Discovery of novel dual-action antidiabetic agents that inhibit glycogen phosphorylase and activate glucokinase

Article abstract of DOI:10.1016/j.ejmech.2012.06.020

Dual-target-directed agents simultaneously inhibiting glycogen phosphorylase (GP) and activating glucokinase (GK) could decelerate the inflow of glucose from glycogenolysis and accelerate the outflow of glucose in the liver, therefore allow for a better control over hyperglycaemia in a synergetic manner. A series of hybrid compounds were designed by structure-assisted and ligand-based strategies. In vitro bioassays found two novel compounds (1j, 6g) worthy of further optimization on balance of dual action to GP and GK. In addition, for single-target activity, two compounds exhibited more potent GP inhibitory activity and four compounds showed better GK activation than their corresponding references.

Full text of DOI:10.1016/j.ejmech.2012.06.020

European Journal of Medicinal Chemistry 58 (2012) 624e639
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Discovery of novel dual-action antidiabetic agents that inhibit glycogen
phosphorylase and activate glucokinase
,
,
,
,
a
a
Lei Zhang a 1, Xiaojie Chen a 1, Jun Liu b 1, Qingzhang Zhu a, Ying Leng a , Xiaomin Luo , Hualiang Jiang ,
*
,
Hong Liu a
*
a State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203, China
b Jiangsu Center for Drug Screening, China Pharmaceutical University, Nanjing 210038, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Dual-target-directed agents simultaneously inhibiting glycogen phosphorylase (GP) and activating
glucokinase (GK) could decelerate the inflow of glucose from glycogenolysis and accelerate the outflow
of glucose in the liver, therefore allow for a better control over hyperglycaemia in a synergetic manner. A
series of hybrid compounds were designed by structure-assisted and ligand-based strategies. In vitro
bioassays found two novel compounds (1j, 6g) worthy of further optimization on balance of dual action
to GP and GK. In addition, for single-target activity, two compounds exhibited more potent GP inhibitory
activity and four compounds showed better GK activation than their corresponding references.
Ó 2012 Elsevier Masson SAS. All rights reserved.
Received 27 February 2012
Received in revised form
27 May 2012
Accepted 9 June 2012
Available online 18 June 2012
Keywords:
Glycogen phosphorylase inhibitors
Glucokinase activators
Type 2 diabetes
Dual-target-directed
Hybrid
1. Introduction
secretion and augmenting hepatic glucose metabolism. In practice,
both GP and GK are promising and validated targets for therapeutic
Type 2 diabetes is characterized by defects in pancreatic insulin
secretion, insulin resistance, and increased hepatic glucose
production [1e7]. Hepatic glucose production consists of glyco-
genolysis (release of monomeric glucose from its polymeric storage
form glycogen) and gluconeogenesis (de novo synthesis of glucose
from lactate, amino acids, and glycerol). One approach to reduce
increased hepatic glucose in type 2 diabetes is to inhibit glycogen
phosphorylase (GP) [8e13]. GP, primarily expressed in the liver,
catalyzes glycogenolysis as the rate-limiting enzyme, thus inhibit-
ing GP will limit glycogenolysis and lower hepatic glucose
production. Glucokinase (GK) [14e23] also plays a crucial role in
intervention in type 2 diabetes. A large number of GP inhibitors
[8e13] and GK activators [14e23] have been developed to modu-
late glucose homeostasis (Fig. 1).
In spite of great efforts of many research groups, most of current
single-target-directed drugs have been proven inadequate to ach-
ieve completely satisfactory glycaemic control. This may attribute
to the complexity and multiple etiologies of diabetes per se. In
recent years, multi-target-directed agents rise as more effective
strategies for diabetes treatment [26e32]. For example, the
combination of PPARg and PPARa agonist activities in a single
compound would result in synergistic improvements in insulin
sensitivity and normalization of glucose metabolism as well as
amelioration of the dyslipidemia associated with type 2 diabetes
[33e40]. Considering the crucial roles of GP and GK in regulating
glucose homeostasis, we envisage that a dual-target-directed
molecule is expected to decelerate the inflow of glucose from
glycogenolysis and accelerate the outflow of glucose in the liver,
therefore allows for a better control over hyperglycaemia in type 2
diabetes (Fig. 2).
regulating blood sugar levels. In pancreatic b-cells, GK is the rate-
limiting enzyme in glucose metabolism and determines the rate
of glucose-induced insulin secretion. In the liver, GK catalyzes the
first step in glucose metabolism and determines the rate of glucose
use and glycogen synthesis [24,25] Therefore, activating GK can
lead to lower glucose levels by increasing pancreatic insulin
In previous work, we have demonstrated the feasibility of this
kind of dual action through a series of benzamide derivatives which
can simultaneously inhibit GP and activate GK [41,42]. Based on
these finding, we further hypothesize that some reported GP
* Corresponding authors. Tel./fax: þ86 21 50807042.
E-mail addresses: yleng@mail.shcnc.ac.cn (Y. Leng), hliu@mail.shcnc.ac.cn
(H. Liu).
1
Authors contributed equally to this work.
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.06.020

L. Zhang et al. / European Journal of Medicinal Chemistry 58 (2012) 624e639
625
Fig. 1. Representative GP inhibitors (left) and GK activators (right).
inhibitors or GK activators may also play a dual role on these two
targets. To the best of our knowledge, no other dual-action
compounds targeting both GP and GK have been reported. In this
article, we report our preliminary attempt to discover novel dual-
target-directed compounds based on GP inhibitors and GK
activators.
The three arms of compounds 7 and 9 fit well in the three pockets of
GP and GK, respectively. The overall structural resemblance shared
by GP inhibitor 7 and GK activator 9 as well as their binding pockets
suggests the feasibility to design dual-target-directed molecules.
Then, we applied ligand-based strategy to design dual-action
compounds by hybridizing GP inhibitors 7 [43] and 8 [46] with
GK activators 9 [44] and 10 [24] (Fig. 4). The indole moiety is crucial
for GP inhibition and the 2-amino-heteroaryl motif is the phar-
macophore of potent GK activators. Therefore, compounds 1e2
were designed by incorporating the 2-amino-heteroaryl motif to
the indole templates. Similarly, compounds 3e6 were designed by
hybridizing GP inhibitors 11 [47] and 12 [48] with GK activators 13
[49] and 14 [50] (Fig. 5). For consistent with the profiles of the three
pockets, group R1 in pocket 1 was designed to contain various
hydrophobic aromatic rings. Cyclohexyl and 4-fluorophenyl groups
were introduced as the hydrophobic ring in pocket 2. 2-Amino-
heteroaryl groups with hydrophilic substitutions were considered
for GP inhibition in pocket 3.
2. Results and discussion
2.1. Design
As an initial attempt to discover novel dual-target-directed
agents towards both GP and GK, we started from seeking surface
similarities between enzymes GP and GK as well as structural
similarities between their binding ligands. It can be observed that
the selected GP inhibitors and GK activators share similar structural
features with three flexible arms containing at least two hydro-
phobic moieties (Fig. 1). Correspondingly, both the ligand-binding
pockets of GP at the dimmer interface site and GK at the allo-
steric site consist of three parts in a Y shape. The three pockets of
GK and GP in Fig. 3A and 3B were notated according to the struc-
tural features of GK activators and GP inhibitors, namely pocket 1
(hydrophobic aryl groups), pocket 2 (hydrophobic benzyl or
cyclohexylmethyl group) and pocket 3 (N-substituted amides).
Representatively, GP inhibitor 7 [43] and GK activator 9 [44] can be
positioned into the three pockets, which resemble a letter Y. The 5-
chloroindole group of GP inhibitor 7 is buried in pocket 1 of GP, the
benzyl group forms hydrophobic interaction with residues in
pocket 2, and pocket 3 is exposed to solvent and allows ligands with
hydrophilic substitutions (Fig. 3A). GK activator 9 is buried in the
three pockets by a large number of hydrophobic interactions,
besides the characteristic hydrogen bonds with Arg63 (Fig. 3B) [45].
2.2. Chemistry
Preparation of compounds 1e6 is summarized in Schemes 1e3.
Compounds 8 [46] and 13 [49] were chosen as the references for GP
inhibition and GK activation respectively.
The intermediates used for synthesis of compounds 1e6 were
obtained by the routes shown in Scheme 1. Vinylation of 5-
bromopyridin-2-amine gave 5-vinylpyridin-2-amine. Carbomet
hoxy-substituted 2-aminothiazole was converted to carboben
zoxy-substituted 2-aminothiazole under microwave irradiation in
the presence of catalytic amount of sulphuric acid. Commercially
available
(S)-2-((tert-butoxycarbonyl)amino)-3-cyclohexylprop
anoic acid, (S)-2-((tert-butoxycarbonyl)amino)-3-phenylpropanoic
Fig. 2. The complex and multifactorial type 2 diabetes can benefit from dual-target-directed molecules that simultaneously inhibiting glycogen phosphorylase and activating
glucokinase.

626
L. Zhang et al. / European Journal of Medicinal Chemistry 58 (2012) 624e639
Fig. 3. Ligand-binding pockets at (A) the dimmer interface site of GP (PDB code 1EXV) and (B) the allosteric site of GK (PDB code 3IMX).
acid, and (S)-2-((tert-butoxycarbonyl)amino)-3-(4-fluorophenyl)
propanoic acid were condensed with various heteroaryl amines 16,
followed by deprotection of the Boc group to yield 17. Reaction of 4-
chloro-benzene-1,2-diamine and trichloromethyl acetimidate, fol-
lowed by hydrolyzation, led to 5-chloro-1H-benzimidazole-2-
carboxylic acid.
to afford desired products 3 and 4. Condensation of 24 and 17
directly provided products 6a-6d. Compounds 5 were synthesized
via reactions of 6a-6d with 1,10-carbonyldiimidazole (CDI). Products
6e, 6f, and 6g were obtained via condensation of commercially
available acids and amines 17. Some of the products among 3, 5 and
6
can be further converted via hydrolyzation with lithium
The synthesis of hybrid molecules 1 and 2 was relatively
straightforward (Scheme 2). Condensation between 17 and acids 18
afforded compounds 1ae1k. In a similar manner as described by
Onda et al. [46], compound 1j was obtained by dihydroxylation of
1k using less toxic potassium osmium(VI) oxide dihydrate and N-
methylmorpholine-N-oxide. Compounds 2ae2h were prepared
directly from corresponding 5-chloroindole-2-carboxylic acid and
amines 16 in one step, and 2b was hydrolyzed to 2d. Compound 2i,
linked by a flexible glycine, was obtained similarly through
condensation and dihydroxylation reactions.
Scheme 3 shows the synthesis of dichloro-substituted deriva-
tives 3, 4, 5, and 6. A key feature of the synthesis is the divergent
strategy that starts from the same material 4,5-dichlorophthalic
acid 19. Treatment of 19 with acetic anhydride and then form-
amide afforded 4,5-dichlorophthalic anhydride 20 and 4,5-
dichlorophthalimide 23 respectively. The resulting 20 was
refluxed with (S)-2-amino-3-cyclohexylpropanoic acid or (S)-2-
amino-3-(4-fluorophenyl)propanoic acid to provide 21 which
were reduced by zinc and acetic acid to 22. Compound 23 was
easily converted to 2-amino-4,5-dichlorobenzoic acid 24.
Compounds 21 and 22 were condensed with differently substituted
thiazol-2-amines, benzothiazol-2-amines and pyridin-2-amines 16
hydroxide (3f, 3m, 3j, 5d, 6h) or reduction to the corresponding
primary alcohols with LiBH4 (3e, 3n).
2.3. Biology
GK activities of the 62 synthesized compounds were assessed
spectrometrically by a coupled reaction with glucose-6-phosphate
dehydrogenase [51e54].
GP activities of the 62 synthesized compounds were assessed for
ability to inhibit rabbit muscle GP-catalyzed release of phosphate
from glucose-1-phosphate according to the reported method [55].
2.4. In vitro activity
For the first step, we tested whether compounds 1 and 2 could
retain the GP inhibitory activity (Table 1). Gratifyingly, in vitro
bioassay indicated that most compounds retained the GP inhibi-
tion activity except 2i. Ten compounds showed IC50 values in
micromolar range and two compounds (1a, 1i) were more potent
than reference compound 8, with IC50 values of 0.34 and
0.91
mM respectively. The substituted 2-aminothiazole and 2-
aminopyridine moieties, which are the key pharmacophores of
Fig. 4. Design strategy for compounds 1 and 2.

