Benzamide derivatives as dual-action hypoglycemic agents that inhibit glycogen phosphorylase and activate glucokinase

Article abstract of DOI:10.1016/j.bmc.2009.08.045

A series of benzamide derivatives which can simultaneously inhibit glycogen phosphorylase (GP) and activate glucokinase (GK) were prepared and evaluated. The structure-activity relationships (SAR) of these compounds were also presented. Among these, compo

Full text of DOI:10.1016/j.bmc.2009.08.045

Bioorganic & Medicinal Chemistry 17 (2009) 7301–7312
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Benzamide derivatives as dual-action hypoglycemic agents that inhibit
glycogen phosphorylase and activate glucokinase
a,b
Lei Zhang a, , Honglin Li b, , Qingzhang Zhu a, , Jun Liu c, Ling Chen a, Ying Leng a, , Hualiang Jiang
,
*
Hong Liu a,
*
a State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences,
555 Zu Chong Zhi Road, Shanghai 201203, PR China
b School of Pharmacy, East China University of Science and Technology, Shanghai 200237, PR China
c Jiangsu Center for Drug Screening, China Pharmaceutical University, Nanjing 210038, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of benzamide derivatives which can simultaneously inhibit glycogen phosphorylase (GP) and
activate glucokinase (GK) were prepared and evaluated. The structure–activity relationships (SAR) of
these compounds were also presented. Among these, compounds 12, 13l, 13q, and 13v showed moderate
Received 17 July 2009
Revised 19 August 2009
Accepted 20 August 2009
Available online 24 August 2009
activities towards both GK and GP. Compound 13h inhibited hLGP with an IC50 of 8.95
GK with an EC50 of 1.87 M. The possible binding modes of compounds 12, 13l, 13h, and 13q with GP and
GK were also explored by molecular docking simulation.
lM and activated
l
Keywords:
Ó 2009 Elsevier Ltd. All rights reserved.
Glucokinase (GK) activators
Glycogen phosphorylase (GP) inhibitors
Type 2 diabetes
Dual action
1. Introduction
vation of GK is expected to improve glycemic control by modulat-
ing hepatic glucose balance and decreasing the threshold for
insulin secretion. Several groups have described various synthetic
small molecules that act as GK activators, which function through
binding to an allosteric site (Fig. 2).4 Among these, Banyu company
has identified a small synthetic compound 6 as GK activator and
published its crystal structure bound to GK.5
Abnormal glucose metabolism is the major pathologic factor of
Type 2 diabetes mellitus. Elevated hepatic glucose production
(HGP) by glycogenolysis and gluconeogenesis is a contributing fac-
tor to hyperglycemia.1 Therefore enzymes that have high control
strength on hepatic glucose metabolism are potential targets for
controlling hepatic glucose balance and thereby modulating blood
glucose levels in Type 2 diabetes. Glucokinase (GK) and glycogen
phosphorylase (GP), two critical enzymes involved in the process
of glucose metabolism, have high control strength over glucose
metabolism (Fig. 1). And both of them are potential therapeutic
targets for Type 2 diabetes.
Glucokinase (hexokinase IV or D) is a cytoplasmic enzyme that
phosphorylates glucose and triggers glucose utilization and metab-
olism. It is predominantly expressed in liver and pancreas, where it
removes glucose from the blood and reacts with adenosine tri-
phosphate (ATP) to form glucose-6-phosphate (Glu-6-P), the first
and rate-limiting biosynthetic step in the conversion of glucose
to its storage form, glycogen.2 Phosphorylation of glucose by GK
in the liver promotes glycogen synthesis, while in the pancreatic
b-cell this results in glucose-sensitive insulin release.3 Both of
these effects in turn reduce plasma glucose levels. Therefore, acti-
Glycogen phosphorylase, the rate determining enzyme of glyco-
gen degradation, catalyzes the breakdown of glycogen to glucose-
1-phosphate.6 In the liver glucose-1-phosphate is metabolised
further to glucose, which is then secreted into the bloodstream.
GP exists in two interconvertible forms, GPa (the phosphorylated
form, high activity, high substrate affinity, predominantly R state)
and GPb (the nonphosphorylated form, low activity, low substrate
affinity, predominantly T state).7 So inhibition of GPa may repre-
sent another useful therapy for the treatment of Type 2 diabetes.
Several structurally different classes of small molecule inhibitors
have been reported (Fig. 3).8–10 Based on screening of specs library
and structural modification, we have recently described a series of
benzamide derivatives as GP inhibitors, among which compound
12 showed a moderate rabbit muscle GPa inhibitory activity with
an IC50 of 2.68
Obviously, GPa inhibitor 12 bears a similar structure with GK
activator because both of them share tetra-substituted
l
M.11
6
a
benzamide scaffold with a heteroaryl thiol moiety. Thus we specu-
late that this kind of compounds can play a dual-action role as
hypoglycemic agents that not only inhibit glycogen phosphorylase
* Corresponding authors. Tel.: +86 21 50807042; fax: +86 21 50807088 (H.L.).
E-mail address: hliu@mail.shcnc.ac.cn (H. Liu).
Authors equally contributed to this work.
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.08.045

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L. Zhang et al. / Bioorg. Med. Chem. 17 (2009) 7301–7312
Glucose
Polyol pathway
Glycogen
Glycogen
Glycogen
synthase
Glucokinase
Glu-6-Pase
Phosphorylase
Pentose phosphate
pathway
Glu-6-P
Glu-1-P
Glycolysis
Gluconeogenesis
Anaerobic metabolism
Lactate
ATP
Pyruvate
Aerobic metabolism
H2O, CO2
ATP
Acetyl-CoA
TCA Cycle
Fats
Mitochondria
Figure 1. Simplified glucose metabolism pathway.
S
N
NH
S
NH
NH
S
S
Cl
O
S
O
O
S
O
O
S
O
N
O
N
N
O
O
3
1
(RO-28-1675)
2
(LY-2121260)
Lily
Prosidion
Hoffman La Roche
O
S
O
S
S
F
N
S
N
O
NH
NH2
N
N
NH NH
S
O
N
NH
O
S
O
Cl
6
4
5
Novartis
Banyu
Novo Nordisk
Figure 2. Structures of representative GK activators.
but also activate glucokinase. It has been reported that CP-320626
developed by Pfizer was a dual-action hypoglycemic and hypocho-
lesterolemic agents that inhibited glycogen phosphorylase and
lanosterol demethylase, which had the potential to favorably affect
both hyperglycemia and hypercholesterolemia in patients with
Type 2 diabetes.12 To verify our hypothesis, we firstly carried out
biological evaluation of compound 6 towards both GK and GP. As
expected, compound 6 showed moderate GPa inhibitory efficacy
5-difluoro-2-nitrobenzoic acid with oxalyl dichloride and DMF
afforded 4, 5-difluoro-2-nitrobenzoyl chloride 16. Then 16 reacted
with appropriate commercially available aniline to give benzam-
ides 17a–x. Reaction of 17a–x with corresponding thiols and tri-
ethylamine provided 18a–x. And compounds 13a–x were
obtained by hydrogenising 18a–x catalyzed by iron power and sat-
urated aqueous NH4Cl (Scheme 1).
The series of piperazine-containing amide analogues 14a–e
were prepared from 1-(2-R2-phenyl) piperazine and 19a–e which
were produced by the reaction of 2-chloroacetyl chloride with ami-
no compounds as shown in Scheme 2.
(GPa IC50 = 43.9
EC50 = 0.06711
l
M) as well as good GK activating efficacy (GK
l
M).
Then we continued our structure–activity relationship (SAR) ef-
forts around compound 12. A series of benzamide compounds as
well as piperazine-containing amides and symmetrical benzamide
analogues were designed and synthesized, and their biological
activities towards both GK and GP were evaluated. Among these,
compounds 12, 13l, and 13q showed moderate activity towards
both GK and GP. Compound 13h inhibited GPa with an IC50 of
The series of symmetrical benzamide analogues 15a–c were
synthesized by isophthaloyl dichloride reacting with aniline in
the presence of pyridine or triethylamine as shown in Scheme 3.
3. Biology
8.95 lM and activated glucokinase with an EC50 of 1.87 lM. The
3.1. Glucokinase enzyme assays
possible binding modes of compounds 12, 13l, 13h, and 13q with
glycogen phosphorylase a and glucokinase were also explored by
molecular docking simulation.
cDNA of human glucokinase (MGC: 1742, purchased from Ori-
Gene Technologies, USA) was subcloned into the pET28a(+) expres-
sion vector, and expressed in Escherichia coli strain BL21(DE3).13–15
The NH2 terminal end of (His)6-tag glucokinase fusion protein was
purified by Ni-NTA metal chelate affinity chromatography and
stored at À80 °C in 50 mM Tris–HCl pH7.4, 1 mM DTT, 50 mM NaCl
and 10% glycerol.16
2. Chemistry
As part of our SAR studies around compound 12, compounds
13a–x were designed and synthesized. In general, treatment of 4,

