Discovery of DS-6930, a potent selective PPARγ modulator. Part II: Lead optimization

Article abstract of DOI:10.1016/j.bmc.2018.09.005

Attempts were made to reduce the lipophilicity of previously synthesized compound (II) for the avoidance of hepatotoxicity. The replacement of the left-hand side benzene with 2-pyridine resulted in the substantial loss of potency. Because poor membrane pe

Full text of DOI:10.1016/j.bmc.2018.09.005

Accepted Manuscript
Discovery of DS-6930, a Potent Selective PPARγ Modulator. Part II: Lead Op-
timization
Tsuyoshi Shinozuka, Tomoharu Tsukada, Kunihiko Fujii, Eri Tokumaru,
Kousei Shimada, Yoshiyuki Onishi, Yumi Matsui, Satoko Wakimoto, Masanori
Kuroha, Tsuneaki Ogata, Kazushi Araki, Jun Ohsumi, Ryoko Sawamura,
Nobuaki Watanabe, Hideki Yamamoto, Kazunori Fujimoto, Yoshiro Tani,
Makoto Mori, Jun Tanaka
PII:
S0968-0896(18)31309-9
https://doi.org/10.1016/j.bmc.2018.09.005
BMC 14530
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
20 July 2018
3 September 2018
5 September 2018
Please cite this article as: Shinozuka, T., Tsukada, T., Fujii, K., Tokumaru, E., Shimada, K., Onishi, Y., Matsui, Y.,
Wakimoto, S., Kuroha, M., Ogata, T., Araki, K., Ohsumi, J., Sawamura, R., Watanabe, N., Yamamoto, H., Fujimoto,
K., Tani, Y., Mori, M., Tanaka, J., Discovery of DS-6930, a Potent Selective PPARγ Modulator. Part II: Lead
Optimization, Bioorganic & Medicinal Chemistry (2018), doi: https://doi.org/10.1016/j.bmc.2018.09.005
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Bioorganic & Medicinal Chemistry
journal homepage: www.elsevi er.c om
Discovery of DS-6930, a Potent Selective PPARγ Modulator. Part II: Lead
Optimization
Tsuyoshi Shinozuka,*,† Tomoharu Tsukada,† Kunihiko Fujii,† Eri Tokumaru,† Kousei Shimada,† Yoshiyuki
Onishi,† Yumi Matsui,‡ Satoko Wakimoto,† Masanori Kuroha,† Tsuneaki Ogata,† Kazushi Araki,† Jun
Ohsumi,† Ryoko Sawamura,† Nobuaki Watanabe,† Hideki Yamamoto,† Kazunori Fujimoto,† Yoshiro Tani,†
Makoto Mori† and Jun Tanaka†
†R&D Division, Daiichi Sankyo Co., Ltd., 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan
‡Daiichi Sankyo RD Novare Co., Ltd., 1-16-13, Kitakasai, Edogawa-ku, Tokyo 134-8630, Japan
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
Attempts were made to reduce the lipophilicity of previously synthesized compound (II) for the
avoidance of hepatotoxicity. The replacement of the left-hand side benzene with 2-pyridine
resulted in the substantial loss of potency. Because poor membrane permeability was responsible
for poor potency in vitro, the adjustment of lipophilicity was examined, which resulted in the
discovery of dimethyl pyridine derivative (I, DS-6930). In preclinical studies, DS-6930
demonstrated high PPARγ agonist potency with robust plasma glucose reduction. DS-6930
maintained diminished PPARγ-related adverse effects upon toxicological evaluation in vivo, and
demonstrated no hepatotoxicity. Cofactor recruitment assay showed that several cofactors, such
as RIP140 and PGC1, were significantly recruited, whereas several canonical factors was not
affected. This selective cofactor recruitment was caused due to the distinct binding mode of DS-
6930. The calcium salt, DS-6930b, which is expected to be an effective inducer of insulin
sensitization without edema, could be evaluated clinically in T2DM patients.
Keywords:
PPARγ
DS-6930
benzimidazole
2009 Elsevier Ltd. All rights reserved.
1. Introduction
reducing lipophilicity, both these critical issues would be
addressed simultaneously. Accordingly, we directed our research
Although eroxisome proliferator-activated receptor γ (PPARγ)
agonists exhibit robust clinical pharmacological efficacy without
hypoglycemic effects,1-3 adverse effects, such as weight gain,
peripheral edema, hepatotoxicity, bone fracture, carcinogenicity,
and cardiovascular risks, limit their use.4-7 If such adverse effects
could be avoided, PPARγ agonists hold good anti-hyperglycemic
potential for the treatment of type 2 diabetes (T2DM). A novel
PPARγ modulator, DS-6930 (I, Figure 1) was identified with
robust plasma glucose (PG) reduction as well as diminished
PPARγ-related adverse effects in vivo. In this paper, we report the
lead optimization of compound II (Figure 1) to identify a novel
potent selective PPARγ agonist, DS-6930, with ideal preclinical
characteristics.
towards the reduction of lipophilicity of compound II.
I; DS-6930
PPAR EC 50: 41 nM
Compound II
PPAR EC 50: 68 nM
γ
γ
PPAR
E
max: 68%
PPAR E max: 82%
γ
γ
Figure 1. Structures of DS-6930 and lead compound II with PPARγ
agonist activities in COS-7 cells.
2. RESULTS and DISCUSSION
2.1. Chemical synthesis
Imidazopyridines 6 listed in Table 1 were synthesized as shown
in Scheme 1. Initially, compound 6b was synthesized from
imidazo[4,5-b]pyridine carbinol 4b as an intermediate. However,
acid promoted imidazo[4,5-b]pyridine ring annulation was not
possible for other amino pyridines 3 because of the weak
nucleophilicity of aromatic amine. Thus, amino pyridines 3 were
Previously, we identified a potent PPARγ intermediate agonist
II (Figure 1).8 Although it possessed excellent pharmacological
effects and a favorable DMPK profile with diminished PPARγ-
related adverse effects, it induced hepatotoxicity. Therefore, we
sought to eliminate this adverse effect and develop a clinically
viable candidate. Hepatotoxicity is known as one of the most
common but severe adverse effects in drug discovery. Numerous
drugs have been withdrawn because of their hepatotoxic effects.9
Hepatotoxicity may be avoided by reducing lipophilicity.10,11
Moreover, compound II exhibited low solubility. Therefore, by
converted
to
amides
8
by
reacting
with
[3-
(methoxycarbonyl)phenoxy]acetic acid,8 followed by the
treatment of 8 with acetic acid to obtain imidazopyridines 5. A
final saponification yielded imidazopyridine derivatives 6.

c
a
d
5b: P = Me
6b: P = H
1
2b: X = NO 2
3b: X = NH 2
4b
e
b
a
f
g
5: P = Me
6a, 6c-6j: P = H
e
1: X= N, Y = CH
7: X= CH, Y = N
2: A = NO 2
3: A = NH 2
8
b
Scheme 1. Synthesis of Compounds 6. Reagents and conditions: (a) NaH, DMF, or THF, 80 °C, 89% (2a), 70% (2e), 92% (2f), 33%
(2h), 85% (2i), 92% (2l); (b) H2, Pd(OH)2 or Pd/C, THF, EtOH; or Fe, NH4Cl, EtOH, H2O, reflux; (c) glycolic acid, HCl, 1,4-dioxane,
reflux, 80% (3 steps); (d) methyl 3-hydroxybenzoate, ADDP, PBu3, toluene, 58%; (e) NaOH, H2O, 1,4-dioxane, reflux, 94% (6a), 61%
(6b), 71% (6c), 92% (6d), 72% (6e), 97% (6f), 85% (6g), 95% (6h), 98% (6i), 74% (6j), 94% (6k), 93% (6l), 74% (6m), 99% (6n); (f)
WSC•HCl, HOBt•H2O, CH2Cl2, 77% (8a, 2 steps), 60% (8c, 3 steps), 23% (8d, 3 steps), 79% (8e, 2 steps), 90% (8f, 2 steps), 97% (8g, 3
steps), 27% (8h, 2 steps), 97% (8i, 2 steps), 34% (8k, 3 steps), 97% (8l, 2 steps), 34% (8m, 3 steps), 77% (8n, 3 steps); (g) AcOH, 80 °C,
76% (5a), 80% (5c), 40% (5d), 76% (5e), 79% (5f), 86% (5g), 75% (5h), 68% (5i), 88% (5j, 4 steps), 39% (5k), 57% (5l), 89% (5m),
69% (5n).
2-Pyridine derivatives 16 listed in Table 2 were synthesized
from versatile intermediate 13 as shown in Scheme 2. The key
intermediate 13 was synthesized from chloride 9, which was
transformed in p-methoxy benzyl ether 10. After reducing nitro
group, acylation with phenoxyacetic acid intermediate8 led to
amide 12. Benzimidazole ring annulation was then performed by
treating amide 12 with HCl to provide compound 13. Under this
reaction condition, p-methoxy benzyl (PMB) ether was cleaved.
The introduction of 2-pyridine ring was accomplished by reacting
13 with 2-bromo- or 2-fluoro-substituted pyridines in the presence
of copper catalyst to yield 14.12,13 In methyl-substituted derivatives
(16l, 16m, 16n, 16o and 16r), halogen atom (Br or Cl) was
transformed to methyl group by Pd-catalyzed reaction to give
compounds 15.
A final saponification of ester provided
compounds 16. DS-6930 (I) was prepared with a high overall yield
using the same synthetic strategy as described above. The key
intermediate 13 was reacted with 3,5-dibromo-2-fluoropyridine,
followed by Pd-catalyzed methylation of bromide 17 to afford
methyl ester 18. A final saponification led to the formation of DS-
6930. This synthetic route provided high yields of DS-6930 (over
200 g scales).
a
c
d
12
13
9
10: X = NO 2
11: X = NH2
b
e
g
(for 15l, 15m, 15n, 15o and 15r)
14: Z = Br or Cl
15: Z = Me
16
X= F or Br
f
e
g
17: Y = Br
18: Y = Me
I (DS-6930)
13
f
Scheme 2. Synthesis of Compounds 16 and DS-6930. Reagents and conditions: (a) PMBOH, NaH, DMF, 80 °C, 99%; (b) Fe, NH4Cl,
EtOH, H2O, reflux, 99%; (c) WSC•HCl, HOBt•H2O, CH2Cl2, 99%; (d) HCl, 1,4-dioxane, 60 °C, 67%; (e) CuI, 1,10-phenanthloline,
Cs2CO3, DMF, 80 °C or Cu, Cs2CO3, DMF, 100 °C, microwave, 14% (14a), 49% (14b), 4.2% (14c), 33% (14d), 58% (14e), 18% (14f),
18% (14g), 15% (14h), 11% (14i), 4.5% (14j), 28% (14k), 11% (14l), 62% (14m), 66% (14n), 54% (14o), 53% (14p), 70% (14q), 91%
(14r), 99% (17); (f) trimethylboroxine, PdCl2 (diphenylphosphino)ferrocene (dppf)•CH2Cl2, K2CO3, DMF, 80 °C, 52% (15l), 80% (15m),
56% (15n), 60% (15o), 75% (15r), 80% (18); (g) NaOH, MeOH, H2O, 48% (16a), 99% (16b), 41% (16c), 94% (16d), 67% (16e), 54%
(16f), 73% (16g), 64% (16h), 98% (16i), 38% (16j), 99% (16k), 96% (16l), 50% (16m), 98% (16n), 95% (16o), 93% (16p), 94% (16q),
51% (16r), 99% (I).
2.2. In vitro Activity, in vitro ADME Profile
nitrogen atom was introduced on the right-hand side 3-benzoic
acid moiety. Picolinic, nicotinic and isonicotinic acid derivatives
were synthesized with unsuccessful results (data not shown).
These results led us to introduce the nitrogen atom in
benzimidazole ring as shown in Table 1.
Because compound II already possesses carboxylic acid, the
incorporation of another polar functional group is challenging.
Consequently, the introduction of a nitrogen atom in each ring
system was explored to reduce the lipophilicity. Initially, the

PPARγ transcriptional activities were evaluated in GAL4-
PPAR-LBD reporter gene assays using COS-7 cells. The
maximum transcriptional activity (Emax) of the test compound was
defined relative to that of rosiglitazone (100%).14 Imidazo[4,5-
b]pyridine 6a exhibited EC50 = 269 nM with Emax = 88% and
showed an attenuated Log D value. Compound 6a maintained a
high stability against human liver microsomes. Consequently,
several imidazo[4,5-b]pyridine derivatives were synthesized as
shown in Table 1. Unsubstituted phenyl derivative 6b showed
diminished potency in vitro, while 4-methyl derivative 6d
exhibited 12-fold higher potency than 6b. Because these SAR
results were similar to those of benzimidazole series,8 several
promising compounds were synthesized to confirm their potencies
in vitro (Compounds 6e–6j). Imidazo[4,5-b]pyridine derivatives
tend to exhibit relatively high PPARα activities, and 4-fluoro and
3-fluoro-4-methyl analogues (Compounds 6c and 6e) exhibited
EC50 < 10 µM. On the other hand, this series of compounds caused
no PPARδ transcriptional activity at 10 µM (Data not shown).
Although lipophilicity was reduced with this modification, these
potent compounds indicated poor aqueous solubility. Among these
derivatives, compounds 6d, 6h and 6j exhibited potent in vitro
potencies as well as acceptable aqueous solubilities. As described
previously,8 the introduction of methyl group is often associated
with diminished human microsomal stability; similarly, certain
methyl-substituted imidazo[4,5-b]pyridine derivatives 6f, 6g and
6h exhibited modest human microsomal stability. We then focused
on imidazo[4,5-c]pyridines. Imidazo[4,5-c]pyridine 6k exhibited
4.4-fold reduced in vitro potency compared to imidazo[4,5-
b]pyridine 6a. Although 4-methyl derivative 6l did not exhibit
significantly enhanced in vitro potency unlike imidazo[4,5-
b]pyridine derivative 6d, indane 6n exhibited high potency in vitro.
Indane 6n exerted intermediate agonist activity (Emax = 55%) and
possessed fair solubility as well as excellent microsomal stability.
Imidazo[4,5-c]pyridine derivatives 6k–6n did not show more than
50% activation of PPARα activity at 10 µM. Membrane
permeability was assessed by a parallel artificial membrane
permeation assay (PAMPA) because the reduction of lipophilicity
is often associated with poor membrane permeability. Compounds
6c, 6l and 6m might exhibit poor potency in vitro because of their
modest permeabilities (Papp < 10 × 10-6 cm/s).
Table 1. PPAR Transcriptional Activities and In Vitro ADME Profiles of Compounds 6
4
3
2
PAMPA
Papp (10-6
cm/s) d
Human
PPARγ
PPARγ
PPARα
PPARα
Solubility
Compound
X
Y
Z
Log Dc
microsomal
EC50 (nM)a
Emax (%)a
EC50 (nM)b
Emax (%)b
(µg/mL) e
stability (%)f
II
CH
N
CH
CH
CH
CH
CH
CH
3-F, 4-Cl
3-F, 4-Cl
H
68 28i
269
82 4.0i
>10000i
>10000
NTh
32 2.7i
2.7
2.2
1.2
1.5
1.7
2.1
38.5 2.0j
0
100
92
6a
6bg
6c
6d
6e
88
61
62
58
80
45
25.6
1.7
160
11
33
5.2
N
550
NTh
55
NTh
100
78
N
4-F
1060
45
NDj
8.6
N
4-Me
>10000
NDj
38
12.6
NTh
N
3-F, 4-Me
50
56
14.8
96
2-Me, 4-
Me
6f
N
N
N
CH
CH
CH
101
41
75
83
74
>10000
>10000
>10000
17
19
30
2.3
2.3
2.2
28.8
17.4
12.4
0.9
0.5
16
63
71
73
3-Me, 5-
Me
6g
6h
3-Me, 4-
Me
90
6i
6j
N
N
CH
CH
25
94
79
89
>10000
>10000
23
20
2.5
1.4
31.5
9.1
4.3
17
76
92
6k
6l
CH
CH
N
N
3-F, 4-Cl
4-Me
1174
746
73
80
>10000
>10000
15
6
1.4
1.0
11.2
6.8
11
98
80
360
6m
6n
CH
CH
N
N
335
20
59
55
>10000
>10000
4
0.6
1.6
3.6
79
70
100
100
22
16.5
aLuciferase activity in COS-7 cells after treatment with the test compound. Values on single experiment run in octuplicate unless
b
otherwise noted. PPARα activity (%) in COS-7 cells. Values on single experiment run in octuplicate unless otherwise noted. The
maximum transcriptional activity (Emax) of the test compound is expressed relative to that of the reference compound as 100%.15
cDistribution coefficients (Log D) were measured after partition between 1-octanol and PBS (pH = 7.4). dPAMPA was performed at pH
7.4. Values on single experiment run in duplicate. eAqueous thermodynamic solubility at pH 6.8. Values on single experiment run in

duplicate. fHuman microsomal stability was assessed based on test compound (%) remaining after 0.5 h of incubation with human liver
microsomes. gHCl salt. hNot tested. iValues represented as mean S.E.M. Values on 13 independent experiments run in octuplicate. jNot
determined.
We then explored the introduction of nitrogen atom in the left-
hand side benzene ring. Although several 3-pyridine and 4-
pyridine derivatives were synthesized, only a few compounds
exhibited moderate potency in vitro (data not shown). Thus, the
derivatization was focused on 2-pyridine derivatives as shown in
Table 2. Unsubstituted 2-pyridine derivative 16a exhibited poor
potency in vitro due to poor membrane permeability owing to its
low lipophilicity (Papp = 2.2 × 10-6 cm/s; Log D, 0.4). Because lead
compound II possessed good membrane permeability due to high
lipophilicity (Papp = 38.5 × 10-6 cm/s; Log D, 2.7), lipophilic
substituents were incorporated in 2-pyridine ring of 16a. Chloro or
methyl group substitution at 4- or 6- position of 2-pyridine ring
was found to be optimal (Compounds 16b–16i). As expected, the
introduction of these substituents increased the lipophilicity that
improved membrane permeability. Chloro-substituted compounds
16b–16e exhibited higher membrane permeabilities than methyl
derivatives 16f–16i and 4-methyl derivative 16g exhibited good
potency (EC50 = 129 nM); therefore Papp > 5.0 × 10-6 was
considered necessary. The introduction of 4-fluoro substituent did
not affect in vitro potency unlike 4-chloro substitution
(Compounds 16c and 16j), while 6-fluoro derivative 16k indicated
better potency than 4-fluoro analogue 16j. Based on this SAR, one
more substituent was incorporated. The introduction of methyl
group at 3, 4, or 6 position in 2-pyridine ring led to the discovery
of 4,6-dimethyl derivative DS-6930 (I). DS-6930 exhibited high
potency in vitro with an intermediate PPARγ agonist activity(EC50
= 41 nM, Emax = 68%). DS-6930 also possessed acceptable
solubility and high PPARα or PPARδ selectivity (13% PPARα
activation at 10 µM and no PPARδ activation at 10 µM).
Although 3,4-dimethyl derivative 16l indicated high solubility, it
exhibited poor in vitro potency. Because 4,6-substitution was
optimal for in vitro potency, several 4,6-substituted compounds
were synthesized. The replacement of methyl group with a fluoro
group retained high potency in vitro (16n and 16o). Although a
further reduction in lipophilicity was achieved in compounds 16n
and 16o (Log D = 1.1), both compounds were less soluble than
DS-6930. To our surprise, compound 16o exhibited a full agonist
profile (Emax = 100%). Dichloro derivative 16p showed fair
solubility with high potency in vitro. The combination of fluoro
and chloro groups provided promising results because compound
16q indicated excellent potency in vitro with fair solubility. The in
vitro potency was further enhanced by 4-chloro-6-methyl
substitution (16r); however, it exhibited poor solubility. All 2-
pyridyl compounds exhibited almost no PPARα activity at 10 µM.
Therefore, we were successful not only in enhancing in vitro
potency with the retention of intermediate agonist activity, but also
in reducing the lipophilicity to discover 4,6-substituted-2-pyridine
derivatives from lead compound II.
Table 2. PPAR Transcriptional Activities and In Vitro ADME Profiles of Compounds 16
5
4
3
6
PAMPA
Papp
Human
PPARγ
PPARγ
PPARα
PPARα
Solubility
Compound
X
Log Dc
microsomal
EC50 (nM)a
Emax (%)a
EC50 (nM)b
Emax (%)b
(µg/mL) e
stability (%)f
(10-6 cm/s) d
38.5 2.0i
2.2 0.15i
7.6
II
3-F, 4-Cl
H
68 28h
1132
736
82 4.0h
74
>10000h
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
32 2.7h
2.7
0.4
1.2
1.2
1.2
1.1
0.8
0.9
0.8
0.9
0.6
0.6
0
100
100
98
16a
16b
16c
16d
16e
16f
16g
16h
16i
16j
16k
1
62
7.7
17
11
15
92
31
33
37
79
7.1
3-Cl
4-Cl
5-Cl
6-Cl
3-Me
4-Me
5-Me
6-Me
4-F
67
13
3
71
66
16.2
100
NTg
99
615
93
11
6
10.3
200
93
8.1
1895
129
78
1
2.7
100
42
4
4.8 0.75i
94
458
41
2
3.5
NTg
100
NTg
100
351
83
8
6.0 1.2i
4.7
808
87
5
6-F
280
80
3
3.9
16l
3-Me, 4-Me
3-Me, 6-Me
176
717
81
>10000
>10000
8
7
1.3
1.4
7.7 0.65i
11 1.8i
190
85
100
NTg
16m
117
DS-6930 (I)
4-Me, 6-Me
4-Me, 6-F
41 10j
43
68 8.0j
74
>10000j
>10000
13 2.0j
4
1.4
1.1
13 3.4i
5.8
31
NTg
100
16n
9.2
16o
4-F, 6-Me
100
100
>10000
2
1.1
8.3
14
93

