Synthesis of Diverse 11C-Labeled PET Radiotracers via Direct Incorporation of [11C]CO2

Article abstract of DOI:10.1021/acs.bioconjchem.6b00163

Three new positron emission tomography (PET) radiotracers of interest to our functional neuroimaging and translational oncology programs have been prepared through new developments in [¹¹C]CO₂ fixation chemistry. [¹¹C]QZ (glutaminyl cyclase) was prepared via a tandem trapping of [¹¹C]CO₂/intramolecular cyclization; [¹¹C]tideglusib (glycogen synthase kinase-3) was synthesized through a tandem trapping of [¹¹C]CO₂ followed by an intermolecular cycloaddition between a [¹¹C]isocyanate and an isothiocyanate to form the 1,2,4-thiadiazolidine-3,5-dione core; [¹¹C]ibrutinib (Brutons tyrosine kinase) was synthesized through a HATU peptide coupling of an amino precursor with [¹¹C]acrylic acid (generated from [¹¹C]CO₂ fixation with vinylmagnesium bromide). All radiochemical syntheses are fully automated on commercial radiochemical synthesis modules and provide radiotracers in 1-5% radiochemical yield (noncorrected, based upon [¹¹C]CO₂). All three radiotracers have advanced to rodent imaging studies and preliminary PET imaging results are also reported.

Full text of DOI:10.1021/acs.bioconjchem.6b00163

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Article
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Synthesis of Diverse C-Labelled PET
11
2
Radiotracers via Direct Incorporation of [C]CO
Andrew V. Mossine, Allen F Brooks, Isaac M Jackson, Carole A. Quesada,
Phillip S. Sherman, Erin L Cole, David Donnelly, Peter J. H. Scott, and Xia Shao
Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.6b00163 • Publication Date (Web): 04 Apr 2016
Downloaded from http://pubs.acs.org on April 7, 2016
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Synthesis of Diverse 11C-Labelled PET Radiotracers via Direct
Incorporation of [11C]CO2
Andrew V. Mossine,† Allen F. Brooks,† Isaac M. Jackson,† Carole A. Quesada,†
Phillip Sherman,† Erin L. Cole,# David J. Donnelly,# Peter J. H. Scott,†,‡
Xia Shao*,†
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† Division of Nuclear Medicine, Department of Radiology, University of Michigan Medical
School, Ann Arbor, MI, USA
‡ The Interdepartmental Program in Medicinal Chemistry, The University of Michigan,
Ann Arbor, MI, USA
# Discovery Chemistry Platforms, PET Radiochemical Synthesis, Bristol-Myers Squibb
Research and Development, Princeton, NJ, USA
* E-mail: xshao@umich.edu
ABSTRACT
Three new positron emission tomography (PET) radiotracers of interest to our
functional neuroimaging and translational oncology programs have been
prepared through new developments in [11C]CO2 fixation chemistry. [11C]QZ
(glutaminyl cyclase) was prepared via a tandem trapping of [11C]CO2 /
intramolecular cyclization; [11C]tideglusib (glycogen synthase kinase-3) was
synthesized through a tandem trapping of [11C]CO2 followed by an intermolecular
cycloaddition between a [11C]isocyanate and an isothiocyanate to form the 1,2,4-
thiadiazolidine-3,5-dione core; [11C]ibrutinib (Bruton’s tyrosine kinase) was
synthesized through a HATU peptide coupling of an amino precursor with
[11C]acrylic acid (generated from [11C]CO2 fixation with vinylmagnesium bromide).
All radiochemical syntheses are fully-automated on commercial radiochemical
synthesis modules and provide radiotracers in 1 – 5% radiochemical yield (non-
corrected, based upon [11C]CO2). All three radiotracers have advanced to rodent
imaging studies and preliminary PET imaging results are also reported.