L. Zhang et al. / Bioorg. Med. Chem. 17 (2009) 7301–7312
7303
F
Ph
O
O
O
OH
Cl
O
CO2H
N
H
N
N
OH
Cl
N
H
N
NH
OH
N
H
NH
O
NH
OH
O
Cl
7
8
(CP-320626)
Pfizer
(CP-368296)
Pfizer
9 (CP-403700)
Pfizer
O
S
Cl
Cl
O
H
N
S
F
N
NH
N
H
NH2
N
N
H
H
N
NH
O
NH2
Cl
O
O
N
12
10
11
SIMM
Merk
AstraZeneca
Figure 3. Selected inhibitors binding at the interface of the GP homodimer.
compounds were tested in the assay, and the fold changes in activ-
ity versus controls were fitted to sigmoidal curve using a four-
parameter logistic model in GraphPad Prism 4.
O
O
O
F
F
F
F
F
F
R1
OH
NO2
b
a
Cl
NO2
N
H
NO2
17a-x
16
3.2. Glycogen phosphorylase enzyme assays
O
O
S
F
R1
d
c
S
F
R1
R2
N
H
NH2
R2
N
Glycogen phosphorylase a (from rabbit muscle), glycogen, glu-
cose-1-phosphate, malachite green, and ammonium molybdate
were purchased from the Sigma–Aldrich Corporation (St. Louis,
MO, USA). Reagents and solvents were obtained from commercial
suppliers and used without further purification. Solvents used
were AR grade.
The enzymatic inhibition of phosphorylase activity was moni-
tored using microplate reader (Bio-Rad) based on the published
methods.17 In brief, GPa activity was measured in the direction of
glycogen synthesis by the release of phosphate from glucose-1-
phosphate. Each compound was dissolved in DMSO and diluted
at different concentrations for IC50 determination. The enzymes
H
NO2
18a-x
13a-x
Scheme 1. Reagents and conditions: (a) oxalyl chloride (1.2 equiv), DMF (two
drops), CH2Cl2, rt, 2 h; (b) R–NH2 (1 equiv), pyridine (2 equiv), CH2Cl2, rt; (c) thiol
(1 equiv), Et3N (3 equiv), CH3CN, mw, 80 °C, 40 min; (d) iron powder (10 equiv),
isopropanol, reflux, 0.5 h.
H
N
N
R1
H
N
a
Cl
b
N
O
Cl
R1
Cl
were added into the 100 lL buffer with compounds dissolved in
containing 50 mM Hepes (pH 7.2), 100 mM KCl, 2.5 mM MgCl2,
0.5 mM glucose-1-phosphate, and 1 mg/mL glycogen in 96-well
O
O
R2
19a-e
14a-e
microplates (costar). After the addition of 150 lL of 1 M HCl con-
Scheme 2. Reagents and conditions: (a) R1–NH2 (1 equiv), K2CO3 (2 equiv), THF,
45 °C, 2 h; (b) 1-(2-R2-phenyl)piperazine (1 equiv), KI (catalyzer), K2CO3 (2 equiv),
THF, 50 °C, 4 h.
taining 10 mg/mL ammonium molybdate and 0.38 mg/mL mala-
chite green, reactions were run at 22 °C for 25 min. And then the
phosphate absorbance was measured at 655 nm. The IC50 values
were estimated by fitting the inhibition data to a dose-dependent
curve using a logistic derivative equation.
O
O
O
O
R
R
a
Cl
Cl
N
H
N
H
4. Results and discussion
15a-c
In this SAR study, we attempted to determine the effect of het-
eroaryl thiol moiety and the substituted benzyl group based on
the central benzamide ring. In addition, according to our previous
work, several piperazine-containing amide analogues and symmet-
rical benzamide analogues with incorporation of substituted benzyl
groups were also presented. For a better comparison, we used the
GK activator 6 developed by Banyu as the reference compound.
Our previous studies indicated that introducing NH2, CN, F, CF3,
and OMe to the phenyl group led to apparent improvement in GPa
inhibitory activity11, we therefore retained these five substitutes.
All these substitutions on the phenyl ring turned out to be tolerated
in the inhibition of GPa. When NH2 was introduced, most of the
compounds displayed good inhibitory activity. Among these com-
pounds, 1-methyl-1H-imidazole thiol derivative 12 showed the
best inhibitory activity while thiazole-2-thiol and 4-methylthia-
zole-2-thiol derivatives (13a, 13b) had slightly decreased activity.
Scheme 3. Reagents and conditions: (a) NH2–R (2 equiv), pyridine or triethylamine
(5 equiv), CHCl3, rt, 12 h.
The GK activity was assessed spectrometrically by a coupled
reaction with glucose-6-phosphate dehydrogenase (G6PDH).14–16
Briefly, GK catalyzes glucose phosphorylation to generate glu-
cose-6-P, which was oxidized by the G6PDH with the concomitant
reduction of NADP. The product NADPH was then monitored by the
increase rate of absorbance at 340 nm in a plate reader (Spectra-
Max 190; Molecular Devices, USA). The assay was performed in
96-well plates in a final volume of 100
pH 7.4, 5 mM glucose, 25 mM KCl, 2 mM MgCl2, 1 mM dithiothre-
itol (DTT), 1 mM ATP, 1 mM NADP, 2.5 U/mL G6PDH, 0.5 g (His)6-
glucokinase. For EC50 determination, different concentrations of
lL containing 50 mM HEPES
l

