Discovery of potent and orally active 3-alkoxy-5-phenoxy-N-thiazolyl benzamides as novel allosteric glucokinase activators

Article abstract of DOI:10.1016/j.bmc.2009.02.038

Identification and synthesis of novel 3-alkoxy-5-phenoxy-N-thiazolyl benzamides as glucokinase activators are described. Removal of an aniline structure of the prototype lead (2a) and incorporation of an alkoxy or phenoxy substituent led to the identifica

Full text of DOI:10.1016/j.bmc.2009.02.038

Bioorganic & Medicinal Chemistry 17 (2009) 2733–2743
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Discovery of potent and orally active 3-alkoxy-5-phenoxy-N-thiazolyl
benzamides as novel allosteric glucokinase activators
*
Tomoharu Iino , Daisuke Tsukahara, Kenji Kamata, Kaori Sasaki, Sumika Ohyama, Hideka Hosaka,
Takuro Hasegawa, Masato Chiba, Yasufumi Nagata, Jun-ichi Eiki, Teruyuki Nishimura
Banyu Tsukuba Research Institute, Banyu Pharmaceutical Co. Ltd, Okubo 3, Tsukuba 300-2611, Ibaraki, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Identification and synthesis of novel 3-alkoxy-5-phenoxy-N-thiazolyl benzamides as glucokinase activa-
tors are described. Removal of an aniline structure of the prototype lead (2a) and incorporation of an alk-
oxy or phenoxy substituent led to the identification of 3-Isopropoxy-5-[4-(methylsulfonyl)phenoxy]-N-
(4-methyl-1,3-thiazol-2-yl)benzamide (27e) as a novel, potent, and orally bioavailable GK activator.
Rat oral glucose tolerance test indicated that 27e exhibited a glucose-lowering effect after 10 mg/kg oral
administration.
Received 15 January 2009
Revised 16 February 2009
Accepted 19 February 2009
Available online 25 February 2009
Keywords:
Glucokinase
Ó 2009 Elsevier Ltd. All rights reserved.
Glucokinase activators
3-Alkoxy-5-phenoxy-N-thiazolyl
benzamides
Glucose-lowering effect
1. Introduction
Compounds that activate GK, by binding to an allosteric pocket
some 20 Å remote from the glucose binding site, have recently
There are three key aspects of Type 2 diabetes pathogenesis
which are the focus of current and future therapies: insulin resis-
tance, defective insulin secretion, and increased hepatic glucose
production. The major classes of oral antidiabetic drugs used to
treat this disorder include thiazolidinediones, biguanides, alpha-
glucosidase inhibitors, sulfonylureas, and meglitinides. Each of
the preceding drugs has documented limitations, and despite these
treatment options, it is difficult to effectively treat Type 2 diabetes
in the long term.1 Currently, no single marketed drug is capable of
achieving enduring blood glucose control in the majority of Type 2
diabetic patients.2,3 Therefore, there is an unmet medical need for
the development of new, safe, and effective antidiabetic therapies
with multiple modes of action.
Glucokinase (GK), a member of the hexokinase family, is ex-
pressed in the liver and in pancreatic b-cells.4 This enzyme cata-
lyzes the key initial step for glucose metabolism, that is,
phosphorylation of glucose to glucose 6-phosphate. In the liver,
GK promotes glycogen synthesis, whilst it enhances insulin secre-
tion from pancreatic b-cells.5,6 Therefore, GK activators (GKAs) can
be expected to function as a hypoglycemic drug, by both increasing
glucose uptake in the liver and the potentiation of insulin secretion
from pancreatic beta-cells.
been discovered.7–9 These GKAs have been shown to engender po-
tent antihyperglycemic actions in rodents, both by increasing pan-
creatic insulin secretion and by augmenting hepatic glucose
metabolism.10
Thus far, several structure classes of GKAs including
RO28167511, piragliatin (Roche)12, LY-2121260 (Lilly)13, GKA50
(AstraZeneca),14 and PSN-GK1 (OSI)15 have been disclosed. Among
them, RO28167511 and piragliatin12 are reported to enter human
clinical trials as prospective Type 2 diabetes therapies.
We have been involved with a GKA program to identify and de-
velop novel, potent and orally bioavailable GKAs for the treatment
of Type 2 diabetes. In previous publications, we described the SARs
of 2-aminobenzamide GKAs, the binding mode of the representa-
tive 2-aminobenzamide GKAs (1 and 2a) with GK, and their
in vitro and/or in vivo profiles (Fig. 1).7,10,16 Using crystal structure
analysis, we revealed that the interaction between the aniline part
of 1 or 2a and Tyr215 of GK played an important role in enhancing
GK activation, in addition to the interaction between the aminothi-
azole moiety of 2a and Arg63 of the hinge region of GK (Fig. 2).
Meanwhile, 2-aminobenzamides (represented by 2a) have an
aniline structure. It is known that aniline compounds sometimes
exert the potential risk for mutagenecity.17,18 Therefore, from the
point of view of drug discovery, it was important to remove this
aniline part from the core structure. However, since N-methyl-
amine derivative (2b) reduced GK activity, alternative approaches
for increasing GK potency are strongly required.
* Corresponding author. Tel.: +81 29 877 2035; fax +81 29 877 2029.
E-mail address: tomoharu_iino@merck.com (T. Iino).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.02.038

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T. Iino et al. / Bioorg. Med. Chem. 17 (2009) 2733–2743
pound 4.16 Des-amination reaction afforded 7a–g, 7k, and 7l. The
2-methanesulfonyl derivative 7h was obtained by mCPBA oxida-
tion after des-amination reaction.
O
S
O
S
S
S
S
F
N
N
N
N
N
N
N
H
N
H
N
N
N
NH2
NH2
Preparation of the 3- and 4-methanesulfonyl derivatives 7i and
7j was started from commercially available methyl 3-hydroxyben-
zoate 8. Coupling reactions using boronic acids in the presence of
Cu(OAc)2 afforded compounds 9i and 9j. Oxidation using mCPBA
gave the 3- and 4-methanesulfonyl derivatives. Saponification of
the methyl esters, followed by a condensation reaction with 2-ami-
no-4-methylthiazole, led to the desired amides 7i and 7j.
Synthesis of methanesulfonyl substituted phenoxy derivatives
16a-c is shown in Scheme 3. A substitution reaction of 10 with
PhOH under 60 °C condition, afforded the nitro compound 11.
Reduction of 11 in the presence of Fe followed by substitution reac-
tion with 2-MeS-C6H4OH at 80 °C followed by an oxidation reac-
tion with mCPBA produced 13. 2-Methanesufonyl derivative 16a
was obtained after des-amination.
1
2a
O
S
O
O
N
N
H
S
N
N
N
H
O
NH
2b
27e
MeO2S
Figure 1. Discovery of 3-alkoxy-5-phenoxy-N-thiazolyl benzamides as novel GK
activators.
Arg63
N
H
O
N
H
N
S
3- or 4-methanesulfonyl substituted derivatives 16b and 16c
were prepared from the phenoxy compound 14. Cross-coupling
with corresponding boronic acids followed by the same manner
as described in Scheme 2 gave 16b and 16c.
S
N
O
N
H
N
N
H
OH
Scheme 4 depicts the preparation of 2-substituted derivatives
20a and 20b. Alkylation of 2-hydroxy-5-iodobenzoate 17 followed
by coupling with 2-methylthiophenol, in the presence of
Cu(OTf)2PhH complex, produced thioether 19. Oxidation using
mCPBA, hydrolysis and an amidation reaction afforded amide
20a. A substitution reaction of methyl 2-bromo-5-hydroxybenzo-
ate 21 with 2-methanesulfonylfluorobenzene, followed by a cou-
pling reaction with phenol in the presence of CuO, produced the
methyl ester 23. Hydrolysis and an amidation reaction gave the
amide 20b.
Tyr215
Figure 2. Binding mode between 2-aminobenzamide GK activator 2a and GK.
Here, we describe the optimization of our lead GKA 2a and dis-
covery of 3-alkoxy-5-phenoxy-N-thiazolyl-benzamide represented
by 27e,19 and in addition, we describe the in vivo profile of 27e,
such as the oral bioavailability and glucose lowering effect in
rodents.
Alkoxy derivatives 27a–e were prepared from the methyl 3,5-
dihydroxybenzoate 24. Treatment of 24 with alkyl halide gave
the mono alkoxy intermediates 25a–c. Substitution reactions of
them with 2-methanesulfonylfluorobenzene afforded sulfone com-
pounds 26a–c. The amide derivatives 27a–c were obtained in the
same manner.
Isopropoxy derivatives 29d and 29e, possessing a methanesul-
fonyl group at the 3- or 4-position on the terminal benzene ring,
were prepared by cross-coupling of 24 with corresponding boronic
acids and alkylation. Oxidation, hydrolysis and an amidation reac-
tion afforded the amide derivatives 27d and 27e (Scheme 5).
2. Chemistry
Preparation of compounds is summarized in Schemes 1–5. Dia-
zonium reaction and Cu2O work-up gave the des-amino derivative
3 (Scheme 1).
Scheme 2 shows synethesis of 3-phenoxybenzamides 7a–l. The
aniline compounds6a–h, 6k, and 6l were prepared from nitro com-
O
S
O
S
a
S
S
N
N
N
N
N
N
N
H
N
H
N
N
NH2
3. Biological results and discussion
2a
3
An in vitro GK assay was conducted at two different glucose
concentrations, 2.5 mM and 10 mM, which were simulated to be
Scheme 1. Synthesis of des-amino derivative 3. Reagents and conditions: (a)
NaNO2, cH2SO4, AcOH followed by Cu2O, EtOH.
