Discovery of 3-(4-methanesulfonylphenoxy)-N-[1-(2-methoxy-ethoxymethyl)-1H- pyrazol-3-yl]-5-(3-methylpyridin-2-yl)-benzamide as a novel glucokinase activator (GKA) for the treatment of type 2 diabetes mellitus

Article abstract of DOI:10.1016/j.bmc.2014.02.009

Novel heteroaryl-containing benzamide derivatives were synthesized and screened using an in vitro assay measuring increases in glucose uptake and glucokinase activity stimulated by 10 mM glucose in rat hepatocytes. From a library of synthesized compounds, 3-(4-methanesulfonylphenoxy)-N-[1-(2-methoxy- ethoxymethyl)-1H-pyrazol-3-yl]-5-(3-methyl pyridin-2-yl)-benzamide (19e) was identified as a potent glucokinase activator with assays demonstrating an EC₅₀ of 315 nM and the induction of a 2.23 fold increase in glucose uptake. Compound 19e exhibited a glucose AUC reduction of 32% (50 mg/kg) in an OGTT study with C57BL/6J mice compared to 28% for metformin (300 mg/kg). Single treatment of the compound in C57BL/J6 and ob/ob mice elicited basal glucose lowering activity, while in a two-week repeated dose study with ob/ob mice, the compound significantly decreased blood glucose levels with no evidence of hypoglycemia risk. In addition, 19e exhibited favorable pharmacokinetic parameters in mice and rats and excellent safety margins in liver and testicular toxicity studies. Compound 19e was therefore selected as a development candidate for the potential treatment of type 2 diabetes.

Full text of DOI:10.1016/j.bmc.2014.02.009

Bioorganic & Medicinal Chemistry 22 (2014) 2280–2293
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Discovery of 3-(4-methanesulfonylphenoxy)-N-[1-(2-methoxy-
ethoxymethyl)-1H-pyrazol-3-yl]-5-(3-methylpyridin-2-yl)-benzamide
as a novel glucokinase activator (GKA) for the treatment of type 2
diabetes mellitus
⇑
Kaapjoo Park , Byoung Moon Lee, Kwan Hoon Hyun, Dong Hoon Lee, Hyun Ho Choi, Hyunmi Kim,
Wonee Chong, Kyeong Bae Kim, Su Youn Nam
Yuhan Research Institute, 25, Tapsil-ro 35beon-gil, Giheung-gu, Yongin-si, Gyeonggi-do, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel heteroaryl-containing benzamide derivatives were synthesized and screened using an in vitro
assay measuring increases in glucose uptake and glucokinase activity stimulated by 10 mM glucose in
rat hepatocytes. From a library of synthesized compounds, 3-(4-methanesulfonylphenoxy)-N-[1-(2-
methoxy-ethoxymethyl)-1H-pyrazol-3-yl]-5-(3-methyl pyridin-2-yl)-benzamide (19e) was identified
as a potent glucokinase activator with assays demonstrating an EC50 of 315 nM and the induction of a
2.23 fold increase in glucose uptake. Compound 19e exhibited a glucose AUC reduction of 32% (50 mg/
kg) in an OGTT study with C57BL/6J mice compared to 28% for metformin (300 mg/kg). Single treatment
of the compound in C57BL/J6 and ob/ob mice elicited basal glucose lowering activity, while in a two-week
repeated dose study with ob/ob mice, the compound significantly decreased blood glucose levels with no
evidence of hypoglycemia risk. In addition, 19e exhibited favorable pharmacokinetic parameters in mice
and rats and excellent safety margins in liver and testicular toxicity studies. Compound 19e was therefore
selected as a development candidate for the potential treatment of type 2 diabetes.
Received 27 December 2013
Revised 5 February 2014
Accepted 9 February 2014
Available online 17 February 2014
Keywords:
Type 2 diabetes mellitus (T2DM)
Glucokinase activator (GKA)
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
(e.g., biguanides, a-glucosidase inhibitors, sulfonylureas, gliptins,
TZDs, DPP-4 inhibitors, incretin mimetics (GLP-1 analogs) and
newly approved SGLT2 inhibitors3 (dapagliflozin4 and canaglifo-
zin5)), these drugs are effective in reducing plasma glucose or gly-
cated hemoglobin A1c (HbA1c), but monotherapy commonly
results in treatment failure, leading to the need for combination
therapy.6 In addition, some drugs exhibit side effect profiles that
include an increased risk of hypoglycemia (sulfonylureas, gliptins),
Hyperglycemia is a condition commonly caused by defects in
the secretion or action of insulin, and triggers the development
of diabetes mellitus. Chronic hyperglycemic conditions typical of
diabetes are associated with macrovascular complications includ-
ing coronary artery disease, peripheral arterial disease and stroke.1
Diabetes can also cause microvascular complications such as dia-
betic retinopathy, nephropathy and neuropathy.
GI complications (biguanides, a-glucosidase inhibitors), cardiovas-
Type 2 diabetes mellitus (T2DM) now affects over 300 million
people worldwide. Rates of diabetes have increased noticeably
over the last 50 years with a concomitant trend in the increasing
rates of obesity, which is thought to be a primary factor in its
cause.2 Although various anti-diabetic drugs are available
cular risk (TZDs), pancreatitis risk (DPP-4 inhibitors, incretin
mimetics) and genital/urinary tract infections (SGLT2 inhibitors).
Thus, there exist interrelated unmet needs in type 2 diabetes that
include a better balance of efficacy of glycemic control with cardio-
vascular safety, improved durability of treatment, hypoglycemia
avoidance, tolerability and ease of compliance.
Glucokinase (GK, also called hexokinase IV or hexokinase D) is a
hexokinase isozyme composed of 465 amino acids (molecular
weight = 50 kD). GK facilitates phosphorylation of glucose to glu-
cose-6-phosphate, which comprises the first step of both glycogen
synthesis and glycolysis. GK is expressed in cells of the liver,
Abbreviations: GK, glucokinase; GKA, glucokinase activator; SU, sulfonylurea;
DPP-4, dipeptidyl peptidase-4; TZD, thiazolidinedione; SGLT2, sodium-glucose
co-transporter 2; GSIS, glucose-stimulated insulin secretion; OGTT, oral glucose
tolerance test; PK, pharmacokinetics; T2DM, Type 2 diabetes mellitus.
⇑
Corresponding author. Tel.: +82 31 899 4274; fax: +82 31 275 6145.
E-mail address: kaapjoo@yuhan.co.kr (K. Park).
http://dx.doi.org/10.1016/j.bmc.2014.02.009
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

K. Park et al. / Bioorg. Med. Chem. 22 (2014) 2280–2293
2281
pancreas, gut, and brain in humans and many other vertebrates. In
comparison to other hexokinases, GK has a lower affinity for glu-
cose and its activity is localized to only a few cell types. Partly as
a result of this reduced affinity, GK activity varies substantially
with the concentration of glucose present. Furthermore, unlike
other hexokinases, GK is not inhibited by its product, glucose-6-
phosphate7 and distinctively, GK shows moderate cooperativity
with glucose, with a Hill coefficient (nH) of approximately 1.7.8
Slow conformational changes between multiple interconverting
states have been proposed to be responsible for this kinetic posi-
tive cooperativity.9 Due to this moderate cooperativity, a half-sat-
uration level S0.5 (the concentration at which the enzyme is 50%
saturated and active) is used for GK instead of using a Km for glu-
cose.10 GK acts as a glucose sensor regulating hepatic glucose
metabolism to provide approximately 95% of the hexokinase activ-
ity in hepatocytes8 and GK activity serves as a key control for glu-
cose-dependent insulin secretion in islet beta cells.11 Glucokinase
activator (GKA) is associated with a dual mechanism for lowering
blood glucose concentrations by enhancing glucose uptake in the
liver and increasing insulin secretion from pancreatic beta cells.
Therefore, GK has been an attractive target for anti-diabetic ther-
apy over the last two decades. Several GKA candidates have ad-
vanced to clinical studies and have been shown to lower both
fasting and postprandial glucose levels in healthy subjects and
T2DM patients. Hypoglycemia and liver or testicular toxicity12
have been revealed as the primary adverse effects of concern for
GKAs. To address the hypoglycemia issue, several clinical strategies
have been utilized including dose titration and more frequent dos-
ing times. Recently, two novel strategies have been employed to
reduce the potential for hypoglycemic induction. One strategy is
the design of partial activators that improve the dependence of
enzymatic activity on various physiological glucose levels.13 The
other is to create liver-selective glucose activators14–16 that restrict
the main enzymatic activity in the liver, since hypoglycemia risk is
postulated to result from increases in pancreatic insulin secretion
at low glucose levels. Initially, we adopted a dose titration clinical
strategy to reduce the possibility of hypoglycemia caused by
GKA.17,18 Later, we employed another strategy involving the mon-
itoring of activation profiles of dose-dependent S0.5, where the
ideal S0.5 could be higher than 2. Through this strategy, we could
demonstrate control of the balance between the potency of GKA
and its adverse effects including hypoglycemia and liver toxicity.
To address the testicular toxicity issue12 that is presumably re-
lated to compound specificity, we performed 7-day tox screen test-
ing in C57Bl/6J mice using high doses of the compounds. This
approach led to the discovery of a potent GKA lead candidate with
good efficacy and a desirable safety margin. There are arguments
disputing the value of GK activation for the treatment of T2DM after
two decades of attempts, that have occurred without medical suc-
cess in GKA development.19 However, several phase I studies have
demonstrated that GKAs can normalize blood sugar in T2DM
patients20 and long-term GKA treatment prevents the development
of diet-induced diabetes mellitus in mice.21 Currently, several small
molecule GKAs are in human clinical studies (phase I and II).
complex27 and other GK–GKA complexes.31 Due to the valuable
structural information generated from research into these com-
plexes, it is now possible to rationally design compounds for further
improvement of molecule-target binding motifs. GKAs hypotheti-
cally bind to the allosteric binding site of the protein to achieve
anti-hyperglycemic effects. Various benzamide derivatives have
been prepared based on binding mode analysis of the X-ray struc-
ture of the allosteric binding site of GK. More recently, Park, et al.,
identified a novel phenylethyl benzamide GKA (9)17,18 which exhib-
its good biological and pharmacological activity with favorable
pharmacokinetics. Herein, we report the discovery of a novel pyra-
zole benzamide glucokinase activator harboring 3-methylpyridine
and 4-phenoxymethyl sulfone groups, as a promising preclinical
candidate for the treatment of T2DM.
2. Results and discussion
2.1. Chemistry
The benzamide scaffold was chosen as a starting point for the
synthesis of selective GKAs due to the existence of unambiguous
structural information from the known co-crystal complexes. Ini-
tially, hundreds of benzamide derivatives with various different
substituents around a central benzene ring were synthesized based
on the binding mode hypothesis shown in Figure 2.
The A-region of the molecule is required to have both a hydrogen
bond donor (NH) and a hydrogen bond acceptor (@N) in order to
bind to Arg63 favorably. The B moiety is required to be of a small size
with potential hydrophobic interactions occurring with Tyr214,
Tyr215 and Leu451. The C-pocket of the enzyme is fairly large and
the end portionof C-moietyhas the potentialfor a hydrogen bonding
interaction with Arg250 in order to increase binding affinity.
Through initial diversification and structure–activity relationship
(SAR) analysis, 3-methylpyridine and 4-phenoxymethyl sulfone
groups have been identified as optimal moieties for the B-region
and C- region, respectively. The A-region was then investigated sys-
tematically by introducing a variety of aryl or hetero-aromatic
groups which led to the identification of thiazole and pyrazole
groups by prioritizing compounds based on in vitro potency and
physicochemical properties. The synthesis of compounds, thiazole
benzamide derivatives and pyrazole benzamide derivatives is out-
lined in Scheme 1.
The reaction of 4-fluorophenyl methyl sulfone and methyl
3,5-dihydroxybenzoate in the presence of Cs2CO3 gave phenoxy
phenol 10. Triflation of phenoxy phenol 10 followed by boration of the
resulting triflate afforded boron compound 11. Pd-catalyzed cou-
pling of 11 with 2-bromo-3-methylpyridine provided 2-(3-methyl
pyridyl)-benzene methyl ester 12 in moderate yield. Hydrolysis of
methyl ester 12 yielded benzoic acid 13. The amide coupling reac-
tion of 13 with ethyl 2-aminothiazole-4-acetate gave thiazole amide
ethyl ester and hydrolysis of the resulting ethyl ester provided acid
14. A subsequent amide coupling reaction of 14 with various amines
afforded final amide products 15a–b. An amide coupling reaction of
acid 13 with 2-aminothiazole or 5-fluoro-2-aminothiazole, or
4-(chloromethyl)-2-aminothiazole produced thiazole amides 16a,
16b, and 17, respectively. 4-(Chloromethyl)-thiazole amide 17 was
further elaborated to generate a variety of aminomethyl thiazoles
18a–k by using routine nucleophilic amine substitution reactions.
In addition, benzoic acid 13 was coupled with various substituted
pyrazoles that were prepared beforehand to make pyrazole amide
derivatives 19a–e as shown in Scheme 2. The prepared thiazole
benzamides and pyrazole benzamides were tested using an
in vitro assay by measuring fold increase of GK activity at 10 mM
glucose concentration. The compounds that exhibited satisfactory
GK is also an attractive target for the treatment of T2DM due to
its dual action resulting in hepatic and pancreatic effects. A number
of allosteric small molecule GKAs have been investigated by numer-
ous research groups over the past decade22–24 (selected representa-
tive small molecule GKAs are shown in Fig. 1). Since the first report
of small molecule allosteric GKAs in 2003,25 a phenylacetamide ser-
ies (2),26 benzamides (1,27 4–728–30,12) and an imidazolylacetamide
(8)14 have also been identified as potent GKAs. A binding mode at an
allosteric site of GK was revealed by researchers in 2009 through
the formation of co-crystal structures of the GK–compound 1