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L. Zhang et al. / Bioorg. Med. Chem. 17 (2009) 7301–7312
Pyridine-2-thiol, 1,3,4-thiadiazole-2-thiol and 4,6-dimethylpyrimi-
dine-2-thiol derivatives (13c–e) displayed even lower inhibition of
GPa activity. The reversed amide analogue 13f showed decreased
activity compared to compound 12. After inserting a methylene
group between the amino nitrogen and the phenyl ring, compound
13g was slightly less potent to GPa. The CF3, F, and CN substituted
series with different heteroaryl thiols moieties shared a similar
trend in potency change towards GPa: 1-methyl-1H-imidazole thi-
ols were more active (13h, 13l, 1q) while the potency decreased for
other heteroaryl thiols. When 3-methoxyl or 4-methoxyl group was
introduced, 13v and 13w exhibited similarly moderate GPa inhibi-
tory activity. Interestingly, incorporation of 3- and 4-methoxyl
groups in the same phenyl ring produced more potent compound
13x possibly due to increased hydrophobic affinity for GP.
In terms of activation of GK, the variation of heteroaryl thiol
moiety indicated a strong preference for 1-methyl-1H-imidazole
thiol group. Replacing the original 1-methyl-1H-imidazole thiol
with other heteroaryl thiols had a detrimental effect on activities
towards GK. Thiazole-2-thiol substituted benzamides showed
weak potency (13a, 13i, 13m) to GK, while 4-methylthiazole-2-thi-
ols (13b, 13j, 13n) led to a slight drop in GK activation. Compound
13u bearing a benzoxazole-2-thiol did not activate GK possibly due
to a steric clash between the bulky benzoxazol group and GK en-
zyme. Compound 13v was more potent compared to 13w, indicat-
ing the preference for 3-methoxyl over 4-methoxyl group in the
phenyl ring. Incorporation of 3- and 4-methoxyl groups in the
same phenyl ring afforded increased GK activator 13x in accor-
dance to the case of GPa inhibition.
In consideration of dual action towards GPa and GK, it turned out
that the substituted benzyl group principally accounted for the
retainment of GPa inhibitory activity while the heteroaryl thiol moi-
etywasresponsiblefortheactivationofGK. Fromthese results, some
compounds were proved to be potent GPa inhibitors but with weak
potency to GK (13a, 13b, 13e, 13t), and some vice versa (13s, 13w).
Satisfactorily, there existed analogues within the series that were
both potent GPa inhibitors and appreciable GK activators (12, 13h,
13l, 13q, 13v). Compounds 12, 13h, 13l, 13q, and 13v demonstrated
a good balance of potency to GK and GPa, and were selected for fur-
ther glucokinase enzyme assay. The EC50 values against GK were also
outlined in Table 1. As shown, 13h and 13l, which inhibited GPa with
determined as the preferred binding poses, as shown in Figures 4
and 5. For RMGP case, these compounds bound at the dimmer
interface site of RMGP (Fig. 4).11 For GK case, these compounds
were buried in a deep pocket in the hinge region of the enzyme re-
mote from the active site in a manner reminiscent of that reported
for GK activator 6 (Fig. 5).5
For further and detailed understanding of the binding modes of
these compounds with GP and GK, 2D pictures of compound 12,
13h and 6 with GP and GK respectively were also created with
the program LIGPLOT based on the above docking simulation (Figs.
6 and 7). Hydrogen bonds and hydrophobic interactions between
compound 12, 13h and 6 with RMGP are exhibited in Figure 6.
As indicated, the substituted benzyl group of compound 12 pro-
trudes into the hydrophobic cavity constituted by residues
Lys191(A), Val40(B), Trp67(A), Pro229(A) and Trp189(A). The het-
eroaryl thiol moiety forms hydrophobic contacts with Phe53(B)
and His57(B). The carbonyl nitrogen and the amino group of the
middle phenyl chelate with the oxygen of Glu190(A). The amino
group in substituted benzyl moiety of compound 12 could form an-
other hydrogen bond with Arg60(A). All these interactions may
provide stable interactions between these benzamide derivatives
and RMGP. Compound 13h demonstrates a similar binding mode
(Fig. 6B) except the loss of a hydrogen bond in the trifluoromethyl
phenyl ring, accounting for the decreased inhibitory activity than
that of compound 12.
Figure 7 represents the main interactions between compound
12, 13h and 6 with GK. These dual-action compounds bind at
the allosteric site of glucokinase. Compound 12 forms hydrophobic
contacts with Pro66(A), Val62(A), Val452(A), Ile159(A), Ile211(A),
Met235(A), Tyr214(A), and Thr65(A) of glucokinase. The carbonyl
oxygen of Arg63(A) and phenolic oxygen of Tyr215(A) anchor
the amide NH and amino nitrogen of these compounds respec-
tively with two hydrogen bonds. The only difference of the binding
mode of compound 12 and 13h compared to that of compound 6
is the loss of a hydrogen bond with Arg63(A). The absence of this
strong interaction may elucidate the significant decrease in the
affinity of these compound for GK with respect to that for com-
pound 6.
6. Conclusions
IC50 values of 8.95
EC50 values of 1.87
l
l
M and 9.87
M and 2.53
l
l
M, showed activation of GK with
M, respectively.
In summary, we have described the ongoing development of a
series of benzamide compounds which can inhibit glycogen phos-
phorylase a and activate glucokinase at the same time. Biochemical
evaluation and SAR of these compounds were also conducted.
Among these compounds, 12, 13l, 13h and 13q showed moderate
activity towards both GK and GP. The possible binding modes of
compounds 12, 13l, 13h, and 13q with glycogen phosphorylase a
and glucokinase were also explored by molecular docking
simulation.
The series of piperazine-containing amide analogues exhibited
least activities to both GK and GPa, probably due to the semi-flexile
moiety of piperazine linker (14a–e). The series of symmetrical
benzamide analogues displayed no potency primarily due to the
rigidity of two amide groups and unfitness into the hydrogen bond
interactions (15a–c) (Table 2).
5. Binding model predicted by molecular docking simulation
In order to gain insight into the binding mode of these dual-ac-
tion compounds against these two targets, molecular simulations
were performed by Maestro v8.5 (Schrödinger, Inc.) with Glide-
XP (extra-precision). Hydrogen atoms and charges were added
during a brief relaxation performed using the Protein Preparation
module in Maestro with the ‘Preparation and refinement’ option.
The grid-enclosing box was centered on the centroid of the bound
ligand, and a scaling factor of 1.0 was set to Van der Waals (VDW)
radii of those receptor atoms with the partial atomic charge less
than 0.25. The Glide scoring function (G-Score) was used to select
the final ten poses for each ligand. The docking simulations were
performed on a Dell Cluster server in parallel.
Based on these results, it can be expected that some reported
GK activators or GPa inhibitors may also play a dual role on these
two targets simultaneously. These dual actions towards GK and
GPa represent a ‘double whammy’ on the hyperglycemia associ-
ated with T2D. The discovery of compounds that act on two targets
provides a new methodology to design anti-diabetic agents.
7. Experimental
Melting points were measured in capillary tube on a SGW X-4
melting point apparatus without correction. The type of analytical
thin-layer chromatography (TLC) was HSGF 254 (0.15–0.2 mm
thickness, Yantai Huiyou Company, China). Nuclear magnetic reso-
nance (NMR) spectra were recorded on a Brucker AMX-300 NMR
(TMS as IS). Chemical shifts were reported in parts per million
The crystal structure of RMGP (PDB entry 2IEI) and GK (PDB en-
try 1V4S) were used in molecular docking simulation. The first
ranked docking poses of compounds 12, 13h, 13l, and 13q were

L. Zhang et al. / Bioorg. Med. Chem. 17 (2009) 7301–7312
7305
Table 1
Activities of compounds 13a–x towards GK and GPa
Compound
Structure
GPa IC50
43.9
(
lM)
GK fold activation at 10
3.97 ( 0.50)a
lM
O
S
S
F
N
N
N
6
N
H
EC50 = 0.06711
lM
N
N
N
NH2
O
S
F
2.09 ( 0.05)a
N
NH2
NH2
12
2.68
5.4
H
EC50 = 12.56
1.21 ( 0.08)
lM
NH2
O
S
F
S
S
13a
N
H
NH2
O
S
F
13b
3.5
1.10 ( 0.11)
N
H
NH2
N
N
NH2
O
S
F
S
13c
13d
13e
13f
17.7
10.6
9.8
1.22 ( 0.09)
1.12 ( 0.10)
1.09 ( 0.06)
1.16 ( 0.04)
1.01 ( 0.13)
N
H
NH2
NH2
N
NH2
O
N
S
F
N
H
NH2
O
N
S
F
N
H
NH2
NH2
N
H
N
S
F
N
N
N
NH2
28.9
25.3
8.95
N
O
NH2
O
S
F
13g
13h
N
H
N
N
NH2
CF3
NH2
O
S
F
N
H
1.47 ( 0.03)a
EC50 = 1.87
l
M
NH2
O
S
F
S
S
13i
13j
13k
N
H
21.1
32.5
18.9
1.15 ( 0.08)
0.89 ( 0.07)
1.14 ( 0.02)
CF3
CF3
N
N
N
NH2
O
S
F
N
H
NH2
O
S
F
S
N
H
CF3
N
NH2
(continued on next page)