O
S
O
S
O
S
R
R
F
O
O
a
b
N
N
N
N
H
N
H
N
H
NO2
NO2
NH2
4
5a-h, 5k, 5l
6a-h, 6k, 6l
c
O
S
O
O
R
O
HO
O
d
e
N
N
O
O
R
H
8
9i: 3-SMe
9j: 4-SMe
7k: 2-OMe
7l: 2-OEt
7a: H 7b: 2-F 7e: 2-Me 7h: 2-SO2Me
7c: 3-F 7f: 3-Me
7i: 3-SO2Me
7j: 4-SO2Me
7d: 4-F 7g: 4-Me
Scheme 2. Synthesis of 3-phenoxybenzamide 7a–l. Reagents and conditions: (a) R-C6H4OH, Et3N or K2CO3, DMF, 80–100 °C; (b) Fe powder, sat-NH4Cl, i-PrOH, reflux; (c) (i)
NaNO2, cH2SO4, AcOH followed by Cu2O, EtOH; (ii) mCPBA, THF, 0 °C (7h); (d) 3-MeS-C6H4B(OH)2 (9i) or 4-MeS-C6H4B(OH)2 (9j), Cu(OAc)2, Et3N, CH2Cl2; (e) (i) mCPBA, THF,
0 °C; (ii) NaOHaq, MeOH; (iii) 2-amino-4-methylthiazole, WSC, HOBT, CHCl3.

T. Iino et al. / Bioorg. Med. Chem. 17 (2009) 2733–2743
MeS
2735
O
S
O
S
MeO2S
O
S
O
S
F
O
O
F
d
a
b, c
N
N
N
N
N
N
N
H
N
H
H
H
NO2
11
NH2
NH2
NO2
PhO
PhO
PhO
13
F
10
12
e
O
S
O
O
R
f
d, g
O
O
HO
N
N
H
O
O
R
OPh
OPh
OPh
14
16a: 2-SO2Me
16b: 3-SO2Me
16c: 4-SO2Me
15b: 3-SMe
15c: 4-SMe
Scheme 3. Synthesis of methanesulfonyl substituted phenoxy derivatives 16a–c. Reagents and conditions: (a) Phenol, K2CO3, DMF, 60 °C; (b) 2-MeS-C6H4OH, K2CO3, DMF,
80 °C; (c) Fe powder, sat-NH4Cl, i-PrOH, reflux; (d) mCPBA, THF, 0 °C; (e) NaNO2, cH2SO4, AcOH followed by Cu2O, EtOH; (f) 3-MeS-C6H4B(OH)2 (15b) or 4-MeS-C6H4B(OH)2
(15c), Cu(OAc)2, Et3N, CH2Cl2; (g) (i) NaOHaq, MeOH; (ii) 2-amino-4-methylthiazole, WSC, HOBT, CHCl3.
O
O
O
MeO2S
O
O
S
MeS
O
O
I
I
O
O
c, d
a
O
O
N
b
N
H
O
OH
17
21
19
MeO2S
20a
MeO2S
18
MeO2S
O
O
S
O
O
HO
O
d
O
O
O
e
f
N
N
H
O
O
Br
OPh
Br
OPh
20b
22
23
Scheme 4. Synthesis of 2-substituted benzamides 20a and 20b. Reagents and conditions: (a) 2-bromopropane, K2CO3, DMF, 60 °C; (b) 2-MeS-C6H4OH, Cu(OTf)2PhH, Cs2CO3,
toluene, EtOAc, 110 °C; (c) mCPBA, THF, 0 °C; (d) (i) NaOHaq, MeOH; (ii) 2-amino-4-methylthiazole, WSC, HOBT, CHCl3; (e) 2-MeSO2-F-benzene, Cs2CO3, DMF, 80 °C; (f)
Phenol, K2CO3, CuO, pyridine, 130 °C.
O
O
MeO2S
O
S
MeO2S
O
HO
HO
O
O
O
O
N
N
H
a
b
O
c
OR1
OR1
OR1
26a-c
OH
27a: R1: Me
24
25a-c
27b: R1: Et
d
27c: R1: i-Pr
O
O
O
O
S
O
e
O
b, c
O
O
R2
N
N
H
R2
R2
OH
O
O
27d: R2: 3-SO2Me
27e: R2: 4-SO2Me
28d: 3-SMe
28e: 4-SMe
29d: 3-SMe
29e: 4-SMe
Scheme 5. Synthesis of 3-alkoxy derivatives 27a–e. Reagents and conditions: (a) MeI (25a) or EtI (25b) or 2-Bromopropane (25c), K2CO3, DMF, 60 °C; (b) 2-MeSO2-F-benzene,
Cs2CO3, DMF, 80 °C; (c) (i) NaOHaq, MeOH; (ii) 2-amino-4-methylthiazole, WSC, HOBT, CHCl3; (d) 3-MeS-C6H4B(OH)2 (28d) or 4-MeS-C6H4B(OH)2 (28e), Cu(OAc)2, Et3N,
CH2Cl2; (e) 2-bromopropane, K2CO3, DMF, 60 °C.
low and high (postprandial) blood glucose conditions, respectively.
GK activity was evaluated as EC50 values and maximal effective re-
sponses (Emax). For comparison of derivatives in each assay, the
EC50 and Emax values of 2a were used as the internal standard (Emax
value of 2a is 1.0). We suppose that not only the EC50 values, but
also the Emax values of GKAs, have an impact on the efficacy of
in vivo experiments.
Prior to the selection of the lead compound from the benzamide
class, we prepared 3 and 7a (which have no aniline structure). The
deletion of the amine part from the core benzene ring resulted in
an approximately 10-fold reduction in the potency, as compared
with that of the aniline compounds 2a and 6a. This reduction
would be explicable by the lack of interaction between 2a (6a)
and Tyr215 of GK.16 In developing the lead compound with no ani-
line structure, we paid attention to 3-phenoxy-benzamide 7a, be-
cause various substituents could be incorporated on the phenoxy
portion at the 3-position of the core benzamide. First, we checked
the effect of the regio-chemical structure of a fluoro atom, a methyl
group or a methanesulfonyl group on GK potency (Table 1). As was
expected16, the GK activity of the 2-substituted derivatives (7b, 7e,
and 7h) was tolerable, in terms of both EC50 and Emax values, whilst
the 3- and 4-substituted derivatives (7c, 7d, 7f, 7g, 7i, and 7j)
showed a significant drop in activity, especially in Emax values.
GK activity of the other 2-substituted derivatives (7k and 7l) was
also tolerable. Among the 2-substituted derivatives (7b, 7e, 7h,
7k, and 7l), the Emax value of 3-(2-methanesulfonyl)phenoxy-benz-
amide 7h was as high as that of the aniline compound 2a, and thus
7h was selected as the lead compound for further modifications.

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T. Iino et al. / Bioorg. Med. Chem. 17 (2009) 2733–2743
Table 1
Table 2
SAR of 3-phenoxy-benzamide derivatives
GK activity of 2- or 3-substituted 5-phenoxy-benzamides
O
S
MeO2S
O
S
X
O
N
N
N
H
N
H
R1
R2
R2
R3
Compd
R1
2.5 mM Glc
10 mM Glc
R2
EC50
(l
M)
Emax
EC50
(lM)
Emax
a
b
a
b
Compd
R3
R2
H
O-iPr
OPh
H
H
H
H
2.5 mM Glc
10 mM Glc
X
S
a
b
a
b
EC50
(l
M)
Emax
EC50
(l
M)
Emax
N
N
2a
3
NH2
0.42
1.0
0.14
1.0
7h
H
H
H
OMe
OEt
O-iPr
OPh
5.5
>30
>30
11
2.4
2.1
1.1
1.0
1.1
1.1
N
N
20a
20b
27a
27b
27c
16a
0.05
0.06
0.75
1.0
0.98
0.81
>30
>30
1.9
2.1
0.44
0.61
0.20
0.14
0.99
1.2
0.91
0.98
N
N
S
H
7.3
0.76
1.5
0.61
6a
7a
7b
7c
7d
7e
7f
7g
7h
7i
Ph
Ph
2-F-C6H4
3-F-C6H4
4-F-C6H4
2-Me-C6H4
3-Me-C6H4
4-Me-C6H4
2-MeSO2-C6H4
3-MeSO2-C6H4
4-MeSO2-C6H4
2-MeO-C6H4
2-EtO-C6H4
O
O
O
O
O
O
O
O
O
O
O
O
O
NH2
H
H
H
H
H
H
H
H
H
H
H
H
0.70
0.91
0.63
0.76
0.55
0.53
0.63
0.45
0.28
1.0
0.48
0.60
0.84
0.53
0.13
0.8
0.45
2.2
4.2
0.82
2.3
6.4
1.1
1.1
3.4
0.95
0.66
0.73
0.74
0.59
0.63
0.69
0.56
1.1
0.48
0.66
0.85
0.69
19
2.4
6.3
a
Values are the means of two or more independent assays. Compound 2a is the
internal standard (EC50: 0.42 0.09 and 0.14 0.04).
b
The maximal activating response elicited the compounds as a ratio of the
21
maximal response evoked by 2a at each concentration independently.
3.2
8.2
5.9
5.5
18
29
2.2
5.4
Table 3
Effects of the position of the methanesulfonyl group on the benzene ring
7j
7k
7l
O
S
0.49
1.9
R1O
N
N
H
a
Values are the means of two or more independent assays. Compound 2a is the
R3
internal standard (EC50: 0.42 0.09 and 0.14 0.04).
b
The maximal activating response elicited the compounds as a ratio of the
Compd
R1
R3
2.5 mM Glc
10 mM Glc
maximal response evoked by 2a at each concentration independently.
a
b
a
b
EC50
(lM)
Emax
EC50
(l
M)
Emax
16a
16b
16c
27c
27d
27e
2-MeSO2-C6H4
3-MeSO2-C6H4
4-MeSO2-C6H4
2-MeSO2-C6H4
3-MeSO2-C6H4
4-MeSO2-C6H4
PhO
PhO
PhO
iPrO
iPrO
iPrO
1.1
1.1
0.42
2.1
1.1
0.81
0.60
0.73
0.97
0.73
0.99
0.61
0.94
0.35
0.44
0.09
0.13
0.98
0.68
0.83
0.91
0.79
1.0
To enhance the GK activity of 7h, we hypothesized that instal-
lation of a substituent into the 2- or 3-position of the core benzam-
ide may result in an alternative noncovalent interaction with the
GK enzyme. Based on this hypothesis, an alkoxy or phenoxy group
was incorporated into the 2- or 3-position of the core structure in
7h (Table 2). Unfortunately, the incorporation of an alkoxy (20a) or
a phenoxy (20b) substituent at the 2-position led to a complete
loss of GK activity, which was probably due to the hydrogen bond-
ing interaction of the N–H of aminothiazole and the oxygen atom
of the alkoxy or phenoxy part. In contrast, the 3-isopropoxy (27c)
and 3-phenoxy (16a) derivatives significantly improved GK po-
tency as compared to the lead compound 7h. The 3-ethoxy com-
pound (27b) had activity comparable to the isoprpoxy analog
(27c), but the 3-methoxy (27a) substituent did not affect the GK
potency of 7h probably due to a lack of bulkiness.
