1.4 Hz), 7.50 (1H, s),
7.83 (1H, d, J = 8.2 Hz). MS m/z: 417.1 (M+H)+.

3202
T. Takai et al. / Bioorg. Med. Chem. 24 (2016) 3192–3206
5.23. Ethyl 8-(3,4-difluorophenoxy)-3-[3-methoxy-4-(2-methyl-
1,3-oxazol-5-yl)phenyl]-5,6,7,8-tetrahydro[1,2,4]triazolo[4,3-a]
pyridine-8-carboxylate (36)
m), 4.84 (1H, br s), 6.52–6.66 (2H, m), 7.07–7.15 (1H, m), 7.23
(1H, dd, J = 8.1, 1.5 Hz), 7.47 (1H, d, J = 1.5 Hz), 7.51 (1H, s),
7.84 (1H, d, J = 8.1 Hz). MS m/z: 513.1 (M+H)+. Anal. Calcd for
C
26H26N4O4ꢁ0.5H2O: C, 59.83; H, 5.21; N, 10.73. Found: C, 59.95;
A mixture of 35 (0.50 g, 1.2 mmol), 3,4-difluorophenol (0.16 g,
1.3 mmol) and K2CO3 (0.50 g, 3.6 mmol) in DMF (5 mL) was stirred
at 100 °C under N2 for 30 min. After being cooled to room temper-
ature, satd NH4Cl aq was added to the reaction mixture, and the
mixture was extracted with EtOAc. The extract was washed with
brine, dried over MgSO4. The residue was purified by NH silica
gel column chromatography (EtOAc/hexane) to give 36 (0.27 g,
44%) as an off-white amorphous. 1H NMR (CDCl3) d: 1.31 (3H, t,
J = 7.1 Hz), 2.16–2.29 (1H, m), 2.34–2.52 (2H, m), 2.57 (3H, s),
2.65 (1H, dd, J = 11.1, 5.6 Hz), 4.03 (3H, s), 4.06–4.19 (1H, m),
4.25–4.46 (3H, m), 6.89–7.14 (3H, m), 7.26–7.29 (1H, m), 7.48
(1H, d, J = 1.1 Hz), 7.51 (1H, s), 7.84 (1H, d, J = 8.0 Hz). MS m/z:
511.2 (M+H)+.
H, 5.37; N, 10.71.
5.28.
2-{3-[3-Methoxy-4-(2-methyl-1,3-oxazol-5-yl)phenyl]-8-
(3,4,5-trifluorophenoxy)-5,6,7,8-tetrahydro[1,2,4]triazolo[4,3-a]
pyridin-8-yl}propan-2-ol (17)
Compound 17 was prepared from 38 by a manner similar to
that described for 7 in 59% yield as a colorless solid, mp 190–
193 °C. 1H NMR (CDCl3) d: 1.29 (3H, s), 1.59 (3H, s), 1.99–2.24
(3H, m), 2.38–2.49 (1H, m), 2.58 (3H, s), 3.97–4.09 (4H, m), 4.26–
4.36 (1H, m), 4.90 (1H, br s), 6.49 (2H, dd, J = 9.1, 6.0 Hz), 7.24
(1H, d, J = 1.4 Hz), 7.47 (1H, d, J = 1.4 Hz), 7.53 (1H, s), 7.86 (1H,
d, J = 8.0 Hz). MS m/z: 515.3 (M+H)+. Anal. Calcd for C26H25F3N4O4:
C, 60.70; H, 4.90; N, 10.89. Found: C, 60.56; H, 5.00; N, 10.81.
5.24. Ethyl 8-(4-chloro-3-fluorophenoxy)-3-[3-methoxy-4-(2-
methyl-1,3-oxazol-5-yl)phenyl]-5,6,7,8-tetrahydro[1,2,4]triazol
o[4,3-a]pyridine-8-carboxylate (37)
5.29. Ethyl 3-[3-methoxy-4-(2-methyl-1,3-oxazol-5-yl)phenyl]-
8-(2-nitrobenzyl)-5,6,7,8-tetrahydro[1,2,4]triazolo[4,3-a]pyridi
ne-8-carboxylate (39)
Compound 37 was prepared from 35 and 4-chloro-4-fluorophe-
nol by a manner similar to that described for 36 in 62% yield as a
colorless amorphous. 1H NMR (CDCl3) d: 1.29 (3H, t, J = 7.0 Hz),
2.11–2.28 (1H, m), 2.34–2.51 (2H, m), 2.55 (3H, s), 2.61–2.71
(1H, m), 4.02 (3H, s), 4.05–4.17 (1H, m), 4.22–4.46 (3H, m), 6.96–
7.03 (1H, m), 7.08 (1H, dd, J = 10.6, 2.9 Hz), 7.17–7.31 (2H, m),
7.46 (1H, d, J = 1.4 Hz), 7.50 (1H, s), 7.83 (1H, d, J = 8.0 Hz). MS
m/z: 527.2 (M+H)+.
