Phosphodiesterase inhibitors. Part 3: Design, synthesis and structure-activity relationships of dual PDE3/4-inhibitory fused bicyclic heteroaromatic-dihydropyridazinones with anti-inflammatory and bronchodilatory activity

Article abstract of DOI:10.1016/j.bmc.2012.01.033

(-)-6-(7-Methoxy-2-trifluoromethylpyrazolo[1,5-a]pyridin-4-yl)-5-methyl-4, 5-dihydro-3-(2H)-pyridazinone (KCA-1490) is a dual PDE3/4 inhibitor that exhibits potent combined bronchodilatory and anti-inflammatory activity. A survey of potential bicyclic heteroaromatic replacement subunits for the pyrazolo[1,5-a]pyridine core of KCA-1490 has identified the 4-methoxy-2- (trifluoromethyl)benzo[d]thiazol-7-yl and 8-methoxy-2-(trifluoromethyl)quinolin- 5-yl analogues as dual PDE3/4-inhibitory compounds that potently suppress histamine-induced bronchoconstriction and exhibit anti-inflammatory activity in vivo.

Full text of DOI:10.1016/j.bmc.2012.01.033

Bioorganic & Medicinal Chemistry 20 (2012) 1644–1658
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Phosphodiesterase inhibitors. Part 3: Design, synthesis and structure–activity
relationships of dual PDE3/4-inhibitory fused bicyclic
heteroaromatic-dihydropyridazinones with anti-inflammatory
and bronchodilatory activity
Koji Ochiai a, Satoshi Takita a, Tomohiko Eiraku a, Akihiko Kojima a, Kazuhiko Iwase a,
Tetsuya Kishi a, Kazunori Fukuchi a, Tokutaro Yasue a, David R. Adams b, Robert W. Allcock b,
Zhong Jiang b, Yasushi Kohno a,
⇑
a Discovery Research Laboratories, Kyorin Pharmaceutical Co., Ltd., 2399-1, Nogi, Nogi-machi, Shimotsuga-gun, Tochigi 329-0114, Japan
b Chemistry Department, School of Engineering and Physical Sciences, Heriot-Watt University, Riccarton, Edinburgh EH14 4AS, United Kingdom
a r t i c l e i n f o
a b s t r a c t
Article history:
(À)-6-(7-Methoxy-2-trifluoromethylpyrazolo[1,5-a]pyridin-4-yl)-5-methyl-4,5-dihydro-3-(2H)-pyridaz-
inone (KCA-1490) is a dual PDE3/4 inhibitor that exhibits potent combined bronchodilatory and
anti-inflammatory activity. A survey of potential bicyclic heteroaromatic replacement subunits for the
pyrazolo[1,5-a]pyridine core of KCA-1490 has identified the 4-methoxy-2-(trifluoromethyl)benzo[d]
thiazol-7-yl and 8-methoxy-2-(trifluoromethyl)quinolin-5-yl analogues as dual PDE3/4-inhibitory com-
pounds that potently suppress histamine-induced bronchoconstriction and exhibit anti-inflammatory
activity in vivo.
Received 13 December 2011
Revised 19 January 2012
Accepted 19 January 2012
Available online 28 January 2012
Keywords:
Phosphodiesterase (PDE)
Dual PDE3/4 inhibitor
Asthma
Ó 2012 Elsevier Ltd. All rights reserved.
COPD
Fused bicyclic heteroaromatics
1. Introduction
for the phosphodiesterase (PDE) 4 enzyme family, and this has
culminated in the deployment of roflumilast (Fig. 1), which, as
Therapeutic management of asthma and chronic obstructive
pulmonary disease (COPD) varies according to the severity of the
symptoms, but for many years corticosteroids and b2-agonists have
been the mainstay of treatment regimens.1 Steroids provide
control of underlying inflammatory dysfunction, whilst the bron-
chodilatory activity of b2-agonists primarily serves to afford symp-
tomatic relief for airway constriction. In recent years combination
treatments comprising an inhaled corticosteroid (ICS) and long-
acting b2-agonist (LABA) have been actively developed, and these
allow effective management of the majority of mild-to-moderate
asthma.1 However, long-term use of corticosteroids in high doses
can produce several adverse side effects, driving an on-going
search for steroid-sparing treatment options.2 Moreover, the safety
of LABAs has also recently come under scrutiny, with the US Food
and Drug Administration ordering a series of post-approval clinical
trials from 2011 to evaluate their potential involvement in serious
adverse outcomes among asthma patients.3
the first PDE4-selective inhibitor to gain regulatory approval, has
recently been launched for the treatment of severe COPD.4 The
potential utility of PDE4 inhibitors for treatment of respiratory
disease derives from their pronounced anti-inflammatory activity,
but they also exhibit modest activity for inducing relaxation of
airway smooth muscle tissue.5 However, PDE3 inhibitors are
known to be superior to PDE4 inhibitors in mediating relaxation
of airway smooth muscle.6 Thus, either the combination of PDE3
and PDE4 inhibitors or the use of dual PDE3/4 inhibitors may offer
enhanced therapeutic potential for asthma and COPD compared to
treatment with individual PDE family selective agents alone, and
this notion is supported by reports that combined inhibition of
PDE3 and PDE4 may function additively or synergistically to
induce airway smooth muscle relaxation.7
In the search for an alternative approach to the ICS/LABA regi-
men for asthma and COPD management, we have also evaluated
and found promising activity with dual PDE3/4-inhibitory
compounds. Thus, we have previously disclosed (À)-6-[7-meth-
oxy-2-(trifluoromethyl)pyrazolo[1,5-a]pyridin-4-yl]-5-methyl-4,5-
dihydro-3(2H)-pyridazinone (KCA-1490, Fig. 1) as a dual PDE3/4
inhibitor with very potent combined bronchodilatory and anti-
inflammatory activity and an improved therapeutic window over
One important avenue explored as a new therapeutic option for
respiratory disease has focused on the development of inhibitors
⇑
Corresponding author.
E-mail address: yasushi.kohno@mb.kyorin-pharm.co.jp (Y. Kohno).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2012.01.033

K. Ochiai et al. / Bioorg. Med. Chem. 20 (2012) 1644–1658
1645
O
N
N
N
selective PDE4 inhibitor
dual PDE3/4 inhibitor
N
CF3
CF3
CF3
CF3
CF3
N
N
N
O
OMe
OMe
OMe
HN
Cl
Cl
3
2
1
N
O
NH
4
7
H
N
N
S
CF3
N
CF3
CF3
N
1
O
N
N
O
OMe
F
O
OMe
OMe
OMe
F
6
4
5
roflumilast
KCA-1490[(–)-form]
IC50 (PDE3A) >100 µM
IC50 (PDE4B) 0.0011 µM
IC50 (PDE3A) 0.36 µM
IC50 (PDE4B) 0.042 µM
CF3
O
S
KCA-1489[(+)-form]
N
CF3
OMe
OMe
OMe
IC50 (PDE3A) 66.9 µM
IC50 (PDE4B) 0.011 µM
8
9
7
Figure 1. Structures of PDE4-selective inhibitor roflumilast and dual PDE3/4-inhibitory KCA-1490 together with target compounds (1–9) derived from KCA-1490. Activity
data quoted for roflumilast, KCA-1490 and KCA-1489 (the enantiomer of KCA-1490) are derived from enzyme inhibition assays using the core catalytic domains for PDE3A
and PDE4B as described previously.8b
roflumilast in a number of in vitro and in vivo models used for
pharmacological profiling.8 Our structure–activity relationship
studies with compound series related to KCA-1490 revealed that
the PDE3-inhibitory activity is heavily dependent on the presence
of the 5-methyldihydropyridazinone, a subunit that is strongly
represented in established PDE3 inhibitors,9,10 and we have ratio-
nalized this dependence in our previously reported8b binding mod-
els (summarized here in Fig. 2). The N(1) nitrogen center and the
oxygen atom of the 7-methoxypyrazolo[1,5-a]pyridine core sub-
unit proved to be critical determinants for potent PDE4 inhibition,
and these centers correspond to the catechol ether oxygen atoms
of roflumilast (Fig. 1) in the role that they play in engaging the pur-
ine-scanning glutamine of the PDE4 catalytic pocket (Fig. 2B).8b
Evaluation of a series of substituents at the pyrazolopyridine 2-po-
sition revealed that a trifluoromethyl group conferred an effica-
cious balance of PDE3 and PDE4 inhibition and was optimal for
activity in our pharmacological models.8a Having defined the
essential structural features required for dual PDE3/4-inhibitory
activity within the confines of our pyrazolo[1,5-a]pyridine-based
series, there remained considerable scope for structural variation
with isosteric replacements for the core bicycle. We therefore set
out to evaluate a series of KCA-1490 analogues with alternative
fused 5–6 bicyclic heteroaromatics in place of the pyrazolo[1,5-
a]pyridine (1–8, Fig. 1) together with the ring expanded analogue,
quinoline 9. The synthesis and activity of these compounds is
presented here.
2. Chemistry
Synthesis of triazolopyridine 1 commenced with the formation
of N-aminopyridinium salt 11 by treatment of parent pyridine 10
with O-mesitylenesulfonylhydroxylamine (MSH) (Scheme 1). Con-
struction of the 2-(trifluoromethyl)-[1,2,4]triazolo[1,5-a]pyridine
bicycle was then accomplished by condensation of salt 11 with
Figure 2. Interaction models for KCA-1490 (orange stick) with PDE3 (panel A) and PDE4 (panel B) based on SAR analysis of our pyrazolopyridine series inhibitors.8 By analogy
to related PDE3 inhibitors,10 KCA-1490 is predicted to possess the (R)-configured 5-methyl-4,5-dihydropyridazinone ring; the antipode (KCA-1489), which has weak PDE3-
inhibitory activity, is predicted to be (S)-configured. The PDE3 binding model is templated on the co-crystal structure (PDB: 1SO2) of (R)-6-(4-{[2-(3-iodobenzyl)-3-
oxocyclohex-1-en-1-yl]amino}phenyl)-5-methyl-4,5-dihydropyridazin-3(2H)-one with the core catalytic domain from PDE3B.10b Residues lining the catalytic pocket of
PDE3B are fully conserved in the core catalytic domain of PDE3A (used in the enzyme inhibition assays of the present study). In this binding pose the pyridazinone ring
hydrogen bonds (dotted lines) the purine-scanning Gln and distal His residues of the PDE3 catalytic pocket. The PDE4 binding model is templated on the co-crystal structure
of zardaverine with the core catalytic domain of PDE4D (PDB: 1XOR, 1MKD).31 Residues lining the catalytic pocket are fully conserved between PDE4D and PDE4B (used in the
assays of the present study). The PDE4 binding model is typical of catechol ether inhibitors and invokes hydrogen bonded engagement of the purine scanning-Gln by the
pyrazolopyridine N(1) and methoxy oxygen centers of KCA-1490. Despite the near 180° rotation in the orientation of the inhibitor in the two models, the pyrazolopyridine
(corresponding to the heterobicyclic subunits of compounds 1–9) occupies the hydrophobic clamp, a conserved feature in PDE3 and PDE4 formed by Ile (yellow surface) and
Phe (green surface) residues on one face of the inhibitor and by the purine-stacking Phe on the other face. The entrance to the catalytic pocket is marked.