L. Zhang et al. / European Journal of Medicinal Chemistry 58 (2012) 624e639
627
Fig. 5. Design strategy for compounds 3e6.
GK activators, were both tolerable to GP inhibition. The 5-
chloroindole was a better moiety than 5-methoxyindole (1b vs
1f) and 5-chloroindazole (1b vs 1g, 1i vs 1l) for GP inhibition. The
substitute position on the 2-aminothiazole fragment slightly
affected the GP inhibitory activity, and results showed that 5-
subutition (1b, 1c) was better than 4-substitution (1d, 1e) on the
2-aminothiazole. In addition, molecules lacking the phenylalanine
linker exhibited decreased GP inhibitory activity (1 vs 2), which
may be due to the lack of the hydrophobic interaction with pocket
2 of GP caused by the phenylalanine.
Next, we tested the activation fold of compounds 1 and 2
towards GK enzyme at concentration of 10 mM (Table 1). Unfortu-
nately, most of the compounds were inactive to GK enzyme. This
might be caused by the transformation of cyclohexylalanine to
phenylalanine and/or that the indole moiety was unfavourable for
GK activity. However, on balance, compound 1j was found to exert
For GP inhibition of compounds 3e6 (Table 2), we found six
compounds (5b, 5d, 6b, 6e, 6f, 6g) exhibited slight inhibitory
activity towards GP. In contrast to GK activation, the hydrophobic 4-
fluorophenyl moiety was required for GP inhibition. For example,
compound 6f showed potent GP inhibition with an IC50 value of
80.9
124.4
balance
m
M while 6e showed reduced potency with an IC50 value of
M. This leads to a contradiction to achieve satisfactory
between GP inhibition and GK activation.
m
Structureeactivity relationships also indicated that the dichloro-
substituted benzamide moiety was favourable for GP inhibition
but unbeneficial to GK activation, while the sulfone-substituted
benzamide per contra (6e vs 6g). On balance of dual action to
enzymes GP and GK, compound 6g with a novel scaffold showed
a strong activity to GK (activation fold ¼ 1.80, EC50 ¼ 0.301
mM) and
a slight activity towards GP inhibition (IC50 ¼ 911.7
mM).
good GP inhibitory activity with an IC50 value of 6.39
mM and slight
2.5. Molecular docking simulation
activity towards GK activation (activation fold ¼ 1.25 at 10
m
M).
For GK activation of compounds 3e6 (Table 2), the results showed
that five compounds (3m, 3n, 4a, 4e, 6g) demonstrated strong acti-
To illustrate the interaction mechanism, compounds 1j and 6g,
as well as their references 8 and 13, were chosen for molecular
simulations (Figs. 6 and 7). The crystal structures of GP (PDB entry
2IEI [56]) and GK (PDB entry 1V4S [57]) were used in the molecular
docking simulation. The binding affinity is predicted by Glide
GScore. The calculated GScore values of 13, 6g, 1j (binding to GK)
and 8, 1j, 6g (binding to GP) are ꢀ7.677, ꢀ5.451, ꢀ5.011
and ꢀ8.306, ꢀ10.353, ꢀ6.269 kcal/mol, respectively.
vation towards GK enzyme (activation fold > 1.5 at 10 mM), three
compounds (3f, 3r, 4a) exhibited slight activity towards GK. Among
the 41 compounds, six compounds showed EC50 values between
0.236 and 1.240
1.95times. The derivativesbearinga 5,6-dichloro-2-isoindolin-1-one
or 5,6-dichloro-2-isoindoline-1,3-dione moiety were active
towards GK enzyme. Unexpectedly, compound 6g also showed
strong GK activationwith an IC50 value of 0.301 M, which to the best
mM, and the activation folds were between 1.4 and
a
For GP inhibition, similar to the binding mode of reference
compound 8 with GP enzyme [46], compound 1j located in the
region of the central cavity of the dimeric GP enzyme (Fig. 6). The
chloroindole moiety of 1j was buried in the hydrophobic pocket 1
(Fig. 3A) surrounded by residues Leu 63, Val64, Trp 67, Lys191, and
Pro 229, and involved in a pi-stacking interaction with the guani-
dinium group of Arg60. The indole NH and the amide NH next to
the indole moiety made hydrogen bonds to the carbonyl oxygen of
m
of our knowledge is a novel GK activator with a novel scaffold.
Comparison of the R2 group indicated a preference for the cyclohexyl
ring over the aromatic 4-fluorophenyl group to maintain GK activa-
tion. For example, compound 4e (EC50 ¼ 0.243
mM) was more potent
than reference 13, but lost activity when the cyclohexyl ring was
replaced with 4-fluorophenyl ring (4h).
Scheme 1. Reagents and conditions: (a) n-Bu3SnCHCH2, Pd(PPh3)4, LiCl, DMF, THF, 80 ꢁC; (b) cat. H2SO4, phenylmethanol, microwave radiation, 120 ꢁC, 20 min; (c) PyBOP, DIPEA,
DMF, rt; (d) CF3COOH, DCM, 0 ꢁC; (e) CH3COOH, rt; (f) 1 N NaOH, then 1 N HCl.

628
L. Zhang et al. / European Journal of Medicinal Chemistry 58 (2012) 624e639
Scheme 2. Reagents and conditions: (a) PyBOP, DIPEA, DMF, rt; (b) OsO4, N-methylmorpholine-N-oxide, THF, H2O; (c) LiOH, THF, rt; (d) CF3COOH, DCM, 0 ꢁC.
Glu190 and the backbone of Thr38. Unlike reference compound 8,
the phenyl ring of compound 1j bound in pocket 2 formed by Phe53
and His57. The pyridine nitrogen and the carbonyl oxygen formed
two hydrogen bonds to the side chain of Lys191. The two hydroxyl
groups were directed towards solvent-exposed pocket 3 at the
subunit interface and may be involved in two hydrogen bonds to
the backbone of Asp50.
For GK activation, compounds 6g and 13 were buried in a similar
orientation at the hinge region of the enzyme (Fig. 7). In pocket 1,
Arg63 anchored the amide NH and adjacent heterocyclic nitrogen
of compounds 6g and 13 with two hydrogen bonds. The cyclohexyl
group of compounds 6g and 13 protruded into hydrophobic pocket
2 defined by residues Val62, Met235, Ile211, Met210, and Tyr214.
The phenolic oxygen of Tyr215 anchored another amide NH of
compound 6g with a hydrogen bond. There was additional
hydrogen bond formation between methylsulfonyl oxygen and
His218. These two hydrogen bonds may account for the significant
increase in GK affinity of compound 6g than reference 13.
A possible difficulty in designing dual-action molecules towards
enzymes GP and GK is the contradictory difference in one of their
ligand-binding pockets, i.e. pocket 2 (Fig. 3). Our research found
that an aliphatic hydrophobic moiety was beneficial for GK
activation but contrarily an aromatic hydrophobic moiety was
favourable for GP inhibition. Further work is currently in progress
to modify analogues of compounds 1j and 6g with greater poten-
cies for the GP inhibition and GK activation.
Glucose homeostasis is the result of a complex interplay of
several neural and endocrine systems. Heterogeneous and complex
type 2 diabetes can benefit from combination therapies that
modulate multiple targets. A dual-action agent targeting both GP
and GK could provide a synergetic effect to decelerate the inflow of
glucose from glycogenolysis and accelerate the outflow of glucose
in the liver, therefore allows for a better control over hyper-
glycaemia. Also, because both GP and GK are primarily expressed in
the liver, we suggest that the development of drugs targeting these
two enzymes could be a more effective way to normalize hepatic
glucose production in type 2 diabetes.
3. Conclusions
In this study, we continued to seek novel dual-target-directed
agents that can simultaneously inhibit glycogen phosphorylase
and activate glucokinase. Based on the enzyme-ligand-binding
profiles as well as structural similarities of GP inhibitors and GK
Scheme 3. Reagents and conditions: (a) Ac2O/165 ꢁC; (b) formamide/180e190 ꢁC; (c) (S)-2-amino-3-cyclohexylpropanoic acid or (S)-2-amino-3-(4-fluorophenyl)propanoic acid,
TEA, toluene; (d) PyBOP, DIPEA, DMF, rt; (e) Zn/AcOH, reflux; (f) LiOH, THF, rt; (g) LiBH4, THF, rt; (h) NaOH, Br2; (i) CDI, DBU, THF, rt.

L. Zhang et al. / European Journal of Medicinal Chemistry 58 (2012) 624e639
629
Table 1
AMX-300 or 400 MHz instrument (TMS as IS). Chemical shifts
were reported in parts per million (ppm, ) downfield from tet-
In vitro activity of compounds 1 and 2 towards enzymes GP and GK.
d
ramethylsilane. Proton coupling patterns were described as
singlet (s), doublet (d), triplet (t), quartet (q), multiplet (m), and
broad (br).
4.1.1. 2-{(S)-2-[(5-chloro-1H-indole-2-carbonyl)-amino]-3-phenyl-
propionylamino}-thiazole-5-carboxylic acid methyl ester (1b)
(i)
A mixture of (2S)-2-{[(tert-butoxy)carbonyl]amino}-3-
Compound R1
X
HetAr
GP IC50 GK fold
M) activation
(
m
phenylpropanoic acid (500 mg, 1.88 mmol), benzotriazol-1-yl-
oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP,
1.63 g, 3.14 mmol) and diisopropylethylamine (DIPEA, 520
at 10 m
Ma
8
13
e
e
e
e
e
e
1.13
NDb
1.03
2.54
(EC50
0.821
0.98
0.78
0.87
0.70
0.75
0.91
0.89
1.05
0.92
1.25
1.04
1.03
0.83
0.54
0.69
1.02
0.85
0.93
0.78
0.64
mL,
3.14 mmol) in DMF was stirred for 30 min, then added methyl 2-
aminothiazole-5-carboxylate (249 mg, 1.57 mmol). After the
reaction was completed monitored by TLC, the reaction mixture
was extracted with EtOAc, washed with water and brine, dried
with anhydrous Na2SO4 and concentrated. The residue was puri-
fied by column chromatography to give methyl 2-[(2S)-2-{[(tert-
butoxy)carbonyl]amino}-3-phenylpropanamido]-1,3-thiazole-5-
carboxylate as a white solid (458 mg, 72%). 1H NMR (300 MHz,
¼
m
M)
1a
1b
1c
1d
1e
1f
Cl
Cl
Cl
Cl
Cl
CH Thiazol-2-yl
0.34
2.61
5.63
5.03
7.34
62.3
67.2
1.35
0.91
CH 5-COOMe-thiazol-2-yl
CH 5-COOBn-thiazol-2-yl
CH 4-COOEt-thiazol-2-yl
CH 4-COOBn-thiazol-2-yl
OMe CH 5-COOMe-thiazol-2-yl
1g
1h
1i
1j
1k
1l
2a
2b
2c
2d
2e
2f
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
N
Thiazol-2-yl
CH Pyridin-2-yl
CH 5-COOMe-pyridin-2-yl
CH 5-CH(OH)CH2OH-pyridin-2-yl 6.39
CDCl3)
d 10.92 (br s, 1H), 8.09 (s, 1H), 7.27e7.19 (m, 3H),
7.14e7.12 (m, 2H), 5.12 (s, 1H), 4.64 (s, 1H), 3.89 (s, 3H),
3.24e3.07 (m, 2H), 1.41 (s, 9H); LRMS (ESI) m/z 406 [M þ H]þ.
(ii) The product in above step (450 mg, 1.11 mmol) was stirred
in dichloromethane (3 mL) and trifluoroacetic acid (3 mL) at
room temperature for 4 h. The resulting solution was basified
with NaHCO3 and extracted with EtOAc. The combined organic
layer was washed with water and brine, dried over anhydrous Na2SO4,
and the solvent was evaporated under reduced pressure to give methyl
2-[(2S)-2-amino-3-phenylpropanamido]-1,3-thiazole-5-carboxylate
CH 5-CH ¼ CH2-pyridin-2-yl
7.76
40.2
57.1
73.7
16.6
90.5
3.27
45.3
36.9
62.9
N
5-COOMe-pyridin-2-yl
CH pyridin-2-yl
CH 5-COOMe-pyridin-2-yl
CH 5-COOBn-pyridin-2-yl
CH 5-COOHepyridin-2-yl
CH 5-CH ¼ CH2-pyridin-2-yl
CH 5-Br-pyridin-2-yl
2g
2h
CH 4-COOEt-thiazol-2-yl
CH 5-COOMe-thiazol-2-yl
as a white solid (339 mg, 93%). 1H NMR (300 MHz, CDCl3)
d 8.12 (s,1H),
7.36e7.29 (m, 3H), 7.27e7.20 (m, 2H), 3.89 (s, 3H), 3.87 (dd, J ¼ 9.6,
3.9 Hz,1H), 3.36 (dd, J ¼ 14.1, 3.9 Hz,1H), 2.85 (dd, J ¼ 14.1, 9.6 Hz, 1H);
LRMS (ESI) m/z 306 [M þ H]þ. (iii) A mixture of 5-chloro-1H-indole-2-
carboxylic acid (100 mg, 0.51 mmol), PyBOP (443 mg, 0.85 mmol) and
2i
ND
1.06
DIPEA (141
methyl
m
L, 0.85 mmol) in DMF was stirred for 30 min, then added
2-[(2S)-2-amino-3-phenylpropanamido]-1,3-thiazole-5-
Bold values of two compounds (1a, 1i) displayed more potent GP activity than
reference compound 8; 1j identifies to display both GP inhibition and GK activation
activities.
carboxylate (130 mg, 0.43 mmol). After the reaction was completed
monitored by TLC, the reaction mixture was extracted with EtOAc,
washed with water and brine, dried with anhydrous Na2SO4 and
concentrated. The residue was purified by column chromatography to
give the title compound as a white solid (109 mg, 53%). 1H NMR
a
Glucokinase activity relative to DMSO control (DMSO: 1).
ND, not determined.
b
activators, sixty-two hybrid compounds were designed bystructure-
assisted and ligand-based strategies. On balance of dual action to
enzymes GP and GK, in vitro biological assay found two novel
compounds (1j, 6g) worthy of further optimization. Compound 1j
(300 MHz, DMSO-d6)
d
13.05 (s, 1H), 11.69 (s, 1H), 9.02 (d, J ¼ 7.5 Hz,
1H), 8.20 (s, 1H), 7.75 (d, J ¼ 2.1 Hz, 1H), 7.44e7.38 (m, 3H), 7.30e7.25
(m, 3H), 7.20e7.16 (m, 2H), 5.04e4.96 (m, 1H), 3.82 (s, 3H), 3.25e3.06
exhibited good GP inhibitory activity (IC50 ¼ 6.39
activation (activation fold ¼ 1.25 at 10 M). Compound 6g with
a novel scaffold showed a strongactivity to GK(activation fold ¼ 1.80,
EC50 ¼ 0.301 M) and a slight GP inhibitoryactivity (IC50 ¼ 911.7 M).
mM) and slight GK
(m, 2H); 13C NMR (100 MHz, DMSO-d6)
d 171.51, 162.12, 161.84, 161.08,
m
145.36, 137.49, 134.92, 132.23, 129.28, 128.22, 128.02, 126.60, 124.33,
123.71,121.23,120.77,113.92,103.21, 54.90, 52.20, 36.58; LRMS (ESI) m/
z 483 [M þ H]þ.
m
m
In addition, for single-target activity, two compounds (1a, 1i)
exhibited more potent GP inhibitory activity and four compounds
(3r, 4a, 4e, 6g) showed better GK activation than their corresponding
references. The possible binding modes of compounds 1j and 6g
were also explored by molecular docking simulation. The novel
conception to restore hepatic glucose levels as well as the design
strategies for dual-target-directed agents in this study may shed
informative lighton the control of hyperglycaemia in type2 diabetes.
4.1.2. (2S)-2-[(5-chloro-1H-indol-2-yl)formamido]-3-phenyl-N-
(1,3-thiazol-2-yl)propanamide (1a)
1H NMR (300 MHz, DMSO-d6)
d 12.55 (s, 1H), 11.73 (s, 1H), 8.95
(d, J ¼ 7.8 Hz, 1H), 7.75 (s, 1H), 7.51 (d, J ¼ 3.6 Hz, 1H), 7.47e7.39 (m,
3H), 7.30e7.20 (m, 4H), 7.18e7.17 (m, 2H), 5.03e4.96 (m, 1H),
3.36e3.05 (m, 2H); LRMS (ESI) m/z 425[M þ H]þ.
4.1.3. Benzyl 2-[(2S)-2-[(5-chloro-1H-indol-2-yl)formamido]-3-
4. Experimental
phenylpropanamido]-1,3-thiazole-5-carboxylate (1c)
1H NMR (300 MHz, DMSO-d6)
d 13.07 (s, 1H), 11.72 (s, 1H), 9.01
4.1. Chemistry
(d, J ¼ 7.8 Hz,1H), 8.24 (s, 1H), 7.92 (s,1H), 7.74 (s, 1H), 7.44e7.33 (m,
8H), 7.30e7.28 (m, 1H), 7.25e7.24 (m, 1H), 7.20e7.17 (m, 2H), 5.23
(s, 2H), 5.02e4.94 (m, 1H), 3.25e3.20 (m, 1H), 3.14e3.09 (m, 1H);
LRMS (ESI) m/z 559 [M þ H]þ.
Chemicals and solvents were used without further purifica-
tion. All target products were characterized by NMR and LRMS.
Nuclear magnetic resonance spectra were recorded on a Brucker