16p
16q
4-Cl, 6-Cl
4-Cl, 6-F
89
24
61
79
>10000
>10000
6
5
1.9
1.3
16.1
11.8
9.9
10
100
90
16r
4-Cl, 6-Me
21
92
>10000
7
1.8
13.7
3.9
85
aLuciferase activity in COS-7 cells after treatment with the test compound. Values on single experiment run in octuplicate unless
b
otherwise noted. PPARα activity (%) in COS-7 cells. Values on single experiment run in octuplicate unless otherwise noted. The
maximum transcriptional activity (Emax) of the test compound is expressed relative to that of the reference compound as 100%.15
cDistribution coefficients (Log D) were measured after partition between 1-octanol and PBS (pH = 7.4). dPAMPA was performed at pH
e
7.4. Values on single experiment run in duplicate. Aqueous thermodynamic solubility at pH 6.8. Values on single experiment run in
duplicate. fHuman microsomal stability was assessed based on test compound (%) remaining after 0.5 h of incubation with human liver
g
h
i
microsomes. Not tested. Values represented as mean S.E.M. Values on 13 independent experiments run in octuplicate. Values
represented as mean S.E.M. Values on two independent experiments run in duplicate. jValues represented as mean S.E.M. Values on
two independent experiments run in octuplicate.
2.3. In Vivo Efficacy in ZDF Rats
exhibited high potency in vitro, but failed to show potent PG
reduction due to its low plasma exposure. Because 2-pyridine
derivatives DS-6930, 16p and 16r exhibited similar plasma
concentrations with high potencies in vitro, these compounds
demonstrated potent PG reduction in ZDF rats. Compounds 16n
and 16q exhibited more potent in vivo efficacy due to their higher
plasma exposures than DS-6930, 16p and 16r. In particular,
compound 16q reduced PG levels by 70.6% with statistical
significance (p < 0.01). As mentioned previously,8 this type of
compounds increases the body weight of ZDF rats similar to the
full agonist rosiglitazone. Body weight gain relates to in vivo
efficacy. Accordingly, compounds DS-6930 and 16r that
exhibited potent activities in vivo induced body weight gain to a
greater extent than the less potent compound, 6d.
Several compounds were assessed for their abilities to reduce
PG in ZDF rats at 3 mg/kg p.o. for 14 days as shown in Table 3 (n
= 5). PK parameters were obtained by an additional administration
of the test compounds on day 15 (Table 3). Imidazo[4,5-
b]pyridines 6d, 6i and 6j exhibited comparable PK profiles,
despite 6i showing diminished rat microsomal stability. Among
them, 6i and 6j remarkably reduced PG levels in ZDF rats. In
particular, compound 6i exhibited more than 70% reduction in PG
with statistical significance (p < 0.01), which was the maximum
effect observed in this study. Imidazo[4,5-c]pyridine 6n failed to
show potent in vivo efficacy, despite exhibiting high in vitro
potency as well as a PK profile comparable to those of
imidazo[4,5-b]pyridines.16,17 Similarly, 2-pyridine derivative 16c
Table 3. Plasma Glucose (PG) Reduction (%) in Zucker Diabetic Fatty (ZDF) Rats After the Oral administration of the Test
Compounds (3 mg/kg) on Day 14 with PK parameters (n = 5)
In vivo efficacies in ZDF ratsb
PK parameters in ZDF ratsc
Rat
Compound
microsomal stability
(%)a
Body weight
gain (%)
AUC0-24 h
(h·µg/mL)
PG reduction (%)
Cmax (µg/mL)
Tmax (h)
rosiglitazone
NTd
100
71
40.3 16.2
37.6 17.5
70.4 1.0**
55.3 12.1**
1.3 7.3
12.6 3.3*
6.8 1.9*
NDe
NDe
NDe
6d
0.51 0.11
2.00 0.00
2.00 0.00
2.00 0.00
2.00 0.00
4.40 2.19
3.20 2.68
2.00 0.00
2.40 0.89
2.00 0.00
2.40 0.89
2.52 0.82
4.08 0.86
3.39 0.78
2.41 0.72
1.01 0.08
2.49 0.21
3.61 0.64
1.40 0.31
3.91 0.70
1.25 0.20
6i
14.0 1.5** 0.60 0.17
6j
89
7.1 5.7
6.3 2.3
0.20 1.8
0.63 0.12
0.41 0.11
0.066 0.01
6n
81
16c
100
92
14.8 3.7*
57.0 10.1*
65.7 9.9**
53.3 13.4**
70.6 2.0**
54.1 9.9**
DS-6930 (I)
16nf
17.4 2.1** 0.18 0.02
10.8 2.1** 0.42 0.12
NTd
NTd
87
16p
10.1 2.8*
0.13 0.04
16q
13.8 1.9** 0.45 0.11
18.5 1.5** 0.12 0.01
16r
81
aRat microsomal stability was assessed based on test compound (%) remaining after 0.5 h of incubation with rat liver microsomes. bPG
reduction (% change in PG level vs vehicle control) and body weight (% change in body weight vs vehicle control) in ZDF rats after the
oral administration of 3 mg/kg of the test compounds in 0.5% methylcellulose on day 15. Data are represented as mean S.E.M. Statistical
significance compared to vehicle treatment is denoted by *p < 0.05 and **p < 0.01 as determined by the Student T-test. cPK parameters
were acquired after the administration of the test compounds to ZDF rats on day 14. Each value represents the mean S.D. dNot tested.
eNot determined. fSodium salt of the compound was administered.
2.4. In Vivo Pharmacological Effects of DS-6930
male ZDF rats once daily at 0.1, 0.3, 1, or 3 mg/kg. After repeated
administration for three weeks, PG levels were assessed (n = 8).
DS-6930 was administered as a calcium salt. As shown in Figure
2, both compounds decreased PG levels in a dose-dependent
In vivo pharmacological effects of DS-6930 are summarized in
Figure 2. DS-6930 or rosiglitazone was orally administered to

manner, and statistically significant (p < 0.05) PG reduction was
observed at 1 and 3 mg/kg. In both compounds, maximum PG
reduction was achieved at 1 mg/kg and higher (PG levels were
around 120 mg/dL). Therefore, DS-6930 was considered to have a
glucose-lowering effect comparable to rosiglitazone in this model.
PK parameters were evaluated after an additional administration
of these compounds after three weeks. PK parameters after
administering 0.3 mg/kg of these compounds are shown in Table
5. Although the PG-lowering effects of DS-6930 and rosiglitazone
were similar at 0.3 mg/kg (47 and 50% PG reduction vs vehicle
control, respectively), the AUC of DS-6930 was 7.6-fold lower
than that of rosiglitazone. Therefore, although DS-6930 exhibited
pharmacological effects comparable to rosiglitazone at the same
dose, it was significantly more potent than rosiglitazone based on
the plasma levels of the two compounds. Along with its superior
in vivo efficacy, it displayed remarkable safety. The higher in vitro
PPARγ potency of DS-6930 supports these superior
pharmacological effects.
Figure 2. Pharmacological effects of DS-6930. PG levels in
ZDF rats after the repeated administration of DS-6930b or
rosiglitazone (0.1, 0.3, 1 and 3 mg/kg) for three weeks. DS-
6930 was administered as a calcium salt. Data are represented
as mean S.E.M. (n = 8). Statistical significance compared to
vehicle treatment is denoted by *p < 0.05, **p < 0.01 as
determined by the Student T-test.
2.5. Monkey PK Profile
The monkey PK profiles of 6j, DS-6930, 16n and 19q are
summarized in Table 4. The compounds were orally administered
to male cynomolgus monkeys at 3 mg/kg (n = 2). The compounds
were also administered to the same monkeys intravenously at 1
mg/kg to calculate total body clearance (CL), distribution volume
at steady state (Vss) and F value (%). As described previously,
compound II exhibited exceptionally excellent PK parameters
with robust monkey microsomal stability.8 Imidazo[4,5-b]pyridine
6j and 2-pyridine derivatives DS-6930, 16n and 16q showed
higher CL and Vss than compound II due to the relatively high
basicity. Imidazo[4,5-b]pyridine 6j showed the highest Vss,
whereas 2-pyridine 16n indicated the highest CL in this series of
compounds; nevertheless, these PK parameters were acceptable
for clinical candidate selection. 2-Pyridine derivative 16n
exhibited the longest T1/2, which is not suitable for conventional
once-daily administration because of the concern of accumulation.
Despite its long T1/2, it exhibited the highest clearance in this series.
Other compounds, 6j, DS-6930 and 16q indicated T1/2 values
suitable for once-daily dosing. Among these derivatives, DS-6930
showed the highest plasma exposure as well as an excellent
bioavailability of 89%, indicating that it is the most preferred
compound in this series.
Table 4. PK Parameters of the test Compounds in Cynomolgus Monkeysa
Compound
Monkey
microsomal
Cmax (µg/mL)
Tmax (h)
T1/2 (h)
AUClast
(h·µg/mL)
F (%)
CL
Vss (L/kg)
stability (%)b
(mL/min/kg)
IIe
100
88
4.44 1.5
1.67 0.6
1.50 0.71
5.00 4.2
4.00 0.00
3.25 3.9
18.3 6.6
13.3 3.3
40.2 4.3
4.91 2.4
25 2.7
27 13
89 15
8.9 2.4
31 18
0.32 0.035
2.73 0.13
2.06 0.21
3.07 0.45
1.66 0.64
0.20 0.090
1.97 0.23
6jd
0.46 0.064
2.25 0.72
0.10 0.014
1.19 0.078
DS-6930 (I)d
16nd
80
13.5 0.42 23.5 5.9
0.36 0.0071
0.88 0.0071
0.50 0.31
NTc
49.8 42
8.84 3.3
1.47 0.40
9.94 5.9
16qd
91
aThe test compounds in 0.5% methylcellulose were administered to male cynomolgus monkeys at 3 mg/kg (p.o.). Total body clearance
(CL), distribution volume at steady state (Vss) and F value were calculated based on the data after intravenous (1 mg/kg) administration
of the test compounds. Each value represents the mean (n = 2) or the mean S.D. (n = 3). bMonkey microsomal stability was assessed
based on test compound (%) remaining after 0.5 h of incubation with monkey liver microsomes. cNot tested. dn = 2. en = 3.
2.6. In Vitro Hepatocytotoxicity Assessment
hepatotoxicity than compound II. Interestingly, 16n affected
mitochondrial enzyme activity more than other optimized
compounds 6j, DS-6930 and 16q, despite being lower lipophilic
(Log D: 1.1). Compound 16n also affected cell viability at the
highest concentration (Figure 3b). Although compounds 6j, DS-
6930 and 16q reduced mitochondrial enzyme activities, no clear
concentration-dependency was established. In particular, DS-6930
exhibited the lowest cell toxicity at 100 µM in both assays. The
derivatization of II yielded compounds with reduced lipophilicity
and hepatocytotoxicity. Despite the reduction of lipophilicity, the
lowest lipophilic compound 16n (Log D = 1.1) induced
hepatocytotoxicity to a greater extent than relatively high
lipophilic derivatives, such as 6j, DS-6930 and 16q, with relatively
high lipophilicities (Log D = 1.3–1.4).
Selected compounds were evaluated for hepatocytotoxicity in
vitro. Hepatocytes derived from F344 rats were treated with the
compounds (10, 30 and 100
µM), and mitochondrial enzyme
activity was evaluated by a WST-8 assay as shown in Figure 3a.
Cell viability was also evaluated at the same concentrations based
on the release of LDH (Figure 3b). Lead compound II reduced
mitochondrial enzyme activity in a concentration-dependent
manner, and exhibited a 19% reduction in mitochondrial enzyme
activity at 100 µM, while II did not affect cell viability. This series
of compounds altered mitochondrial enzyme activity at high
concentrations, but did not attenuate cell viability based on the
LDH release assay. Owing to their reduced lipophilicities,
compounds 6j, DS-6930, 16n and 16q induced lower

a)
b)
Figure 3. In vitro hepatocytotoxicity assessment. (a) Mitochondrial enzyme activity (WST-8 assay) after treatment with the test
compounds at 10, 30 and 100 M (n = 4). Values on single experiment run in quadruplicate. Each value represents the mean
µ
S.D. (b) Cell viability (LDH release) after treatment with the test compounds at 10, 30 and 100
represents the mean S.D.
µM (n = 4). Each value
2.7. In Vivo Toxicological Profile of DS-6930
whereas the administration of DS-6930 increased body weight by
6.9% without statistical significance compared to vehicle control,
even at 1000 mg/kg (Figure 4c). Therefore, it can be concluded
that although DS-6930 causes body weight gain, it is much lower
than that caused by rosiglitazone. DS-6390 induced hemodilution
similar to compound II.8 As shown in Figure 4d, DS-6930 was
found to decrease red blood cell (RBC) count (3.8, 6.6 and 5.6%
reduction at 100, 300 and 1000 mg/kg, respectively), while
rosiglitazone caused remarkable reduction (13%) in RBC count,
even at 50 mg/kg (p < 0.01). Similar results were observed upon
hematocrit analysis (Figure 4e). Heart weight gain is thought to be
associated with hemodilution,19-21 and the administration of 50
mg/kg of rosiglitazone significantly increased heart weight (% of
body weight) by 18% (p < 0.01). On the other hand, the
administration of DS-6930 caused no remarkable change in
relative heart weight, even at 1000 mg/kg (Figure 4f). At 50
mg/kg, rosiglitazone also affected relative weights of kidney and
ovary, whereas the administration of DS-6930 caused no
remarkable change at any dose (data not shown). These results
clearly indicate that DS-6930 possesses an excellent safety profile.
The in vivo toxicological profile of HCl salt of DS-6930 was
assessed as shown in Figure 4.18 Adverse effects were assessed
after the oral administration of DS-6930 (100, 300 and 1000
mg/kg) to female F344 rats for four weeks (n = 5). Rosiglitazone
(50 mg/kg) was evaluated as a control. TK parameters were
obtained on day 28 from a satellite group of female rats as shown
in Table 5 (n = 2). In this assessment, both compounds displayed
dose-dependent increases in plasma exposure. Based on
pharmacological studies, the therapeutically effective dosage of
both compounds was found to be around 0.3 mg/kg. Based on
AUC, the exposure margins of DS-6930 at 100, 300 and 1000
mg/kg were estimated as 460-, 1100-, 1300-fold higher,
respectively, whereas those of rosiglitazone at 50 mg/kg was 80-
fold higher. No death or clinical abnormality was observed during
the administration period in any test group. As expected in the
hepatocytotoxicity assessment in vitro, DS-6930 did not affect any
liver enzyme activities in vivo (Figure 4a, 4b).
The administration of rosiglitazone (50 mg/kg) caused
significant (p < 0.01) body weight gain (10% vs vehicle control),
a)
b)
c)
d)

e)
f)
Figure 4. In vivo toxicological profile of DS-6930. (a) aspartate aminotransferase (AST) level, (b) alanine aminotransferase
(ALT) level, (c) Body weight, (d) RBC count, (e) hematocrit analysis and (f) heart weight (% of body weight) in F344/DuCrlCrlj
rats after the repeated administration of DS-6930 (100, 300 and 1000 mg/kg) or rosiglitazone (50 mg/kg) for four weeks. DS-
6930 was administered as an HCl salt. Data are represented as mean S.D. (n = 5). Statistical significance compared to vehicle
treatment is denoted by *p < 0.05 and **p < 0.01 as determined by the Student T-test.
Table 5. PK and TK Parameters of DS-6930 and Rosiglitazone in Ratsa
Compd
DS-6930 Ca salt
DS-6930 HCl
rosiglitazone
Dose (mg/kg/day)
Cmax (µg/mL)
Tmax (h)
0.3a
100b
300b
1000b
0.3a
50b
0.0792 0.0121
1.8 0.5
92.8 17.3
0.50 0.0
206 6.4
0.50 0.0
273 44.5
1.0 0.0
1.04 0.20
1.6 0.7
67.6 5.4
1.0 0.0
AUC0-24h
(h·µg/mL)
0.861 0.092
399 38.9
933 15.6
1080 259
6.55 1.25
525 114
aPK parameters were obtained after the administration of the test compounds to ZDF rats on day 22 (n = 8). Each value represents the
b
mean S.D. TK parameters were obtained after the administration of the test compounds to F344 rats on day 28 (n = 2). Each value
represents the mean S.D.
2.8. X-ray Crystal Structure of DS-6930 Bound to PPARγ-LBD
1-Methylbenzimidazole moiety of DS-6930 forms a water-
mediated hydrogen bond with the main-chain nitrogen of Ser342.
Additional lipophilic interactions of dimethylpyridyl group in DS-
6930 were also observed.
The binding mode of DS-6930 to PPARγ-LBD was determined
at 1.8 Å resolution. As shown in Figure 5, it displayed the same
binding mode as compound II.8 DS-6930 forms hydrophobic and
hydrophilic interactions with PPARγ-LBD, including two direct
hydrogen bonds, two water-mediated hydrogen bonds and van der
Waals contacts (Figure 5a). The benzoic acid group of DS-6930
forms direct hydrogen bonds with side chains of Tyr327 and
Lys367 as well as a water-mediated hydrogen bond with Ser289.
As shown in Figure 5b, DS-6930 do not directly interact with
helix12. This indicates that the additional lipophilic interactions of
dimethylpyridyl group with a β-sheet, helix 2’ and helix 3,
combined with no direct interaction with helix 12 may relate to the
intermediate agonist activity of DS-6930.8
a)
b)

Figure 5. X-ray crystal structure of DS-6930 bound to PPARγ-LBD (PDB 5Z6S). (a) Details of the binding model of DS-6930
to PPARγ-LBD. Residues of PPARγ-LBD involved in the binding of DS-6930, residue 473 on helix 12 and DS-6930 are shown
as stick models. Hydrogen bonds are marked as red dotted lines. (b) The binding mode of DS-6930 to PPARγ-LBD. DS-6930
lacks the direct interaction with Tyr473.
2.9. Cofactor Recruitment by DS-6930
particular, DS-6930 promotes the recruitment of RIP140e184b,
which is an amino-terminal RIP140 domain. AUCsigmoid values for
DS-6930 are normalized against the respective values for
rosiglitazone (Figure 6b). DS-6930 did not induce the recruitment
of TRAP220, CBP, SRC1 and NCoA3, unlike rosiglitazone. In
contrast, it preferentially promotes the recruitment of
RIP140e184b, PGC1, SHP and RAP250 constructs.
Gene expression studies on DS-6930 were performed with 11
selected cofactors. Yeast 2-hybrid dose-response experiments
were carried out with these 11 cofactors and AUC values for
sigmoid curves (AUCsigmoid; not shown) were calculated for
analyzing the cofactor profiles (Figure 6a). DS-6930 induces the
selective recruitment of cofactors similar to rosiglitazone. In
a)
b)
Figure 6. PPARγ cofactor profiles on the basis of AUC values. Values on single experiment run in quadruplicate (a) AUC values
for sigmoid curves (AUCsigmoid) of selected cofactors. (b) AUCsigmoid values normalized against the respective values for
rosiglitazone (AUCrosiglitazone = 1).
PK evaluation combined with in vitro hepatocytotoxicity
assessment led to the discovery of a potent selective PPARγ
modulator, DS-6930. In preclinical studies, DS-6930
demonstrated promising pharmacological efficacy with an
excellent DMPK profile as well as diminished PPARγ-related
adverse effects without any signs of hepatotoxicity. Its distinct
binding mode facilitates the selective recruitment of PPARγ
cofactors. The calcium salt, DS-6930b was selected as a clinical
candidate, which is expected to significantly induce insulin
sensitization without edema in T2DM patients.
3. CONCLUSIONS
We attempted to attenuate the hepatotoxicity of compound II
to develop a clinical candidate. Because reducing the lipophilicity
was hypothesized to be the best way in order to avoid such adverse
effects, the introduction of a nitrogen atom was carried out.
Initially, we synthesized imidazo[4,5-b]pyridines that exerted high
in vitro potency as well as robust in vivo efficacy, while
imidazo[4,5-c]pyridine derivatives showed poor PG reduction,
despite exhibiting high in vitro potencies and plasma exposures.
Although unsubstituted 2-pyridine derivative showed poor
potency in vitro due to poor membrane permeability, substituted
2-pyridines exhibited potent in vitro potencies as well as in vivo
efficacies. These modifications also improved the solubility of
compounds, another limitation of compound II. Consequently, we
synthesized compounds that exerted enhanced in vitro potencies
with the retention of intermediate agonist affinities as well as
reduced lipophilicity. In vivo pharmacological screening, monkey
4. EXPERIMENTAL SECTION
4.1. General Procedures
Starting reagents were purchased from commercial suppliers
and were used without further purification unless otherwise
specified. Chromatographic elution was carried under continuous
monitoring by TLC using silica gel 60F254 (Merck & Co., Inc.) as

