Design, synthesis, and activity evaluation of GK/PPARγ Dual-target-directed ligands as hypoglycemic agents

Article abstract of DOI:10.1002/cmdc.201400009

Based on the multi-target strategy to treat type 2 diabetes mellitus (T2DM), glucokinase/peroxisome proliferator-activated receptor γ (GK/PPARγ) dual-target molecules were constructed by the rational combination of pharmacophores from known GK activators

Full text of DOI:10.1002/cmdc.201400009

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DOI: 10.1002/cmdc.201400009
Design, Synthesis, and Activity Evaluation of GK/PPARg
Dual-Target-Directed Ligands as Hypoglycemic Agents
Jianxun Lu,[a] Lei Lei,[b] Yi Huan,[b] Yongqiang Li,[b] Lijing Zhang,[b] Zhufang Shen,[b]
Wenxiang Hu,[a] and Zhiqiang Feng*[b]
Based on the multi-target strategy to treat type 2 diabetes
mellitus (T2DM), glucokinase/peroxisome proliferator-activated
receptor g (GK/PPARg) dual-target molecules were constructed
by the rational combination of pharmacophores from known
GK activators and PPARg agonists. A series of dual-target
agents were designed and synthesized, and their capacities to
induce GK and PPARg transcriptional activity were evaluated.
Three of these compounds showed particularly high potency
toward GK, moderate activity toward PPARg, and their struc-
ture–activity relationships were preliminarily analyzed. The pu-
tative binding modes of one of the most promising com-
pounds were also explored by molecular docking simulations
with GK and PPARg.
secretion, whereas in the liver, GK is required for glucose me-
tabolism and glycogen synthesis.[4] GK therefore contributes to
whole-body glucose disposal and has been suggested to act
as the glucose sensor, playing an important role in both insulin
secretion and hepatic glucose metabolism.[5] In recent years,
several amide GK activators (Figure 1), including RO-28-1675
Type 2 diabetes mellitus (T2DM) is a progressive heterogene-
ous disease that affected more than 366 million patients
worldwide in 2011. It is characterized by insulin resistance, ex-
cessive hepatic glucose production, and decreased glucose-
triggered insulin secretion from pancreatic b cells. Although
a number of T2DM-pathogenesis-targeting drugs have been
marketed as anti-diabetic therapies, no single current available
drug is capable of achieving sustained blood glucose control.
Modulating multiple targets in the biological network simulta-
neously is recognized as being beneficial for treating a range
of diseases, including cancer and T2DM.[1] At present, with the
development of molecular biology, pathophysiology, and phar-
macology, it has become feasible to develop multi-target
therapies for T2DM to improve efficacy and to minimize side
effects.[2] There is therefore a need for new and effective
broad-spectrum pharmacological agents in this regard.
Figure 1. Representative structures of glucokinase activators.
(Roche),[6] piragliatin (Roche),[7] GKA22 (AstraZeneca),[8]AZD-
1656 (AstraZeneca),[9] LY2121260 (Lilly),[10] and PSN010 (Prosi-
dion/OSI)[11] have been disclosed, and some of these com-
pounds have entered clinical trials.
Glucokinase (GK) catalyzes the phosphorylation of glucose
to glucose-6-phosphate as the first step in glycolysis, playing
a crucial role in maintaining normoglycemia.[3] GK is predomi-
nantly expressed in the liver and pancreas. In pancreatic islets,
GK catalyzes the rate-limiting step in glucose-stimulated insulin
Peroxisome proliferator-activated receptors (PPARs) are
members of the nuclear receptor superfamily and are present
as three isoforms: PPARa, PPARg, and PPARb. PPARg is specifi-
cally expressed in adipose tissue and is a vital regulator of glu-
cose and lipid metabolism. Rosiglitazone and pioglitazone
(Figure 2) are the only approved PPARg agonists for reversing
insulin resistance and are currently in use for treating T2DM as
insulin sensitizers.[12]
[a] Dr. J. Lu, Dr. W. Hu
Capital Normal University
105 North Xisanhuan Road, Beijing, 100048 (China)
[b] L. Lei, Y. Huan, Y. Li, Dr. L. Zhang, Z. Shen, Z. Feng
Beijing Key Laboratory of Active Substance Discovery
and Druggability Evaluation, Institute of Materia Medica
Chinese Academy of Medical Sciences & Peking Union Medical College
100050 (China)
Based on the multi-target strategy to treat T2DM, this study
was aimed at the design of an agonist that can activate both
GK and PPARg simultaneously. A wide range of biological ef-
fects from this dual-target agonist was expected, including
promotion of insulin secretion and hepatic glucose metabo-
lism, and restoration of insulin sensitization. A series of de-
E-mail: fengzq@imm.ac.cn
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201400009.