630
L. Zhang et al. / European Journal of Medicinal Chemistry 58 (2012) 624e639
Table 2
In vitro activity of compounds 3, 4, 5, and 6 towards enzymes GK and GP.
Compound
NR3
R2
HetAr
GP IC50
(
m
M)
GK fold activation at 10
0.99
m
Ma
8
13
3a
e
e
e
e
1.13
NDb
ND
e
e
2.54 (EC50 ¼ 0.821
m
M)
Cyclohexyl
Thiazol-2-yl
1.29
3b
3c
3d
3e
3f
3g
3h
3i
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
4-Fluorophenyl
4-Fluorophenyl
5-Me-thiazol-2-yl
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
1.00
0.91
0.86
1.07
1.46
1.07
1.37
0.83
5-COOMe-thiazol-2-yl
5-COOEt-thiazol-2-yl
5-CH2OHethiazol-2-yl
5-COOHethiazol-2-yl
4-Me-thiazol-2-yl
4-COOMe-thiazol-2-yl
4-COOEt-thiazol-2-yl
4-COOHethiazol-2-yl
4-CH2COOEt-thiazol-2-yl
4-CH2COOMe-thiazol-2-yl
4-CH2COOHethiazol-2-yl
4-CH2CH2OHethiazol-2-yl
Benzothiazol-2-yl
3j
3k
3l
1.36
1.21
1.29
3m
3n
3ꢁ
3p
3q
3r
3s
3t
1.95 (EC50 ¼ 0.999
m
m
M)
M)
1.79 (EC50 ¼ 1.240
0.84
6-Cl-benzothiazol-2-yl
4-COOMe-pyridin-2-yl
5-COOMe-pyridin-2-yl
5-COOMe-thiazol-2-yl
4-CH2COOMe-thiazol-2-yl
0.60
0.80
1.40 (EC50 ¼ 0.403
m
m
M)
M)
0.82
0.83
4a
Cyclohexyl
thiazol-2-yl
ND
1.52 (EC50 ¼ 0.236
4b
4c
4d
4e
4f
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
4-Fluorophenyl
4-Me-thiazol-2-yl
ND
ND
ND
ND
ND
ND
ND
1.26
0.87
1.18
4-COOEt-thiazol-2-yl
4-COOMe-thiazol-2-yl
5-COOMe-thiazol-2-yl
5-COOEt-thiazol-2-yl
benzothiazol-2-yl
1.64 (EC50 ¼ 0.243
m
M)
1.27
1.05
0.82
4g
4h
5-COOMe-thiazol-2-yl
5a
Cyclohexyl
4-Me-thiazol-2-yl
ND
0.88
5b
5c
5d
5e
Cyclohexyl
Cyclohexyl
Cyclohexyl
4-Fluorophenyl
5-COOMe-thiazol-2-yl
5-COOEt-thiazol-2-yl
5eCOOHethiazol-2-yl
5-COOMe-thiazol-2-yl
445.7
ND
917.1
ND
1.01
0.80
1.35
0.81
6a
Cyclohexyl
4-Me-thiazol-2-yl
ND
1.35
6b
6c
6d
Cyclohexyl
Cyclohexyl
4-Fluorophenyl
5-COOMe-thiazol-2-yl
5-COOEt-thiazol-2-yl
5-COOMe-thiazol-2-yl
987.3
ND
ND
0.78
0.96
0.80
6e
6f
Cyclohexyl
5-COOMe-pyridin-2-yl
5-COOMe-pyridin-2-yl
5-COOMe-pyridin-2-yl
5-COOH-pyridin-2-yl
124.4
80.9
911.7
ND
1.07
4-Fluorophenyl
Cyclohexyl
0.89
6g
6h
1.80 (EC50 [ 0.301 mM)
Cyclohexyl
1.26
Bold value signifies to display both GP inhibition and GK activation activities.
a
Glucokinase activity relative to DMSO control (DMSO: 1).
ND, not determined.
b

L. Zhang et al. / European Journal of Medicinal Chemistry 58 (2012) 624e639
631
Fig. 6. Binding details of glycogen phosphorylase complexed (A) with compound 1j and (B) with reference compound 8. The figures were prepared using PyMOL (http://www.
pymol.org).
4.1.4. 2-{(S)-2-[(5-chloro-1H-indole-2-carbonyl)-amino]-3-phenyl-
the addition (10 min), the reaction mixture was kept at room
temperature for 1 h. The reaction mixture was extracted with CH2Cl2,
washed with water, dried with anhydrous Na2SO4 and concentrated.
The residue was purified by column chromatography to provide 5-
chloro-2-trichloromethyl-1H-benzimidazole (805 mg, 85%). LRMS
(ESI) m/z 269 [M þ H]þ. (ii) 5-Chloro-2-trichloromethyl-1H-benz-
imidazole (700 mg, 2.61 mmol) was added to a cooled solution of
sodiumhydroxide(60 mL,1 N). The resulting solutionwasfiltered and
the filtrate was acidified with hydrochloric acid (1 N) to pH 4. The
precipitate was filtered off, washed twice with a solution of water and
acetonitrile (ratio 3:1) and twice with ether. After drying, this led to 5-
chloro-1H-benzimidazole-2-carboxylic acid (484 mg, 95%). LRMS
(ESI) m/z 195 [M ꢀ H]ꢀ. (iii) A mixture of 5-chloro-1H-benzimidazole-
2-carboxylic acid (100 mg, 0.51 mmol), PyBOP (441 mg, 0.85 mmol)
propionylamino}-thiazole-4-carboxylic acid ethyl ester (1d)
1H NMR (300 MHz, DMSO-d6)
d 12.98 (s,1H), 11.72 (s,1H), 8.95 (d,
J ¼ 8.1 Hz,1H), 8.08(s,1H), 7.75(s,1H),7.45e7.39 (m, 3H), 7.31e7.25 (m,
3H), 7.20e7.16 (m, 2H), 4.95e4.88 (m, 1H), 4.28 (q, J ¼ 7.5 Hz, 2H),
3.25e3.06 (m, 2H),1.30 (t, J¼ 7.5 Hz, 3H); LRMS (ESI) m/z497[Mþ H]þ.
4.1.5. Benzyl 2-[(2S)-2-[(5-chloro-1H-indol-2-yl)formamido]-3-
phenylpropanamido]-1,3-thiazole-4-carboxylate (1e)
1H NMR (300 MHz, DMSO-d6)
d 12.97 (s, 1H), 11.71 (s, 1H), 8.93
(d, J ¼ 8.1 Hz, 1H), 8.15 (s, 1H), 7.75 (s, 1H), 7.47e7.38 (m, 7H),
7.29e7.15 (m, 5H), 5.32 (s, 2H), 4.91 (m, 1H), 3.32e2.49 (m, 2H);
LRMS (ESI) m/z 559 [M þ H]þ.
4.1.6. 2-{(S)-2-[(5-methoxy-1H-indole-2-carbonyl)-amino]-3-
phenyl-propionylamino}-thiazole-5-carboxylic acid methyl ester
(1f)
and DIPEA (140
mL, 0.85 mmol) in DMF was stirred for 30 min, then
added (2S)-2-amino-3-phenyl-N-(1,3-thiazol-2-yl)propanamide
(105 mg, 0.42 mmol). After the reaction was completed monitored by
TLC, the reaction mixture was extracted with EtOAc, washed with
water and brine, dried with anhydrous Na2SO4 and concentrated. The
residue was purified by column chromatography to give the title
compound as a white solid (99 mg, 52%). 1H NMR (300 MHz, DMSO-
1H NMR (300 MHz, DMSO-d6)
d 13.04 (s, 1H), 11.36 (s, 1H), 8.86
(d, J ¼ 7.8 Hz,1H), 8.20 (s,1H), 7.45 (s,1H), 7.42 (s,1H), 7.30e7.25 (m,
3H), 7.20e7.16 (m, 2H), 7.11 (d, J ¼ 2.4 Hz, 1H), 6.83 (dd, J ¼ 9.0,
2.1 Hz, 1H), 5.02e4.95 (m, 1H), 3.82 (s, 3H), 3.77 (s, 3H), 3.24e3.07
(m, 2H); LRMS (ESI) m/z 479 [M þ H]þ.
d6)
d
13.44 (d, J ¼ 20.4 Hz, 1H), 12.56 (s, 1H), 9.17 (t, J ¼ 8.4 Hz, 1H),
7.84e7.78(m,1H), 7.56e7.52 (m, 2H), 7.41e7.36(m, 2H), 7.34e7.24 (m,
4.1.7. (2S)-2-[(5-chloro-1H-1,3-benzodiazol-2-yl)formamido]-3-
phenyl-N-(1,3-thiazol-2-yl)propanamide (1g)
3H), 7.20e7.16 (m,1H), 5.09e5.02 (m,1H), 3.26 (d, J ¼ 6.9 Hz, 2H); 13
C
NMR (100 MHz, DMSO-d6) tautomers d 169.52, 158.52,157.81, 146.27,
(i) Methyl 2,2,2-trichloroacetimidate (480
added dropwise to a cooled solution of 4-chloro-benzene-1,2-
diamine (500 mg, 3.51 mmol) in acetic acid (20 mL). At the end of
m
L, 3.86 mmol) was
145.93, 143.22, 141.21, 137.81, 137.36, 135.11, 133.24, 129.25, 128.68,
128.21, 127.08, 126.57, 124.66, 123.25, 121.52, 119.33, 114.05, 113.89,
112.16, 54.73, 36.63; LRMS (ESI) m/z 426 [M þ H]þ.