INTRODUCTION
Positron emission tomography (PET) imaging is a type of functional molecular
imaging using probes, known as radiotracers, composed of a bioactive molecule
conjugated with a positron-emitting radionuclide.1 The functional information
garnered from a PET scan, when combined with the anatomical information from
the co-registered CT or MRI scan, provide unprecedented insight into
biochemical pathways, mechanisms of disease pathology, and in vivo behavior of
drug molecules. Reflecting this, PET imaging is having far reaching impact on
personalized medicine2 and drug discovery.3
Commonly used PET radionuclides include carbon-11 (t1/2 = 20 min),
fluorine-18 (t1/2 = 110 min), and gallium-68 (t1/2 = 68 min). The choice of
radionuclide depends on a number of factors ranging from synthetic
considerations about how it will be incorporated into the bioactive molecule of
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choice, to practical aspects associated with intended application (e.g. the short
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half-life of C allows patients to receive multiple PET scans in a single hospital
visit, while the half-life of 18F facilitates radiotracer distribution from centralized
manufacturing facilities).
Carbon-11 is an attractive choice of PET radionuclide because multiple
scans can be conducted in series during a single patient visit (e.g. scans with 2
different radiotracers, baseline and intervention studies with 1 tracer). Moreover,
it can be frequently incorporated into bioactive or endogenous molecules without
any structural modification to the original (non-radioactive) molecule, which may
or may not be the case with other radionuclides (e.g. use of radioactive metal
ions such as 68Ga require decoration of the bioactive molecule with a suitable
metal-chelating group prior to radiolabeling). Carbon-11 is produced by a
cyclotron, reacting with oxygen added to the cyclotron target gas to generate
[11C]CO2, which is delivered to the radiochemistry laboratory and used to
synthesize radiotracers. The short half-life of carbon-11 is advantageous for the
reasons outlined above, but it presents challenges. Most notably, the short half-
life necessitates that all reactions used to synthesize 11C-radiotracers are
reasonably high yielding over a very short time course (e.g. 2-10 min) so that
they provide usable amounts of radiotracer, thereby limiting the number of
reactions that are practical. Typically, [11C]CO2 is converted into a secondary
synthon such as [11C]CH3I, [11C]CH3OTf or [11C]KCN, which is then reacted with
a suitable precursor to yield the 11C-labeled compound. Such radiochemical
reactions have been used to great effect to synthesize 11C-radiotracers (for
recent reviews of carbon-11 radiochemistry, see: 4 ,5 , 6 ). However, there are
limitations in the types of radiotracers that can be accessed from such synthons.
For example, there must be a place to introduce a methyl group if [11C]CH3I or
[11C]CH3OTf are to be used for labeling. Given the prevalence of carbonyl groups
in bioactive molecules (e.g. many of the best-selling drugs contain one or more
C=O bonds7), there is significant interest in developing methods that enable
incorporation of a 11C-carbonyl unit into bioactive molecules to increase the
number and diversity of available PET radiotracers. One such approach involves
synthesis of PET radiotracers directly from [11C]CO2.
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The electrophilic carbon in [11C]CO2 means it can be used as a carbonyl
source, and can be trapped by an appropriate nucleophilic component. For
example, this approach can be used to synthesize radiolabeled carboxylic acids,
such as [11C]acetate and [11C]palmitate, by reacting [11C]CO2 with an appropriate
Grignard reagent.8 New advances in the synthesis of [11C]carboxylic acids
involve treating organoboron precursors with [11C]CO2 in the presence of a
copper catalyst.9,10 More recently, there has also been a surge in [11C]CO2
fixation chemistry (for a review of current developments, see: 11). For example,
[11C]CO2 fixation chemistry has recently been employed in the synthesis of
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[11C]ureas (both symmetrical
and unsymmetrical
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[11C]carbamates.14,17,18,19,20,21 In an interesting variant of the latter, Miller also
demonstrated that analogous reactions with [11C]CS2 can be employed to
generate [11C]dithiocarbamates.22
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These impressive new developments in [11C]CO2 fixation chemistry were
of particular interest to us because they have greatly opened up the synthetic
transformations possible with carbon-11, and we believed that we could now
employ [11C]CO2 fixation to synthesize three radiotracers of interest to our
neuroimaging and translational oncology programs that would be extremely
challenging to prepare by other means (Figure 1). From a neuroimaging
perspective, we were interested in accessing [11C]3-(3-(1H-imidazol-1-
yl)propyl)quinazoline-2,4(1H,3H)-dione ([11C]QZ, 1) and [11C]tideglusib (2) as
potential radiotracers for glutaminyl cyclase (QC) and glycogen synthase kinase-
3 (GSK-3), respectively.23,24 In our growing translational oncology program, we
were also interested in a method for synthesizing [11C]ibrutinib (3), a radiolabeled
version of the anticancer drug targeting B-cell malignancies.25 We reasoned that
all three radiotracers could be synthesized from labeled building blocks
accessible using [11C]CO2 fixation. Herein we report three new variants of
[11C]CO2 fixation chemistry to generate intermediates that can be used in
intramolecular cyclization-, intermolecular cycloaddition- and peptide coupling-
reactions to generate [11C]QZ, [11C]tideglusib and [11C]ibrutinib, respectively.