4.6. General Procedure for Benzimidazole Ring Annulation to
Prepare 5 (General Procedure E).
the stationary phase; the mobile phase was the elution solvent used
in column chromatography. A UV detector was used for detection.
Silica gel SK-85 (230–400 mesh) or silica gel SK-34 (70–230
mesh), manufactured by Merck & Co., Inc., or Chromatorex NH
(200–350 mesh), manufactured by Fuji Silysia Chemical Ltd., was
A solution of 8 (3.55 mmol) in AcOH (20 mL) was stirred at 80
°C for 4 h. Water was added to the cooled mixture, and the mixture
was extracted with EtOAc several times. The combined organic
layers were washed with saturated NaHCO3 solution several times
and dried over anhydrous Na2SO4. After concentration under
reduced pressure, the residue was purified by silica gel
chromatography to obtain 5.
1
used as the column packing silica gel. H NMR spectra were
obtained on Varian Unity 400- and 500-MHz spectrometers.
Spectra were recorded in the indicated solvent at ambient
temperature; chemical shifts are reported in ppm (δ) relative to the
solvent peak. Resonance patterns are represented with the
following notations: br (broad signal), s (singlet), d (doublet), t
(triplet), q (quartet) and m (multiplet). MS analysis was carried out
by FAB, EI, or ESI. HRMS was carried out using an LC-MS
system composed of a Waters Xevo Q-ToF MS and an Acquity
UHPLC system. Elemental analyses were carried out on a
Microcorder JM10 and a Dionex ICS-1500. The purity was
assessed by reversed-phase HPLC analysis (column, Inertsil ODS-
3, 4.6 × 250 mm; eluent, MeCN/0.1% Et3N•HCl aqueous solution;
flow rate, 1 mL/min; wavelength, 254 nm). All assay compounds
were ≥ 95% pure.
4.7. General Procedure for Hydrolysis to Prepare 6 or 16
(General Procedure F).
A solution of 5, 14, or 15 (5.80 mmol) and 2 M NaOH (10 mL,
10 mmol) in 1,4-dioxane (20 mL) was stirred at 80 °C for 2 h. The
cooled reaction mixture was neutralized by adding 1 M HCl, and
the precipitated solid was collected by filtration to obtain 6 or 16.
4.8. General Procedure for Copper-catalyzed Coupling Reaction
to Prepare 14 (General Procedure G).
A solution of 13 (3.12 g, 10.0 mmol), 2-fluoro or 2-bromo
substituted pyridine (11.0 mmol), CuI (0.19 g, 1.00 mmol), 1,10-
phenanthroline (0.18 g, 1.00 mmol) and Cs2CO3 (9.77 g, 30 mmol)
in DMF (50 mL) was stirred under N2 at 80 °C for 2 h. Aqueous
NH4Cl solution (10%) was added to the cooled reaction mixture,
and the mixture was extracted with EtOAc several times. The
combined organic layers were washed with water and dried over
anhydrous Na2SO4. After concentration under reduced pressure,
the residue was purified by silica gel chromatography to obtain 14.
4.2. General Procedure for SNAr Reaction to Prepare 2 (General
Procedure A).
NaH (12.0 mmol) was added to a solution of 1 or 722 (1.88 g,
10.0 mmol) and substituted phenol (12.0 mmol) in DMF (50 mL)
at room temperature under N2, and the mixture was stirred at 80
°C for 10 h. Water was added to the cooled mixture, and the
mixture was extracted with EtOAc several times. The combined
organic layers were washed with water, dried over anhydrous
Na2SO4, and concentrated under reduced pressure. The crude
residue was purified by silica gel column chromatography to
obtain purified compound 2, or crude 2 was used directly for the
next reaction without further purification.
4.9. General Procedure for Alternative Copper-catalyzed
Coupling Reaction to Prepare 14 (General Procedure H).
A solution of 13 (625 mg, 2.00 mmol), 2-bromo substituted
pyridine (2.40 mmol), copper (25.4 mg, 0.400 mmol) and Cs2CO3
(1.95 g, 6.00 mmol) in DMF (2.0 mL) was stirred at 100 °C under
microwave irradiation for 20 min. Water was added to the cooled
reaction mixture, and the mixture was extracted with EtOAc
several times. The combined organic layers were washed with
water twice and dried over anhydrous Na2SO4. After concentration
under reduced pressure, the residue was purified by silica gel
chromatography to obtain 14.
4.3. General Procedure for Reduction of Nitro Group with Iron
to Prepare 3 (General Procedure B).
A solution of 2 (18.1 mmol), iron powder (4.84 g, 90.5 mmol)
and NH4Cl (0.48 g, 9.05 mmol) in water (50 mL) and EtOH (100
mL) was stirred under reflux for 5 h. The cooled reaction mixture
was filtered through a pad of celite and the filtrate was extracted
with EtOAc several times. The combined organic layers were
dried over anhydrous Na2SO4 and concentrated under reduced
pressure to obtain crude 3, which was used directly for the next
reaction without further purification.
4.10. General Procedure for Pd-Catalyzed Methylation to
Prepare 15 (General Procedure I).
A solution of 13 (625 mg, 2.00 mmol), 2-bromo substituted pyr
A solution of 14 (1.41 mmol), trimethylboroxine (50% solution in
THF, 0.39 mL, 1.41 mmol), PdCl2(dppf)•CH2Cl2 complex (120
mg, 0.140 mmol) and K2CO3 (390 mg, 2.82 mmol) in DMF (5.0
mL) was stirred under N2 at 80 °C for 24 h. Water was added to
the cooled reaction mixture, and the mixture was extracted with
EtOAc several times. The combined organic layers were washed
with water twice and dried over anhydrous Na2SO4. After
concentration under reduced pressure, the residue was purified by
silica gel chromatography to obtain 15.
4.4. General Procedure for Reduction of Nitro Group by
Catalytic Hydrogenation to Prepare 3 (General Procedure C).
A solution of 2 (38.8 mmol) and Pd/C (10%, 0.53 g) in EtOH
(100 mL) was stirred under H2 for 6 h. The reaction mixture was
filtered through a pad of celite and the filtrate was concentrated
under reduced pressure to obtain crude 3, which was used directly
for the next reaction without further purification.
4.5. General Procedure for Amidation to Prepare 8 (General
Procedure D).
4.10.1. 6-(4-Chloro-3-fluorophenoxy)-N-methyl-3-
nitropyridin-2-amine (2a).
A
solution
of
3
(4.49
mmol),
[3-
(methoxycarbonyl)phenoxy]acetic acid8 (0.94 g, 4.49 mmol),
WSC•HCl (0.86 g, 4.49 mmol) and HOBt•H2O (0.61 g, 4.49
mmol) in CH2Cl2 (100 mL) was stirred at room temperature for 18
h under N2. Water was added to the reaction mixture and the
mixture was extracted with CH2Cl2 several times. The combined
organic layers were dried over anhydrous Na2SO4 and
concentrated under reduced pressure. The residue was purified by
silica gel chromatography to obtain 8.
Compound 2a was prepared according to general procedure A
1
(yield, 89%). H-NMR (500 MHz, CDCl3) δ 2.90 (3H, d, J = 4.9
Hz), 6.24 (1H, d, J = 8.8 Hz), 6.97 (1H, ddd, J = 1.5, 2.4, 8.8 Hz),
7.08 (1H, dd, J = 2.4, 9.8 Hz), 7.43 (1H, t, J = 8.8 Hz), 8.45 (1H,
d, J = 9.3 Hz), 8.46 (1H, br).
4.10.2. 6-(3-Fluoro-4-methylphenoxy)-N-methyl-3-
nitropyridin-2-amine (2e).

Compound 2e was prepared according to general procedure A
(yield, 70%). 1H-NMR (400 MHz, DMSO-d6) δ 2.25 (3H, s), 2.76
(3H, d, J = 4.7 Hz), 6.30 (1H, d, J = 9.0 Hz), 7.02 (1H, dd, J = 2.4,
8.6 Hz), 7.18 (1H, dd, J = 2.0, 10.6 Hz), 7.35 (1H, t, J = 8.6 Hz),
8.44 (1H, d, J = 9.0 Hz), 8.75 (1H, br).
2.4, 8.8 Hz), 7.04 (1H, dd, J = 2.9, 10.3 Hz), 7.26–7.28 (1H, m),
7.39 (2H, dt, J = 4.9, 8.3 Hz), 7.69–7.72 (2H, m), 8.06 (1H, d, J =
8.8 Hz).
4.10.9. Methyl 3-[(3-methyl-5-phenoxy-3H-
imidazo[4, 5-b]pyridin-2-yl)methoxy]benzoate 2HCl
(5b). 2 3
4.10.3. 6-(2,4-Dimethylphenoxy)-N-methyl-3-
nitropyridin-2-amine (2f).
A solution of 4b (380 mg, 1.50 mmol), n-Bu3P (610 mg, 3.00
mmol), ADDP (760 mg, 3.00 mmol) and methyl 3-
hydroxybenzoate (340 mg, 2.30 mmol) in toluene (30 mL) was
stirred for 10 h under N2. After concentration of the reaction
mixture under reduced pressure, the residue was purified by silica
gel chromatography (hexane/EtOAc, 1:2) to obtain the free form
of 5b. HCl in 1,4-dioxane (4.0 M, 10 mL) was added to the free
form of 5b, and the precipitated solid was collected by filtration to
Compound 2f was prepared according to general procedure A
(yield, 92%). 1H-NMR (400 MHz, CDCl3) δ 2.15 (3H, s), 2.35 (3H,
s), 2.90 (3H, d, J = 5.1 Hz), 6.11 (1H, d, J = 9.0 Hz), 6.95 (1H, d,
J = 8.2 Hz), 7.08 (1H, d, J = 8.2 Hz), 7.07 (1H, d, J = 0.8 Hz), 7.39
(1H, d, J = 9.8 Hz), 8.45 (1H, br).
4.10.4. 6-(3,4-Dimethylphenoxy)-N-methyl-3-
nitropyridin-2-amine (2h).
1
obtain 5b (0.40 g, 58%). H-NMR (400 MHz, DMSO-d6) δ 3.72
Compound 2h was prepared according to general procedure A
(yield, 33%). 1H-NMR (500 MHz, CDCl3) δ 2.28 (6H, s), 2.96 (3H,
d, J = 4.9 Hz), 6.11 (1H, d, J = 8.8 Hz), 6.91 (1H, dd, J = 2.4, 7.8
Hz), 6.96 (1H, d, J = 2.9 Hz), 7.15 (1H, d, J = 8.3 Hz), 7.38 (1H,
d, J = 8.8 Hz), 8.46 (1H, br).
(3H, s), 3.86 (3H, s), 5.54 (2H, s), 6.96 (1H, d, J = 8.6 Hz), 7.18
(1H, d, J = 7.5 Hz), 7.22 (1H, t, J = 7.3 Hz), 7.43 (2H, m), 7.45
(1H, dd, J = 7.6, 8.2 Hz), 7.61 (1H, dd, J = 1.5, 7.6 Hz), 7.66 (1H,
dd, J = 1.5, 2.3 Hz), 8.18 (1H, d, J = 8.6 Hz); HRMS (ESI) m/z:
[M + H]+ calcd for C22H20N3O4, 390.1454; found 390.1469.
4.10.5. 6-(2,3-Dihydro-1H-inden-5-yloxy)-N-
methyl-3-nitropyridin-2-amine (2i).
4.10.10. Methyl 3-{[5-(4-fluorophenoxy)-3-methyl-
3H-imidazo[4,5-b]pyridin-2-yl]methoxy}benzoate
(5c).
Compound 2i was prepared according to general procedure A
(yield, 85%). 1H-NMR (500 MHz, CDCl3) δ 2.13 (2H, quint., J =
7.3 Hz), 2.93 (4H, t, J = 7.3 Hz), 2.97 (3H, d, J = 5.4 Hz), 6.11
(1H, d, J = 8.3 Hz), 6.93 (1H, dd, J = 1.5, 8.3 Hz), 7.02 (1H, s),
7.23 (1H, d, J = 7.8 Hz), 7.38 (1H, dd, J = 1.0, 9.3 Hz), 8.46 (1H,
br).
Compound 5c was prepared according to general procedure E
(yield, 80%). 1H-NMR (400 MHz, CDCl3) δ 3.80 (3H, s), 3.92 (3H,
s), 5.37 (2H, s), 6.81 (1H, d, J = 8.6 Hz), 7.06–7.16 (4H, m), 7.25–
7.28 (1H, m), 7.37 (1H, t, J = 8.2 Hz), 7.69 (1H, dt, J = 1.2, 7.8
Hz), 7.71 (1H, dd, J = 1.2, 2.7 Hz), 8.02 (1H, d, J = 8.6 Hz).
4.10.6. N-Methyl-2-(4-methylphenoxy)-5-
nitropyridin-4-amine (2l).
4.10.11. Methyl 3-{[3-methyl-5-(4-methylphenoxy)-
3H-imidazo[4,5-b]pyridin-2-yl]methoxy}benzoate
(5d).
Compound 2l was prepared according to general procedure A
(yield, 92%). 1H-NMR (400 MHz, CDCl3) δ 2.37 (3H, s), 2.99 (3H,
d, J = 5.1 Hz), 6.10 (1H, s), 7.02 (2H, d, J = 8.2 Hz), 7.22 (2H, d,
J = 8.6 Hz), 8.07 (1H, br), 8.96 (1H, s).
Compound 5d was prepared according to general procedure E
(yield, 40%). 1H-NMR (400 MHz, CDCl3) δ 2.37 (3H, s), 3.83 (3H,
s), 3.92 (3H, s), 5.38 (2H, s), 6.77 (1H, d, J = 8.6 Hz), 7.06 (2H, d,
J = 8.2 Hz), 7.19 (2H, d, J = 8.6 Hz), 7.25–7.28 (1H, m), 7.37 (1H,
t, J = 7.4 Hz), 7.68–7.72 (2H, m), 8.00 (1H, d, J = 8.6 Hz).
4.10.7. (3-Methyl-5-phenoxy-3H-imidazo[4,5-
b]pyridin-2-yl)methanol (4b).
1 (20.0 g, 107 mmol) was added to a solution of NaH (60%,
5.12 g, 128 mmol) and phenol (12.1 g, 128 mmol) in THF (200
mL) at room temperature under N2, and the mixture was stirred at
80 °C for 4 h. Water was added to the cooled reaction mixture, and
the mixture was extracted with EtOAc. The organic layer was
sequentially washed with 1 M KOH solution and water, dried over
anhydrous Na2SO4, and concentrated under reduced pressure to
obtain crude 2b. A solution of crude 2b and Pd(OH)2 (500 mg) in
THF (50 mL) and EtOH (50 mL) was stirred under H2 overnight.
The catalyst was removed by filtration, and the solvent was
evaporated under reduced pressure to obtain crude 3b. A solution
of crude 3b, glycolic acid (24.4 g, 321 mmol) in 4 M HCl in 1,4-
dioxane (150 mL) was stirred under reflux for 10 h. Saturated
Na2CO3 solution was added to the cooled reaction mixture, and the
mixture was extracted with EtOAc. The organic layer was
sequentially washed with water and brine, dried over anhydrous
Na2SO4, and concentrated under reduced pressure. The generated
solid was washed with i-Pr2O to obtain 4b (21.8 g, 80%; 3 steps).
1H-NMR (400 MHz, DMSO-d6) δ 3.67 (3H, s), 4.69 (2H, d, J =
5.0 Hz), 5.61 (1H, t, J = 5.0 Hz), 6.84 (1H, d, J = 8.0 Hz), 7.12–
7.23 (3H, m), 7.39–7.45 (2H, m), 8.06 (1H, d, J = 8.0 Hz).
4.10.12. Methyl 3-{[5-(3-fluoro-4-methylphenoxy)-
3-methyl-3H-imidazo[4,5-b]pyridin-2-
yl]methoxy}benzoate (5e).
Compound 5e was prepared according to general procedure E
1
(yield, 76%). H-NMR (400 MHz, CDCl3) δ 2.29 (3H, d, J = 1.6
Hz), 3.84 (3H, s), 3.93 (3H, s), 5.38 (2H, s), 6.83 (1H, d, J = 8.6
Hz), 6.86–6.91 (2H, m), 7.18 (1H, t, J = 8.6 Hz), 7.26–7.29 (1H,
m), 7.38 (1H, t, J = 7.8 Hz), 7.69–7.72 (2H, m), 8.03 (1H, d, J =
8.6 Hz).
4.10.13. Methyl 3-{[5-(2,4-dimethylphenoxy)-3-
methyl-3H-imidazo[4,5-b]pyridin-2-
yl]methoxy}benzoate (5f).
Compound 5f was prepared according to general procedure E
(yield, 79%). 1H-NMR (400 MHz, CDCl3) δ 2.18 (3H, s), 2.35 (3H,
s), 3.83 (3H, s), 3.92 (3H, s), 5.37 (2H, s), 6.84 (1H, d, J = 8.6 Hz),
6.96 (1H, d, J = 8.2 Hz), 7.03 (1H, dd, J = 1.6, 8.2 Hz), 7.09 (1H,
s), 7.27 (1H, ddd, J = 0.8, 2.7, 8.2 Hz), 7.38 (1H, t, J = 7.8 Hz),
7.69 (1H, dt, J = 1.2, 7.8 Hz), 7.71 (1H, dd, J = 1.6, 2.7 Hz), 7.96
(1H, d, J = 8.6 Hz).
4.10.14. Methyl 3-{[5-(3,5-dimethylphenoxy)-3-
methyl-3H-imidazo[4,5-b]pyridin-2-
yl]methoxy}benzoate (5g).
4.10.8. Methyl 3-{[5-(4-chloro-3-fluorophenoxy)-3-
methyl-3H-imidazo[4,5-b]pyridin-2-
yl]methoxy}benzoate (5a).
Compound 5g was prepared according to general procedure E
(yield, 86%). 1H-NMR (400 MHz, DMSO-d6) δ 2.32 (6H, s), 3.86
(3H, s), 3.93 (3H, s), 5.38 (2H, s), 6.71–6.81 (3H, m), 6.84 (1H, s),
7.29 (1H, dd, J = 1.0, 2.5 Hz), 7.38 (1H, t, J = 7.8 Hz), 7.67–7.71
Compound 5a was prepared according to general procedure E
(yield, 76%). 1H-NMR (500 MHz, CDCl3) δ 3.82 (3H, s), 3.92 (3H,
s), 5.38 (2H, s), 6.89 (1H, d, J = 8.3 Hz), 6.93 (1H, ddd, J = 1.5,