632
L. Zhang et al. / European Journal of Medicinal Chemistry 58 (2012) 624e639
Fig. 7. Binding details of glucokinase complexed (A) with compound 6g and (B) with reference compound 13. The figures were prepared using PyMOL (http://www.pymol.org).
4.1.8. (2S)-2-[(5-chloro-1H-indol-2-yl)formamido]-3-phenyl-N-
concentrated and the residue was partitioned between EtOAc
(30 mL) and 10% NaHSO3 (10 mL). The EtOAc layer was washed with
water and brine, dried and concentrated. The residue was purified
by column chromatography to give the title compound as a colour-
(pyridin-2-yl)propanamide (1h)
1H NMR (300 MHz, DMSO-d6)
d 11.72 (s, 1H), 10.85 (s, 1H), 8.86
(d, J ¼ 8.4 Hz,1H), 8.34 (d, J ¼ 4.2 Hz,1H), 8.10 (d, J ¼ 8.1 Hz,1H), 7.79
(t, J ¼ 7.2 Hz, 1H), 7.73 (s, 1H), 7.49e7.38 (m, 3H), 7.28e7.23 (m, 3H),
7.18e7.09 (m, 3H), 5.02 (m, 1H), 3.37e3.03 (m, 2H); LRMS (ESI) m/z
419 [M þ H]þ.
less solid (67 mg, 31%). 1H NMR (400 MHz, CD3OD)
d 8.27 (s,1H), 8.10
(d, J ¼ 8.4 Hz, 1H), 7.77 (d, J ¼ 8.8 Hz, 1H), 7.56 (s, 1H), 7.35 (d,
J ¼ 8.8 Hz, 1H), 7.26 (s, 1H), 7.25 (s, 1H), 7.16e7.12 (m, 3H), 7.09e7.05
(m, 2H), 5.17e5.12 (m,1H), 4.68 (t, J ¼ 6.0 Hz,1H), 3.64e3.62 (m, 2H),
3.30e3.29 (m, 1H), 3.16e3.11 (m, 1H); 13C NMR (100 MHz, CD3OD)
4.1.9. Methyl 6-[(2S)-2-[(5-chloro-1H-indol-2-yl)formamido]-3-
phenylpropanamido]pyridine-3-carboxylate (1i)
d 172.44,163.54, 151.94, 147.30, 138.23, 137.85, 136.62, 135.44,133.01,
1H NMR (300 MHz, DMSO-d6)
d
11.74 (s,1H),11.34 (s,1H), 8.91 (s,
130.31, 129.81, 129.43, 127.77, 126.75, 125.47, 121.96, 115.11, 114.41,
1H), 8.32 (d, J ¼ 5.7 Hz, 1H), 8.25 (d, J ¼ 6.6 Hz, 1H), 7.76 (s, 1H), 7.52
(s, 1H), 7.49 (s, 1H), 7.42 (d, J ¼ 6.6 Hz, 1H), 7.30e7.19 (m, 3H), 7.18 (s,
1H), 7.16 (s, 1H), 5.07e5.03 (m, 1H), 3.87 (s, 3H), 3.26e3.07 (m, 2H);
LRMS (ESI) m/z 477 [M þ H]þ.
104.55, 73.19, 68.06, 57.08, 38.91; LRMS (ESI) m/z 477 [M ꢀ H]ꢀ.
4.1.12. Methyl 6-[(2S)-2-[(5-chloro-1H-1,3-benzodiazol-2-yl)
formamido]-3-phenylpropanamido]pyridine-3-carboxylate (1l)
1H NMR (300 MHz, DMSO-d6)
d
13.44 (d, J ¼ 20.4 Hz, 1H), 11.36
4.1.10. (2S)-2-[(5-chloro-1H-indol-2-yl)formamido]-N-(5-
ethenylpyridin-2-yl)-3-phenylpropanamide (1k)
(s, 1H), 9.15e9.09 (m, 1H), 8.33 (d, J ¼ 9.0 Hz, 1H), 8.21 (d, J ¼ 9.0 Hz,
1H), 7.84e7.77 (m, 1H), 7.55e7.52 (m, 1H), 7.43e7.16 (m, 6H),
5.10e5.13 (m, 1H), 3.87 (s, 3H), 3.26 (d, J ¼ 7.2 Hz, 2H); LRMS (ESI)
m/z 478 [M þ H]þ.
1H NMR (300 MHz, CD3OD)
d
8.26 (s, 1H), 8.13 (d, J ¼ 8.7 Hz, 1H),
7.83 (d, J ¼ 8.4 Hz, 1H), 7.62 (s, 1H), 7.50 (d, J ¼ 8.4 Hz, 1H), 7.41e7.36
(m, 2H), 7.28e7.19 (m, 4H), 7.04 (s, 1H), 6.67 (dd, J ¼ 17.4 Hz, 11.1 Hz,
1H), 5.78 (d, J ¼ 17.7 Hz, 1H), 5.34 (d, J ¼ 10.8 Hz, 1H), 5.07 (t,
J ¼ 7.2 Hz, 1H), 3.41e3.35 (m, 1H), 3.23e3.16 (m, 1H); LCeMS (ESI)
m/z 445 [M þ H]þ.
4.1.13. 5-Chloro-N-(pyridin-2-yl)-1H-indole-2-carboxamide (2a)
A mixture of 5-chloro-1H-indole-2-carboxylic acid (100 mg,
0.51 mmol), PyBOP (443 mg, 0.85 mmol) and DIPEA (141
mL,
0.85 mmol) in DMF was stirred for 30 min, then added pyridin-2-
amine (40 mg, 0.43 mmol). After the reaction was completed
monitored by TLC, the reaction mixture was extracted with EtOAc,
washed with water and brine, dried with anhydrous Na2SO4 and
concentrated. The residue was purified by column chromatography
to give the title compound as a white solid (74 mg, 64%). 1H NMR
4.1.11. (2S)-2-[(5-chloro-1H-indol-2-yl)formamido]-N-[5-(1,2-
dihydroxyethyl)pyridin-2-yl]-3-phenylpropanamide (1j)
Compound 1k (200 mg, 0.45 mmol) in THF/H2O (4:1,10 mL) were
added potassium osmate (VI) dihydrate (33 mg, 0.089 mmol) and N-
methylmorpholine-N-oxide (79 mg, 0.67 mmol), and the mixture
was stirred at room temperature for 22 h. The resulting mixture was
(300 MHz, DMSO-d6)
d
12.03 (s, 1H), 10.93 (s, 1H), 8.41 (d, J ¼ 5.1 Hz,

L. Zhang et al. / European Journal of Medicinal Chemistry 58 (2012) 624e639
633
1H), 8.24 (d, J ¼ 8.4 Hz, 1H), 7.87 (t, J ¼ 8.4 Hz, 1H), 7.75 (s, 1H), 7.64
(s, 1H), 7.50 (d, J ¼ 9.0 Hz, 1H), 7.25 (d, J ¼ 8.4 Hz, 1H), 7.19 (t,
was stirred for 30 min, then added 5-vinylpyridin-2-amine
(286 mg, 2.38 mmol). After the reaction was completed moni-
tored by TLC, the reaction mixture was extracted with EtOAc,
washed with water and brine, dried with anhydrous Na2SO4 and
concentrated. The residue was purified by column chromatography
to give tert-butyl N-{[(5-ethenylpyridin-2-yl)carbamoyl]methyl}
carbamate as a white solid (574 mg, 87%). LRMS (ESI) m/z 278
[M þ H]þ. (ii) The product in the above step (500 mg, 1.80 mmol)
was stirred in dichloromethane (4 mL) and trifluoroacetic acid
(4 mL) at room temperature for 4 h. The resulting solution was
basified with NaHCO3 and extracted with EtOAc. The combined
organic layer was washed with water and brine, dried over anhy-
drous Na2SO4, and the solvent was evaporated under reduced
pressure to obtain 2-amino-N-(5-vinylpyridin-2-yl)acetamide as
a white solid (288 mg, 90%). LRMS (ESI) m/z 178 [M þ H]þ. (iii) 2-
Amino-N-(5-vinylpyridin-2-yl)acetamide (250 mg, 1.41 mmol)
and 5-chloro-1H-indole-2-carboxylic acid (330 mg, 1.69 mmol)
were condensed with PyBOP (1.46 g, 2.81 mmol) and DIPEA
J ¼ 5.4 Hz, 1H); 13C NMR (100 MHz, DMSO-d6)
d 159.76, 151.94,
148.01, 138.24, 135.39, 132.31, 128.06, 124.47, 124.21, 121.00, 119.81,
114.67, 114.06, 104.71; LRMS (ESI) m/z 272 [M þ H]þ.
4.1.14. Methyl 6-(5-chloro-1H-indole-2-amido)pyridine-3-
carboxylate (2b)
1H NMR (300 MHz, DMSO-d6)
d 12.05 (s, 1H), 11.35 (s, 1H), 8.94
(s, 1H), 8.38 (s, 2H), 7.75 (s, 1H), 7.68 (s, 1H), 7.49 (d, J ¼ 9.0 Hz, 1H),
7.26 (d, J ¼ 8.7 Hz, 1H), 3.88 (s, 3H); LRMS (ESI) m/z 230 [M þ H]þ.
4.1.15. Benzyl 6-(5-chloro-1H-indole-2-amido)pyridine-3-
carboxylate (2c)
1H NMR (300 MHz, DMSO-d6)
d 12.06 (s, 1H), 11.38 (s, 1H), 8.96
(s, 1H), 8.39 (s, 2H), 7.74 (s, 1H), 7.69 (s, 1H), 7.51e7.37 (m, 6H), 7.26
(d, J ¼ 8.7 Hz, 1H), 5.39 (s, 2H); LRMS (ESI) m/z 230 [M þ H]þ.
4.1.16. 6-(5-Chloro-1H-indole-2-amido)pyridine-3-carboxylic acid
(2d)
(466 mL, 2.81 mmol) in DMF at room temperature. After the reaction
was completed monitored by TLC, the reaction mixture was
extracted with EtOAc, washed with water and brine, dried with
anhydrous Na2SO4 and concentrated. The residue was purified by
column chromatography to give 2-[(5-chloro-1H-indol-2-yl)for-
Compound 2b (100 mg, 0.30 mmol) in THF/H2O (v/v, 1:1) was
added LiOH monohydrate (127 mg, 3.0 mmol) and stirred at room
temperature. After the reaction was completed monitored by TLC,
The reaction mixture was diluted with water, acidified with 1 N HCl,
extracted with EtOAc, washed with water and brine, dried with
anhydrous Na2SO4 and concentrated. The residue was purified by
column chromatography to give the title compound as a white solid
mamido]-N-(5-ethenylpyridin-2-yl)acetamide as
a white solid
(334 mg, 67%). LRMS (ESI) m/z 355 [M þ H]þ. (iv) Compound in step
iii (200 mg, 0.56 mmol) in THF/H2O (4:1, 10 mL) were added
potassium osmate (VI) dihydrate (61 mg, 0.11 mmol) and N-
methylmorpholine-N-oxide (99 mg, 0.85 mmol), and the mixture
was stirred at room temperature for 24 h. The resulting mixture
was concentrated and the residue was partitioned between EtOAc
(30 mL) and 10% NaHSO3 (10 mL). The EtOAc layer was washed with
water and brine, then dried and concentrated. The residue was
purified by column chromatography to give the title compound as
a colourless solid (68 mg, 31%). 1H NMR (400 MHz, DMSO-d6)
(89 mg, 93%). 1H NMR (300 MHz, DMSO-d6)
d 12.06 (s, 1H), 11.29 (s,
1H), 8.92 (s, 1H), 8.36e8.35 (m, 2H), 7.75 (s, 1H), 7.68 (s, 1H), 7.50 (d,
J ¼ 8.7 Hz, 1H), 7.26 (d, J ¼ 8.7 Hz, 1H); 13C NMR (100 MHz, DMSO-
d6)
d 166.98, 159.73, 135.47, 132.30, 131.71, 131.55, 128.68, 128.03,
124.47, 124.24, 120.99, 114.13, 104.94; LRMS (ESI) m/z 314 [M ꢀ H]ꢀ.
4.1.17. 5-Chloro-N-(5-ethenylpyridin-2-yl)-1H-indole-2-
carboxamide (2e)
d 11.89 (s, 1H), 10.56 (s, 1H), 8.98 (s, 1H), 8.26 (s, 1H), 7.99 (d,
1H NMR (300 MHz, DMSO-d6)
d
12.02 (s, 1H), 11.03 (s, 1H), 8.48
J ¼ 7.2 Hz, 1H), 7.73 (s, 2H), 7.44 (d, J ¼ 8.4 Hz, 1H), 7.20e7.16 (m,
(s, 1H), 8.23 (d, J ¼ 8.7 Hz, 1H), 8.04 (d, J ¼ 8.7 Hz, 1H), 7.74 (s, 1H),
7.63 (s, 1H), 7.49 (d, J ¼ 8.7 Hz, 1H), 7.24 (d, J ¼ 8.7 Hz, 1H), 6.77 (d,
J ¼ 17.7,11.1 Hz,1H), 5.93 (d, J ¼ 17.7 Hz,1H), 5.34 (d, J ¼ 11.1 Hz,1H);
LRMS (ESI) m/z 298 [M þ H]þ.
2H), 5.39 (br s, 1H), 4.82 (br s, 1H), 4.54 (m, 1H), 4.15 (s, 2H), 3.47 (m,
2H); 13C NMR (100 MHz, DMSO-d6)
d 168.36, 161.22, 150.75, 146.19,
136.34, 134.93, 134.37, 132.89, 128.14, 124.28, 123.54, 120.70, 113.98,
112.71, 102.65, 71.38, 66.87, 42.80; LRMS (ESI) m/z 387 [M ꢀ H]ꢀ.
4.1.18. N-(5-bromopyridin-2-yl)-5-chloro-1H-indole-2-
4.1.22. (2S)-3-cyclohexyl-2-(5,6-dichloro-1-oxo-2,3-dihydro-1H-
isoindol-2-yl)-N-(1,3-thiazol-2-yl)propanamide (3a)
carboxamide (2f)
1H NMR (300 MHz, DMSO-d6)
d
12.04 (s, 1H), 11.14 (s, 1H), 8.54
(i) 4,5-Dichlorophthalic acid (5.0 g, 21.27 mmol) was added to
acetic anhydride (20 mL) and the solution was refluxed for 5 h. The
acetic anhydride was removed in vacuo and crude 4,5-
dichlorophthalic anhydride 20 was washed with petroleum ether
and collected by filtration (4.43 g 96%). LRMS (ESI) m/z 218 [M þ H]þ.
(s, 1H), 8.24 (d, J ¼ 9.3 Hz, 1H), 8.09 (d, J ¼ 9.3 Hz, 1H), 7.74 (s, 1H),
7.64 (s, 1H), 7.49 (d, J ¼ 8.7 Hz, 1H), 7.25 (d, J ¼ 8.7 Hz, 1H); LRMS
(ESI) m/z 350 [M þ H]þ.
4.1.19. 2-[(5-Chloro-1H-indole-2-carbonyl)-amino]-thiazole-4-
(ii) A suspension of (2S)-2-amino-3-cyclohexylpropanoic acid
carboxylic acid ethyl ester (2g)
(3.16 g, 18.43 mmol), triethylamine (7.67 mL, 55.30 mmol) and 4,5-
dichlorophthalic anhydride (4.0 g, 18.43 mmol) in toluene (30 mL)
was refluxed overnight under DeaneStark conditions. The reaction
solvent was evaporated and then purified by column chromatog-
raphy to give (S)-3-cyclohexyl-2-(5,6-dichloro-1,3-dioxoisoindolin-
2-yl)propanoic acid as a white solid (6.28 g, 92%). 1H NMR (400 MHz,
1H NMR (300 MHz, DMSO-d6)
d 13.25 (br s, 1H), 12.20 (s, 1H),
8.18 (s, 1H), 7.83 (d, J ¼ 2.1 Hz, 1H), 7.74 (s, 1H), 7.55 (d, J ¼ 8.7 Hz,
1H), 7.32 (dd, J ¼ 8.7, 1.5 Hz, 1H), 4.36 (q, J ¼ 7.2 Hz, 2H), 1.37 (t,
J ¼ 7.2 Hz, 3H); LRMS (ESI) m/z 350 [M þ H]þ.
4.1.20. 2-[(5-Chloro-1H-indole-2-carbonyl)-amino]-thiazole-5-
DMSO-d6) d 13.19 (br s, 1H), 8.18 (s, 2H), 4.82e4.78 (m, 1H),
carboxylic acid methyl ester (2h)
2.13e2.05 (m, 1H), 1.92e1.84 (m, 1H), 1.78e1.76 (m, 1H), 1.59e1.50
(m, 4H), 1.15e1.02 (m, 4H), 0.95e0.81 (m, 2H); LCeMS (ESI) m/z
368 [M ꢀ H]ꢀ. (iii) The acid in step ii (3 g, 8.1 mmol) was dissolved in
20 mL of acetic acid glacial. After addition of Zn dust (2.65 g,
40.52 mmol), the reaction mixture was refluxed under nitrogen
atmosphere. After the reaction was completed monitored by TLC,
the reaction mixture was filtered by suction, concentrated and
purified by column chromatography to provide (S)-3-cyclohexyl-2-
(5,6-dichloro-1-oxoisoindolin-2-yl)propanoic acid as a white solid
1H NMR (300 MHz, DMSO-d6)
d 13.26 (br s,1H),12.22 (s,1H), 8.26
(s,1H), 7.79 (d, J ¼ 2.1 Hz,1H), 7.69 (s,1H), 7.49 (d, J ¼ 8.7 Hz,1H), 7.28
(dd, J ¼ 8.7, 1.8 Hz, 1H), 3.84 (s, 3H); LRMS (ESI) m/z 336 [M þ H]þ.
4.1.21. 2-[(5-Chloro-1H-indol-2-yl)formamido]-N-[5-(1,2-
dihydroxyethyl)pyridin-2-yl]acetamide (2i)
(i) A mixture of Boc-protected glycine (500 mg, 2.85 mmol),
PyBOP (2.48 g, 4.76 mmol) and DIPEA (788 mL, 4.76 mmol) in DMF