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N
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[11C]QZ
[11C]Tideglusib
[11C]Ibrutinib
(1)
(2)
(3)
Figure 1: New radiotracers labeled by [11C]CO2 fixation (* = 11C)
RESULTS AND DISCUSSION
Automation and Specific Activity Considerations
We initially wished to have a means of automating [11C]CO2 fixation chemistry
using our General Electric (GE) TRACERLab FXC-Pro systems and made
modifications to the synthesis modules accordingly (see ESI for detailed
description). Briefly, because of the harsh, often sparingly soluble, reagents used
in [11C]CO2 fixation chemistry, we initially replaced the reagent reservoirs of the
synthesis module with disposable v-vials to enable handling of air sensitive
reagents and to prevent cross contamination between syntheses. Moreover,
valves were modified or installed to allow delivery of [11C]CO2 directly to the
TRACERLab reaction vessel. [11C]CO2 was produced with a GE PETTrace
cyclotron via the 14N(p,α)11C reaction. Carbon-11 produced in this manner has
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very high specific activity (for example, previously reported specific activities for
11C-labeled tracers produced in our laboratory range from 1 – 40 Ci/µmol26).
While high specific activities are required for potent radiotracers such as
[11C]carfentanil (low specific activity doses could elicit unwanted pharmacological
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responses), in our hands very high specific activity C has proven problematic
for [11C]CO2 fixation chemistry. For example, we have previously reported the
need to include carrier [12C]CO2 when producing [11C]acetate to avoid
contaminating doses with [11C]acetone and [11C]tert-butanol resulting from over
alkylation of [11C]CO2 with the Grignard reagent.8 No-carrier-added (NCA)
reactions proved to be an issue in the present work too (vide infra). For example,
while [11C]ibrutinib could be prepared carrier-free, [11C]tideglusib could not. The
synthesis of [11C]ibrutinib was also notably cleaner and higher yielding when
carrier [12C]CO2 was added. Our standard protocol is to introduce 0.5 – 1.0 mL
(~22.5 – 45 µmoles) of [12C]CO2 to the molecular sieves prior to delivery of
[11C]CO2 from the cyclotron.
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[11C]QZ (1)
Increased levels of glutaminyl cyclase (QC) in AD are thought to result in the
formation of pyroglutamate (pGlu)-modified Aβ peptides. These derivatives of Aβ
are suspected to be more neurotoxic and resistant to clearance than regular
amyloid-β, and they are prion-like, functioning as a template for misfolding and as
a nucleus for aggregation with unmodified amyloid-β. In a post-mortem study of
human neocortical brain samples it was found AD patients had upregulated
levels of QC mRNA compared to patients without dementia, while
immunohistochemistry suggests 20-30% higher expression of QC in all cortical
layers of AD patients compared to controls.27,28 We postulate that upregulated
QC could therefore be a prodromal marker for AD and were extremely interested
to determine if this upregulation could be detected using PET. Our initial efforts to
develop a PET radiotracer for QC concentrated on [11C]PBD150.29 However,
despite strong literature precedent for the compound (PBD150 is the only QC
inhibitor to date that has been shown to reduce the formation of Aβ aggregates in
vitro and in vivo27,30), we were disappointed to discover that [11C]PBD150 does
not cross the blood-brain barrier in rodents. Given the importance of QC as both
a therapeutic and diagnostic target in AD, our efforts to develop a radiotracer for
QC continue. It is unclear why [11C]PBD150 did not enter the CNS. P-
glycoprotein transporter involvement was ruled out,29 but one potential
explanation is the compound is too hydrophilic to cross the BBB (CLogP = 0.9).