(1H, m), 7.72 (1H, dd, J = 1.4, 2.5 Hz), 7.99 (1H, d, J = 8.6 Hz);
MS (FAB) m/z: 418 [M + H]+.
d, J = 2.0 Hz), 7.22 (1H, d, J = 7.8 Hz), 7.29 (1H, dd, J = 1.2, 2.4
Hz), 7.39 (1H, t, J = 8.2 Hz), 7.69–7.72 (2H, m), 8.71 (1H, d, J =
0.8 Hz).
4.10.15. Methyl 3-{[5-(3,4-dimethylphenoxy)-3-
methyl-3H-imidazo[4,5-b]pyridin-2-
yl]methoxy}benzoate (5h).
4.10.22. 3-{[5-(4-Chloro-3-fluorophenoxy)-3-
methyl-3H-imidazo[4,5-b]pyridin-2-
yl]methoxy}benzoic acid (6a).
Compound 5h was prepared according to general procedure E
(yield, 75%). 1H-NMR (400 MHz, CDCl3) δ 2.27 (3H, s), 2.27 (3H,
s), 3.85 (3H, s), 3.92 (3H, s), 5.37 (2H, s), 6.75 (1H, d, J = 8.6 Hz),
6.90 (1H, dd, J = 2.3, 7.8 Hz), 6.95 (1H, d, J = 2.3 Hz), 7.14 (1H,
d, J = 8.6 Hz), 7.25–7.29 (2H, m), 7.38 (1H, t, J = 7.8 Hz), 7.67–
7.73 (2H, m).
Compound 6a was prepared according to general procedure F
(yield, 94%). Mp, 213–215 °C. 1H-NMR (400 MHz, DMSO-d6) δ
3.72 (3H, s), 5.49 (2H, s), 7.00 (1H, d, J = 8.6 Hz), 7.09 (1H, ddd,
J = 1.6, 2.7, 9.0 Hz), 7.37 (1H, ddd, J = 0.8, 3.5, 8.2 Hz), 7.40 (1H,
dd, J = 2.7, 10.6 Hz), 7.45 (1H, t, J = 7.8 Hz), 7.58 (1H, dt, J = 1.6,
7.8 Hz), 7.60–7.65 (2H, m), 8.19 (1H, d, J = 8.6 Hz); MS (FAB)
m/z: 428 [M + H]+; Anal. calcd for C21H15ClFN3O4•0.25H2O: C,
58.34; H, 3.61; N, 9.72; F, 4.39; Cl, 8.20; found C, 58.44; H, 3.51;
N, 9.79; F, 4.56; Cl, 8.42.
4.10.16. Methyl 3-{[5-(2,3-dihydro-1H-inden-5-
yloxy)-3-methyl-3H-imidazo[4, 5-b]pyridin-2-
yl]methoxy}benzoate (5i).
Compound 5i was prepared according to general procedure E
(yield, 68%). 1H-NMR (400 MHz, CDCl3) δ 2.12 (2H, quint., J =
7.4 Hz), 2.92 (4H, t, J = 7.4 Hz), 3.85 (3H, s), 3.92 (3H, s), 5.38
(2H, s), 6.75 (1H, d, J = 8.6 Hz), 6.93 (1H, dd, J = 2.4, 8.2 Hz),
7.02 (1H, d, J = 2.4 Hz), 7.22 (1H, d, J = 7.8 Hz), 7.28 (1H, ddd,
J = 1.2, 2.7, 8.2 Hz), 7.38 (1H, t, J = 7.8 Hz), 7.69 (1H, dt, J = 0.8,
7.8 Hz), 7.72 (1H, dd, J = 1.6, 2.7 Hz), 7.98 (1H, d, J = 8.6 Hz).
4.10.23. 3-[(3-Methyl-5-phenoxy-3H-imidazo[4,5-
b]pyridin-2-yl)methoxy]benzoic acid•2HCl (6b).
Compound 6b was prepared according to general procedure F
(yield, 61%). 1H-NMR (400 MHz, DMSO-d6) δ 3.73 (3H, s), 5.53
(2H, s), 6.96 (1H, d, J = 8.6 Hz), 7.18 (1H, d, J = 8.7 Hz), 7.22
(1H, t, J = 7.5 Hz), 7.38 (1H, ddd, J = 1.1, 2.6, 8.2 Hz), 7.42 (2H,
m), 7.46 (1H, dd, J = 7.6, 8.2 Hz), 7.59 (1H, ddd, J = 1.1, 1.3, 7.6
Hz), 7.64 (1H, dd, J = 1.3, 2.6 Hz), 8.18 (1H, d, J = 8.6 Hz);HRMS
(ESI) m/z: [M + H]+ calcd for C21H18N3O4, 376.1297; found
376.1295.
4.10.17. Methyl 3-{[5-(2,3-dihydro-1-benzofuran-6-
yloxy)-3-methyl-3H-imidazo[4, 5-b]pyridin-2-
yl]methoxy}benzoate (5j).
Compound 5j was prepared according to general procedure E
(yield, 88%, 4 steps). 1H-NMR (400 MHz, CDCl3) δ 3.22 (2H, t, J
= 8.6 Hz), 3.85 (3H, s), 3.92 (3H, s), 4.64 (2H, t, J = 9.0 Hz), 5.38
(2H, s), 6.62–6.65 (2H, m), 6.78 (1H, d, J = 8.6 Hz), 7.17 (1H, d,
J = 7.0 Hz), 7.25–7.28 (1H, m), 7.38 (1H, t, J = 7.8 Hz), 7.68–7.72
(2H, m), 7.99 (1H, d, J = 8.6 Hz); MS (FAB) m/z: 432 [M + H]+.
4.10.24. 3-{[5-(4-Fluorophenoxy)-3-methyl-3H-
imidazo[4, 5-b]pyridin-2-yl]methoxy}benzoic acid
(6c).
Compound 6c was prepared according to general procedure F
(yield, 71%). 1H-NMR (500 MHz, DMSO-d6) δ 3.69 (3H, s), 5.47
(2H, s), 6.91 (1H, d, J = 8.3 Hz), 7.22–7.28 (4H, m), 7.37 (1H, dd,
J = 2.4, 8.3 Hz), 7.44 (1H, t, J = 7.8 Hz), 7.58 (1H, d, J = 7.3 Hz),
7.62 (1H, s), 8.14 (1H, d, J = 8.8 Hz), 13.04 (1H, s); MS (FAB)
m/z: 393 [M+·]; Anal. calcd for C21H16FN3O4•0.20H2O: C, 63.54;
H, 4.16; N, 10.59 F, 4.79; found C, 63.61; H, 3.92; N, 10.57; F,
4.82.
4.10.18. Methyl 3-{[6-(4-chloro-3-fluorophenoxy)-
1-methyl-1H-imidazo[4,5-c]pyridin-2-
yl]methoxy}benzoate (5k).
Compound 5k was prepared according to general procedure E
(yield, 39%). 1H-NMR (400 MHz, CDCl3) δ 3.88 (3H, s), 3.93 (3H,
s), 5.42 (2H, s), 6.87 (1H, ddd, J = 1.6, 3.2, 9.0 Hz), 6.91 (1H, d, J
= 0.8 Hz), 6.95 (1H, dd, J = 2.7, 10.2 Hz), 7.26–7.28 (1H, m), 7.39
(2H, q, J = 8.6 Hz), 7.70–7.72 (2H, m), 8.71 (1H, s).
4.10.25. 3-{[3-Methyl-5-(4-methylphenoxy)-3H-
imidazo[4, 5-b]pyridin-2-yl]methoxy}benzoic acid
(6d).
4.10.19. Methyl 3-{[1-methyl-6-(4-methylphenoxy)-
1H-imidazo[4,5-c]pyridin-2-yl]methoxy}benzoate
(5l).
Compound 6d was prepared according to general procedure F
(yield, 92%). Mp, 209–216 °C. 1H-NMR (400 MHz, DMSO-d6) δ
2.32 (3H, s), 3.70 (3H, s), 5.47 (2H, s), 6.86 (1H, d, J = 8.6 Hz),
7.06 (2H, d, J = 8.2 Hz), 7.22 (2H, d, J = 7.8 Hz), 7.35–7.39 (1H,
m), 7.45 (1H, t, J = 7.8 Hz), 7.62 (1H, dd, J = 1.4, 2.5 Hz), 7.58
(1H, dt, J = 1.3, 7.5 Hz), 8.12 (1H, d, J = 8.6 Hz), 13.05 (1H, br);
MS (FAB) m/z: 389 [M+·]; Anal. calcd for C22H19N3O4: C, 67.86;
H, 4.92; N, 10.79; found C, 67.69; H, 4.71; N, 10.72.
Compound 5l was prepared according to general procedure E
(yield, 57%). 1H-NMR (400 MHz, CDCl3) δ 2.36 (3H, s), 3.82 (3H,
s), 3.92 (3H, s), 5.39 (2H, s), 6.78 (1H, s), 7.02 (2H, d, J = 8.2 Hz),
7.19 (2H, d, J = 8.6 Hz), 7.29 (1H, dd, J = 1.2, 2.4 Hz), 7.39 (1H,
t, J = 8.2 Hz), 7.69–7.74 (2H, m), 8.70 (1H, s).
4.10.20. Methyl 3-{[6-(2,3-dihydro-1-benzofuran-6-
yloxy)-1-methyl-1H-imidazo[4, 5-c]pyridin-2-
yl]methoxy}benzoate (5m).
4.10.26. 3-{[5-(3-Fluoro-4-methylphenoxy)-3-
methyl-3H-imidazo[4,5-b]pyridin-2-
yl]methoxy}benzoic acid (6e).
Compound 5m was prepared according to general procedure E
Compound 6e was prepared according to general procedure F
(yield, 72%). Mp, 205–207 °C. 1H-NMR (400 MHz, DMSO-d6) δ
2.23 (3H, s), 3.70 (3H, s), 5.41 (2H, s), 6.91 (1H, d, J = 8.2 Hz),
6.91-.6.94 (1H, m), 7.05 (1H, dd, J = 2.4, 11.0 Hz), 7.15 (1H, d, J
= 9.0 Hz), 7.28 (1H, d, J = 7.8 Hz), 7.32 (1H, d, J = 8.2 Hz), 7.51
(1H, d, J = 7.4 Hz), 7.57 (1H, dd, J = 1.2, 2.4 Hz), 8.14 (1H, d, J
= 8.6 Hz); MS (FAB) m/z: 408 [M + H]+; Anal. calcd for
C22H18FN3O4•0.20H2O: C, 64.29; H, 4.51; N, 10.22; found C,
64.41; H, 4.31; N, 10.30.
1
(yield, 89%). H-NMR (400 MHz, CDCl3) δ 3.20 (2H, t, J = 9.0
Hz), 3.84 (3H, s), 3.93 (3H, s), 4.62 (2H, t, J = 8.6 Hz), 5.39 (2H,
s), 6.57 (1H, d, J = 2.4 Hz), 6.61 (1H, dd, J = 2.4, 8.2 Hz), 6.81
(1H, s), 7.17 (1H, d, J = 7.8 Hz), 7.29 (1H, t, J = 2.0 Hz), 7.39 (1H,
t, J = 8.2 Hz), 7.69–7.72 (2H, m), 8.71 (1H, d, J = 0.8 Hz).
4.10.21. Methyl 3-{[6-(2,3-dihydro-1H-inden-5-
yloxy)-1-methyl-1H-imidazo[4, 5-c]pyridin-2-
yl]methoxy}benzoate (5n).
Compound was prepared according to general procedure E
(yield, 69%). 1H-NMR (400 MHz, CDCl3) δ 2.12 (2H, quint., J =
7.0 Hz), 2.89-2.93 (4H, m), 3.83 (3H, s), 3.93 (3H, s), 5.39 (2H, s),
6.79 (1H, d, J = 0.8 Hz), 6.90 (1H, dd, J = 2.0, 7.8 Hz), 6.98 (1H,
4.10.27. 3-{[5-(2,4-Dimethylphenoxy)-3-methyl-3H-
imidazo[4, 5-b]pyridin-2-yl]methoxy}benzoic acid
(6f).

Compound 6f was prepared according to general procedure F
(yield, 97%). Mp, 209–216 °C. 1H-NMR (400 MHz, DMSO-d6) δ
2.09 (3H, s), 2.30 (3H, s), 3.67 (3H, s), 5.46 (2H, s), 6.78 (1H, d,
J = 8.6 Hz), 6.97 (1H, d, J = 8.2 Hz), 7.05 (1H, dd, J = 2.0, 8.2 Hz),
7.14 (1H, d, J = 2.0 Hz), 7.37 (1H, ddd, J = 1.2, 2.7, 8.2 Hz), 7.45
(1H, t, J = 7.8 Hz), 7.58 (1H, dt, J = 1.2, 7.8 Hz), 7.62 (1H, dd, J
= 1.6, 2.7 Hz), 8.09 (1H, d, J = 8.6 Hz), 13.06 (1H, s); MS (FAB)
m/z: 404 [M + H]+; Anal. calcd for C23H21N3O4: C, 68.47; H, 5.25;
N, 10.42; found C, 68.15; H, 5.20; N, 10.32.
1.6, 2.7 Hz), 8.61 (1H, d, J =0.8 Hz), 13.09 (1H, br); MS (FAB)
m/z: 428 [M + H]+; Anal. calcd for C21H15ClFN3O4•0.25H2O: C,
58.34; H, 3.61; N, 9.72; F, 4.39; Cl, 8.20; found C, 58.53; H, 3.56;
N, 9.69; F, 4.60; Cl, 8.13.
4.10.33. 3-{[1-Methyl-6-(4-methylphenoxy)-1H-
imidazo[4, 5-c]pyridin-2-yl]methoxy}benzoic acid
(6l).
Compound 6l was prepared according to general procedure F
(yield, 93%). 1H-NMR (400 MHz, DMSO-d6) δ 2.30 (3H, s), 3.84
(3H, s), 5.51 (2H, s), 6.95 (2H, d, J = 8.6 Hz), 7.18 (2H, d, J = 8.2
Hz), 7.24 (1H, d, J = 0.8 Hz), 7.39 (1H, ddd, J = 0.8, 2.4, 8.2 Hz),
7.46 (1H, t, J = 7.8 Hz), 7.59 (1H, dt, J = 1.2, 7.4 Hz), 7.64 (1H,
dd, J = 1.2, 2.4 Hz), 8.85 (1H, d, J = 1.2 Hz), 13.08 (1H, br); MS
(FAB) m/z: 390 [M + H]+; Anal. calcd for C22H19N3O4•0.33H2O:
C, 66.83; H, 5.01; N, 10.63; found C, 66.71; H, 5.10; N, 10.58.
4.10.28. 3-{[5-(3,5-Dimethylphenoxy)-3-methyl-3H-
imidazo[4,5-b]pyridin-2-yl]methoxy}benzoic acid
(6g).
Compound 6g was prepared according to general procedure F
(yield, 85%). 1H-NMR (400 MHz, DMSO-d6) δ 2.27 (6H, s), 3.72
(3H, s), 5.48 (2H, s), 6.75 (2H, s), 6.84 (2H, s), 7.34–7.41 (1H, m),
7.45 (1H, t, J = 7.8 Hz), 7.58 (1H, d, J = 7.4 Hz), 7.63 (1H, s), 8.12
(1H, d, J = 8.6 Hz); MS (FAB) m/z: 404 [M + H]+; Anal. calcd for
C23H21N3O4•0.33H2O: C, 67.47; H, 5.33; N, 10.26; found C,
67.69; H, 5.30; N, 10.28.
4.10.34. 3-{[6-(2,3-Dihydro-1-benzofuran-6-yloxy)-
1-methyl-1H-imidazo[4,5-c]pyridin-2-
yl]methoxy}benzoic acid (6m ).
Compound 6m was prepared according to general procedure F
1
4.10.29. 3-{[5-(3,4-Dimethylphenoxy)-3-methyl-3H-
imidazo[4,5-b]pyridin-2-yl]methoxy}benzoic acid
(6h).
(yield, 74%). H-NMR (400 MHz, DMSO-d6) δ 3.15 (2H, t, J =
9.0 Hz), 3.84 (3H, s), 4.57 (2H, t, J = 8.6 Hz), 5.51 (2H, s), 6.47
(1H, d, J = 2.0 Hz), 6.50 (1H, dd, J = 2.0, 7.8 Hz), 7.18 (1H, d, J
= 7.8 Hz), 7.24 (1H, s), 7.38 (1H, ddd, J = 1.2, 2.7, 8.2 Hz), 7.46
(1H, t, J = 7.8 Hz), 7.59 (1H, d, J = 7.4 Hz), 7.63 (1H, dd, J = 1.6,
2.4 Hz), 8.56 (1H, d, J = 0.8 Hz), 13.07 (1H, s); MS (FAB) m/z:
418 [M + H]+; Anal. calcd for C23H19N3O5•0.20H2O: C, 65.61; H,
4.64; N, 9.98; found C, 65.76; H, 4.51; N, 9.95.
Compound 6h was prepared according to general procedure F
(yield, 95%). 1H-NMR (400 MHz, DMSO-d6) δ 2.22 (3H, s), 2.22
(3H, s), 3.71 (3H, s), 5.47 (2H, s), 6.82 (1H, d, J = 8.6 Hz), 6.88
(1H, dd, J = 2.7, 8.2 Hz), 6.96 (1H, d, J = 2.7 Hz), 7.16 (1H, d, J
= 7.8 Hz), 7.37 (1H, ddd, J = 1.2, 2.7, 8.2 Hz), 7.45 (1H, t, J = 7.8
Hz), 7.56–7.59 (1H, m), 7.62–7.64 (1H, m), 8.10 (1H, d, J = 8.2
Hz), 13.03 (1H, s); MS (FAB) m/z: 404 [M + H]+; Anal. calcd for
C23H21N3O4: C, 68.47; H, 5.25; N, 10.42; found C, 68.07; H, 5.09;
N, 10.35.
4.10.35. 3-{[6-(2,3-Dihydro-1H-inden-5-yloxy)-1-
methyl-1H-imidazo[4,5-c]pyridin-2-
yl]methoxy}benzoic acid (6n).
Compound 6n was prepared according to general procedure F
1
4.10.30. 3-{[5-(2,3-Dihydro-1H-inden-5-yloxy)-3-
methyl-3H-imidazo[4,5-b]pyridin-2-
yl]methoxy}benzoic acid (6i).
(yield, 99%). H-NMR (400 MHz, DMSO-d6) δ 2.05 (2H, quint.,
J = 7.4 Hz), 2.84 (4H, t, J = 7.4 Hz), 3.84 (3H, s), 5.50 (2H, s),
6.81 (1H, dd, J = 2.4, 8.2 Hz), 6.90 (1H, d, J = 2.0 Hz), 7.20 (1H,
d, J = 8.2 Hz), 7.23 (1H, d, J = 1.2 Hz), 7.39 (1H, ddd, J = 1.2, 2.7,
8.2 Hz), 7.45 (1H, t, J = 7.4 Hz), 7.59 (1H, dt, J = 1.6, 7.8 Hz),
7.64 (1H, dd, J = 1.6, 2.4 Hz), 8.54 (1H, d, J = 0.8 Hz), 13.06 (1H,
Compound 6i was prepared according to general procedure F
1
(yield, 98%). H-NMR (400 MHz, DMSO-d6) δ 2.05 (2H, quint.,
J = 7.0 Hz), 2.86 (4H, t, J = 7.4 Hz), 3.71 (3H, s), 5.47 (2H, s),
6.83 (1H, d, J = 8.2 Hz), 6.92 (1H, dd, J = 2.4, 7.8 Hz), 7.01 (1H,
d, J = 2.4 Hz), 7.24 (1H, d, J = 8.2 Hz), 7.38 (1H, ddd, J = 1.2, 2.7,
8.2 Hz), 7.45 (1H, t, J = 7.4 Hz), 7.58 (1H, dt, J = 1.5, 7.8 Hz),
7.63 (1H, dd, J = 1.2, 2.4 Hz), 8.11 (1H, d, J = 8.2 Hz), 13.06 (1H,
s); MS (FAB) m/z: 416 [M + H]+; Anal. calcd for C24H21N3O4: C,
69.39; H, 5.10; N, 10.11; found C, 69.34; H, 5.07; N, 10.13.
s); MS (FAB) m/z: 416 [M
+
H]+; Anal. calcd for
C24H21N3O4•0.25H2O: C, 68.64; H, 5.16; N, 10.01; found C, 68.71;
H, 5.11; N, 9.97.
4.10.36. Methyl 3-(2-{[6-(4-chloro-3-
fluorophenoxy)-2-(methylamino)pyridin-3-
yl]amino}-2-oxoethoxy)benzoate (8a).
4.10.31. 3-{[5-(2,3-Dihydro-1-benzofuran-6-yloxy)-
3-methyl-3H-imidazo[4,5-b]pyridin-2-
yl]methoxy}benzoic acid (6j).
Compound 8a was prepared according to general procedure C,
followed by D (yield, 77%; 2 steps). 1H-NMR (400 MHz, CDCl3)
δ 2.81 (3H, s), 3.94 (3H, s), 4.73 (2H, s), 6.14 (1H, d, J = 8.2 Hz),
6.93 (1H, ddd, J = 1.6, 2.7, 9.0 Hz), 7.03 (1H, dd, J = 2.0, 10.2 Hz),
7.21 (1H, ddd, J = 0.8, 2.7, 8.2 Hz), 7.35 (1H, d, J = 8.6 Hz), 7.40
(1H, d, J = 8.2 Hz), 7.45 (1H, t, J = 7.8 Hz), 7.67 (1H, dd, J = 1.6,
2.7 Hz), 7.77 (1H, s), 7.78 (1H, dt, J = 1.2, 7.8 Hz).
Compound 6j was prepared according to general procedure F
1
(yield, 74%). H-NMR (400 MHz, DMSO-d6) δ 3.18 (2H, t, J =
9.0 Hz), 3.72 (3H, s), 4.59 (2H, t, J = 8.6 Hz), 5.48 (2H, s), 6.58–
6.60 (2H, m), 6.85 (1H, d, J = 8.6 Hz), 7.23 (1H, d, J = 8.6 Hz),
7.38 (1H, ddd, J = 1.2, 2.7, 8.2 Hz), 7.45 (1H, t, J = 7.8 Hz), 7.58
(1H, dt, J = 1.6, 7.8 Hz), 7.63 (1H, dd, J = 1.2, 2.4 Hz), 8.12 (1H,
d, J = 8.6 Hz), 13.07 (1H, s); MS (FAB) m/z: 418 [M + H]+; Anal.
calcd for C23H19N3O5•0.20H2O: C, 65.61; H, 4.64; N, 9.98; found
C, 65.77; H, 4.55; N, 9.90.
4.10.37. Methyl 3-(2-{[6-(4-fluorophenoxy)-2-
(methylamino)pyridin-3-yl]amino}-2-
oxoethoxy)benzoate (8c).
Compound 8c was prepared according to general procedure A,
1
followed by procedures C and D (yield, 60%; 3 steps). H-NMR
4.10.32. 3-{[6-(4-Chloro-3-fluorophenoxy)-1-
methyl-1H-imidazo[4,5-c]pyridin-2-
yl]methoxy}benzoic acid (6k).
(500 MHz, CDCl3) δ 2.82 (3H, s), 3.94 (3H, s), 4.72 (2H, s), 6.02
(1H, d, J = 8.3 Hz), 7.05 (2H, t, J = 8.8 Hz), 7.11–7.13 (2H, m),
7.20 (1H, dd, J = 2.9, 8.3 Hz), 7.35 (1H, d, J = 8.3 Hz), 7.44 (1H,
t, J = 7.8 Hz), 7.66 (1H, dd, J = 1.5, 2.4 Hz), 7.74 (1H, br), 7.77
(1H, dt, J = 1.5, 7.8 Hz).
Compound 6k was prepared according to general procedure F
(yield, 94%). 1H-NMR (400 MHz, DMSO-d6) δ 3.68 (3H, s), 5.53
(2H, s), 6.96 (1H, ddd, J = 1.2, 2.7, 8.6 Hz), 7.25 (1H, dd, J = 3.1,
11.0 Hz), 7.39 (1H, ddd, J = 1.2, 2.7, 8.2 Hz), 7.42 (1H, d, J = 0.8
Hz), 7.46 (1H, t, J = 7.4 Hz), 7.56–7.60 (2H, m), 7.64 (1H, dd, J =
4.10.38. Methyl 3-(2-{[2-(methylamino)-6-(4-
methylphenoxy)pyridin-3-yl]amino}-2-
oxoethoxy)benzoate (8d).