2282
K. Park et al. / Bioorg. Med. Chem. 22 (2014) 2280–2293
O
O
O
S
H
N
S
S
N
H
N
N
F
N
H
O
S
O
Cl
O
S
N
N
O
N
N
N
NH2
O
O
1 (Banyu-Merck)
3 PSN-GK1
2 Piragliatin
OH
O
O
O
O
N
N
O
O
O
N
N
N
H
HO
N
N
H
HO
O
N
N
H
O
O
F
O
O
S
O
N
O
F
6 GKA60
4 AZD1092
5 MK-0941
CO2H
N
S
O
O
O
O
O
N
H
N
N
O
O
S
O
N
H
H
F
N
N
N
F
O
O
OH
F
N
O
7 GKA71
8 (Pfizer)
Figure 1. Representative structures of GKAs.
9 YH-GKA
mice were orally administered with the compound or vehicle alone
(0.5% methylcellulose solution).
2.3. Structure–activity relationships (SAR)
The biological activity data of selected thiazole benzamide
derivatives have been summarized in Table 1. The introduction of
a fluorine group at the R2 position in the thiazole ring decreased
the potency of the enzyme EC50 by 2.5-fold (16b vs 16a). Thus,
while the R2 position in the thiazole ring was fixed with H, the
R1 position on the thiazole ring was systematically substituted
with various acetyl amides and methyl amines. Cyclopropyl amide
15a maintained similar potency to 16a and ethyl amide 15b
showed a 19-fold increase in potency as compared with 16a. Other
acetyl amides containing large substituents at the amide position
had a tendency to exhibit weak potency (unpublished data). For
methylamine derivatives, the potency varied with substituent pat-
terns. In particular, some alkyl amine moieties, as shown by com-
pounds 18a, 18f, 18k, decreased the potency significantly.
However, other alkyl amines, 18b–c and 18h–j, exhibited similar
enzyme potency to 16a. Among the methyl amine derivatives,
compound 18g significantly improved the enzyme potency by
12-fold with an EC50 of 8 nM. The potency improvement appears
to result from the electrostatic interaction with the pocket and/or
the size effect of the moiety. Most of the active compounds in
the GK enzyme assay showed good glucose uptake activity with
>1.7 fold increase in the presence of 5.6 mM glucose, except for
18c. The compounds that exhibited sub-micromolar EC50 values
of GK activity and a higher than 1.7 fold increase in the glucose up-
take assay were chosen for in vivo OGTT experiments. Among the
compounds measured by in vivo OGTT, compound 16a exhibited a
29.8% AUC reduction at 50 mg/kg. For pyrazole benzamide deriva-
tives summarized in Table 2, the morpholine ethyl moiety (19d)
and the methoxyethylmethoxy moiety (19e) at the R1 position im-
proved enzyme potency by 2-fold and 2.5-fold respectively, com-
pared to the compound with R1 = CH3 (19a). Both compounds
exhibited good glucose uptake activity with >2-fold increase. Com-
pound 19e was tested using in vivo OGTT and found to exhibit a
32.3% AUC reduction at 50 mg/kg. Therefore, compound 19e was
chosen for further studies including additional in vivo activity, a
basal glucose lowering test, DMPK, and toxicity tests (Table 3).
Figure 2. Binding mode hypothesis for benzamide GKAs.
enzyme activity were tested in a glucose uptake assay with hepato-
cyte cells. Subsequently, the selected compoundsthat demonstrated
good glucose uptake activity were evaluated using an in vivo oral
glucose tolerance test (OGTT). The biological results of thiazole
amides, 16a–b and 18a–k, and pyrazole benzamides 19a–e have
been summarized in Tables 1 and 2.
2.2. Biological assays
An enzymatic glucokinase assay using purified recombinant hu-
man pancreatic glucokinase was used to evaluate the compounds.
Selectivity against hexokinase I and II was tested using enzymatic
assays. Glucose uptake assays using rat primary hepatocytes and
an insulin secretion assay in Min6 cells or in rat pancreatic islets
were performed to evaluate the effect of glucokinase activity on
glucose-dependent insulin secretion. Anti-diabetic effects of se-
lected compounds were evaluated using glucose tolerance tests
in normal and diabetic db/db mice. To investigate efficacy with
an oral glucose tolerance test (OGTT), eight-week old male
C57BL/6 mouse (OrientBio Inc, Republic of Korea) and ten-week
old male C57BLKS/J db/db mice (SLC Inc, Japan) were fasted over-
night (with free access to water) before performing the test. The

K. Park et al. / Bioorg. Med. Chem. 22 (2014) 2280–2293
2283
ꢀ
O
O
O
O
B
F
HO
O
N
a
b
c
O
O
O
O
S
O
85%
72%
66%
O
O
O
4-Fluorophenyl
methyl sulfone
O
O
O
S
S
O
S
O
O
10
11
O
12
R1R2
N
O
O
OH
O
N
O
N
d
e
f
OH
N
N
S
N
N
S
N
95%
95%
46~71%
O
O
O
O
O
S
O
O
S
O
S
O
13
15a : NR1R2 =
15b : NR1R2 =
14
HN
HN
86%
h
g
50~64%
R1R2
R1
N
Cl
O
N
O
O
N
N
R2
N
S
i
40~75%
N
N
S
N
N
S
N
O
O
O
O
S
O
O
S
O
O
S
O
(R1=R2=H)
16a
16b
17
18a-k
(R1=H, R2=F)
Scheme 1. Reagents and conditions: (a) Methyl 3,5-dihydroxybenzoate, Cs2CO3, DMF, reflux; (b) (i) Tf2O, TEA, CH2Cl2, (ii) KOAc, PdCl2(dppf), Bis(pinacolato)diboron, 85 °C,
1,4-dioxane; (c) 2-bromo-3-methylpyridine, Pd(PPh3)4, Na2CO3, DMF/H2O; (d) NaOH, THF/MeOH/H2O; (e) (i) ethyl 2-aminothiazole-4-acetate, HOBT, EDAC, TEA, CH2Cl2,
(ii) NaOH, THF/MeOH/H2O; (f) R1R2NH, HOBT, EDAC, TEA, CH2Cl2; (g) 2-aminothiazole or 5-fluoro-2-aminothiazole, HOBT, EDAC, TEA, CH2Cl2; (h) (i) SOCl2, reflux,
(ii) 4-(chloromethyl)-2-thiazolamine, DIEA, CH2Cl2; (i) R1R2NH, K2CO3, KI, DMF.
O
O
R1
N
a
N
N
OH
N
N
b
R1
N
R1
N
X
+
N
50~75%
N
H2N
H2N
50%
O
O
O
O
S
S
O
O
13
19a-e
Scheme 2. Reagents and conditions: (a) 1-substituted 3-aminopyrazole, HOBT, EDAC, TEA, CH2Cl2; (b) K2CO3, DMF.
2.4. Molecular docking of 19e
and ligand. In addition, consensus scores represented by D_score,
PMF_score, G_score and CHEM_socre were also validated. Consen-
sus scoring integrated a number of popular scoring functions for
ranking the affinity of ligands bound to the active site of the recep-
tor. The highest scoring docked pose of 19e structure is shown in
Figure 3. According to binding mode analysis of the Surflex-Dock
results, the aminopyrazole moiety elicited strong hydrogen bonds
with Arg63 and the methyl-pyridine moiety was directed to the
hydrophobic pocket surrounded by Tyr214, Tyr215 and Gly97.
Methanesulfonyl phenoxy groups oriented toward the solvent ex-
posed region with hydrogen bonding to Arg250. These critical
interactions underscored the potent glucokinase activation poten-
tial of the 19e ligand.
To confirm the binding mode of glucokinase structure with dia-
ryl benzamide derivatives, a crystal structure of glucokinase re-
trieved from RCSB Protein Data Bank (PDB entry code: 3A0I)27
was used. A representative compound of 2-pyridinecarboxamide
derivatives was co-crystallized with the binding site of glucoki-
nase. The 3A0I-co-crystallized ligand showed good glucokinase po-
tency (EC50 = 0.25 lM). According to analysis of the binding mode
of the 3A0I-co-crystallized ligand, the 4-fluorophenylthio group
occupied the binding pocket by eliciting hydrophobic interactions
with Tyr214 and Gly97. The aminothiazole region exhibited prefer-
able hydrogen bonding to Arg63.
To predict the binding mode of 19e with the glucokinase struc-
ture, analysis with the Surflex-Dock program was conducted.35
Compound 19e docking conformers into the binding site of gluco-
kinase were generated based on the scores. Total scores for the 19e
docked structure were 9.5, which represented a hydrogen bond, io-
nic, aromatic, and lipophilic interaction between the glucokinase
2.5. Biological activity of 19e
Pyrazole benzamide 19e exhibited human pancreatic glucoki-
nase activity of EC50 = 315 nM at 10 mM glucose with a half max-
imal saturation concentration (S0.5
) of 2.33 mM glucose and

2284
K. Park et al. / Bioorg. Med. Chem. 22 (2014) 2280–2293
Table 1
In vitro glucokinase activity of thiazole benzamide derivatives 15a–b, 16a–b and 18a–k
R1
O
N
R2
N
S
N
O
O
S
O
R1
R2
S0.5 (mM)
EC50 at10 mM glucose, (
lM)
Glucose uptake at 5.6 mM glucose, (fold)
a
b
Compounds
16a
16b
H
H
H
F
2.01
1.95
0.095
0.238
1.98
1.75
N
15a
15b
H
H
1.78
4.35
0.103
0.005
1.80
2.54
O
N
O
N
N
N
N
18a
18b
18c
H
H
H
5.79
3.24
3.44
>50
O
N
O
0.212
0.688
2.10
1.19
18d
H
5.43
2.43
2.02
1.46
O
O
N
N
N
18e
18f
H
H
4.56
3.08
7.46
>50
N
O
18g
H
2.27
0.008
1.72
1.92
N
N
O
O
18h
18i
H
H
0.57
3.98
0.033
0.463
1.47
2.35
N
N
18j
H
H
2.21
4.51
0.106
>50
18k
a
Recombinant human pancreatic glucokinase was used and the activity was measured at 1
Data represented as the mean values of data obtained from three duplicate runs.
lM concentration.
b
maximum reaction rate (Vmax) of 150%. Treatment with 19e also
enhanced glucose uptake in rat primary hepatocytes by 2.23-fold
and did not affect hexokinase I and II. The compound increased
insulin secretion by 9-fold from MIN-6 cells and 3-fold from rat
pancreatic islets in the presence of 11 mM glucose. It also im-
proved oral glucose tolerance in C57BL/J6 mice in a dose-depen-
dent manner (Fig. 4). An oral glucose tolerance test (OGTT) of
19e at 50 mg/kg in C57Bl/6J mice revealed a blood glucose area un-
der the curve (AUC) reduction of 32.3%, closely comparable to that
of 29.9% for metformin at 300 mg/kg. As shown in Figure 4, com-
pound 19e showed no indication of hypoglycemia risk at
150 mg/kg. In addition, a combination of 19e (50 mg/kg) and met-
formin (100 mg/kg) further increased the AUC reduction to 41.2%.
In a two-week repeated dose study with db/db mice, 19e showed
significant decrease in blood glucose levels with 39.8% AUC reduc-
tion in OGTT (Fig. 5) and no adverse effects on serum lipid profiles
or body weight.
to fasting without glucose challenge. Eight-week old male C57BL/
6 mice (OrientBio Inc, Republic of Korea) were fasted for 6 h with
free access to water, before being orally administered with the
compound or vehicle alone (0.5% methylcellulose solution). Glu-
cose levels were measured at 0, 30, 60, 90, 120, 180, 240, 300
and 360 min from the tail tip (Fig. 6). While sitagliptin, a DPP-4
inhibitor marketed by Merck & Co. under the trade name Januvia,
has never been demonstrated to affect blood glucose levels under
fasting conditions, 19e exerted a glucose-lowering effect in the ab-
sence of glucose challenge. However, glucose levels after 60 min
were maintained above 60 mg/dl, the minimum glucose level de-
fined as non-hypoglycemic. GKA50, a potent GKA from AstraZene-
ca,32 was used as a reference. These study results strongly support
the notion that 19e is a promising candidate for study in humans
as a potential therapeutic agent for type 2 diabetes mellitus.
2.6. Physicochemical properties and pharmacokinetics of 19e
It has been demonstrated that stimulators of insulin secretion
(e.g., sulfonylureas, glinides, GKAs) can induce hypoglycemia. We
therefore investigated the effect of 19e on glucose lowering due
Compound 19e possesses good aqueous solubility at 122
phosphate buffer (pH 7.4) and a PAMPA permeability score of 2.62
lM in