0.33
a
Values are the means of two or more independent assays. Compound 2a is the
internal standard (EC50: 0.42 0.09 and 0.14 0.04).
b
The maximal activating response elicited the compounds as a ratio of the
maximal response evoked by 2a at each concentration independently.
4. Pharmacological results and discussion
To assess the lead potential of this structure class, in vivo effi-
cacy studies on the identified potent GKAs, the phenoxy-(16c)
and isopropoxy (27e) derivatives, were conducted. In the normal
fed mice, 27e significantly reduced the plasma glucose AUC level
after 30 mg/kg oral dosing, while 16c did not show a significant
glucose-lowering effect at 30 mg/kg (Fig. 3).
Next, we conducted the PK study and oral glucose tolerance test
(OGTT) in rats, using 27e. The data indicated that 27e had a mod-
erate PK profile in SD rats (Table 4). In the OGTT, 27e exhibited glu-
This result suggested that the introduction of the bulky substi-
tution onto the 3-position of the benzene ring was quite effective
in increasing GK activity. Then, a 3,5-bis substituted benzamide,
such as compounds 27c and 16a, was found to be a promising tem-
plate, showing good GK potency without an aniline moiety in the
structure.
Finally, further optimization on the substituent (R1) of the benz-
amides 27c and 16a was performed (Table 3). The EC50 values of
the 4-(methanesulfonyl)phenoxy derivatives (16c and 27e) were
superior to those of 2- (27c and 16a) or 3-(methanesulfonyl)phen-
oxy derivatives (16b and 27d), although the Emax value of 16c was
relatively low. It is noteworthy that this trend that the incorpora-
tion of 4-(methanesulfonyl)phenoxy group in 3,5-disubstituted
benzamides (16c and 27e) is effective in increasing GK potency,
is opposite to that seen in the 3-monosubstituted benzamides
(7h–j). We speculate that the binding mode of the 3,5-disubsti-
tuted benzamides in GK is slightly different from that of the 3-
monosubstituted benzamides and, as a result, the 4-(methanesul-
fonyl)phenoxy group of 16c or 27e occupies the additional interac-
tion space of GK. To confirm this hypothesis, we are attempting to
co-crystallize these derivatives with GK.
cose-lowering effects in
a
dose-dependent manner using
concentrations of 3 to 30 mg/kg (Figs. 4–1 and 4–2). In particular,
the decrease in glucose AUC seen with 27e administration was al-
most equivalent to that of the original compound with an aniline
moiety (2a).
5. Conclusion
In conclusion, modifications performed on the class of 2-amino-
benzamide derivatives with the aim to identify a potent and orally
active GK activator without an aniline structure, led to the discov-
ery of compound 27e. This compound was potent and orally bio-
available, and exhibited significant glucose-lowering efficacy in
the OGTT study in rats, at 10 and 30 mg/kg oral administration.
This was achieved by the deletion of the amine part of the benzam-

T. Iino et al. / Bioorg. Med. Chem. 17 (2009) 2733–2743
2737
Figure 4-2. Glucose AUC of 27e and 2a in rat OGTT.
Figure 3. Glucose AUC of 16c and 27e in normal fed mouse.
Table 4
Pharmacokinetic parameters of compound 27e in SD rat
a Waters micromass ZQ, micromass Quattro II or micromass Q-
Tof-2 instrument. Flash chromatography was carried out with pre-
packed silica gel columns (KP-Sil silica) from Biotage or (Purif-
Pack) from Moritex. Preparative thin-layer chromatography (TLC)
was performed on a TLC Silica Gel 60 F (Merck KGaA). Preparative
HPLC purification was carried out on a YMC-Pack Pro C18 (YMC,
50 mm ꢀ 30 mm id), eluting with a gradient of CH3CN/0.1% aq
CF3CO2H) 10:90–50:50 over 8 min at a flow rate of 40 mL/min.
High resolution mass spectra were recorded with electron-spray
ionization on a micromass Q-Tof-2 instrument. HPLC analysis
was performed on a SPELCO Ascentis Express (4.6 ꢀ 150 mm id),
eluting with a gradient of (a) 5:95–90:10 CH3CN: 0.1% aqueous
H3PO4, linear gradient over 7 min followed by 90:10 isocratic over
1 min and (b) 5:95–80:20 CH3CN: 10 mM potassium phosphate
buffer, linear gradient over 7 min followed by 80:20 isocratic over
1 min (detection at 210 nm). All solvent and reagent were obtained
from commercial sources and used without purification.
O
S
O
N
N
H
O
MeO2S
AUC0-1
M h)
CLtot (mL/
min/kg)
T1/2
(h)
Vdss
(L/kg)
Cmax
M)
Tmax
(h)
F
(%)
(
l
(
l
IV (1 mg/kg,
60% PEG)
9.7
4.1
1.1
0.4
PO (3 mg/kg,
0.5% MC)
12
2.9
1.0
40
F: oral bioavailability.
PEG: polyethylene glycol 400.
MC: methylcellulose.
6.2. N-(4-Methyl-1,3-thiazol-2-yl)-3-[(4-methyl-4H-1,2,4-
triazol-3-yl)sulfanyl]benzamide (3)
To a solution of NaNO2 (13.0 mg, 0.19 mmol) in concd. H2SO4
(0.085 mL) was added 2a (24.7 mg, 0.071 mmol) in AcOH
(0.3 mL), and the mixture was stirred at room temperature for
20 min. To the reaction mixture was added Cu2O (12 mg,
0.084 mmol) in EtOH (0.3 mL), and the mixture was stirred at
100 °C for 30 min. After cooling, the reaction was quenched with
saturated NaHCO3 solution and the resulting mixture was ex-
tracted with CHCl3. The organic phase was washed with brine,
dried over MgSO4, and evaporated under reduced pressure. The
residue was purified by preparative TLC on silica gel (10% MeOH/
CHCl3) to give
3 (5.6 mg, 24%) as a
colorless oil. 1H NMR
Figure 4-1. Blood glucose levels of 27e and 2a in rat OGTT.
(400 MHz, CDCl3) d 2.16 (3H, d, J = 1.0 Hz), 3.64 (3H, s), 6.56 (1H,
d, J = 1.0 Hz), 7.44 (1H, t, J = 7.7 Hz), 7.78 (1H, ddd, J = 1.0, 1.8,
7.7 Hz), 7.85 (1H, ddd, J = 1.0, 1.8, 7.7 Hz), 7.96 (1H, t, J = 1.8 Hz),
8.29 (1H, s); MS (ESI) m/z = 332 [M+H]+; HRMS (ESI) calcd for
C14H14N5OS2 332.0640; found 332.0645 [M+H]+.
ide core structure, followed by the incorporation of an isopropoxy
moiety at the 3-position. Further SAR study of this class is under-
way to identify a development candidate.
6. Experimental
6.1. Chemistry
6.3. 5-(2-Fluorophenoxy)-N-(4-methyl-1,3-thiazol-2-yl)-2-
nitrobenzamide (5b)
To a solution of 4 (204 mg, 0.73 mmol) in DMF (2.0 mL) were
added Et3N (1.21 mL, 8.71 mmol) and 2-fluorophenol (0.39 mL,
4.35 mmol), and the mixture was stirred at 100 °C overnight. After
cooling, the reaction was evaporated under reduced pressure. The
residue was purified by silica gel column chromatography (33%
EtOAc/hexane) to give 5b (244 mg, 90%) as a yellow oil. 1H NMR
(300 MHz, CDCl3) d 1.98 (3H, br s), 6.56 (1H, s), 6.98 (1H, s), 7.07–
7.24 (5H, m), 8.11 (1H, d, J = 9.0 Hz); MS (ESI) m/z = 374 [M+H]+.
In general, reagents and solvents were used as purchased and
without further purification. The 1H NMR spectra were obtained
at 300 MHz on a Gemini-300, 400 MHz on a MERCURY-400
(Varian) or 400 MHz on a JMN-AL400 (JEOL) spectrometer, with
chemical shift (d, ppm) expressed relative to TMS as an internal
standard. Mass spectra were recorded with electronspray ioniza-
tion (ESI) or atmospheric pressure chemical ionization (APCI) on

2738
T. Iino et al. / Bioorg. Med. Chem. 17 (2009) 2733–2743
6.4. 2-Amino-5-(2-fluorophenoxy)-N-(4-methyl-1,3-thiazol-2-
6.9. 3-(2-Methylphenoxy)-N-(4-methyl-1,3-thiazol-2-yl)benz-
yl)benzamide (6b)
amide (7e)
To a solution of 5b (240 mg, 0.64 mmol) in iPrOH (4.0 mL)
were added saturated NH4Cl solution (3.0 mL) and Fe (2.0 g),
and the mixture was stirred at 100 °C for 20 min. After cooling,
the mixture was filtered through a pad of Celite and the filtrate
was evaporated under reduced pressure. The residue was diluted
with water and EtOAc, and the organic phase was washed with
brine, dried over MgSO4, and evaporated. The residue was puri-
fied by silica gel column chromatography (25% EtOAc/hexane) to
give 6b (196 mg, 89%) as colorless foam. 1H NMR (300 MHz,
CD3OD) d 2.27 (3H, s), 6.61 (1H, s), 6.83 (1H, d, J = 9.0 Hz),
6.94–7.19 (5H, m), 7.42 (1H, d, J = 2.8 Hz); MS (ESI) m/z = 344
[M+H]+.
