Development of [18F]Maleimide-Based Glycogen Synthase Kinase-3β Ligands for Positron Emission Tomography Imaging

Article abstract of DOI:10.1021/acsmedchemlett.6b00405

Dysregulation of glycogen synthase kinase-3β (GSK-3β) is implicated in the pathogenesis of neurodegenerative and psychiatric disorders. Thus, development of GSK-3β radiotracers for positron emission tomography (PET) imaging is of paramount importance, because such a noninvasive imaging technique would allow better understanding of the link between the activity of GSK-3β and central nervous system disorders in living organisms, and it would enable early detection of the enzyme’s aberrant activity. Herein, we report the synthesis and biological evaluation of a series of fluorine-substituted maleimide derivatives that are high-affinity GSK-3β inhibitors. Radiosynthesis of a potential GSK-3β tracer [¹⁸F]10a is achieved. Preliminary in vivo PET imaging studies in rodents show moderate brain uptake, although no saturable binding was observed in the brain. Further refinement of the lead scaffold to develop potent [¹⁸F]-labeled GSK-3 radiotracers for PET imaging of the central nervous system is warranted.

Full text of DOI:10.1021/acsmedchemlett.6b00405

Letter
pubs.acs.org/acsmedchemlett
18
Development of [ F]Maleimide-Based Glycogen Synthase Kinase-3β
Ligands for Positron Emission Tomography Imaging
†
‡
§
†
†
Kongzhen Hu, Debasis Patnaik, Thomas Lee Collier, Katarzyna N. Lee, Han Gao,
Matthew R. Swoyer, Benjamin H. Rotstein, Hema S. Krishnan, Steven H. Liang, Jin Wang,†
†
§
§
§
⊥
∥,#
§
‡
,†
Zhiqiang Yan, Jacob M. Hooker, Neil Vasdev, Stephen J. Haggarty, and Ming-Yu Ngai*
†Department of Chemistry, and Institute of Chemical Biology and Drug Discovery, Stony Brook University, Stony Brook, New York
1794-3400, United States
1
‡
Chemical Neurobiology Laboratory, Center for Genomic Medicine, Departments of Neurology & Psychiatry, Massachusetts General
Hospital and Harvard Medical School, Boston Massachusetts 02114, United States
§
Gordon Center for Medical Imaging & Nuclear Medicine and Molecular Imaging, Massachusetts General Hospital & Department of
Radiology, Harvard Medical School, Boston, Massachusetts 02114, United States
∥
Division of Nuclear Medicine and Molecular Imaging, Department of Radiology, Massachusetts General Hospital, Boston,
Massachusetts 02114, United States
#
Athinoula A. Martinos Center for Biomedical Imaging, Massachusetts General Hospital and Harvard Medical School, Charlestown,
Massachusetts 02129, United States
⊥
State Key Laboratory of Electroanalytical Chemistry Changchun Institute of Applied Chemistry, Chinese Academy of Sciences,
Changchun 130022, Jilin, China
*
S Supporting Information
ABSTRACT: Dysregulation of glycogen synthase kinase-3β
GSK-3β) is implicated in the pathogenesis of neuro-
(
degenerative and psychiatric disorders. Thus, development of
GSK-3β radiotracers for positron emission tomography (PET)
imaging is of paramount importance, because such a
noninvasive imaging technique would allow better under-
standing of the link between the activity of GSK-3β and central
nervous system disorders in living organisms, and it would
enable early detection of the enzyme’s aberrant activity.
Herein, we report the synthesis and biological evaluation of a series of fluorine-substituted maleimide derivatives that are
18
high-affinity GSK-3β inhibitors. Radiosynthesis of a potential GSK-3β tracer [ F]10a is achieved. Preliminary in vivo PET
imaging studies in rodents show moderate brain uptake, although no saturable binding was observed in the brain. Further
1
8
refinement of the lead scaffold to develop potent [ F]-labeled GSK-3 radiotracers for PET imaging of the central nervous system
is warranted.
KEYWORDS: Glycogen synthase kinase-3, PET imaging, radiofluorination, maleimide
lycogen synthase kinase-3 (GSK-3) is a serine-threonine
protein kinase family consisting of two highly homolo-
gous isoforms (GSK-3α and GSK-3β). This enzyme is
emerged as an important target for development of new
drugs for the treatment of these diseases.