Compound 39 was prepared from 33 and 2-nitrobenzyl bro-
mide by a manner similar to that described for 34 in 60% yield as
a
J = 7.1 Hz), 1.65–1.77 (1H, m), 1.94–2.04 (2H, m), 2.36–2.47 (1H,
m), 2.55 (3H, s), 3.74–3.90 (1H, m), 3.97 (1H, d, J = 14.0 Hz), 4.04
(3H, s), 4.05–4.15 (1H, m), 4.17–4.27 (3H, m), 7.19 (1H, dd,
J = 8.0, 1.4 Hz), 7.32–7.45 (2H, m), 7.46–7.49 (2H, m), 7.80 (1H, d,
J = 8.0 Hz), 7.85 (1H, dd, J = 7.7, 1.6 Hz). MS m/z: 518.4 (M+H)+.
pale yellow amorphous. 1H NMR (CDCl3) d: 1.26 (3H, t,
5.25. Ethyl 3-[3-methoxy-4-(2-methyl-1,3-oxazol-5-yl)phenyl]-
8-(3,4,5-trifluorophenoxy)-5,6,7,8-tetrahydro[1,2,4]triazolo
[4,3-a]pyridine-8-carboxylate (38)
5.30. Ethyl 3-[3-methoxy-4-(2-methyl-1,3-oxazol-5-yl)phenyl]-
8-(2-nitrophenyl)-5,6,7,8-tetrahydro[1,2,4]triazolo[4,3-a]pyridi
ne-8-carboxylate (40)
Compound 38 was prepared from 35 and 3,4,5-trifluorophenol
by a manner similar to that described for 36 in 43% yield as a col-
orless amorphous. 1H NMR (CDCl3) d: 1.31 (3H, t, J = 7.1 Hz), 2.17–
2.31 (1H, m), 2.34–2.52 (2H, m), 2.57 (3H, s), 2.65 (1H, dd, J = 11.0,
5.5 Hz), 4.04 (3H, s), 4.07–4.19 (1H, m), 4.26–4.44 (3H, m), 6.96
(2H, dd, J = 9.1, 6.0 Hz), 7.29 (1H, d, J = 1.4 Hz), 7.49 (1H, d,
J = 1.1 Hz), 7.52 (1H, s), 7.85 (1H, d, J = 8.0 Hz). MS m/z: 529.2
(M+H)+.
To a mixture of 33 (2.0 g, 5.2 mmol) and 1-fluoro-2-nitroben-
zene (1.0 g, 7.3 mmol) in DMF (10 mL) was added NaH (60% in
oil, 0.21 g, 5.2 mmol) at room temperature. After being stirred for
5 h at 40 °C, the reaction mixture was diluted with satd NaHCO3
aq and extracted with EtOAc. The extract was washed with water
and brine, dried over MgSO4, and concentrated in vacuo. The resi-
due was purified by silica gel column chromatography (EtOAc/hex-
ane) to give 40 (1.2 g, 46%) as a colorless solid. 1H NMR (CDCl3) d:
1.24 (3H, t, J = 7.0 Hz), 1.80–1.95 (1H, m), 2.16–2.34 (1H, m), 2.56
(3H, s), 2.58–2.69 (1H, m), 3.16–3.25 (1H, m), 4.06 (3H, s), 4.16–
4.32 (4H, m), 6.77–6.85 (1H, m), 7.31–7.38 (1H, m), 7.44–7.58
(4H, m), 7.87 (1H, d, J = 7.9 Hz), 8.11 (1H, dd, J = 7.7, 2.1 Hz). MS
m/z: 504.5 (M+H)+.
5.26. 2-{8-(3,4-Difluorophenoxy)-3-[3-methoxy-4-(2-methyl-1,
3-oxazol-5-yl)phenyl]-5,6,7,8-tetrahydro[1,2,4]triazolo[4,3-a]
pyridin-8-yl}propan-2-ol (15)
Compound 15 was prepared from 36 by a manner similar to
that described for 7 in 58% yield as a colorless solid, mp 171–
174 °C. 1H NMR (CDCl3) d: 1.29 (3H, s), 1.61 (3H, s), 2.03–2.17
(3H, m), 2.37–2.49 (1H, m), 2.57 (3H, s), 3.94–4.09 (4H, m), 4.21–
4.31 (1H, m), 4.86 (1H, br s), 6.52–6.60 (1H, m), 6.63–6.70 (1H,
m), 6.83–6.96 (1H, m), 7.19–7.24 (1H, m), 7.48 (1H, d, J = 1.1 Hz),
7.53 (1H, s), 7.85 (1H, d, J = 8.0 Hz). MS m/z: 497.2 (M+H)+. Anal.
Calcd for C26H26F2N4O4: C, 62.90; H, 5.28; N, 11.28. Found: C,
62.80; H, 5.39; N, 11.16.
5.31. Ethyl 3-[3-methoxy-4-(2-methyl-1,3-oxazol-5-yl)phenyl]-
8-[(2-nitrobenzyl)oxy]-5,6,7,8-tetrahydro[1,2,4]triazolo[4,3-a]
pyridine-8-carboxylate (41)
To a solution of 33 (1.0 g, 2.6 mmol) in DMF (13 mL) was added
NaH (60% in oil, 0.12 g, 2.9 mmol) at room temperature. After being
stirred for 1 h at room temperature, NaH (60% in oil, 0.12 g,
2.9 mmol) was added to the reaction mixture. After being stirred
for 10 min at room temperature, 2-nitrobenzyl bromide was added
to the reaction mixture. After being stirred for 1 h at room temper-
ature, the mixture was diluted with sat. NH4Cl aq and EtOAc. After
addition of sat. NaHCO3 aq, the mixture was extracted with EtOAc.