Compound 8d was prepared according to general procedure A,
followed by procedures C and D (yield, 23%; 3 steps). H-NMR
Compound 8j was prepared according to general procedure A,
followed by procedures C and D. Crude 8j was used for the next
reaction without further purification. 1H-NMR (400 MHz, CDCl3)
δ 2.92 (3H, d, J = 5.1 Hz), 3.21 (2H, t, J = 9.4 Hz), 3.95 (3H, s),
4.53 (1H, d, J = 4.7 Hz), 4.63 (2H, t, J = 8.6 Hz), 4.73 (2H, s), 5.98
(1H, d, J = 8.2 Hz), 6.62–6.66 (2H, m), 7.15 (1H, d, J = 7.8 Hz),
7.21 (1H, ddd, J = 1.2, 2.7, 8.2 Hz), 7.33 (1H, d, J = 7.8 Hz), 7.45
(1H, t, J = 7.8 Hz), 7.67 (1H, dd, J = 1.2, 2.7 Hz), 7.75 (1H, br),
7.78 (1H, dt, J = 1.2, 7.8 Hz).
1
(400 MHz, CDCl3) δ 2.35 (3H, s), 2.88 (3H, s), 3.94 (3H, s), 4.72
(2H, s), 5.96 (1H, d, J = 8.2 Hz), 7.02–7.08 (2H, m), 7.16 (2H, d,
J = 8.2 Hz), 7.20 (1H, ddd, J = 1.2, 2.7, 8.2 Hz), 7.32 (1H, d, J =
8.2 Hz), 7.45 (1H, t, J = 8.0 Hz), 7.66 (1H, dd, J = 1.4, 2.5 Hz),
7.73 (1H, s), 7.77 (1H, dt, J = 1.0, 1.2, 7.6 Hz); MS (FAB) m/z:
422 [M + H]+.
4.10.39. Methyl 3-(2-{[6-(3-fluoro-4-
methylphenoxy)-2-(methylamino)pyridin-3-
yl]amino}-2-oxoethoxy)benzoate (8e).
4.10.45. Methyl 3-(2-{[6-(4-chloro-3-
fluorophenoxy)-4-(methylamino)pyridin-3-
yl]amino}-2-oxoethoxy)benzoate (8k).
Compound 8e was prepared according to general procedure B,
1
followed by procedure D (yield, 79%; 2 steps). H-NMR (400
Compound 8k was prepared according to general procedure A,
1
MHz, CDCl3) δ 2.27 (3H, d, J = 2.0 Hz), 2.86 (3H, s), 3.95 (3H,
s), 4.53 (1H, br), 4.73 (2H, s), 6.05 (1H, d, J = 8.2 Hz), 6.85–6.90
(2H, m), 7.15 (1H, t, J = 9.0 Hz), 7.21 (1H, ddd, J = 0.8, 2.7, 8.2
Hz), 7.36 (1H, d, J = 7.8 Hz), 7.46 (1H, t, J = 7.8 Hz), 7.67 (1H,
dd, J = 1.1, 2.4 Hz), 7.74 (1H, s), 7.78 (1H, dt, J = 1.1, 7.8 Hz).
followed by procedures C and D (yield, 34%; 3 steps). H-NMR
(400 MHz, CDCl3) δ 2.88 (3H, d, J = 5.1 Hz), 3.94 (3H, s), 4.66
(1H, d, J = 5.1 Hz), 4.75 (2H, s), 6.18 (1H, s), 6.89 (1H, ddd, J =
1.2, 2.7, 8.6 Hz), 6.98 (1H, dd, J = 2.7, 10.2 Hz), 7.21 (1H, ddd, J
= 1.2, 2.7, 8.2 Hz), 7.37 (1H, t, J = 8.2 Hz), 7.45 (1H, t, J = 7.8
Hz), 7.67 (1H, dd, J = 1.6, 2.7 Hz), 7.75 (1H, s), 7.77 (1H, dt, J =
1.2, 7.8 Hz), 7.82 (1H, br).
4.10.40. Methyl 3-(2-{[6-(2,4-dimethylphenoxy)-2-
(methylamino)pyridin-3-yl]amino}-2-
oxoethoxy)benzoate (8f).
4.10.46. Methyl 3-(2-{[4-(methylamino)-6-(4-
methylphenoxy)pyridin-3-yl]amino}-2-
oxoethoxy)benzoate (8l).
Compound 8f was prepared according to general procedure C,
1
followed by procedure D (yield, 90%; 2 steps). H-NMR (400
MHz, CDCl3) δ 2.17 (3H, s), 2.33 (3H, s), 2.88 (3H, s), 3.94 (3H,
s), 4.72 (2H, s), 5.80 (1H, d, J = 8.2 Hz), 6.96 (1H, t, J = 8.2 Hz),
7.00 (1H, dd, J = 2.0, 8.2 Hz), 7.05 (1H, s), 7.19 (1H, dd, J = 2.7,
8.2 Hz), 7.28 (1H, d, J = 8.2 Hz), 7.44 (1H, t, J = 7.8 Hz), 7.66
(1H, dd, J = 1.6, 2.7 Hz), 7.74 (1H, s), 7.76 (1H, t, J = 7.8 Hz).
Compound 8l was prepared according to general procedure C,
followed by procedure D (yield, 97%; 2 steps). H-NMR (500
MHz, CDCl3) δ 2.34 (3H, s), 2.83 (3H, d, J = 4.9 Hz), 3.94 (3H,
s), 4.57 (1H, d, J = 4.9 Hz), 4.73 (2H, s), 6.12 (1H, s), 7.01 (2H, d,
J = 8.3 Hz), 7.17 (2H, d, J = 7.8 Hz), 7.20 (1H, ddd, J = 2.0, 2.9,
8.3 Hz), 7.44 (1H, t, J = 7.8 Hz), 7.66 (1H, dd, J = 1.5, 2.4 Hz),
7.74 (1H, s), 7.76 (1H, dt, J = 1.0, 7.8 Hz), 7.78 (1H, br).
1
4.10.41. Methyl 3-(2-{[6-(3,5-dimethylphenoxy)-2-
(methylamino)pyridin-3-yl]amino}-2-
oxoethoxy)benzoate (8g).
4.10.47. Methyl 3-(2-{[6-(2, 3-dihydro-1-
benzofuran-6-yloxy)-4-(methylamino)pyridin-3-
yl]amino}-2-oxoethoxy)benzoate (8m ).
Compound 8g was prepared according to general procedure A,
1
followed by procedures C and D (yield, 97%; 3 steps). H-NMR
(400 MHz, CDCl3) δ 2.31 (6H, s), 2.89 (3H, d, J = 4.7 Hz), 3.94
(3H, s), 4.50 (1H, br), 4.72 (2H, s), 5.96 (1H, d, J = 7.8 Hz), 6.78
(2H, s), 6.81 (1H, s), 7.20 (1H, ddd, J = 1.2, 2.7, 8.6 Hz), 7.32 (1H,
d, J = 8.2 Hz), 7.45 (1H, t, J = 7.8 Hz), 7.66 (1H, dd, J = 1.6, 2.7
Hz), 7.74 (1H, br), 7.77 (1H, dt, J = 1.2, 7.8 Hz).
Compound 8m was prepared according to general procedure A,
followed by procedures C and D (yield, 34%; 3 steps). H-NMR
1
(400 MHz, CDCl3) δ 2.84 (3H, d, J = 5.1 Hz), 3.18 (2H, t, J = 8.6
Hz), 3.94 (3H, s), 4.60 (1H, br), 4.60 (2H, t, J = 8.6 Hz), 4.73 (2H,
s), 6.13 (1H, s), 6.58 (1H, d, J = 2.0 Hz), 6.60 (1H, dd, J = 2.0, 8.2
Hz), 7.15 (1H, d, J = 7.8 Hz), 7.20 (1H, ddd, J = 0.8, 2.4, 8.2 Hz),
7.44 (1H, t, J = 7.8 Hz), 7.66 (1H, dd, J = 1.2, 2.7 Hz), 7.75 (1H,
s), 7.75–7.77 (1H, m), 7.83 (1H, br).
4.10.42. Methyl 3-(2-{[6-(3,4-dimethylphenoxy)-2-
(methylamino)pyridin-3-yl]amino}-2-
oxoethoxy)benzoate (8h).
4.10.48. Methyl 3-(2-{[6-(2, 3-dihydro-1H-inden-5-
yloxy)-4-(methylamino)pyridin-3-yl]amino}-2-
oxoethoxy)benzoate (8n).
Compound 8h was prepared according to general procedure B,
followed by procedure D (yield, 27%; 2 steps). H-NMR (400
1
MHz, CDCl3) δ 2.25 (3H, s), 2.25 (3H, s), 2.89 (3H, s), 3.94 (3H,
s), 4.72 (2H, s), 5.93 (1H, d, J = 8.2 Hz), 6.89 (1H, dd, J = 2.7, 8.2
Hz), 6.94 (1H, d, J = 2.3 Hz), 7.11 (1H, d, J = 7.8 Hz), 7.19 (1H,
dd, J = 2.7, 8.2 Hz), 7.31 (1H, d, J = 8.2 Hz), 7.44 (1H, t, J = 8.0
Hz), 7.64–7.67 (1H, m), 7.73–7.80 (2H, m).
Compound 8n was prepared according to general procedure C,
1
followed by procedure D (yield, 77%; 3 steps). H-NMR (400
MHz, CDCl3) δ 2.09 (2H, quint., J = 7.0 Hz), 2.84 (3H, d, J = 5.1
Hz), 2.90 (4H, q, J = 7.8 Hz), 3.94 (3H, s), 4.58 (1H, d, J = 5.1
Hz), 4.73 (2H, s), 6.14 (1H, s), 6.88 (1H, dd, J = 2.4, 8.2 Hz), 6.97
(1H, s), 7.19 (2H, dd, J = 2.7, 7.8 Hz), 7.74 (1H, t, J = 7.8 Hz),
7.66 (1H, dd, J = 1.7, 2.7 Hz), 7.75 (1H, s), 7.77 (1H, d, J = 6.7
Hz), 7.81 (1H, br).
4.10.43. Methyl 3-(2-{[6-(2,3-dihydro-1H-inden-5-
yloxy)-2-(methylamino)pyridin-3-yl]amino}-2-
oxoethoxy)benzoate (8i).
Compound 8i was prepared according to general procedure C,
1
4.10.49. tert-Butyl {5-[(4-methoxybenzyl)oxy]-2-
nitrophenyl}methylcarbamate (10).
followed by procedure D (yield, 97%; 2 steps). H-NMR (400
MHz, CDCl3) δ 2.11 (2H, quint., J = 7.0 Hz), 2.90 (4H, q, J = 7.0
Hz), 2.91 (3H, s), 3.94 (3H, s), 4.72 (2H, s), 5.92 (1H, d, J = 8.2
Hz), 6.82 (1H, dd, J = 2.4, 8.2 Hz), 7.01 (1H, d, J = 2.4 Hz), 7.18–
7.21 (2H, m), 7.31 (1H, d, J = 8.2 Hz), 7.45 (1H, t, J = 8.2 Hz),
7.66 (1H, dd, J = 1.6, 2.7 Hz), 7.75–7.78 (2H, m).
NaH (63%, 25.0 g, 656 mmol) was added to a solution of 4-
methoxybenzyl alcohol (90.3 g, 654 mmol) and tert-butyl (5-
chloro-2-nitrophenyl)methylcarbamate (9, 156 g, 544 mmol) in
DMF (1.4 L) at room temperature under N2, and the mixture was
stirred at 80 °C for 4 h. Water (1.5 L) was added to the cooled
reaction mixture. The precipitated solid was collected by filtration
to obtain 10 (209 g, 99%) as a yellow solid. 1H-NMR (400 MHz,
CDCl3) δ 1.28 (9H, s), 3.25 (3H, s), 3.81 (3H, s), 5.04 (2H, s),
6.79–6.95 (4H, m), 7.29–7.37 (2H, m), 7.91–8.03 (1H, m).
4.10.44. Methyl 3-(2-{[6-(2,3-dihydro-1-
benzofuran-6-yloxy)-2-(methylamino)pyridin-3-
yl]amino}-2-oxoethoxy)benzoate (8j).

4.10.50. tert-Butyl {2-amino-5-[(4-
methoxybenzyl)oxy]phenyl}methylcarbamate (11).
1.2, 7.8 Hz), 7.73 (1H, dd, J = 1.6, 2.7 Hz), 7.78 (1H, d, J = 8.6
Hz).
A solution of 10 (209 g, 538 mmol), iron powder (150 g, 2.69
mol) and NH4Cl (15.0 g, 280 mmol) in EtOH (1.2 L) and water
(1.0 L) was stirred under reflux for 5 h. The cooled reaction
mixture was filtered through a pad of celite. The filtrate was
concentrated under reduced pressure, and the residue was
extracted with toluene (1.0 L) twice. The combined organic layers
were dried over anhydrous Na2SO4 and concentrated under
reduced pressure to obtain 11 (193 g, 99%) as a brown oil, which
was used directly for the next reaction without further purification.
1H-NMR (400 MHz, CDCl3) δ 1.38 (9H, br), 3.15 (3H, s), 3.49
(2H, br), 3.82 (3H, s), 4.91 (2H, s), 6.70 (1H, d, J = 8.6 Hz), 6.68–
6.71 (1H, m), 6.76 (1H, dd, J = 2.4, 8.6 Hz), 6.91 (2H, d, J = 8.6
Hz), 7.34 (2H, d, J = 8.6 Hz).
4.10.55. Methyl 3-({6-[(5-chloropyridin-2-yl)oxy]-
1-methyl-1H-benzimidazol-2-yl}methoxy)benzoate
(14c).
Compound 14c was prepared according to general procedure H
using 2-bromo-5-chloropyridine (yield, 4.2%. 1H-NMR (400 MHz,
CDCl3) δ 3.85 (3H, s), 3.92 (3H, s), 5.42 (2H, s), 6.91 (1H, d, J =
8.6 Hz), 7.05 (1H, dd, J = 2.4, 9.0 Hz), 7.16 (1H, d, J = 2.4 Hz),
7.29 (1H, ddd, J = 1.2, 2.7, 8.2 Hz), 7.37 (1H, t, J = 8.2 Hz), 7.66
(1H, dd, J = 2.7, 8.6 Hz), 7.69 (1H, dt, J = 1.2, 7.4 Hz), 7.72 (1H,
dd, J = 1.2, 2.7 Hz), 7.79 (1H, d, J = 8.6 Hz), 8.11 (1H, d, J = 2.4
Hz).
4.10.56. Methyl 3-({6-[(4-chloropyridin-2-yl)oxy]-
1-methyl-1H-benzimidazol-2-yl}methoxy)benzoate
(14d).
4.10.51. Methyl 3-[2-({2-[(tert-
butoxycarbonyl)(methyl)amino]-4-[(4-
methoxybenzyl)oxy]phenyl}amino)-2-
oxoethoxy]benzoate (12).
Compound 14d was prepared according to general procedure G
using 4-chloro-2-fluoropyridine (yield, 33%). 1H-NMR (400 MHz,
CDCl3) δ 3.86 (3H, s), 3.93 (3H, s), 5.42 (2H, s), 6.95 (1H, d, J =
2.4 Hz), 7.00 (1H, dd, J = 2.0, 5.5 Hz), 7.06 (1H, dd, J = 2.4, 8.6
Hz), 7.17 (1H, d, J = 2.4 Hz), 7.29 (1H, ddd, J = 1.2, 2.7, 8.6 Hz),
7.38 (1H, t, J = 7.8 Hz), 7.69 (1H, dt, J = 1.2, 7.8 Hz), 7.72 (1H,
dd, J = 1.6, 2.7 Hz), 7.79 (1H, d, J = 8.6 Hz), 8.08 (1H, d, J = 5.5
Hz).
A solution of [3-(Methoxycarbonyl)phenoxy]acetic acid8 (121
g, 577 mmol), 11 (193 g, 538 mmol), HOBt•H2O (77.7 g, 575
mmol) and WSC•HCl (110 g, 575 mmol) in CH2Cl2 (1.0 L) was
stirred at room temperature for 3 h under N2. Water (1.0 L) was
added to the reaction mixture, and the precipitated solid was
removed by filtration. The separated organic layer was dried over
anhydrous Na2SO4, and concentrated under reduced pressure. The
residue was recrystallized from a solvent mixture of i-Pr2O/EtOAc
4.10.57. Methyl 3-({6-[(3-chloropyridin-2-yl)oxy]-
1-methyl-1H-benzimidazol-2-yl}methoxy)benzoate
(14e).
1
(20:1) to obtain 12 (296 g, 99%) as a white solid. H-NMR (400
MHz, CDCl3) δ 1.43 (9H, br), 3.12 (3H, s), 3.83 (3H, s), 3.93 (3H,
s), 4.67 (2H, s), 4.98 (2H, s), 6.80 (1H, br), 6.93 (4H, d, J = 8.6
Hz), 7.21–7.27 (2H, m), 7.35 (2H, d, J = 8.6 Hz), 7.42 (1H, t, J =
8.2 Hz), 7.64 (1H, s), 7.75 (1H, d, J = 8.2 Hz).
Compound 14e was prepared according to general procedure G
using 3-chloro-2-fluoropyridine (yield, 58%). 1H-NMR (400 MHz,
CDCl3) δ 3.87 (3H, s), 3.93 (3H, s), 5.42 (2H, s), 6.98 (1H, dd, J =
4.7, 7.4 Hz), 7.10 (1H, dd, J = 2.4, 9.0 Hz), 7.22 (1H, d, J = 2.0
Hz), 7.30 (1H, dd, J = 1.0, 8.6 Hz), 7.38 (1H, t, J = 7.8 Hz), 7.70
(1H, d, J = 7.4 Hz), 7.72–7.73 (1H, m), 7.79 (1H, dd, J = 1.6, 7.4
Hz), 7.80 (1H, d, J = 8.6 Hz), 8.02 (1H, dd, J = 1.6, 4.7 Hz).
4.10.52. Methyl 3-[(6-hydroxy-1-methyl-1H-
benzimidazol-2-yl)methoxy]benzoate (13).
A solution of 12 (296 g, 538 mmol) in 2 M HCl in 1,4-dioxane
(1.0 L) was stirred at 60 °C for 2 h. After cooling the reaction
mixture, the precipitated solid was collected by filtration to obtain
13 as an HCl salt. An aqueous solution (1.0 L) of imidazole (59.0
g, 867 mmol) was added to a suspension of the HCl salt of 13 in
EtOAc (1.0 L). The precipitated solid was collected by filtration to
4.10.58. Methyl 3-({1-methyl-6-[(6-methylpyridin-
2-yl)oxy]-1H-benzimidazol-2-yl}methoxy)benzoate
(14f).
Compound 14f was prepared according to general procedure G
using 2-bromo-6-methylpyridine (yield, 18%). 1H-NMR (400
MHz, CDCl3) δ 2.46 (3H, s), 3.85 (3H, s), 3.93 (3H, s), 5.41 (2H,
s), 6.56 (1H, d, J = 8.2 Hz), 6.87 (1H, d, J = 7.4 Hz), 7.08 (1H, dd,
J = 2.0, 8.6 Hz), 7.16 (1H, d, J = 2.4 Hz), 7.30 (1H, dd, J = 3.1,
7.4 Hz), 7.38 (1H, t, J = 7.4 Hz), 7.53 (1H, t, J = 7.4 Hz), 7.69 (1H,
dt, J = 1.6, 9.0 Hz), 7.73 (1H, dd, J = 1.6, 3.1 Hz), 7.76 (1H, d, J
= 8.6 Hz).
1
obtain 13 (113 g, 67%). H-NMR (400 MHz, DMSO-d6) δ 3.74
(3H, s), 3.85 (3H, s), 5.40 (2H, s), 6.71 (1H, dd, J = 2.4, 8.6 Hz),
6.83 (1H, d, J = 2.0 Hz), 7.40–7.43 (2H, m), 7.47 (1H, t, J = 7.4
Hz), 7.58 (1H, dt, J = 1.6, 7.8 Hz), 7.63–7.64 (1H, m), 9.36 (1H,
s).
4.10.53. Methyl 3-{[1-methyl-6-(pyridin-2-yloxy)-
1H-benzimidazol-2-yl]methoxy}benzoate (14a).
4.10.59. Methyl 3-({1-methyl-6-[(5-methylpyridin-
2-yl)oxy]-1H-benzimidazol-2-yl}methoxy)benzoate
(14g).
Compound 14a was prepared according to general procedure G
using 2-bromopyridine (yield, 14%). 1H-NMR (400 MHz, CDCl3)
δ 3.85 (3H, s), 3.93 (3H, s), 5.42 (2H, s), 6.94 (1H, d, J = 8.6 Hz),
6.98–7.01 (2H, m), 7.08 (1H, dd, J = 2.4, 8.6 Hz), 7.18 (1H, d, J =
2.0 Hz), 7.31 (1H, d, J = 3.1 Hz), 7.37 (1H, t, J = 7.8 Hz), 7.69
(1H, dt, J = 2.0, 8.2 Hz), 7.72–7.73 (1H, m), 7.78 (1H, d, J = 9.0
Hz), 8.19 (1H, ddd, J = 0.8, 2.0, 5.1 Hz).
Compound 14g was prepared according to general procedure G
using 2-bromo-5-methylpyridine (yield, 18%). 1H-NMR (400
MHz, CDCl3) δ 2.28 (3H, s), 3.84 (3H, s), 3.92 (3H, s), 5.41 (2H,
s), 6.84 (1H, d, J = 8.6 Hz), 7.05 (1H, dd, J = 2.0, 8.6 Hz), 7.15
(1H, d, J = 2.0 Hz), 7.29 (1H, ddd, J = 0.8, 2.4, 9.0 Hz), 7.37 (1H,
t, J = 7.8 Hz), 7.51 (1H, dd, J = 3.1, 9.0 Hz), 7.68 (1H, dt, J = 1.2,
7.8 Hz), 7.72 (1H, dd, J = 1.6, 2.7 Hz), 7.76 (1H, d, J = 8.6 Hz),
8.00 (1H, dd, J = 0.8, 1.6 Hz).
4.10.54. Methyl 3-({6-[(6-chloropyridin-2-yl)oxy]-
1-methyl-1H-benzimidazol-2-yl}methoxy)benzoate
(14b).
4.10.60. Methyl 3-({1-methyl-6-[(4-methylpyridin-
2-yl)oxy]-1H-benzimidazol-2-yl}methoxy)benzoate
(14h).
Compound 14b was prepared according to general procedure G
using 2-chloro-6-fluoropyridine (yield, 49%). 1H-NMR (400 MHz,
CDCl3) δ 3.86 (3H, s), 3.93 (3H, s), 5.42 (2H, s), 6.75 (1H, dd, J =
8.2 Hz), 7.04 (1H, dd, J = 0.8, 8.2 Hz), 7.08 (1H, dd, J = 2.4, 8.6
Hz), 7.18 (1H, d, J = 2.4 Hz), 7.30 (1H, ddd, J = 1.2, 2.7, 8.2 Hz),
7.38 (1H, t, J = 7.8 Hz), 7.62 (1H, t, J = 7.4 Hz), 7.69 (1H, dt, J =
Compound 14h was prepared according to general procedure G
using 2-bromo-4-methylpyridine (yield, 15%). 1H-NMR (400
MHz, CDCl3) δ 2.35 (3H, s), 3.85 (3H, s), 3.92 (3H, s), 5.41 (2H,