Reflecting this, we were interested in next evaluating QC radiotracers that were
more lipophilic. To this end, we selected [11C]QZ (1) as a 2nd generation QC
radiotracer for further evaluation (CLogP = 1.5). QZ is a closely related
desmethyl analog of a recently reported QC inhibitor (IC50 = 123 nM), Figure 2.23
Our strategy for accessing [11C]QZ (1) was through an intramolecular
[11C]CO2 fixation reaction with precursor 5 (Scheme 1). Precursor 5 was
synthesized in 39% yield from isatoic anhydride (4) by coupling with 1-(3-
aminopropyl)imidazole. Treatment with [11C]CO2/[12C]CO2 and BEMP, followed
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by POCl3, generated carrier-added [11C]QZ (CA-[11C]QZ, 1). Purification by semi-
preparative HPLC provided 2 mCi of the isolated and formulated radiotracer
(non-decay corrected radiochemical yield (RCY) based upon [11C]CO2 = 1%,
radiochemical purity (RCP) >99% and specific activity = 3 Ci/mmol).
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R
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O
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R = H (QZ, 1)
R = Me (QC Inhibitor, IC50 = 123 nM23)
Figure 2. QC Inhibitors
1) [11C]CO2, BEMP
H
N
NH2
H
N
Amine Side
O
MeCN
O
Chain
THF, rt, 18 h
(39%)
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[11C]QZ (1)
(1% RCY based
upon [11C]CO2)
Scheme 1. Synthesis of [11C]QZ (1)
A preliminary rodent microPET imaging study was conducted with CA-
[11C]QZ (Figure 3). CA-[11C]QZ was administered to a female Sprague Dawley
rat and dynamic PET scans were acquired for 60 min post-injection of the
radiotracer (n = 1). Unfortunately, like [11C]PBD150 previously,29 analysis of
summed frame images showed no brain uptake and early frames showed no first
pass uptake in the brain. Given these findings, at this time we do not recommend
the imidazolyl propyl urea or thiourea scaffolds for further development of a
radiotracer for quantifying brain QC activity without further work to address brain
permeability of the compounds.
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Brain
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Figure 3: Summed sagittal rat PET image with [11C]QZ (1) (0 – 60 min after tail-
vein injection of the radiotracer)
[11C]Tideglusib (2)
Glycogen synthase kinase-3β (GSK-3β) dysregulation is hypothesized to play an
important role in a number of disease states, and inhibitors of GSK-3 are being
explored as potential therapeutics.31 Notably, increased levels of the active form
of the GSK-3β isoform are linked to hyperphosphorylation and subsequent toxic
aggregation of tau protein in Alzheimer’s disease and related tauopathies.32,33 In
connection with our program developing radiotracers for PET imaging of GSK-
3β,34,35 we were interested in radiolabeling tideglusib, an irreversible inhibitor of
GSK-3β that was shown to reduce tau hyperphosphorylation in murine models
and has recently been used in phase 2 clinical trials for treatment of progressive
supranuclear palsy (PSP)36 and AD.37 Radiolabeled tideglusib could also be used
as a companion diagnostic during development of the therapeutic allowing, for
example, study of brain uptake, target engagement, receptor occupancy and
biodistribution, as well as potentially serving as a high affinity GSK-3β probe for
future research studies.