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L. Zhang et al. / Bioorg. Med. Chem. 17 (2009) 7301–7312
Table 1 (continued)
Compound
Structure
GPa IC50
9.87
(l
M)
GK fold activation at 10
1.95 ( 0.04)a
lM
O
S
F
N
13l
N
H
CN
EC50 = 2.53
l
M
N
N
N
N
NH2
O
S
F
S
S
13m
13n
13o
13p
13q
N
H
34.5
18.9
44.3
19.3
30.9
1.20 ( 0.13)
0.99 ( 0.10)
1.20 ( 0.10)
1.01 ( 0.04)
2.04 ( 0.04)a
CN
CN
NH2
O
S
F
N
H
NH2
O
S
F
S
N
H
CN
N
NH2
O
N
S
F
N
H
CN
N
NH2
O
S
F
N
N
H
F
EC50 = 2.13 lM
N
NH2
O
S
F
S
S
13r
N
H
25.9
1.25 ( 0.15)
F
F
N
N
NH2
O
S
F
13s
13t
13u
N
H
33.6
7.2
1.36 ( 0.06)
1.10 ( 0.08)
0.83 ( 0.07)
N
NH2
O
N
S
F
N
F
H
NH2
O
S
F
O
21.6
N
H
F
N
NH2
O
O
13v
S
F
23.9
2.01 ( 0.52)a
EC50 = 1.36 lM
N
N
N
H
NH2
N
N
O
S
F
N
13w
13x
20.2
9.3
1.15 ( 0.01)
1.32 ( 0.19)
O
H
NH2
O
O
O
S
F
N
N
H
NH2
N
a
GK max fold activation.

L. Zhang et al. / Bioorg. Med. Chem. 17 (2009) 7301–7312
7307
Table 2
Activities of compounds 14a–e, 15a–c towards GK and GPa
Compound
Structure
GPa IC50
25.8
(l
M)
GK fold activation at 10
1.08 ( 0.13)
lM
14a
N
HN
NH2
F
N
O
14b
14c
14d
78.5
18.7
12.5
1.06 ( 0.07)
1.01 ( 0.07)
0.96 ( 0.04)
N
HN
HN
HN
CF3
F
N
N
N
O
O
O
N
F
F
N
CN
F
O
F
O
14e
35.9
1.01 ( 0.14)
N
HN
O
F
N
O
O
15a
15b
NDa
77.4
NDa
1.02 ( 0.13)
0.81 ( 0.08)
0.89 ( 0.12)
N
N
H
H
O
O
O
O
O
O
N
H
N
H
O
O
O
O
15c
N
H
N
H
a
ND, not determined.
(ppm, d) downfield from tetramethylsilane. Proton coupling pat-
terns were described as singlet (s), doublet (d), triplet (t), quartet
(q), multiplet (m), and broad (br). Low- and high-resolution mass
spectra (LRMS and HRMS) were given with electric (EI) produced
by Finnigan MAT-95 mass spectrometer.
the obtained benzamide (190 mg, 0.68 mmol), 1-methyl-1H-imid-
azole-2-thiol (78 mg, 0.68 mmol), and triethylamine (2.05 mmol)
in acetonitrile was heated under microwave at 80 °C for 40 min.
Upon cooling, the yellow precipitate was filtered and washed with
water (5 mL). Then deoxidizing the precipitate by iron power (2 g)
and saturated aqueous NH4Cl (2 mL) in isopropanol gave com-
pound 12 as a white solid (157 mg, 45% total yield); mp 144–
148 °C; 1H NMR (DMSO) d 7.85 (d, J = 7.8 Hz, 1H), 7.25 (s, 1H),
6.95 (t, J = 18.9 Hz, 2H), 6.90 (s, 1H), 6.78 (t, J = 15.3 Hz, 1H), 6.57
(d, J = 11.4 Hz, 1H), 6.31 (d, J = 8.1 Hz, 1H), 3.63 (s, 3H); MS (EI,
m/z) 357 [M]+; HRMS (EI) m/z calcd for C17H16FN5OS (M+)
357.1060, found 357.1052.
7.1. 2-Amino-N-(3-amino-phenyl)-4-fluoro-5-(1-methyl-1H-
imidazol-2-ylsulfanyl)-benzamide (12)
4,5-Difluoro-2-nitrobenzoic acid (200 mg, 0.98 mmol) reacted
with oxalyl dichloride (0.2 mL) and DMF (two drops) at room tem-
perature for 2 h. After concentration of the reaction mixture, the
residue was dissolved in CH2Cl2 and then added dropwise into
the mixture of 3-nitroaniline (91 mg, 0.98 mmol) and pyridine
(1.96 mmol) in CH2Cl2. The mixture was stirred vigorously for
12 h at room temperature and washed with 0.1 M aqueous hydro-
chloric acid solution (2 Â 50 mL), a saturated aqueous sodium
bicarbonate solution (2 Â 50 mL) and a saturated aqueous sodium
chloride solution (2 Â 50 mL) sequentially, and then dried over so-
dium sulfate, filtered, and concentrated in vacuum. A mixture of
7.2. 2-Amino-N-(3-amino-phenyl)-4-fluoro-5-(thiazol-2-
ylsulfanyl)-benzamide (13a)
Compound 13a was prepared as a white solid in a similar man-
ner as described for compound 12. Yield: 44%; mp 168–172 °C; 1H
NMR (CDCl3) d 7.99 (br s, 1H), 7.85 (d, J = 7.8 Hz, 1H), 7.64 (d,
J = 3.3 Hz, 1H), 7.20 (d, J = 3.3 Hz, 1H), 7.12 (t, J = 7.8 Hz, 2H), 6.80

7308
L. Zhang et al. / Bioorg. Med. Chem. 17 (2009) 7301–7312
Figure 4. Stereoview of the binding poses of compounds 12, 13h, 13l, and 13q at the dimmer interface site of RMGP (left) and enlarged view of binding site (right) with some
key residues. The RMGP structure is shown in cartoon (chain A in green and chain B in cyan), and the residues in the binding site are shown in line. The coloring for these
compounds is as follows: compound 12 (carbons in green); compound 13h (carbons in magenta); compound 13l (carbons in yellow); and compound 13q (carbons in slate);
oxygen atoms, red; nitrogen atoms, blue; sulfur atoms, orange; fluorine, palecyan; respectively. Hydrogen atoms have been omitted for clarity. All structure figures were
prepared using PyMol (http://pymol.sourceforge.net/).
Figure 5. Stereoview of the binding poses of compounds 12, 13h, 13l, and 13q at the allosteric site of glucokinase (left) and enlarged view of allosteric site (right) with some
key residues. The GK structure is shown in cartoon and the residues in the binding site are shown in line. The coloring for these compounds is as follows: compound 12
(carbons in green); compound 13h (carbons in magenta); compound 13l (carbons in yellow) and compound 13q (carbons in slate); oxygen atoms, red; nitrogen atoms, blue;
sulfur atoms, orange; fluorine, palecyan; respectively. Hydrogen atoms have been omitted for clarity.
(d, J = 7.5 Hz, 1H), 6.48 (d, J = 10.2 Hz, 2H), 6.10 (br s, 2H); MS (EI,
m/z) 360 [M]+; HRMS (EI) calcd for C16H13OS2N4F [M]+ 360.0515,
found 360.0523.
(d, J = 7.2 Hz, 1H), 6.71 (d, J = 11.4 Hz, 1H), 6.33 (d, J = 8.1 Hz, 1H),
5.22 (br s, 2H), 2.29 (s, 3H); MS (EI, m/z) 374 [M]+; HRMS (EI) calcd
for C17H15OS2N4F [M]+ 374.0671, found 374.0687.
7.3. 2-Amino-N-(3-amino-phenyl)-4-fluoro-5-(4-methyl-
7.4. 2-Amino-N-(3-amino-phenyl)-4-fluoro-5-(1,3,4-thiadiazol-
thiazol-2-ylsulfanyl)-benzamide (13b)
2-ylsulfanyl)-benzamide (13c)
Compound 13b was prepared as a white solid in a similar man-
ner as described for compound 12. Yield: 59%; mp 175–178 °C; 1H
NMR (DMSO) d 10.36 (s, 1H), 9.86 (s, 1H), 8.12 (d, J = 8.1 Hz, 1H),
7.24 (s, 1H), 7.11 (s, 1H), 7.00 (s, 1H), 6.95(d, J = 8.4 Hz, 1H), 6.80
Compound 13c was prepared as a white solid in a similar man-
ner as described for compound 12. Yield: 49%; mp 228 °C; 1H NMR
(DMSO) d 9.85 (s, 1H), 9.46 (s, 1H), 8.17 (d, J = 8.1 Hz, 1H), 7.29 (s,
2H), 6.98 (s, 1H), 6.96 (t, J = 8.1 Hz, 1H), 6.79 (d, J = 9.0 Hz, 1H), 6.73