Compound 7e was prepared from 4 in a similar manner as de-
scribed for compound 6b and 3 as colorless foam. Yield: 51%; 6e;
1H NMR (300 MHz, CDCl3) d 2.26 (6H, s), 6.52 (1H, s), 6.71–6.76
(2H, m), 6.99–7.10 (4H, m), 7.21 (1H, d, J = 6.7 Hz); MS (ESI) m/
z = 340 [M+H]+; 7e; 1H NMR (300 MHz, CDCl3) d 2.05 (3H, s), 2.18
(3H, s), 6.53 (1H, s), 6.85 (1H, dd, J = 1.0, 7.9 Hz), 7.05–7.25 (4H,
m), 7.33 (1H, t, J = 1.0 Hz), 7.41 (1H, t, J = 7.9 Hz), 7.54 (1H, d,
J = 7.9 Hz); MS (ESI) m/z = 325 [M+H]+; HRMS (ESI) calcd for
C18H17N2O2S 325.1011; found 325.1021 [M+H]+.
6.10. 3-(3-Methylphenoxy)-N-(4-methyl-1,3-thiazol-2-yl)benz-
amide (7f)
Compound 7f was prepared from 4 in a similar manner as de-
scribed for compound 6b and 3 as colorless foam. Yield: 16%; 6f;
1H NMR (300 MHz, CDCl3) d 2.32 (6H, s), 6.53 (1H, s), 6.71–6.77
(3H, m), 6.86–6.89 (1H, m), 7.08 (1H, dd, J = 2.7, 8.9 Hz), 7.16–
7.21 (2H, m); MS (ESI) m/z = 340 [M+H]+; 7f; 1H NMR (300 MHz,
CDCl3) d 2.20–2.24 (3H, m), 2.34 (3H, s), 6.55 (1H, s), 6.79–6.85
(2H. m), 6.97 (1H, dd, J = 0.8, 7.6 Hz), 7.21–7.26 (2H, m), 7.44
(1H, t, J = 7.6 Hz), 7.48–7.50 (1H, m), 7.58 (1H, d, J = 7.6 Hz); MS
(ESI) m/z = 325 [M+H]+; HRMS (ESI) calcd for C18H17N2O2S
325.1011; found 325.1017 [M+H]+.
6.5. 3-(2-Fluorophenoxy)-N-(4-methyl-1,3-thiazol-2-yl)benza-
mide (7b)
Compound 7b was prepared from 6b in a similar manner as
described for compound 3 as colorless foam. Yield: 25%; 1H
NMR (300 MHz, CDCl3) d 2.09 (3H, d, J = 1.0 Hz), 6.54 (1H, d,
J = 1.0 Hz), 7.05–7.24 (5H, m), 7.39 (1H, s), 7.45 (1H, t,
J = 7.8 Hz), 7.58 (1H, d, J = 7.8 Hz); MS (ESI) m/z = 329 [M+H]+;
HRMS (ESI) calcd for C17H14N2O2FS 329.0760; found 329.0769
[M+H]+.
6.11. 3-(4-Methylphenoxy)-N-(4-methyl-1,3-thiazol-2-yl)benz-
amide (7g)
6.6. N-(4-Methyl-1,3-thiazol-2-yl)-3-phenoxybenzamide (7a)
Compound 7a was prepared from 4 in a similar manner as
described for compound 6b and 3 as colorless foam. Yield:
16%; 6a; 1H NMR (300 MHz, CDCl3) d 2.21–2.24 (3H, m), 5.53
(1H, s), 6.51 (1H, s), 6.75 (1H, d, J = 8.8 Hz), 6.86–6.89 (5H, m),
7.03–7.14 (3H, m), 7.25–7.30 (2H, m); MS (ESI) m/z = 326
Compound 7g was prepared from 4 in a similar manner as de-
scribed for compound 6b and 3 as colorless foam. Yield: 8%; 6g;
1H NMR (400 MHz, CDCl3) d 2.16 (3H, s), 2.24 (3H, s), 5.48 (2H,
s), 6.50 (1H, s), 6.72–6.75 (3H, m), 7.02–7.09 (4H, m); MS (ESI)
m/z = 340 [M+H]+; 7g; 1H NMR (400 MHz, CDCl3) d 2.13 (3H, br
s), 2.29 (3H, s), 6.55 (1H, s), 6.85–6.89 (2H. m), 7.13–7.16 (2H,
m), 7.23–7.26 (1H, m), 7.38 (1H, s), 7.41–7.46 (1H, m), 7.56–7.58
(1H, m); MS (ESI) m/z = 325 [M+H]+; HRMS (ESI) calcd for
C18H17N2O2S 325.1011; found 325.1021 [M+H]+.
[M+H]+; 7a; 1H NMR (400 MHz, CDCl3)
d 2.04 (3H, d,
J = 1.0 Hz), 6.54 (1H, d, J = 1.0 Hz), 6.98 (2H, d, J = 7.8 Hz), 7.12–
7.25 (3H, m), 7.26–7.45 (3H, m), 7.57–7.58 (1H, m); MS (ESI)
m/z = 311 [M+H]+; HRMS (ESI) calcd for C17H15N2O2S 311.0854;
found 311.0864 [M+H]+.
6.12. 3-(2-Methoxyphenoxy)-N-(4-methyl-1,3-thiazol-2-yl)benz-
amide (7k)
6.7. 3-(3-Fluorophenoxy)-N-(4-methyl-1,3-thiazol-2-yl)benz-
amide (7c)
Compound 7k was prepared from 4 in a similar manner as de-
scribed for compound 6b and 3 as colorless foam. Yield: 4%; 6k;
1H NMR (300 MHz, CDCl3) d 2.25–2.27 (3H, m), 3.86 (3H, s), 6.51
(1H, s), 6.73 (1H, d, J = 9.2 Hz), 6.80–6.90 (2H, m), 7.04–7.10 (4H,
m); MS (ESI) m/z = 356 [M+H]+; 7k; 1H NMR (300 MHz, CDCl3) d
2.02–2.10 (3H, m), 3.79 (3H, s), 6.53 (1H, s), 6.92–7.01 (3H, m),
7.12–7.22 (2H, m), 7.33–7.44 (2H, m), 7.53 (1H, d, J = 7.6 Hz); MS
(ESI) m/z = 341 [M+H]+; HRMS (ESI) calcd for C18H17N2O3S
341.0960; found 341.0965 [M+H]+.
Compound 7c was prepared from 4 in a similar manner as de-
scribed for compound 6b and 3 as colorless foam. Yield: 6%; 6c;
1H NMR (300 MHz, CDCl3) d 2.28 (3H, s), 6.52 (1H, d, J = 1.0 Hz),
6.59–6.79 (4H, m), 7.08 (1H, dd, J = 2.7, 8.8 Hz), 7.16–7.26 (2H,
m); MS (ESI) m/z = 344 [M+H]+; 7c; 1H NMR (300 MHz, CDCl3) d
2.25 (3H, s), 6.56 (1H, s), 6.72–6.86 (3H, m), 7.25–7.32 (2H, m),
7.49 (1H, t, J = 8.0 Hz), 7.56 (1H, s), 7.65 (1H, d, J = 8.0 Hz); MS
(ESI) m/z = 329 [M+H]+; HRMS (ESI) calcd for C17H14N2O2FS
329.0760; found 329.0766 [M+H]+.
6.13. 3-(2-Ethoxyphenoxy)-N-(4-methyl-1,3-thiazol-2-yl)benza-
6.8. 3-(4-Fluorophenoxy)-N-(4-methyl-1,3-thiazol-2-yl)benz-
mide (7l)
amide (7d)
Compound 7l was prepared from 4 in a similar manner as de-
scribed for compound 6b and 3 as colorless foam. Yield: 47%; 6l;
1H NMR (300 MHz, CDCl3) d 1.34 (3H, t, J = 7.0 Hz), 2.05–2.27
(3H, m), 4.07 (2H, q, J = 7.0 Hz), 5.44 (1H, s), 6.50 (1H, s), 6.73
(1H, d, J = 9.4 Hz), 6.81–6.86 (2H, m), 6.93–7.08 (4H, m); MS (ESI)
m/z = 370 [M+H]+; 7l; 1H NMR (300 MHz, CDCl3) d 1.23 (3H, t,
J = 7.1 Hz), 2.17–2.27 (3H, m), 4.01 (2H, q, J = 7.1 Hz), 6.54 (1H, d,
J = 1.0 Hz), 6.94–7.05 (3H, m), 7.12–7.20 (2H, m), 7.38–7.43 (2H,
m), 7.53 (1H, d, J = 7.7 Hz); MS (ESI) m/z = 355 [M+H]+; HRMS
(ESI) calcd for C19H19N2O3S 355.1116; found 355.1127 [M+H]+.
Compound 7d was prepared from 4 in a similar manner as de-
scribed for compound 6b and 3 as colorless foam. Yield: 6%; 6d; 1H
NMR (400 MHz, CDCl3) d 2.14 (3H, s), 5.53 (2H, s), 6.51 (1H, s), 6.75
(1H, d, J = 9.2 Hz), 6.78–6.83 (2H, m), 6.93–6.97 (2H, m), 7.03–7.06
(2H, m); MS (ESI) m/z = 344 [M+H]+; 7d; 1H NMR (400 MHz, CDCl3)
d 2.09 (3H, s), 6.55 (1H, s), 6.94–7.07 (4H, m), 7.19–7.22 (1H, m),
7.40–7.44 (2H, m), 7.57 (1H, d, J = 8.0 Hz); MS (ESI) m/z = 329
[M+H]+; HRMS (ESI) calcd for C17H14N2O2FS 329.0760; found
329.0767 [M+H]+.

T. Iino et al. / Bioorg. Med. Chem. 17 (2009) 2733–2743
2739
6.14. 3-[2-(Methylsulfonyl)phenoxy]-N-(4-methyl-1,3-thiazol-
2-yl)benzamide (7h)
6.16. 3-[3-(Methylsulfonyl)phenoxy]-N-(4-methyl-1,3-thiazol-
2-yl)benzamide (7i)
To a solution of 4 (427 mg, 1.52 mmol) in DMF (5.0 mL) were
added K2CO3 (2.50 g, 18.2 mmol) and 2-(methylthio)phenol
(0.80 mL, 9.10 mmol), and the mixture was stirred at 100 °C over-
night. After cooling, the mixture was partitioned between water
and EtOAc. The organic phase was washed with brine, dried over
MgSO4, and evaporated under reduced pressure. The residue was
purified by silica gel column chromatography (50% EtOAc/hexane)
to give 5h (607 mg, 99%) as a colorless oil. 1H NMR (300 MHz,
CDCl3) d 2.06 (3H, br s), 2.41 (3H, s), 6.56 (1H, s), 6.91–6.98 (2H,
m), 7.07–7.32 (4H, m), 8.10–8.13 (1H, m); MS (ESI) m/z = 402
[M+H]+.