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moiety as a PPARg agonist. The dual-target ligands were con-
structed by combining 2-(4-((2,4-dioxothiazolidin-5-yl)methyl)-
phenyl)acetyl or 2-(4-((2,4-dioxothiazolidin-5-ylidene)methyl)-
phenyl)acetyl and various 2-amino-nitrogen heteroaromatic
systems. Molecular docking of these compounds to GK and
PPARg was carried out to select the ligand structure with good
matching and high binding energy for both GK and PPARg.
Synthesis of (E)-3-((2,4-dioxothiazolidin-5-ylidene)methyl
benzamide derivatives 4a and 4b was carried out as depicted
in Scheme 1. The intermediate 2, from the condensation of
3-methoxyformyl benzaldehyde 1 and 2,4-thiazolidinedione,
Figure 2. Structures of marketed PPARg agonists rosiglitazone and pioglita-
zone.
signed dual-target ligand structures were synthesized, their ac-
tivities in GK activation and PPARg transcriptional activation
were estimated, and the structure–activity relationship was an-
alyzed.
Although complex diseases such as T2DM can benefit from
complex treatment that modulates multiple targets, designing
dual-target ligands with predefined biological profiles presents
a new challenge. Dual-target ligand design has attracted much
recent attention.[13] For target structure-based design, sequen-
tial docking and multiple cross-docking are straightforward
strategies, which are all computationally demanding. Com-
pared with this, ligand-based dual-target ligand design takes
advantage of the chemical structures of known ligands, and at-
tempts to combine them. The chances of deriving compounds
that are active for one of the targets are high, while it is diffi-
cult to retain another desirable activity at the same time. Our
rational design of GK/PPARg dual-target ligands was mainly
ligand-based, while molecular docking of the designed com-
pounds to both GK and PPARg was also performed to compare
their interaction affinities. We started from the fusion of
2-amino-5-(4-methyl-4H-1,2,4-triazol-3-ylthio)-N-(4-methylthia-
zol-2-yl)benzamide (Figure 3), a GK activator reported by re-
Scheme 1. Reagents and conditions: a) thiazolidinedione, piperidine, AcOH,
toluene, reflux, 4 h; b) LiOH, MeOH, reflux, 5 h, toluene, (COCl)2, reflux, 8 h;
c) EDCI, DIPEA, DMF, 8 h.
was hydrolyzed by lithium hydroxide and treated with oxalyl
dichloride to afford compound 3. Compound 3 was then treat-
ed with pyridin-2-amine and isopropyl-6-aminonicotinate to
give target compounds 4a and 4b, respectively. (E)-4-((2,4-di-
oxothiazolidin-5-ylidene)methyl)benzamide derivatives 8a–
e were prepared from 4-formylbenzoic acid 5, avoiding the
basic hydrolysis step, as depicted in Scheme 2.
(E)-2-(3-((2,4-dioxothiazolidin-5-ylidene)methyl)phenoxy)acet-
amide derivatives 13a,b were synthesized as depicted in
Scheme 3. The condensation of 3-hydroxybenzaldehyde (9)
Figure 3. Design of GK/PPARg dual-target ligands from a GK activator and
a PPARg agonist.
searchers at Merck-Banyu,[14] and 5-((2,4-dioxothiazolidin-5-yl)-
methyl)-2-methoxy-N-(4-(trifluoromethyl)benzyl)benzamide or
MK-767 (Figure 3), a PPARg agonist also developed at Merck-
Banyu.[15,16] Although these two compounds are different, they
are similar in overall shape. They share the same benzamide
scaffold and have definite pharmacophore structure: an alkaryl
amidine for the GK activator, and a 2,4-thiazolidinedione
Scheme 2. Reagents and conditions: a) thiazolidinedione, piperidine, AcOH,
toluene, reflux, 4 h; b) LiOH, MeOH, reflux, 5 h, toluene, (COCl)2, reflux, 8 h;
c) DIEPA, DMF, 8 h.
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the target compounds 26a–l;
additional reduction with Pd/C
and hydrogen in methanol gave
target compounds 27a–c.