634
L. Zhang et al. / European Journal of Medicinal Chemistry 58 (2012) 624e639
(2.05 g, 71%). (iv) A mixture of (S)-3-cyclohexyl-2-(5,6-dichloro-1-
oxoisoindolin-2-yl)propanoic acid (100 mg, 0.28 mmol), PyBOP
pressure and the concentrate was purified by column chromatog-
raphy to give the title compound as a white solid (99 mg, 85%). 1H
(244 mg, 0.47 mmol) and DIPEA (78
mL, 0.47 mmol) in DMF was
NMR (400 MHz, DMSO-d6) d 13.04 (br s, 1H), 8.07 (s, 1H), 7.98 (s,
stirred for 30 min, then added thiazol-2-amine (24 mg, 0.24 mmol).
After the reaction was completed monitored by TLC, the reaction
mixture was extracted with EtOAc, washed with water and brine,
dried with anhydrous Na2SO4 and concentrated. The residue was
purified by column chromatography to give the title compound as
a colourless solid (80 mg, 78%). 1H NMR (400 MHz, DMSO-d6)
1H), 7.94 (s, 1H), 5.23 (dd, J ¼ 10.8, 4.8 Hz, 1H), 4.82 (d, J ¼ 18.4 Hz,
1H), 4.58 (d, J ¼ 18.4 Hz, 1H), 1.94e1.78 (m, 3H), 1.70e1.56 (m, 4H),
1.14e1.05 (m, 4H), 1.01e0.92 (m, 2H); 13C NMR (100 MHz, DMSO-
d6)
d 170.77, 165.94, 162.96, 161.27, 144.05, 142.52, 134.57, 132.01,
131.22, 126.22, 124.77, 51.81, 47.04, 36.67, 33.78, 33.10, 31.23, 25.97,
25.64, 25.42; LRMS (ESI) m/z 480 [M ꢀ H]ꢀ.
d
12.63 (s, 1H), 7.95 (s, 1H), 7.91 (s, 1H), 7.49 (s, 1H), 7.24 (s, 1H),
5.22e5.21 (m, 1H), 4.82 (d, J ¼ 18.0 Hz, 1H), 4.55 (d, J ¼ 18.0 Hz, 1H),
4.1.28. (2S)-3-cyclohexyl-2-(5,6-dichloro-1-oxo-2,3-dihydro-1H-
1.88e1.76 (m, 3H), 1.69e1.55 (m, 4H), 1.19e0.93 (m, 6H); 13C NMR
isoindol-2-yl)-N-(4-methyl-1,3-thiazol-2-yl)propanamide (3g)
(100 MHz, DMSO-d6)
d
169.97, 165.90, 157.49, 142.50, 137.82, 134.50,
1H NMR (400 MHz, CDCl3)
d 8.41 (s, 1H), 7.59 (s, 1H), 6.53 (s, 1H),
132.08, 131.19, 126.17, 124.73, 114.01, 51.71, 47.01, 36.82, 33.80, 33.10,
5.66 (t, J ¼ 7.6 Hz, 1H), 4.81 (d, J ¼ 17.6 Hz, 1H), 4.51 (d, J ¼ 17.6 Hz,
1H), 2.37 (s, 3H), 2.09e2.02 (m, 1H), 1.87e1.80 (m, 3H), 1.65e1.61
(m, 3H), 1.19e1.09 (m, 4H), 1.01e0.94 (m, 2H); LRMS (ESI) m/z
452 [M þ H]þ.
31.26, 25.96, 25.64, 25.42; LRMS (ESI) m/z 438 [M þ H]þ.
4.1.23. (2S)-3-cyclohexyl-2-(5,6-dichloro-1-oxo-2,3-dihydro-1H-
isoindol-2-yl)-N-(5-methyl-1,3-thiazol-2-yl)propanamide (3b)
1H NMR (400 MHz, CDCl3)
d
8.22 (s, 1H), 7.58 (s, 1H), 7.38 (s, 1H),
4.1.29. Methyl 2-[(2S)-3-cyclohexyl-2-(5,6-dichloro-1-oxo-2,3-
dihydro-1H-isoindol-2-yl)propanamido]-1,3-thiazole-4-carboxylate
(3h)
5.45 (t, J ¼ 8.8 Hz, 1H), 4.86 (d, J ¼ 17.6 Hz, 1H), 4.48 (d, J ¼ 18.0 Hz,
1H), 2.42 (s, 3H), 1.99e1.1.80 (m, 3H), 1.69e1.60 (m, 4H), 1.17e1.09
(m, 4H), 0.99e0.86 (m, 2H); LRMS (ESI) m/z 452 [M þ H]þ.
1H NMR (300 MHz, CDCl3)
d 10.47 (br s, 1H), 8.03 (s, 1H), 7.84 (s,
1H), 7.58 (s, 1H), 5.40e5.35 (m, 1H), 4.62 (d, J ¼ 17.4 Hz, 1H), 4.43 (d,
J ¼ 17.1 Hz, 1H), 3.89 (s, 3H), 2.04e1.99 (m, 1H), 1.92e1.83 (m, 2H),
1.74e1.67 (m, 4H), 1.19e1.09 (m, 4H), 1.03e0.92 (m, 2H); LRMS (ESI)
m/z 496 [M þ H]þ.
4.1.24. Methyl 2-[(2S)-3-cyclohexyl-2-(5,6-dichloro-1-oxo-2,3-
dihydro-1H-isoindol-2-yl)propanamido]-1,3-thiazole-5-carboxylate
(3c)
1H NMR (400 MHz, CDCl3)
d 8.59 (s, 1H), 8.22 (s, 1H), 7.60 (s, 1H),
5.68 (t, J ¼ 8.0 Hz, 1H), 4.80 (d, J ¼ 17.6 Hz, 1H), 4.54 (d, J ¼ 18.0 Hz,
1H), 3.89 (s, 3H), 2.05e2.00 (m, 1H), 1.93e1.84 (m, 1H), 1.79e1.76
(m, 1H), 1.68e1.60 (m, 4H), 1.26e1.12 (m, 4H), 1.01e0.95 (m, 2H);
LRMS (ESI) m/z 496 [M þ H]þ.
4.1.30. Ethyl 2-[(2S)-3-cyclohexyl-2-(5,6-dichloro-1-oxo-2,3-
dihydro-1H-isoindol-2-yl)propanamido]-1,3-thiazole-4-carboxylate
(3i)
1H NMR (400 MHz, CDCl3)
d 10.83 (br s, 1H), 8.05 (s, 1H), 7.84 (s,
1H), 7.58 (s, 1H), 5.49e5.45 (m, 1H), 4.68 (d, J ¼ 17.2 Hz, 1H), 4.44 (d,
J ¼ 17.2 Hz,1H), 4.37 (q, J ¼ 7.2 Hz, 2H), 2.07e2.01 (m,1H),1.93e1.88
(m, 1H), 1.86e1.82 (m, 1H), 1.73e1.61 (m, 4H), 1.36 (t, J ¼ 7.2 Hz, 3H),
1.21e1.13 (m, 4H), 1.04e0.90 (m, 2H); LRMS (ESI) m/z 510 [M þ H]þ.
4.1.25. Ethyl 2-[(2S)-3-cyclohexyl-2-(5,6-dichloro-1-oxo-2,3-
dihydro-1H-isoindol-2-yl)propanamido]-1,3-thiazole-5-carboxylate
(3d)
1H NMR (400 MHz, CDCl3)
d 12.15 (br s, 1H), 8.57 (s, 1H), 8.21 (s,
1H), 7.59 (s,1H), 5.68 (t, J ¼ 8.0 Hz,1H), 4.80 (d, J ¼ 18.0 Hz,1H), 4.54
(d, J ¼ 17.6 Hz, 1H), 4.34 (q, J ¼ 7.2 Hz, 2H), 2.08e2.01 (m, 1H),
1.92e1.84 (m, 1H), 1.79e1.76 (m, 1H), 1.67e1.60 (m, 4H), 1.36 (t,
J ¼ 7.2 Hz, 3H), 1.22e1.09 (m, 4H), 1.04e0.96 (m, 2H); LRMS (ESI) m/
z 510 [M þ H]þ.
4.1.31. 2-[(2S)-3-cyclohexyl-2-(5,6-dichloro-1-oxo-2,3-dihydro-
1H-isoindol-2-yl)propanamido]-1,3-thiazole-4-carboxylic acid (3j)
1H NMR (300 MHz, DMSO-d6)
d 13.72 (br s, 1H), 13.01 (br s, 1H),
7.98 (s, 1H), 7.93 (s, 1H), 7.72 (s, 1H), 5.32 (m, 1H), 4.80 (d,
J ¼ 18.0 Hz, 1H), 4.60 (d, J ¼ 18.0 Hz, 1H), 1.90e1.55 (m, 7H),
1.23e1.94 (m, 6H); LRMS (ESI) m/z 480 [M ꢀ H]ꢀ.
4.1.26. (2S)-3-cyclohexyl-2-(5,6-dichloro-1-oxo-2,3-dihydro-1H-
isoindol-2-yl)-N-[5-(hydroxymethyl)-1,3-thiazol-2-yl]propanamide
(3e)
Compound 3c (100 mg, 0.20 mmol) in THF (10 mL) was added
LiBH4 (18 mg, 0.81 mmol). After the reaction was completed
monitored by TLC, the reaction mixture was quenched with
methanol, concentrated and purified by column chromatography to
provide the title compound as a white solid (86 mg, 91%). 1H NMR
4.1.32. Ethyl 2-{2-[(2S)-3-cyclohexyl-2-(5,6-dichloro-1-oxo-2,3-
dihydro-1H-isoindol-2-yl)propanamido]-1,3-thiazol-4-yl}acetate
(3k)
1H NMR (400 MHz, CDCl3)
d 11.20 (br s, 1H), 8.32 (s, 1H), 7.59 (s,
1H), 6.83 (s, 1H), 5.59 (t, J ¼ 8.4 Hz, 1H), 4.77 (d, J ¼ 18.0 Hz,1H), 4.49
(d, J ¼ 17.6 Hz,1H), 4.13 (q, J ¼ 7.2 Hz, 2H), 3.75 (s, 2H), 2.08e2.01 (m,
1H), 1.88e1.77 (m, 3H), 1.68e1.61 (m, 3H), 1.23 (t, J ¼ 7.2 Hz, 3H),
1.18e1.09 (m, 4H), 1.01e0.94 (m, 2H); LRMS (ESI) m/z 524 [M þ H]þ.
(300 MHz, CDCl3)
d 7.79 (s, 1H), 7.53 (s, 1H), 7.27 (s, 1H), 4.48 (m,
1H), 4.45 (d, J ¼ 17.7 Hz, 1H), 4.28 (d, J ¼ 17.7 Hz, 1H), 3.87e3.67 (m,
2H), 3.06 (br s, 1H), 1.89e1.57 (m, 7H), 1.14e1.11 (m, 4H), 1.01e0.86
4.1.33. Methyl 2-{2-[(2S)-3-cyclohexyl-2-(5,6-dichloro-1-oxo-2,3-
dihydro-1H-isoindol-2-yl)propanamido]-1,3-thiazol-4-yl}acetate
(3l)
(m, 2H); 13C NMR (100 MHz, CDCl3)
d 167.72, 140.65, 135.97, 132.91,
132.69, 125.47, 124.98, 64.42, 51.93, 46.43, 36.80, 34.33, 33.96,
32.85, 26.51, 26.31, 26.13; LRMS (ESI) m/z 468 [M þ H]þ.
1H NMR (300 MHz, CDCl3)
d 11.41 (br s, 1H), 8.30 (s, 1H), 7.59 (s,
1H), 6.82 (s, 1H), 5.60 (t, J ¼ 7.5 Hz,1H), 4.79 (d, J ¼ 17.7 Hz, 1H), 4.49
(d, J ¼ 18.3 Hz, 1H), 3.76 (s, 2H), 3.67 (s, 3H), 2.08e1.99 (m, 1H),
1.89e1.77 (m, 3H), 1.64 (m, 3H), 1.28e1.12 (m, 4H), 1.05e0.94 (m,
2H); LRMS (ESI) m/z 510 [M þ H]þ.
4.1.27. 2-[(2S)-3-cyclohexyl-2-(5,6-dichloro-1-oxo-2,3-dihydro-
1H-isoindol-2-yl)propanamido]-1,3-thiazole-5-carboxylic acid (3f)
Compound 3c (120 mg, 0.24 mmol) in THF/H2O (v/v, 1:1) was
added LiOH monohydrate (102 mg, 2.42 mmol) and stirred at room
temperature. After the reaction was completed monitored by TLC,
the reaction mixture was diluted with water, acidified with 1 N HCl,
extracted with EtOAc, washed with water and brine, dried over
anhydrous Na2SO4. The solvent was evaporated under reduced
4.1.34. 2-{2-[(2S)-3-cyclohexyl-2-(5,6-dichloro-1-oxo-2,3-dihydro-
1H-isoindol-2-yl)propanamido]-1,3-thiazol-4-yl}acetic acid (3m)
1H NMR (400 MHz, CD3OD)
d
7.88 (s, 1H), 7.80 (s, 1H), 6.90 (s,
1H), 5.29e5.25 (m, 1H), 4.85 (d, J ¼ 18.4 Hz, 1H), 4.57 (d, J ¼ 18.4 Hz,