To a solution of 5h (310 mg, 0.77 mmol) in iPrOH (5 mL) were
added saturated NH4Cl solution (6.0 mL) and Fe (3.0 g), and the
mixture was stirred at 100 °C for 20 min. After cooling, the mixture
was filtered through a pad of Celite and the filtrate was evaporated
under reduced pressure. The residue was diluted with water and
EtOAc, and the organic phase was washed with brine, dried over
MgSO4, and evaporated. The residue was purified by silica gel col-
umn chromatography (30% EtOAc/hexane) to give 6h (137 mg,
48%) as a colorless oil. 1H NMR (300 MHz, CDCl3) d 2.33 (3H, d,
J = 1.0 Hz), 2.46 (3H, s), 6.51 (1H, d, J = 1.0 Hz), 6.74 (1H, d,
J = 9.3 Hz), 7.05–7.11 (4H, m), 7.23–7.26 (1H, m); MS (ESI) m/
z = 372 [M+H]+.
To a suspension of NaNO2 (28.0 mg, 0.40 mmol) in concentrated
H2SO4 (0.2 mL) was added a solution of 6h (58.0 mg, 0.15 mmol) in
AcOH (3.0 mL), and the mixture was stirred at room temperature
for 20 min. To the mixture was added Cu2O (28.0 mg) in EtOH
(1.0 mL), and the mixture was further stirred at 50 °C for 30 min.
After cooling, the mixture was partitioned between saturated NaH-
CO3 solution and CHCl3. The organic phase was washed with brine,
dried over MgSO4, and evaporated. The residue was purified by
preparative TLC on silica gel (60% EtOAc/hexane) to give 3-[2-
(methylsulfanyl)phenoxy]-N-(4-methyl-1,3-thiazol-2-yl)benzam-
ide (18.0 mg) as a colorless oil.
To a solution of 9i (195 mg, 0.71 mmol) in CHCl3 (10 mL) was
added mCPBA (613 mg, 3.55 mmol) at 0 °C, and the mixture was
stirred for 2 h. The reaction was quenched with saturated Na2S2O3
solution and the resulting mixture was extracted with EtOAc. The
organic phase was washed with saturated NaHCO3 solution and
brine, dried over MgSO4, and evaporated to provide methanesulfo-
nyl derivative as colorless foam.
To a solution of the above obtained sulfone compound in a mix-
ture of CHCl3 (1.0 mL) and MeOH (3.0 mL) was added 5 N NaOH
solution (0.85 mL, 4.26 mmol), and the mixture was stirred at
room temperature overnight. The mixture was neutralized with
5 N HCl solution and extracted with CHCl3. The organic phase
was washed with brine, dried over MgSO4, and evaporated. To a
solution of the above–obtained carboxylic acid in CHCl3 (3.0 mL)
were added 2-amino-4-methylthiazole (162 mg, 1.42 mmol),
HOBt-hydrate (109 mg, 0.71 mmol) and EDCI (273 mg, 1.42 mmol),
and the mixture was stirred at room temperature overnight. The
mixture was evaporated and the residue was purified by silica
gel column chromatography (80% EtOAc/hexane) to give 7i
(172 mg, 62%) as colorless foam. 1H NMR (400 MHz, CDCl3) d
2.24 (1H, d, J = 1.0 Hz), 3.08 (3H, s), 6.58 (1H, d, J = 1.0 Hz), 7.28–
7.29 (2H, m), 7.50–7.60 (4H, m), 7.66–7.68 (1H, m), 7.71–7.73
(1H, m), 10.31 (1H, br s); MS (ESI) m/z = 389 [M+H]+; HRMS (ESI)
calcd for C18H17N2O4S2 389.0630; found 389.0628 [M+H]+.
6.17. Methyl 3-[4-(methylsulfanyl)phenoxy]benzoate (9j)
Compound 9j was prepared from 8 in a similar manner as de-
scribed for compound 9i as colorless foam. Yield: 9%; 1H NMR
(400 MHz, CDCl3) d 2.49 (3H, s), 3.90 (3H, s), 6.95–6.97 (1H, m),
7.20 (1H, dd, J = 1.3, 8.2 Hz), 7.26–7.30 (2H, m), 7.40 (1H, t,
J = 8.2 Hz), 7.63 (1H, dd, J = 1.3, 2.4 Hz), 7.77 (1H, dd, J = 1.3,
8.2 Hz); MS (ESI) m/z = 275 [M+H]+.
To a solution of the above obtained compound (16.0 mg,
0.042 mmol) in CHCl3 (2.0 mL) was added mCPBA (36.0 mg,
0.19 mmol) at 0 °C, and the mixture was stirred for 50 min. The
reaction was quenched with saturated Na2S2O3 solution and the
resulting mixture was extracted with EtOAc. The organic phase
was washed with saturated NaHCO3 solution and brine, dried over
MgSO4, and evaporated. The residue was purified by preparative
TLC on silica gel (50% EtOAc/hexane) to provide 7h (19.2 mg,
47%) as colorless foam. 1H NMR (300 MHz, CDCl3) d 2.14 (3H, d,
J = 1.0 Hz), 3.23 (3H, s), 6.56 (1H, d, J = 1.0 Hz), 6.94 (1H, dd,
J = 0.9, 8.2 Hz), 7.28–7.33 (2H, m), 7.47–7.59 (2H, m), 7.63 (1H, d,
J = 1.2 Hz), 7.70 (1H, dd, J = 1.2, 7.6 Hz), 8.08 (1H, dd, J = 1.4,
7.8 Hz); MS (ESI) m/z = 389 [M+H]+; HRMS (ESI) calcd for
C18H17N2O4S2 389.0630; found 389.0628 [M+H]+.
6.18. 3-[4-(Methylsulfonyl)phenoxy]-N-(4-methyl-1,3-thiazol-
2-yl)benzamide (7j)
Compound 7j was prepared from 9j in a similar manner as de-
scribed for compound 7i as colorless foam. Yield: 36%; 1H NMR
(400 MHz, CDCl3) d 2.31 (3H, d, J = 1.0 Hz), 3.09 (3H, s), 6.59 (1H,
d, J = 1.0 Hz), 7.14 (2H, dd, J = 2.0, 7.3 Hz), 7.33 (2H, dd, J = 2.0,
7.3 Hz), 7.57 (1H, t, J = 8.0 Hz), 7.64 (1H, t, J = 2.2 Hz), 7.72 (1H, d,
J = 8.0 Hz), 7.94 (1H, dd, J = 2.0, 7.3 Hz); MS (ESI) m/z = 389
[M+H]+; HRMS (ESI) calcd for C18H17N2O4S2 389.0630; found
389.0631 [M+H]+.
6.19. 3,5-Difluoro-N-(4-methyl-1,3-thiazol-2-yl)-2-nitrobenz-
amide (10)
6.15. Methyl 3-[3-(methylsulfanyl)phenoxy]benzoate (9i)
To 3,5-difluorobenzoic acid (1.00 g, 6.33 mmol) was added
fuming HNO3 (3.0 mL), and the mixture was stirred at room
temperature for 3 days. The mixture was poured into ice-cold
water (150 mL) and extracted with CHCl3. The organic phase
was washed with brine, dried over MgSO4, and evaporated. To
a solution of the residual compound in CHCl3 (15 mL) were
added 2-amino-4-methylthiazole (720 mg, 6.30 mmol), Et3N
(2.20 mL, 15.8 mmol) and 2-chloro-1,3-dimethylimidazolinium
chloride (1.33 g, 7.88 mmol), and the mixture was stirred at
room temperature overnight. The mixture was partitioned be-
tween water and CHCl3. The organic phase was washed with
brine, dried over MgSO4, and evaporated. The residue was puri-
fied by silica gel column chromatography (50% EtOAc/hexane) to
give 10 (485 mg, 26%) as yellow foam. 1H NMR (300 MHz, CDCl3)
To
a solution of 8 (1.0 g, 6.57 mmol) and [3-(methyl-
thio)phenyl]boronic acid (1.22 g, 7.23 mmol) in CHCl3 (50 mL)
were added Cu(OAc)2 (1.31 g, 7.23 mmol) and Et3N (4.58 mL,
32.9 mmol), and the mixture was stirred at room temperature
overnight. The mixture was filtered through a pad of Celite, and
the filtrate was concentrated. The residue was purified by silica
gel column chromatography (20% EtOAc/hexane) to give 9i
(198 mg, 11%) as a colorless oil. 1H NMR (400 MHz, CDCl3) d 2.46
(3H, s), 3.90 (3H, s), 6.74–6.76 (1H, m), 6.90 (1H, t, J = 2.0 Hz),
7.01–7.02 (1H, m), 7.21–7.22 (1H, m), 7.26–7.27 (1H, m), 7.41
(1H, d, J = 8.0 Hz), 7.66 (1H, dd, J = 1.5, 2.0 Hz), 7.78–7.80 (1H,
m); MS (ESI) m/z = 275 [M+H]+.

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T. Iino et al. / Bioorg. Med. Chem. 17 (2009) 2733–2743
d 2.28 (3H, d, J = 1.1 Hz), 6.50 (1H, d, J = 1.1 Hz), 7.13 (1H, ddd,
J = 2.5, 7.6, 9.4 Hz), 7.42 (1H, ddd, J = 1.8, 2.5, 8.0 Hz); MS (ESI)
m/z = 300 [M+H]+.
thio)phenyl]boronic acid (160 mg, 0.94 mmol) in CHCl3 (10 mL)
were added Cu(OAc)2 (140 mg, 0.79 mmol) and Et3N (0.55 mL,
3.93 mmol), and the mixture was stirred at room temperature
overnight. The mixture was filtered through a pad of Celite, and
the filtrate was concentrated. The residue was purified by silica
gel column chromatography (20% EtOAc/hexane) to give 15b
(81.0 mg, 28%) as a colorless oil. 1H NMR (300 MHz, CDCl3) d 2.46
(3H, s), 3.86 (3H, s), 6.76–6.79 (1H, m), 6.86–6.92 (2H, m), 6.96–
7.05 (3H, m), 7.14–7.28 (2H, m), 7.34–7.39 (4H, m).