K. Park et al. / Bioorg. Med. Chem. 22 (2014) 2280–2293
2285
Table 2
In vitro glucokinase activity of pyrazole benzamide derivatives 19a–e
O
R1
N
N
N
N
O
O
S
O
a
Compounds
R1
S0.5 (mM)
EC50bat 10 mM glucose, (
0.807
lM)
Glucose uptake at 5.6 mM glucose, (fold)
19a
CH3
3.89
1.74
19b
>50
O
N
N
19c
>50
O
19d
19e
3.54
2.33
0.441
0.315
1.86
2.23
O
O
a
Recombinant human pancreatic glucokinase was used and the activity was measured at 1
Data represented as the mean values of data obtained from three duplicate runs.
lM concentration.
b
Table 3
suggested that 19e has a low risk of drug–drug interactions in hu-
mans and a highly attractive pharmacokinetic profile for an oral
anti-diabetic agent.
In vivo OGTT results for the selected benzamide derivatives
Compounds
OGTT at 50 mg/kg,
(AUC reduction, %)
2.7. Toxicity evaluation of 19e
16a
16b
15a
15b
18c
18d
18i
19a
19d
19e
29.8⁄⁄
22.6⁄⁄
ꢀ3.1
ꢀ0.3
4.4
Since Waring et al., recently reported that GKAs containing pyr-
idine-5-carboxylic acid (e.g., GKA60 (6) and GKA50) might cause
testicular toxicity,12 we carefully monitored organ toxicity for
our GKA compounds. To address the testicular toxicology liability
during the optimization of GKA compounds, we developed a
7-day tox screen assay using high doses of compounds (normally
300 and 500 mg/kg) and found that testicular toxicity might be
related to the structure of compounds based on assessment of
the results. An oral dose toxicity study was carried out in rats to
evaluate the testicular toxicity of 19e when administered daily
via oral gavage to rats for 14 days (Table 5). The dose dependent
increase of systemic exposures of 19e was observed in the dose
range of 250 to 1000 mg/kg in rats. The systemic exposure of
19e was generally increased less than dose-proportionally in the
dose range of 250–1000 mg/kg in both sexes on Day 1 and Day
14. No consistent sex differences were observed in systemic expo-
sure and no accumulation of 19e was observed in all dose groups in
both sexes. The systemic exposure of 19e after repeated dose was
generally decreased in the 500 and 1000 mg/kg dose groups.
Assessment of toxicity was based on observations for mortality,
clinical signs, body weight and food consumption data, clinical
pathology, organ weights and macroscopic/microscopic examina-
tions. Under the conditions of the current study, administration
of 19e was well tolerated to 1000 mg/kg and no test article-related
toxicity findings were observed in any other organs.
20.2⁄⁄
2.2
24.1⁄⁄
17.8⁄
32.3⁄⁄
Sample size = 6; significantly different from the vehicle-treated control ⁄p <0.05 and
⁄⁄p <0.01.
(Pe 10ꢀ6 cm/s). In vitro metabolism studies for CYP inhibition and
CYP induction were conducted using human liver microsomes
and a PXR reporter gene assay. Metabolic stability studies were
also performed using liver microsomes from mouse, dog and hu-
man. The pharmacokinetics of 19e in mice and rats was deter-
mined following single intravenous and oral administration
(Table 4).
Compound 19e showed acceptable metabolic stability across
species with more than 60% of the compound remaining after 1 h
incubation. In vitro CYP inhibition (CYP1A2, 2B6, 2C8, 2C19, 2D6,
3A) and CYP induction (CYP3A4) studies for 19e suggested no
inhibitory effect except for 2C9 (IC50 = 5.2
effect up to 25–50 M concentration. Time-dependent inhibition
lM) with no induction
l
(TDI) studies for 19e were performed using human liver micro-
somes and no TDI was observed. Pharmacokinetic studies of 19e
in mice (Table 4) showed that the volume of distribution at steady
state (Vdss) was 0.18 L/kg, elimination half-life (t1/2) was 1.4 h, and
bioavailability (F) was 24%. Overall, 19e exhibited moderate bio-
availability and a relatively short half-life in mice. However these
PK data might support a b.i.d. (twice daily) or t.i.d. (three times dai-
ly) prescription strategy for GKA in humans to control postprandial
glucose levels effectively and to avoid possible hypoglycemic risk.
In vitro examination of CYP inhibition/induction and PK results
For the histopathological examination of GKAs, test article-re-
lated microscopic findings included seminiferous tubule degenera-
tion, seminiferous tubule vacuolation, and multinucleated giant
cells in the testis. No test article-related microscopic findings for
19e were observed in the testis of the high-dose group in this
study. An escalating oral gavage dose range-finding study was per-
formed to evaluate the potential testicular and liver toxicity of 19e
when administered in a repeated escalating-dose program (i.e., re-
peated doses of 100, 300, and 1000 mg/kg on Days 1, 4, and 7,

2286
K. Park et al. / Bioorg. Med. Chem. 22 (2014) 2280–2293
O
N
O
S
N
N
N
O
N
N
S
O
N
N
N
O
S
O
S
O
N
F
3A0I-cocrystalized ligand
19e
Figure 3. The docked conformer of 19e (purple color based carbon type) at the active site of glucokinase as generated by Surflex-Dock, compared with the X-ray structure of
3A0I-co-crystallized ligand (orange color-based carbon type). Specific binding of 19e to glucokinase shows potential hydrogen bonds in red dotted lines.
Control
19e 15 mg/kg
19e 30 mg/kg
300
19e 150 mg/kg
Metformin 100 mg/kg
250
19e + Metformin 50+100 mg/kg
200
150
100
50
0
-30
0
30
60
120
180
glucose (p.o.)
Time (min)
compound (p.o.)
Figure 4. Plasma blood glucose lowering effect of 19e in C57BL/J6 mice OGTT. (po: per os (oral administration); no of animals = 6; error bars represent standard deviation).

K. Park et al. / Bioorg. Med. Chem. 22 (2014) 2280–2293
2287
Control (normal)
Control (db/db)
19e 30 mg/kg
19e 100 mg/kg
Metformin 60mg/kg
Glimepiride 30mg/kg
800
700
600
500
400
300
200
100
0
-30
0
30
60
120
180
Time (min)
glucose (p.o.)
compound (p.o.)
Figure 5. Plasma blood glucose lowering effect of 19e in db/db mice OGTT. (po: per os (oral administration); no of animals = 6; error bars represent standard deviation).
160
140
120
100
80
Control
19e 50 mg/kg
GKA-50 10 mg/kg
Sitagliptin 30 mg/kg
60
40
20
0
-30
0
30
60
90
120
180
240
300
360
Time (min)
compound (P.O)
Figure 6. Glucose lowering activity of 19e in C57BL/J6 mice in fasting conditions (no of animals = 6; error bars represent standard deviation).
Table 4
PK parameters of 19e in mouse and rat
Species
Clearance (L/h/kg)
Vdss (L/kg)
MRT (h)
T1/2 (h)
AUC (lg h/mL)
Cmax
(l
g/mL)
Tmax (h)
F (%)
Mouse
Rat
0.20
0.59
0.18
0.21
0.92
0.37
1.4
0.3
11.9
3.8
11.7
3.7
0.25
0.42
24
22
Table 5
Mean toxicokinetic parameter of 19e on Day 1 and Day 14 after oral administration of 19e for 14 days at the dose levels of 250, 500 and 1000 mg/kg in rats
Parameter
Sex
Male
Female
Dose
250 mg/kg
500 mg/kg
1000 mg/kg
250 mg/kg
500 mg/kg
1000 mg/kg
Cmax
(ng/mL)
Tmax
Day1
Day 14
Day1
3055
1816.3
2.7
4100.3
2795.5
3.7
4250.3
3093.7
4.0
3451.3
5201.3
3.7
4297.7
3917.3
3.3
7590.7
5213.0
2.7
(h)
AUC0–t
(ng h/mL)
Day 14
Day1
Day 14
3
3
5
1.3
13727.9
17567.2
1.3
19750.3
16293.9
2.7
29243.1
23978.8
13145.8
8177.6
16934.7
13593.8
35445.8
25043.8