s), 6.74 (1H, s), 6.82 (1H, d, J = 5.5 Hz), 7.06 (1H, dd, J = 2.4, 8.6
Hz), 7.15 (1H, d, J = 2.4 Hz), 7.30 (1H, ddd, J = 0.8, 2.7, 8.2 Hz),
7.37 (1H, t, J = 7.8 Hz), 7.68 (1H, dt, J = 1.2, 7.8 Hz), 7.72 (1H,
dd, J = 1.2, 2.7 Hz), 7.77 (1H, d, J = 8.6 Hz), 8.04 (1H, d, J = 5.1
Hz).
br), 7.23 (1H, br), 7.31 (1H, d, J = 8.3 Hz), 7.38 (1H, t, J = 7.8 Hz),
7.67 (1H, dd, J = 2.0, 8.8 Hz), 7.70 (1H, d, J = 7.8 Hz), 7.73 (1H,
s), 7.80 (1H, br), 7.96 (1H, d, J = 2.0 Hz).
4.10.67. Methyl 3-({6-[(3-bromo-5-fluoropyridin-2-
yl)oxy]-1-methyl-1H-benzimidazol-2-
yl}methoxy)benzoate (14o).
4.10.61. Methyl 3-({1-methyl-6-[(3-methylpyridin-
2-yl)oxy]-1H-benzimidazol-2-yl}methoxy)benzoate
(14i).
Compound 14o was prepared according to general procedure G
1
using 3-bromo-2,5-difluoropyridine (yield, 54%). H-NMR (500
Compound 14i was prepared according to general procedure G
using 2-bromo-3-methylpyridine (yield, 11%). 1H-NMR (400
MHz, CDCl3) δ 2.40 (3H, s), 3.84 (3H, s), 3.92 (3H, s), 5.41 (2H,
s), 6.91 (1H, dd, J = 4.7, 7.0 Hz), 7.05 (1H, dd, J = 2.4, 8.6 Hz),
7.16 (1H, d, J = 2.4 Hz), 7.30 (1H, ddd, J = 1.2, 2.4, 9.0 Hz), 7.37
(1H, t, J = 7.8 Hz), 7.54 (1H, ddd, J = 0.8, 2.0, 7.0 Hz), 7.68 (1H,
dt, J = 1.2, 7.8 Hz), 7.72 (1H, dd, J = 1.6, 2.7 Hz), 7.77 (1H, d, J
= 8.6 Hz), 7.97 (1H, dd, J = 2.0, 4.3 Hz).
MHz, DMSO-d6) δ 3.83 (3H, s), 3.85 (3H, s), 5.50 (2H, s), 7.00
(1H, dd, J = 2.4, 8.6 Hz), 7.43 (1H, ddd, J = 1.2, 2.4, 8.2 Hz), 7.45–
7.50 (2H, m), 7.59 (1H, dt, J = 1.2, 7.8 Hz), 7.65–7.67 (2H, m),
8.13 (1H, d, J = 2.7 Hz), 8.38 (1H, dd, J = 2.7, 7.4 Hz).
4.10.68. Methyl 3-({6-[(3,5-dichloropyridin-2-
yl)oxy]-1-methyl-1H-benzimidazol-2-
yl}methoxy)benzoate (14p).
Compound 14p was prepared according to general procedure G
1
4.10.62. Methyl 3-({6-[(5-fluoropyridin-2-yl)oxy]-
1-methyl-1H-benzimidazol-2-yl}methoxy)benzoate
(14j).
using 3,5-dichloro-2-fluoropyridine (yield, 53%). H-NMR (400
MHz, CDCl3) δ 3.87 (3H, s), 3.93 (3H, s), 5.42 (2H, s), 7.08 (1H,
dd, J = 2.0, 8.6 Hz), 7.20 (1H, s), 7.29–7.31 (1H, m), 7.38 (1H, t,
J = 8.2 Hz), 7.70 (1H, d, J = 7.4 Hz), 7.73 (1H, s), 7.80 (1H, d, J
= 2.4 Hz), 7.82 (1H, br), 7.96 (1H, d, J = 2.4 Hz).
Compound 14j was prepared according to general procedure G
using 2-bromo-5-fluoropyridine (yield, 4.5%). 1H-NMR (400
MHz, CDCl3) δ 3.85 (3H, s), 3.92 (3H, s), 5.42 (2H, s), 6.94 (1H,
dd, J = 3.5, 9.0 Hz), 7.05 (1H, dd, J = 2.4, 9.0 Hz), 7.15 (1H, d, J
= 2.4 Hz), 7.30 (1H, dd, J = 1.2, 8.2 Hz), 7.37 (1H, t, J = 7.8 Hz),
7.43-7.47 (1H, m), 7.69 (1H, dt, J = 0.8, 7.8 Hz), 7.72 (1H, t, J =
1.6 Hz), 7.78 (1H, d, J = 8.6 Hz), 8.02 (1H, d, J = 3.1 Hz).
4.10.69. Methyl 3-({6-[(5-chloro-3-fluoropyridin-2-
yl)oxy]-1-methyl-1H-benzimidazol-2-
yl}methoxy)benzoate (14q).
Compound 14q was prepared according to general procedure G
1
using 5-chloro-2,3-difluoropyridine (yield, 70%). H-NMR (500
4.10.63. Methyl 3-({6-[(3-fluoropyridin-2-yl)oxy]-
1-methyl-1H-benzimidazol-2-yl}methoxy)benzoate
(14k).
MHz, CDCl3) δ 3.87 (3H, s), 3.93 (3H, s), 5.42 (2H, s), 7.09 (1H,
dd, J = 2.4, 8.8 Hz), 7.21 (1H, d, J = 2.0 Hz), 7.30 (1H, ddd, J =
1.0, 2.4, 8.3 Hz), 7.38 (1H, t, J = 8.3 Hz), 7.54 (1H, dd, J = 2.0,
8.8 Hz), 7.70 (1H, dt, J = 1.0, 7.8 Hz), 7.73 (1H, dd, J = 1.5, 2.4
Hz), 7.80 (1H, d, J = 8.8 Hz), 7.88 (1H, d, J = 2.0 Hz).
Compound 14k was prepared according to general procedure G
using 2,3-difluoropyridine (yield, 28%). 1H-NMR (400 MHz,
CDCl3) δ 3.86 (3H, s), 3.93 (3H, s), 5.42 (2H, s), 7.00 (1H, ddd, J
= 3.1, 5.1, 8.2 Hz), 7.11 (1H, dd, J = 2.4, 8.6 Hz), 7.22 (1H, d, J =
2.4 Hz), 7.30 (1H, ddd, J = 1.2, 2.7, 8.2 Hz), 7.38 (1H, t, J = 7.2
Hz), 7.50 (1H, ddd, J = 1.6, 7.8, 9.8 Hz), 7.69 (1H, dt, J = 1.2, 7.8
Hz), 7.72 (1H, dd, J = 1.6, 2.4 Hz), 7.80 (1H, d, J = 8.6 Hz), 7.91
(1H, dd, J = 1.6, 5.1 Hz).
4.10.70. Methyl 3-({6-[(3-bromo-5-chloropyridin-2-
yl)oxy]-1-methyl-1H-benzimidazol-2-
yl}methoxy)benzoate (14r).
Compound 14r was prepared according to general procedure G
1
using 3-bromo-5-chloro-2-fluoropyridine (yield, 91%). H-NMR
(400 MHz, CDCl3) δ 3.83 (3H, s), 3.89 (3H, s), 5.38 (2H, s), 6.96–
7.01 (1H, m), 7.09–7.11 (1H, m), 7.23–7.27 (1H, m), 7.30–7.36
(1H, m), 7.48–7.50 (1H, m), 7.62–7.66 (1H, m), 7.67–7.70 (1H,
m), 7.72–7.75 (1H, m), 7.85–7.87 (1H, m).
4.10.64. Methyl 3-({6-[(5-bromo-6-methylpyridin-2-
yl)oxy]-1-methyl-1H-benzimidazol-2-
yl}methoxy)benzoate (14l).
Compound 14l was prepared according to general procedure G
using 3-bromo-6-fluoro-2-methylpyridine (yield, 11%). 1H-NMR
(400 MHz, CDCl3) δ 2.54 (3H, s), 3.86 (3H, s), 3.93 (3H, s), 5.42
(2H, s), 6.53 (1H, d, J = 8.6 Hz), 7.07 (1H, dd, J = 2.4, 8.6 Hz),
7.15 (1H, d, J = 2.0 Hz), 7.31 (1H, ddd, J = 1.2, 2.7, 8.2 Hz), 7.39
(1H, t, J = 7.4 Hz), 7.69–7.73 (2H, m), 7.77 (1H, d, J = 8.6 Hz).
4.10.71. Methyl 3-({6-[(5,6-dimethylpyridin-2-
yl)oxy]-1-methyl-1H-benzimidazol-2-
yl}methoxy)benzoate (15l).
Compound 15l was prepared according to general procedure I
(yield, 52%). 1H-NMR (400 MHz, CDCl3) δ 2.24 (3H, s), 2.42 (3H,
s), 3.85 (3H, s), 3.93 (3H, s), 5.41 (2H, s), 6.51 (1H, d, J = 8.2 Hz),
7.07 (1H, dd, J = 2.0, 8.6 Hz), 7.13 (1H, d, J = 2.0 Hz), 7.29 (1H,
ddd, J = 1.2, 3.1, 8.6 Hz), 7.38 (1H, d, J = 7.8 Hz), 7.39 (1H, t, J
= 7.8 Hz), 7.69–7.76 (3H, m).
4.10.65. Methyl 3-({6-[(3-bromo-6-chloropyridin-2-
yl)oxy]-1-methyl-1H-benzimidazol-2-
yl}methoxy)benzoate (14m ).
Compound 14m was prepared according to general procedure
G using 3-bromo-6-chloro-2-fluoropyridine (yield, 62%). 1H-
NMR (400 MHz, DMSO-d6) δ 3.85 (3H, s), 3.86 (3H, s), 5.50 (2H,
s), 7.05 (1H, dd, J = 2.4, 8.6 Hz), 7.21 (1H, d, J = 7.8 Hz), 7.44
(1H, ddd, J = 1.2, 2.7, 8.2 Hz), 7.49 (1H, t, J = 7.4 Hz), 7.54 (1H,
d, J = 2.4 Hz), 7.60 (1H, dt, J = 1.6, 9.0 Hz), 7.67 (1H, dd, J = 1.6,
2.4 Hz), 7.70 (1H, d, J = 9.0 Hz), 8.25 (1H, d, J = 8.2 Hz).
4.10.72. Methyl 3-({6-[(3,6-dimethylpyridin-2-
yl)oxy]-1-methyl-1H-benzimidazol-2-
yl}methoxy)benzoate (15m ).
Compound 15m was prepared according to general procedure I
(yield, 80%). 1H-NMR (400 MHz, CDCl3) δ 2.30 (3H, s), 2.33 (3H,
s), 3.83 (3H, s), 3.92 (3H, s), 5.40 (2H, s), 6.81 (1H, d, J = 7.4 Hz),
7.01 (1H, dd, J = 2.4, 8.6 Hz), 7.10 (1H, d, J = 2.0 Hz), 7.28–7.30
(1H, m), 7.37 (1H, d, J = 7.8 Hz), 7.44 (1H, d, J = 8.2 Hz), 7.68
(1H, dt, J = 1.6, 7.8 Hz), 7.70–7.73 (2H, m).
4.10.66. Methyl 3-({6-[(5-bromo-3-fluoropyridin-2-
yl)oxy]-1-methyl-1H-benzimidazol-2-
yl}methoxy)benzoate (14n).
4.10.73. Methyl 3-({6-[(3-fluoro-5-methylpyridin-2-
yl)oxy]-1-methyl-1H-benzimidazol-2-
yl}methoxy)benzoate (15n).
Compound 14n was prepared according to general procedure G
using 5-bromo-2,3-difluoropyridine (yield, 66%). H-NMR (500
MHz, CDCl3) δ 3.87 (3H, s), 3.93 (3H, s), 5.41 (2H, br), 7.10 (1H,
1