6.20. 5-Fluoro-N-(4-methyl-1,3-thiazol-2-yl)-2-nitro-3-
phenoxybenzamide (11)
To a solution of 10 (130 mg, 0.43 mmol) in DMF (4.0 mL) were
added phenol (289 mg, 3.03 mmol) and Et3N (0.85 mL, 6.07 mmol),
and the mixture was stirred at 120 °C for 11 h. After cooling, the mix-
ture was concentrated in vacuo. The residue was purified by pre-
parative TLC on silica gel (40% EtOAc/hexane) to give 11 (110 mg,
68%) as yellow foam. 1H NMR (300 MHz, CDCl3) d 2.18 (3H, d,
J = 1.0 Hz), 6.55 (1H, d, J = 1.0 Hz), 6.71 (1H, dd, J = 2.6, 9.0 Hz), 7.04
(1H, dd, J = 2.6, 7.8 Hz), 7.09 (2H, d, J = 7.6 Hz), 7.26 (1H, t,
J = 7.6 Hz), 7.43 (2H, d, J = 7.6 Hz); MS (ESI) m/z = 374 [M+H]+.
6.25. 3-[3-(Methylsulfonyl)phenoxy]-N-(4-methyl-1,3-thiazol-
2-yl)-5-phenoxybenzamide (16b)
Compound 16b was prepared from 15b in a similar manner as
described for compound 7h as colorless foam. Yield: 77%; 1H
NMR (400 MHz, CDCl3) d 2.29 (3H, s), 3.08 (3H, s), 6.56 (1H, s),
6.92 (1H, d, J = 2.2 Hz), 7.07 (2H, d, J = 7.6 Hz), 7.20–7.22 (3H, m),
7.30–7.42 (3H, m), 7.56–7.62 (2H, m), 7.74 (1H, d, J = 7.6 Hz); MS
(ESI) m/z = 481 [M+H]+; HRMS (ESI) calcd for C24H21N2O5S2
481.0892; found 481.0897 [M+H]+.
6.21. 2-Amino-N-(4-methyl-1,3-thiazol-2-yl)-5-[2-
(methylthio)phenoxy]-3-phenoxybenzamide (12)
To a solution of 11 (85.0 mg, 0.23 mmol) in DMF (4.0 mL) were
added K2CO3 (315 mg, 2.28 mmol) and 2-(methylthio)phenol
(0.10 mL, 1.14 mmol), and the mixture was stirred at 100 °C over-
night. After cooling, the mixture was diluted with water and EtOAc.
The organic phase was washed with brine, dried over MgSO4, and
evaporated. The residue was purified by silica gel column chroma-
tography (50% EtOAc/hexane) to give the 5-[2-(methylthio)phen-
6.26. Methyl 3-[4-(methylthio)phenoxy]-5-phenoxy-benzoate
(15c)
Compound 15c was prepared from 14 in a similar manner as de-
scribed for compound 15b. Yield: 33%; 1H NMR (300 MHz, CDCl3) d
2.05 (3H, s), 2.45 (3H, s), 6.53 (1H, s), 6.89–7.36 (12H, m); MS (ESI)
m/z = 449 [M+H]+.
oxy]-2-nitro-3-phenoxybenzamide
colorless foam.
derivative
(78.0 mg)
as
Compound 12 was prepared from the above obtained com-
pound in a similar manner as described for compound 7h as color-
less foam. Yield: 53%; 1H NMR (300 MHz, CDCl3) d 2.27 (3H, s), 2.43
(3H, s), 6.53 (1H, s), 6.72–6.75 (1H, m), 6.77 (1H, d, J = 2.5 Hz), 6.90
(1H, d, J = 2.5 Hz), 7.03–7.07 (4H, m), 7.11–7.23 (2H, m), 7.33–7.38
(2H, m); MS (ESI) m/z = 464 [M+H]+.
6.27. 3-[4-(Methylsulfonyl)phenoxy]-N-(4-methyl-1,3-thiazol-
2-yl)-5-phenoxybenzamide (16c)
Compound 16c was prepared from 15c in a similar manner as
described for compound 7h as colorless foam. Yield: 54%; 1H
NMR (300 MHz, CDCl3) d 2.29 (3H, s), 3.08 (3H, s), 6.56 (1H, s),
6.92 (1H, d, J = 2.2 Hz), 7.07 (2H, d, J = 7.6 Hz), 7.20–7.22 (3H, m),
7.30–7.42 (3H, m), 7.56–7.62 (2H, m), 7.74 (1H, d, J = 7.6 Hz); MS
(ESI) m/z = 481 [M+H]+; HRMS (ESI) calcd for C24H21N2O5S2
481.0892; found 481.0900 [M+H]+.
6.22. 2-Amino-5-[2-(methylsulfonyl)phenoxy]-N-(4-methyl-
1,3-thiazol-2-yl)-3-phenoxybenzamide (13)
Compound 13 was prepared from 12 in a similar manner as de-
scribed for compound 7h as colorless foam. Yield: 20%; 1H NMR
(300 MHz, CDCl3) d 2.33 (3H, s), 3.28 (3H, s), 6.54 (1H, s), 6.81
(1H, d, J = 2.4 Hz), 6.86 (1H, d, J = 7.8 Hz), 7.05 (2H, d, J = 8.2 Hz),
7.16–7.22 (2H, m), 7.19 (1H, d, J = 2.4 Hz), 7.37 (2H, t, J = 8.2 Hz),
7.50 (1H, dt, J = 1.4, 7.8 Hz), 8.04 (1H, dd, J = 1.4, 7.9 Hz); MS (ESI)
m/z = 496 [M+H]+; HRMS (ESI) calcd for C24H22N3O5S2 496.1001;
found 496.0991 [M+H]+.
6.28. Methyl 5-iodo-2-(propan-2-yloxy)benzoate (18)
To a solution of methyl 17 (2.0 g, 7.19 mmol) in DMF (15 mL)
were added K2CO3 (1.49 g, 10.8 mmol) and 2-bromopropane
(0.88 mL, 9.35 mmol), and the mixture was stirred at 60 °C over-
night. After cooling, the mixture was partitioned between water
and EtOAc. The organic phase was washed with brine, dried over
MgSO4, and evaporated. The residue was purified by silica gel col-
umn chromatography (20% EtOAc/hexane) to give 18 (2.30 g, 93%)
as a colorless solid. 1H NMR (400 MHz, CDCl3) d 1.32–1.40 (6H, m),
3.87 (3H, s), 4.52–4.59 (1H, m), 6.75 (1H, d, J = 8.8 Hz), 7.68 (1H, dd,
J = 2.6, 8.8 Hz), 8.02 (1H, d, J = 2.6 Hz).
6.23. 3-[2-(Methylsulfonyl)phenoxy]-N-(4-methyl-1,3-thiazol-
2-yl)-5-phenoxybenzamide (16a)
Compound 16a was prepared from 13 in a similar manner as
described for the synthesis of 3 as colorless foam. Yield: 56%; 1H
NMR (300 MHz, CDCl3) d 2.17 (3H, s), 3.27 (3H, s), 6.55 (1H, s),
6.99 (1H, t, J = 2.1 Hz), 6.99 (1H, d, J = 7.8 Hz), 7.02 (2H, d,
J = 8.1 Hz), 7.17 (1H, t, J = 7.8 Hz), 7.31 (1H, d, J = 2.1 Hz), 7.30–
7.39 (1H, m), 7.34 (1H, d, J = 2.1 Hz), 7.36 (1H, t, J = 8.1 Hz), 7.59
(1H, t, J = 7.8 Hz), 8.08 (1H, d, J = 7.8 Hz); MS (ESI) m/z = 481
[M+H]+; HRMS (ESI) calcd for C24H21N2O5S2 481.0892; found
481.0885 [M+H]+.
6.29. Methyl 5-[2-(methylsulfanyl)phenoxy]-2-(propan-2-
yloxy)benzoate (19)
To a solution of 18 (540 mg, 1.69 mmol) in toluene (30 mL) were
added 2-(methylsulfanyl)phenol (473 mg, 3.37 mmol), copper(I)
trifluoromethanesulfonate benzene complex (1.69 g, 3.37 mmol)
and Cs2CO3 (1.65 g, 5.06 mmol) and EtOAc (3 drops), and the mix-
ture was stirred at 120 °C overnight. After cooling, the reaction mix-
ture was quenched with water and filtered through a pad of Celite.
The filtrate was evaporated and the residue was partitioned be-
tween water and EtOAc. The organic phase was washed with brine,
dried over MgSO4, and evaporated. The residue was purified by silica
gel column chromatography (10% EtOAc/hexane) to give 19
6.24. Methyl 3-[3-(methylthio)phenoxy]-5-phenoxy-benzoate
(15b)
To a suspension of methyl 3-hydroxy-5-phenoxybenzoate 14
(190 mg,
0.79 mmol),
MS4A
(1.0 g)
and
[3-(methyl-

T. Iino et al. / Bioorg. Med. Chem. 17 (2009) 2733–2743
2741
(34.0 mg, 6%) as a colorless oil. 1H NMR (400 MHz, CDCl3) d 1.36 (6H,
d, J = 6.3 Hz), 2.46 (3H, s), 3.85 (3H, s), 4.46–4.52 (1H, m), 6.79–6.87
(1H, m), 6.96 (1H, d, J = 9.3 Hz), 7.07–7.13 (3H, m), 7.25–7.28 (1H,
m), 7.41 (1H, d, J = 2.9 Hz; MS (ESI) m/z = 333 [M+H]+.
3.57 mmol) and iodomethane (0.22 mL, 3.57 mmol), and the mix-
ture was stirred at 60 °C overnight. After cooling, the mixture was
partitioned between water and EtOAc. The organic phase was
washed with brine, dried over MgSO4, and evaporated. The residue
was purified by silica gel column chromatography (30% EtOAc/hex-
ane) to give 25a (42.9 mg, 13%) as a colorless solid. 1H NMR
(400 MHz, CDCl3) d 3.83 (3H, s), 3.90 (3H, s), 5.17 (1H, s), 6.61 (1H,
t, J = 2.2 Hz), 7.12 (1H, dd, J = 1.5, 2.2 Hz), 7.16–7.17 (1H, m).