L. Zhang et al. / European Journal of Medicinal Chemistry 58 (2012) 624e639
635
1H), 3.66 (s, 2H),1.94e1.87 (m, 3H),1.77e1.61 (m, 4H),1.20e1.13 (m,
4H), 1.08e0.99 (m, 2H); LRMS (ESI) m/z 496 [M þ H]þ.
3H), 3.63 (s, 1H), 3.54e3.45 (m, 1H), 3.25e3.15 (m, 1H); LRMS (ESI)
m/z 522 [M þ H]þ.
4.1.35. (2S)-3-cyclohexyl-2-(5,6-dichloro-1-oxo-2,3-dihydro-1H-
isoindol-2-yl)-N-[4-(2-hydroxyethyl)-1,3-thiazol-2-yl]propanamide
(3n)
4.1.42. (2S)-3-cyclohexyl-2-(5,6-dichloro-1,3-dioxo-2,3-dihydro-
1H-isoindol-2-yl)-N-(1,3-thiazol-2-yl)propanamide (4a)
A
mixture
dioxoisoindolin-2-yl)propanoic acid (100 mg, 0.27 mmol), PyBOP
(234 mg, 0.45 mmol) and DIPEA (75 L, 0.45 mmol) in DMF was
of
(S)-3-cyclohexyl-2-(5,6-dichloro-1,3-
1H NMR (400 MHz, CD3OD)
d 7.89 (s, 1H), 7.81 (s, 1H), 6.75 (s,
1H), 5.28e5.24 (m, 1H), 4.85 (d, J ¼ 17.6 Hz, 1H), 4.57 (d, J ¼ 18.0 Hz,
1H), 3.81 (t, J ¼ 6.8 Hz, 2H), 2.84 (t, J ¼ 6.8 Hz, 2H), 1.94e1.87 (m,
3H), 1.78e1.62 (m, 4H), 1.24e1.14 (m, 4H), 1.05e0.99 (m, 2H); LRMS
(ESI) m/z 482 [M þ H]þ.
m
stirred for 30 min, then added thiazol-2-amine (23 mg, 0.23 mmol).
After the reaction was completed monitored by TLC, the reaction
mixture was extracted with EtOAc, washed with water and brine,
dried with anhydrous Na2SO4 and concentrated. The residue was
purified by column chromatography to give the title compound as
4.1.36. (2S)-N-(1,3-benzothiazol-2-yl)-3-cyclohexyl-2-(5,6-
dichloro-1-oxo-2,3-dihydro-1H-isoindol-2-yl)propanamide (3o)
a colourless solid (76 mg, 75%). 1H NMR (300 MHz, DMSO-d6)
d 9.15
1H NMR (400 MHz, DMSO-d6)
d
12.88 (s, 1H), 8.10e7.96 (m,
(s, 2H), 8.34 (d, J ¼ 3.3 Hz, 1H), 8.12 (d, J ¼ 3.9 Hz,1H), 5.97e5.92 (m,
2H), 7.92 (s, 1H), 7.75 (d, J ¼ 7.6 Hz, 1H), 7.43 (t, J ¼ 7.6 Hz, 1H),
7.30 (t, J ¼ 7.6 Hz, 1H), 5.26e5.24 (m, 1H), 4.83 (d, J ¼ 18.0 Hz,
1H), 5.57 (d, J ¼ 18.0 Hz, 1H), 1.94e1.79 (m, 3H), 1.70e1.55 (m,
4H), 1.19e1.07 (m, 4H), 1.01e0.95 (m, 2H); LRMS (ESI) m/z 488
[M þ H]þ.
1H), 2.97e2.88 (m, 2H), 2.74e2.70 (m, 1H), 2.54e2.47 (m, 4H),
2.15e1.74 (m, 6H); 13C NMR (100 MHz, DMSO-d6)
d 165.74, 137.54,
131.60,125.52,113.81, 50.62, 35.52, 33.55, 33.36, 31.28, 26.00, 25.65,
25.39; LRMS (ESI) m/z 450 [M ꢀ H]ꢀ.
4.1.43. (2S)-3-cyclohexyl-2-(5,6-dichloro-1,3-dioxo-2,3-dihydro-
4.1.37. (2S)-N-(6-chloro-1,3-benzothiazol-2-yl)-3-cyclohexyl-2-
(5,6-dichloro-1-oxo-2,3-dihydro-1H-isoindol-2-yl)propanamide
(3p)
1H-isoindol-2-yl)-N-(4-methyl-1,3-thiazol-2-yl)propanamide (4b)
1H NMR (300 MHz, CDCl3)
d 7.92 (s, 2H), 6.51 (s, 1H), 5.13e5.06
(m, 1H), 2.36e2.26 (m, 1H), 2.29 (s, 3H), 2.02e1.93 (m, 1H),
1.85e1.81 (m, 1H), 1.66e1.62 (m, 4H), 1.14e1.11 (m, 4H), 0.99e0.84
(m, 2H); MS (EI, m/z) 466 [M þ H]þ.
1H NMR (400 MHz, CDCl3)
d 11.58 (br s, 1H), 8.41 (s, 1H), 7.77 (s,
1H), 7.74 (d, J ¼ 8.8 Hz, 1H), 7.62 (s, 1H), 7.37 (d, J ¼ 8.8 Hz, 1H),
5.66e5.60 (m, 1H), 4.80 (d, J ¼ 17.6 Hz, 1H), 4.53 (d, J ¼ 18.0 Hz, 1H),
2.10e2.03 (m, 1H), 1.92e1.86 (m, 1H), 1.82e1.78 (m, 1H), 1.66e1.59
(m, 4H), 1.19e1.09 (m, 4H), 1.04e0.94 (m, 2H); LRMS (ESI) m/z 522
[M þ H]þ.
4.1.44. Ethyl 2-[(2S)-3-cyclohexyl-2-(5,6-dichloro-1,3-dioxo-2,3-
dihydro-1H-isoindol-2-yl)propanamido]-1,3-thiazole-4-carboxylate
(4c)
1H NMR (400 MHz, CDCl3)
d 10.69 (br s, 1H), 7.93 (s, 2H), 7.79 (s,
4.1.38. Methyl 2-[(2S)-3-cyclohexyl-2-(5,6-dichloro-1-oxo-2,3-
dihydro-1H-isoindol-2-yl)propanamido]pyridine-4-carboxylate
(3q)
1H), 5.19e5.15 (m, 1H), 4.41e4.36 (q, J ¼ 7.2 Hz, 2H), 2.30e2.21 (m,
1H), 1.99e1.92 (m, 1H), 1.84e1.81 (m, 1H), 1.70e1.60 (m, 4H), 1.38 (t,
J ¼ 7.2 Hz, 3H), 1.15e1.08 (m, 4H), 0.89e0.79 (m, 2H); MS (EI, m/z)
524 [M þ H]þ.
1H NMR (300 MHz, CDCl3)
d 9.36 (br s, 1H), 8.68 (s, 1H), 8.42 (d,
J ¼ 4.8 Hz, 1H), 8.07 (s, 1H), 7.61 (d, J ¼ 5.1 Hz, 1H), 7.59 (s, 1H), 5.25
(t, J ¼ 8.7 Hz, 1H), 4.68 (d, J ¼ 17.4 Hz, 1H), 4.45 (d, J ¼ 17.7 Hz, 1H),
3.94 (s, 3H), 2.06e1.99 (m, 1H), 1.91e1.82 (m, 2H), 1.74e1.64 (m,
4H), 1.20e1.16 (m, 4H), 1.08e0.96 (m, 2H); LRMS (ESI) m/z 490
[M þ H]þ.
4.1.45. Methyl 2-[(2S)-3-cyclohexyl-2-(5,6-dichloro-1,3-dioxo-2,3-
dihydro-1H-isoindol-2-yl)propanamido]-1,3-thiazole-4-carboxylate
(4d)
1H NMR (400 MHz, CDCl3)
d 10.74 (s, 1H), 7.91 (s, 2H), 7.78 (s,
1H), 5.16 (dd, J ¼ 10.8, 4.4 Hz, 1H), 3.91 (s, 3H), 2.24e2.17 (m, 1H),
2.01e1.94 (m, 1H), 1.85e1.82 (m, 1H), 1.69e1.60 (m, 4H), 1.15e1.07
(m, 4H), 0.97e0.83 (m, 2H); MS (EI, m/z) 510 [M þ H]þ.
4.1.39. Methyl 6-[(2S)-3-cyclohexyl-2-(5,6-dichloro-1-oxo-2,3-
dihydro-1H-isoindol-2-yl)propanamido]pyridine-3-carboxylate
(3r)
1H NMR (300 MHz, CDCl3)
d
9.06 (br s, 1H), 8.89 (s, 1H), 8.28 (d,
4.1.46. Methyl 2-[(2S)-3-cyclohexyl-2-(5,6-dichloro-1,3-dioxo-2,3-
dihydro-1H-isoindol-2-yl)propanamido]-1,3-thiazole-5-carboxylate
(4e)
J ¼ 9.0 Hz, 1H), 8.19 (d, J ¼ 8.7 Hz, 1H), 7.99 (s, 1H), 7.59 (s, 1H),
5.18e5.10 (m, 1H), 4.59 (d, J ¼ 17.4 Hz, 1H), 4.42 (d, J ¼ 17.1 Hz, 1H),
3.92 (s, 3H), 2.04e1.96 (m, 1H), 1.92e1.83 (m, 2H), 1.72e1.63 (m,
4H), 1.22e1.14 (m, 4H), 1.05e0.96 (m, 2H); LRMS (ESI) m/z 490
[M þ H]þ.
1H NMR (400 MHz, CDCl3)
d 10.75 (s, 1H), 8.02 (s, 1H), 7.92 (s,
2H), 5.13 (dd, J ¼ 11.2, 5.2 Hz, 1H), 3.89 (s, 3H), 2.35e2.27 (m, 1H),
2.02e1.95 (m, 1H), 1.81 (d, J ¼ 11.6 Hz, 1H), 1.65e1.62 (m, 4H),
1.13e1.10 (m, 4H), 0.89e0.83 (m, 2H); MS (EI, m/z) 510 [M þ H]þ.
4.1.40. Methyl 2-[(2S)-2-(5,6-dichloro-1-oxo-2,3-dihydro-1H-
isoindol-2-yl)-3-(4-fluorophenyl)propanamido]-1,3-thiazole-5-
carboxylate (3s)
4.1.47. Ethyl 2-[(2S)-3-cyclohexyl-2-(5,6-dichloro-1,3-dioxo-2,3-
dihydro-1H-isoindol-2-yl)propanamido]-1,3-thiazole-5-carboxylate
(4f)
1H NMR (300 MHz, DMSO-d6)
d 13.08 (br s, 1H), 8.19 (s, 1H), 7.95
(s, 1H), 7.86 (s, 1H), 7.35e7.30 (m, 2H), 7.11e7.05 (m, 2H), 5.41e5.37
(m, 1H), 4.69e4.56 (m, 2H), 3.82 (s, 3H), 3.37e3.23 (m, 2H); LRMS
(ESI) m/z 508 [M þ H]þ.
1H NMR (400 MHz, CDCl3)
d 8.02 (s, 1H), 7.92 (s, 2H), 5.14 (dd,
J ¼ 10.8, 4.4 Hz, 1H), 4.35 (q, J ¼ 7.2 Hz, 2H), 2.32 (m, 1H), 2.02e1.96
(m, 1H), 1.82e1.79 (m, 1H), 1.65e1.62 (m, 4H), 1.37 (t, J ¼ 7.2 Hz, 3H),
1.13e1.10 (m, 4H), 0.87e0.83 (m, 2H); MS (EI, m/z) 524 [M þ H]þ.
4.1.41. Methyl 2-{2-[(2S)-2-(5,6-dichloro-1-oxo-2,3-dihydro-1H-
isoindol-2-yl)-3-(4-fluorophenyl)propanamido]-1,3-thiazol-4-yl}
acetate (3t)
4.1.48. (2S)-N-(1,3-benzothiazol-2-yl)-3-cyclohexyl-2-(5,6-
dichloro-1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl)propanamide (4g)
1H NMR (300 MHz, CDCl3)
d
11.27 (br s, 1H), 8.14 (s, 1H), 7.56 (s,
1H NMR (300 MHz, CDCl3)
d
7.83 (s, 2H), 7.78 (d, J ¼ 7.8 Hz, 1H),
1H), 7.18e7.11 (m, 2H), 6.89e6.82 (m, 3H), 5.72 (t, J ¼ 8.1 Hz, 1H),
7.68 (d, J ¼ 8.1 Hz, 1H), 7.43 (t, J ¼ 7.8 Hz, 1H), 7.32 (t, J ¼ 7.2 Hz, 1H),
4.75 (d, J ¼ 17.4 Hz, 1H), 4.51 (d, J ¼ 17.7 Hz, 1H), 3.68 (s, 1H), 3.64 (s,
5.18e5.13 (m, 1H), 2.33e2.26 (m, 1H), 2.08e1.99 (m, 1H), 1.86e1.77