1646
K. Ochiai et al. / Bioorg. Med. Chem. 20 (2012) 1644–1658
TBSO
TBSO
COOEt
NH2
COOEt
NH2
COOEt
N
N
N
N
N
a
c, d
b
e
CF3
CF3
CF3
N+
N
N
N
N
N
-
NH2
SO3
I
12
13
14
10
11
O
f-h
COOt-Bu
HN
N
O
O
CHO
N
N
N
N
N
N
l, m
i, j
k
CF3
CF3
CF3
CF3
N
N
N
N
N
N
OMe
OMe
OMe
OMe
16
1
17
15
Scheme 1. Reagents and conditions: (a) MSH/CH2Cl2, rt (92%); (b) TFAA, Et3N/MeOH, rt (79%); (c) DIBAL-H/THF, À10 °C; (d) TBSCl, imidazole/DMF, rt (92%, 2 steps); (e) n-
BuLi/THF, À78 °C then 1,2-diiodoethane, À78 °C (94%); (f) TBAF/THF, rt; (g) MnO2/CHCl3, 60 °C; (h) NaOMe/MeOH, reflux (35%, 3 steps); (i) EtMgBr/THF, rt; (j) SO3–pyridine,
Et3N/DMSO, rt (45%, 2 steps); (k) LiHMDS/THF, À78 °C then BrCH2COOBut, À78 °C to rt; (l) TFA/CH2Cl2, rt; (m) H2N–NH2, AcOH/EtOH, reflux (40%, 3 steps).
TFAA to afford 12 in moderate yield. DIBAL-H reduction of the ester
followed by TBS protection of the intermediate primary alcohol
gave 13. Iodination at the 5-position of the triazolopyridine,
removal of the TBS group and MnO2 oxidation followed by
substitution of the iodide using NaOMe furnished aldehyde 15.
The pyridazinone ring was then constructed by conversion of 15
into ketone 16 through a two-step sequence consisting of Grignard
reaction and Parikh–Doering oxidation. Reaction of the enolate
from 19 before constructing the dihydropyridazinone ring using
a strategy similar to that applied in the synthesis of triazolopyri-
dine 1. However, in the case of the imidazopyridine, formation of
the dihydropyridazinone ring in the final step was unsuccessful
and starting material was recovered. This suggested that the reac-
tivity of the carbonyl group at the 5-position of the imidazopyri-
dine was low. We therefore revised the synthetic route to install
the pyridazinone ring on pyridine 19 before completing construc-
tion of the imidazopyridine ring. Thus, Boc protection of 19 fol-
lowed by bromination at the benzylic position afforded bromide
21. Oxidation in two steps gave carboxylic acid 23 via aldehyde
22. The carboxylic acid was coupled with N,O-dimethylhydroxyl-
amine to give the Weinreb amide (24). Treatment of 24 with
EtMgBr resulted in removal of one Boc group as well as formation
of the desired ethyl ketone functionality. Reinstallation of the Boc
derived from 16 with bromoacetate then led to
c-ketoester 17
and thence to target 1 by condensation with hydrazine following
cleavage of the tert-butyl ester.
Target imidazopyridine 2 was prepared from pyridine deriva-
tive 18 (Scheme 2). Methylation of the phenolic hydroxyl group
followed by reduction of the nitro group gave amine 19. Initially
we attempted to complete the imidazo[1,2-a]pyridine scaffold
Scheme 2. Reagents and conditions: (a) MeI, K2CO3/DMF, rt; (b) H2 (1 atm), 10% Pd–C/EtOAc, rt (95%, 2 steps); (c) (Boc)2O, Et3N, DMAP/MeCN, rt (79%); (d) NBS, Bz2O2/CCl4,
reflux (69%); (e) NMO, MS4A/MeCN, rt (79%); (f) NaClO2, NaH2PO4, 2-methyl-2-butene/t-BuOH, H2O, rt (quant.); (g) N,O-dimethylhydroxylamine, EDCI, HOBt, DIEA/DMF, rt
(88%); (h) EtMgBr/THF, À78 °C to rt; (i) (Boc)2O, Et3N, DMAP/MeCN, rt (85%, 2 steps); (j) LiHMDS/THF, 0 °C then BrCH2COOEt, rt (63%); (k) KOH aq/MeOH, 60 °C; (l) H2N–NH2,
AcOH/EtOH, reflux; (m) TFA/CH2Cl2, rt (89%, 3 steps); (n) 3-bromo-1,1,1-trifluoroacetone/EtOH, 75 °C (19%).

K. Ochiai et al. / Bioorg. Med. Chem. 20 (2012) 1644–1658
1647
OH
OH
O
c
b
CF3
N
NO2
NH2
OR
OMe
OMe
28 R = H
30
31
a
29
R = Me
d
O
Br
HN
N
O
O
O
O
N
O
N
f-i
e
CF3
N
CF3
CF3
OMe
OMe
33
OMe
3
32
Scheme 5. Reagents and conditions: (a) KOH aq reflux (b) PPSE, TFA, 95 °C (69%, 2
steps); (c) AlCl3, propionyl chloride/MeNO2, rt (quant.); (d) LiHMDS/THF, 0 °C then
BrCH2COOBut, 0 °C to rt; (e) TFA/CH2Cl2, rt; (f) H2N–NH2/EtOH, reflux (30%, 3 steps).
Scheme 3. Reagents and conditions: (a) MeI, K2CO3/DMF, rt (32%); (b) H2 (1 atm),
10% Pd–C/EtOH, rt (98%); (c) TFAA, TsOH/PhCH3, reflux (59%); (d) AlCl3, propionyl
chloride/CH2Cl2, rt (63%); (e) CuBr2/EtOAc, reflux (61%); (f) di-t-butyl malonate,
NaH/DMF, rt; (g) TFA/CH2Cl2, rt; (h) xylene, 150 °C; (i) BocNHNH2, TsOH/xylene,
reflux (31%, 4 steps).
Scheme 6. Reagents and conditions: (a) DPPA, Et3N/t-BuOH, reflux (98%); (b) TFA/
CH2Cl2, rt (91%); (c) H2 (1 atm), 10% Pd–C/EtOH, EtOAc, AcOH, rt; (d) TFA, reflux
(90%, 2 steps); (e) NBS/CHCl3, rt (63%); (f) n-BuLi/THF, À78 °C then N,N-dimethyl-
propionamide, À78 °C to rt (46%); (g) LiHMDS/THF, À78 °C to -20 °C then
BrCH2COOBut, À78 °C to rt; (h) TFA/CH2Cl2, rt; (i) H2N–NH2/EtOH, reflux (68%, 3
steps).
Scheme 4. Reagents and conditions: (a) TFAA/PhCH3, reflux (96%); (b) TsOH/PhCH3,
reflux (86%); (c) TiCl4, propionyl chloride/CH2Cl2, rt (80%); (d) CuBr2/EtOAc, reflux
(94%); (e) di-t-butyl malonate, NaH/DMF, rt; (f) TFA/CH2Cl2, rt; (g) xylene, 150 °C;
(h) BocNHNH2, TsOH/xylene, reflux (45%, 4 steps).
group was therefore undertaken to obtain the di-Boc derivative
(25) prior to treatment with base and bromoacetate, affording
ketoester 26. Formation of the dihydropyridazinone ring was then
completed by hydrolysis of the ester and condensation with hydra-
zine. Removal of the Boc protection with TFA gave aminopyridine
27 which was then treated with 3-bromo-1,1,1-trifluoroacetone
to furnish target imidazopyridine 2.
Our synthetic route to the 4-methoxybenzoxazole analogue (3)
of KCA-1490 is presented in Scheme 3. Monomethylation of resor-
cinol 28 followed by reduction of the nitro group gave aniline 30.
Construction of the 2-(trifluoromethyl)benzoxazole scaffold was
then accomplished by reaction with TFAA under acidic conditions
to afford 31. Friedel–Crafts acylation of 31 afforded 7-propionyl-
benzoxazole 32. Application of our standard procedure for forma-
tion of the pyridazinone precursor—by treatment of 32 with
LiHMDS followed by bromoacetate—generated a complex product
was unstable to the alkylation conditions used, and we therefore
employed an alternative strategy for preparation of the dihydropy-
ridazinone ring precursor that avoided exposure to the strong base.
Thus, bromination of ketone 32 with copper(II) bromide gave
a-bromoketone 33. The latter was converted into a c-ketoacid
dihydropyridazinone precursor through a three-step sequence
consisting of nucleophilic substitution with di-tert-butyl malonate,
deprotection and then decarboxylation. In the final step, our usual
conditions for formation of the dihydropyridazinone ring (hydra-
zine, AcOH/EtOH, reflux) gave only a trace of target 3 due to com-
peting nucleophilic attack of hydrazine at the 2-position of the
benzoxazole. Through further investigation we found that the
undesired side reaction could be suppressed by treating the keto-
acid with BocNHNH2 under acidic conditions, allowing formation
of target 3 in moderate yield.11
The 7-methoxybenzoxazole target (4) was prepared using a
similar strategy to that used for construction of the isomeric
4-methoxybenzoxazole analogue (3), this time commencing with
mixture with only a trace of the desired
c-ketoester. This sug-
gested that the 4-methoxy-2-(trifluoromethyl)benzoxazole ring

1648
K. Ochiai et al. / Bioorg. Med. Chem. 20 (2012) 1644–1658
Scheme 9. Reagents and conditions: (a) n-BuLi/THF, À78 °C then propionic
anhydride, À78 °C (34%); (b) LiHMDS/THF, 0 °C then BrCH2COOBut, rt; (c) TFA/
CH2Cl2, rt; (d) H2N–NH2, AcOH/EtOH, reflux (43%, 3 steps).
Curtius rearrangement in the presence of tert-butanol afforded
N-Boc aniline 43;14 removal of the Boc group, hydrogenation and
condensation of the intermediate phenylenediamine with TFA
completed construction of the 2-(trifluoromethyl)benzimidazole
scaffold in 45. Bromination at the 7-position of 45 gave bromide
46. Bromine–lithium exchange and reaction of the resulting
benzimidazolyllithium with N,N-dimethylpropionamide then pro-
vided ethyl ketone 47, which was finally converted into target 6
using the reaction sequence developed for construction of the
dihydropyridazinone ring in compound 1. As with the benzothia-
zole (5), target 6 was also accessible through a Suzuki cross-
coupling strategy following bromine–lithium exchange on 46 and
boronation (Supplementary data).
The benzofuran target (7) was prepared from the known phos-
phonium salt 4815 (Scheme 7). Reaction of the latter with TFAA fur-
nished the benzofuran core structure in 49. Bromobenzofuran 49
was then converted into 7 in a similar manner to the synthesis of
6 from bromobenzimidazole 46 (Scheme 6).
Synthesis of the benzothiophene analogue (8), Scheme 8, was
carried out in much the same way as for benzofuran 7 but using
the thiophenol equivalent (55) of the phenolic phosphonium bro-
mide salt (48). Thus, thiocarbamoylation of phenol 51, thermal rear-
rangement of the resulting thiocarbamate to 53 and then hydrolysis
followed by reduction of the aldehyde afforded benzyl alcohol 54.
The latter was easily converted into the requisite phosphonium salt
(55) by treatment with triphenylphosphine hydrobromide, and 55
was then transformed into target benzothiophene 8 using the reac-
tion sequence previously applied in the synthesis of benzofuran 7
from phosphonium bromide salt 48.
Scheme 7. Reagents and conditions: (a) TFAA, Et3N/PhCH3, reflux (87%); (b) n-BuLi/
THF, À78 °C then N,N-dimethylpropionamide, rt (35%); (c) LiHMDS/THF, À78 °C
then BrCH2COOBut, À78 °C to rt; (d) TFA/CH2Cl2, rt; (e) H2N–NH2, AcOH/EtOH,
reflux (37%, 3 steps).
aniline 34 (Scheme 4). N-acylation of the latter with TFAA followed
by cyclization with catalytic TsOH in hot toluene gave 7-methoxy-
2-(trifluoromethyl)benzoxazole (36) in good yield. Intermediate 36
was then converted into target 4 following a similar six-step se-
quence to that developed for synthesis of 3 from 4-methoxy-2-(tri-
fluoromethyl)benzoxazole (31).
Our synthetic route to the 4-methoxybenzothiazole analogue
(5) of KCA-1490 is shown in Scheme 5. Hydrolysis of 2-aminoben-
zothiazole 3912 to open the azole ring followed by thiazole
reconstruction with TFA in the presence of trimethylsilyl polyphos-
phate (PPSE)13 gave the parent 4-methoxy-2-(trifluoromethyl)ben-
zothiazole bicycle (40). Friedel–Crafts acylation of 40 with
propionyl chloride afforded the key ethyl ketone intermediate
(41) in quantitative yield. Ketone 41 was then converted into target
5 through a reaction sequence similar to that used for synthesis of 1
from ketone 16 (Scheme 1). Compound 5 was also accessible from
intermediate 40 by boronation at C-7 followed by Suzuki cross-
coupling with a 6-chloro-5-methylpyridazinone derivative (Sup-
plementary data).
Our synthetic route to the 8-methoxyquinoline derivative (9) is
outlined in Scheme 9. Bromoquinoline 5816 was converted into
ethyl ketone 59 by bromine–lithium exchange followed by
The benzimidazole target (6) was synthesized from commer-
cially available benzoic acid 42 (Scheme 6). DPPA-mediated
Scheme 8. Reagents and conditions: (a) dimethylthiocarbamoyl chloride, DABCO/DMF, rt (81%); (b) Ph2O, 200 °C (57%); (c) NaOH aq/i-PrOH, 60 °C; (d) NaBH4/MeOH, rt
(quant., 2 steps); (e) Ph3PHBr/MeCN, reflux; (f) TFAA, Et3N/PhCH3, reflux (82%, 2 steps); (g) n-BuLi/THF, À78 °C then N,N-dimethylpropionamide, À78 °C to rt (15%); (h)
LiHMDS/THF, 0 °C then BrCH2COOBut, À78 °C to rt; (i) TFA/CH2Cl2, rt; (j) H2N–NH2, AcOH/EtOH, reflux (72%, 3 steps).