All target compounds pre-
pared herein were evaluated for
GK activity at 10 mm, with a glu-
cose concentration of 4 mm. The
glucose concentration simulates
moderate blood glucose condi-
tions. The GK activity of com-
pounds is expressed as GK fold
activation; compound GKA22,
a GK activator developed by As-
traZeneca, was used as positive
Scheme 3. Reagents and conditions: a) thiazolidinedione, piperidine, AcOH, toluene, reflux, 4 h; b) ethyl chloroace-
tate, acetone, K2CO3, reflux, 8 h; c) LiOH, THF, reflux, 8 h; d) EDCI, DIPEA, DMF, 8 h.
and 2,4-thiazolidinedione in toluene afforded intermediate 10,
which was then treated with ethyl 2-bromoacetic acid to pro-
vide compound 11, followed by hydrolysis to produce com-
pound 12. Treatment with isopropyl 6-aminonicotinate or
ethyl 6-aminonicotinate in the presence of EDCI and DIPEA af-
forded the desired target compounds 13a,b as depicted in
Scheme 3. (E)-2-(4-((2,4-dioxothiazolidin-5-ylidene)methyl)phe-
noxy)acetamide derivatives 18a,b were prepared from 4-hy-
droxybenzaldehyde 14, as depicted in Scheme 4.
Synthesis of (E)-2-(4-((2,4-dioxothiazolidin-5-ylidene)methyl)-
2-nitrophenoxy)acetamide derivatives 23a,b was carried out as
shown in Scheme 5. The condensation of 4-chloro-3-nitrobenz-
aldehyde (19) and 2,4-thiazolidinedione in toluene afforded
the intermediate 20, which was
control in the activity assays. Potent compounds in GK activa-
tion were selected for further testing in a PPARg transcription
cell-based assay. The effect of tested compounds on the activi-
ty of PPARg transcription was evaluated by luciferase reporter
gene assay, and results are reported relative to the activity of
rosiglitazone.
To evaluate the effect of the relative positions of the GK and
PPARg pharmacophores on the benzene core in GK activation,
a series of meta- and para-oriented compounds (4a,b and 8a–
e) were first assayed (Table 1). Compared with meta-oriented
compounds 4a,b, the corresponding para-oriented com-
pounds 8a–b displayed improved potency for both GK and
PPARg, and 2-aminopyridine derivatives 8a, 8b, and 8d exhib-
then treated with ethyl 2-bromo-
acetic acid to provide compound
21. Compound 21 was hydro-
lyzed by lithium hydroxide to
afford intermediate 22. Then an
intermolecular condensation be-
tween compound 22 and isopro-
pyl 6-aminonicotinate or ethyl
6-aminonicotinate yielded target
compounds 23a,b.
The preparation of (E)-2-(2-
nitro-4-((2,4-dioxothiazolidin-5-
ylidene)methyl)pheny)acetamide
derivatives 27a–l and (E)-2-(2-
amino-4-((2,4-dioxothiazolidin-5-
ylidene)methyl)phenyl)acetamide
derivatives 28a–c was carried
out as depicted in Scheme 6.
The treatment of (E)-5-(4-chloro-
3-nitrobenzylidene)thiazolidine-
2,4-dione (20) with diethyl malo-
nate and sodium hydride in
DMSO afforded intermediate 24,
followed by acidic hydrolysis
and decarboxylation to produce
intermediate 25. This was then
reacted with various 2-amino-
Scheme 4. Reagents and conditions: a) thiazolidinedione, piperidine, AcOH, toluene, reflux, 4 h; b) ethyl chloroace-
tate, acetone, K2CO3, reflux, 8 h; c) LiOH, THF, reflux, 8 h; d) EDCI, DIEPA, DMF, 8 h.
Scheme 5. Reagents and conditions: a) thiazolidinedione, piperidine, AcOH, toluene, reflux, 4 h; b) ethyl hydroxy-
nitrogen heteroaromatics to give acetate, acetone, K2CO3, reflux, 8 h; c) LiOH, THF, reflux, 8 h; d) EDCI, DIEPA, DMF, 8 h.
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Table 1. Activities of synthetic target compounds toward GK and PPARg.
Compd
R
GK [fold][a]
1.9
PPARg [%][b]
Compd
R
GK [fold][a]
2.4
PPARg [%][b]
4a
53
26c
91.3
4b
8a
8b
8c
8d
2.0
2.4
2.2
1.6
1.8
40
56
26d
26e
26 f
26g
26h
2.9
2.9
2.5
2.5
2.6
47.1
25.6
46.7
62
47
ND
ND
38.9
8e
1.6
2.5
2.0
2.5
1.9
2.4
1.7
2.9
2.7
ND
43
26i
1.7
2.5
2.3
2.3
2.8
3.3
2.6
2.9
–
ND
55.9
33.9
34.9
22.8
17.1
20.2
–
13a
13b
18a
18b
23a
23b
26a
26b
26j
51
26k
37
26l
63
27a
45
27b
ND
48.0
47.4
27c
GKA22
rosiglitazone
100
[a] GK fold activation values were obtained at a Glc concentration of 4 mm. [b] The effect of test compounds (at 10 mm) on PPARg transcription was evalu-
ated by luciferase reporter gene assay, and values reflect the percent activity relative to that of rosiglitazone; ND: not determined. Each test was performed
in triplicate, and the data in this table are the mean of triplicate measurements.