The extract was washed with water and brine, dried over MgSO4,
and concentrated in vacuo. The residue was purified by NH silica
gel column chromatography (EtOAc/hexane) to give 41 (0.48 g,
35%) as a colorless oil. 1H NMR (CDCl3) d: 1.32 (3H, t, J = 7.2 Hz),
5.27. 2-{8-(4-Chloro-3-fluorophenoxy)-3-[3-methoxy-4-(2-me
thyl-1,3-oxazol-5-yl)phenyl]-5,6,7,8-tetrahydro[1,2,4]triazolo
[4,3-a]pyridin-8-yl}propan-2-ol (16)
Compound 16 was prepared from 37 by a manner similar to
that described for 7 in 66% yield as a colorless amorphous. 1H
NMR (CDCl3) d: 1.28 (3H, s), 1.59 (3H, s), 2.02–2.18 (3H, m),
2.38–2.49 (1H, m), 2.56 (3H, s), 3.91–4.07 (4H, m), 4.20–4.30 (1H,

T. Takai et al. / Bioorg. Med. Chem. 24 (2016) 3192–3206
3203
2.06–2.16 (1H, m), 2.19–2.48 (3H, m), 2.56 (3H, s), 3.95–4.08 (4H,
m), 4.22–4.46 (3H, m), 5.04–5.30 (2H, m), 7.29 (1H, dd, J = 8.0,
1.5 Hz), 7.38–7.45 (1H, m), 7.46–7.53 (2H, m), 7.53–7.61 (1H, m),
7.65–7.71 (1H, m), 7.85 (1H, d, J = 8.3 Hz), 7.88–7.94 (1H, m).
534.6 (M+H)+.
7.34 (1H, m), 7.45 (1H, s), 7.51 (1H, s), 7.86 (1H, d, J = 7.9 Hz),
8.64 (1H, s). MS m/z: 428.4 (M+H)+.
5.36. 30-[3-Methoxy-4-(2-methyl-1,3-oxazol-5-yl)phenyl]-1,5,60,
70-tetrahydro-2H,50H-spiro[4,1-benzoxazepine-3,80-[1,2,4]triazo
lo[4,3-a]pyridin]-2-one (46)
5.32. Ethyl 8-[(2-chloro-6-nitrobenzyl)oxy]-3-[3-methoxy-4-(2-
methyl-1,3-oxazol-5-yl)phenyl]-5,6,7,8-tetrahydro[1,2,4]triazo
lo[4,3-a]pyridine-8-carboxylate (42)
A mixture of 41 (0.34 g, 0.64 mmol) and PtO2 (0.020 g) in MeOH
(6 mL) was hydrogenated under balloon pressure at room temper-
ature for 2 h. The catalyst was removed by filtration and the filtrate
was concentrated in vacuo to give the aniline, which was subjected
to the next reaction without further purification. After the aniline
was diluted with 1 M NaOH aq (2 mL) and THF (4 mL), the mixture
was stirred at room temperature for 4 h, neutralized with 1 M HCl
aq and concentrated in vacuo to afford the aminobenzoic acid,
which was subjected to the next reaction without further purifica-
tion. After the aminobenzoic acid was azeotropically dried with
toluene, DMF (25 mL), Et3N (0.44 mL, 3.1 mmol) and HATU
(0.29 g, 0.77 mmol) were added to the residue. After being stirred
at room temperature for 30 min, the mixture was diluted with
water and extracted with EtOAc. The extract was washed with
water and brine, dried over MgSO4, and concentrated in vacuo.
The residue was purified by NH silica gel column chromatography
(MeOH/EtOAc) to give 46 (0.17 g, 57%) as a colorless oil. 1H NMR
(CDCl3) d: 2.09–2.53 (4H, m), 2.56 (3H, s), 4.04 (3H, s), 4.07–4.17
(1H, m), 4.19–4.29 (1H, m), 4.48 (1H, d, J = 14.6 Hz), 5.80 (1H, d,
J = 14.0 Hz), 6.95 (1H, d, J = 7.1 Hz), 7.02–7.13 (1H, m), 7.15–7.22
(1H, m), 7.27–7.34 (2H, m), 7.45 (1H, d, J = 1.4 Hz), 7.51 (1H, s),
7.85 (1H, d, J = 8.0 Hz), 7.99 (1H, s). MS m/z: 458.5 (M+H)+.
Compound 42 was prepared from 33 and 2-(bromomethyl)-1-
chloro-3-nitrobenzene by a manner similar to that described for
41 in 64% yield as a colorless amorphous. 1H NMR (CDCl3) d: 1.26
(3H, t, J = 7.2 Hz), 1.97–2.07 (1H, m), 2.19–2.41 (3H, m), 2.56 (3H,
s), 3.91–4.07 (4H, m), 4.25–4.51 (3H, m), 4.98 (1H, d, J = 11.1 Hz),
5.62 (1H, d, J = 10.9 Hz), 7.29–7.41 (2H, m), 7.48–7.55 (2H, m),
7.60 (2H, d, J = 7.7 Hz), 7.86 (1H, d, J = 8.1 Hz). MS m/z: 568.1
(M+H)+.
5.33. Ethyl 3-[3-methoxy-4-(2-methyl-1,3-oxazol-5-yl)phenyl]-
8-{[2-nitro-6-(trifluoromethyl)benzyl]oxy}-5,6,7,8-tetrahydro
[1,2,4]triazolo[4,3-a]pyridine-8-carboxylate (43)
Compound 43 was prepared from 33 and 2-(bromomethyl)-1-
nitro-3-(trifluoromethyl)benzene by a manner similar to that
described for 41 in 39% yield as a colorless amorphous. 1H NMR
(CDCl3) d: 1.34 (3H, t, J = 7.0 Hz), 2.10–2.41 (4H, m), 2.56 (3H, s),
3.91–4.10 (1H, m), 4.05 (3H, s), 4.27–4.48 (3H, m), 5.13 (1H, d),
5.50 (1H, d, J = 11.7 Hz), 7.31–7.40 (1H, m), 7.47–7.63 (3H, m),
7.77–7.94 (3H, m). MS m/z: 602.1 (M+H)+.