K. Ochiai et al. / Bioorg. Med. Chem. 20 (2012) 1644–1658
1649
reaction with propionic anhydride. Application of our standard
route for installation of the dihydropyridazinone ring then afforded
target quinoline 9 in three steps from 59.
form, assaying for inhibitory activity against the core catalytic do-
mains from PDE3A and PDE4B using previously reported proto-
cols.18 Activity data for these compounds and for KCA-14508a
(the racemate corresponding to KCA-1490 and its enantiomer
KCA-1489) are presented in Table 1.
3. Results and discussion
For the majority of the compounds (1–9) replacement of the
KCA-1450 pyrazolopyridine subunit resulted in activity loss
against PDE3—an activity loss that was maximal in the case of
the benzimidazole (6), where a 10-fold reduction in inhibitory
potency was observed. Only two of the compounds exhibited
improved PDE3-inhibitory potency over KCA-1450, namely quino-
line 9 and benzothiazole 5, with 2.5-fold and 16-fold enhance-
ments, respectively. In principle the reduced PDE3-inhibitory
activity observed for benzimidazole 6 might reflect a conforma-
tional bias arising from establishment of an intramolecular hydro-
gen bond between the imidazole ring and the dihydropyridazinone
Having previously identified8a (À)-6-[7-methoxy-2-(trifluoro-
methyl)pyrazolo[1,5-a]pyridin-4-yl]-5-methyl-4,5-dihydro-3(2H)-
pyridazinone (KCA-1490) as a dual PDE3/4 inhibitor that exhibited
potent combined bronchodilatory and anti-inflammatory activity,
we set out to evaluate a range of related compounds (1–9) in which
the pyrazolo[1,5-a]pyridine subunit of the parent structure was
replaced by an alternative heterobicycle. For the majority of com-
pounds in the target set we chose to replace the pyrazolopyridine
by an isosteric 5–6 fused bicycle (1–8). Quinoline 9 was included in
order to provide a structure with an expanded 6–6 fused replace-
ment bicycle, and structurally-related 8-methoxyquinoline carbox-
amides have also been evaluated as PDE4-selective inhibitors by
other groups.17 For initial assessment the target compounds
(1–9) in the present study were prepared and tested in racemic
N(1)-center for the 1H-benzo[d]imidazol-7-yl tautomer of
6
(shown in Scheme 6). Alternatively an intramolecular hydrogen
bond of this type might partially compromise the ligand’s interac-
tion with the purine-scanning glutamine because the X-ray
Table 1
SAR survey for optimization of heterobicycle-pyridazinones (in vitro)
a
a
Compound
Fused heterobicycle
Inhibition IC50
PDE3A
(
l
M)
Compound
Fused heterobicycle
Inhibition IC50
PDE3A
(lM)
PDE4B
PDE4B
KCA-1450b (racemic form of KCA-1490)
2.38c
10.2
1.45
5.74
6.43
0.15
0.070c
6
22.2
19.8
2.85
0.97
20.7
11.3c
0.30
1
2
3
4
5
0.43
0.26
1.01
1.52
0.021
7
0.38
0.28
0.35
3.49
0.47c
8
9
60b
61b
Enzyme assays were performed using the core catalytic domains from the PDE3A and PDE4B isoforms according to previously procedures.18 Data reported are the mean of
a
at least three experiments. Roflumilast was used as a positive control [IC50(PDE4B core cat. domain) 0.0011
l
M; IC50 (PDE3A core cat. domain) >100
lM].
b
c
KCA-1450, 60 and 61 were prepared according to the literature (experimental detail is provided in the Supplementary data).8a,21
Pharmacological data have been described in the literature.8a

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K. Ochiai et al. / Bioorg. Med. Chem. 20 (2012) 1644–1658
Table 2
SAR survey for optimization of heterobicycle-pyridazinones (in vivo)
Compound
Fused heterobicycle
Inhibitory effect on bronchoconstrictiona
LPS modelb
iv (%)
po (%) (1 mg/kg)
po (1 mg/kg)
KCA-1450 (racemic form of KCA-1490)
97
++
100
62
2
5
8
9
99
97
17
99
+
++
NTc
++
80
NTc
68
a
The inhibitory activity of compound on histamine-induced bronchoconstriction in guinea pigs was measured according to an established procedure.22 iv testing was
carried out with compounds dosed at 0.1 mg/kg and is expressed as % inhibition of constriction relative to control. po testing was carried out with compounds at 1 mg/kg
dosing; (++) and (+) denote respectively P70% inhibition and 30–69% inhibition of bronchoconstriction relative to control.
b
Anti-inflammatory activity was assessed in the rat LPS model according to an established procedure.23
NT = Not tested.
c
co-crystal structure of another 5-methyldihydropyridazinone
inhibitor bound to PDE3 (PDB: 1SO2)10b suggests formation of a
loose hydrogen bond between N(1) and the purine-scanning gluta-
non-bonded SÁ Á ÁN interaction20 between the benzothiazole sulfur
and dihydropyridazinone N(1) center may also need to be consid-
ered in rationalizing the PDE3-inhibitory performance of 5. In prin-
ciple, such an interaction might result in controlled conformation
and improved fit to the PDE3 catalytic pocket, but the nature of
the conformational control would likely be similar to that arising
from the possible intramolecular hydrogen bond discussed above
for benzimidazole 6, where the inhibitory potency for PDE3 is
148-fold weaker.
Our earlier SAR data for the pyrazolo[1,5-a]pyridine series re-
lated to KCA-1450 had strongly implicated a binding mode for
the compounds in PDE4 that was distinct from PDE3 (Fig. 2).8
Key hydrogen bond acceptor roles were invoked for the N(1) center
of the pyrazolopyridine subunit and the methoxy group in engag-
ing the side chain amide NH2 of the purine-scanning glutamine.
Thus it is postulated that in PDE4 it is the pyrazolopyridine as op-
posed to the pyridazinone ring that anchors the inhibitor to this
important residue in the catalytic pocket, and it was for this reason
that we focused on pyrazolopyridine replacements in our target
compound set (1–9) that conserve a heteroatom at the position
corresponding to the pyrazolopyridine N(1) center in KCA-1450.
The importance of this heteroatom for the PDE4-inhibitory activity
of the compounds is clearly illustrated with the 2-ethyl-5-methoxy-
indolizine analogue of KCA-1450 (60, Table 1), a compound that we
mine Ne center (cf. binding model, Fig. 2A). However, relative to
KCA-1450, benzofuran 7 exhibits nearly as great a reduction in
PDE3-inhibitory activity as benzimidazole 6. Thus a simple ratio-
nalization that links the weaker performance of the benzimidazole
as a PDE3 inhibitor to internal hydrogen bonding may not be valid.
Interestingly the benzothiophene analogue (8) of benzofuran 7 re-
tains essentially all of the PDE3-inhibitory potency of KCA-1450.
Taken together with the enhanced activity of quinoline 9 and ben-
zothiazole 5 this may actually indicate that the performance of
these compounds is critically affected by the interaction between
the inhibitor and the ‘hydrophobic clamp’19 of the PDE catalytic
pocket, a conserved feature in PDE3 and PDE4 comprising phenyl-
alanine and isoleucine residues (marked in Fig. 2) that facilitate
substrate binding by packing against the two faces of the cyclic
nucleotide’s purine subunit. Thus, the expanded ring system of
the quinoline analogue (9) may provide enhanced surface contact
between ligand and protein in this region, while the position, size
and polarisability of the sulfur center in benzothiazole 5 may
potentially allow optimal interaction, thereby accounting for the
marked improvement of this compound’s in vitro PDE3-inhibitory
activity over the parent structure, KCA-1450. An intramolecular