Compound 15n was prepared according to general procedure I
(yield, 56%). 1H-NMR (500 MHz, CDCl3) δ 2.31 (3H, s), 3.86 (3H,
s), 3.93 (3H, s), 5.42 (2H, s), 7.09 (1H, dd, J = 2.0, 8.8 Hz), 7.19
(1H, d, J = 2.0 Hz), 7.30 (1H, ddd, J = 1.0, 2.9, 8.3 Hz), 7.32–7.35
(1H, m), 7.38 (1H, t, J = 7.8 Hz), 7.69 (1H, dt, J = 1.5, 7.3 Hz),
7.72–7.73 (2H, m), 7.78 (1H, d, J = 8.8 Hz).
Compound 16d was prepared according to general procedure F
(yield, 94%). Mp, 246–252 °C. 1H-NMR (400 MHz, DMSO-d6) δ
3.84 (3H, s), 5.49 (2H, s), 7.02 (1H, dd, J = 2.4, 8.6 Hz), 7.20 (1H,
d, J = 1.6 Hz), 7.26 (1H, dd, J = 1.6, 5.5 Hz), 7.39 (1H, ddd, J =
0.8, 2.4, 7.8 Hz), 7.45 (1H, t, J = 7.4 Hz), 7.47 (1H, s), 7.58 (1H,
dt, J = 1.2, 7.4 Hz), 7.64 (1H, dd, J = 1.2, 2.4 Hz), 7.68 (1H, d, J
= 9.0 Hz), 8.12 (1H, d, J = 5.5 Hz), 13.05 (1H, br); Anal. calcd for
C21H16ClN3O4: C, 61.54; H, 3.94; N, 10.25; Cl, 8.65; found C,
61.44; H, 3.92; N, 9.97; Cl, 8.24.
4.10.74. Methyl 3-({6-[(5-fluoro-3-methylpyridin-2-
yl)oxy]-1-methyl-1H-benzimidazol-2-
yl}methoxy)benzoate (15o).
4.10.80. 3-({6-[(3-Chloropyridin-2-yl)oxy]-1-
methyl-1H-benzimidazol-2-yl}methoxy)benzoic acid
(16e).
Compound 15o was prepared according to general procedure I
(yield, 60%). 1H-NMR (500 MHz, CDCl3) δ 2.41 (3H, s), 3.85 (3H,
s), 3.93 (3H, s), 5.41 (2H, s), 7.03 (1H, dd, J = 1.5, 8.8 Hz), 7.13
(1H, d, J = 2.0 Hz), 7.30 (1H, dd, J = 2.9, 8.3 Hz), 7.34 (1H, dd, J
= 1.5, 7.3 Hz), 7.37 (1H, t, J = 8.3 Hz), 7.69 (1H, d, J = 7.3 Hz),
7.73 (1H, s), 7.77 (1H, d, J = 8.8 Hz), 7.81 (1H, d, J = 2.9 Hz).
Compound 16e was prepared according to general procedure F
(yield, 67%). Mp, 264–266 °C. 1H-NMR (400 MHz, DMSO-d6) δ
3.83 (3H, s), 5.49 (2H, s), 7.01 (1H, dd, J = 2.4, 9.0 Hz), 7.15 (1H,
dd, J = 5.1, 7.8 Hz), 7.37–7.40 (1H, m), 7.45 (1H, t, J = 7.4 Hz),
7.49 (1H, d, J = 2.4 Hz), 7.57 (1H, d, J = 7.8 Hz), 7.63–7.64 (1H,
m), 7.67 (1H, d, J = 8.6 Hz), 8.03 (1H, dd, J = 2.0, 5.1 Hz), 8.06
(1H, dd, J = 1.6, 7.8 Hz), 13.06 (1H, br); Anal. calcd for
C21H16ClN3O4: C, 61.54; H, 3.94; N, 10.25; Cl, 8.65; found C,
61.40; H, 3.86; N, 10.17; Cl, 8.61.
4.10.75. Methyl 3-({6-[(5-chloro-3-methylpyridin-
2-yl)oxy]-1-methyl-1H-benzimidazol-2-
yl}methoxy)benzoate (15r).
Compound 15r was prepared according to general procedure I
(yield, 75%). 1H-NMR (400 MHz, CDCl3) δ 2.41 (3H, s), 3.82 (3H,
s), 3.89 (3H, s), 5.38 (2H, s), 6.98–7.00 (1H, m), 7.10–7.10 (1H,
m), 7.22–7.27 (1H, m), 7.31–7.37 (1H, m), 7.49–7.50 (1H, m),
7.64–7.66 (1H, m), 7.64–7.69 (1H, m), 7.74 (1H, d, J = 8.6 Hz),
7.84–7.87 (1H, m).
4.10.81. 3-({1-Methyl-6-[(6-methylpyridin-2-
yl)oxy]-1H-benzimidazol-2-yl}methoxy)benzoic acid
(16f).
Compound 16f was prepared according to general procedure F
(yield, 54%). 1H-NMR (500 MHz, DMSO-d6) δ 2.30 (3H, s), 3.83
(3H, s), 5.48 (2H, s), 6.72 (1H, d, J = 7.8 Hz), 6.96–6.98 (2H, m),
7.38–7.41 (2H, m), 7.46 (1H, t, J = 7.8 Hz), 7.58 (1H, dd, J = 1.0,
7.3 Hz), 7.65 (1H, s), 7.66 (1H, d, J = 8.3 Hz), 7.70 (1H, t, J = 7.8
Hz), 13.03 (1H, br); MS (FAB) m/z: 390 [M + H]+; Anal. calcd for
C22H19N3O4: C, 67.86; H, 4.92; N, 10.79; found C, 67.58; H, 4.82;
N, 10.67.
4.10.76. 3-{[1-Methyl-6-(pyridin-2-yloxy)-1H-
benzimidazol-2-yl]methoxy}benzoic acid (16a).
Compound 16a was prepared according to general procedure F
(yield, 48%). 1H-NMR (500 MHz, DMSO-d6) δ 3.83 (3H, s), 5.48
(2H, s), 6.99 (2H, t, J = 8.8 Hz), 7.11 (1H, dd, J = 5.9, 6.8 Hz),
7.39 (1H, d, J = 8.3 Hz), 7.43 (1H, s), 7.45 (1H, t, J = 7.3 Hz), 7.58
(1H, d, J = 7.8 Hz), 7.64 (1H, s), 7.66 (1H, d, J = 8.8 Hz), 7.83
(1H, t, J = 6.4 Hz), 8.14 (1H, d, J = 6.4 Hz), 13.02 (1H, br); MS
(FAB) m/z: 376 [M + H]+; Anal. calcd for C21H17N3O4•0.67H2O:
C, 65.11; H, 4.77; N, 10.85; found C, 65.26; H, 4.47; N, 10.71.
4.10.82. 3-({1-Methyl-6-[(5-methylpyridin-2-
yl)oxy]-1H-benzimidazol-2-yl}methoxy)benzoic acid
(16g).
4.10.77. 3-({6-[(6-Chloropyridin-2-yl)oxy]-1-
methyl-1H-benzimidazol-2-yl}methoxy)benzoic acid
(16b).
Compound 16g was prepared according to general procedure F
(yield, 73%). 1H-NMR (500 MHz, DMSO-d6) δ 2.24 (3H, s), 3.82
(3H, s), 5.48 (2H, s), 6.91 (1H, d, J = 8.3 Hz), 6.96 (1H, dd, J =
2.0, 8.3 Hz), 7.38–7.40 (2H, m), 7.45 (1H, t, J = 7.8 Hz), 7.58 (1H,
d, J = 7.3 Hz), 7.63–7.67 (3H, m), 7.95 (1H, s), 13.02 (1H, br); MS
(FAB) m/z: 390 [M + H]+; Anal. calcd for C22H19N3O4•0.25H2O:
C, 67.08; H, 4.99; N, 10.67; found C, 67.26; H, 4.87; N, 10.45.
Compound 16b was prepared according to general procedure F
(yield, 99%). 1H-NMR (400 MHz, DMSO-d6) δ 3.85 (3H, s), 5.49
(2H, s), 6.98 (1H, d, J = 7.4 Hz), 7.03 (1H, dd, J = 2.0, 8.6 Hz),
7.23 (1H, d, J = 7.4 Hz), 7.40 (1H, ddd, J = 1.2, 2.7, 8.6 Hz), 7.46
(1H, t, J = 7.8 Hz), 7.51 (1H, d, J = 2.0 Hz), 7.58 (1H, dt, J = 1.2,
7.4 Hz), 7.65 (1H, dd, J = 1.2, 2.7 Hz), 7.70 (1H, d, J = 9.0 Hz),
7.89 (1H, t, J = 7.8 Hz), 13.07 (1H, br); Anal. calcd for
C21H16ClN3O4•0.50H2O: C, 60.22; H, 4.09; N, 10.03; Cl, 8.46;
found C, 60.26; H, 3.91; N, 10.02; Cl, 8.67.
4.10.83. 3-({1-Methyl-6-[(4-methylpyridin-2-
yl)oxy]-1H-benzimidazol-2-yl}methoxy)benzoic acid
(16h).
Compound 16h was prepared according to general procedure F
(yield, 64%). 1H-NMR (500 MHz, DMSO-d6) δ 2.31 (3H, s), 3.83
(3H, s), 5.48 (2H, s), 6.81 (1H, s), 6.95 (1H, d, J = 6.4 Hz), 6.97
(1H, dd, J = 2.0, 8.8 Hz), 7.38–7.40 (2H, m), 7.45 (1H, t, J = 7.3
Hz), 7.58 (1H, d, J = 7.3 Hz), 7.64–7.66 (2H, m), 7.99 (1H, d, J =
5.4 Hz), 13.03 (1H, br); MS (FAB) m/z: 390 [M + H]+; Anal. calcd
for C21H16ClN3O4•0.25H2O: C, 67.08; H, 4.99; N, 10.67; found C,
67.33; H, 4.93; N, 10.40.
4.10.78. 3-({6-[(5-Chloropyridin-2-yl)oxy]-1-
methyl-1H-benzimidazol-2-yl}methoxy)benzoic acid
(16c).
Compound 16c was prepared according to general procedure F
(yield, 41%). Mp, 251–253 °C. 1H-NMR (400 MHz, DMSO-d6) δ
3.83 (3H, s), 5.48 (2H, s), 7.01 (1H, dd, J = 2.0, 8.6 Hz), 7.08 (1H,
d, J = 8.2 Hz), 7.39 (1H, ddd, J = 1.2, 2.7, 8.2 Hz), 7.43–7.47 (2H,
m), 7.58 (1H, dt, J = 1.2, 7.4 Hz), 7.64 (1H, dd, J = 1.6, 2.7 Hz),
7.67 (1H, d, J = 8.6 Hz), 7.95 (1H, dd, J = 2.7, 9.0 Hz), 8.19 (1H,
4.10.84. 3-({1-Methyl-6-[(3-methylpyridin-2-
yl)oxy]-1H-benzimidazol-2-yl}methoxy)benzoic acid
(16i).
d,
J
=
2.7 Hz), 13.03 (1H, br); Anal. calcd for
Compound 16i was prepared according to general procedure F
(yield, 98%). 1H-NMR (400 MHz, DMSO-d6) δ 2.35 (3H, s), 3.83
(3H, s), 5.48 (2H, s), 6.97 (1H, ddd, J = 0.8, 2.4, 8.6 Hz), 7.02 (1H,
dd, J = 5.1, 7.4 Hz), 7.38–7.41 (2H, m), 7.45 (1H, t, J = 7.8 Hz),
7.58 (1H, d, J = 7.4 Hz), 7.63–7.65 (2H, m), 7.71 (1H, ddd, J = 0.8,
2.0, 7.4 Hz), 7.89 (1H, dd, J = 2.0, 4.7 Hz), 13.06 (1H, br); MS
C21H16ClN3O4•0.20H2O: C, 61.01; H, 4.00; N, 10.16; Cl, 8.58;
found C, 61.22; H, 3.82; N, 10.16; Cl, 8.78.
4.10.79. 3-({6-[(4-Chloropyridin-2-yl)oxy]-1-
methyl-1H-benzimidazol-2-yl}methoxy)benzoic acid
(16d).

(FAB) m/z: 390 [M + H]+; Anal. calcd for C22H19N3O4: C, 67.86;
H, 4.92; N, 10.79; found C, 67.86; H, 5.10; N, 10.42.
Compound 16o was prepared according to general procedure F
(yield, 95%). Mp, 255–264 °C. 1H-NMR (400 MHz, DMSO-d6) δ
2.35 (3H, s), 3.81 (3H, s), 5.47 (2H, s), 6.95 (1H, dd, J = 2.4, 8.6
Hz), 7.36–7.38 (2H, m), 7.44 (1H, t, J = 7.4 Hz), 7.57 (1H, d, J =
7.4 Hz), 7.62–7.64 (2H, m), 7.75 (1H, dd, J = 2.7, 8.6 Hz), 7.89
(1H, dd, J = 0.8, 2.7 Hz), 13.04 (1H, br); Anal. calcd for
C22H18FN3O4•0.50H2O: C, 63.46; H, 4.60; N, 10.09; found C,
63.74; H, 4.26; N, 10.26.
4.10.85. 3-({6-[(5-Fluoropyridin-2-yl)oxy]-1-
methyl-1H-benzimidazol-2-yl}methoxy)benzoic acid
(16j).
Compound 16j was prepared according to general procedure F
(yield, 38%). 1H-NMR (400 MHz, DMSO-d6) δ 3.83 (3H, s), 5.48
(2H, s), 6.99 (1H, dd, J = 2.4, 8.6 Hz), 7.09 (1H, dd, J = 3.5, 9.0
Hz), 7.37–7.40 (1H, m), 7.43–7.43 (1H, m), 7.45 (1H, t, J = 7.4
Hz), 7.58 (1H, dd, J = 1.2, 7.4 Hz), 7.64 (1H, dd, J = 1.2, 2.0 Hz),
7.65 (1H, t, J = 8.6 Hz), 7.78–7.83 (1H, m), 8.13 (1H, d, J = 3.1
Hz), 13.02 (1H, br); Anal. calcd for C21H16ClN3O4•0.20H2O: C,
61.01; H, 4.00; N, 10.16; Cl, 8.58; found C, 61.22; H, 3.82; N,
10.16; Cl, 8.78.
4.10.91. 3-({6-[(3,5-Dichloropyridin-2-yl)oxy]-1-
methyl-1H-benzimidazol-2-yl}methoxy)benzoic acid
(16p).
Compound 16p was prepared according to general procedure F
(yield, 93%). 1H-NMR (400 MHz, DMSO-d6) δ 3.83 (3H, s), 5.49
(2H, s), 7.04 (1H, dd, J = 2.4, 9.0 Hz), 7.39 (1H, dd, J = 2.4, 8.2
Hz), 7.45 (1H, t, J = 7.8 Hz), 7.51 (1H, d, J = 2.4 Hz), 7.57 (1H, d,
J = 7.4 Hz), 7.64 (1H, s), 7.68 (1H, d, J = 9.0 Hz), 8.12 (1H, dd, J
= 0.8, 2.4 Hz), 8.36 (1H, d, J = 2.4 Hz), 13.04 (1H, br); Anal. calcd
for C21H15Cl2N3O4•0.25H2O: C, 56.20; H, 3.48; N, 9.36; found C,
56.20; H, 3.30; N, 9.53.
4.10.86. 3-({6-[(3-Fluoropyridin-2-yl)oxy]-1-
methyl-1H-benzimidazol-2-yl}methoxy)benzoic acid
(16k).
Compound 16k was prepared according to general procedure F
(yield, 99%). 1H-NMR (400 MHz, DMSO-d6) δ 3.84 (3H, s), 5.49
(2H, s), 7.04 (1H, dd, J = 2.4, 9.0 Hz), 7.18 (1H, ddd, J = 3.1, 4.7,
8.2 Hz), 7.39 (1H, ddd, J = 1.2, 2.7, 8.2 Hz), 7.45 (1H, t, J = 7.4
Hz), 7.50 (1H, d, J = 2.4 Hz), 7.58 (1H, dt, J = 1.2, 7.4 Hz), 7.64
(1H, dd, J = 1.2, 1.6 Hz), 7.67 (1H, d, J = 8.6 Hz), 7.87 (1H, ddd,
J = 1.2, 7.8, 10.6 Hz), 7.91 (1H, dd, J = 1.2, 5.1 Hz), 13.05 (1H,
br); Anal. calcd for C21H16FN3O4: C, 64.12; H, 4.10; N, 10.68;
found C, 63.95; H, 4.12; N, 10.49.
4.10.92. 3-({6-[(5-Chloro-3-fluoropyridin-2-
yl)oxy]-1-methyl-1H-benzimidazol-2-
yl}methoxy)benzoic acid (16q).
Compound 16q was prepared according to general procedure F
(yield, 94%). Mp, 245–262 °C. 1H-NMR (400 MHz, DMSO-d6) δ
3.83 (3H, s), 5.49 (2H, s), 7.06 (1H, ddd, J = 1.2, 2.4, 8.6 Hz),
7.37–7.40 (1H, m), 7.45 (1H, t, J = 7.4 Hz), 7.52 (1H, d, J = 2.4
Hz), 7.58 (1H, dd, J = 1.6, 7.8 Hz), 7.64 (1H, t, J = 1.2 Hz), 7.68
(1H, d, J = 8.6 Hz), 8.02 (1H, dd, J = 1.2, 2.4 Hz), 8.23 (1H, ddd,
J = 1.2, 2.0, 9.8 Hz), 13.06 (1H, s); Anal. calcd for C21H15ClFN3O4:
C, 58.96; H, 3.53; N, 9.82; found C, 58.73; H, 3.40; N, 9.74.
4.10.87. 3-({6-[(5,6-Dimethylpyridin-2-yl)oxy]-1-
methyl-1H-benzimidazol-2-yl}methoxy)benzoic acid
(16l).
Compound 16l was prepared according to general procedure F
(yield, 96%). Mp, 234–241 °C. 1H-NMR (400 MHz, DMSO-d6) δ
2.19 (3H, s), 2.25 (3H, s), 3.82 (3H, s), 5.47 (2H, s), 6.66 (1H, d,
J = 7.8 Hz), 6.94 (1H, dd, J = 2.4, 8.6 Hz), 7.37 (1H, d, J = 2.0 Hz),
7.38 (1H, ddd, J = 1.2, 2.7, 8.2 Hz), 7.45 (1H, t, J = 7.4 Hz), 7.54
(1H, d, J = 8.2 Hz), 7.58 (1H, dt, J = 1.6, 6.3 Hz), 7.63–7.65 (2H,
m), 13.03 (1H, br); Anal. calcd for C23H21N3O4•0.33H2O:C, 67.47;
H, 5.33; N, 10.26; found C, 67.40; H, 5.26; N, 10.27.
4.10.93. 3-({6-[(5-Chloro-3-methylpyridin-2-
yl)oxy]-1-methyl-1H-benzimidazol-2-
yl}methoxy)benzoic acid (16r).
Compound 16r was prepared according to general procedure F
(yield, 51%). 1H-NMR (400 MHz, DMSO-d6) δ 2.32 (3H, s), 3.79
(3H, s), 5.45 (2H, s), 6.94 (1H, dd, J = 2.2. 8.8 Hz), 7.37–7.41 (3H,
m), 7.53–7.55 (1H, m), 7.60–7.62 (2H, m), 7.83–7.85 (1H, m),
7.89–7.92 (1H, m), 13.01 (1H, s); MS (FAB) m/z: 424 [M + H]+;
Anal. calcd for C22H18ClN3O4: C, 58.96; H, 3.53; N, 9.82; found
C, 58.73; H, 3.40; N, 9.74.
4.10.88. 3-({6-[(3,6-Dimethylpyridin-2-yl)oxy]-1-
methyl-1H-benzimidazol-2-yl}methoxy)benzoic acid
(16m).
4.10.94. Methyl 3-({6-[(3,5-dibromopyridin-2-
yl)oxy]-1-methyl-1H-benzimidazol-2-
yl}methoxy)benzoate (17).
Compound 16m was prepared according to general procedure
F (yield, 50%). Mp, 215–220 °C. 1H-NMR (400 MHz, DMSO-d6)
δ 2.19 (3H, s), 2.28 (3H, s), 3.82 (3H, s), 5.47 (2H, s), 6.88 (1H, d,
J = 7.0 Hz), 6.93 (1H, dd, J = 2.4, 8.6 Hz), 7.35 (1H, d, J = 2.4 Hz),
7.37–7.39 (1H, m), 7.45 (1H, t, J = 7.8 Hz), 7.57–7.65 (4H, m),
13.04 (1H, br); Anal. calcd for C23H21N3O4•0.20H2O: C, 67.87; H,
5.30; N, 10.32; found C, 68.14; H, 5.17; N, 10.32.
A solution of 13 (275 g, 881 mmol), 3,5-dibromo-2-
fluoropyridine (247 g, 969 mmol), CuI (16.8 g, 88.2 mmol), 1,10-
phenanthroline (15.9 g, 88.2 mmol) and Cs2CO3 (861 g, 2.64 mol)
in DMF (4.0 L) was stirred at 80 °C for 24 h under N2. Aqueous
NH4Cl solution (10%, 5.0 L) was added to the cooled reaction
mixture, and the precipitated solid was collected. The solid was
dissolved in CH2Cl2 (17.0 L). The organic layer was washed with
water (2.0 L) and dried over anhydrous Na2SO4. After
concentration under reduced pressure, the residue was purified by
silica gel chromatography (Chromatorex NH; CH2Cl2) to obtain 17
4.10.89. 3-({6-[(3-Fluoro-5-methylpyridin-2-
yl)oxy]-1-methyl-1H-benzimidazol-2-
yl}methoxy)benzoic acid (16n).
Compound 16n was prepared according to general procedure F
(yield, 98%). Mp, 266–268 °C. 1H-NMR (500 MHz, DMSO-d6) δ
2.27 (3H, s), 3.82 (3H, s), 5.47 (2H, s), 7.00 (1H, dd, J = 2.4, 8.8
Hz), 7.38 (1H, dd, J = 1.5, 7.8 Hz), 7.42 (1H, d, J = 2.4 Hz), 7.44
(1H, t, J = 8.3 Hz), 7.56 (1H, dt, J = 1.5, 7.3 Hz), 7.63–7.65 (2H,
m), 7.73 (1H, dd, J = 1.5, 11.2 Hz), 7.75 (1H, s), 13.01 (1H, br);
Anal. calcd for C22H18FN3O4•0.20H2O: C, 64.29; H, 4.51; N,
10.22; found C, 64.28; H, 4.41; N, 10.20.
1
(476 g, 99%) as a white solid. H-NMR (400 MHz, DMSO-d6) δ
3.83 (3H, s), 3.85 (3H, s), 5.50 (2H, s), 7.02 (1H, dd, J = 2.4, 8.6
Hz), 7.43 (1H, ddd, J = 1.2, 2.7, 8.2 Hz), 7.46–7.50 (2H, m), 7.59
(1H, dt, J = 1.6, 7.4 Hz), 7.66–7.68 (2H, m), 8.20 (1H, d, J = 2.0
Hz), 8.52 (1H, d, J = 2.4 Hz).
4.10.95. Methyl 3-({6-[(3,5-dimethylpyridin-2-
yl)oxy]-1-methyl-1H-benzimidazol-2-
yl}methoxy)benzoate (18).
4.10.90. 3-({6-[(5-Fluoro-3-methylpyridin-2-
yl)oxy]-1-methyl-1H-benzimidazol-2-
yl}methoxy)benzoic acid (16o).
A solution of 17 (238 g, 435 mmol), trimethylboroxine (50%
solution in THF, 500 mL, 1.77 mol), PdCl2(dppf)•CH2Cl2 complex

