Identification of lead small molecule inhibitors of glycogen synthase kinase-3 beta using a fragment-linking strategy

Article abstract of DOI:10.1016/j.bmcl.2016.10.060

Glycogen synthase kinase-3 beta (GSK3β) kinase serves as a promising therapeutic target for the treatment of various human diseases, such as diabetes, obesity, and Alzheimer's disease. In this study, we report lead GSK3β inhibitors identified using a fragment-linking strategy. Through the systematic exploration, a six-atom chain unit bearing the rigid double bond was found to be a suitable linker connecting two fragments, which enables favorable contacts with backbone groups of residues in the pockets. As a consequence, potent GSK3β inhibitor 9i was found with IC₅₀values of 19 nM. The binding mode analysis indicates that the activities of the inhibitors appear to be achieved by the establishment of multiple hydrogen bonds and hydrophobic interactions in the ATP-binding site of GSK3β. The good biochemical potencies and structural uniqueness of the inhibitors support consideration in the further study to optimize the biological activity.

Full text of DOI:10.1016/j.bmcl.2016.10.060

Accepted Manuscript
Identification of Lead Small Molecule Inhibitors of Glycogen Synthase Kin-
ase-3 beta Using a Fragment-Linking Strategy
Jinhee Kim, Yonghoon Moon, Sungwoo Hong
PII:
S0960-894X(16)31098-8
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.10.060
BMCL 24363
Reference:
Bioorganic & Medicinal Chemistry Letters
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Revised Date:
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29 September 2016
19 October 2016
21 October 2016
Please cite this article as: Kim, J., Moon, Y., Hong, S., Identification of Lead Small Molecule Inhibitors of Glycogen
Synthase Kinase-3 beta Using a Fragment-Linking Strategy, Bioorganic & Medicinal Chemistry Letters (2016),
doi: http://dx.doi.org/10.1016/j.bmcl.2016.10.060
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Bioorganic & Medicinal Chemistry Letters
Identification of Lead Small Molecule Inhibitors of Glycogen Synthase Kinase-3
beta Using a Fragment-Linking Strategy
Jinhee Kim,a Yonghoon Moon,b,a and Sungwoo Honga,b
aCenter for Catalytic Hydrocarbon Functionalization, Institute for Basic Science (IBS), Daejeon 305-701, Korea
bDepartment of Chemistry, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 305-701, Korea
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
Glycogen synthase kinase-3 beta (GSK3) kinase serves as a promising therapeutic target for
the treatment of various human diseases, such as diabetes, obesity, and Alzheimer’s disease. In
this study, we report lead GSK3 inhibitors identified using a fragment-linking strategy.
Through the systematic exploration, a six-atom chain unit bearing the rigid double bond was
found to be a suitable linker connecting two fragments, which enables favorable contacts with
backbone groups of residues in the pockets. As a consequence, potent GSK3 inhibitor 9i was
found with IC50 values of 19 nM. The binding mode analysis indicates that the activities of the
inhibitors appear to be achieved by the establishment of multiple hydrogen bonds and
hydrophobic interactions in the ATP-binding site of GSK3. The good biochemical potencies
and structural uniqueness of the inhibitors support consideration in the further study to optimize
the biological activity.
Keywords:
glycogen Synthase Kinase-3 beta
inhibitor
fragment-linking strategy
7-azaindole
kinase
2015 Elsevier Ltd. All rights reserved.
Glycogen synthase kinase-3 (GSK3) protein is
a
Fragment-based drug discovery (FBDD) approach presents an
appealing alternative to the current drug discovery process by
either growing or linking two fragments.8 Fragment-linking
approach is conceptually most fascinating in fragment-based
drug discovery approaches and can lead to a rapid buildup of
fragments using an appropriate linker moiety to generate new
high-affinity ligands.9 Once two or more fragments are identified
in high affinity regions to determine favorable spots, they are
chemically linked in an optimal fashion to benefit from a
superadditivity effect.10 In this strategy, even non-selective initial
serine/threonine kinase, which was originally identified as a
regulator of glycogen metabolism.1 This kinase subfamily
includes two homologous isoforms GSK3α and GSK3β which
are 95% identical at the amino acid level.2 GSK3 kinase plays an
important role in a variety of physiological processes, including
cell cycle progression, microtubule stability, apoptosis, signaling,
and transcription.3 Among two different isoforms, GSK3β
potentiates to modulate blood glucose level by impairing the
catalytic activity of glycogen synthase through phosphorylation.4
Besides its role in the conversion of glucose to glycogen, GSK3β
is a key kinase to obtain abnormal hyperphosphorylation on tau ,
which causes destabilization of microtubules, subsequent
dissociation, and aggregation of tau to form neurofibrillary
tangles (NFTs).5 Taken together, regulation of GSK3β activity
would be a promising therapeutic strategy for the treatment of
various human diseases, such as diabetes, obesity, and
Alzheimer’s disease.
A variety of GSK3β inhibitors, including lithium, natural
products, and synthetic molecules, have been developed in recent
decades through intensive drug discovery programs.6 Despite the
achievements in both high potency and chemical structure
diversity, GSK3targeted therapeutic approaches by small
molecule inhibitors have often been unsuccessful because of the
selectivity issues against other related kinases.6h,6j,7 Off-target
kinase inhibition can cause toxicity due to cross-interaction of
multiple pathways.
 Corresponding authors. Tel: +82 42 350 2811 Fax: +82 42 350 2812
E-mail addresses: hongorg@kaist.ac.kr (S. Hong).
Figure 1. Structure and schematic binding mode of 1.
———