K. Ochiai et al. / Bioorg. Med. Chem. 20 (2012) 1644–1658
1651
have previously described.21 Indolizine 60 and KCA-1450 are not
fully isosteric, but a direct comparison can be made between 60
and pyrazolopyridine 61 (Table 1),8a in which the trifluoromethyl
group of KCA-1450 is replaced by ethyl. This reveals a 7.4-fold loss
in PDE4-inhibitory activity associated with deletion of the pyrazol-
opyridine N(1) center; PDE3 inhibition is less severely compro-
mised by this structural change, however, and indolizine 60 is
1.8-fold less active than pyrazolopyridine 61 for inhibition of
PDE3 in our assays. Interestingly, with the exception of benzothia-
zole 5, all of the pyrazolopyridine replacement analogues in our
target set (1–4, 6–9) show reduced PDE4-inhibitory potency rela-
tive to KCA-1450 (at least as measured against the PDE4B core cat-
alytic domain). Compounds 1, 2, 6, 7, 8 and 9 exhibit remarkably
similar levels of PDE4 inhibition with IC50s in the range 0.26–
Fig. 1). As we have discussed elsewhere,8b the contrasting eudismic
ratios for inhibition of PDE3 and PDE4 likely reflect a preference for
distinct binding modes in the interaction of the pyrazolopyridine-
pyridazinone structures within the catalytic pockets of PDE3 and
PDE4 enzymes (Fig. 2). Our observation of a high eudismic ratio
for PDE3 inhibition is consistent with the findings of others, where
a strong preference for an (R)-configured 5-methyldihydropyridaz-
inone inhibitor subunit has been noted.10a Of the heterocycles
evaluated in the current study, the benzothiazole (in compound
5) and quinoline (in compound 9) showed greatest promise in
in vitro enzyme inhibition assays undertaken using the core cata-
lytic domains of PDE3A and PDE4B. Both compounds 5 and 9 po-
tently suppress histamine-induced bronchoconstriction in
rodents and show promising anti-inflammatory activity in our pre-
liminary screens. Isosteric benzoxazole, triazolopyridine, imidazo-
pyridine, benzofuran, benzimidazole and benzothiophene
replacements for the 5–6 fused pyrazolopyridine core of the parent
compound showed less promise. The activity of quinoline 9 high-
lights the potential of 6–6 fused bicyclic heteroaromatic replace-
ments for the pyrazolopyridine subunit of KCA-1490 as well as
benzothiazole replacement. Thus, further studies with the enanti-
omers of benzothiazole 5 and 6–6 fused bicyclic heteroaromatics,
such as quinoline derivatives, are now in progress.
0.43 lM; the two benzoxazoles (3 and 4) perform rather less well.
It is difficult to rationalize the structure–activity relationships for
PDE4 inhibition with this compound set, but the three-fold
enhancement in the activity of benzothiazole 5 (relative to KCA-
1450) may also reflect improved interactions within the ‘hydro-
phobic clamp’ of the PDE4 catalytic pocket as proposed for PDE3
(vide supra). In the context of the heterobicycle-dihydropyridazi-
none series, benzothiazole may therefore constitute a privileged
scaffold for construction of compounds with dual PDE3/4-inhibi-
tory activity.
5. Experimental
5.1. General
We have already demonstrated that the parent pyrazolo[1,5-
a]pyridine structure, KCA-1450, exhibits potent combined bron-
chodilatory and anti-inflammatory activity, which appears to be
linked to the capacity to inhibit both PDE3 and PDE4.8a In particu-
lar, the (À)-enantiomer (KCA-1490) of this structure, with which
the PDE3-inhibitory activity is primarily associated (Fig. 1),
strongly outperformed roflumilast in several in vitro and in vivo
models used for pharmacological profiling of drugs targeted to
respiratory disease.8a Based on this experience, four compounds
(2, 5, 8 and 9) were chosen from the target set presented here
and evaluated in an initial screen to assess their capacity to sup-
press histamine-induced bronchoconstriction (0.1 mg/kg, iv in gui-
nea pig)22 (Table 2). Of these four compounds, imidazopyridine 2,
benzothiazole 5 and quinoline 9 all performed well (affording a
97–99% suppression of bronchoconstriction vs control), whereas
the benzothiophene (8) had lower activity. Thus, we selected three
compounds (2, 5 and 9) for further evaluation against histamine-
induced bronchoconstriction (1 mg/kg, po in guinea pig)22 and in
an LPS model used to assess anti-inflammatory activity (1 mg/kg,
po in rat).23 Both the benzothiazole (5) and quinoline (9) were
effective for suppression of histamine-induced bronchoconstric-
tion under po administration. Imidazopyridine 2 was significantly
less effective in this regard however. In the LPS model, benzothia-
zole 5 showed more potent anti-inflammatory activity than 2 or 9,
although both of these compounds exhibited significant levels of
activity. However, benzothiazole 5 was rather less effective than
KCA-1450, perhaps because of the vulnerability of the benzothia-
zole scaffold to metabolism30 under po administration.
1H NMR spectra were measured with a JEOL JNM-ECA-400
or -ECX-400 (400 MHz) spectrometer. The chemical shifts are ex-
pressed in parts per million (d value) downfield from tetramethyl-
silane, using tetramethylsilane (d = 0) and/or residual solvents such
as chloroform (d = 7.26) as an internal standard. Splitting patterns
are indicated as s, singlet; d, doublet; t, triplet; q, quartet; m, mul-
tiplet; br s, broad singlet. Measurements of mass spectra were per-
formed with a JEOL JMS-SX102X mass spectrometer. Data for
elemental analyses are within 0.3% of the theoretical values,
and were determined by a Yanaco CHN-corder MT-6. Unless other-
wise noted, all the experiments were carried out using anhydrous
solvents. Throughout this study, Merck precoated TLC plates (Silica
gel 60 F254, 0.25 mm) were used for thin layer chromatographic
(TLC) analysis, and all of the spots were visualized using UV light
followed by coloring with phosphomolybdic acid or anisaldehyde
reagents. Silica gel 60 N (40–50 lm, neutral; Kanto Chemical Co.,
Inc., Tokyo, Japan) or ChromatorexÒ NH DM2035 (200–350 mesh;
Fuji Silysia Chemical, Ltd., Aichi, Japan) was used for the flash col-
umn chromatography. Alternative synthetic routes to 5 and 6 that
exploit Suzuki cross-coupling strategy to install the pyridazinone
ring on the benzothiazole and benzimidazole core structures are
presented in the accompanying Supplementary data. Experimental
procedures for synthesis of KCA-1450, 60 and 61 are also detailed
in the Supplementary data. A number of the synthetic routes in-
volved N-amination reactions of pyridine substrates using
O-mesitylenesulfonylhydroxylamine (MSH).24 CAUTION: MSH is
explosively unstable as a dry powder and is best handled in CH2Cl2
solution.25
4. Conclusions
In this study we have surveyed the potential of several hetero-
bicycles as replacement subunits for the pyrazolo[1,5-a]pyridine
core of our dual PDE3/4 inhibitor, KCA-1490. Our initial evaluation
has been undertaken with the compounds in racemic form,
although their PDE3-inhibitory activity is likely associated primar-
ily with only one of the two enantiomers. Thus in our previous
work with the parent pyrazolopyridine8 the (À)-enantiomer
(KCA-1490), to which the pharmacological activity is principally
linked, was found to possess 185-fold greater potency over its anti-
pode (KCA-1489) for inhibition of PDE3. Inhibitory potency against
PDE4 was comparable for the two enantiomers however (see
5.2. Synthetic Chemistry
5.2.1. 1,2-Diamino-3-(ethoxycarbonyl)pyridinium 2,4,6-
trimethylbenzenesulfonate (11)
To a stirred solution of ethyl O-mesitylenesulfonylacetohydrox-
amate24 (28.3 g, 99.3 mmol) in dioxane (40 mL) was added 70%
HClO4 (14 mL) at 0 °C. After stirring for 0.5 h at the same temper-
ature, ice-water was added to the reaction mixture and the result-
ing precipitate collected by filtration. The wet filter cake of MSH

1652
K. Ochiai et al. / Bioorg. Med. Chem. 20 (2012) 1644–1658
(CAUTION) was dissolved in CH2Cl2; the organic layer was sepa-
rated and quickly dried over MgSO4. The resulting solution of
MSH was slowly added to a stirred solution of 10 (13.8 g,
82.7 mmol) in CH2Cl2 (40 mL) at 0 °C. After stirring for 1 h at room
temperature, the reaction mixture was concentrated in vacuo.
Diethyl ether was added to the residue and the resulting precipi-
tate was collected by filtration to give 11 (29.0 g, 76.1 mmol,
92%) as a yellow solid. 1H NMR (DMSO-d6, 400 MHz): d 1.34 (3H,
t, J = 7.3 Hz), 2.17 (3H, s), 2.50 (6H, s), 4.37 (2H, q, J = 7.3 Hz),
6.74 (2H, s), 6.96 (2H, br s), 7.00 (1H, t, J = 6.7 Hz), 8.41 (1H, dd,
J = 6.7, 1.2 Hz), 8.53 (1H, d, J = 6.7 Hz), 8.75 (2H, br s).
0 °C. After stirring for 1 h at room temperature, the reaction was
quenched with water and the resulting mixture was extracted with
EtOAc. The organic layer was washed with brine, dried over
Na2SO4, and concentrated in vacuo. The residue was dissolved in
CHCl3 (150 mL) and MnO2 (14.5 g, 167 mmol) was added. The reac-
tion mixture was stirred for 5 h at 60 °C and filtered through a pad
on CeliteÒ. The filtrate was concentrated in vacuo. The residue was
dissolved in MeOH (100 mL) and NaOMe (3.61 g, 66.8 mmol) was
added. After stirring for 2 h under reflux conditions, the reaction
was quenched with satd NH4Cl aq and the resulting mixture was
extracted with EtOAc. The organic layer was washed with brine,
dried over Na2SO4, and concentrated in vacuo. The residue was
purified by silica gel column chromatography (n-hexane–
EtOAc = 1:1) to give 15 (1.43 g, 5.85 mmol, 35% over 3 steps) as a
yellow solid. 1H NMR (CDCl3, 400 MHz): d 4.34 (3H, s), 6.66 (1H,
d, J = 7.9 Hz), 8.36 (1H, d, J = 7.9 Hz), 10.59 (1H, s).
5.2.2. Ethyl 2-(trifluoromethyl)-[1,2,4]triazolo[1,5-a]pyridine-8-
carboxylate (12)
To a stirred solution of 11 (10.0 g, 26.2 mmol) in toluene
(75 mL) were added triethylamine (12.5 mL, 89.7 mmol) and TFAA
(5.60 mL, 39.3 mmol). After stirring for 13 h under reflux condi-
tions, the mixture was concentrated in vacuo. The residue was dis-
solved in EtOAc and the resulting mixture was washed with satd
NaHCO3 aq followed by brine, dried over Na2SO4, and concentrated
in vacuo. The residue was purified by silica gel column chromatog-
raphy (n-hexane–EtOAc = 1.5:1) to give 12 (5.34 g, 20.7 mmol,
79%) as a white solid. 1H NMR (CDCl3, 400 MHz): d 1.47 (3H, t,
J = 7.3 Hz), 4.54 (2H, q, J = 7.3 Hz), 7.30 (1H, t, J = 7.3 Hz), 8.39
(1H, dd, J = 7.3, 1.2 Hz), 8.81 (1H, dd, J = 7.3, 1.2 Hz).
5.2.6. 1-(5-Methoxy-2-(trifluoromethyl)-[1,2,4]triazolo[1,5-
a]pyridin-8-yl)propan-1-one (16)
To a stirred solution of 15 (682 mg, 2.78 mmol) in THF (25 mL)
was slowly added EtMgBr (0.970 mol/L in THF; 3.44 mL,
3.34 mmol) at À78 °C under an argon atmosphere. After stirring
for 3 h at room temperature, the reaction was quenched with satd
NaHCO3 aq and the resulting mixture was extracted with EtOAc.
The organic layer was washed with brine, dried over Na2SO4, and
concentrated in vacuo. The residue was dissolved in DMSO
(9 mL); triethylamine (2.50 mL, 17.9 mmol) and SO3-pyridine
(1.42 g, 8.94 mmol) were then added. After stirring for 1 h at the
room temperature, water was added to the reaction mixture and
the resulting precipitate was collected by filtration to give 16
(343 mg, 1.25 mmol, 45% over 2 steps) as a white solid. 1H NMR
5.2.3. 8-(((tert-Butyldimethylsilyl)oxy)methyl)-2-
(trifluoromethyl)-[1,2,4]triazolo[1,5-a]pyridine (13)
To a stirred solution of 12 (5.03 g, 19.4 mmol) in THF (150 mL)
was slowly added DIBAL-H (0.950 mol/L in n-hexane; 40.9 mL,
38.8 mmol) at À10 °C under an argon atmosphere. After stirring
for 1 h at the same temperature, the reaction was quenched with
1 mol/L HCl and the resulting mixture was extracted with EtOAc.
The organic layer was washed with brine, dried over Na2SO4, and
concentrated in vacuo. The residue was dissolved in DMF
(100 mL) and imdiazole (3.30 g, 48.5 mmol) and TBSCl (3.51 g,
23.3 mmol) were added at 0 °C. After stirring for 1 h at room tem-
perature, the reaction was quenched with water and the resulting
mixture was extracted with EtOAc. The organic layer was washed
with brine, dried over Na2SO4, and concentrated in vacuo. The res-
idue was purified by silica gel column chromatography (n-hexane–
EtOAc = 30:1) to give 13 (5.90 g, 17.9 mmol, 92% over 2 steps) as a
white solid. 1H NMR (CDCl3, 400 MHz): d 0.17 (6H, s), 0.99 (9H, s),
5.17 (2H, s), 7.22 (1H, t, J = 6.7 Hz), 7.80 (1H, dd, J = 6.7, 1.2 Hz),
8.53 (1H, dd, J = 6.7, 1.2 Hz).
(CDCl3, 400 MHz):
d 1.27 (3H, t, J = 7.3 Hz), 3.50 (2H, q,
J = 7.3 Hz), 4.30 (3H, s), 6.60 (1H, d, J = 8.6 Hz), 8.47 (1H, d,
J = 8.6 Hz).
5.2.7. 6-(5-Methoxy-2-(trifluoromethyl)-[1,2,4]triazolo[1,5-a]
pyridin-8-yl)-5-methyl-4,5-dihydropyridazin-3(2H)-one (1)
To a stirred solution of 16 (342 mg, 1.25 mmol) in THF (10 mL)
was slowly added LiHMDS (1.00 mol/L in THF; 1.34 mL,
1.34 mmol) at À78 °C under an argon atmosphere. After stirring
for 0.5 h at the same temperature, tert-butyl bromoacetate
(277 lL, 1.88 mmol) was added in one portion and the reaction
mixture was stirred for 1 h at room temperature. The reaction
was quenched with water and the resulting mixture was extracted
with EtOAc. The organic layer was washed with brine, dried over
Na2SO4, and concentrated in vacuo. The residue was dissolved in
CH2Cl2 (15 mL) and treated with TFA (5 mL). The reaction mixture
was stirred for 2 h at room temperature and concentrated in vacuo.
The residue was dissolved in EtOH (10 mL) and hydrazine monohy-
5.2.4. 8-(((tert-Butyldimethylsilyl)oxy)methyl)-5-iodo-2-
(trifluoromethyl)-[1,2,4]triazolo[1,5-a]pyridine (14)
To a stirred solution of 13 (5.90 g, 17.8 mmol) in THF (120 mL)
was slowly added n-BuLi (2.71 mol/L in n-hexane; 7.23 mL,
19.6 mmol) at À78 °C under an argon atmosphere. After stirring
for 0.5 h at the same temperature, 1,2-diiodoethane (5.52 g,
19.6 mmol) was added and the reaction mixture was stirred for
2.5 h at À78 °C. The reaction was quenched with satd NaHCO3 aq
and the resulting mixture was extracted with EtOAc. The organic
layer was washed with brine, dried over Na2SO4, and concentrated
in vacuo. The residue was purified by silica gel column chromatog-
raphy (n-hexane–EtOAc = 30:1) to give 14 (7.64 g, 16.7 mmol, 94%)
as a yellow solid. 1H NMR (CDCl3, 400 MHz): d 0.17 (6H, s), 0.96
(9H, s), 5.14 (2H, d, J = 1.2 Hz), 7.55 (1H, dt, J = 7.9, 1.2 Hz), 7.68
(1H, d, J = 7.9 Hz).
drate (182 lL, 3.75 mmol) and AcOH (472 lL, 8.25 mmol) were
added. After stirring for 7 h under reflux conditions, the reaction
was quenched with water and the resulting mixture was extracted
with EtOAc. The organic layer was washed with brine, dried over
Na2SO4, and concentrated in vacuo. The residue was purified by sil-
ica gel column chromatography (n-hexane–EtOAc = 3:1) to give 1
(166 mg, 0.500 mmol, 40% over 3 steps) as a yellow solid. 1H
NMR (CDCl3, 400 MHz): d 1.27 (3H, d, J = 7.3 Hz), 2.54 (1H, dd,
J = 17.1, 1.8 Hz), 2.84 (1H, dd, J = 17.1, 6.7 Hz), 4.27 (3H, s), 4.27–
4.31 (1H, m), 6.56 (1H, d, J = 8.2 Hz), 8.19 (1H, d, J = 8.2 Hz), 8.58
(1H, br s). HRMS (EI): calcd for C13H12F3N5O2 ([M]+) 327.0943,
found 327.0943. Anal. calcd for C13H12F3N5O2: C, 47.71; H, 3.70;
N, 21.40. Found: C, 47.73; H, 3.82; N, 21.10.
5.2.5. 5-Methoxy-2-(trifluoromethyl)-[1,2,4]triazolo[1,5-
a]pyridine-8-carbaldehyde (15)
5.2.8. 2-Amino-3-methoxy-6-methylpyridine (19)26
To a stirred solution of 14 (7.64 g, 16.7 mmol) in THF (100 mL)
was added TBAF (1.00 mol/L in THF; 33.4 mmol, 33.4 mmol) at
To a stirred solution of 18 (9.76 g, 63.3 mmol) in DMF (120 mL)
were added K2CO3 (14.0 g, 101 mmol) and MeI (5.91 mL,