5.37. 6-Chloro-30-[3-methoxy-4-(2-methyl-1,3-oxazol-5-yl)phen
yl]-1,5,60,70-tetrahydro-2H,50H-spiro[4,1-benzoxazepine-3,80-[1,
2,4]triazolo[4,3-a]pyridin]-2-one (47)
5.34. 30-[3-Methoxy-4-(2-methyl-1,3-oxazol-5-yl)phenyl]-1,4,60,
70-tetrahydro-2H,50H-spiro[quinoline-3,80-[1,2,4]triazolo[4,3-a]
pyridin]-2-one (44)
To a mixture of 42 (0.70 g, 1.2 mmol) and FeCl3ꢁ6H2O (0.067 g,
0.25 mmol) in MeOH (20 mL) and THF (20 mL) were added acti-
vated charcoal (0.10 g) and a solution of hydrazine monohydrate
(0.37 g, 7.4 mmol) in MeOH at 60 °C. The mixture was stirred at
60 °C for 1 h, and concentrated in vacuo. The residue was diluted
with EtOAc, washed with brine, dried over MgSO4, and concen-
trated in vacuo. The residue was purified by NH silica gel column
chromatography (MeOH/EtOAc) to give the aniline. Compound 47
was prepared from the aniline by a manner similar to that
described for 46 in 45% yield as a colorless amorphous. 1H NMR
(DMSO-d6) d: 2.08 (2H, d, J = 4.5 Hz), 2.25–2.45 (2H, m), 3.33 (3H,
s), 4.03 (3H, s), 4.17–4.39 (2H, m), 4.99 (1H, d, J = 14.7 Hz), 5.29
(1H, d, J = 14.7 Hz), 7.12–7.38 (3H, m), 7.42–7.62 (3H, m), 7.82
(1H, d, J = 7.9 Hz), 10.75 (1H, s). MS m/z: 492.4 (M+H)+.
A mixture of 39 (0.16 g, 0.31 mmol) and 10% Pd/C (50% water,
0.016 g) in MeOH (2 mL) was hydrogenated under balloon pressure
at room temperature for 9 h. After the reaction mixture was diluted
with EtOAc, the catalyst was removed by filtration and the filtrate
was concentrated in vacuo. After the residue was dissolved with
EtOH (5 mL), the mixture was stirred at 60 °C overnight and con-
centrated in vacuo. The residue was recrystallized from MeOH/
IPE to give 44 (0.059 g, 43%) as a colorless solid, mp 251–253 °C.
1H NMR (DMSO-d6) d: 1.64–1.85 (1H, m), 1.89–2.10 (3H, m), 2.49
(3H, s), 3.02 (1H, d, J = 16.5 Hz), 3.79 (1H, d, J = 16.8 Hz), 4.01
(3H, s), 4.06–4.17 (1H, m), 4.17–4.28 (1H, m), 6.88–7.03 (2H, m),
7.15–7.26 (2H, m), 7.41–7.51 (3H, m), 7.78 (1H, d, J = 8.0 Hz),
10.49 (1H, s). MS m/z: 442.3 (M+H)+. Anal. Calcd for C25H23N5O3ꢁ0.5
H2O: C, 66.65; H, 5.77; N, 15.55. Found: C, 66.63; H, 5.38; N, 15.42.
5.38. 30-[3-Methoxy-4-(2-methyl-1,3-oxazol-5-yl)phenyl]-6-(trif
luoromethyl)-1,5,60,70-tetrahydro-2H,50H-spiro[4,1-benzoxazepi
ne-3,80-[1,2,4]triazolo[4,3-a]pyridin]-2-one (48)
5.35. 30-[3-Methoxy-4-(2-methyl-1,3-oxazol-5-yl)phenyl]-60,70-
dihydro-50H-spiro[indole-3,80-[1,2,4]triazolo[4,3-a]pyridin]-2
(1H)-one (45)
Compound 48 was prepared from the aniline by a manner sim-
ilar to that described for 47 in 69% yield as a pale yellow amor-
phous. 1H NMR (CDCl3) d: 2.28–2.54 (4H, m), 2.56 (3H, s), 4.05
(3H, s), 4.08–4.18 (1H, m), 4.18–4.31 (1H, m), 4.93 (1H, d,
J = 15.1 Hz), 5.68 (1H, d, J = 15.1 Hz), 7.23–7.55 (6H, m), 7.86 (1H,
d, J = 7.9 Hz), 8.94 (1H, s). MS m/z: 526.4 (M+H)+.
To a solution of 40 (0.60 g, 1.2 mmol) in AcOH (10 mL) and EtOH
(10 mL) was added iron (0.30 g, 5.4 mmol) at 90 °C. The reaction
mixture was refluxed for 3 h and concentrated in vacuo. The resi-
due was diluted with 2 M NaOH aq and extracted with EtOAc.