636
L. Zhang et al. / European Journal of Medicinal Chemistry 58 (2012) 624e639
(m, 1H), 1.66e1.59 (m, 4H), 1.18e1.09 (m, 4H), 0.97e0.95 (m, 2H);
MS (EI, m/z) 502 [M þ H]þ.
4.1.54. (2S)-3-cyclohexyl-2-(6,7-dichloro-2,4-dioxo-1,2,3,4-
tetrahydroquinazolin-3-yl)-N-(4-methyl-1,3-thiazol-2-yl)
propanamide (5a)
4.1.49. Methyl 2-[(2S)-2-(5,6-dichloro-1,3-dioxo-2,3-dihydro-1H-
isoindol-2-yl)-3-(4-fluorophenyl)propanamido]-1,3-thiazole-5-
carboxylate (4h)
To a solution of 6a (100 mg, 0.22 mmol) and 1,10-carbon-
yldiimidazole (72 mg, 0.44 mmol) in THF (10 mL) was added l,8-
diazabicyclo[5.4.0]undec-7-ene (DBU, 66
mL, 0.44 mmol). The
1H NMR (300 MHz, CDCl3)
d
11.26 (br s, 1H), 7.88 (s, 1H), 7.79 (s,
mixture was stirred at room temperature for 16 h. The reaction
mixture was diluted with EtOAc and washed with water. The
organic layer was dried over anhydrous Na2SO4. The solvent was
evaporated under reduced pressure, and the concentrate was
purified by column chromatography to give the title compound as
2H), 7.07 (dd, J ¼ 8.4, 5.7 Hz, 2H), 6.86 (t, J ¼ 8.7 Hz, 2H), 5.29e5.23
(m, 1H), 3.89 (s, 3H), 3.66e3.45 (m, 2H); MS (EI, m/z) 522 [M þ H]þ.
4.1.50. (2S)-2-[(2-amino-4,5-dichlorophenyl)formamido]-3-
cyclohexyl-N-(4-methyl-1,3-thiazol-2-yl)propanamide (6a)
a white solid (91 mg, 86%). 1H NMR (300 MHz, CDCl3)
d 10.56 (br s,
(i) 4,5-Dichlorophthalic anhydride 20 (5 g, 23.0 mmol) was
heated in formamide (30 mL) for 3 h under reflux. After cooling,
the brown precipitate was filtered, washed with water and dried in
vacuum to give 4, 5-dichlorophthalimide 23 in quantitative yield.
(ii) Aqueous sodium hydroxide (25%, 20 mL) was cooled to 0 ꢁC,
bromine (0.96 mL, 18.52 mmol) was added and the mixture was
1H), 8.07 (s, 1H), 7.13 (s, 1H), 6.45 (s, 1H), 5.74e5.69 (m, 1H),
2.38e2.25 (m, 1H), 2.25 (s, 3H), 2.04e1.92 (m, 2H), 1.69e1.62 (m,
4H), 1.30e1.13 (m, 4H), 0.99e0.86 (m, 2H); 13C NMR (100 MHz,
CDCl3) d 168.21, 160.70, 151.12, 145.96, 140.32, 137.41, 129.87, 128.09,
116.99,113.73,108.78, 53.41, 36.23, 34.64, 33.74, 32.89, 26.48, 26.24,
26.08, 16.85; MS (EI, m/z) 481 [M þ H]þ.
stirred to form
a
yellow solution of NaOBr. 4,5-
dichlorophthalimide 23 (4 g, 18.52 mmol) was dissolved in 5%
aqueous sodium hydroxide (40 mL), the solution was cooled to 0 ꢁC
and added to the cold solution of NaOBr. The mixture was stirred
vigorously for 5 min and then heated to 80 ꢁC for 2 min. The
reaction mixture was cooled and neutralized with concentrated
hydrochloric acid. The precipitate was filtered off, washed with ice
water and recrystallized to give 2-amino-4,5-dichlorobenzoic acid
24 (2.67 g, 72%). (iii) A mixture of 24 (200 mg, 0.96 mmol), PyBOP
4.1.55. Methyl 2-[(2S)-3-cyclohexyl-2-(6,7-dichloro-2,4-dioxo-
1,2,3,4-tetrahydroquinazolin-3-yl)propanamido]-1,3-thiazole-5-
carboxylate (5b)
1H NMR (300 MHz, CD3OD)
d 8.10 (s, 1H), 8.00 (s, 1H), 7.35 (s,
1H), 5.76e5.71 (m, 1H), 3.86 (s, 3H), 2.35e2.25 (m, 1H), 2.03e1.88
(m, 2H), 1.74e1.67 (m, 4H), 1.32e1.18 (m, 4H), 1.03e0.87 (m, 2H);
MS (EI, m/z) 525 [M þ H]þ.
(842 mg, 1.62 mmol), DIPEA (270
m
L, 1.62 mmol) and (S)-2-amino-
4.1.56. Ethyl 2-[(2S)-3-cyclohexyl-2-(6,7-dichloro-2,4-dioxo-
1,2,3,4-tetrahydroquinazolin-3-yl)propanamido]-1,3-thiazole-5-
carboxylate (5c)
3-cyclohexyl-N-(4-methylthiazol-2-yl)propanamide (216 mg,
0.82 mmol) in DMF was stirred at room temperature. After the
reaction was completed monitored by TLC, the reaction mixture
was extracted with EtOAc, washed with water and brine, dried
with anhydrous Na2SO4 and concentrated. The residue was puri-
fied by column chromatography to give the title compound as
1H NMR (300 MHz, CD3OD)
d 11.75 (br s, 1H), 9.92 (br s, 1H), 8.01
(s, 1H), 7.82 (s, 1H), 7.26 (s, 1H), 7.09 (s, 1H), 5.73 (m, 1H), 4.40e4.34
(m, 2H), 2.43e2.34 (m, 1H), 1.95e1.83 (m, 2H), 1.68e1.66 (m, 4H),
1.41e1.37 (m, 3H), 1.20e1.16 (m, 4H), 0.98e0.88 (m, 2H); MS (EI, m/
z) 539 [M þ H]þ.
a colourless solid (240 mg, 65%). 1H NMR (400 MHz, CDCl3)
(d, J ¼ 7.6 Hz, 1H), 7.46 (s, 1H), 6.68 (s, 1H), 6.56 (s, 1H), 5.52 (br s,
2H), 4.91 (m, 1H), 2.35 (s, 3H), 1.79e1.70 (m, 3H), 1.61e1.60 (m, 4H),
1.22e1.06 (m, 4H), 0.91e0.78 (m, 2H); 13C NMR (100 MHz, CDCl3)
d 7.48
4.1.57. 2-[(2S)-3-cyclohexyl-2-(6,7-dichloro-2,4-dioxo-1,2,3,4-
tetrahydroquinazolin-3-yl)propanamido]-1,3-thiazole-5-carboxylic
acid (5d)
d
171.38, 168.06, 157.61, 148.26, 147.22, 136.60, 129.04, 119.24,
118.25, 114.18, 108.82, 51.84, 39.54, 34.24, 33.71, 32.46, 26.36, 26.19,
Compound 5b (100 mg, 0.18 mmol) in THF/H2O (v/v, 1:1) was
added LiOH monohydrate (78 mg, 1.85 mmol) and stirred at room
temperature. After the reaction was completed monitored by TLC,
the reaction mixture was diluted with water, acidified with 1 N HCl,
extracted with EtOAc, washed with water and brine, dried over
anhydrous Na2SO4. The solvent was evaporated under reduced
pressure and the concentrate was purified by column chromatog-
raphy to give the title compound as a white solid (85 mg, 90%). 1H
26.01, 17.10; LRMS (ESI) m/z 455 [M þ H]þ.
4.1.51. Methyl 2-[(2S)-2-[(2-amino-4,5-dichlorophenyl)
formamido]-3-cyclohexylpropanamido]-1,3-thiazole-5-carboxylate
(6b)
1H NMR (400 MHz, CDCl3)
d 8.21 (s, 1H), 7.42 (s, 1H), 7.07e6.99
(m, 1H), 6.71 (s, 1H), 4.92e4.86 (m, 1H), 3.89 (s, 3H), 1.83e1.79 (m,
2H), 1.74e1.64 (m, 5H), 1.24e1.09 (m, 4H), 0.99e0.90 (m, 2H); MS
(EI, m/z) 499 [M þ H]þ.
NMR (400 MHz, CD3OD)
d 8.12 (s, 1H), 7.83 (s, 1H), 7.38 (s, 1H),
5.73e5.69 (m, 1H), 2.30e2.25 (m, 1H), 2.01e1.93 (m, 2H), 1.70e1.67
(m, 4H), 1.23e1.16 (m, 4H), 1.03e0.93 (m, 2H); 13C NMR (100 MHz,
4.1.52. Ethyl 2-[(2S)-2-[(2-amino-4,5-dichlorophenyl)formamido]-
CD3OD) d 170.45, 162.93, 162.61, 151.54, 142.69, 140.42, 140.24,
3-cyclohexylpropanamido]-1,3-thiazole-5-carboxylate (6c)
130.87, 130.79, 130.42, 127.79, 118.12, 115.71, 53.86, 37.09, 35.86,
1H NMR (400 MHz, CDCl3)
d
8.21 (s, 1H), 7.44 (s, 1H), 7.20 (d,
34.93, 33.90, 27.56, 27.38, 27.20; MS (EI, m/z) 509 [M ꢀ H]ꢀ.
J ¼ 7.2 Hz, 1H), 6.70 (s, 1H), 4.94e4.88 (m, 1H), 4.35 (d, J ¼ 7.2 Hz,
2H), 1.83e1.79 (m, 2H), 1.74e1.64 (m, 5H), 1.36 (t, J ¼ 7.2 Hz,
3H),1.24e1.08 (m, 4H), 0.99e0.84 (m, 2H); MS (EI, m/z) 513
[M þ H]þ.
4.1.58. Methyl 2-[(2S)-2-(6,7-dichloro-2,4-dioxo-1,2,3,4-
tetrahydroquinazolin-3-yl)-3-(4-fluorophenyl)propanamido]-1,3-
thiazole-5-carboxylate (5e)
1H NMR (400 MHz, CDCl3)
d 11.82 (br s,1H), 9.77 (br s,1H), 7.88 (s,
4.1.53. Methyl 2-[(2S)-2-[(2-amino-4,5-dichlorophenyl)
formamido]-3-(4-fluorophenyl)propanamido]-1,3-thiazole-5-
carboxylate (6d)
1H), 7.73 (s,1H), 7.11 (m, 2H), 7.02 (s,1H), 6.89e6.85 (m, 2H), 5.84 (m,
1H), 3.87 (s, 3H), 3.67(m,1H), 3.33 (m,1H); MS (EI, m/z) 537 [M þ H]þ.
1H NMR (300 MHz, DMSO-d6)
d
12.99 (s, 1H), 8.83 (d, J ¼ 10.0 Hz,
4.1.59. Methyl 6-[(2S)-3-cyclohexyl-2-[(3,4-dichlorophenyl)
formamido]propanamido]pyridine-3-carboxylate (6e)
A mixtureof3,4-dichlorobenzoic acid (100 mg, 0.52mmol), PyBOP
(454 mg, 0.87 mmol), DIPEA (145 mL, 0.87 mmol) and (S)-methyl 6-(2-
1H), 8.19 (s,1H), 7.83 (s,1H), 7.43 (t, J ¼ 8.7 Hz, 2H), 7.13 (t, J ¼ 8.7 Hz,
2H), 6.93 (s, 1H), 6.67 (s, 2H), 4.89e4.82 (m, 1H), 3.82 (s, 3H),
3.20e3.02 (m, 2H); LRMS (ESI) m/z 511 [M þ H]þ.