K. Ochiai et al. / Bioorg. Med. Chem. 20 (2012) 1644–1658
1653
95.0 mmol). After stirring for 2 h at room temperature, the reaction
5.2.13. 6-Di(tert-butoxycarbonyl)amino-N,5-dimethoxy-N-
methylpicolinamide (24)
was quenched with water and the resulting mixture was extracted
with EtOAc. The organic layer was washed with water followed by
brine, dried over Na2SO4, and concentrated in vacuo. The residue
was purified by silica gel column chromatography (n-hexane–
EtOAc = 4:1) to give 3-methoxy-6-methyl-2-nitropyridine (10.1 g)
as a white solid.
10% Pd–C (1 g) was added to a solution of 3-methoxy-6-methyl-
2-nitropyridine (10.1 g) in EtOAc (300 mL) and the reaction mix-
ture was stirred for 4 h under hydrogen atmosphere (1 atm). The
reaction mixture was filtered through a pad of CeliteÒ. The filtrate
was concentrated in vacuo to give 19 (8.28 g, 58.9 mmol, 95% over
2 steps) as a white solid. 1H NMR (CDCl3, 400 MHz): d 2.33 (3H, s),
3.81 (3H, s), 4.59 (2H, br s), 6.45 (1H, d, J = 7.9 Hz), 6.82 (1H, d,
J = 7.9 Hz)
To a stirred solution of 23 (1.62 g, 4.40 mmol) in DMF (50 mL)
were added EDCIÁHCl (1.27 g, 6.60 mmol), N,O-dimethylhydroxyl-
amine hydrochloride (644 mg, 6.60 mmol), HOBtÁH2O (1.01 g,
6.60 mmol), and DIEA (3.45 mL, 19.8 mmol) and the reaction mix-
ture was stirred for 2 h at room temperature. The reaction was
then quenched with water and the resulting mixture was extracted
with EtOAc. The organic layer was washed with water followed by
brine, dried over Na2SO4, and concentrated in vacuo. The residue
was purified by silica gel column chromatography (n-hexane–
EtOAc = 1:1 to EtOAc) to give 24 (1.60 g, 3.87 g, 88%) as a colorless
amorphous powder. 1H NMR (CDCl3, 400 MHz): d 1.40 (18H, s),
3.42 (3H, br s), 3.81 (3H, s), 3.90 (3H, s), 7.29 (1H, d, J = 8.6 Hz),
7.82 (1H, br s).
5.2.9. 2-Di(tert-butoxycarbonyl)amino-3-methoxy-6-
methylpyridine (20)
5.2.14. 1-(6-Di(tert-butoxycarbonyl)amino-5-methoxypyridin-
2-yl)propan-1-one (25)
To a stirred solution of 19 (3.00 g, 21.7 mmol) in MeCN
(100 mL) were added (Boc)2O (28.4 g, 130 mmol), triethylamine
(6.05 mL, 43.4 mmol), and DMAP (100 mg, 0.82 mmol). After stir-
ring for 8 h at room temperature, the reaction mixture was concen-
trated in vacuo. The residue was dissolved in EtOAc and water was
added. The organic layer was separated, washed with brine, dried
over Na2SO4, and concentrated in vacuo. The residue was purified
by silica gel column chromatography (n-hexane–EtOAc = 3:1) to
give 20 (5.80 g, 17.1 mmol, 79%) as a white solid. 1H NMR (CDCl3,
400 MHz): d 1.41 (18H, s), 2.48 (3H, s), 3.81 (3H, s), 7.07 (1H, d,
J = 8.6 Hz), 7.14 (1H, d, J = 8.6 Hz).
To a stirred solution of 24 (1.60 g, 3.89 mmol) in THF (25 mL)
was slowly added EtMgBr (0.970 mol/L in THF; 12.0 mL,
11.7 mmol) at À78 °C under an argon atmosphere and the reaction
mixture was allowed to gradually warm to room temperature over
3 h. The reaction was quenched with satd NH4Cl aq and the result-
ing mixture was extracted with EtOAc. The organic layer was
washed with water followed by brine, dried over Na2SO4, and con-
centrated in vacuo. The residue was dissolved in MeCN (50 mL),
and treated with (Boc)2O (3.39 g, 15.6 mmol), triethylamine
(1.08 mL, 7.78 mmol) and DMAP (20 mg, 0.164 mmol). After stir-
ring for 2 h at room temperature, the reaction mixture was concen-
trated in vacuo. The residue was purified by silica gel column
chromatography (n-hexane–EtOAc = 3:1 to EtOAc) to give 25
(1.26 g, 3.31 mmol, 85% over 2 steps) as a pale yellow solid. 1H
NMR (CDCl3, 400 MHz): d 1.19 (3H, t, J = 7.3 Hz), 1.41 (18H, s),
3.14 (2H, q, J = 7.3 Hz), 3.92 (3H, s), 7.29 (1H, d, J = 8.6 Hz), 8.06
(1H, d, J = 8.6 Hz).
5.2.10. 6-Bromomethyl-2-di(tert-butoxycarbonyl)amino-3-
methoxypyridine (21)
To a stirred solution of 20 (6.34 g, 18.7 mmol) in CCl4 (50 mL)
were added NBS (3.67 g, 20.6 mmol) and benzoyl peroxide
(20.0 mg, 82.5 lmol) and the reaction mixture was stirred for 4 h
under reflux conditions. The resulting precipitate was filtered off
and the filtrate was concentrated in vacuo. The resulting solid
was recrystallized from EtOAc–n-hexane to give 21 (6.33 g,
12.9 mmol, 69%) as a white solid. 1H NMR (CDCl3, 400 MHz): d
1.40 (18H, s), 3.86 (3H, s), 4.53 (2H, s), 7.21 (1H, d, J = 8.6 Hz),
7.37 (1H, d, J = 8.6 Hz).
5.2.15. Ethyl 4-(6-di(tert-butoxycarbonyl)amino-5-
methoxypyridin-2-yl)-3-methyl-4-oxobutanoate (26)
To a stirred solution of 25 (108 mg, 0.284 mmol) in THF (5 mL)
was slowly added LiHMDS (1.00 mol/L in THF; 0.312 mL,
0.312 mmol) at 0 °C under an argon atmosphere. After stirring for
0.5 h at the same temperature, ethyl bromoacetate (39.4 mL,
0.36 mmol) was added at À78 °C and the reaction mixture was al-
lowed to gradually warm to room temperature over 3 h. The reac-
tion was quenched with satd NH4Cl aq and the resulting mixture
was extracted with EtOAc. The organic layer was washed with
water followed by brine, dried over Na2SO4, and concentrated in
vacuo. The residue was purified by silica gel column chromatogra-
phy (n-hexane–EtOAc = 3:1 to EtOAc) to give 26 (84.0 mg,
179 mmol, 63%) as a pale yellow oil. 1H NMR (CDCl3, 400 MHz): d
1.20 (3H, t, J = 7.3 Hz), 1.23 (3H, d, J = 7.3 Hz), 1.40 (18H, s), 2.48
(1H, dd, J = 16.5, 6.1 Hz), 2.89 (1H, dd, J = 16.5, 8.6 Hz), 3.93 (3H,
s), 4.03–4.16 (2H, m), 4.25–4.37 (1H, m), 7.30 (1H, d, J = 8.6 Hz),
8.07 (1H, d, J = 8.6 Hz).
5.2.11. 6-Di(tert-butoxycarbonyl)amino-5-methoxypyridine-2-
carbaldehyde (22)
To a stirred suspension of NMO (3.55 g, 30.3 mmol) and 4 Å
molecular sieves (powder, 5 g) in MeCN (80 mL) was added a solu-
tion of 21 (6.33 g, 15.2 mmol) in MeCN (20 mL). After stirring for
4 h at room temperature, the reaction mixture was filtered through
a pad of silica gel. The filtrate was concentrated in vacuo. The res-
idue was purified by silica gel column chromatography (n-hexane–
EtOAc = 3:1) to give 22 (5.80 g, 17.1 mmol, 79%) as a white solid. 1H
NMR (CDCl3, 400 MHz): d 1.42 (18H, s), 3.96 (3H, s), 7.35 (1H, d,
J = 8.5 Hz), 8.00 (1H, d, J = 8.5 Hz), 9.94 (1H, s).
5.2.12. 6-Di(tert-butoxycarbonyl)amino-5-methoxypyridine-2-
carboxylic acid (23)
5.2.16. 6-(6-amino-5-methoxypyridin-2-yl)-5-methyl-4,5-
dihydropyridazin-3(2H)-one (27)
NaClO2 (80%; 2.48 g, 27.4 mmol), NaH2PO4Á2H2O (1.22 g,
7.83 mmol), and 2-methyl-2-butene (3.70 mL, 35.2 mmol) were
added to a solution of 22 (2.76 g, 7.83 mmol) in tert-butanol
(80 mL) and water (20 mL). After stirring for 4 h at room tempera-
ture, the reaction mixture was acidified with 0.5 mol/L HCl and ex-
tracted with EtOAc. The organic layer was washed with water
followed by brine, dried over Na2SO4, and concentrated in vacuo
to give 23 (2.95 g, 7.83 mmol, quant.) as a white solid. 1H NMR
(CDCl3, 400 MHz): d 1.41 (18H, s), 3.97 (3H, s), 7.42 (1H, d,
J = 8.6 Hz), 8.22 (1H, d, J = 8.6 Hz), 10.24 (1H, br s).
To a stirred solution of 26 (733 mg, 1.57 mmol) in MeOH
(30 mL) was added 4 mol/L KOH aq (1.57 mL, 6.29 mmol) and the
reaction mixture was stirred for 4 h at 60 °C. The reaction mixture
was concentrated in vacuo, acidified with 1 mol/L HCl and ex-
tracted with EtOAc. The organic layer was washed with water fol-
lowed by brine, dried over Na2SO4, and concentrated in vacuo. The
residue was dissolved in EtOH (50 mL) and treated with hydrazine
monohydrate (0.230 mL, 4.71 mmol). After stirring for 3 h under
reflux conditions, the reaction mixture was concentrated in vacuo,