The extract was washed with water and brine, dried over MgSO4,
and concentrated in vacuo. After addition of IPE to the residue,
the precipitate was collected by filtration and washed with IPE to
give 45 (0.20 g, 39%) as a colorless solid. 1H NMR (CDCl3) d:
2.13–2.31 (2H, m), 2.38–2.52 (1H, m), 2.56 (3H, s), 2.65–2.85
(1H, m), 4.04 (3H, s), 4.16–4.41 (2H, m), 6.85 (1H, d, J = 7.5 Hz),
6.96–7.07 (1H, m), 7.10–7.16 (1H, m), 7.16–7.24 (1H, m), 7.28–
5.39. 30-[3-Methoxy-4-(2-methyl-1,3-oxazol-5-yl)phenyl]-1-(2,
2,2-trifluoroethyl)-1,4,60,70-tetrahydro-2H,50H-spiro[quinoline-
3,80-[1,2,4]triazolo[4,3-a]pyridin]-2-one (18)
To a suspension of 44 (0.066 g, 0.15 mmol) in DMF (1 mL) was
added NaH (60% in oil, 7.2 mg, 0.18 mmol) at 0 °C under N2. After

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T. Takai et al. / Bioorg. Med. Chem. 24 (2016) 3192–3206
the mixture was stirred at 0 °C for 30 min, 2,2,2-trifluoroethyl tri-
fluoromethanesulfonate (26 L, 0.18 mmol) was added to the mix-
5.44. N-[2-(Benzyloxy)phenyl]-8-(hydroxymethyl)-3-[3-methox
y-4-(2-methyl-1,3-oxazol-5-yl)phenyl]-5,6,7,8-tetrahydro[1,2,4]
triazolo[4,3-a]pyridine-8-carboxamide (50)
l
ture. After being stirred at room temperature for 1 h, the mixture
was diluted with water. The precipitate was collected by filtration,
and purified by silica gel column chromatography (MeOH/EtOAc)
and preparative HPLC (L-Column 2 ODS, eluted with H2O in ace-
tonitrile containing 0.1% TFA) to give 18 (0.031 g, 40%) as an off-
white solid. 1H NMR (CDCl3) d: 1.66–1.82 (1H, m), 1.94–2.27 (3H,
m), 2.56 (3H, s), 3.01 (1H, d, J = 16.2 Hz), 3.89–4.02 (1H, m), 4.04
(3H, s), 4.16–4.31 (2H, m), 4.36 (1H, d, J = 15.9 Hz), 4.93–5.14
(1H, m), 7.07–7.19 (2H, m), 7.22–7.29 (2H, m), 7.31–7.39 (1H,
m), 7.46 (1H, d, J = 1.4 Hz), 7.50 (1H, s), 7.83 (1H, d, J = 8.0 Hz).
MS m/z: 524.3 (M+H)+. Purity: 91%.
To a suspension of NaH (60% in oil, 0.035 g, 0.86 mmol) in DMF
(3 mL) was added 33 (0.30 g, 0.78 mmol) at 0 °C under Ar. After the
mixture was stirred at 0 °C for 15 min, paraformaldehyde (0.028 g)
was added to the reaction mixture at 0 °C. After the mixture was stir-
red at 0 °C for 15 min, water (0.014 mL, 0.78 mmol) was added to the
mixture at room temperature. The mixture was stirred at room tem-
perature for 1 h, and dried over Na2SO4 (0.11 g, 0.78 mmol). To the
reaction mixture were added 2-benzyloxyaniline (0.19 g, 0.94 mmol)
and HATU (0.31 g, 1.2 mmol) at 0 °C. After being stirred at room tem-
perature for 30 min, the mixture was diluted with EtOAc, washed
with water and brine, dried over Na2SO4 and concentrated in vacuo.
The residue was purified by silica gel column chromatography
(MeOH/EtOAc) to give 50 (0.24 g, 55%) as an off-white amorphous.
1H NMR (CDCl3) d: 1.78–1.91 (1H, m), 2.01–2.21 (2H, m), 2.56 (3H,
s), 2.75–2.86 (1H, m), 3.92–4.05 (5H, m), 4.07–4.21 (2H, m), 5.15
(2H, s), 6.87–7.05 (3H, m), 7.20–7.24 (2H, m), 7.29–7.38 (3H, m),
7.43–7.49 (2H, m), 7.50 (1H, s), 7.82 (1H, d, J = 8.0 Hz), 8.29 (1H, dd,
J = 8.0, 1.9 Hz), 9.52 (1H, s). MS m/z: 566.1 (M+H)+.
5.40. 30-[3-Methoxy-4-(2-methyl-1,3-oxazol-5-yl)phenyl]-1-(2,2,
2-trifluoroethyl)-60,70-dihydro-50H-spiro[indole-3,80-[1,2,4]triaz
olo[4,3-a]pyridin]-2(1H)-one (19)
Compound 19 was prepared from 45 by a manner similar to
that described for 18 in 73% yield as a colorless solid. 1H NMR
(CDCl3) d: 2.16–2.32 (2H, m), 2.35–2.47 (1H, m), 2.56 (3H, s),
2.71–2.88 (1H, m), 4.03 (3H, s), 4.06–4.27 (2H, m), 4.30–4.43 (1H,
m), 4.66 (1H, dq, J = 15.5, 9.0 Hz), 7.05 (1H, d, J = 7.9 Hz), 7.10–
7.23 (2H, m), 7.30 (1H, dd, J = 7.9, 1.5 Hz), 7.38 (1H, td, J = 7.6,
1.7 Hz), 7.44 (1H, s), 7.51 (1H, s), 7.86 (1H, d, J = 7.9 Hz). MS m/z:
510.5 (M+H)+.