2288
K. Park et al. / Bioorg. Med. Chem. 22 (2014) 2280–2293
respectively) via oral gavage to beagle dogs for 8 days. No test arti-
cle-related microscopic findings for 19e were observed in the
testis.
carbon, hydrogen and nitrogen (CHN analysis) was accomplished
by combustion analysis.
A mutagenicity study was carried out according to standard
Ames test procedures (Maron and Ames). Two different Salmonella
typhimurium strains (TA98 and TA100) were used in the reverse
mutation test both in the presence and absence of S9 mix. Cytotox-
icity caused by 19e was not observed at all dose levels and there
were no increases in revertants per plate at any dose level for
the TA98 and TA100 strains in the presence and absence of S9
mix (please see the Section 4). Taken together, these bacterial as-
say results suggest that 19e is not mutagenic.
4.1.1. 3-Hydroxy-5-(4-methanesulfonylphenoxy) benzoic acid
methyl ester (10)
To
a solution of methyl 3, 5-dihydroxybenzoate (20.5 g,
0.13 mol) in DMF (130 mL) were added 4-fluorophenylmethyl sul-
fone (21.2 g, 0.12 mol) and cesium carbonate (59.3 g, 0.18 mol).
The reaction mixture was heated at 180 °C for 3 h. The mixture
was cooled to room temperature, and then water was added to
the mixture. The reaction mixture was filtered through Celite pad
and washed with ethyl acetate. The filtrate was extracted with
ethyl acetate and the combined organic layers were washed with
brine, dried over MgSO4, and concentrated under reduced pressure.
The residue was purified by flash column chromatography (EA/
Pet.Ether = 1:5 ꢂ 1:1) to afford 11.6 g (85% yield) of 10 as a white
solid. 1H NMR (400 MHz, CDCl3) d 7.91–7.89 (d, J = 8.8 Hz, 2H),
7.42 (s, 1H), 7.28–7.26 (m, 1H), 7.12–7.10 (d, J = 8.8 Hz, 2H),
6.80–6.79 (t, J = 4.0 Hz, 1H), 3.90 (s, 3H), 3.08 (s, 3H); TLC Rf 0.50
(50% EtOAc in petroleum ether).
3. Conclusion
Various benzamide derivatives have been synthesized by
modifying three main regions around a benzamide scaffold. Lead
optimization led to the identification of 3-methylpyridine and
4-phenoxymethyl sulfone groups as optimal moieties for the
B-region and C-region, respectively. Consequently, the A-region
was optimized to identify thiazole and pyrazole groups as optimal
moieties in terms of in vitro potency and physicochemical proper-
ties. Among the thiazole and pyrazole benzamide derivatives,
Compound 19e was found to be an active GKA with an EC50 of
315 nM and induced a glucose reduction of 32.3% (50 mg/kg) in
an OGTT study, equivalent to 300 mg/kg metformin. Single treat-
ment in C57BL/J6 and db/db mice elicited basal glucose lowering
activity. In addition, 19e exhibited favorable bioavailability and
an appropriate half-life for a b.i.d or t.i.d dosing strategy. More
importantly, 19e exhibits a good safety profile without any
evidence of mutagenicity or liver/testicular toxicity.
In conclusion, the pyrazole benzamide GKA 19e is a promising
preclinical lead candidate with good efficacy and an excellent
safety profile, with low risk hypoglycemic risk for the treatment
of T2DM. To further improve the efficacy and safety profile with
additional benefits, the lead optimization of a novel GKA scaffold
is continuing with an innovative and translational screening
strategy.
4.1.2. 3-(4-Methanesulfonylphenoxy)-5-(4,4,5,5-tetramethyl-
[1,3,2]dioxaborolan-2-yl) benzoic acid methyl ester (11)
To a solution of 10 (11.5 g, 0.036 mol) in dichloromethane
(150 mL) were added trifluoromethanesulfonic anhydride
(11.5 mL, 0.054 mol) and triethylamine (12.3 mL, 0.089 mol) at
ꢀ78 °C bath. The reaction mixture was stirred for 2 h at ꢀ78 °C
and then at room temperature overnight. The mixture was washed
with saturated NH4Cl aqueous solution and washed with brine. The
organic layer was dried over MgSO4, filtered, and concentrated un-
der reduced pressure. The residue was purified by flash column
chromatography (EA/Pet.Ether = 1:8 ꢂ 1:4) to yield 14.0 g (86%
yield) of 3-(4-methanesulfonylphenoxy)-5-trifluoromethanesulfo-
nyloxy benzoic acid methyl ester as a white solid. 1H NMR
(400 MHz, CDCl3) d 7.99–7.97 (q, J = 8.0 Hz, 2H), 7.77–7.73 (m,
2H), 7.23–7.21 (m, 1H), 7.18–7.16 (d, J = 8.8 Hz, 2H), 3.95 (s, 3H),
3.09 (s, 3H); TLC Rf 0.60 (50% EtOAc in petroleum ether).
To a mixture of [1,10-bis(diphenylphosphino) ferrocene]dichlo-
ropalladium (ll) (724 mg, 0.88 mmol), KOAc (5.8 g, 59.1 mmol)
and bis(pinacolato) diboron (11.2 g, 44.3 mmol) in 1,4-dioxane
(200 mL) was added 3-(4-methanesulfonylphenoxy)-5-trif-
luoromethanesulfonyloxy benzoic acid methyl ester (13.4 g,
29.5 mmol) at room temperature under nitrogen. The reaction
mixture was heated at 85 °C overnight before it was cooled to
room temperature, washed with saturated NH4Cl aqueous solu-
tion, and extracted with ethyl acetate. The combined organic layer
was washed with brine, dried over MgSO4, filtered, and concen-
trated under reduced pressure. The residue was purified by flash
column chromatography (EA/Pet.Ether = 1:12 ꢂ 1:5) to yield
4. Experimental
4.1. Chemistry
All reagents and solvents were used as received from commer-
cial sources. 1H NMR spectra were recorded on a Bruker, Avance
400 Spectrometer 400 MHz nuclear magnetic resonance spectrom-
eter. 1H NMR spectra were recorded in CDCl3, CD3OD, or DMSO-d6,
and chemical shifts are reported relative to a TMS internal standard
to the residual solvent peak (abbreviation in spectra: s = singlet,
d = doublet, t = triplet, q = quartet, quin = quintet, m = multiplet).
High-resolution mass spectra (HRMS) were obtained on a Waters
Q-TOF Premier machine using electrospray ionization (ESI). Purity
of all final analogues for biological testing were confirmed to be
>95% as determined by UPLC analysis and inspection of NMR spec-
tra. UPLC analysis was performed using an Acquity UPLC BEHsR
11.6 g (91% yield) of 11 as
a
pale yellow solid. 1H NMR
(400 MHz, CDCl3) d 8.33 (s, 1H), 7.90–7.88 (q, J = 4.8 Hz, 2H),
7.83–7.82 (m, 1H), 7.69 (s, 1H), 7.08–7.05 (d, J = 8.8 Hz, 2H), 3.70
(s, 3H), 3.06 (s, 3H), 1.35 (s, 12H); TLC Rf 0.50 (50% EtOAc in petro-
leum ether).
4.1.3. 3-(4-Methanesulfonyl-phenoxy)-5-(3-methyl-pyridin-2-
yl)-benzoic acid methyl ester (12)
C18 (1.7
l
m, 2.1 ꢁ 100 mM) with 5% acetonitrile in water (0–
1 min), 15–90% acetonitrile in water (1–8.5 min), 90% acetonitrile
(8.5–10 min), with both solvents containing 0.1% formic acid as a
modifier with a flow of 0.4 mL/min; and UV detection at 230 nM.
Purification by flash chromatography was carried out using either
Zeochem C-gel 560 (0.060–0.200 mM, Zeochem) or MPLC with a
Teledyne Isco CombiFlash Rf with RediSep Flash columns using a
gradient of ethyl acetate in n-hexanes or methanol in CH2Cl2, or
similar instrument or reverse phase preparative HPLC. TLC analysis
was performed on silica gel 60 F254 plates. Elemental analysis for
To a solution of 11 (11.5 g, 26.6 mmol) in DMF (100 mL) were
added 2-bromo-3-methylpyridine (5.9 g, 34.58 mmol), tetrakis(tri-
phenylphosphine) palladium (1.53 g, 1.33 mmol) and sodium
bicarbonate (8.4 g, 79.8 mmol) in water (10 mL). The reaction mix-
ture was heated at 80 °C for 18 h. The mixture was cooled to room
temperature, diluted with water, and extracted with ethyl acetate.
The combined organic layers were washed with brine, dried over
MgSO4, filtered, and concentrated under reduced pressure. The res-
idue was purified by flash column chromatography (EA/

K. Park et al. / Bioorg. Med. Chem. 22 (2014) 2280–2293
2289
Pet.Ether = 1:5 ꢂ 1:2) to afford 7.0 g (66% yield) of 12 as a yellow
solid. 1H NMR (400 MHz, CDCl3) d 8.53–8.52 (d, J = 8.8 Hz, 1H),
8.09 (s, 1H), 7.93–7.91 (q, J = 4.8 Hz, 2H), 7.77–7.77 (m, 1H),
7.69–7.61 (m, 1H), 7.48–7.44 (m, 1H), 7.25–7.22 (m, 1H), 7.18–
7.15 (m, 2H), 3.93 (s, 3H), 3.06 (s, 3H), 2.38 (s, 3H); TLC Rf 0.40
(50% EtOAc in petroleum ether).
J = 3.6 Hz, 1H), 8.18 (s, 1H), 8.04–8.03 (d, J = 4.0 Hz, 1H), 7.98–
7.93 (m, 3H), 7.78–7.76 (d, J = 7.2 Hz, 1H), 7.59 (s, 1H), 7.37–7.30
(m, 3H), 6.95 (s, 1H), 3.43 (s, 2H), 3.22 (s, 3H), 2.62–2.60 (t,
J = 7.2 Hz, 1H), 2.39 (s, 3H), 0.61–0.57 (m, 2H), 0.40–0.36 (m, 2H);
HRMS (ESI-TOF) m/z for C28H27N4O5S2 [M+H]+ calcd 563.1423,
found 563.1422; TLC Rf 0.50 (5% MeOH in dichloromethane). Anal.
Calcd for C28H26N4O5S2: C, 59.77; H, 4.66; N, 9.96. Found: C, 58.68;
H, 4.67; N, 9.97.
4.1.4. 3-(4-Methanesulfonylphenoxy)-5-(3-methylpyridin-2-yl)
benzoic acid (13)
To a solution of 12 (7.0 g, 17.6 mmol) in tetrahydrofuran
(40 mL) and methanol (40 mL) was added NaOH (2.1 g, 52.9 mmol)
in water (40 mL) and the reaction mixture was stirred at room
temperature overnight. The reaction mixture was then concen-
trated under reduced pressure, diluted with water, acidified by
1 N HCl aqueous solution to pH = 6, and extracted with dichloro-
methane. The combined organic layers were dried over MgSO4, fil-
tered, and concentrated to afford 7.0 g (95% yield) of 13 as a pale
white solid. 1H NMR (400 MHz, CDCl3) d 8.52–8.50 (q, J = 5.2 Hz,
1H), 7.99 (s, 1H), 7.52–7.50 (m, 3H), 7.77–7.75 (q, J = 8.8 Hz, 1H),
7.65–7.60 (m, 3H), 7.57 (br s, 1H), 7.36–7.33 (m, 1H), 7.30–7.28
(m, 2H), 3.22 (s, 3H), 2.36 (s, 3H); TLC Rf 0.40 (5% MeOH in
dichloromethane).
4.1.7. N-(4-Ethylcarbamoylmethyl-thiazol-2-yl)-3-(4-
methanesulfonyl-phenoxy)-5-(3-methyl-pyridine-2-yl)-
benzamide (15b)
15b was prepared according to the procedure described for the
synthesis of 15a using ethylamine instead of cyclopropylamine.
46% yield, white solid. 1H NMR (400 MHz, CDCl3) d 8.56 (s, 1H),
7.96–7.94 (d, J = 8.8 Hz, 2H), 7.72 (s, 1H), 7.67–7.65 (d, J = 6.4 Hz,
2H), 7.51 (s, 1H), 7.21–7.19 (m, 2H), 6.99 (s, 1H), 6.82 (s, 1H),
3.73 (s, 2H), 3.32–3.29 (t, 2H), 3.27–3.23 (m, 4H), 3.08 (s, 3H),
2.41 (s, 3H); HRMS (ESI-TOF) m/z for C27H27N4O5S2 [M+H]+ calcd
551.1423, found 551.1409. Anal. Calcd for
C27H26N4O5S2: C,
58.89; H, 4.76; N, 10.17. Found: C, 58.79; H, 4.79; N, 10.19.
4.1.8. 3-(4-Methanesulfonyl-phenoxy)-5-(3-methyl-pyridin-2-
yl)-N-thiazol-2-yl-benzamide (16a)
4.1.5. {2-[3-(4-Methanesulfonylphenoxy)-5-(3-methyl-
pyridine-2-yl)-benzoylamino]-thiazol-4-yl}-acetic acid (14)
To a solution of 13 (3.7 g, 9.62 mmol) in dichloromethane
(60 mL) were added ethyl 2-aminothiazole-4-acetate (1.97 g,
10.57 mmol), HOBT (1.49 g, 11.06 mmol), EDAC (2.11 g,
11.06 mmol) and triethylamine (2.47 g, 11.06 mmol) and the reac-
tion mixture was stirred at room temperature overnight. The reac-
tion mixture was then washed with 1 N HCl aqueous solution,
saturated NaHCO3 aqueous solution, and brine, before the organic
layer was dried over MgSO4, filtered, and concentrated under
reduced pressure. The residue was purified by flash column chro-
matography (EA/Pet.Ether = 1:5 ꢂ 1:1) to afford 5.1 g (95% yield)
of {2-[3-(4-methanesulfonylphenoxy)-5-(3-methyl-pyridine-2-yl)-
benzoylamino]-thiazol-4-yl}-acetic acid ethyl ester as a white
solid. TLC Rf 0.50 (5% MeOH in dichloromethane).
To a solution of 13 (1.00 g, 2.6 mmol) in dichloromethane
(20 mL) were added 2-aminothiazole (313 mg, 3.1 mmol), HOBT
(705 mg, 5.2 mmol), EDAC (1000 mg, 5.2 mmol) and TEA
(0.73 mL, 5.2 mmol) and the mixture was stirred overnight at room
temperature. The reaction mixture was washed with 1 N HCl solu-
tion, washed with saturated NaHCO3 solution, and brine. The or-
ganic layer was dried over MgSO4, concentrated under reduced
pressure and the residue was purified by flash column chromatog-
raphy (EA/n-HX = 1:3 ꢂ 2:1) to yield 387 mg (32% yield) of 16a as a
white solid. 1H NMR (400 MHz, CDCl3) d 11.46 (s, 1H), 8.55–8.54 (d,
J = 4.0 Hz, 1H), 7.95–7.91 (m, 2H), 7.71–7.61 (m, 3H), 7.56–7.44 (m,
2H), 7.29–7.27 (m, 1H), 7.26–7.16 (m, 2H), 6.93–6.90 (m, 1H), 3.08
(s, 3H), 2.38 (s, 3H); HRMS (ESI-TOF) m/z for C23H20N3O4S2 [M+H]+
calcd 466.0895, found 466.0887; TLC Rf 0.38 (33% n-HX in EtOAc).
Anal. Calcd for C23H19N3O4S2: C, 59.34; H, 4.11; N, 9.03. Found: C,
59.25; H, 4.12; N, 8.99.
To
a
solution of {2-[3-(4-methanesulfonylphenoxy)-5-(3-
acid
methyl-pyridine-2-yl)-benzoylamino]-thiazol-4-yl}-acetic
ethyl ester (4.0 g, 7.26 mmol) in THF (20 mL)/MeOH (20 mL) was
added 3 N LiOH aqueous solution (7.26 mL, 21.78 mmol) and the
reaction mixture was stirred at room temperature overnight. The
reaction mixture was acidified by 1 N HCl aqueous solution to pH
6, The resulting solution was extracted with DCM and washed with
brine. The organic layer was dried over MgSO4, filtered and concen-
trated to yield 3.2 g (84% yield) of 14 as a yellow solid. 1H NMR
(400 MHz, DMSO) d 12.63 (br s, 2H), 8.53–8.52 (d, J = 3.6 Hz, 1H),
8.20 (s, 1H), 7.99–7.92 (m, 3H), 7.79–7.77 (d, J = 7.6 Hz, 1H),
7.65–7.56 (m, 4H), 7.38–7.26 (m, 3H), 7.05 (s, 1H), 3.65 (s, 3H),
2.51 (s, 3H); TLC Rf 0.50 (10% MeOH in dichloromethane).
4.1.9. N-(5-Fluoro-thiazol-2-yl)-3-(4-methanesulfonyl-
phenoxy)-5-(3-methyl-pyridin-2-yl)-benzamide (16b)
To a solution of 13 (228 mg, 0.59 mmol) in dichloromethane
(5 mL) were added 5-fluoro-2-aminothiazole (184 mg, 1.19 mmol),
HOBT (161 mg, 1.19 mmol), EDAC (228 mg, 1.19 mmol) and TEA
(0.17 mL, 1.19 mmol) and the reaction mixture was stirred at room
temperature overnight. The reaction mixture was then washed
with 1 N HCl aqueous solution, saturated NaHCO3 aqueous solu-
tion and brine. The organic layer was dried over MgSO4, filtered
and concentrated under reduced pressure, and purified by flash
column chromatography (EA/n-Hex = 1:2 ꢂ 2:1) to yield 142 mg
(50% yield) of 16b as a yellow solid. 1H NMR (400 MHz, CDCl3) d
11.39 (br s, 1H), 8.54–8.53 (d, J = 4.4 Hz, 1H), 7.98 (s, 1H),
7.91–7.89 (d, J = 9.2 Hz, 2H), 7.61–7.45 (m, 3H), 7.15–7.12 (d,
J = 11.2 Hz, 2H), 6.91–6.90 (d, J = 2.8 Hz, 1H), 3.06 (s, 3H), 2.37 (s,
4.1.6. N-(4-Cyclopropylcarbamoylmethyl-thiazol-2-yl)-3-(4-
methanesulfonyl-phenoxy)-5-(3-methyl-pyridine-2-yl)-
benzamide (15a)
To a solution of 14 (100 mg, 0.19 mmol) in DCM (10 mL) were
added cyclopropylamine (10.9 mg, 0.19 mmol), HOBT (29.6 mg,
0.23 mmol), EDAC (42.2 mg, 0.23 mmol) and triethylamine
(49.3 mg, 0.29 mmol) and the reaction mixture was stirred at room
temperature overnight. The reaction mixture was then washed
with 1 N HCl aqueous solution, saturated NaHCO3 aqueous solu-
tion, and brine. The organic layer was dried over MgSO4, filtered,
and concentrated under reduced pressure. The residue was puri-
fied by MPLC (MeOH/DCM = 5:95) to yield 75.9 mg (71% yield) of
15a as a white solid. 1H NMR (400 MHz, DMSO) d 8.52–8.51 (d,
3H); HRMS (ESI-TOF) m/z for
C
23H19FN3O4S2 [M+H]+ calcd
484.0801, found 484.0788; TLC Rf 0.20 (33% n-HX in EtOAc). Anal.
Calcd for C23H18FN3O4S2: C, 57.13; H, 3.75; F, 3.93; N. Found:
C, 57.08; H, 3.76; N, 3.96.
4.1.10. N-(4-Chloromethyl-thiazol-2-yl)-3-(4-methanesulfonyl-
phenoxy)-5-(3-methyl-pyridine-2-yl)-benzamide (17)
Compound 13 (3.3 g, 8.60 mmol) was dissolved in SOCl2
(10 mL), then the mixture was stirred at reflux for 1 h. The reaction