G
Positron emission tomography (PET) is a noninvasive in vivo
imaging technique using radiotracers to characterize, visualize,
ubiquitously expressed in a variety of tissues and is particularly
20
1
and quantify physiological processes at the molecular level. In
addition, due to the high penetrating power of γ photons (511
keV) generated in the annihilation process, PET is especially
suitable for brain imaging, which renders it a powerful tool to
study GSK-3β dysregulation in neurological disorders. Thus,
development of a selective GSK-3β PET tracer may enable
early detection of GSK-3β aberrant activity and accelerate GSK-
abundant in the central nervous system (CNS). GSK-3 is
associated with a number of cellular functions and physiological
events, such as cell proliferation, stem cell renewal, neuro-
2
−5
pathological events, and apoptosis.
A growing body of
literature has linked dysregulation of GSK-3β activity to
6
numerous severe pathologies, such as diabetes, neurodegener-
7
,8
ative conditions, such as Alzheimer’s disease (AD) and
9
Parkinson disease, psychiatric disorders, such as bipolar
Received: October 13, 2016
Accepted: January 26, 2017
1
0−12
13,14
15−17
disorder and major depression,
and cardiac hypertrophy.
pain,
cancer,
1
8,19
Accordingly, GSK-3β has
©
XXXX American Chemical Society
A
DOI: 10.1021/acsmedchemlett.6b00405
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ACS Medicinal Chemistry Letters
β therapeutic drug discovery. To date, several PET radio-
Letter
3
g) (Scheme 1 and Scheme 2) in a few synthetic steps. We
focused our efforts on the synthesis of maleimide derivatives
tracers for GSK-3 were reported, but only a few of them were
1
1
successfully used in PET imaging (Figure 1). [ C]Methoxy-
a
Scheme 1. General Synthesis of Compounds 10a−k
Figure 1. Structures of published GSK-3 PET radiotracers.
2
1,22
11
AR-A014418 (IC = 104 nM)
4
and [ C]PyrATP-1 (IC =
a
50
50
Reagents and conditions: (a) (i) (COCl) , Et O, 0 °C to rt, 1 h; (ii)
2
3
2
2
.9 nM) were radiolabeled and evaluated in rodents.
NaOMe, MeOH, −78 °C to rt, 2 h; (b) NaH, DMF, R X, 0 °C to rt,
1
Unfortunately, both of them lacked sufficient brain penetration
overnight; (c) (i) MeOH, H SO , reflux, 5 h; (ii) NH (aq), rt,
2
4
3
21,23
and thus were unsuitable for neuroimaging purposes.
In
overnight; (d) K CO , DMF, NaI, 1-bromo-2-fluoroethane, 60 °C, 20
2 3
1
1
2
=
015, Zhang et al. reported two PET radiotracers, [ C]1 (IC
66 nM) and [ C]3 (IC = 42 nM), that could pass the
50
24
h; (e) t-BuOK, THF, 0 °C, 4 h.
50
11
a
blood-brain-barrier (BBB). In the same year, Li showed that
Scheme 2. General Synthesis of Compounds 14a−i
1
1
[
C]SB-216763 (IC = 34 nM) was able to cross the BBB and
50
25
enter the CNS in both rodents and nonhuman primates.
11
Most recently, [ C]PF-04082367 (IC = 2.1 nM) was
5
0
reported to be a selective GSK-3 PET tracer with reasonable
brain permeability and specificity in a nonhuman primate
2
6
brain.
Despite the recent promising attempts to study GSK-3β in
the CNS, further efforts to quantify GSK-3β activity in vivo
using brain-penetrating radiotracers are needed. A major
drawback of the current PET radiotracers is the use of a
short-lived carbon-11 (t1/2 = 20.4 min), which limits their
applications to PET imaging centers equipped with an in-house
cyclotron. In contrast, the longer half-life of fluorine-18 (t1/2
=
1
09.7 min) compared with that of carbon-11 offers the
potential advantage of facilitating widespread use and
distribution of the tracer as well as longer imaging protocols
to be carried out. In addition, F radioisotope emits positrons
1
8
a
with a short positron range, which offers a better spatial
resolution and image quality. Thus, development of [ F]-
Reagents and conditions: (a) NaH, DMF, 1-bromo-2-fluoroethane, 0
C to rt, overnight; (b) (i) SOCl , CH Cl , rt to reflux, 2 h; (ii)
18
°
2
2
2
NH (aq), rt; overnight; (c) t-BuOK, THF, 0 °C, 4 h.
labeled brain-penetrating GSK-3β PET tracers is highly
3
2
7−29
desired.