6.30. 5-[2-(Methylsulfonyl)phenoxy]-N-(4-methyl-1,3-thiazol-
2-yl)-2-(propan-2-yloxy)benzamide (20a)
Compound 20a was prepared from 19 in a similar manner as de-
scribed for the synthesis of 7i as colorless foam. Yield: 68%; 1H NMR
(400 MHz, CDCl3) d 1.58 (6H, d, J = 6.3 Hz), 2.38 (3H, d, J = 1.0 Hz),
3.31 (3H, s), 4.79–4.85 (1H, m), 6.56 (1H, s), 6.92 (1H, dd, J = 1.0,
8.3 Hz), 7.09 (1H, d, J = 9.3 Hz), 7.23–7.27 (1H, m), 7.31 (1H, dd,
J = 3.2, 9.3 Hz), 7.51–7.55 (1H, m), 8.05–8.07 (2H, m), 11.34 (1H, s);
MS (ESI) m/z = 447 [M+H]+; HRMS (ESI) calcd for C21H23N2O5S2
447.1048; found 447.1049 [M+H]+.
6.35. Methyl 3-ethoxy-5-hydroxybenzoate (25b)
Compound 25b was prepared from 24 in a similar manner as
described for compound 25a. Yield: 99%; 1H NMR (400 MHz,
CDCl3)
d 1.41 (3H, t, J = 6.9 Hz), 3.88 (3H, s), 4.05 (2H, q,
J = 6.9 Hz), 5.10 (1H, s), 6.60 (1H, t, J = 2.2 Hz), 7.10 (1H, dd,
J = 1.5, 2.2 Hz), 7.15 (1H, dd, J = 1.5, 2.2 Hz).
6.31. Methyl 2-bromo-5-[2-(methylsulfonyl)phenoxy]benzoate
(22)
6.36. Methyl 3-hydroxy-5-isopropoxybenzoate (25c)
Compound 25c was prepared from 24 as described for compound
25a. Yield: 99%; 1H NMR (400 MHz, CDCl3) d 1.32–1.33 (6H, m), 3.88
(3H, s), 4.52–4.61 (1H, m), 5.50 (1H, s), 6.60 (1H, t, J = 2.2 Hz), 7.12
(1H, dd, J = 1.5, 2.2 Hz), 7.15 (1H, dd, J = 1.5, 2.2 Hz).
To a solution of 21 (1.08 g, 4.67 mmol) in DMF (15 mL) were
added Cs2CO3 (3.05 g, 9.35 mmol) and 2-fluorophenymethylsulf-
one (1.22 g, 7.01 mmol), and the mixture was stirred at 80 °C
for 6 h. After cooling, the mixture was partitioned between
water and EtOAc. The organic phase was washed with brine,
dried over MgSO4, and evaporated. The residue was purified by
silica gel column chromatography (50% EtOAc/hexane) to give
22 (1.06 g, 59%) as a colorless oil. 1H NMR (400 MHz, CDCl3) d
3.29 (3H, s), 3.92 (3H, s), 6.96 (1H, dd, J = 1.4, 8.5 Hz), 7.08
(1H, dd, J = 2.9, 8.5 Hz), 7.30–7.33 (1H, m), 7.55 (1H, d,
J = 2.9 Hz), 7.59 (1H, dd, J = 1.4, 8.5 Hz), 7.67 (1H, d, J = 8.5 Hz),
8.09 (1H, dd, J = 1.4, 8.5 Hz).
6.37. Methyl 3-methoxy-5-[2-(methylsulfonyl)phenoxy]-
benzoate (26a)
To a solution of 25a (40.0 mg, 0.22 mmol) in DMF (1.5 mL) were
added Cs2CO3 (215 mg, 0.66 mmol) and 2-fluorophenymethylsulf-
one (76.0 mg, 0.44 mmol), and the mixture was stirred at 130 °C
for 2 h. After cooling, the mixture was partitioned between water
and EtOAc. The organic phase was washed with brine, dried over
MgSO4, and evaporated. The residue was purified by silica gel col-
umn chromatography (50% EtOAc/hexane) to give 26a (54.2 mg,
73%) as a colorless oil. 1H NMR (400 MHz, CDCl3) d 3.30 (3H, s),
3.86 (3H, s), 3.90 (3H, s), 6.87 (1H, d, J = 2.2 Hz), 6.96 (1H, d,
J = 8.0 Hz), 7.24–7.30 (1H, m), 7.35 (1H, d, J = 1.5 Hz), 7.43 (1H,
dd, J = 1.5, 2.2 Hz), 7.54–7.58 (1H, m), 8.08 (1H, dd, J = 1.5,
8.0 Hz); MS (ESI) m/z = 337 [M+H]+.
6.32. Methyl 5-[2-(methylsulfonyl)phenoxy]-2-
phenoxybenzoate (23)
To a solution of 22 (400 mg, 1.04 mmol) in pyridine (2.5 mL) were
added phenol (195 mg, 2.08 mmol), K2CO3 (287 mg, 2.08 mmol) and
CuO (165 mg, 2.08 mmol), and the mixture was stirred at 130 °C
overnight. After cooling, the mixture was diluted with saturated
NH4Cl solution and filtered through a pad of Celite. The filtrate
was evaporated and the residue was diluted with water and EtOAc.
The organic phase was washed with brine, dried over MgSO4, and
evaporated. The residue was purified by silica gel column chroma-
tography (50% EtOAc/hexane) to give 23 (172 mg, 42%) as yellow
foam. 1H NMR (400 MHz, CDCl3) d 3.32 (3H, s), 3.80 (3H, s), 6.96–
6.98 (3H, m), 7.03 (1H, d, J = 9.3 Hz), 7.09–7.12 (1H, m), 7.21–7.30
(2H, m), 7.33–7.36 (2H, m), 7.54–7.59 (1H, m), 7.68 (1H, d,
J = 2.9 Hz), 8.08–8.09 (1H, m); MS (ESI) m/z = 399 [M+H]+.
6.38. Methyl 3-ethoxy-5-[2-(methylsulfonyl)phenoxy]-
benzoate (26b)
Compound 26b was prepared from 25b in a similar manner as
described for compound 26a. Yield: 91%; 1H NMR (400 MHz,
CDCl3) d 1.41–1.44 (3H, m), 3.29 (3H, s), 3.90 (3H, s), 4.04–4.11
(2H, m), 6.85 (1H, d, J = 2.2 Hz), 6.96 (1H, d, J = 8.0 Hz), 7.27–7.29
(1H, m), 7.34 (1H, d, J = 1.5 Hz), 7.42 (1H, d, J = 1.5 Hz), 7.53–7.58
(1H, m), 8.08 (1H, dd, J = 1.5, 8.0 Hz); MS (ESI) m/z = 351 [M+H]+.
6.33. 5-[2-(Methylsulfonyl)phenoxy]-N-(4-methyl-1,3-thiazol-
2-yl)-2-phenoxybenzamide (20b)
6.39. Methyl 3-isopropoxy-5-[2-(methylsulfonyl)phenoxy]-
benzoate (26c)
Compound 20b was prepared from 23 in a similar manner as
described for the synthesis of 7i as colorless foam. Yield: 55%; 1H
NMR (400 MHz, CDCl3) d 2.35 (3H, d, J = 1.0 Hz), 3.31 (3H, s), 6.57
(1H, d, J = 1.0 Hz), 6.86 (1H, d, J = 8.8 Hz), 6.97–6.99 (1H, m),
7.19–7.21 (3H, m), 7.27–7.32 (2H, m), 7.45–7.48 (2H, m), 7.54–
7.58 (1H, m), 8.07–8.09 (2H, m), 10.91 (1H, s);.MS (ESI) m/z = 481
[M+H]+; HRMS (ESI) calcd for C24H21N2O5S2 481.0892; found
482.0882 [M+H]+.
Compound 26c was prepared from 25c in a similar manner as
described for compound 26a. Yield: 99%; 1H NMR (400 MHz,
CDCl3) d 1.33–1.35 (6H, m), 3.30 (3H, s), 3.88 (3H, s), 4.56–4.62
(1H, m), 6.83 (1H, d, J = 2.2 Hz), 6.97 (1H, dd, J = 1.5, 8.0 Hz),
7.24–7.29 (1H, m), 7.32 (1H, d, J = 1.5, 2.2 Hz), 7.41 (1H, dd,
J = 1.5, 2.2 Hz), 7.53–7.58 (1H, m), 8.07 (1H, dd, J = 1.5, 8.0 Hz);
MS (ESI) m/z = 365 [M+H]+.
6.40. 3-Methoxy-5-[2-(methylsulfonyl)phenoxy]-N-(4-methyl-
6.34. Methyl 3-hydroxy-5-methoxybenzoate (25a)
1,3-thiazol-2-yl)benzamide (27a)
To a solution of methyl 3,5-dihydroxybenzoate 24 (300 mg,
To a solution of 26a (50.0 mg, 0.15 mmol) in a mixture of CHCl3
1.78 mmol) in DMF (1.0 mL) were added K2CO3 (490 mg,
(1.0 mL) and MeOH (1.0 mL) was added 5 N NaOH solution

2742
T. Iino et al. / Bioorg. Med. Chem. 17 (2009) 2733–2743
(0.15 mL, 0.74 mmol), and the mixture was stirred at room temper-
ature overnight. The mixture was neutralized with 5 N HCl solu-
tion and extracted with CHCl3. The organic phase was washed
with brine, dried over MgSO4, and evaporated. To a solution of
the above obtained carboxylic acid in CHCl3 (3.0 mL) were added
2-amino-4-methylthiazole (33.3 mg, 0.29 mmol), HOBt-hydrate
(67.0 mg, 0.44 mmol) and EDCI (55.9 mg, 0.29 mmol), and the mix-
ture was stirred at room temperature overnight. The mixture was
evaporated and the residue was purified by silica gel column chro-
matography (70% EtOAc/hexane) to give 27a (42.4 mg, 70%) as col-
orless foam. 1H NMR (400 MHz, CDCl3) d 2.23–2.30 (3H, m), 3.29
(3H, s), 3.84 (3H, s), 6.57 (1H, d, J = 1.0 Hz), 6.88 (1H, d,
J = 2.2 Hz), 6.99–7.01 (1H, m), 7.16 (1H, t, J = 1.7 Hz), 7.28–7.29
(1H, m), 7.30–7.33 (1H, m), 7.56–7.60 (1H, m), 8.08 (1H, dd,
J = 1.7, 8.0 Hz), 10.01 (1H, br s); MS (ESI) m/z = 419 [M+H]+; HRMS
(ESI) calcd for C19H19N2O5S2 419.0735; found 419.0728 [M+H]+.