2290
K. Park et al. / Bioorg. Med. Chem. 22 (2014) 2280–2293
mixture was concentrated under reduced pressure to yield 3.46 g
(100% yield) of yellow solid, which was used for the following step
without further purification. To a solution of 3-(4-methanesulfo-
nyl-phenoxy)-5-(3-methyl-pyridin-2-yl)-benzoyl chloride (3.46 g,
8.60 mmol) in DCM (50 mL) was added 4-(chloromethyl)-2-thia-
zolamine (1.75 g, 9.50 mmol) and N,N-diisopropylamine (6.67 g,
51.6 mmol) and the reaction mixture was stirred at room temper-
ature overnight. The reaction mixture was washed with 1 N HCl
aqueous solution, saturated NaHCO3 aqueous solution, and brine.
The organic layer was dried over MgSO4, filtered and concentrated
under reduced pressure, and purified by flash column chromatog-
raphy (EA/Pet.Ether = 1:5 ꢂ 1:1) to afford 3.8 g (86% yield) of 17 as
a brown solid. 1H NMR (400 MHz, DMSO) d 8.53–8.51 (d, J = 4.4 Hz,
1H), 8.20 (s, 1H), 7.98–7.95 (m, 3H), 7.79–7.77 (d, J = 7.6 Hz, 1H),
7.61 (s, 1H), 7.38–7.31 (m, 4H), 4.78 (s, 2H), 3.22 (s, 4H), 2.40 (s,
3H), 1.91 (s, 2H), 1.35 (s, 1H), 1.29–1.23 (m, 2H); HRMS (ESI-
4.1.14. N-(4-{[Bis-(2-methoxy-ethyl)-amino]-methyl}-thiazol-2-
yl)-3-(4-methanesulfonyl-phenoxy)-5-(3-methyl-pyridin-2-yl)-
benzamide (18d)
41% yield, white solid. 1H NMR (400 MHz, CDCl3) d 8.61–8.60 (d,
J = 4.4 Hz, 1H), 8.21 (s, 1H), 7.94–7.92 (t, J = 8.4 Hz, 3H), 7.81–7.79
(d, J = 7.6 Hz, 1H), 7.50 (s, 1H), 7.41–7.38 (m, 1H), 7.22–7.20 (d,
J = 8.4 Hz, 2H), 7.05 (s, 1H), 4.44 (s, 2H), 3.82 (s, 3H), 3.35 (s, 1H),
3.33 (s, 6H), 3.07 (s, 4H), 2.62 (s, 3H), 2.47 (s, 3H); HRMS (ESI-
TOF) m/z for
C
30H35N4O6S2 [M+H]+ calcd 611.1998, found
611.1985. Anal. Calcd for C30H34N4O6S2: C, 59.00; H, 5.61; N,
9.17. Found: C, 58.88; H, 5.60; N, 9.19.
4.1.15. 3-(4-Methanesulfonyl-phenoxy)-N-(4-{[(2-methoxy-
ethyl)-methyl-amino]-methyl}-thiazol-2-yl)-5-(3-methyl-
pyridin-2-yl)-benzamide (18e)
45% yield, white solid. 1H NMR (400 MHz, CDCl3) d 8.48–8.47 (q,
J = 6.0 Hz, 1H), 7.97 (s, 1H), 7.88–7.85 (q, J = 8.8 Hz, 2H), 7.68–7.67
(t, J = 3.6 Hz, 1H), 7.58–7.56 (q, J = 8.0 Hz, 1H), 7.44–7.43 (q,
J = 3.6 Hz, 1H), 7.20–7.11 (m, 3H), 6.75 (s, 1H), 3.54 (s, 2H), 3.48–
3.45 (t, 2H), 3.28 (s, 3H), 3.01 (s, 3H), 2.68–2.59 (t, J = 11.2 Hz,
2H), 2.35 (s, 3H), 2.22 (s, 3H); HRMS (ESI-TOF) m/z for C28H31N4O5-
S2 [M+H]+ calcd 567.1736, found 567.1743. Anal. Calcd for C28H30-
N4O5S2: C, 59.35; H, 5.34; N, 9.89. Found: C, 59.22; H, 5.35; N, 9.91.
TOF) m/z for
C
24H27ClN3O4S2 [M+H]+ calcd 519.1052, found
519.1053; TLC Rf 0.50 (5% EtOAc in petroleum ether). Anal. Calcd
for C24H26ClN3O4S2: C, 56.08; H, 3.92; N, 8.17. Found: C, 55.98; H,
3.94; N, 8.20.
4.1.11. N-(4-Diethylaminomethyl-thiazol-2-yl)-3-(4-
methanesulfonyl-phenoxy)-5-(3-methyl-pyridin-2-yl)-
benzamide (18a)
4.1.16. 3-(4-Methanesulfonyl-phenoxy)-5-(3-methyl-pyridin-2-
yl)-N-(4-piperazin-1-ylmethyl-thiazol-2-yl)-benzamide (18f)
40% yield, white solid. 1H NMR (400 MHz, CDCl3) d 8.47–8.46 (q,
J = 5.6 Hz, 1H), 7.91–7.85 (m, 3H), 7.63–7.63 (t, J = 3.6 Hz, 1H),
7.57–7.55 (q, J = 8.0 Hz, 1H), 7.43–7.42 (q, J = 4.0 Hz, 1H), 7.19–
7.17 (m, 1H), 7.13–7.10 (q, J = 8.8 Hz, 2H), 6.72 (s, 1H), 3.44 (s,
2H), 3.01 (s, 4H), 2.87–2.84 (t, J = 9.2 Hz, 4H), 2.41 (s, 4H), 2.33 (s,
To a solution of 17 (150 mg, 0.29 mmol) in DMF (5 mL) were
added diethylamine (43 mg, 0.58 mmol), K2CO3 (54 mg,
0.58 mmol) and KI (4.8 mg, 0.03 mmol) and the reaction mixture
was stirred at room temperature overnight. The reaction mixture
was then washed with water and brine. The organic layer was
dried over MgSO4, filtered and concentrated under reduced pres-
sure, and purified by MPLC (MeOH/DCM = 15:85) to afford 64 mg
(42% yield) of 18a as a white solid. 1H NMR (400 MHz, CDCl3) d
8.48–8.47 (q, J = 4.8 Hz, 1H), 7.91–7.90 (m, 1H), 7.88–7.85 (m,
2H), 7.62–7.62 (t, J = 3.6 Hz, 1H), 7.58–7.56 (t, 1H), 7.43–7.42 (q,
J = 7.6 Hz, 1H), 7.19–7.17 (m, 1H), 7.13–7.10 (m, 2H), 6.72 (s, 1H),
3.55 (s, 2H), 3.01 (s, 3H), 2.53–2.47 (dd, J = 7.6, 7.2 Hz, 4H), 2.34
(s, 3H), 1.00–0.96 (t, J = 14.4 Hz, 6H); HRMS (ESI-TOF) m/z for C28-
H31N4O4S2 [M+H]+ calcd 551.1786, found 551.1782. Anal. Calcd
for C28H30N4O4S2: C, 61.07; H, 5.49; N, 10.17. Found: C, 60.98; H,
5.47; N, 10.19.
3H); HRMS (ESI-TOF) m/z for
564.1740, found 564.1733. Anal. Calcd for
C
28H30N5O4S2 [M+H]+ calcd
28H29N5O4S2: C,
C
59.66; H, 5.19; N, 12.42. Found: C, 59.58; H, 5.20; N, 12.45.
4.1.17. 3-(4-Methanesulfonyl-phenoxy)-N-{4-[(2-methoxy-1-
methyl-ethylamino)-methyl]-thiazol-2-yl}-5-(3-methyl-
pyridin-2-yl)-benzamide (18g)
52% yield, white solid. 1H NMR (400 MHz, CDCl3) d 8.52–8.51 (d,
J = 4.4 Hz, 1H), 8.09 (s, 1H), 7.91–7.89 (d, J = 8.8 Hz, 2H), 7.81 (s,
1H), 7.72–7.70 (d, J = 7.6 Hz, 1H), 7.37 (s, 1H), 7.32–7.29 (m, 1H),
7.17–7.15 (d, J = 8.4 Hz, 2H), 6.83 (s, 1H), 4.13 (s, 2H), 3.58–3.56
(d, J = 6.0 Hz, 2H), 3.49–3.44 (m, 1H), 3.29 (s, 3H), 3.06 (s, 3H),
2.61 (s, 3H), 2.39 (s, 3H); HRMS (ESI-TOF) m/z for C28H31N4O5S2
[M+H]+ calcd 567.1736, found 567.1736. Anal. Calcd for C28H30N4-
O5S2: C, 59.35; H, 5.34; N, 9.89. Found: C, 59.27; H, 5.36; N, 9.91.
The following compounds (18b–k) were prepared according to
the procedure described above using suitable amines.
4.1.12. 3-(4-Methanesulfonyl-phenoxy)-5-(3-methyl-pyridin-2-
yl)-N-(4-morpholin-4-ylmethyl-thiazol-2-yl)-benzamide (18b)
70% yield, white solid. 1H NMR (400 MHz, CDCl3) d 8.51–8.49 (t,
J = 4.8 Hz, 1H), 7.90–7.86 (m, 3H), 7.61–7.60 (q, J = 3.6 Hz, 1H),
7.58–7.56 (q, J = 8.4 Hz, 1H), 7.44–7.43 (q, J = 3.6 Hz, 1H), 7.20–
7.18 (m, 1H), 7.13 (s, 1H), 7.11 (s, 1H), 6.74 (s, 1H), 3.66–3.64 (t,
J = 9.2 Hz, 4H), 3.45 (s, 2H), 3.01 (s, 3H), 2.41–2.39 (t, J = 8.8 Hz,
4H), 2.34 (s, 3H); HRMS (ESI-TOF) m/z for C28H29N4O5S2 [M+H]+
calcd 565.1580, found 565.1586. Anal. Calcd for C28H28N4O5S2: C,
59.56; H, 5.00; N, 9.92. Found: C, 59.48; H, 4.98; N, 9.96.
4.1.18. N-{4-[(2,2-Dimethoxy-ethylamino)-methyl]-thiazol-2-
yl}-3-(4-methanesulfonyl-phenoxy)-5-(3-methyl-pyridin-2-yl)-
benzamide (18h)
75% yield, white solid. 1H NMR (400 MHz, MeOD) d 8.70–8.69
(d, J = 4.8 Hz, 1H), 8.43–8.41 (d, J = 8.0 Hz, 1H), 8.12 (s, 1H), 8.01–
7.98 (m, 3H), 7.66–7.66 (t, J = 3.2 Hz, 1H), 7.35–7.30 (m, 3H),
4.66–4.63 (t, J = 8.8 Hz, 3H), 4.26 (s, 2H), 3.44 (s, 6H), 3.19–3.18
(d, J = 4.8 Hz, 2H), 3.12 (s, 3H), 2.49 (s, 3H); HRMS (ESI-TOF) m/z
for C28H31N4O6S2 [M+H]+ calcd 583.1685, found 583.1679. Anal.
Calcd for C28H30N4O6S2: C, 57.72; H, 5.19; N, 9.61. Found: C,
57.59; H, 5.18; N, 9.64.
4.1.13. 3-(4-Methanesulfonyl-phenoxy)-N-[4-(4-methyl-
piperazin-1-ylmethyl)-thiazol-2-yl]-5-(3-methyl-pyridin-2-yl)-
benzamide (18c)
51% yield, brown solid. 1H NMR (400 MHz, CDCl3) d 8.59–8.58 (d,
J = 4.0 Hz, 1H), 8.08 (s, 1H), 7.96–7.94 (d, J = 8.4 Hz, 2H), 7.79 (s, 1H),
7.75–7.73 (d, J = 7.2 Hz, 1H), 7.54 (s, 1H), 7.35–7.35 (m, 1H), 7.22–
7.20 (d, J = 9.2 Hz, 2H), 6.90 (s, 1H), 3.79(s, 2H), 3.17–3.10 (m, 3H),
3.08 (s, 2H), 3.02 (br s, 3H), 2.77 (s, 3H), 2.62 (s, 3H), 2.39 (s, 3H);
HRMS (ESI-TOF) m/z for C29H32N5O4S2 [M+H]+ calcd 578.1896,
found 578.1901. Anal. Calcd for C29H31N5O4S2: C, 60.29; H, 5.41;
N, 12.12. Found: C, 60.18; H, 5.43; N, 12.15.
4.1.19. N-[4-(Isopropylamino-methyl)-thiazol-2-yl]-3-(4-
methanesulfonyl-phenoxy)-5-(3-methyl-pyridin-2-yl)-
benzamide (18i)
65% yield, white solid. 1H NMR (400 MHz, CDCl3) d 8.58–8.57 (d,
J = 4.8 Hz, 1H), 8.12 (s, 1H), 7.92–7.90 (d, J = 8.4 Hz, 2H), 7.86–7.82
(m, 2H), 7.42–7.37 (m, 2H), 7.20–7.18 (d, J = 8.4 Hz, 2H), 6.74 (s, 1H),
3.95 (s, 2H), 3.37–3.35 (m, 1H), 3.07 (s, 3H), 2.62 (s, 6H), 2.41 (s, 3H);