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Scheme 6. Reagents and conditions: a) CH2(COOEt)2, NaH, 1008C, 1 h; b) 4n
HCl, reflux, 12 h; c) EDCI, DIPEA, DMF, 12 h; d) Pd/C, H2, MeOH, 50 psi.
ited better potency in GK activation than 2-amino-nitrogen
five-member heteroaromatic ring compounds 8c and 8e.
Additionally, compounds 13a and 18a, each two bond
lengths longer than the respective structures of compounds
4b and 8b, still showed slightly more potency than 4b and
8b for GK. Ethyl esters 13b and 18b displayed lower potency
than the corresponding isopropyl esters 13a and 18a for GK,
but higher potency than the corresponding isopropyl esters
13a and 18a for PPARg, suggesting that ester size may impact
the effect balance between GK and PPARg. Furthermore, com-
pounds 23a and 23b, with a nitro substituent on the benzene
core of 18a and 18b, displayed slightly lower potency than
18a and 18b for GK, but slightly higher potency than 18a and
18b for PPARg.
As expected, compounds 26a and 26b, shorter by one
bond length relative to the base chain structure of compounds
23a and 23b, displayed conspicuously improved potency over
corresponding compounds 23a and 23b in GK activation, and
slightly improved potency for PPARg. In addition, other
2-amino-nitrogen heteroaromatic amides exhibited improved
potency for GK, such as compounds 26d and 26e, showing
significant enhancement in potency with fold activation of 2.9,
which is similar to that of GKA22, which displayed fold activa-
tion of 2.9 in our assay as well. However, compounds 27a and
27b, from reduced products of 26a and 26j at the nitro
group and double bond, showed a noticeable decrease in po-
tency for PPARg.
Figure 4. a) Putative binding mode of compound 26b in GK (PDB ID: 3H1V).
b) Putative binding mode of compound 26b in PPARg (PDB ID: 1FM6).
Arg63 on GK. The clear difference between the binding mode
of compound 26b in GK and that of the Merck-Banyu com-
pound is that the aniline NH of Merck-Banyu additionally inter-
acts with the hydroxy group of Tyr215 by hydrogen bonding,
but the 2,4-thiazolidinedione moiety of compound 26b can
make two hydrogen bonding interactions with Gln98 of GK
(Figure 4a), which may explain the potent GK activation ob-
served. Figure 4b suggests the possible interactions between
compound 26b and PPARg. The 2,4-thiazolidinedione moiety
in compound 26b occupies the only substantially polar cavity
of the PPARg ligand binding domain, similar to the binding
mode of rosiglitazone, making a network of hydrogen bonds
with the side chains of Tyr473 and His323. The main difference
between the binding mode of 26b and that of rosiglitazone
may be the loss of two hydrogen bonds with His449 and
Ser289 (Figure 4b). The absence of this strong interaction may
be behind the significant decrease in the activity of PPARg
transcription.
For further understanding of the binding mode of the ob-
tained dual-target active compound 26b with these two tar-
gets, images of compound 26b with GK and PPARg were cre-
ated by molecular docking simulations. Figure 4a suggests
possible interactions between compound 26b and GK. The
compound might bind at the allosteric site of GK similar to the
binding mode observed in the co-crystal structure of Merck-
Banyu compound (Figure 3) in complex with GK.[14] There is
also a bidentate hydrogen bond formed between the pyridine
nitrogen atom and the NH group of the amide in compound
26b with both the backbone NH and carbonyl oxygen atom of
In summary, we have described the ongoing development
of a series of dual-target ligand compounds which can activate
GK and PPARg simultaneously. Biochemical evaluation and SAR
of these compounds were also conducted preliminarily.
Among these compounds, 26a, 26b, and 26d showed high
potency for GK, and moderate activity toward PPARg. The pos-
sible binding modes of compound 26b with GK and PPARg
were also explored preliminarily by molecular docking simula-
tions. Based on these results, it can be expected that some
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Keywords: activators · agonists · dual targeting · glucokinase ·
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Received: January 2, 2014
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Two birds, one stone: GK/PPARg dual-
target ligands were constructed by ra-
tional combination of pharmacophores
from known GK activators and PPARg
agonists. Their ability to induce GK and
PPARg transcriptional activity were eval-
uated, and the putative binding modes
of one of the most promising com-
pounds were also explored by molecu-
lar docking simulations with GK and
PPARg.
J. Lu, L. Lei, Y. Huan, Y. Li, L. Zhang,
Z. Shen, W. Hu, Z. Feng*
&& – &&
Design, Synthesis, and Activity
Evaluation of GK/PPARg Dual-Target-
Directed Ligands as Hypoglycemic
Agents
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