Scheme 1. Preparation of azaindole-based derivatives.
Reagents and conditions: (a) HOBt, DCC, DCM, overnight, 75%; H2, Pd/C, MeOH, rt, overnight. (b) 4, HOBt, DCC, DCM or DMF, rt, overnight, up to
66%.
o
Reagents and conditions: (d) methyl acrylate, Pd(PPh3)2Cl2, Et3N, DMF, 140 C, N2, 1 h; 10% HCl(aq), dioxane/H2O = 3:1, for 8-10 h. (e) methyl acrylate,
Pd(PPh3)2Cl2, Et3N, DMF, 140 oC, N2, 1 h; 4 N KOH(aq), dioxane, rt, for 10 h. (f) RNH2, DIPEA, HBTU, DMF, rt; KOH, dioxane/H2O = 3:1, rt, for 8-10 h.
(g) RNH2, DIPEA, HBTU, DMF, rt, for 8-10 h.
core could be developed into a highly selective inhibitor by
linking fragments in two neighboring sites. Fragment-linking
strategy is, however, considerably more challenging than
growing strategy because the linker should not disrupt any
important functionality of the fragments and even subtle change
of the conformation can cause the loss of key interactions
between the initial fragment and biological target. Therefore, the
optimally linked molecules should completely retain the key
interactions of the individual fragments in an additive manner
and maintain the optimum binding geometries of the original
fragment hits. In this study, we have designed and identified
promising lead GSK3 inhibitors using a fragment-linking
strategy in which two fragments were connected with an
optimum linker-length.
azaindole and indole fragments in compound 1 likely to be
fixed in folded conformation, probably caused by
a
intramolecular pi-stacking interaction. This spatial restriction
may prevent the indole moiety of 1 from accessing Region 3 by
distorting the linker. With the binding mode set in this way, we
speculated that more rigid linkers can possibly increase the
interactions with the residue by forcing the indole moiety to
extend into Region 3 and make favorable contacts with residues.
The availability of the substantial 3D-structural information of
GSK3 has enhanced opportunities for the identification of
potent and selective inhibitors utilizing structure-based drug
design.6 Through our concurrent program supporting the
development of GSK3 inhibitors, we recently identified hit
compound 1 (IC50 = 1.45 μM) by a systematic fragment-based de
novo design (FBDD) procedure. Compound 1 is comprised of
three parts in order to maximize the interactions of an inhibitor
with three binding regions in the active site of GSK3β. Figure 1
presents the schematic binding mode of 1 in the ATP binding site
of GSK3β. The 7-azaindole and indole fragments were linked
together by an amide linkage to investigate the main interactions
in Region 2 and 3. The aprotic nitrogen on the central 7-
azaindole moiety and the neighboring pyrrolic nitrogen accept
and donate a hydrogen bond in the bidentate form with the back
bone of Asp133 and Val135 in the hinge region. According to
proposed binding mode, the phenyl group in 7-azaindole is
directed toward the polar binding pocket near the salt bridge
comprised of Lys85 and Glu97 and Asp200 in the DFG motif,
which has been known as a pocket for potency and selectivity.6
Based on these premises, we turned to our attention to linker
design and modifications to increase inhibitory activity. As the
starting point for generating derivatives, the lowest-energy
conformation of 1 was analyzed to estimate the energy penalty
resulting from the different geometry of the bioactive
conformation as show in Figure 2. The systematic evaluation of
the conformational features indicates that the geometry of 7-
Figure 2. Calculated conformation of 1.
With a goal to develop a new structural class of potent GSK3
inhibitors, our initial round of analogues was focused around the
incorporation of suitable linkers while fixing 7-azaindole and
indole fragments (Table 1). The general synthetic route for the
preparation of the target compounds is shown in Scheme 1. The
synthesis commenced with DCC/HOBt mediated coupling of
Cbz protected glycine (2) and indole-3-ethylamine (3), followed
by Cbz deprotection under catalytic hydrogenation conditions to
afford the amine 4. The resulting amine 4 was coupled with
azaindoles 511 to furnish the target compounds 1, 6a, 6b, 6c, and
6d. To build compounds bearing acrylamide as a linking group,
target compounds 9 were prepared by a two step sequence,
shown in Scheme 1b. Thus, the methyl acrylate group was
installed by a palladium-catalyzed cross-coupling with 5-bromo-
7-azaindole derivatives 712 to furnish the ester compounds, which
were hydrolyzed under either acidic or basic conditions. Finally,
amides 9 were produced from the resulting carboxylic acids 8
and appropriate amines in the presence an amide coupling
reagent (HBTU). For the synthesized derivatives, IC50 (50%
inhibitory concentration) values were tested over GSK3β in
enzymatic activity assay (Reaction Biology Corp.)
Table 1 shows structure-activity relationship (SAR) of
derivatives. The phenyl group at the R1 was found to be essential