L. Zhang et al. / Bioorg. Med. Chem. 17 (2009) 7301–7312
7309
Figure 6. Two-dimensional representations for the interacting modes of compounds 12 (A), 13h (B) and 6 (C) with GP. Dotted lines represent the hydrogen bonds and the half
circles represent the hydrophobic contacts. The hydrogen bonds and hydrophobic interactions were calculated by LIGPLOT.18
Figure 7. Two-dimensional representations for the interacting modes of compounds 12 (A), 13h (B) and 6 (C) with GK. Dotted lines represent the hydrogen bonds and the half
circles represent the hydrophobic contacts. The hydrogen bonds and hydrophobic interactions were calculated by LIGPLOT.18
(d, J = 11.7 Hz, 1H), 6.32 (d, J = 8.4 Hz, 1H), 5.08 (s, 2H); MS (EI, m/z)
361 [M]+; HRMS (EI) calcd for C15H12OS2N5F [M]+ 361.0467, found
361.0472.
J = 7.5 Hz, 1H), 5.06 (s, 2H), 2.28 (s, 6H); MS (EI, m/z) 383 [M]+;
HRMS (EI) calcd for C19H18OSN5F [M]+ 383.1216, found 383.1225.
7.7. 3-Amino-N-[2-amino-4-fluoro-5-(1-methyl-1H-imidazol-2-
ylsulfanyl)-phenyl]-benzamide (13f)
7.5. 2-Amino-N-(3-amino-phenyl)-4-fluoro-5-(pyridin-2-
ylsulfanyl)-benzamide (13d)
Compound 13f was prepared as a white solid in a similar man-
ner as described for compound 12. Yield: 48%; mp 202–205 °C; 1H
NMR (DMSO) d 9.42 (s, 1H), 7.29 (s, 1H), 7.15–7.09 (m, 4H), 6.92 (s,
1H), 6.73 (d, J = 7.5, 1H), 6.59 (d, J = 7.5, 1H), 5.46 (s, 2H), 5.27 (s,
2H), 3.66 (s, 3 Hz,); MS (EI, m/z) 357 [M]+; HRMS (EI) calcd for
C17H16OSN5F [M]+ 357.1060, found 357.1050.
Compound 13d was prepared as a white solid in a similar man-
ner as described for compound 12. Yield: 35%; mp 180–182 °C; 1H
NMR (DMSO) d 9.89 (s, 1H), 8.43 (s, 1H), 8.04 (d, J = 8.1 Hz, 1H),
7.70 (t, J = 6.9 Hz, 1H), 7.15 (s, 3H), 7.04 (s, 1H), 7.00 (s, 1H), 6.98
(s, 1H), 6.83 (d, J = 8.4 Hz, 1H), 6.74 (d, J = 11.1 Hz, 1H), 6.36 (d,
J = 7.5 Hz, 1H), 5.16 (br s, 2H); MS (EI, m/z) 354 [M]+; HRMS (EI)
calcd for C18H15OSN4F [M]+ 354.0951, found 354.0960.
7.8. 2-Amino-N-(3-aminomethyl-phenyl)-4-fluoro-5-(1-
methyl-1H-imidazol-2-ylsulfanyl)-benzamide (13g)
7.6. 2-Amino-N-(3-amino-phenyl)-5-(4,6-dimethyl-pyrimidin-
2-ylsulfanyl)-4-fluoro-benzamide (13e)
Compound 13g was prepared as a white solid in a similar man-
ner as described for compound 12. Yield: 46%; mp 80–83 °C; 1H
NMR (DMSO) d 10.15 (s, 1H), 7.93 (d, J = 8.1 Hz, 1H), 7.68 (s, 1H),
7.53 (d, J = 8.1 Hz, 1H), 7.27 (s, 1H), 7.09 (d, J = 8.1 Hz, 1H), 6.92
(s. 1H), 6.89 (s. 1H), 6.57 (d, J = 11.7 Hz, 1H), 3.77 (s, 2H); MS (EI,
Compound 13e was prepared as a white solid in a similar man-
ner as described for compound 12. Yield: 61%; mp 115–118 °C; 1H
NMR (DMSO) d 9.81 (s, 1H), 7.91 (d, J = 7.5 Hz, 1H), 7.03–6.90 (m,
5H), 6.76 (d, J = 8.1 Hz, 1H), 6.63 (d, J = 12.0 Hz, 1H), 6.29 (d,