Herein, we report our efforts in development of
1
8
a [ F]-labeled maleimide-based radiotracer for GSK-3β PET
imaging.
with different aryl/heteroaryl substituents at the C3′ and C4′
positions (Figure 2). We also incorporated a fluoroethyl group
in an array of compounds synthesized with a fluorine atom at a
We selected substituted maleimides as a platform for
development of high-affinity GSK-3β tracers because this
class of compounds were used as scaffolds for ATP competitive
GSK-3 inhibitors and showed an exceptional potency and
3
0−40
selectivity profile for GSK-3.
It was recently demonstrated
1
1
that radiolabeled maleimide [ C]SB-216763 (Figure 1) is able
2
5
to cross the BBB, which provides evidence that maleimides
show sufficient brain uptake to potentially study GSK-3β
activity in the CNS. Additionally, the maleimide scaffold is
highly modular and allows fast assembly of a wide range of
molecules through condensation of methylindole-3-glyoxalates
(6a−k, 11a−c) and (hetero)arylacetamide fragments (9, 13a−
Figure 2. Synthetic modifications of maleimide scaffold.
B
DOI: 10.1021/acsmedchemlett.6b00405
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ACS Medicinal Chemistry Letters
Letter
1
8
position to which [ F]fluoride could be incorporated in the
PET radiotracer.
Table 1. In Vitro Evaluation of GSK-3β Inhibition by
a
Maleimides 10a−k and 14a−i
Target compounds (10a−k) were synthesized according to
literature procedures as depicted in Scheme 1.
3
5,41,42
Commercially available indole derivatives 4a−d were first
treated with oxalyl chloride and then with sodium methoxide to
afford methylindole-3-glyoxalates 5a−d. N-Alkylation of 5a−d
with alkyl halides in the presence of sodium hydride afforded
6
a−k. Preparation of the coupling partner, 2-(3-(2-
fluoroethoxy)phenyl)acetamide 9 began with an acid catalyzed
esterification of 2-(3-hydroxyphenyl)acetic acid 7 followed by
amidation of the resulting ester with aqueous ammonia to give
acetamide 8. O-Alkylation of 8 with 1-bromo-2-fluoroethane in
the presence of potassium carbonate and sodium iodide
afforded 9. With 6a−k and 9 in hand, we performed a
condensation reaction using potassium tert-butoxide in
tetrahydrofuran at 0 °C to provide the desired fluorinated
maleimides 10a−k in 35−69% yield. It is noteworthy that
maintaining reaction temperature at 0 °C and dropwise
addition of the potassium tert-butoxide solution (in THF) are
critical in minimizing formation of an alkene side product (HF
elimination) and obtaining the desired product in good yields.
The synthetic route to inhibitors containing different
aromatic and heteroaromatic moieties at the C4′ position of
the maleimide (14a−i) is illustrated in Scheme 2. N-
Fluoroethylindole derivatives 11a−c were prepared from 5a−
c and 1-bromo-2-fluoroethane in the presence of sodium
hydride in DMF. The amide coupling partners 13a−g were
synthesized from the corresponding carboxylic acids 12a−g in a
two-step procedure: 12a−g were treated with thionyl chloride
to produce acyl chloride intermediates, which were sub-
sequently treated with ammonia to afford acetamides 13a−g.
Condensation of N-fluoroethylindole derivatives 11a−c with
substituted acetamides 13a−g afforded 14a−i in 10−65% yield.
With fluorine-substituted maleimides in hand, we first
investigated the relationship between indole rings with different
substitution patterns and the inhibitory activity of the
maleimides (10a−k), while keeping the fluoroethyl substituent
fixed on the aromatic ring (Figure 2A). As indicated in Table
a
These IC50 values were experimentally determined under our
1
1
identical assay conditions as used for the GSK-3 tracer [ C]PF-
04082367. Kinase reactions were performed with 9 μM GSP-2, 15
μM ATP, and 10 nM GSK-3β in reaction buffer (50 mM Tris pH 7.5,
4
4
5
mM MgCl , 0.01% Brij-35, 3 mM DTT) in the presence of 0−30
2
μM of the maleimides. For comparison purposes, the IC values of
the known GSK-3β inhibitor CHIR-99021 in the same assay are
shown.
5
0
4
5
the 5-position of the indole improves inhibition of GSK-3β is
consistent with the previously reported data.