control to investigate the role of the 7-azaindole moiety, which
resulted in a complete loss of activity (IC50 >50 M). The result
reveals the indispensable nature of the central 7-azaindole
scaffold in maintaining activity.
Table 1. Exploration of the groups and linker.
Building upon the success observed with the trans olefin
linker, we next focused our design attempts to evaluate the effect
of fragment at the R position to expand the structure-activity
relationship (Table 2). With this agenda, fragments were varied
to include morpholine, imidazole, phenyl and pyridyl groups,
which were connected to the linker through the formation of
amide bond. In general, the incorporation of small-sized
heterocyclic ring systems, such as morpholine and imidazole led
to about 7-fold increase in the inhibitory activity. In contrast,
derivative 9d bearing the phenyl ring featured about 4-fold
reduction in potency (IC50 = 248 nM) compared with morpholine,
or imidazole analogues (9b and 9c). Attempts to further increase
potency by installing substituents into the ethylene linker, as
exemplified by compounds 9e and 9f, did not provide any
advantages compared to 9d. We observed that the 4-pyridyl
group at this position was effective in enhancing inhibitory
activity (IC50 = 44 nM), which prompted further investigation
about other pyridyl groups. The potency was slightly increased
when the 4-pyridyl group was replaced with 3-pyridyl group (9h,
IC50 = 32 nM). Notably, analogue incorporating 2-pyridyl group
(9i) is a highly potent GSK3 inhibitor with an IC50 of 19 nM.
GSK3βIC50
(μM)
entry
R1
R2
1
-Ph
1.45
>50
>50
15.7
>50
-H
-Ph
6a
6ba
6c
-COPh
-Ph
6db
Table 2. Structure-activity relationship against GSK3
6e
-Ph
2.61
9a
10
-Ph
-Ph
0.356
1.03
entry
R
GSK3βIC50 (nM)
9a
356
a 2-phenyl group instead of 3-phenyl group. b indole instead of central 7-
azaindole core.
for enhanced potency, and replacement of phenyl group with
hydrogen (6a) or benzoyl group (6c) resulted in a significant loss
of the inhibitory activity over GSK3(IC50 >50 M). The results
imply that the aryl group at the C3 position is allocated to
generate stabilizing hydrophobic interactions in Region 1 and
important for enhanced potency. We also examined the
significance of the position of the phenyl group and observed that
the activity was dramatically reduced when the phenyl group of
compound 1 was moved to the C2 position (6b). Next, the effect
of linkers for bridging two fragments were explored using –
(CH2)6–, and –(CH2)7– linker length on the grounds that the
distances between Region 2 and Region 3 in the ATP-binding
site of GSK3. Because the phenyl group at the R1 position
proved to be effective for high activity, we tentatively fixed the
phenyl group in this region. Of particular significance is the
observation that a profound potency on GSK3 was achieved by
incorporating a rigid linker containing trans olefin (9a), which is
consistent with our assumption in the previous conformation
study (Figure 1). We further attempted to investigate the
structural and functional relevance of linker flexibility. Thus,
importance of the linker rigidity was demonstrated by reducing
the trans oflefin in the linker unit in derivative 10, which led to a
significant loss of inhibitory activity. Because the six-atom chain
unit bearing the rigid double bond appeared to be the superior
linker, it was selected for further optimization to embark upon in-
depth structural modification. Separately, 6d was prepared as the
46
49
9b
9c
9d
248
9e
9f
180
752
9g
9h
9i
44
32
19
To obtain structural insight into the inhibitory mechanisms of
the identified GSK3 inhibitor, its binding modes and the
detailed interactions responsible for stabilizing inhibitor 9i were
investigated in a comparative fashion, as calculated using the