7310
L. Zhang et al. / Bioorg. Med. Chem. 17 (2009) 7301–7312
m/z) 371 [M]+; HRMS (EI) calcd for C18H18OSN5F [M]+ 371.1216,
found 371.1215.
(d, J = 4.5 Hz, 1H), 7.32 (s, 2H), 6.75 (d, J = 11.7 Hz, 1H); MS (EI,
m/z) 370 [M]+; HRMS (EI) calcd for C17H11FN4OS2 [M]+ 370.0358,
found 370.0357.
7.9. 2-Amino-4-fluoro-5-(1-methyl-1H-imidazol-2-ylsulfanyl)-
N-(3-trifluoromethyl-phenyl)-benzamide (13h)
7.15. 2-Amino-N-(3-cyano-phenyl)-4-fluoro-5-(4-methyl-
thiazol-2-ylsulfanyl)-benzamide (13n)
Compound 13h was prepared as a white solid in a similar man-
ner as described for compound 12. Yield: 34%; mp 95–100 °C; 1H
Compound 13n was prepared as a white solid in a similar man-
ner as described for compound 12. Yield: 45%; mp 162–166 °C; 1H
NMR (DMSO) d 8.19 (s, 1H), 8.17 (s, 1H), 7.96 (br, 1H), 7.61–7.55
(m, 3H), 7.10 (s, 1H), 6.75 (d, J = 11.7 Hz, 1H), 3.29 (s, 3H); MS
(EI, m/z) 384 [M]+; HRMS (EI) calcd for C18H13FN4OS2 [M]+
384.0515, found 384.0508.
NMR (DMSO)
d 8.13 (s, 1H), 7.90–7.97 (m, 2H), 7.56 (t,
J = 16.5 Hz, 1H), 7.42 (d, J = 7.8 Hz, 1H), 7.25 (s, 1H), 6.89 (s, 1H),
6.58 (d, J = 11.7 Hz, 1H), 3.66 (s, 3H); MS (EI, m/z) 410 [M]+; HRMS
(EI) m/z calcd for C18H14F4N4OS (M+) 410.0824, found 410.0818.
7.10. 2-Amino-4-fluoro-5-(thiazol-2-ylsulfanyl)-N-(3-
trifluoromethyl-phenyl)-benzamide (13i)
7.16. 2-Amino-N-(3-cyano-phenyl)-4-fluoro-5-(1,3,4-
thiadiazol-2-ylsulfanyl)-benzamide (13o)
Compound 13i was prepared as a white solid in a similar man-
ner as described for compound 12. Yield: 66%; mp 182–183 °C; 1H
NMR (DMSO) d 10.42 (br s, 1H), 8.23 (d, J = 8.1 Hz, 1H), 8.16 (s, 1H),
7.97 (d, J = 8.1 Hz, 1H), 7.68 (d, J = 3.6 Hz, 1H), 7.58 (dd, J = 9.0,
5.7 Hz, 2H), 7.44 (d, J = 8.1 Hz, 1H), 7.34 (s, 1H), 6.75 (d,
J = 11.4 Hz, 1H); MS (EI, m/z) 413 [M]+; HRMS (EI) calcd for
C17H11OS2N3F4 [M]+ 413.0280, found 413.0285.
Compound 13o was prepared as a white solid in a similar man-
ner as described for compound 12. Yield: 55%; mp 213–216 °C; 1H
NMR (DMSO) d 10.39 (s, 1H), 9.47 (s, 1H), 8.23 (d, J = 8.1 Hz, 1H),
8.17 (s, 1H), 7.94 (br, 1H), 7.57 (d, J = 5.1 Hz, 2H), 7.36 (s, 2H),
6.76 (d, J = 11.7 Hz, 1H); MS (EI, m/z) 371 [M]+; HRMS (EI) calcd
for C16H10FN5OS2 [M]+ 371.0311, found 371.0309.
7.11. 2-Amino-4-fluoro-5-(4-methyl-thiazol-2-ylsulfanyl)-N-(3-
trifluoromethyl-phenyl)-benzamide (13j)
7.17. 2-Amino-N-(3-cyano-phenyl)-5-(4,6-dimethyl-pyrimidin-
2-ylsulfanyl)-4-fluoro-benzamide (13p)
Compound 13j was prepared as a white solid in a similar man-
ner as described for compound 12. Yield: 57%; mp 183–185 °C; 1H
NMR (DMSO) d 10.48 (br s, 1H), 8.26 (d, J = 7.8 Hz, 1H), 8.18 (s, 1H),
7.99 (d, J = 7.8 Hz, 1H), 7.58 (t, J = 7.8 Hz, 1H), 7.44 (d, J = 8.1 Hz,
1H), 7.37 (s, 1H),7.11 (s, 1H), 6.78 (d, J = 11.7 Hz, 1H), 2.29 (s,
3H); MS (EI, m/z) 427 [M]+; HRMS (EI) calcd for C18H13OS2N3F4
[M]+ 427.0436, found 427.0451.
Compound 13p was prepared as a white solid in a similar man-
ner as described for compound 12. Yield: 57%; mp 246 °C; 1H NMR
(DMSO) d 10.36 (s, 1H), 8.17 (s, 1H), 7.99 (d, J = 7.8 Hz, 1H), 7.96 (m,
1H), 7.55 (d, J = 5.1 Hz, 2H), 7.13 (s, 1H), 6.98 (s, 1H), 6.67 (d,
J = 11.7 Hz, 1H), 2.29 (s, 6H); MS (EI, m/z) 393 [M]+; HRMS (EI)
calcd for C20H16FN5OS [M]+ 393.1060, found 393.1052.
7.12. 2-Amino-4-fluoro-5-(1,3,4-thiadiazol-2-ylsulfanyl)-N-(3-
trifluoromethyl-phenyl)-benzamide (13k)
7.18. 2-Amino-4-fluoro-N-(3-fluoro-phenyl)-5-(1-methyl-1H-
imidazol-2-ylsulfanyl)-benzamide (13q)
Compound 13k was prepared as a white solid in a similar man-
ner as described for compound 12. Yield: 61%; mp 193 °C; 1H NMR
(DMSO) d 10.38 (br s, 1H), 9.47 (br s, 1H), 8.26 (d, J = 8.1 Hz, 1H),
8.16 (s, 1H), 7.96 (d, J = 7.8 Hz, 1H), 7.59 (t, J = 7.8 Hz, 1H), 7.45
(d, J = 7.5 Hz, 1H), 7.37 (s, 2H), 6.77 (d, J = 11.7 Hz, 1H); MS (EI,
m/z) 414 [M]+; HRMS (EI) calcd for C16H10OS2N4F4 [M]+ 414.0232,
found 414.0229.
Compound 13q was prepared as a white solid in a similar man-
ner as described for compound 12. Yield: 49%; mp 190–193 °C; 1H
NMR (CD3OD) d 7.92 (d, J = 7.5 Hz, 1H), 7.64 (d, J = 11.7 Hz, 1H),
7.46 (d, J = 7.5 Hz, 1H), 7.35–7.40 (m, 1H), 7.25 (s, 1H), 6.90–6.94
(m, 2H), 6.59 (d, J = 11.7 Hz, 1H), 3.68 (s, 3H); MS (EI, m/z) 360
[M]+; HRMS (EI) m/z calcd for C17H14F2N4OS (M+) 360.0856, found
360.0833.
7.13. 2-Amino-N-(3-cyano-phenyl)-4-fluoro-5-(1-methyl-1H-
imidazol-2-ylsulfanyl)-benzamide (13l)
7.19. 2-Amino-4-fluoro-N-(3-fluoro-phenyl)-5-(thiazol-2-
ylsulfanyl)-benzamide (13r)
Compound 13l was prepared as a white solid in a similar man-
ner as described for compound 12. Yield: 55%; mp 172–174 °C; 1H
NMR (DMSO) d 7.92 (d, J = 8.0 Hz, 1H), 7.84 (s, 1H), 7.68 (d,
J = 8.3 Hz, 1H), 7.34 (t, J = 16.3 Hz, 1H), 7.24 (s, 1H), 7.18 (d,
J = 7.3 Hz, 1H), 6.88 (s, 1H), 6.57 (d, J = 11.7 Hz, 1H), 3.66 (s, 3H);
MS (EI, m/z) 367 [M]+; HRMS (EI) m/z calcd for C18H14FN5OS (M+)
367.0903, found 367.0977.
Compound 13r was prepared as a white solid in a similar man-
ner as described for compound 12. Yield: 36%; 1H NMR (DMSO) d
10.27 (s, 1H), 8.17 (d, J = 8.4 Hz, 1H), 7.68 (d, J = 3.3 Hz, 1H), 7.64
(s, 1H), 7.57 (d, J = 3.3 Hz, 1H), 7.47 (d, J = 8.1 Hz, 1H), 7.36 (dd,
J = 15.0, 8.1 Hz, 1H), 7.29 (s, 1H), 6.92 (td, J = 9.0, 2.7 Hz, 1H), 6.73
(d, J = 11.7 Hz, 1H); MS (EI, m/z) 363 [M]+; HRMS (EI) calcd for
C16H11F2N3OS2 [M]+ 363.0312, found 363.0311.
7.14. 2-Amino-N-(3-cyano-phenyl)-4-fluoro-5-(thiazol-2-
ylsulfanyl)-benzamide (13m)
7.20. 2-Amino-4-fluoro-N-(3-fluoro-phenyl)-5-(1,3,4-
thiadiazol-2-ylsulfanyl)-benzamide (13s)
Compound 13m was prepared as a white solid in a similar man-
ner as described for compound 12. Yield: 30%; mp 213–215 °C; 1H
NMR (DMSO) d 10.38 (s, 1H), 8.21 (d, J = 8.1 Hz, 1H), 8.17 (s, 1H),
7.95 (br, 1H), 7.68 (d, J = 3.0 Hz, 1H), 7.58 (d, J = 3.0 Hz, 1H), 7.56
Compound 13s was prepared as a white solid in a similar man-
ner as described for compound 12. Yield: 43%; mp 215–218 °C; 1H
NMR (DMSO) d 10.28 (s, 1H), 9.46 (s, 1H), 8.20 (d, J = 8.1 Hz, 1H),