1654
K. Ochiai et al. / Bioorg. Med. Chem. 20 (2012) 1644–1658
the residue redissolved in EtOAc and the solution was filtered
through a pad of silica gel. The filtrate was concentrated in vacuo.
The residue was dissolved in CH2Cl2 (15 mL), and treated with TFA
(15 mL). After stirring for 12 h at room temperature, the reaction
mixture was concentrated in vacuo and neutralized with satd NaH-
CO3 aq. The resulting precipitate was collected by filtration to give
27 (328 mg, 1.40 mmol, 89% over 3 steps) as a white solid. 1H NMR
(CDCl3, 400 MHz): d 1.21 (3H, d, J = 7.3 Hz), 2.43 (1H, d, J = 16.5 Hz),
2.69 (1H, dd, J = 16.5, 6.7 Hz), 3.61–3.73 (1H, m), 3.88 (3H, s), 5.04
(2H, br s), 6.93 (1H, d, J = 7.9 Hz), 7.21 (1H, d, J = 7.9 Hz), 9.17 (1H,
br s).
white solid. 1H NMR (CDCl3, 400 MHz): d 4.08 (3H, s), 6.90 (1H,
d, J = 7.9 Hz), 7.25 (1H, d, J = 8.6 Hz), 7.46 (1H, t, J = 8.6, 7.9 Hz).
LRMS (EI): 217 ([M]+).
5.2.21. 1-(4-Methoxy-2-(trifluoromethyl)benzo[d]oxazol-7-
yl)propan-1-one (32)
To a stirred suspension of AlCl3 (2.21 g, 16.6 mmol) in CH2Cl2
(55 mL) were added propionyl chloride (1.45 mL, 16.7 mmol) and
31 (1.20 g, 4.39 mmol). After stirring for 3 days at room tempera-
ture, 5% HCl was added and the resulting mixture was extracted
with CH2Cl2. The organic layer was dried over MgSO4 and concen-
trated in vacuo. The residue was purified by silica gel column
chromatography (n-hexane–EtOAc = 4:1) to give 32 (955 mg,
5.2.17. 5-Methyl-6-(2-(trifluoromethyl)imidazo[1,2-a]pyridin-
5-yl)-4,5-dihydropyridazin-3(2H)-one (2)
3.50 mmol, 63%) as
a
pale yellow solid. 1H NMR (CDCl3,
To a stirred solution of 27 (328 mg, 1.40 mmol) in EtOH (50 mL)
was added 3-bromo-1,1,1-trifluoroacetone (535 mg, 2.80 mmol)
and the reaction mixture was stirred for 5 h at 70 °C. Additional
3-bromo-1,1,1-trifluoroacetone (535 mg, 2.80 mmol) was added
and the reaction mixture was stirred for a further 72 h at 70 °C.
The reaction mixture was then concentrated in vacuo; CHCl3 and
1 mol/L HCl were added, and the organic layer was separated.
The organic extract was washed with satd NaHCO3 aq followed
by brine, dried over Na2SO4, and concentrated in vacuo. The result-
ing solid was recrystallized from CHCl3-i-Pr2O to give 2 (87.1 mg,
400 MHz): d 1.29 (3H, t, J = 7.3 Hz), 3.17 (2H, q, J = 7.3 Hz), 4.15
(3H, s), 6.98 (1H, d, J = 8.6 Hz), 8.17 (1H, d, J = 8.6 Hz). LRMS (EI):
273 ([M]+).
5.2.22. 2-Bromo-1-(4-methoxy-2-(trifluoromethyl)
benzo[d]oxazol-7-yl)propan-1-one (33)
To a stirred solution of 32 (92.0 mg, 0.337 mmol) in EtOAc
(4.2 mL) was added CuBr2 (150 mg, 0.672 mmol). After stirring
for 2 h under reflux conditions, the reaction mixture was filtered
through a pad of CeliteÒ. The filtrate was concentrated in vacuo.
The residue was purified by silica gel column chromatography
266 l
mol, 19%) as a white solid. 1H NMR (CDCl3, 400 MHz): d
1.35 (3H, d, J = 7.3 Hz), 2.58 (1H, dd, J = 17.1, 1.2 Hz), 2.80 (1H,
dd, J = 17.1, 6.7 Hz), 3.37–3.48 (1H, m), 4.11 (3H, s), 6.66 (1H, d,
J = 7.9 Hz), 7.26 (1H, d, J = 7.9 Hz), 8.77 (1H, br s), 9.34 (1H, s). LRMS
(EI): 326 ([M]+). Anal. calcd for C14H13F3N4O2: C, 51.54; H, 4.02; N,
17.17. Found: C, 51.34; H, 3.95; N, 17.04.
(n-hexane–EtOAc = 3:1) to give 33 (72.3 mg, 205 lmol, 61%) as a
pale yellow solid. 1H NMR (CDCl3, 400 MHz): d 1.97 (3H, d, J
=6.7 Hz), 4.18 (3H, s), 5.45 (1H, q, J = 6.7 Hz), 7.03 (1H, d,
J = 8.6 Hz), 8.25 (1H, d, J = 8.6 Hz). LRMS (EI): 351 ([M]+).
5.2.23. 6-(4-Methoxy-2-(trifluoromethyl)benzo[d]oxazol-7-yl)-
5-methyl-4,5-dihydropyridazin-3(2H)-one (3)
5.2.18. 3-Methoxy-2-nitrophenol (29)27
To a stirred solution of 28 (25.0 g, 161 mmol) in DMF (500 mL)
were added K2CO3 (6.80 g, 49.2 mmol) and MeI (11.0 mL,
177 mmol). After stirring for 7 h at room temperature, the reaction
mixture was concentrated in vacuo and diluted with water. The
resulting mixture was washed with EtOAc; the aqueous layer
was acidified with 1 mol/L HCl and extracted with EtOAc. The or-
ganic layer was washed with brine, dried over Na2SO4, and concen-
trated in vacuo. The residue was purified by silica gel column
chromatography (n-hexane–EtOAc = 2:1) to give 29 (8.78 g,
51.9 mmol, 32%) as a yellow oil. 1H NMR (CDCl3, 400 MHz): d
3.95 (3H, s), 6.54 (1H, dd, J = 8.6, 1.2 Hz), 6.71 (1H, dd, J = 8.6,
1.2 Hz), 7.40 (1H, t, J = 8.6 Hz), 10.22 (1H, s).
To a stirred solution of di-tert-butyl malonate (856 mg,
3.96 mmol) in DMF (15 mL) was added 60% NaH (198 mg,
4.95 mmol) at 0 °C under an argon atmosphere. After stirring for
0.5 h at room temperature, a solution of 33 (541 mg, 1.54 mmol)
in DMF (15 mL) was slowly added at 0 °C. The reaction mixture
was then stirred for 1 h at room temperature and quenched with
satd NH4Cl aq The resulting mixture was extracted with EtOAc.
The organic layer was washed with water followed by brine, dried
over Na2SO4, and concentrated in vacuo. The residue was purified
by silica gel column chromatography (n-hexane–EtOAc = 3:1) to
give the intermediate di-tert butyl ester which was subsequently
dissolved in CH2Cl2 (10 mL) and treated with TFA (5 mL). After stir-
ring for 1 h at room temperature, the reaction mixture was concen-
trated in vacuo. The residue was then dissolved in xylene (20 mL)
and the resulting solution was stirred for 2 h at 150 °C. p-Toluene-
sulfonic acid monohydrate (296 mg, 1.56 mmol) and tert-butyl car-
bazate (618 mg, 4.68 mmol) were then added. After stirring for 3 h
under reflux conditions with a Dean-Stark trap. The reaction mix-
ture was concentrated in vacuo. The residue was purified by silica
gel column chromatography (n-hexane–acetone = 3:2) to give 3
(157 mg, 0.480 mmol, 31% over 4 steps) as a white solid. 1H NMR
(CDCl3, 400 MHz): d 1.32 (3H, d, J = 7.3 Hz), 2.55 (1H, dd, J = 17.1,
1.2 Hz), 2.80 (1H, dd, J = 17.1, 6.7 Hz), 3.51–3.59 (1H, m), 4.12
(3H, s), 6.96 (1H, d, J = 9.2 Hz), 7.86 (1H, d, J = 9.2 Hz), 8.65 (1H,
s). LRMS (FAB): 328 ([M+H]+). Anal. calcd for C14H12F3N3O: C,
51.38; H, 3.70; N, 12.84. Found: C, 51.08; H, 3.69; N, 12.68.
5.2.19. 2-Amino-3-methoxyphenol (30)28
To a stirred solution of 29 (8.78 g, 51.9 mmol) in EtOH (250 mL)
was added 10% Pd–C (1 g) and the reaction mixture was stirred for
2.5 h at room temperature under a hydrogen atmosphere (1 atm).
The reaction mixture was filtered through a pad of CeliteÒ. The fil-
trate was concentrated in vacuo to give 30 (7.09 g, 51.0 mmol, 98%)
as a yellow solid. 1H NMR (CDCl3, 400 MHz): d 3.85 (3H, s), 6.46
(1H, d, J = 7.9 Hz), 6.48 (1H, d, J = 7.9 Hz), 7.40 (1H, t, J = 7.9 Hz),
10.22 (1H, s).
5.2.20. 4-Methoxy-2-(trifluoromethyl)benzo[d]oxazole (31)
To a stirred solution of 30 (2.00 g, 14.4 mmol) in toluene
(53 mL) was added TFAA (2.20 mL, 15.6 mmol). After stirring for
5 h under reflux conditions, p-toluenesulfonic acid monohydrate
(274 mg, 1.44 mmol) was added and the reaction mixture was
stirred for 2 h under reflux conditions with a Dean-Stark trap.
The mixture was then cooled to room temperature, quenched with
satd NaHCO3 aq and extracted with EtOAc. The organic layer was
washed with brine, dried over Na2SO4, and concentrated in vacuo.
The residue was purified by silica gel column chromatography
(n-hexane–EtOAc = 4:1) to give 31 (1.84 g, 8.47 mmol, 59%) as a
5.2.24. 2,2,2-Trifluoro-N-(2-hydroxy-3-methoxyphenyl)
acetamide (35)
To a stirred solution of 34 (1.92 g, 13.8 mmol) in toluene
(50 mL) was added TFAA (2.10 mL, 15.2 mmol). After stirring for
12 h under reflux conditions, water was added and the resulting
mixture was extracted with EtOAc. The organic layer was washed
with water followed by brine, dried over Na2SO4, and concentrated