L. Zhang et al. / European Journal of Medicinal Chemistry 58 (2012) 624e639
637
amino-3-cyclohexylpropanamido)picolinate (133 mg, 0.44 mmol) in
DMF was stirred at room temperature. After the reaction was
completed monitoredbyTLC,thereactionmixturewasextractedwith
EtOAc, washed with water and brine, dried with anhydrous Na2SO4
and concentrated. The residue was purified by column chromatog-
raphytogivethetitle compoundasacolourlesssolid(146mg, 70%).1H
0.5 mM glucose-1-phosphate, and 1 mg/mL glycogen in 96-well
microplates (Costar). After the addition of 150 L of 1 M HCl con-
m
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.
NMR(300MHz, CDCl3)d9.76(brs,1H), 8.88(s,1H), 8.40e8.31(m, 2H),
7.99(s,1H), 7.73e7.69 (m, 1H), 7.52 (d, J¼8.4Hz,1H), 6.85(d,J¼ 6.9Hz,
1H), 4.91e4.84 (m, 1H), 3.95 (s, 3H), 1.92e1.67 (m, 7H), 1.27e1.15 (m,
4.2.2. In vitro glucokinase enzyme assay
4H), 0.99e0.88 (m, 2H); 13C NMR (100 MHz, CDCl3)
d
171.23, 165.67,
cDNA of human glucokinase (MGC: 1742, purchased from Ori-
Gene Technologies, USA) was subcloned into the pET28a(þ)
expression vector, and expressed in Escherichia coli strain
BL21(DE3). The NH2 terminal end of (His)6-tag glucokinase fusion
protein was purified by Ni-NTA metal chelate affinity chromatog-
raphy and stored at ꢀ80 ꢁC in 50 mM Tris/HCl pH 7.4, 1 mM DTT,
50 mM NaCl and 10% glycerol. The GK activity was assessed spec-
trometrically by a coupled reaction with glucose-6-phosphate
dehydrogenase (G6PDH) [55]. Briefly, GK catalyzes glucose phos-
phorylation to generate glucose-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 (SpectraMax 190; Molecular Devices,
USA). The assay was performed in 96-well plates in a final volume
165.39, 153.98, 150.12, 140.03, 136.74, 133.42, 133.19, 130.86, 129.66,
126.37, 122.57, 113.27, 52.82, 52.45, 39.82, 34.40, 33.81, 32.83, 26.39,
26.23, 26.08; LRMS (ESI) m/z 478 [M þ H]þ.
4.1.60. Methyl 6-[(2S)-3-cyclohexyl-2-[(3,4-dichlorophenyl)
formamido]propanamido]pyridine-3-carboxylate (6f)
1H NMR (300 MHz, DMSO-d6)
d
11.35 (s, 1H), 8.90 (d, J ¼ 7.8 Hz,
1H), 8.88 (s, 1H), 8.31 (d, J ¼ 8.7 Hz, 1H), 8.21 (d, J ¼ 8.7 Hz, 1H), 8.05
(s, 1H), 7.75 (s, 2H), 7.48 (t, J ¼ 7.8 Hz, 2H), 7.10 (t, J ¼ 8.7 Hz, 2H),
4.96 (m, 1H), 3.86 (s, 3H), 3.19 (d, J ¼ 10.5 Hz, 1H), 3.05 (d,
J ¼ 11.7 Hz, 1H); LRMS (ESI) m/z 488 [M ꢀ H]ꢀ.
4.1.61. Methyl 6-[(2S)-3-cyclohexyl-2-[(4-methanesulfonylphenyl)
formamido]propanamido]pyridine-3-carboxylate (6g)
of 100
KCl, 2 mM MgCl2, 1 mM dithiothreitol (DTT),1 mM ATP,1 mM NADP,
2.5 U/mL G6PDH, 0.5 g (His)6-glucokinase. For EC50 determination,
different concentrations of compounds were tested in the assay,
and the fold changes in activity vs controls were fitted to sigmoidal
curve using a four-parameter logistic model in GraphPad Prism 4.
mL containing 50 mM HEPES pH 7.4, 5 mM glucose, 25 mM
1H NMR (300 MHz, CDCl3)
d 9.53 (br s, 1H), 8.86 (s, 1H),
8.31e8.23 (m, 2H), 8.04e7.95 (m, 4H), 7.36 (d, J ¼ 7.8 Hz, 1H),
5.05e4.98 (m, 1H), 3.93 (s, 3H), 3.07 (s, 3H), 1.91e1.81 (m, 3H),
1.74e1.66 (m, 4H), 1.26e1.14 (m, 4H), 1.05e0.86 (m, 2H); LRMS (ESI)
m/z 488 [M þ H]þ.
m
4.1.62. 6-[(2S)-3-cyclohexyl-2-[(4-methanesulfonylphenyl)
formamido]propanamido]pyridine-3-carboxylic acid (6h)
4.3. Molecular docking
Compound 6g (100 mg, 0.20 mmol) in THF/H2O (v/v, 1:1) was
added LiOH monohydrate (86 mg, 2.05 mmol) and stirred at room
temperature. After the reaction was completed monitored by TLC,
the reaction mixture was diluted with water, acidified with 1 N HCl,
extracted with EtOAc, washed with water and brine, dried over
anhydrous Na2SO4. The solvent was evaporated under reduced
pressure and the concentrate was purified by column chromatog-
raphy to give the title compound as a white solid (80 mg, 82%). 1H
The crystal structures of RMGP (PDB entry 2IEI) and GK (PDB
entry 1V4S) were used in the molecular docking simulation. The
protein structures were prepared using Protein Preparation module
of Maestro (Schrödinger, Inc.) with standard parameter settings.
The LigPrep module was used to set up and start ligand preparation
calculations. The binding poses of all compounds were modelled
using Glide in standard precision (SP) mode. Final scoring was
carried out on the energy-minimized poses. By default, Schrö-
dinger’s proprietary GlideScore multi-ligand scoring function was
used to score the poses. The grid-enclosing box was centred 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. All calculations and docking simula-
tions were performed by using Maestro on a Dell Cluster server.
NMR (400 MHz, DMSO-d6)
d 11.02 (s, 1H), 8.86 (s, 1H), 8.43 (d,
J ¼ 7.6 Hz, 1H), 8.29 (d, J ¼ 8.8 Hz, 1H), 8.20 (d, J ¼ 8.4 Hz, 1H), 7.69
(d, J ¼ 8.0 Hz,1H), 7.26 (s, 1H), 7.14e7.02 (m, 2H), 4.80e4.75 (m, 1H),
3.95 (s, 3H),1.80e1.77 (m,1H),1.71e1.59 (m, 6H),1.20e1.08 (m, 4H),
0.99e0.87 (m, 2H); 13C NMR (100 MHz, DMSO-d6)
d 172.60, 164.84,
164.15, 157.76, 155.02, 149.58, 139.43, 136.68, 131.81, 121.73, 121.05,
120.66, 112.71, 112.66, 56.66, 52.26, 52.17, 38.90, 33.96, 33.30, 31.72,
26.02, 25.90, 25.71; LRMS (ESI) m/z 472 [M ꢀ H]ꢀ.
Acknowledgements
4.2. Biology
We gratefully acknowledge financial support from National
Basic Research Program of China (Grants 2009CB940903 and
2012CB518000), the National Natural Science Foundation of China
(Grants 20721003 and 81025017), National S&T Major Projects
(2012ZX09103-101-072) and Silver Project (260644).
4.2.1. In vitro glycogen phosphorylase enzyme assay
Glycogen phosphorylase a (from rabbit muscle), glycogen,
glucose-1-phosphate, malachite green, and ammonium molybdate
were purchased from the SigmaeAldrich Corporation. 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 monitored
using microplate reader (Bio-Rad) based on the published methods
[51e54]. In brief, GP 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
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, athttp://dx.doi.org/10.1016/j.ejmech.2012.06.020.
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were added into the 100
containing 50 mM Hepes (pH 7.2), 100 mM KCl, 2.5 mM MgCl2,
mL buffer with compounds dissolved in

638
L. Zhang et al. / European Journal of Medicinal Chemistry 58 (2012) 624e639
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