L. Zhang et al. / Bioorg. Med. Chem. 17 (2009) 7301–7312
7311
7.65 (d, J = 12.0 Hz, 1H), 7.48–7.33 (m, 4H), 6.93 (t, J = 7.8 Hz, 1H),
6.75 (d, J = 11.7 Hz, 1H); MS (EI, m/z) 364 [M]+; HRMS (EI) calcd for
C15H10F2N4OS2 [M]+ 364.0246, found 364.0258.
with 2-chloroacetyl chloride (339 mg, 3 mmol) dropwise. Then the
mixture was stirred under reflux for 2 h. After evaporation THF, the
residue was dissolved with water and the precipitate was collected
by filtration. A mixture of the obtained acetamide (232 mg,
1.32 mmol), 1-(2-fluorophenyl)piperazine (213 mg, 1.32 mmol),
and anhydrous potassium carbonate (273 mg, 1.98 mmol) in THF
was reacted under reflux for 4 h using potassium iodide as cata-
lyzer. The reaction mixture was evaporated and water was added
subsequently, and then the pH value was adjusted to neutral using
1 M aqueous hydrochloric acid solution. 2-[4-(2-Fluoro-phenyl)-
piperazin-1-yl]-N-(3-nitro-phenyl)-acetamide was collected by
filtration, water washing, and purification by flash column chroma-
tography. The obtained acetamide was then dissolved in methanol
(20 mL) containing 10% palladium on carbon (25 mg) and the sus-
pension was stirred in an atmosphere of hydrogen for 2 h. The cat-
alyst was then filtered and the solvent evaporated. Compound 14a
was obtained as a white solid by flash column chromatography.
Yield: 67%; mp 121–123 °C; 1H NMR (DMSO) d 9.42 (s, 1H),
7.16–7.05 (m, 3H), 7.02–6.89 (m, 3H), 6.71 (d, J = 7.8 Hz, 1H),
6.28 (d, J = 7.5 Hz, 1H), 5.07 (br s, 2H), 3.15 (s, 2H), 3.07 (t,
J = 4.5 Hz, 4H), 2.68 (t, J = 4.2 Hz, 4H); MS (EI, m/z) 328 [M]+; HRMS
(EI) calcd for C18H21FN4O [M]+ 328.1699, found 381.1701.
7.21. 2-Amino-4-fluoro-N-(3-fluoro-phenyl)-5-(pyridin-2-
ylsulfanyl)-benzamide (13t)
Compound 13t was prepared as a white solid in a similar man-
ner as described for compound 12. Yield: 38%; mp 183–185 °C; 1H
NMR (DMSO) d 10.25 (s, 1H), 8.37 (d, J = 3.9 Hz, 1H), 8.05 (d,
J = 7.8 Hz, 1H), 7.67 (d, J = 7.8 Hz, 1H), 7.64 (s, 1H), 7.48 (d,
J = 8.4 Hz, 1H), 7.36 (dd, J = 12.0, 7.8 Hz, 1H), 7.13 (m, 2H), 6.93
(m, 2H), 6.72 (d, J = 12.0 Hz, 1H); MS (EI, m/z) 357 [M]+; HRMS
(EI) calcd for C18H13F2N3OS [M]+ 357.0747, found 357.0755.
7.22. 2-Amino-5-(benzoxazol-2-ylsulfanyl)-4-fluoro-N-(3-
fluoro-phenyl)-benzamide (13u)
Compound 13u was prepared as a white solid in a similar man-
ner as described for compound 12. Yield: 23%; mp 190–194 °C; 1H
NMR (DMSO) d 10.30 (s, 1H), 8.16 (d, J = 7.8 Hz, 1H), 7.68–7.61 (m,
2H), 7.48- 7.26 (m, 6H), 6.92 (t, J = 9.0 Hz, 1H), 6.76 (d, J = 11.4 Hz,
1H); MS (EI, m/z) 397 [M]+; HRMS (EI) calcd for C20H13F2N3O2S [M]+
397.0697, found 397.0698.
7.27. 2-[4-(2-Fluoro-phenyl)-piperazin-1-yl]-N-(3-
trifluoromethyl-phenyl)-acetamide (14b)
7.23. 2-Amino-4-fluoro-N-(4-methoxy-phenyl)-5-(1-methyl-
1H-imidazol-2-ylsulfanyl)-benzamide (13v)
Compound 14b was prepared as a white solid in a similar man-
ner as described for compound 14a. Yield: 75%; mp 93–95 °C; 1H
NMR (DMSO) d 8.16 (s, 1H), 7.90 (d, J = 8.3 Hz, 1H), 7.56 (t,
J = 15.6 Hz, 1H), 7.41 (d, J = 7.7 Hz, 1H), 7.05–7.16 (m, 3H), 6.95–
6.99 (m, 1H), 3.24 (s, 2H), 3.09 (t, J = 9.2 Hz, 4H), 2.70 (t,
J = 8.8 Hz, 4H); MS (EI, m/z) 381 [M]+; HRMS (EI) m/z calcd for
C19H19F4N3O (M+) 381.1464, found 381.1472.
Compound 13v was prepared as a white solid in a similar man-
ner as described for compound 12. Yield: 22%; mp 95–100 °C; 1H
NMR (300 Hz, CD3OD) d 7.91 (d, J = 8.1 Hz, 1H), 7.51 (d, J = 9.0 Hz,
2H), 7.17 (s, 1H), 6.99 (s, 1H), 6.92 (d, J = 9.3 Hz, 2H), 6.49 (d,
J = 11.7 Hz, 1H), 3.80 (d, J = 2.1 Hz, 6H); MS (EI, m/z) 372 [M]+;
HRMS (EI) m/z calcd for C18H17FN4O2S (M+) 372.1056, found
372.1056.
7.28. N-(3-Fluoro-phenyl)-2-[4-(2-fluoro-phenyl)-piperazin-1-
yl]-acetamide (14c)
Compound 14c was prepared as a white solid in a similar man-
ner as described for compound 14a. Yield: 77%; mp 100–101 °C; 1H
NMR (DMSO) d 9.98 (s, 1H), 7.64 (d, J = 12.0 Hz, 1H), 7.42–7.30 (m,
2H), 7.16 (m, 5H), 3.21 (s, 2H), 3.08 (t, J = 4.2 Hz, 4H), 2.68 (t,
J = 4.5 Hz, 4H); MS (EI, m/z) 331 [M]+; HRMS (EI) calcd for
C18H19F2N3O [M]+ 331.1496, found 331.1501.
7.24. 2-Amino-4-fluoro-N-(3-methoxy-phenyl)-5-(1-methyl-
1H-imidazol-2-ylsulfanyl)-benzamide (13w)
Compound 13w was prepared as a white solid in a similar man-
ner as described for compound 12. Yield: 32%; mp 72–73 °C; 1H
NMR (300 Hz, DMSO) d 7.95 (d, J = 8.0 Hz, 1H), 7.40 (s, 1H), 7.27–
7.30 (m, 3H), 6.94 (s, 1H), 6.72 (d, J = 7.2 Hz, 1H), 6.63 (d,
J = 11.7 Hz, 1H), 3.78 (s, 3H), 3.72 (s, 3H); MS (EI, m/z) 372 [M]+;
HRMS (EI) m/z calcd for C18H17FN4O2S (M+) 372.1056, found
372.1059.
7.29. N-(3-Cyano-phenyl)-2-[4-(2-fluoro-phenyl)-piperazin-1-
yl]-acetamide (14d)
Compound 14d was prepared as a white solid in a similar man-
ner as described for compound 14a. Yield: 71%; mp 140–141 °C; 1H
NMR (DMSO) d 10.11 (s, 1H), 8.16 (s, 1H), 7.95 (s, 1H), 7.53 (d,
J = 4.5 Hz, 2H), 7.16–6.96 (m, 4H), 3,24 (s, 2H), 3.09 (s, 4H), 2.69
(s, 4H); MS (EI, m/z) 338 [M]+; HRMS (EI) calcd for C19H19FN4O
[M]+ 338.1543, found 338.1554.
7.25. 2-Amino-N-(3,4-dimethoxy-phenyl)-4-fluoro-5-(1-
methyl-1H-imidazol-2-ylsulfanyl)-benzamide (13x)
Compound 13x was prepared as a white solid in a similar man-
ner as described for compound 12. Yield: 63%; mp 101–104 °C; 1H
NMR (DMSO) d 10.01 (s, 1H), 7.91 (d, J = 8.1 Hz, 1H), 7.35 (s, 1H),
7.26 (s, 1H), 7.23 (d, J = 8.1 Hz, 1H), 6.91 (m. 3H), 6.57 (d,
J = 11.7 Hz, 1H), 3.75 (s. 3H), 3.74 (s. 3H), 3.35 (s. 3H); MS (EI, m/
z) 402 [M]+; HRMS (EI) calcd for C19H19FN4O3S [M]+ 402.1162,
found 402.1166.
7.30. 2-[4-(2,4-Difluoro-phenyl)-piperazin-1-yl]-N-(3,4-
dimethoxy-phenyl)-acetamide (14e)
Compound 14e was prepared as a white solid in a similar man-
ner as described for compound 14a. Yield: 69%; mp 142–145 °C; 1H
NMR (CDCl3) d 9.59 (s, 1H), 7.34 (d, J = 2.4 Hz, 1H), 7.23–7.15 (m,
2H), 7.13–6.96 (m, 3H), 6.88 (d, J = 8.7 Hz, 1H), 3.73 (s, 3H), 3.72
(s, 3H), 3.17 (s, 2H), 3.04 (t, J = 4.5 Hz, 4H), 2.68 (t, J = 4.5 Hz, 4H);
MS (EI, m/z) 391 [M]+; HRMS (EI) calcd for C20H23F2N3O3 [M]+
391.1707, found 391.1709.
7.26. N-(3-Amino-phenyl)-2-[4-(2-fluoro-phenyl)-piperazin-1-
yl]-acetamide (14a)
A solution of 3-nitroaniline (276 mg, 2 mmol) and anhydrous
potassium carbonate (415 mg, 3 mmol) in THF (20 mL) was added