K. Ochiai et al. / Bioorg. Med. Chem. 20 (2012) 1644–1658
1655
in vacuo. The residue was purified by silica gel column chromatog-
raphy (n-hexane–EtOAc = 3:1) to give 35 (3.11 g, 13.3 mmol, 96%)
as a colorless oil. 1H NMR (CDCl3, 400 MHz): d 3.92 (3H, s), 5.78
(1H, s), 6.76 (1H, dd, J = 8.8, 1.2 Hz), 6.91 (1H, t, J = 8.8 Hz), 7.87
(1H, dd, J = 8.8, 1.2 Hz), 8.35 (1H, br s).
The residue was purified by silica gel column chromatography
(n-hexane–EtOAc = 30:1) to give 40 (44.7 g, 192 mmol, 69%
over 2 steps) as a white solid. 1H NMR (CDCl3, 400 MHz): d 4.08
(3H, s), 6.92 (1H, d, J = 8.6 Hz), 7.44–7.76 (2H, m).
5.2.30. 1-(4-Methoxy-2-(trifluoromethyl)benzo[d]thiazol-7-
yl)propan-1-one (41)
5.2.25. 7-Methoxy-2-(trifluoromethyl)benzo[d]oxazole (36)
To a stirred solution of 35 (2.60 g, 11.1 mmol) in toluene
(120 mL) was added p-toluenesulfonic acid monohydrate (2.10 g,
11.1 mmol). After stirring for 16 h under reflux conditions with a
Dean-Stark trap. The reaction mixture was cooled to room temper-
ature, poured into satd NaHCO3 aq and extracted with EtOAc. The
organic layer was washed with water followed by brine, dried over
Na2SO4, and concentrated in vacuo. The residue was purified by sil-
ica gel column chromatography (n-hexane–EtOAc = 4:1) to give 36
To a stirred solution of AlCl3 (18.9 g, 142 mmol) in nitrometh-
ane (100 mL) was added propionyl chloride (14.2 mL, 163 mmol)
at 0 °C followed by a solution of 40 (11.0 g, 47.2 mmol) in nitro-
methane (10 mL) at room temperature. After stirring for 11 h at
room temperature, the reaction mixture was poured into ice. The
resulting precipitate was collected by filtration, washed with
water, and dried in vacuo. The solid was triturated with n-hexane
and filtered to give 41 (13.7 g, 47.2 mmol, quant.) as a white solid.
1H NMR (CDCl3, 400 MHz): d 1.31 (3H, t, J = 7.3 Hz), 3.14 (2H, q,
J = 7.3 Hz), 4.18 (3H, s), 7.08 (1H, d, J = 8.6 Hz), 8.20 (1H, d,
J = 8.6 Hz).
(2.06 g, 9.55 mmol, 86%) as
400 MHz): d 4.06 (3H, s), 7.02 (1H, dd, J = 8.0, 1.2 Hz), 7.39 (1H, t,
J = 8.0 Hz), 7.46 (1H, dd, J = 8.0, 1.2 Hz).
a
white solid. 1H NMR (CDCl3,
5.2.26. 1-(7-Methoxy-2-(trifluoromethyl)benzo[d]oxazol-4-
yl)propan-1-one (37)
5.2.31. 6-(4-Methoxy-2-(trifluoromethyl)benzo[d]thiazol-7-yl)-
5-methyl-4,5-dihydropyridazin-3(2H)-one (5)
To a stirred solution of TiCl4 (1.00 mol/L in CH2Cl2; 2.90 mL,
2.90 mmol) were added propionyl chloride (0.254 mL, 2.91 mmol)
and a solution of 36 (420 mg, 1.93 mmol) in CH2Cl2 (5 mL). After
stirring for 2 days at room temperature, 5% HCl was added and
the resulting mixture was extracted with CH2Cl2. The organic layer
was dried over MgSO4 and concentrated in vacuo. The residue was
purified by silica gel column chromatography (n-hexane–
EtOAc = 5:1) to give 37 (422 mg, 1.55 mmol, 80%) as a white solid.
1H NMR (CDCl3, 400 MHz): d 1.26 (3H, t, J = 7.3 Hz), 3.39 (2H, q,
J = 7.3 Hz), 4.11 (3H, s), 7.08 (1H, d, J = 8.6 Hz), 8.13 (1H, d,
J = 8.6 Hz). LRMS (EI): 273 ([M]+).
Compound 5 was prepared from 41 by the method described for
the preparation of compound 1 from 16. Yield: 30% over 3 steps
(white solid). 1H NMR (CDCl3, 400 MHz):
d 1.34 (3H, d,
J = 7.3 Hz), 2.59 (1H, d, J = 17.1 Hz), 2.81 (1H, dd, J = 17.1, 6.7 Hz),
3.55–3.58 (1H, m), 4.16 (3H, s), 7.09 (1H, d, J = 8.6 Hz), 7.76 (1H,
d, J = 8.6 Hz), 8.68 (1H, s). HRMS (EI): calcd for C14H12F3N3O2S
([M]+) 343.0602, found 343.0594.
5.2.32. tert-Butyl 3-methoxy-2-nitrophenylcarbamate (43)
To a stirred solution of 42 (10.0 g, 50.7 mmol) in tert-butanol
(50 mL) were added DPPA (11.5 mL, 53.4 mmol) and triethylamine
(7.40 mL, 53.1 mmol). After stirring for 10 h under reflux condi-
tions, the reaction mixture was concentrated in vacuo. The residue
was dissolved in EtOAc; the resulting mixture was washed with
satd NaHCO3 aq followed by brine, dried over Na2SO4, and concen-
trated in vacuo. The resulting precipitate was triturated with n-
hexane and filtered to give 43 (13.3 g, 49.6 mmol, 98%) as a yellow
solid. 1H NMR (CDCl3, 400 MHz): d 1.50 (9H, s), 3.90 (3H, s), 6.71
(1H, dd, J = 8.6, 1.2 Hz), 7.39 (1H, t, J = 8.6 Hz), 7.55 (1H, br s),
7.77 (1H, dd, J = 8.6, 1.2 Hz).
5.2.27. 2-Bromo-1-(7-methoxy-2-
(trifluoromethyl)benzo[d]oxazol-4-yl)propan-1-one (38)
Compound 38 was prepared from 37 by the method described
for the preparation of compound 33 from 32. Yield: 94% (white so-
lid). 1H NMR (CDCl3, 400 MHz): d 1.95 (3H, d, J = 6.7 Hz), 4.13 (3H,
s), 6.20 (1H, q, J = 6.7 Hz), 7.12 (1H, d, J = 8.6 Hz), 8.22 (1H, d,
J = 8.6 Hz). LRMS (EI): 351 ([M]+).
5.2.28. 6-(7-Methoxy-2-(trifluoromethyl)benzo[d]oxazol-4-yl)-
5-methyl-4,5-dihydropyridazin-3(2H)-one (4)
5.2.33. 3-Methoxy-2-nitroaniline (44)
Compound 4 was prepared from 38 by the method described for
the preparation of compound 3 from 33. Yield: 45% over 4 steps
To a stirred solution of 43 (13.3 g, 49.6 mmol) in CH2Cl2
(100 mL) was added TFA (20 mL) and the reaction mixture was
stirred for 4 h at room temperature. The mixture was then concen-
trated in vacuo to a residue that was dissolved in EtOAc. The result-
ing solution was poured into satd NaHCO3 aq and the organic layer
was separated, washed with brine, dried over Na2SO4, and concen-
trated in vacuo. The resulting precipitate was triturated with n-
hexane and filtered to give 44 (7.55 g, 44.9 mmol, 91%) as a yellow
solid. 1H NMR (CDCl3, 400 MHz): d 3.88 (3H, s), 6.31 (1H, dd, J = 8.6,
1.2 Hz), 7.36 (1H, dd, J = 8.6, 1.2 Hz), 7.16 (1H, t, J = 8.6 Hz).
(white solid). 1H NMR (CDCl3, 400 MHz):
d 1.27 (3H, d,
J = 7.3 Hz), 2.53 (1H, dd, J = 17.1, 1.2 Hz), 2.82 (1H, dd, J = 17.1,
7.3 Hz), 4.06–4.16 (1H, m), 4.09 (3H, s), 7.06 (1H, d, J = 8.6 Hz),
7.91 (1H, d, J = 8.6 Hz), 8.65 (1H, s). LRMS (EI): 327 ([M]+). Anal.
calcd for C14H12F3N3O: C, 51.38; H, 3.70; N, 12.84. Found: C,
51.54; H, 3.65; N, 12.88.
5.2.29. 4-Methoxy-2-(trifluoromethyl)benzo[d]thiazole (40)
A mixture of 39 (50.0 g, 277 mmol) in 1 mol/L KOH aq (275 mL,
275 mmol) was stirred for 25 h under reflux conditions under an
argon atmosphere. Ice water was added and the mixture was
washed with CH2Cl2. The aqueous layer was acidified with conc.
HCl (to approximately pH 3) and extracted with CH2Cl2. The organ-
ic layer was dried over MgSO4 and concentrated in vacuo to give
2-amino-3-methoxybenzenethiol (36.3 g). To a mixture of 2-ami-
no-3-methoxybenzenethiol (36.3 g) in TFA (50 mL) was added
trimethylsilyl polyphosphate (50 mL). After stirring for 2 h at
95 °C, the reaction mixture was concentrated in vacuo; water
was added, and the resulting mixture was extracted with EtOAc.
The organic layer was washed with satd NaHCO3 aq, water
followed by brine, dried over Na2SO4, and concentrated in vacuo.
5.2.34. 4-Methoxy-2-(trifluoromethyl)-1H-benzo[d]imidazole
(45)
To a stirred solution of 44 (7.75 g, 44.9 mmol) in EtOAc
(100 mL), EtOH (100 mL) with a few drops of AcOH was added
10% Pd–C (775 mg). After stirring for 11 h at room temperature un-
der a hydrogen atmosphere (1 atm), the reaction mixture was fil-
tered through a pad of CeliteÒ. The filtrate was concentrated in
vacuo to give the intermediate diamine (6.49 g) which was then
dissolved in TFA (75 mL) initially at 0 °C. The resulting mixture
was subsequently stirred for 4 h under reflux conditions before
concentration to a residue in vacuo. The residue was dissolved in
EtOAc and the resulting solution was poured into satd NaHCO3