K. Park et al. / Bioorg. Med. Chem. 22 (2014) 2280–2293
2291
HRMS (ESI-TOF) m/z for C27H29N4O4S2 [M+H]+ calcd 537.1630, found
537.1637. Anal. Calcd for C27H28N4O4S2: C, 60.43; H, 5.26; N, 10.44.
Found: C, 60.37; H, 5.24; N, 10.47.
7.81 (d, J = 7.2 Hz, 1H), 7.75 (s, 1H), 7.51 (s, 2H), 7.41–7.39 (t,
J = 7.6 Hz, 1H), 7.28 (s, 2H), 6.40 (s, 1H), 4.29–4.26 (t, J = 7.2 Hz,
2H), 3.12 (s, 3H), 2.65 (s, 6H), 2.38 (s, 3H), 1.00 (s, 6H); HRMS
(ESI-TOF) m/z for C29H34N5O4S [M+H]+ calcd 548.2332, found
548.2321. Calcd for C29H33N5O4S: C, 66.60; H, 6.07; N, 12.79.
Found: C, 66.50; H, 6.06; N, 12.77.
4.1.20. 3-(4-Methanesulfonyl-phenoxy)-5-(3-methyl-pyridin-2-
yl)-N-(4-prop-2-ynylaminomethyl-thiazol-2-yl)-benzamide
(18j)
54% yield, white solid. 1H NMR (400 MHz, CDCl3) d 8.56–8.55 (d,
J = 4.4 Hz, 1H), 8.10 (s, 1H), 7.93–7.91 (d, J = 8.8 Hz, 2H), 7.85 (s,
1H), 7.76–7.74 (d, J = 7.6 Hz, 1H), 7.44 (s, 1H), 7.35–7.35 (m, 1H),
7.20–7.18 (d, J = 8.8 Hz, 2H), 6.79 (s, 1H), 4.17 (s, 2H), 3.83 (s,
2H), 3.14–3.11 (m, 1H), 3.07 (s, 3H), 2.42 (s, 3H); HRMS(ESI-TOF)
m/z for C27H25N4O4S2 [M+H]+ calcd 533.1317, found 533.1322.
Anal. Calcd for C27H24N4O4S2: C, 60.89; H, 4.54; N, 10.52. Found:
C, 60.80; H, 4.55; N, 10.55.
4.1.25. 3-(4-Methanesulfonylphenoxy)-5-(3-methylpyridin-2-
yl)-N-[1-(2-morpholin-4-yl)-ethyl]-1H-pyrazol-3-yl]-benzamide
(19d)
53% yield, white solid. 1H NMR (400 MHz, CDCl3) d 8.55–8.54 (d,
J = 4.8 Hz, 1H), 8.44 (s, 1H), 7.94–7.92 (d, J = 8.4 Hz, 2H), 7.88 (s,
1H), 7.66–7.62 (m, 2H), 7.45 (s, 1H), 7.40 (s, 1H), 7.19–7.17 (d,
J = 8.8 Hz, 2H), 6.80 (s, 1H), 4.14–4.11 (t, J = 13.2 Hz, 2H), 3.70–
3.67 (t, J = 9.2 Hz, 2H), 3.07 (s, 3H), 2.78–2.75 (m, 2H), 2.47–2.45
(t, J = 8.8 Hz, 2H), 2.39 (s, 3H); HRMS (ESI-TOF) m/z for C29H32N5O5-
S [M+H]+ calcd 562.2124, found 562.2131. Anal. Calcd for C29H31-
N5O5S: C, 62.02; H, 5.56; N, 12.47. Found: C, 61.96; H, 5.54; N,
12.50.
4.1.21. 3-(4-Methanesulfonyl-phenoxy)-5-(3-methyl-pyridin-2-
yl)-N-(4-propylaminomethyl-thiazol-2-yl)-benzamide (18k)
70% yield, white solid. 1H NMR (400 MHz, CDCl3) d 9.44 (br s,
1H), 8.61–8.60 (d, J = 4.8 Hz, 1H), 8.12 (s, 1H), 7.94–7.87 (m, 4H),
7.51–7.48 (t, J = 12.8 Hz, 1H), 7.36 (s, 1H), 7.20–7.18 (d, J = 8.4 Hz,
2H), 6.83 (s, 1H), 3.99 (s, 2H), 3.07 (s, 3H), 2.91–2.87 (m, 2H),
2.62 (s, 3H), 2.42 (s, 3H), 1.74–1.67 (dd, J = 7.6 Hz, 7.6 Hz, 2H);
HRMS (ESI-TOF) m/z for C27H29N4O4S2 [M+H]+ calcd 537.1630,
found 537.1624. Anal. Calcd for C27H28N4O4S2: C, 60.43; H, 5.26;
N, 10.44. Found: C, 60.36; H, 5.27; N, 10.47.
4.1.26. 3-(4-Methanesulfonylphenoxy)-N-[1-(2-methoxy-
ethoxymethyl)-1H-pyrazol-3-yl]-5-(3-methylpyridin-2-yl)-
benzamide (19e)
To a solution of 13 (2.2 g, 5.74 mmol) in DMF (20 mL) were
added 1-(2-methoxyethoxymethyl)-3-amino-1H-pyrazole (1.2 g,
7.01 mmol), HATU (3.27 g, 8.60 mol) and DIEA (1.48 g, 11.5 mmol)
and the reaction mixture was stirred at room temperature over-
night. The reaction mixture was then washed with 1 N HCl aque-
ous solution, saturated NaHCO3 aqueous solution, and brine. The
organic layer was dried over MgSO4, filtered and concentrated un-
der reduced pressure, and purified by flash column chromatogra-
phy (EA/Pet.Ether = 2:1 ꢂ 1:1) to afford 1.8 g (75% yield) of 19e
as a white solid. 1H NMR (400 MHz, CDCl3) d 8.66 (s, 1H), 8.55 (s,
1H), 7.94–7.92 (d, J = 8.4 Hz, 2H), 7.89 (s, 1H), 7.65–7.62 (br s,
2H), 7.52 (s, 1H), 7.45 (s, 1H), 7.26 (s, 1H), 7.19–7.17 (d,
J = 8.4 Hz, 2H), 6.92 (s, 1H), 5.39 (s, 2H), 3.61 (s, 2H), 3.51–3.50
(br s, 2H), 3.36 (s, 3H), 3.07 (s, 3H), 2.39 (s, 3H); HRMS (ESI-TOF)
m/z for C27H29N4O6S [M+H]+ calcd 537.1808, found 537.1799;
TLC Rf 0.35 (50% EtOAc in petroleum ether). Anal. Calcd for C27H28-
N4O6S: C, 60.43; H, 5.26; N, 10.44. Found: C, 60.36; H, 5.27; N,
10.47.
4.1.22. 3-(4-Methanesulfonylphenoxy)-N-(1-methyl-1H-
pyrazol-3-yl)-5-(3-methylpyridin-2-yl)-benzamide (19a)
To a solution of 13 (187 mg, 0.49 mmol) in dichloromethane
(5 mL) were added 1-methyl-1H-pyrazol-3-ylamine (71 mg,
0.73 mmol), HOBT (132 mg, 0.98 mmol), EDAC (188 mg,
0.98 mmol) and TEA (0.14 mL, 0.98 mmol) and the reaction mix-
ture was stirred at room temperature overnight. The reaction mix-
ture was then washed with 1 N HCl aqueous solution, saturated
NaHCO3 aqueous solution and brine. The organic layer was dried
over MgSO4, filtered and concentrated under reduced pressure,
and purified by flash column chromatography (EA/n-
Hex = 1:2 ꢂ 2:1) to yield 79 mg (35% yield) of 19a as a white solid.
1H NMR (400 MHz, CDCl3) d 8.72 (s, 1H), 8.54–8.53 (d, J = 4.4 Hz,
1H), 7.98–7.88 (m, 3H), 7.66–7.61 (m, 2H), 7.45 (s, 1H), 7.28–
7.23 (m, 1H), 7.19–7.16 (m, 2H), 6.80 (s, 1H), 3.79 (s, 3H), 3.07 (s,
3H), 2.38 (s, 3H); HRMS (ESI-TOF) m/z for C24H23N4O4S [M+H]+
calcd 463.1440, found 463.1425; TLC Rf 0.20(33% n-Hx in EtOAc).
Anal. Calcd for C24H22N4O4S2: C, 62.32; H, 4.79; N, 12.11. Found:
C, 62.38; H, 4.77; N, 12.14.
4.2. Molecular docking
A docking study was carried out using standard protocols with a
Surflex-Dock35 module in SYBYL-X 2.0 (Tripos Inc, St. Louis, MO,
USA) modeling package. The crystal structure of glucokinase was
retrieved from the RCSB Protein Data Bank (PDB entry code:
3A0I).27 According to the protocol of Surflex-Dock, substructures
including glucose, sodium ion, and water were deleted from the
crystal structure modeling and the ligand substructure of the ac-
tive site was extracted. The glucokinase protein structure was pre-
pared by repairing the backbone and side chains. Hydrogen atoms
were then added to the protein structure and ligand. Atom types
and charges were also assigned using AMBER7 FF99 force field
for protein and Gasteiger–Huckel charge for the ligand. With a
standard staged energy minimization protocol, the glucokinase
structure was generated with energy minimization using AMBER7
F99 force field with the Powell energy minimization algorithm.
Using the extracted ligand, a ‘protomol’ which is a representation
of an idealized ligand to which putative ligands can be aligned,
was generated. To predict the appropriate binding conformation
for glucokinase activators, co-crystallized ligand was used as a
template reference molecule. Docking simulations of compounds
were conducted with binding to the active site of glucokinase
by an empirical scoring function to score the ligand and
The following compounds (19b–e) were prepared according to
the procedure described for the synthesis of 19e using appropriate
pyrazoles.
4.1.23. N-(1-Ethoxymethyl-1H-pyrazol-3-yl)-3-(4-
methanesulfonylphenoxy)-5-(3-methylpyridin-2-yl)-
benzamide (19b)
52% yield, white solid. 1H NMR (400 MHz, CDCl3) d 8.91 (s, 1H),
8.55–8.54 (d, J = 5.2 Hz, 1H), 7.95–7.93 (d, J = 8.4 Hz, 1H), 7.85 (s, 1H),
7.69–7.64 (m, 3H), 7.56–7.53 (t, 1H), 7.48–7.44 (m, 2H), 7.21–7.18 (d,
J = 8.8 Hz, 1H), 6.72 (s, 1H), 5.54 (s, 2H), 3.62–3.57 (dd, J = 6.8, 6.8 Hz,
2H), 3.07 (s, 3H), 2.41 (s, 3H); HRMS (ESI-TOF) m/z for C26H27N4O5S
[M+H]+ calcd 507.1702, found 507.1692. Anal. Calcd for C26H26N4O5S:
C, 61.65; H, 5.17; N, 11.06. Found: C, 61.57; H, 5.16; N, 11.09.
4.1.24. N-[1-(2-Diethylaminoethyl-1H-pyrazol-3-yl)-3-(4-
methanesulfonylphenoxy)-5-(3-methylpyridin-2-yl)-
benzamide (19c)
50% yield, white solid. 1H NMR (400 MHz, MeOD) d 8.47–8.46
(d, J = 4.4 Hz, 1H), 8.01–7.97 (d, J = 8.8 Hz, 1H), 7.93 (s, 1H), 7.83–