7312
L. Zhang et al. / Bioorg. Med. Chem. 17 (2009) 7301–7312
7.31. N,N’-Bis-(2-methoxy-phenyl)-isophthalamide (15a)
search from CAS, and the 863 Hi-Tech Program of China (Grants
2006AA020402, 2006AA020602).
To a solution of 2-methoxyaniline (123 mg, 1 mmol) and pyri-
dine (2.5 mmol) in CHCl3 (10 mL) was added isophthaloyl dichlo-
ride (82 mg, 0.5 mmol) dropwise. After the mixture was stirred
Supplementary data
overnight,
a saturated aqueous sodium bicarbonate solution
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2009.08.045.
(5 mL) was added in and reacted for another 30 min. Then com-
pound 15a was collected by filtration, water washing, and purifica-
tion as a white solid. Yield: 82%; mp 173–176 °C; 1H NMR (CDCl3) d
8.63 (s, 2H, 8.52 (dd, J = 6.0, 1.5 Hz, 2H), 8.42 (t J = 1.2 Hz, 1H), 8.08
(dd, J = 6.0, 1.8 Hz, 2H), 7.64 (t, J = 7.8 Hz, 1H), 7.12 (t, J = 7.1 Hz,
2H), 7.04 (t, J = 6.3 Hz, 2H), 6.94 (d, J = 7.8 Hz, 2H), 3.94 (s, 6H);
MS (EI, m/z) 376 [M]+; HRMS (EI) calcd for C22H20N2O4 [M]+
376.1423, found 376.1421.
References and notes
1. Hampson, L. J.; Arden, C.; Agius, L.; Ganotidis, M.; Kosmopoulou, M. N.; Tiraidis,
C.; Elemes, Y.; Sakarellos, C.; Leonidas, D. D.; Oikonomakos, N. G. Bioorg. Med.
Chem. 2006, 14, 7835.
2. Matschinsky, F. M. Diabetes 1996, 45, 223.
3. Matschinsky, F. M. Diabetes 2002, 51, S394.
4. (a) Pal, M. Drug Discovery Today, in press, doi:10.1016/j.drudis.2009.05.013.;
(b) Coghlan, M.; Leighton, B. Exp. Opin. Invest. Drugs 2008, 17, 145; (c) Sarabu,
R.; Taub, R.; Grimsby, J. Drug Discovery Today: Therapeutic Strategies 2007, 4,
111; (d) Guertin, K. R.; Grimsby, J. Curr. Med. Chem. 2006, 13, 1839; (e) Johnson,
T. O.; Humphries, P. S.; Anthony, W.. In Annual Reports in Medicinal Chemistry;
Academic Press, 2006; Vol. 41. p 141.
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429.
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47, 3537.
7.32. N,N’-Bis-(3-methoxy-phenyl)-isophthalamide (15b)
Compound 15b was prepared as a white solid in a similar man-
ner as described for compound 15a. Yield: 83%; mp 302–303 °C; 1H
NMR (DMSO) d 10.39 (s, 2H), 8.51 (s, 1H), 8.14 (d, J = 7.8 Hz, 2H),
7.69 (t, J = 7.5 Hz, 1H), 7.49 (s, 2H), 7.40 (d, J = 7.8 Hz, 2H), 7.27
(t, J = 8.1 Hz, 2H), 6.71 (dd, J = 7.8, 2.1 Hz, 2H), 3.77 (s, 6H); MS
(EI, m/z) 376 [M]+; HRMS (EI) calcd for C22H20N2O4 [M]+
376.1423, found 376.1428.
7. Oikonomakos, N. G.; Tiraidis, C.; Leonidas, D. D.; Zographos, S. E.;
Kristiansen, M.; Jessen, C. U.; Norskov-Lauritsen, L.; Agius, L. J. Med.
Chem. 2006, 49, 5687.
8. Treadway, J. L.; Mendys, P.; Hoover, D. J. Exp. Opin. Invest. Drugs 2001, 10, 439.
9. Oikonomakos, N. G.; Somsák, L. Curr. Opin. Invest. Drugs 2008, 9, 9379.
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11. Chen, L.; Li, H.; Liu, J.; Zhang, L.; Liu, H.; Jiang, H. Bioorg. Med. Chem. 2007, 15,
6763.
12. Harwood, H. J.; Petras, S. F.; Hoover, D. J.; Mankowski, D. C.; Soliman, V. F.;
Sugarman, E. D.; Hulin, B.; Kwon, Y.; Gibbs, E. M.; Mayne, J. T.; Treadway, J. L. J.
Lipid Res. 2005, 46, 547.
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Nishimura, T.; Eiki, J.; Nagata, Y. J. Biol. Chem. 2006, 281, 37668.
14. Brocklehurst, K. J.; Payne, V. A.; Davies, R. A.; Carroll, D.; Vertigan, H. L.;
Wightman, H. J.; Aiston, S.; Waddell, I. D.; Leighton, B.; Coghlan, M. P.; Agius, L.
Diabetes 2004, 53, 535.
7.33. N,N’-Bis-(4-methoxy-phenyl)-isophthalamide (15c)
Compound 15c was prepared as a white solid in a similar man-
ner as described for compound 15a. Yield: 79%; mp 242–245 °C; 1H
NMR (DMSO) d10.29 (s, 2H), 8.50 (s, 1H), 8.11 (dd, J = 7.5, 1.2 Hz,
2H), 7.71–7.64 (m, 5H), 6.94 (m, 4H), 3.75 (6H, s); MS (EI, m/z)
376 [M]+; HRMS (EI) calcd for C22H20N2O4 [M]+ 376.1423, found
376.1419.
Acknowledgments
15. Efanov, A. M.; Barrett, D. G.; Brenner, M. B.; Briggs, S. L.; Delaunois, A.; Durbin, J.
D.; Giese, U.; Guo, H.; Radloff, M.; Gil, G. S.; Sewing, S.; Wang, Y.; Weichert, A.;
Zaliani, A.; Gromada, J. Endocrinology 2005, 146, 3696.
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Sulpice, T.; McCormack, J. G.; Procter, M. J.; Reynet, C.; Widdowson, P. S.;
Wong-Kai-In, P. Diabetologia 2007, 50, 1277.
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Danley, D. E.; Stevenson, R. W.; Barrett, E. J.; Treadway, J. L. Proc. Natl. Acad. Sci.
U.S.A. 1998, 95, 1776.
We gratefully acknowledge generous support from the National
Natural Science Foundation of China (Grants 20372069, 29725203,
20803022, and 20472094), the Basic Research Project for Talent
Research Group from the Shanghai Science and Technology Com-
mission, the Key Project from the Shanghai Science and Technology
Commission (Grant 02DJ14006), the Key Project for New Drug Re-
18. Wallace, A. C.; Laskowski, R. A.; Thornton, J. M. Protein Eng. 1995, 8, 127.