(35.0 g, 43.5 mmol) and K2CO3 (241 g, 1.74 mol) in DMF (4.5 L)
was stirred at 80°C for 17 h under N2. Water (20 L) was added to
the cooled reaction mixture, and the mixture was extracted with
EtOAc (17.0 L). The organic layer was washed with brine (3.0 L)
and dried over anhydrous Na2SO4. After concentration under
reduced pressure, the residue was purified by silica gel
chromatography (EtOAc) to obtain 18 (145 g, 80%) as a white
nonlinear curve fitting using GraphPad Prism 4.0 (GraphPad
Software Inc.). Data represent single experiment run in
octuplicate, except for DS-6930, which are from two independent
experiments run in octuplicate.
4.12. Measurement of Log D.
Equal amounts of PBS and 1-octanol were shaken and left
overnight. The upper layer (1-octanol) and lower layer (PBS) were
collected individually. Each test compound was dissolved in 1-
octanol or PBS (200 mM). The same amount of either PBS or 1-
octanol was added and the mixture was shaken vigorously for 30
min at room temperature followed by centrifugation at 2100g for
5 min at room temperature. Then, both phases were separated and
assayed by HPLC and LC-MS. Log D7.4 was calculated by the
following equation:
1
solid. H-NMR (400 MHz, CDCl3) δ 2.25 (3H, s), 2.36 (3H, s),
3.84 (3H, s), 3.93 (3H, s), 5.41 (2H, s), 7.02 (1H, dd, J = 2.0, 8.6
Hz), 7.12 (1H, d, J = 2.0 Hz), 7.28–7.31 (1H, m), 7.35–7.39 (2H,
m), 7.69 (1H, dt, J = 1.2, 7.4 Hz), 7.72–7.79 (3H, m).
4.10.96. 3-({6-[(3,5-Dimethylpyridin-2-yl)oxy]-1-
methyl-1H-benzimidazol-2-yl}methoxy)benzoic acid
(I, DS-6930).
A solution of 18 (296 g, 709 mmol) and 1 M NaOH (1.8 L) in
MeOH (3.5 L) was stirred at 80 °C for 2 h. The reaction mixture
was neutralized by adding 1 M HCl at 0 °C, and the precipitated
solid was collected by filtration to obtain I (281 g, 99%) as a white
solid. Mp, 243–247 °C. 1H-NMR (400 MHz, DMSO-d6) δ 2.20
(3H, s), 2.30 (3H, s), 3.81 (3H, s), 5.47 (2H, s), 6.92 (1H, dd, J =
2.4, 8.6 Hz), 7.32 (1H, d, J = 2.4 Hz), 7.36 (1H, dd, J = 1.6, 7.4
Hz), 7.44 (1H, t, J = 7.4 Hz), 7.54–7.63 (4H, m), 7.72 (1H, s),
13.04 (1H, br); Anal. calcd for C23H21N3O4: C, 68.47; H, 5.25; N,
10.42; Cl, 0.00; Na, 0.00; found C, 68.29; H, 5.17; N, 10.41; Cl,
0.00; Na,0.00.
Log D7.4 = log (peak area of compound in 1-octanol/peak area
of compound in PBS).
4.13. Solubility Assay.
After lyophilization of 10 mM DMSO solution of the test
compounds, aqueous neutral solution (pH 6.8) was added, stirred,
and allowed to stand at room temperature for at least 4 hours. After
allowing to filter by suction through Uni Filter (Uni Filter), the
concentration of the filtrate was measured by HPLC-UV
methodologies. Data represent single experiment run in duplicate.
4.11. Measurement of PPAR Transcriptional Activity.
4.14. Parallel Artificial Membrane Permeation Assay (PAMPA).
GAL4-human PPARγ chimera receptor expression vector, pM-
h PPARγ, expressed the LBD of PPARγ as a fusion protein with
the DNA-DB of yeast transcription factor GAL4. The pG5luc
vector (included in CheckMate Mammalian Two-Hybrid System,
Promega Corporation), which contains five tandem repeats of a
GAL4-binding DNA sequence upstream of minimal TATA to
facilitate the detection of firefly luciferase activity induced by a
GAL4-h PPARγ fusion protein, was used as a GAL4-dependent
reporter vector. Dulbecco’s Modified Eagle Medium (DMEM,
Invitrogen Corporation) containing 10% (v/v) heat-inactivated
fetal bovine serum (FBS, Invitrogen Corporation) was prepared as
a culture medium. The COS-7 cells were cultured in the culture
medium at 37 °C with 5% CO2 (5% CO2, 95% air). The COS-7
cells were cultured to the confluent in 75-cm2 culture flasks. Cells
were transfected with 4.8 µg of the pM-h PPARγ and 19.2 µg of
pG5luc using Lipofectamine 2000 (Invitrogen Corporation) in
Opti-MEM I reduced serum medium (Opti-MEM I, Invitrogen
Corporation) according to the manufacturer’s instruction. After the
transfection, the COS-7 cells were harvested and re-seeded in 96-
well white plates and cultured for about 24 h in a CO2 incubator.
The pM-h PPARα expression vector was used for the GAL4-h
PPARα-LBD reporter gene assay.
DMSO solution of the test compounds (10 mM) was diluted
with buffer (consisting of PRISMA HT (pION) and DMSO) to
obtain donor solutions (pH 7.4) with concentration of 5 µM. GIT-
0 lipid solution (pION) was applied onto acceptor plate filters
(Stirwell PAMPA Sandwich, pION). And the donor solutions of
pH 7.4 were added to the donor plate, and the acceptor sink buffer
(pION) to the acceptor plate, respectively. The donor plate was
placed on the top of the acceptor plate and incubated at 25 °C for
4 hours. The donor and acceptor solutions were collected, and the
concentrations of each compound were determined by LC-MS/MS.
The apparent permeability coefficient (Papp, 10-6 cm/sec) was
calculated by PAMPA Evolution DP (pION) from the area ratio
and incubation time to the internal standard. Data represent single
experiment run in duplicate unless otherwise noted.
4.15. Hypoglycemic effect in ZDF rats.
All experimental procedures were performed in accordance
with the in-house guideline of the Institutional Animal Care and
Use Committee of Daiichi Sankyo Co., Ltd. Six-week-old male
Zucker diabetic fatty rats were purchased from Charles River
Japan, Inc. and then were fed until 8 weeks old. Before the study,
the each PG level was measured by Glucoloader GXT (A&T
Corp.), and individuals having a PG level of about 300 mg/dL or
more were selected. The test compound (0.5%methylcellulose)
was orally administered to ZDF rats once daily for 14 days (n = 5).
The body weight was measured and blood was collected from the
tail vein to measure the PG level. The glucose lowering rate was
determined by the following formula.
The serial dilutions of the test compounds (1, 3, 10, 30, 100,
300, 1000, 3000, 10000 nM) and the control solution (0.1%
DMSO) were added into the individual wells in the 96-well plates,
and the cells were further incubated for about 24 h in a CO2
incubator. A luciferase assay was performed using a Picagene LT
2.0 Luminescence Reagent (TOYO INK, Co., Ltd.) according to
the manufacturer’s instruction. The light intensity in each well was
measured using a Multimode Microplate Reader (Analyst GT,
Molecular Devices, Inc.). Rosiglitazone and 2-(4-tert-
Butylphenoxy)-3-[4-[2-[(4-pyridin-2-
PG reduction (%) = [(PG level (Control group) - PG level
(Compound-administered group))/ PG level (Control group)] x
100
ylbenzoyl)amino]ethoxy]phenyl]propionic acid15 were used as
positive references for the PPARγ and PPARα, respectively. The
maximum transcriptional activity of the test compound alone was
defined as the maximum efficacy (Emax, %). The concentration of
the test compound indicating a half value of Emax was defined as
the EC50 value. Values of each parameter were determined by
4.16. In Vivo Pharmacological Effects of DS-6930.
All experimental procedures were performed in accordance
with the in-house guideline of the Institutional Animal Care and
Use Committee of Daiichi Sankyo Co., Ltd. Six-week old male
ZDF rats were purchased from Charles River Laboratories Japan,

Inc. and acclimatized for one week. Before the study, the PG level
was measured, and individuals having a PG level of about 240
mg/dL or more were selected. The test compound was orally
administered to ZDF rats once daily for 21 days (n = 8). Water was
administered orally to the control group. Body weight was
measured and blood was collected from the tail vein to measure
the PG level.
Serial dilutions of the test compounds and the control solution
(0.25% DMSO) were added into the individual wells of 96-well
plates. The plate was incubated for 24 or 48 h in an incubator at
28 °C and fluorescence was measured upon excitation at 355 nm
wavelength and emission at 460 nm wavelength (Perkin Elmer
VictorTMX4 instrument). Data represent single experiment run in
quadruplicate.
4.17. Monkey Pharmacokinetic Study.
4.21. Protein Crystallography Method.
All experimental procedures were performed in accordance
with the in-house guideline of the Institutional Animal Care and
Use Committee of Daiichi Sankyo Co., Ltd. For the determination
of test compound exposures in male cynomolgus monkeys, blood
samples were taken at several time points postdose. The plasma
Histidine-tagged human PPARγ-LBD was expressed and
purified as described previously.24 A synthetic peptide with a
sequence derived from PGC-1 (QEAEEPSLLKKLLLAPANT)
was purchased from Sigma-Aldrich. Before crystallization,
PPARγ-LBD was concentrated to 22 mg/mL and mixed with DS-
6930 and PGC-1 in a molar ratio of 1:4:4. Crystals were obtained
by the hanging drop vapor diffusion technique with a reservoir
solution of 24% (w/v) PEG4000, 200 mM NaSCN and 100 mM
Tris-HCl (pH 8.5). Prior to data collection, crystals were
transiently soaked in the reservoir solution containing additional
8% (v/v) PEG400 as a cryoprotectant. X-ray diffraction data were
collected using an X-ray generator FR-E with detector R-AXIS IV
(RIGAKU). The diffraction data were integrated, scaled using
HKL2000,25 and converted to structure factors using the CCP4
software suite.26 The structure of PPARγ-LBD derived from the
structure 3V9T.pdb27 was used as an initial model. Several rounds
of manual rebuilding with O28 followed by refinement with CNX29
(Accerlys) were carried out. A Ramachandran plot for the final
model was calculated with RAMPAGE.30 Figures were created
with PyMOL (v.1.7; Schrödinger). Authors will release the atomic
coordinates and experimental data upon article publication.
Coordinates are available from PDB using accession code 5Z6S.
−70 °C
was separated from blood by centrifugation, and stored at
until use for measurement of plasma concentration. The
determination of the plasma concentration of the compound was
performed by LC-MS/MS method using API 4000QTRAP
(Applied Biosystems/MDS SCIEX). PK parameters were
calculated using a non-compartmental analysis techniques by the
computer software WinNonlin Professional version 4.0.1.
(Pharsight Corporation).
4.18. In Vitro hepatotoxicity Evaluation.
Rat primary cultured hepatocytes were harvested from male
F344/DuCrlCrlj rats (> 8 weeks age) and maintained with
sandwich culture between extracellular matrix layers. Test
compounds were dissolved with DMSO, diluted with medium to
be 0.5% as final DMSO concentration, and exposed for 24 hrs.
Cytotoxicity was evaluated with LDH leakage assay and water-
soluble tetrazolium based assay (WST-8 assay). Data represent
single experiment run in quadruplicate.
5. AUTHOR INFORMATION
4.19. In Vivo Toxicological Evaluation.
*Tel: +81-3-3492-3131. Fax: +81-3-5436-8563.
All experimental procedures were performed in accordance
with the in-house guideline of the Institutional Animal Care and
Use Committee of Daiichi Sankyo Co., Ltd. Six-week-old female
F344/DuCrlCrlj SPF rats were purchased from Charles River
Japan, Inc. and then were housed for one week. Rats (n = 5) were
orally dosed once daily by gavage with vehicle or test compounds
at indicated doses in 0.5% methylcellulose for 28 days. Animals
were euthanized on the next day of the last dose, and the indicated
tissues and organs were collected for organ weight assessment and
histopathological examination Blood samples were exsanguinated
via the abdominal aorta and assayed for serum chemistry.
E-mail:
sinozu.xf6@gmail.com;
shinozuka.tsuyoshi.s5@daiichisankyo.co.jp.
6. ORCID ID
Tsuyoshi Shinozuka: 0000-0002-7785-6080
7. Notes
The authors declare no competing financial interest.
8. REFERENCES
4.20. Yeast 2-Hybrid (Y2H) Assay.
1. Yki-Järvinen H. N. Engl. J. Med. 2004, 351, 1106-1118.
2. Eldor, R.; DeFronzo, R. A.; Abdul-Ghani, M. Diabetes Care
2013, 36, S162-S174.
3. Pirat, C.; Farce, A.; Lebègue, N.; Renault, N.; Furman, C.; Millet,
R.; Yous, S.; Speca, S.; Berthelot, P.; Desreumaux, P.; Chavatte,
P. J. Med. Chem. 2012, 55, 4027-4061.
4. Nesto, R. W.; Bell, D.; Bonow, R. O.; Fonseca, V.; Grundy, S. M.;
Horton, E. S.; Winter, M. L.; Porte, D.; Semenkovich, C. F.;
Smith, S.; Young, L. H.; Kahn, R. Diabetes Care 2004, 27, 256-
263.
5. Home, P. D.; Pocock, S. J.; Beck-Nielsen, H.; Curtis, P. S.;
Gomis, R.; Hanefeld, M.; Jones, N. P.; Komajda, M.; McMurray,
J. J.; RECORD Study Team. Lancet 2009, 373, 2125-2135.
6. Mudaliar, S.; Chang, A. R.; Henry, R. R. Endocr. Pract. 2003, 9,
406-416.
Two expression plasmids were used allowing expression of
distinct hybrid proteins. The fusion protein expressed from pGBT9
(Clontech)-based vector expressing the NR or cofactor C-
terminally fused to the DNA BD of the GAL4 transcription factor
is referred to as BAIT. The fusion protein espressed from
pGAD424 (Clontech)-based vector expressing the NR or cofactor
C-terminally fused to the transcriptional activation domain of the
GAL4 transcription factor is referred to as PREY. The BAIT
expression plasmids were transformed into yeast strain AH109a
(Clontech), and the PREY expression plasmids into strain
Y187alpha
(Clontech)
using
a
standard
lithium
7. Loke, Y. K.; Singh, S.; Furberg, C. D. CMAJ 2009, 180, 32-39.
8. Shinozuka, T.; Tsukada, T.; Fujii, K.; Tokumaru, E.; Shimada, K.;
Onishi, Y.; Matsui, Y.; Wakimoto, S.; Kuroha, M.; Ogata, T.;
Araki, K.; Ohsumi, J.; Sawamura, R.; Watanabe, N.; Yamamoto,
H.; Fujimoto, K.; Tani, Y.; Mori, M.; Tanaka, J. Submitted to
Bioorg. Med. Chem.
acetate/PEG/heatshock procedure. Yeast strains were mated by
mixing equal volumes of the haploid yeast strains in selective
medium supplemented with 0.5 volumes of rich YPDA medium.
Cells were incubated for 5-7 h at 28 °C. After mating, a small
aliquot of the yeast cell suspension was transferred into the
medium selective for diploid cells and grown to saturation. The
cells were diluted in the medium selective for interacting clones.
9. Issa, A. M.; Phillips, K. A.; Van Bebber, S.; Nidamarthy, H. G.;
Lasser, K. E.; Haas, J. S.; Alldredge, B. K.; Wachter, R. M.;
Bates, D. W. Curr. Drug Saf. 2007, 2, 177-185.

10. Chen, M.; Borlak, J.; Tong, W. Hepatology 2013, 58, 388-396.
11. Zitko, J.; Paterova, P.; Kubicek, V.; Mandikova, J.; Trejtnar, F.;
Kunes, J.; Dolezal, M. Bioorg. Med. Chem. Lett. 2013, 23, 476-
479.
12. Wolter, M.; Nordmann, G.; Job, G. E.; Buchwald, S. L. Org. Lett.
2002, 4, 973-976.
13. Marcoux, J. F.; Doye, S.; Buchwald, S. L. J. Am. Chem. Soc.
1997, 119, 10539-10540.
14. Rosiglitazone exhibited EC50 = 396 nM in this reporter assay.
15. Takamura, M.; Sakurai, M.; Yamada, E.; Fujita, S.; Yachi, M.;
Takagi, T.; Isobe, A.; Hagisawa, Y.; Fujiwara, T.; Yanagisawa, H.
Bioorg. Med. Chem. 2004, 12, 2419-2439.
16. The inhibition of PPARγ phosphorylation by Cdk5 is reported to
be involved in the mechanism of antidiabetic efficacy of PPARγ
modulator rather than PPARγ transcriptional activity.17 The
inhibition of PPARγ phosphorylation could have explained for 6n
not showing potent in vivo efficacy. The inhibitory activities of
this series of compounds against PPARγ phosphorylation were not
evaluated.
17. Choi, J. H.; Banks, A. S.; Estall, J. L.; Kajimura, S.; Bostrom, P.;
Laznik, D.; Ruas, J. L.; Chalmers, M. J.; Kamenecka, T. M.;
Bluher, M.; Griffin, P. R.; Spiegelman, B. M. Nature 2010, 466,
451-456.
18. Preliminary rodent toxicological study in vivo was performed with
HCl salt of DS-6930 (DS-6930HCl), because it was one of the
formulation candidates of DS-6930 in clinical studies. For the
kinetic solubility of DS-6930, see supporting information.
19. Acton III, J. J.; Akiyama, T. E.; Chang, C. H.; Colwell, L.;
Debenham, S.; Doebber, T.; Einstein, M.; Liu, K.; McCann, M.
E.; Moller, D. E.; Muise, E. S.; Tan, Y.; Thompson, J. R.; Wong,
K. K.; Wu, M.; Xu, L.; Meinke, P. T.; Berger, J. P.; Wood, H. B.
J. Med. Chem. 2009, 52, 3846-3854.
20. Liu, W.; Lau, F.; Liu, K.; Wood, H. B.; Zhou, G.; Chen, Y.; Li,
Y.; Akiyama, T. E.; Castriota, G.; Einstein, M.; Wang, C.;
McCann, M. E.; Doebber, T. W.; Wu, M.; Chang, C. H.;
McNamara, L.; McKeever, B.; Mosley, R. T.; Berger, J. P.;
Meinke, P. T. J. Med. Chem. 2011, 54, 8541-8554.
21. Furukawa, A.; Arita, T.; Fukuzaki, T.; Mori, M.; Honda, T.;
Satoh, S.; Matsui, Y.; Wakabayashi, K.; Hayashi, S.; Nakamura,
K.; Araki, K.; Kuroha, M.; Tanaka, J.; Wakimoto, S.; Suzuki, O.;
Ohsumi, J. Eur. J. Med. Chem. 2012, 54, 522-533.
22. Menet, C.; Schmitt, B.; Geney, R.; Doyle, K.; Peach, J.; Palmer,
N.; Jones, G.; Hardy, D.; Duffy, J. PCT Int. Appl. WO
2013117649 A1, 2013.
23. The HCl salt of 5b was synthesized to evaluate in vitro activity,
which indicated EC50 (Emax) = 12.4 µM (65%).
24. Wakabayashi, K.; Hayashi, S.; Matsui, Y.; Matsumoto, T.;
Furukawa, A.; Kuroha, M.; Tanaka, N.; Inaba, T.; Kanda, S.;
Tanaka, J.; Okuyama, R.; Wakimoto, S.; Ogata, T.; Araki, K.;
Ohsumi, J. Biol. Pharm. Bull. 2011, 34, 1094−1104.
25. Otwinowski, Z.; Minor, W. Methods Enzymol. 1997, 276, 307-
326.
26. Winn, M. D.; Ballard, C. C.; Cowtan, K. D.; Dodson, E. J.;
Emsley, P.; Evans, P. R.; Keegan, R. M.; Krissinel, E. B.; Leslie,
A. G. W.; McCoy, A.; McNicholas, S. J.; Murshudov, G. N.;
Pannu, N. S.; Potterton, E. A.; Powell, H. R.; Read, R. J.; Vagin,
A.; Wilson, K. S. Acta Crystallogr.,Sect. D: Biol. Crystallogr.
2011, 67, 235-242.
27. Furukawa, A., Arita, T., Fukuzaki, T., Satoh, S., Mori, M., Honda,
T., Matsui, Y., Wakabayashi, K., Hayashi, S., Araki, K., and
Ohsumi, J. Bioorg. Med. Chem. Lett. 2012, 22, 1348-1351.
28. Jones, T. A.; Zou, J. Y.; Cowan, S. W.; Kjeldgaard, M. Acta
Crystallogr. 1991, A47, 110-119.
29. Brunger, A. T. Nat. Protoc. 2007, 2, 2728-2733.
30. Lovell S. C.; Davis I. W.; Arendall W. B.; de Bakker P. I. W.;
Word J. M.; Prisant M. G.; Richardson J. S.; Richardson D. C.
Proteins 2003, 50, 437–450.

Graphical Abstract
To create your abstract, type over the instructions in the
template box below.
Fonts or abstract dimensions should not be changed or altered.
Discovery of DS-6930, a Potent Selective
PPARγ Modulator. Part II: Lead
Optimization
Leave this area blank for abstract info.
Tsuyoshi Shinozuka,*,† Tomoharu Tsukada,† Kunihiko Fujii,† Eri Tokumaru,† Kousei Shimada,† Yoshiyuki
Onishi,† Yumi Matsui,‡ Satoko Wakimoto,† Masanori Kuroha,† Tsuneaki Ogata,† Kazushi Araki,† Jun Ohsumi,†
Ryoko Sawamura,† Nobuaki Watanabe,† Hideki Yamamoto,† Kazunori Fujimoto,† Yoshiro Tani,† Makoto Mori†
and Jun Tanaka†