7.02 (1H, m), 7.21–7.23 (1H, m), 7.26–7.27 (1H, m), 7.29–7.31 (1H,
m); MS (ESI) m/z = 333 [M+H]+.
6.46. 3-Isopropoxy-5-[3-(methylsulfonyl)phenoxy]-N-(4-
methyl-1,3-thiazol-2-yl)benzamide (27d)
Compound 27d was prepared from 29d in a similar manner as
described for compound 7h as colorless foam. Yield: 20%; 1H
NMR (400 MHz, CDCl3) d 1.36 (6H, d, J = 6.3 Hz), 2.30 (3H, s), 3.08
(3H, s), 4.55–4.61 (1H, m), 6.58 (1H, s), 6.77 (1H, t, J = 2.0 Hz),
7.08 (1H, t, J = 2.0 Hz), 7.23 (1H, t, J = 2.0 Hz), 7.30 (1H, dd, J = 2.0,
7.9 Hz), 7.56–7.59 (2H, m), 7.72 (1H, d, J = 7.9 Hz); MS (ESI) m/
z = 447 [M+H]+; HRMS (ESI) calcd for C21H23N2O5S2 447.1048;
found 447.1047 [M+H]+.
6.47. 3-Isopropoxy-5-[4-(methylsulfonyl)phenoxy]-N-(4-
methyl-1,3-thiazol-2-yl)benzamide (27e)
6.41. 3-Ethoxy-5-[2-(methylsulfonyl)phenoxy]-N-(4-methyl-
1,3-thiazol-2-yl)benzamide (27b)
Compound 27e was prepared from 29e in a similar manner as
described for compound 7h as colorless foam. Yield: 56%; 1H
NMR (300 MHz, CDCl3) d 1.34 (6H, d, J = 6.0 Hz), 2.22 (3H, d,
J = 0.7 Hz), 3.08 (3H, s), 4.53–4.57 (1H, m), 6.57 (1H, d, J = 0.7 Hz),
6.80 (1H, t, J = 2.0 Hz), 7.11 (1H, d, J = 2.0 Hz), 7.12 (2H, d,
J = 8.8 Hz), 7.27 (1H, d, J = 2.0 Hz), 7.92 (2H, d, J = 8.8 Hz); MS
(ESI) m/z = 447 [M+H]+; HRMS (ESI) calcd for C21H23N2O5S2
447.1048; found 447.1053 [M+H]+; HPLC (a) 99.6%; (b) 99.7%.
Compound 27b was prepared from 26b in a similar manner as
described for compound 27a as colorless foam. Yield: 67%; 1H
NMR (400 MHz, CDCl3) d 1.42 (3H, t, J = 7.1 Hz), 2.29 (3H, s), 3.27
(3H, s), 4.06 (1H, q, J = 7.1 Hz), 6.57 (1H, s), 6.86 (1H, d,
J = 2.2 Hz), 6.99–7.01 (1H, m), 7.16 (1H, t, J = 1.7 Hz), 7.26–7.27
(1H, m), 7.30–7.34 (1H, m), 7.56–7.60 (1H, m), 8.09 (1H, dd,
J = 1.7, 7.0 Hz), 10.00 (1H, br s); MS (ESI) m/z = 433 [M+H]+; HRMS
(ESI) calcd for C20H21N2O5S2 433.0892; found 483.0888 [M+H]+.
7. Biology
7.1. In vitro GK assay
6.42. 3-Isopropoxy-5-[2-(methylsulfonyl)phenoxy]-N-(4-
methyl-1,3-thiazol-2-yl)benzamide (27c)
The recombinant human liver GK used in this assay was ex-
pressed in Escherichia coli as a FLAG fusion protein. GK activity
was measured by the glucose-6-phosphate dehydrogenase coupled
continuous spectrophotometric assay. GK was incubated with
DMSO solution in assay buffer containing 25 mM Hepes, pH 7.2,
1 mM dithiothreitol, 0.5 mM thionicotinamide adenine dinucleo-
tide, 2 mM MgCl2, 1 mM ATP, 2 U/mL glucose-6-phosphate dehy-
drogenase and 2.5 or 10 mM glucose at 30 °C. Reaction velocities
were obtained from the rate of increase in absorbance at 405 nm
after 5 min of reaction. The OD values were measured at each con-
centration of the evaluated compound, using the OD value of the
Compound 27c was prepared from 26c in a similar manner as
described for compound 27a as colorless foam. Yield: 52%; 1H
NMR (400 MHz, CDCl3) d 1.35 (6H, s), 2.29 (3H, s), 3.29 (3H, s),
4.55–4.61 (1H, m), 6.57 (1H, s), 6.85 (1H, d, J = 2.2 Hz), 7.00 (1H,
d, J = 8.0 Hz), 7.12–7.13 (1H, m), 7.25 (1H, s), 7.31 (1H, d,
J = 8.0 Hz), 7.56–7.60 (1H, m), 8.09 (1H, dd, J = 1.7, 7.0 Hz), 10.00
(1H, br s); MS (ESI) m/z = 447 [M+H]+; HRMS (ESI) calcd for
C21H23N2O5S2 447.1048; found 447.1042 [M+H]+.
6.43. Methyl 3-hydroxy-5-[4-(methylthio)phenoxy]-benzoate
(28e)
DMSO control as 100%. The EC50 (lM) values were calculated from
the OD value at each concentration, and used as indices of GK acti-
vator potency of the compound.
Compound 28e was prepared from 24 as described for com-
pound 15b. Yield: 22%; 1H NMR (300 MHz, CD3OD) d 2.46 (3H, s),
3.82 (3H, s), 6.58 (1H, t, J = 1.0 Hz), 6.97 (2H, d, J = 8.8 Hz), 7.01
(1H, t, J = 1.0 Hz), 7.14 (1H, t, J = 1.0 Hz), 7.30 (2H, d, J = 8.8 Hz).
7.2. In vivo assay in mice
Ten-to-eleven-week old male ICR mice (n = 4ꢁ5) were freely
fed prior to performing the test. The mice were orally adminis-
trated compounds 16c, 27e or vehicle alone (0.5% methylcellulose
solution). Blood glucose concentrations were measured just prior
to and following oral dosing (0.5, 1, 1.5, 2, 3 and 4 h). AUC values
were calculated from the data (from 0 h to 4 h).
6.44. Methyl 3-isopropoxy-5-[4-(methylthio)phenoxy]-
benzoate (29e)
Compound 29e was prepared from 28e as described for the
synthesis of 25a. Yield: 89%; 1H NMR (300 MHz, CDCl3) d 1.32
(6H, s), 2.48 (3H, s), 3.88 (3H, s), 4.52–4.61 (1H, m), 6.70 (1H,
d, J = 1.0 Hz), 6.96 (2H, d, J = 8.8 Hz), 7.18 (1H, t, J = 1.0 Hz),
7.26–7.31 (3H, m).
7.3. Oral glucose tolerance test (OGTT)
Nine-week old male Wistar rats (n = 5) were fasted overnight
before performing the test. The rats were orally administrated
compounds 27e, 2a or vehicle alone (0.5% methylcellulose solu-
tion), followed 30 min later by an oral glucose challenge (2 g/kg).
Plasma glucose concentrations were measured just prior to and fol-
6.45. Methyl 3-isopropoxy-5-[3-(methylthio)phenoxy]-
benzoate (29d)
Compound 29d was prepared from 24 as described for the syn-
thesis of 15b and 25a. Yield: 11%; 1H NMR (400 MHz, CDCl3) d 1.34
(6H, d, J = 6.3 Hz), 2.46 (3H, s), 3.88 (3H, s), 4.55–4.61 (1H, m), 6.72
(1H, d, J = 2.1 Hz), 4.75–4.78 (1H, m), 6.91 (1H, t, J = 2.1 Hz), 7.00–
ICR mice: mice used for general purposes and established at The Institute for
Cancer Research.

T. Iino et al. / Bioorg. Med. Chem. 17 (2009) 2733–2743
2743
8. Guertin, K. R.; Grimsby, J. Curr. Med. Chem. 2006, 13, 1839.
9. Coghlan, M.; Leighton, B. Exp. Opin. Invest. Drugs 2008, 17, 145.
10. Futamura, M.; Hosaka, H.; Kadotani, A.; Shimazaki, H.; Shimazaki, H.; Sasaki,
K.; Ohyama, S.; Nishimura, T.; Eiki, J.; Nagata, Y. J. Biol. Chem. 2006, 281, 37668.
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lowing the glucose challenge (30, 60, 90, and 120 min). AUC values
were calculated from the data (from ꢁ30 min to 120 min).
Acknowledgments
12. Daniewski, A.R.; Liu, W.; Radinov, R.N. WO Patent 2007/115968 .
13. Efanov, A. M.; Barrett, D. G.; Brenner, B. M.; Briggs, S. L.; Delaunois, A.; Durbin, J.
D.; Giese, U.; Guo, H.; Radloff, M.; Sanz, G. G.; Sewing, S.; Wang, Y.; Weichert,
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The authors are grateful to Hirokazu Ohsawa and Miyoko
Miyashita for the analytical and spectral studies and Hiroshi Takeh-
ana for the PK study in rats. We would also like to thank Dr. J. Sakaki
and Dr. N. Ohtake for critical reading of the manuscript.
15. Bertram, L. S.; Black, D.; Briner, P. H.; Chatfield, R.; Cooke, A.; Fyfe, M. C. T.;
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