2292
K. Park et al. / Bioorg. Med. Chem. 22 (2014) 2280–2293
protomol-guided docking in Surflex-Dock. CScore calculations of
the docking ligands were also performed.
Eight-week old male C57BL/6 mice (OrientBio Inc, Republic of Kor-
ea) or ten-week old male C57BLKS/J db/db mice (SLC Inc, Japan)
were fasted overnight (with free access to water) before perform-
ing the test. The mice were orally administered with the compound
or vehicle alone (0.5% methylcellulose solution). After 30 min, the
mice were administered with an oral glucose challenge (2 g/kg).
Blood glucose concentrations were measured by GlucoDr
AMG-3000 (Allmedicus Inc., Republic of Korea) just prior to and
following the glucose challenge (30, 60, 90, 120 and 180 min) from
the tail tip. AUC values of the time–glucose curve were calculated
from the data.
4.3. Bioassays
For experiments involving the use of animals, all procedures
were carried out in accordance with the guidelines of the Institu-
tional Animal Care and Use Committee of Yuhan Research Institute.
4.3.1. Glucokinase enzymatic assay
An enzymatic glucokinase assay using purified recombinant hu-
man pancreatic glucokinase was used to evaluate the compounds.
Glucokinase activity was assessed spectrometrically by a coupled
reaction with glucose-6-phosphate dehydrogenase (G6PDH).33
Briefly, GK catalyzes glucose phosphorylation to generate glu-
cose-6-P, which is oxidized by G6PDH with the concomitant reduc-
tion of NADPH. The resultant NADPH is then monitored via an
increase in the rate of absorbance at 340 nm using a plate reader
(Spectra-Max 384 plus, Molecular Devices, CA, USA). All com-
pounds were prepared in DMSO and the assay was performed in
4.4. Measurement of physiochemical properties
Aqueous solubility of the compounds in phosphate buffer (pH
7.4) was determined. To investigate permeability of the com-
pounds, the Papp, PAMPA values (via parallel artificial membrane per-
meability assay (PAMPA)) were measured experimentally using a
BD Gentest™ Pre-coated PAMPA Plate System. In vitro metabolism
studies for CYP inhibition and time-dependent inhibition (TDI)
were performed using human liver microsomes and incubation
conditions were similar to those previously described.34 Briefly,
the incubation mixtures containing pooled human liver micro-
somes (0.25 mg/mL), CYP-selective substrates, and each test com-
96-well plates in a final volume of 100
pH 7.4, 10 mM glucose, 25 mM KCl, 1 mM MgCl2, 1 mM DTT, 1 mM
ATP, 1 mM NADP, 2.5 U/mL G6PDH, 1 mM glucose, 0.1 g glucoki-
lL containing 25 mM HEPES
l
nase, and the test compounds. The fold activation of the enzyme
was assessed by comparing with controls (GK activation in DMSO
only was considered as 100%). For EC50 determination, 12 different
concentrations of the compounds were tested in the assay, and the
fold changes in activity versus controls were fitted to a sigmoidal
curve using a dose–response variable slope model in GraphPad
Prism 5.
pound (up to 25 lM) were preincubated at 37 °C. The reaction
was initiated by adding an NADPH-generating system (1.3 mM
NADP, 3.3 mM glucose 6-phosphate, 3.3 mM MgCl2, and 1.0 unit/
mL glucose-6-phosphate dehydrogenase). After incubation, the
reaction was stopped by placing the tubes on ice and adding
ice-cold acetonitrile solution. The incubation mixtures were then
centrifuged (10,000g for 5 min at 4 °C). Supernatant aliquots were
injected into an LC–MS/MS system.
4.3.2. Glucose uptake in rat primary hepatocytes
Hepatocytes were isolated by in situ liver perfusion with colla-
genase. The viability of isolated hepatocytes were determined to be
over 85% via Trypan blue exclusion assay. Cells were suspended in
MEM supplemented with 10% fetal bovine serum (v/v), 100 U/mL
4.5. Pharmacokinetics (PK) protocol
ICR male mice and SD rats (7-weeks old) received an intrave-
nous bolus (3 mg/kg) and oral gavage doses (10 mg/kg) of the test
compound. Blood samples were obtained at 0.08 (iv only), 0.25, 0.5,
1, 2, 4, and 7 h after drug administration. Plasma samples were ob-
tained following centrifugation of blood at 4 °C and stored at
ꢀ20 °C for analysis. To a 0.1 mL aliquot of mouse or rat plasma,
0.2 mL of acetonitrile was added. The mixture was vortexed for
10 min and then centrifuged for a further 10 min. The supernatant
was then tested for test compound concentration via HPLC. Phar-
macokinetic variables were evaluated by noncompartmental anal-
ysis using WinNonlin Professional Version 5.2 (Pharsight, MO,
USA).
penicillin, and 100
l
g/mL streptomycin. 1 ꢁ 106 cells/well were
planted onto collagen 1-coated 12-well plates. After 36 h incuba-
tion (37 °C; 5% CO2/95% air v/v) to allow cell attachment, the med-
ium was replaced with serum-free media for a further 12 h culture.
2-Deoxy-
cytes. Cells were washed and incubated in serum-free MEM con-
taining 5.5 mM glucose, and 2 Ci/mL 2-deoxy-
-[3H]-glucose
D
-[3H]-glucose uptake was assessed in cultured hepato-
l
D
with or without the test compounds for 4 h. The reaction was ter-
minated and washed three times with ice-cold phosphate buffered
saline (PBS), and then cells were lysed with 0.1 N NaOH. Portions of
cell lysates were used for scintillation counting, and the results
were recorded as increased percentage versus control.
4.6. Toxicity study
4.3.3. Insulin secretion in rat pancreatic islets
4.6.1. 14-Day repeated oral dose toxicity study in rats
An insulin secretion assay was performed using pancreatic is-
lets isolated from male Sprague–Dawley rats. Insulin release was
determined during static islet incubation. Briefly, groups of five is-
lets were placed in incubation wells. After a 30-min pre-incubation
with HEPES-buffered Krebs–Ringer buffer (pH 7.4) containing
5 mm glucose, islets were transferred to wells containing 2 mL
HEPES-buffered Krebs–Ringer buffer and varying concentrations
of glucose and the test compounds. The studies were performed
at 37 °C in a waterbath shaker with an atmosphere of 5% CO2. Sam-
ples of incubation buffer were collected at 1 h for insulin determi-
nation using a mouse ELISA kit (Mercodia, Uppsala, Sweden).
Results were recorded as the increased percentage versus control.
Specific pathogen-free Sprague–Dawley rats of each gender (20
males and 20 females) at 5 weeks of age were obtained from ORI-
ENTBIO INC. (Republic of Korea). These animals were housed in a
room maintained at a temperature of 22 2 °C and a relative
humidity of 50 15%, with artificial lighting from 08:00 to 20:00
(150–300 Lux) and 10–15 air changes per hour. Each animal was
housed in separate stainless steel cages (W260 ꢁ D350 ꢁ
H210 mm). All rats had free access to tap water and food (Teklad
Certified Irradiated Global 18% Protein Rodent Diet 2918C, Harlan
Laboratories Inc., USA). Male and female rats were assigned to
Groups 1 through 4 and the compound was administered at
dose levels of 0, 250, 500, and 1000 mg/kg body weight/day
(mg/kg/day) for 14 days. Group 1 received vehicle control
(0.5% methylcellulose solution) and animals were dosed at a vol-
ume of 10 mL/kg. After 14 days of treatment, all surviving animals
were sacrificed and necropsied by Day 15. Liver from each animal
4.3.4. In vivo oral glucose tolerance test (OGTT) assay
Anti-diabetic effects of the compounds were evaluated by a glu-
cose tolerance test (OGTT) using normal and diabetic db/db mice.