We investigated the synthesis of [11C]tideglusib (2) from naphthalen-1-
amine (6), Scheme 2. We hypothesized that carbon-11 fixation chemistry could
be used to generate [11C]naphthylisocyanate 7, and that the 1,2,4-thiadiazolidine-
3,5-dione core of [11C]tideglusib could then be accessed through an
intermolecular
cycloaddition
between
[11C]isocyanate
7
and
(isothiocyanatomethyl)benzene (8), analogous to reported cold syntheses of
such compounds.38 Initially treating 6 with carrier-added [11C]CO2/[12C]CO2 and
BEMP followed by POCl3 generated CA-[11C]naphthylisocyanate 7. Gratifyingly,
exposing 7 to (isothiocyanatomethyl)benzene (8) in the presence of additional
POCl3, followed by hydrolysis with water/atmospheric oxygen, promoted
cyclization to CA-[11C]tideglusib (2) as expected. However, yields using this
method were poor (0.2% non-decay corrected RCY based upon [11C]CO2), likely
due to inefficient chlorination of 8 with POCl3 and, consequently, a poor
cyclization yield. Considering alternative chlorinating agents identified in the
39
literature,
we investigated whether N-chlorosuccinimide would be more
effective at promoting the cyclization. This proved to be the case, and CA-
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[11C]tideglusib (2) was synthesized in 2% non-decay corrected RCY based upon
[11C]CO2. Attempts to synthesize non-carrier-added [11C]tideglusib (NCA-
[11C]tideglusib) from [11C]CO2 were unsuccessful, possibly because of competing
side reactions of NCA-[11C]7, and studies to address this will be conducted in due
course. We therefore focused upon automating the procedure using CA-[11C]CO2
on a GE TRACERLab FXC-Pro synthesis module (see SI for further details), which
included purification by semi-preparative HPLC. However, initial attempts at
automation employing a 60% acetonitrile buffered eluent for semi-preparative
HPLC purification led to rapid decomposition of 2 and a RCP of only 25% at 10
min post-end-of-synthesis (EOS). Additional investigation of this issue revealed
that decomposition was likely occurring due to radiolysis and not chemical
instability of 2, as dilute samples (0.0001mg/mL) of non-radioactive reference
standard did not show decomposition in this eluent system after 48 hours. The
addition of ascorbic acid did not retard the decomposition of CA-[11C]tideglusib,
so the eluent composition was switched from acetonitrile to ethanol. A 70%
ethanol buffered eluent led to stabilization of CA-[11C]tideglusib, and 1 – 2 mCi
were obtained following semi-preparative purification. The isolated product,
formulated in 70% ethanolic HPLC mobile phase, exhibited stability over time
with no decrease in RCP after ~30 min. Reformulation of a 550 µCi sample
provided 150 µCi of CA-[11C]tideglusib in 10% ethanol in USP saline suitable for
preclinical use (>99% RCP, specific activity: 37 Ci/mmol). No change in RCP of
the reformulated sample was observed over 60 min, indicating radiolytic stability
of CA-[11C]tideglusib in the injectable formulation at these levels of radioactivity.
CA-[11C]Tideglusib was advanced to a microPET imaging study in a
female Sprague Dawley rat. CNS permeability was observed with peak brain
uptake of 200 nCi/cc, corresponding to a peak SUV of 0.7, after 3 minutes,
followed by washout (Figure 4). In the whole brain time-radioactivity curve
(Figure 4B), there is an apparent increase in SUV during the last two time points.
We see this frequently in our data analysis, independent of radiotracer, and
attribute it to increased noise resulting from the low residual counts that remain in
the brain during the late frames of the PET scans. The imaging results are
somewhat inconsistent with reported pharmacokinetic data, which suggests that
tideglusib is an irreversible inhibitor of GSK-3β (Ki= 60nM in vitro,
[35S]tideglusib/GSK-3β binding study), albeit with a time-dependent inhibition
profile (IC50 = 5 nM with 60 min pre-incubation, 105 nM without pre-incubation in
a double ATP and inhibitor study).40 Thus, it was expected that brain uptake
should increase, or at least not decrease, over time. It is uncertain whether
washout was due to A) the low specific activity of the radiotracer resulting in
GSK-3β saturation, B) rapid metabolism to a non-GSK-3β active agent, or C) that
tideglusib PK in vivo differ from PK in vitro for another reason. As such, studies
are currently underway to improve RCY and specific activity of [11C]tideglusib, so
that a better understanding of the in vivo characteristics of this clinically-relevant
pharmaceutical agent can be realized.