5.45. 30-[3-Methoxy-4-(2-methyl-1,3-oxazol-5-yl)phenyl]-5-(2,2,
2-trifluoroethyl)-60,70-dihydro-50H-spiro[1,5-benzoxazepine-
3,80-[1,2,4]triazolo[4,3-a]pyridin]-4(5H)-one (21)
A mixture of 50 (0.44 g, 0.78 mmol) and 10% Pd/C (50% water,
0.060 g) in MeOH (2 mL) and THF (1 mL) was hydrogenated under
balloon pressure at room temperature for 6 h. The catalyst was
removed by filtration and the filtrate was concentrated in vacuo to
give the deprotected compound, which was subjected to the next
reaction without further purification. To a mixture of the depro-
tected compound and triphenylphosphine (0.079 g, 0.30 mmol)
was added diethyl azodicarboxylate (40% in toluene, 0.14 mL) at
0 °C under Ar. After the mixture was stirred at room temperature
5.41. 30-[3-Methoxy-4-(2-methyl-1,3-oxazol-5-yl)phenyl]-1-(2,2
,2-trifluoroethyl)-1,5,60,70-tetrahydro-2H,50H-spiro[4,1-benzoxa
zepine-3,80-[1,2,4]triazolo[4,3-a]pyridin]-2-one (20)
Compound 20 was prepared from 46 by a manner similar to
that described for 18 in 60% yield as a colorless solid. 1H NMR
(CDCl3) d: 1.99–2.37 (4H, m), 2.55 (3H, s), 3.97–4.09 (5H, m),
4.30–4.50 (1H, m), 4.73 (1H, d, J = 13.2 Hz), 4.97–5.15 (1H, m),
5.44 (1H, d, J = 13.2 Hz), 7.22 (1H, dd, J = 7.9, 1.5 Hz), 7.24–7.29
(1H, m), 7.30–7.52 (5H, m), 7.81 (1H, d, J = 7.9 Hz). MS m/z: 540.4
(M+H)+.
for 2 h, tributylphosphine (75 l
L, 0.30 mmol) and 1,10-(azodicar-
bonyl)dipiperidine (0.076 g, 0.30 mmol) were added to the mixture
at room temperature. After being stirred at room temperature for
4 days, the mixture was concentrated in vacuo. The residue was
purified by silica gel column chromatography (MeOH/EtOAc) to give
51as an off-white oil, which was subjected to thenext reaction with-
out further purification. To a solution of 51 in DMF (1 mL) was added
NaH (60% in oil, 8.1 mg, 0.20 mmol) at 0 °C under Ar. After the mix-
ture was stirred at 0 °C for 20 min, 2,2,2-trifluoroethyl trifluo-
5.42. 6-Chloro-30-[3-methoxy-4-(2-methyl-1,3-oxazol-5-yl)phe
nyl]-1-(2,2,2-trifluoroethyl)-1,5,60,70-tetrahydro-2H,50H-spiro
[4,1-benzoxazepine-3,80-[1,2,4]triazolo[4,3-a]pyridin]-2-one
(22)
romethanesulfonate (38 lL, 0.25 mmol) was added to the mixture
at 0 °C. After being stirred at room temperature for 20 min, the mix-
ture was diluted with EtOAc, washed with water and brine, dried
over Na2SO4 and concentrated in vacuo. The residue was purified
by silica gel column chromatography (MeOH/EtOAc) and NH silica
gel column chromatography (EtOAc/hexane) to give 21 (0.024 g,
5.7%) as a colorless amorphous. 1H NMR (CDCl3) d: 2.08–2.35 (2H,
m), 2.47–2.66 (5H, m), 3.97–4.09 (4H, m), 4.14 (1H, d, J = 6.4 Hz),
4.19–4.29 (1H, m), 4.35–4.47 (2H, m), 4.81 (1H, d, J = 6.4 Hz), 6.87
(1H, dd, J = 7.9, 1.5 Hz), 7.03–7.16 (2H, m), 7.22–7.26 (1H, m), 7.43
(1H, d, J = 1.5 Hz), 7.51 (1H, s), 7.84 (1H, d, J = 8.3 Hz), 8.08 (1H, dd,
J = 7.9, 1.9 Hz). MS m/z: 540.1 (M+H)+.
Compound 22 was prepared from 47 by a manner similar to
that described for 18 in 43% yield as a colorless amorphous. 1H
NMR (CDCl3) d: 1.97–2.38 (4H, m), 2.54 (3H, s), 3.94–4.17 (5H,
m), 4.34–4.51 (1H, m), 4.96–5.13 (1H, m), 5.16 (1H, d,
J = 13.6 Hz), 5.40 (1H, d, J = 13.6 Hz), 7.16–7.44 (5H, m), 7.49 (1H,
s), 7.81 (1H, d, J = 7.9 Hz). MS m/z: 574.6 (M+H)+.