K. Park et al. / Bioorg. Med. Chem. 22 (2014) 2280–2293
2293
was preserved in 10% neutral-buffered formalin and testis was
preserved in modified Davidson’s fixative. The tissue was embed-
ded in paraffin wax, sectioned, stained with hematoxylin and eosin
(H&E) and examined microscopically. Histopathological examina-
tion was performed only on control and high-dose groups of both
genders.
Hee-Sook Jun at Gachon University of Medicine & Science for their
helpful discussion. We also thank Lee Farrand for proofreading the
manuscript.
References and notes
1. Fowler, M. J. Clin. Diab. 2008, 26, 77.
2. Kahn, S. E.; Hull, R. L.; Utzschneider, K. M. Nature 2006, 444, 840.
3. Bouchie, A. Nat. Biotechnol. 2013, 31, 469.
4. Han, S.; Hagan, D. L.; Taylor, J. R.; Xin, L.; Meng, W.; Biller, S. A.; Wetterau, J. R.;
Washburn, W. N.; Whaley, J. M. Diabetes 2008, 57, 1723.
4.6.2. Escalating oral gavage dose range-finding study in beagle
dogs
Male and female beagle dogs (1 each) were obtained from Mar-
shall Biotech. Co., Ltd (Beijing, China). The dogs were 5 months old
and weighed 6.1 kg (male) and 5.98 kg (female) at the initiation of
the study. Dogs were acclimated to laboratory conditions for
14 days, and were housed in stainless steel cages in a room main-
tained at 23 3 °C and a relative humidity of 28–67%, with artifi-
cial lighting from 08:00 to 20:00 (150–300 Lux) and 10–15 air
changes per hour. All dogs were fed Purina Certified Lab Canine
Diet (Agribrands Purina Korea Inc.) with filtered tap water avail-
able ad libitum. Animals were administered with the test com-
pound at repeated escalating dose levels of 100, 300, and
1000 mg/kg of body weight/day (mg/kg/day) for 8 days (at Days
3, 6, and 8 for each dose). The vehicle control was 0.5% methylcel-
lulose solution and the animals were dosed at a volume of 5 mL/kg.
Necropsies were performed on the two animals at the termination
of the study. Test is were preserved in modified Davidson’s fixative.
The tissue was embedded in paraffin wax, sectioned, stained with
hematoxylin and eosin (H&E), and examined microscopically.
5. Stenlöf, K.; Cefalu, W. T.; Kim, K. A.; Alba, M.; Usiskin, K.; Tong, C.; Canovatchel,
W.; Meininger, G. Diabetes Obes. Metab. 2013, 15, 372.
6. Joslin’s Diabetes Mellitus; Lebovitz, H. E., Ed., 14th ed.; Lippincott Williams &
Wilkins: Philadelphia, PA, 2005; p 687.
7. Ralph, E. C.; Thomson, J.; Almaden, J.; Sun, S. Biochemistry 2008, 47, 5028.
8. Matschinsky, F. M. Diabetes 1996, 45, 223.
9. Antoine, M.; Boutin, J. A.; Ferry, G. Biochemistry 2009, 48, 5466.
10. Heredia, W.; Thomson, J.; Nettleton, D.; Sun, S. Biochemistry 2006, 45, 7553.
11. Matschinsky, F. M.; Glaser, B.; Magnuson, M. A. Diabetes 1998, 47, 307.
12. Waring, M. J.; Brogan, I. J.; Coghlan, M.; Johnstone, C.; Jones, H. B.; Leighton, B.;
Mckerrecher, D.; Pike, K. G.; Robb, G. R. Med. Chem. Commun. 2011, 2, 771.
13. Pfefferkorn, J. A.; Guzman-Perez, A.; Oates, P. J.; Litchfield, J.; Aspnes, G.; Basak,
A., et al Med. Chem. Commun. 2011, 2, 828.
14. Pfefferkorn, J. A.; Guzman-Perez, A.; Litchfield, J.; Aiello, R.; Treadway, J. L.;
Pattersen, J.; Minich, M. L.; Filipski, K. J.; Jones, C. S.; Tu, M.; Aspnes, G.; Sweet,
L.; Liras, S.; Rolph, T. P., et al J. Med. Chem. 2012, 55, 1318.
15. Massa, M. L.; Gagliardino, J. J.; Francini, F. Life 2011, 63, 1.
16. Bebernitz, G. R.; Beaulieu, V.; Dale, B. A.; Deacon, R.; Duttaroy, A.; Gao, J.;
Grondine, M. S.; Gupta, R. C.; Kakmak, M.; Kavana, M.; Kirman, L. C.; Liang, J.;
Maniara, W. M.; Munshi, S.; Nadkarni, S. S.; Schuster, H. F.; Stams, T.; St Denny,
I.; Taslimi, P. M.; Vash, B.; Caplan, S. L. J. Med. Chem. 2009, 52, 6142.
17. Park, K.; Lee, B.-M.; Kim, Y.-H.; Han, T.; Yi, W.; Lee, D.-H.; Choi, H.-H.; Chong,
W.; Lee, C.-H. Bioorg. Med. Chem. Lett. 2013, 23, 537.
18. Park, K. Arch. Pharmacal Res. 2012, 35, 2029.
19. Matschinsky, F. M. Trends Pharmacol. Sci. 2013, 34, 90.
20. Verspohl, E. J. Pharmacol. Rev. 2012, 2, 188.
21. Winzell, M. S.; Coghlan, M.; Leighton, B.; Frangioudakis, G.; Smith, D. M.;
Storlien, L. H.; Ahrén, B. Eur. J. Pharmacol. 2011, 663, 80.
22. (a) Fyfe, M. C. T.; Procter, M. J. Drugs Future 2009, 34, 641; (b) Matschinsky, F.
Nat. Rev. Drug Disc. 2009, 8, 399.
23. Iino, T.; Hashimoto, N.; Hasegawa, T.; Chiba, M.; Eiki, J.; Nishimura, T. Bioorg.
Med. Chem. Lett. 2010, 20, 1619.
24. Sidduri, A.; Grimsby, J. S.; Corbett, W. L.; Sarabu, R.; Grippo, J. F.; Lou, J.; Kester,
R. F.; Dvorozniak, M.; Marcus, L.; Spence, C.; Racha, J. K.; Moore, D. J. Bioorg.
Med. Chem. Lett. 2010, 20, 5673.
25. Grimsby, J.; Sarabu, R.; Corbett, W. L.; Hayne, N. E.; Bizzarro, F. T.; Coffey, J. W.;
Guertin, K. R.; Hilliard, D. W.; Kester, R. F.; Mahaney, P. E.; Marcus, L.; Qi, L. D.;
Soence, C. L.; Tengi, J.; Magnuson, M. A.; Chu, C. A.; Dvorozniak, M. T.;
Matschinsky, F. M.; Grippo, J. F. Science 2003, 301, 370.
26. Sarabu, R.; Tilley, J. W.; Grimsby, J. RSC Drug Discov. Series 2011, 4, 51.
27. Mitsuya, M.; Kamata, K.; Bamba, M.; Watanabe, H.; Sasaki, Y.; Sasaki, K.;
Ohyama, S.; Hosaka, H.; Nagata, Y.; Eiki, J.; Nishimura, T. Bioorg. Med. Chem.
Lett. 2009, 19, 2718.
28. Waring, M. J.; Johnstone, C.; McKerrecher, D.; Pike, K. G.; Robb, G. R. Med. Chem.
Commun. 2011, 2, 775.
29. Meininger, G. E.; Scott, R.; Alba, R.; Shentu, Y.; Luo, E.; Amin, H.; Davies, M. J.;
Kaufman, K. D.; Goldstein, B. J. Diabetes Care 2011, 34, 2560.
30. Pike, K. G.; Allen, J. V.; Caulkett, P. W.; Clarke, D. S.; Donald, C. S.; Fenwick, M. L.;
Johnson, K. M.; Johnstone, C.; McKerrecher, D.; Rayner, J. W.; Walker, R. P.;
Wilson, I. Bioorg. Med. Chem. Lett. 2011, 11, 3467.
31. (a) Iino, T.; Sasaki, Y.; Bamba, M.; Mitsuya, M.; Ohno, A.; Kamata, K.; Hosaka,
H.; Maruki, H.; Futamura, M.; Yoshimoto, R.; Ohyama, S.; Sasaki, K.; Chiba, M.;
Ohtake, N.; Nagata, Y.; Eiki, J.; Nishimura, T. Bioorg. Med. Chem. Lett. 2009, 19,
5531; (b) Takahashi, K.; Hashimoto, N.; Nakama, C.; Kamata, K.; Sasaki, K.;
Yoshimoto, R.; Ohyama, S.; Hosaka, H.; Maruki, H.; Nagata, Y.; Eiki, J.;
Nishimura, T. Bioorg. Med. Chem. 2009, 17, 7042; (c) Nishimura, T.; Iino, T.;
Mitsuya, M.; Bamba, M.; Watanabe, H.; Tsukahara, D.; Kamata, K.; Sasaki, K.;
Ohyama, S.; Hosaka, H.; Futamura, M.; Nagata, Y.; Eiki, J. Bioorg. Med. Chem.
Lett. 2009, 19, 1357; (d) Ishikawa, M.; Nonoshita, K.; Ogino, Y.; Nagae, Y.;
Tsukahara, D.; Hosaka, H.; Maruki, H.; Ohyama, S.; Yoshimoto, R.; Sasaki, K.;
Nagata, Y.; Eiki, J.; Nishimura, T. Bioorg. Med. Chem. Lett. 2009, 19, 4450; (e)
Iino, T.; Hashimoto, N.; Sasaki, K.; Ohyama, S.; Yoshimoto, R.; Hosaka, H.;
Hasegawa, T.; Chiba, M.; Nagata, Y.; Eiki, J.; Nishimura, T. Bioorg. Med. Chem.
2009, 17, 3800.
4.6.3. Mutagenicity study
Mutagenicity testing was carried out according to standard Ames
test procedures (Maron and Ames). Two Salmonella typhimurium
strains (TA98 and TA100) were used in a reverse mutation test both
with and without S9 mix (Aroclor 1254-induced male Sprague–
Dawley rat liver (MOLTOXTM, USA) and Cofactor-I (Wako Chemicals,
Japan). The designated 19e concentrations in this test were 1.5, 5, 15,
50, 150, 500, 1500, 5000 lg/plate. The treatment mixture and plate
conditions were checked for the formation of precipitation and cyto-
toxicity and the test strains were exposed using a direct plate incor-
poration method. A small amount of bacterial growth on each
master plate was transferred to a flask containing 20 mL of liquid
medium (2.5% Oxoid Nutrient Broth No.2). Inoculated flasks were
incubated for 10 h in a shaker/incubator (37 2 °C, 120 rpm). Viable
cell counts from overnight cultures were determined by optical den-
sitometry (OD) at 600 nM, and the cultures were stored at 4 °C. For
the plating assay, 0.5 mL of S9 mix, 0.1 mL of bacterial culture and
0.1 mL of 19e dissolved in DMSO were added to each sterile culture
tube containing 2 mL of top agar held at 45 2 °C in a dry bath. After
the top agar solidified, plates were inverted and incubated at
37 2 °C for 50 2 h and then revertant colonies were counted.
For cultures without S9 mix, sodium azide (0.5 lg/plate) and
4-nitroquinoline-N-oxide (0.5 g/plate) were used as positive con-
trols for TA100 and TA98, respectively. In cultures containing S9
mix, 2-aminoanthracene (1
lg/plate) for TA100 and benzo[a]pyrene
(1 g/plate) for TA98 were used as positive controls. The evaluation
l
criteria defined a positive result as reproducible exhibiting a more
than 2 fold dose-related increase in the number of revertants per
plate in at least one strain with or without a metabolic activation
system.
32. Johnson, D.; Shepherd, R. M.; Gill, D.; Gorman, T.; Smith, D. M.; Dunne, M. J.
Diabetes 2007, 56, 1694.
33. Fyfe, M. C.; White, J. R.; Taylor, A.; Chatfield, R.; Wargent, E.; Printz, R. L.;
Sulpice, T.; McCormack, J. G.; Procter, M. J.; Reynet, C.; Widdowson, P. S.;
Wong-Kai-In, P. Diabetologia 2007, 50, 1277.
34. (a) Bae, S. K.; Cao, S.; Seo, K. A.; Kim, H.; Kim, M. J.; Shon, J. H.; Liu, K. H.; Zhou,
H. H.; Shin, J. G. Drug Metab. Dispos. 2008, 36, 1679; (b) Parkinson, A.; Kazmi, F.;
Buckley, D. B.; Yerino, P.; Paris, B. L.; Holsapple, J.; Toren, P.; Otradovec, S. M.;
Ogilvie, B. W. Drug Metab. Dispos. 2011, 39, 1370.
Acknowledgements
This study was supported by a Grant from the Chungcheong
Leading Industry Office for Medicine & Bio Projects, Ministry of
Knowledge Economy, Republic of Korea (C-2-1, 500108,
7007618). The authors would like to thank Professor Woo-Chan
Son at the University of Ulsan College of Medicine and Professor
35. Jain, A. N. J. Med. Chem. 2003, 46, 499.