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derived from docking simulations, the inhibitory activity of 9i is
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hydrogen-bonding pattern with backbones of Asp133 and Val
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bonding with the sidechain of Arg141 at the interface of the
solvent front.
In conclusion, the initial hit 1 was modified by applying a
fragment-linking strategy to obtain potent inhibitors for GSK3β.
Through the systematic exploration, the six-atom chain unit
bearing the rigid double bond was found to be an optimum linker
connecting two fragments, which enables favorable contacts with
residues in the pockets. As a consequence of these modifications,
potent GSK3 inhibitor 9i with an IC50 of 19 nM was identified.
The good biochemical potencies and structural uniqueness of the
GSK3β inhibitors support consideration in the further study to
optimize the biological activity.
7.
Acknowledgments
Vougogiannopoulou,
K.;
Ferandin,
Y.;
Bettayeb,
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This research was supported by Institute for Basic Science
(IBS-R010-G1).
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ethynylbenzene (0.24 mL, 2.2 mmol), Pd(dppf)Cl2 (81 mg, 0.1
mmol), CuI (38 mg, 0.2 mmol), tryethylamine (1mL), toluene (2
mL), THF (1 mL), N2, 80 oC, 2 h, 91%. (ii) methyl 6-amino-5-
(phenylethynyl)nicotinate (230 mg, 0.91 mmol), KOtBu (205 mg,
1.82 mmol), NMP, 80 oC, 1 h.; 4 N KOH (aq), methanol, reflux,
overnight. (b) preparation of 3-benzoyl-1H-pyrrolo[2,3-b]pyridine-5-
carboxylic acid 5c: (i) 5-bromo-7-azaindol (100 mg, 0.507 mmol),
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benzoyl chloride (0.3 mL, 2.54 mmol), room temp. 3 h. 24%. (ii) 5-
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0.156 mmol), Zn(CN)2, Pd(PPh3)4, DMF, 150 oC, microwave, 30
min, 59%. (iii) 3-benzoyl-1H-pyrrolo[2,3-b]pyridine-5-carbonitrile
(20 mg, 0.081 mmol), conc. HCl (0.06 mL), dioxane, reflux, 31%.
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3531.

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Jinhee Kim, Yonghoon Moon, and Sungwoo Hong