In the next set of SAR modifications, the fluoroethyl group
was fixed at indole’s nitrogen, while the substituent at the C4′
position of the maleimide scaffold was varied (Table 1b).
Previous studies showed that incorporation of a 3-methox-
yphenyl group to the structure of maleimide analogues resulted
34
1
a, most of the compounds tested showed potent GSK-3β
4
3
inhibitory activity. The maleimide derivative with an ethyl
group on the indole nitrogen and a fluorine atom at the C5-
position of the indole ring (10a) was found to be the most
potent GSK-3β inhibitor among the whole series, with an IC50
value of 1.70 nM, which is of equivalent potency to the known
34
in potent GSK-3β inhibitors (IC < 10 nM). Accordingly, 3-
50
and highly selective GSK-3β inhibitor CHIR-99021 (IC50
=
methoxyphenyl maleimide analogues 14a and 14b were very
potent inhibitors, with IC50 values of 14.7 and 8.80 nM,
respectively. Replacement of the 3-methoxyphenyl substituent
with either a 1-naphthalenyl (14c) or a 1H-indol-3-yl (14d)
moiety was found to be deleterious for the inhibitory activity.
Substitution of 3-methoxyphenyl with 2-thiophenyl (14e)
resulted in an approximately 2-fold decrease in potency. 2,4-
Dichlorophenyl analogues (14f and 14g) significantly dimin-
ished GSK-3β inhibition. Replacement of 3-methoxyphenyl
with a phenyl substituent (14h, IC50 = 6.90 nM) rendered an
inhibitor with the highest potency in the series. An inhibitor
1
.50 nM). We observed that both shortening (10b, IC = 45.6
50
nM) and lengthening of the alkyl chain (10c, IC50 = 63.9 nM;
0d, IC50 = 144 nM, 10e, IC50 > 10,000 nM) diminished the
1
GSK-3β inhibition. Chemical modifications were carried out to
decrease the lipophilicity of the maleimides, which is commonly
accomplished by introduction of an oxygen- or nitrogen-
containing functional group. The aminoethyl (10f, IC50 = 169
nM) and 2-morpholinylethyl (10g, IC50 = 50.0 nM) analogues
were 99- and 29-fold less potent than the ethyl analogue 10a,
respectively. Substitution of ethyl by a 2-methoxyethyl group at
the indole nitrogen (10h, IC50 = 5.60 nM) resulted in an
approximately 3-fold decrease in potency. Comparison of the
substituents at the 5-position of indole showed that 5-halogen
analogues (10b, IC = 45.6 nM; 10j, IC = 27.0 nM; and 10k,
with a 2-chlorophenyl group at the C4′ position (14i, IC
=
50
81.0 nM) was almost 10-fold less potent than the 3-
methoxyphenyl analogue 14b.
Based on the in vitro pharmacological results, compound 10a
50
50
IC50 = 127 nM) were about 1−6-fold higher in potency than
(IC = 1.70 nM) was selected as the potential PET radioligand
for in vivo imaging of GSK-3β. The required precursor 15 was
50
the unsubstituted indole analogue 10i (IC = 164 nM). The
5
0
observation that the presence of a chlorine or fluorine atom at
prepared (Scheme S1, SI), and fully automated radiosynthesis
C
DOI: 10.1021/acsmedchemlett.6b00405
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ACS Medicinal Chemistry Letters
Letter
1
8
of [ F]10a was performed on a GE Tracerlab FX synthesis
radiochemical purity and high specific activity. Thus, using
FN
1
8
module. Treatment of precursor 15 with [ F]K/K complex
conventional chemical transformations to quickly and reliably
2
22
1
8
18
at 90 °C for 10 min afforded the desired [ F]10a in 36%
radiochemical yield (non-decay-corrected) with excellent
radiochemical purity (>99%) and high specific activity (>148
GBq/μmol) at the end of the synthesis (Figure 3a). These
access the [ F]-labeled maleimide-based radiotracers is one of
the advantages of the reported chemistry. In vivo PET-CT
18
imaging studies in rodents showed that [ F]10a enters the
rodent brain. Further refinement of this scaffold to improve the
potency of the lead compounds is underway in our laboratories
18
in an attempt to discover optimized [ F]-labeled GSK-3
inhibitors for PET imaging of the central nervous system.
ASSOCIATED CONTENT
Supporting Information
■
*
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsmedchem-
lett.6b00405.