5.43. 30-[3-Methoxy-4-(2-methyl-1,3-oxazol-5-yl)phenyl]-1-(2,2,
2-trifluoroethyl)-6-(trifluoromethyl)-1,5,60,70-tetrahydro-2H,50
H-spiro[4,1-benzoxazepine-3,80-[1,2,4]triazolo[4,3-a]pyridin]-
2-one (23)
5.46. 2-[(8R)-3-[3-Methoxy-4-(2-methyl-1,3-oxazol-5-yl)phen yl]
-8-(3,4,5-trifluorophenoxy)-5,6,7,8-tetrahydro[1,2,4]triazolo[4,3-
a]pyridin-8-yl]propan-2-ol ((R)-17) and 2-[(8S)-3-[3-Methoxy-4-
(2-methyl-1,3-oxazol-5-yl)phenyl]-8-(3,4,5-trifluorophenoxy)-5, 6,7,
8-tetrahydro[1,2,4]triazolo[4,3-a]pyridin-8-yl]propan-2-ol ((S)-17)
Compound 23 was prepared from 48 by a manner similar to
that described for 18 in 54% yield as a colorless amorphous. 1H
NMR (CDCl3) d: 1.97–2.13 (1H, m), 2.14–2.40 (3H, m), 2.54 (3H,
s), 3.94–4.18 (5H, m), 4.35–4.52 (1H, m), 5.01–5.19 (2H, m), 5.37
(1H, d, J = 13.9 Hz), 7.19 (1H, dd, J = 7.9, 1.5 Hz), 7.38 (1H, s), 7.49
(1H, s), 7.54–7.68 (3H, m), 7.81 (1H, d, J = 8.3 Hz). MS m/z: 608.6
(M+H)+.
Compound 17 was separated by HPLC (column: CHIRALPAK AD
50 mmID ꢂ 500 mmL, manufactured by DAICEL CHEMICAL INDUSTRIES,

T. Takai et al. / Bioorg. Med. Chem. 24 (2016) 3192–3206
3205
LTD, mobile phase: hexane/isopropyl alcohol 700:300) to give
(R)-17 with a shorter retention time and (S)-17 with a longer
retention time. (R)-17: mp 188 °C. 1H NMR (CDCl3) d: 1.27 (3H, s),
1.57 (3H, s), 1.99–2.23 (3H, m), 2.37–2.46 (1H, m), 2.56 (3H, s),
3.96–4.09 (4H, m), 4.25–4.35 (1H, m), 4.91 (1H, s), 6.42–6.54
(2H, m), 7.24 (1H, dd, J = 8.0, 1.4 Hz), 7.45 (1H, d, J = 1.3 Hz), 7.52
(1H, s), 7.85 (1H, d, J = 8.1 Hz). 13C NMR (DMSO-d6) d: 13.6, 20.3,
25.0, 26.0, 27.7, 44.1, 55.9, 75.6, 83.5, 108.0, 108.2, 111.0, 117.7,
120.6, 125.2, 127.0, 127.2, 146.2, 151.6, 152.6, 155.0, 160.4. MS
pared. All incubations were made in duplicate. Test compound in
the reaction mixture was measured by an HPLC system equipped
with a UV detector or LC/MS/MS. For the determinations of the
metabolic clearance, chromatograms were analyzed for parent
compound disappearance from the reaction mixtures.
5.51. Pharmacokinetics study
Compound (R)-17 (6 mg/kg) was orally administered to
C57BL/6J mice (male, 9 w.o., fed, n = 3) using 0.5% methylcellulose.
At 3 h after oral administration, blood and cortex samples were
collected. The blood samples were centrifuged to obtain the
plasma fraction. Cortex was homogenized in saline to obtain the
brain homogenate. The plasma and brain homogenate samples
were deproteinized with MeCN containing an internal standard.
After centrifugation, the supernatants were decanted into micro-
plates, and were diluted with 0.01 mol/L ammonium formate and
acetonitrile (7:3, v/v) containing 0.2% formic acid. After another
centrifugation, the supernatants were injected into an LC/MS/MS
system to measure the compound concentrations.
m/z: 515.3 (M+H)+. Anal. Calcd for C26H25F3N4O4: C, 60.70; H,
20
4.90; N, 10.89. Found: C, 60.74; H, 4.93; N, 10.84. [
a
]
D
+6.9 (c
1.00, MeOH). (S)-17: 1H NMR (CDCl3) d: 1.27 (3H, br s), 1.57 (3H,
br s), 1.97–2.24 (3H, m), 2.42 (1H, d, J = 9.6 Hz), 2.56 (3H, br s),
4.04 (4H, br s), 4.29 (1H, d, J = 9.3 Hz), 4.90 (1H, br s), 6.41–6.55
(2H, m), 7.17–7.24 (1H, m), 7.39–7.55 (2H, m), 7.84 (1H, d,
J = 7.1 Hz). MS m/z: 515.3 (M+H)+.
5.47. Ab ELISA
Ab40, Ab42 or total Ab was quantified by two-site sandwich
ELISA.11 BNT-77 (Ab11–28) was used as the capture antibody and
horseradish peroxidase-coupled BA-27, BC-05 or BAN50 was used
as the detector antibody for Ab40, Ab42 or total Ab, respectively.
5.52. Multiple treatment study
Compound (R)-17 (3 and 6 mg/kg) was orally administered for
58 days using 0.5% methylcellulose in Tg2576 mice (female, 6 m.
o.). On the 45th day, mice were provided a novel object recognition
test and then brain from them were collected to quantify Ab by
ELISA 3 h after administration on the 58th day. Cortex was isolated
from Tg2576 mice brain and immediately frozen on dry ice and
stored at ꢀ80 °C. Cortex was homogenized in ice-cold tris-extrac-
tion buffer (50 mmol/L Tris–HCl, pH 7.2, 200 mmol/L sodium chlo-
ride, 2% (w/v) protease-free bovine serum albumin, and 0.01% (v/v)
sodium merthiolate) containing protease inhibitor cocktails
(Roche, Basel, Switzerland). After centrifugation at 15,000 rpm for
15 min, the supernatants were subjected to two-site sandwich
ELISA to measure amount of soluble Ab. For assessment of insol-
uble Ab, the pellets were homogenized in guanidine extraction buf-
fer (5 mol/L guanidine, 1 mol/L Tris–HCl (pH 7.2)) and centrifuged
at 15,000 rpm for 15 min. The supernatants were diluted 19-fold
with tris-extraction buffer and applied for Ab ELISA.