1656
K. Ochiai et al. / Bioorg. Med. Chem. 20 (2012) 1644–1658
aq The organic layer was separated, washed with brine, dried over
Na2SO4, and concentrated in vacuo. The resulting powder was trit-
urated with n-hexane and filtered to give 45 (8.69 g, 40.2 mmol,
90% over 2 steps) as a brown solid. 1H NMR (CD3OD, 400 MHz): d
4.01 (3H, s), 6.89 (1H, d, J = 8.0 Hz), 7.24 (1H, d, J = 8.0 Hz), 7.32
(1H, t, J = 8.0 Hz).
5.2.40. 6-(7-Methoxy-2-(trifluoromethyl)benzofuran-4-yl)-5-
methyl-4,5-dihydropyridazin-3(2H)-one (7)
Compound 7 was prepared from 50 by the method described for
the preparation of compound 1 from 16. Yield: 37% over 3 steps
(white solid). 1H NMR (CDCl3, 400 MHz):
d 1.30 (3H, d,
J = 7.3 Hz), 2.52 (1H, dd, J = 16.8, 0.6 Hz), 2.77 (1H, dd, J = 16.8,
6.7 Hz), 3.40–3.48 (1H, m), 4.07 (3H, s), 6.93 (1H, d, J = 8.3 Hz),
7.45 (1H, d, J = 8.3 Hz), 7.89–7.90 (1H, m), 8.56 (1H, s). HRMS
(EI): calcd for C15H13F3N2O3 ([M]+) 326.0878, found 326.0886. Anal.
calcd for C15H13F3N2O3: C, 55.22; H, 4.02; N, 8.59. Found: C, 55.13;
H, 4.02; N, 8.42.
5.2.35. 7-Bromo-4-methoxy-2-(trifluoromethyl)-1H-
benzo[d]imidazole (46)
To a stirred solution of 45 (5.54 g, 25.6 mmol) in CHCl3 (130 mL)
was added NBS (5.02 g, 28.2 mmol). After stirring for 2 h at room
temperature, the reaction was quenched with satd NaHCO3 aq
and the organic layer was separated. The organic extract was dried
over Na2SO4 and concentrated in vacuo. The residue was purified
by silica gel column chromatography (n-hexane–EtOAc = 4:1) to
give 46 (4.72 g, 16.0 mmol, 63%) as a brown solid. 1H NMR (CDCl3,
400 MHz): d 4.00 (3H, s), 6.71 (1H, d, J = 8.6 Hz), 7.46 (1H, d,
J = 8.6 Hz), 10.1 (1H, br s).
5.2.41. O-(3-Bromo-2-formyl-6-methoxyphenyl)
dimethylcarbamothioate (52)
To a stirred solution of 51 (231 mg, 1.00 mmol) in DMF (4 mL)
were added DABCO (224 mg, 2.00 mmol) and dimethylthiocarba-
moyl chloride (247 mg, 2.00 mmol). After stirring for 12 h at room
temperature, the reaction mixture was concentrated in vacuo.
EtOAc and water were added, and the organic layer was separated.
The organic extract was washed with brine, dried over Na2SO4, and
concentrated in vacuo. The resulting solid was triturated with
diisopropyl ether and filtered to give 52 (258 mg, 0.810 mmol,
81%) as a pale yellow solid. 1H NMR (CDCl3, 400 MHz): d 3.40
(3H, s), 3.45 (3H, s), 3.86 (3H, s), 7.05 (1H, d, J = 8.6 Hz), 7.51 (1H,
d, J = 8.6 Hz), 10.20 (1H, s). LRMS (EI): 317 ([M]+).
5.2.36. 1-(4-Methoxy-2-(trifluoromethyl)-1H-benzo[d]imidazol-
7-yl)propan-1-one (47)
To a stirred solution of 46 (800 mg, 2.71 mmol) in THF (20 mL)
was slowly added n-BuLi (1.58 mol/L in n-hexane; 3.90 mL,
6.16 mmol) at À78 °C under an argon atmosphere. After stirring
for 1 h at the same temperature, N,N-dimethylpropionamide
(0.890 mL, 8.10 mmol) was added in one portion at À78 °C and
the reaction mixture was allowed to gradually warm to room tem-
perature over 3 h. The reaction was quenched with satd NH4Cl aq
and the resulting mixture was extracted with EtOAc. The organic
layer was washed with brine, dried over Na2SO4, and concentrated
in vacuo. The residue was purified by silica gel column chromatog-
raphy (CHCl3) to give 47 (342 mg, 1.26 mmol, 46%) as a white solid.
1H NMR (CDCl3, 400 MHz): d 1.29 (3H, t, J = 7.3 Hz), 3.08 (2H, q,
J = 7.3 Hz), 4.15 (3H, s), 6.80 (1H, d, J = 8.6 Hz), 7.96 (1H, d,
J = 8.6 Hz), 11.40 (1H, br s). LRMS (EI): 272 ([M]+).
5.2.42. S-3-Bromo-2-formyl-6-methoxyphenyl
dimethylcarbamothioate (53)
A solution of 52 (5.78 g, 18.2 mmol) in diphenyl ether (57 mL)
was stirred for 0.5 h at 200 °C. The reaction mixture was cooled
to room temperature and purified by silica gel column chromatog-
raphy (n-hexane–EtOAc = 1:1) to give 53 (3.28 g, 10.4 mmol, 57%)
as a white solid. 1H NMR (CDCl3, 400 MHz): d 3.00 (3H, br s),
3.16 (3H, br s), 3.89 (3H, s), 6.97 (1H, d, J = 9.2 Hz), 7.64 (1H, d,
J = 9.2 Hz), 10.25 (1H, s). LRMS (EI): 317 ([M]+).
5.2.43. (6-Bromo-2-mercapto-3-methoxyphenyl)methanol (54)
To a stirred solution of 53 (2.44 g, 7.67 mmol) in i-PrOH (60 mL)
was added 1 mol/L NaOH aq (15.3 mL, 15.3 mmol). After stirring
for 0.5 h at 60 °C, the reaction mixture was concentrated in vacuo.
The residue was acidified with 1 mol/L HCl and extracted with
EtOAc. The organic layer was washed with brine, dried over MgSO4,
and concentrated in vacuo. The residue was dissolved in MeOH
(60 mL) and treated with NaBH4 (580 mg, 15.3 mmol) at 0 °C. After
stirring for 0.5 h at room temperature, the reaction was quenched
with 1 mol/L HCl and the resulting mixture was concentrated in
vacuo. The residue was extracted with EtOAc. The organic layer
was washed with brine, dried over Na2SO4 and concentrated in va-
cuo to give 54 (1.95 g, 7.67 mmol, quant. over 2 steps) as a light
purple oil. 1H NMR (CDCl3, 400 MHz): d 1.99 (1H, br s), 3.90 (3H,
s), 4.46 (1H, s), 4.93 (2H, s), 6.71 (1H, d, J = 8.6 Hz), 7.33 (1H, d,
J = 8.6 Hz). LRMS (EI): 248 ([M]+).
5.2.37. 6-(4-Methoxy-2-(trifluoromethyl)-1H-benzo[d]imidazol-
7-yl)-5-methyl-4,5-dihydropyridazin-3(2H)-one (6)
Compound 6 was prepared from 47 by the method described for
the preparation of compound 1 from 16. Yield: 68% over 3 steps
(pale yellow solid). 1H NMR (CDCl3, 400 MHz): d 1.33 (3H, d,
J = 7.3 Hz), 2.57 (1H, d, J = 17.1 Hz), 2.79 (1H, dd, J = 17.1, 6.7 Hz),
3.50–3.57 (1H, m), 4.11 (3H, s), 6.81 (1H, d, J = 8.6 Hz), 7.55 (1H,
d, J = 8.6 Hz), 8.79 (1H, br s), 11.30 (1H, br s). HRMS (EI): calcd
for C14H13F3N4O2 ([M]+) 326.0991, found 326.0961.
5.2.38. 4-Bromo-7-methoxy-2-(trifluoromethyl)benzofuran (49)
To a stirred suspension of 4815,29 (2.20 g, 3.94 mmol) in toluene
(20 mL) were added TFAA (1.62 mL, 11.5 mmol) and triethylamine
(1.64 mL, 11.8 mmol). After stirring for 5 h under reflux conditions,
the reaction was quenched with water and the resulting mixture
was extracted with EtOAc. The organic layer was washed with
brine, dried over Na2SO4, and concentrated in vacuo. The residue
was purified by silica gel column chromatography (n-hexane–
EtOAc = 10:1) to give 49 (1.01 g, 3.42 mmol, 87%) as a pale yellow
solid. 1H NMR (CDCl3, 400 MHz): d 4.01 (3H, s), 6.82 (1H, d,
J = 8.6 Hz), 7.20–7.21 (1H, m), 7.38 (1H, d, J = 8.6 Hz).
5.2.44. 4-Bromo-7-methoxy-2-(trifluoromethyl)
benzo[b]thiophene (56)
To a stirred solution of 54 (1.95 g, 7.83 mmol) in MeCN (15 mL)
was added triphenylphosphine hydrobromide (2.90 g, 8.45 mmol)
and the reaction mixture was stirred for 17 h under reflux condi-
tions. Solvent was concentrated in vacuo and EtOAc was added.
The resulting powder was filtered to give phosphonium salt 55
(4.39 g) as a white solid. To a stirred suspension of 55 (4.39 g) in tol-
uene (60 mL) were added triethylamine (3.17 mL, 22.7 mmol) and
TFAA (1.18 mL, 8.43 mmol). After stirring for 3 h under reflux condi-
tions, water was added and the resulting mixture was extracted
with EtOAc. The organic layer was washed with brine, dried over
Na2SO4, and concentrated in vacuo. The residue was purified by
5.2.39. 1-(7-Methoxy-2-(trifluoromethyl)benzofuran-4-
yl)propan-1-one (50)
Compound 50 was prepared from 49 by the method described
for the preparation of compound 47 from 46. Yield: 35% (white so-
lid). 1H NMR (CDCl3, 400 MHz): d 1.26 (3H, t, J = 7.3 Hz), 3.05 (2H, q,
J = 7.3 Hz), 4.10 (3H, s), 6.93 (1H, d, J = 8.6 Hz), 7.90 (1H, d,
J = 8.6 Hz), 8.01 (1H, q, J = 1.2 Hz).

K. Ochiai et al. / Bioorg. Med. Chem. 20 (2012) 1644–1658
1657
silica gel column chromatography (n-hexane–EtOAc = 10:1) to give
References and notes
56 (2.00 g, 6.42 mmol, 82% in 2 steps) as a white solid. 1H NMR
(CDCl3, 400 MHz): d 4.00 (3H, s), 6.75 (1H, d, J = 8.6 Hz), 7.53 (1H,
d, J = 8.6 Hz), 7.79 (1H, q, J = 1.2 Hz). LRMS (EI): 310 ([M]+).
1. (a) Giembycz, M. A.; Kaur, M.; Leigh, R.; Newton, R. Br. J. Pharmacol. 2008, 153,
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5.2.45. 1-(7-Methoxy-2-(trifluoromethyl)benzo[b]thiophen-4-
yl)propan-1-one (57)
Compound 57 was prepared from 56 by the method described
for the preparation of compound 47 from 46. Yield: 15% (white so-
lid). 1H NMR (CDCl3, 400 MHz): d 1.27 (3H, t, J = 7.3 Hz), 3.07 (2H, q,
J = 7.3 Hz), 4.09 (3H, s), 6.89 (1H, d, J = 8.6 Hz), 8.05 (1H, d,
J = 8.6 Hz), 8.80 (1H, q, J = 1.2 Hz). LRMS (EI): 288 ([M]+).
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5.2.46. 6-(7-Methoxy-2-(trifluoromethyl)benzo[b]thiophen-4-
yl)-5-methyl-4,5-dihydropyridazin-3(2H)-one (8)
Compound 8 was prepared from 57 by the method described for
the preparation of compound 1 from 16. Yield: 72% over 3 steps
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(white solid). 1H NMR (CDCl3, 400 MHz):
d 1.30 (3H, d,
J = 7.3 Hz), 2.53 (1H, dd, J = 17.1, 1.8 Hz), 2.80 (1H, dd, J = 17.1,
6.7 Hz), 3.37–3.47 (1H, m), 4.06 (3H, s), 6.90 (1H, d, J = 8.6 Hz),
7.58 (1H, d, J = 8.6 Hz), 8.54 (1H, q, J = 1.2 Hz), 8.62 (1H, s). LRMS
(EI): 342 ([M]+). Anal. calcd for C15H13F3N2O2S: C, 52.62; H, 3.83;
N, 8.18. Found: C, 52.46; H, 3.76; N, 8.12.
5.2.47. 1-(8-Methoxy-2-trifluoromethylquinolin-5-yl)propan-1-
one (59)
To a stirred solution of 5816 (3.86 g, 12.6 mmol) in THF (100 mL)
was slowly added n-BuLi (2.71 mol/L in n-hexane, 5.20 mL,
14.1 mmol) at À78 °C under an argon atmosphere. After stirring
for 1 h at the same temperature, propionic anhydride (3.50 mL,
27.2 mmol) was added. After stirring for 2.5 h at À78 °C, the reac-
tion was quenched with satd NH4Cl aq and extracted with EtOAc.
The organic layer was washed with brine, dried over Na2SO4, and
concentrated in vacuo. The residue was purified by silica gel col-
umn chromatography (n-hexane–EtOAc = 5:1) to give 59 (1.21 g,
4.27 mmol, 34%) as a yellow solid. 1H NMR (CDCl3, 400 MHz): d
1.29 (3H, t, J = 7.3 Hz), 3.11 (2H, q, J = 7.3 Hz), 4.18 (3H, s), 7.12
(1H, d, J = 8.6 Hz), 7.88 (1H, d, J = 8.6 Hz), 8.23 (1H, d, J = 8.6 Hz),
9.65 (1H, d, J = 8.6 Hz).
5.2.48. 6-(8-Methoxy-2-trifluoromethylquinolin-5-yl)-5-
methyl-4,5-dihydropyridazin-3(2H)-one (9)
Compound 9 was prepared from 59 by the method described for
the preparation of compound 1 from 16. Yield: 43% over 3 steps
18. Gibson, L. C. D.; Hastings, S. F.; McPhee, I.; Clayton, R. A.; Darroch, C. E.;
Mackenzie, A.; MacKenzie, F. L.; Nagasawa, M.; Stevens, P. A.; MacKenzie, S. J.
Eur. J. Pharmacol. 2006, 538, 39.
(yellow solid). 1H NMR (CDCl3, 400 MHz):
d 1.24 (3H, t,
J = 7.3 Hz), 2.58 (1H, dd, J = 16.8, 3.4 Hz), 2.90 (1H, dd, J = 16.8,
6.7 Hz), 3.29–3.32 (1H, m), 4.15 (3H, s), 7.16 (1H, d, J = 8.3 Hz),
7.70 (1H, d, J = 8.3 Hz), 7.82 (1H, d, J = 9.2 Hz), 8.65 (1H, br s),
8.97 (1H, d, J = 9.2 Hz). HRMS (EI): calcd for C16H14F3N3O2 ([M]+)
337.1038, found 337.1041. Anal. calcd for C16H14F3N3O2: C,
56.97; H, 4.18; N, 12.46. Found: C, 56.67; H, 4.13; N, 12.36.
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Supplementary data
Supplementary data (alternative synthetic routes for prepara-
tion of 5 and 6; synthetic routes to 60, 61 and KCA-1450) associ-
ated with this article can be found, in the online version, at
doi:10.1016/j.bmc.2012.01.033.

1658
K. Ochiai et al. / Bioorg. Med. Chem. 20 (2012) 1644–1658
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