Full experimental details and copies of NMR spectra for
all compounds synthesized. Procedures for in vitro
evaluation of GSK-3β inhibitors, radiochemical synthe-
ses, quality control, and in vivo PET-CT imaging in
rodents (PDF)
AUTHOR INFORMATION
Corresponding Author
E-mail: ming-yu.ngai@stonybrook.edu. Tel: +1 (631) 632-
641. Fax: +1 (631) 632-7960.
■
*
2
ORCID
Benjamin H. Rotstein: 0000-0001-9707-9357
Steven H. Liang: 0000-0003-1413-6315
Ming-Yu Ngai: 0000-0002-3055-6662
Present Address
Kongzhen Hu’s current address is Department of PET
Center, Nanfang Hospital, Guangzhou, China.
1
8
18
Figure 3. (a) Radiosynthesis of [ F]10a: (i) [ F]KF, K complex,
222
MeCN, 90 °C, 10 min. Ts = toluenesulfonyl, RCY = radiochemical
yield. (b) Time-course of [ F]10a activity of the brain and tongue of
the rat without (red) and with (blue) pretreatment of nonradioactive
tracer 10a. (c) Brain PET-CT scans of rat with [ F]10a. Images were
converted to units of Bq/mL, and a color scale running from 35k-150k
Bq/mL was used. Rat 2 was pretreated with nonradioactive tracer 10a.
18
∇
1
8
Funding
This work was partially supported by National Institute of
General Medical Sciences (R35GM119652 to M.-Y.N.) and
National Institute on Aging of the National Institute of Health
and (R01AG054473 to N.V.), start-up funds from Stony Brook
University (M.-Y.N.), and National Natural Science Foundation
of China (Grant no. 21403208 to Z.Y.). K.N.L. received a
graduate fellowship from an NIH Chemical-Biology training
grant (T32GM092714). S.J.H. received support from the Tau
Consortium. S.H.L is a recipient of NIH career development
award from the National Institute on Drug Abuse (DA038000).
The content is solely the responsibility of the authors and does
not necessarily represent the official views of the National
Institutes of Health.
results demonstrate the feasibility of synthesizing our radio-
tracer using a fully automated module, which thereby facilitated
our preclinical studies with [ F]10 and should be readily
1
8
1
8
translatable to structurally related [ F]-analogs.
Preliminary PET imaging was conducted in Sprague−Dawley
rats at baseline (n = 2) and after pretreatment with
nonradioactive 10a (1 mg/kg, iv, n = 2) to determine brain
uptake and binding saturability (Figure 3). Radiotracer uptake
in whole-brain was reasonable, reached a peak >0.4%ID/cc at
1
−2 min postinjection, and cleared slowly over 60 min.
Interestingly, greater uptake (>0.5%ID/cc) was observed at
baseline in certain loci outside of the brain, including harderian
glands and tissue near the jaws. Pretreatment did not
measurably affect whole-brain radiotracer uptake, but did
diminish uptake in other VOIs to ∼0.4%ID/cc. Although
Notes
The authors declare no competing financial interest.
DEDICATION
■
1
8
This paper is dedicated to Prof. Barry M. Trost on the occasion
of his 75th birthday.
[
F]10a did not display saturable binding in vivo in the rodent
brain, our efforts reveal the first report with biological
evaluation and imaging studies attempting to develop a [ F]-
labeled GSK-3β PET tracer for the central nervous system.
18
46
ABBREVIATIONS
■
In conclusion, we synthesized and evaluated a series of
maleimide-based GSK-3β inhibitors containing a fluoroethyl
group as candidates for in vivo PET imaging. Through structural
modifications of the maleimide scaffold at the C3′ and C4′
positions, we identified a number of highly potent GSK-3β
inhibitors with nanomolar IC values. Radiosynthesis of tracer
GSK-3, glycogen synthase kinase-3; CNS, central nervous
system; AD, Alzheimer’s disease; PET, positron emission
tomography; CT, computed tomography; SAR, structure−
activity relationship; NMR, nuclear magnetic resonance; RCY,
radiochemical yield; ATP, adenosine triphosphate; DTT,
dithiothreitol; K222, 4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo-
[8.8.8]hexacosane; VOI, volume of interest
5
0
1
8
[
F]10a on an automated module was achieved with excellent
D
DOI: 10.1021/acsmedchemlett.6b00405
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ACS Medicinal Chemistry Letters
Letter
(
22) Hicks, J. W.; Wilson, A. A.; Rubie, E. A.; Woodgett, J. R.; Houle,
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F
DOI: 10.1021/acsmedchemlett.6b00405
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