5.48. Primary rat neuronal cells assay
Cerebral cortical neurons in rat primary culture were prepared
from rat fetal brain at 17 days of gestation (SD; JCL) using neural
cell dispersion kit (SUMILON). The dissociated cell suspensions
were plated at a density of 5.0 ꢂ 104 cells/well on poly-
L-lysine
coated 96-well plates (Sumitomo Bakelite). After incubation for
7 days in neurobasal medium (Invitrogen) including 1% peni-
cillin–streptomycin (Invitrogen), 2% B-27 supplement (Invitrogen),
and 2 mM L-glutamine (Invitrogen), the cells were treated with or
without compounds for 3 days. Then the culture media were sub-
jected to measurement of Ab40 and Ab42 by ELISA.
5.49. Plasma and brain Ab assay
Hippocampi and plasma were isolated from C57BL/6J mice, 3 h
after compound was orally administrated using a solubilizing vehi-
cle (10% DMSO/10% Cremophor EL/30% PEG400/60% 0.2 M citrate
solution) for 13, 16, 17, 22 and 23, or 0.5% methylcellulose for
(R)-17. Samples were homogenized in ice-cold Tris-extraction buf-
fer (50 mmol/L Tris–HCl, pH 7.2, 200 mmol/L sodium chloride, 2%
protease-free bovine serum albumin, and 0.01% sodium merthio-
late) containing protease inhibitor cocktails (Roche, Basel, Switzer-
land). After centrifugation at 15,000 rpm for 15 min, the
supernatants were subjected to ELISA to measure amount of sol-
uble Ab.
5.53. Novel object recognition test (NORT)
The box and objects used were purchased from BrainScience
Idea (Osaka, Japan). The box was 30 ꢂ 30 ꢂ 30 cm and gray col-
ored. The two objects used were aluminum, silver-colored cylinder
(object A and A0) and ceramic, white-colored trigonal pyramid
(object B). In the acquisition test, the identical objects (objects A
and A0) were symmetrically placed in the box. Each animal was
placed in the box for 5 min followed by a 24 h retention interval
in the home cage. Mice were placed at the corner of the box turn-
ing their heads toward the wall. After replacing one of the objects
with a novel object (object B), the mouse was reintroduced into the
box as a 5 min retention test. Time spent in exploring the objects
were measured during both tests. Retention was represented by
how long the animals explored the novel object versus the familiar
object.
5.50. In vitro metabolic clearance in mouse hepatic microsomes
Hepatic microsomes from mice were purchased from Xenotech,
LLC (Lenexa, KS). An incubation mixture consisted of microsomal
protein in 50 mmol/L KH2PO4–K2HPO4 phosphate buffer (pH 7.4)
and 1 lmol/L each compound. The concentration of hepatic micro-
somal protein was 0.2 mg/mL. An NADPH-generating system con-
taining 25 mmol/L MgCl2, 25 mmol/L glucose-6-phosphate,
2.5 mmol/L b-NADP+, and 7.5 unit/mL glucose-6-phosphate dehy-
drogenase was added to the incubation mixture with a 20% volume
of the reaction mixture to initiate the enzyme reaction. After the
addition of the NADPH-generating system, the reaction mixture
was incubated at 37 °C. The reaction was terminated by the addi-
tion of MeCN equivalent to the volume of the reaction mixture.
As a control, the mixture without 37 °C-incubation was also pre-
Acknowledgments
We thank Mr. Yasutaka Hoashi, Dr. Yoshihide Tomata,
Mr. Masataka Murakami and Mr. Etsuo Kotani for compound
synthesis and helpful discussion. We are indebted to Mr. Motoo
Iida and Ms. Mie Itou for structural determination by single-crystal
X-ray analysis. We express our gratitude to Dr. Takanobu Kuroita
for helpful discussion during the preparation of the manuscript.

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T. Takai et al. / Bioorg. Med. Chem. 24 (2016) 3192–3206
Neurol. 2008, 1031, 65; (e) Bateman, R. J.; Siemers, E. R.; Mawuenyega, K. G.;
Wen, G.; Browning, K. R.; Sigurdson, W. C.; Yarasheski, K. E.; Friedrich, S. W.;
Demattos, R. B.; May, P. C.; Paul, S. M.; Holtzman, D. M. Ann. Neurol. 2009, 66,
48; (f) Carlson, C.; Estergard, W.; Oh, J.; Suhy, J.; Jack, C. R., Jr; Siemers, E.;
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We also thank Dr. Takashi Horiguchi, Dr. Hideki Takahashi,
Dr. Nobuyuki Koyama, Dr. Shigeru Morita and Dr. Shinichi Kondo
for their contribution in the biological evaluation and helpful
discussion, and the members of our laboratory for discussion and
support.
5. (a) Oehlrich, D.; Berthelot, D. J.; Gijsen, H. J. J. Med. Chem. 2011, 54, 669; (b)
Pettersson, M.; Kauffman, G. W.; am Ende, C. W.; Patel, N. C.; Stiff, C.; Tran, T. P.;
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crystallography. The crystal structure has been deposited at the Cambridge
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