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Scheme 2. Synthesis of [11C]Tideglusib (2)
0.7
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Figure 4. A: Summed sagittal rat PET image with CA-[11C]tideglusib (2) (0 – 60
min after tail-vein injection of the radiotracer); B: whole brain time-radioactivity
curve.
[11C]Ibrutinib (3)
Bruton's tyrosine kinase (BTK) is a mediator of the B-cell-receptor signaling
pathway implicated in the pathogenesis of B-cell cancers. Early-stage clinical
trials found ibrutinib, a BTK inhibitor, showed antitumor activity in several types of
B cell lymphoma (for a review of the clinical use of ibrutinib, see: 25), and is
approved by the U.S. Food and Drug Administration (FDA) for the treatment of
mantle cell lymphoma and chronic lymphocytic leukemia. Radiolabeled ibrutinib
has the potential to function as a companion diagnostic for predicting response to
ibrutinib therapy (using, for example, comparable approaches to those recently
employed for [11C]docetaxel41), as well as to support future drug discovery efforts
around the BTK pathway.
Synthesis of [11C]ibrutinib (Scheme 3) commenced from vinylmagnesium
bromide (8). The Grignard reagent was treated with [11C]CO2 directly. In our first
approach, we then generated [11C]acryloyl chloride 10 by treatment with
phthaloyl dichloride, per a literature procedure.42 We next attempted to distill 10
into a solution of precursor 11 and 2,6-di-tert-butylpyridine (DTBP), and were
encouraged to generate [11C]ibrutinib (3), albeit in low yields (15 mCi, ~0.5%
non-decay corrected RCY based upon [11C]CO2). Analysis of reaction mixtures
suggested that ~90% of the [11C]acryloyl chloride polymerized during distillation.
To avoid this polymerization issue at elevated temperatures, we next used oxalyl
chloride to generate 10, and then transferred the entire [11C]acryloyl chloride
reaction mixture into a vial containing amino precursor 11 and DTBP at room
temperature. We manually produced injectable doses of 30mCi (~1% non-decay
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corrected RCY, based upon [11C]CO2) with >90% RCP, but had a difficult time
adapting this method to the automated radiochemistry synthesis modules. Both
the Grignard reagent and oxalyl chloride are very reactive and are sensitive to
air, or can react with each other. In addition, any excess of either reagent would
negatively impact the next step of the reaction, so quantitative quenching was
required for each step. Although the synthetic method works, we anticipated that
these issues would make the method practically unreliable and complicate
routine automated production.
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To address these issues, we next explored the possibility of generating
[11C]acrylic acid 12 and then using it in a peptide coupling with precursor 11
(Scheme 3). Quenching the carboxylate, resulting from treatment of
vinylmagnesium bromide with [11C]CO2 (diluted with 0.5 mL of 12CO2), using HCl
efficiently generated CA-[11C]acrylic acid (12). We selected HATU as the peptide-
coupling reagent, and efficient coupling of precursor 11 with CA-[11C]acrylic acid
12 generated CA-[11C]ibrutinib (3). Purification by semi-preparative HPLC was
followed by reconstitution into ethanolic saline using a C18 Sep-Pak cartridge
and gave 150 mCi of isolated and formulated product (5% non-decay corrected
RCY based upon [11C]CO2, RCP >99% and specific activity ~55 mCi/µmol). Total
synthesis time was 70 minutes from end of beam and the procedure was fully
automated on a GE TRACERLab FXC-Pro synthesis module. We also tried this
method no-carrier added, obtaining similar amounts (60 – 150 mCi) of isolated
and formulated NCA-[11C]ibrutinib (2 – 5% non-decay corrected RCY based upon
[11C]CO2) but with higher specific activities (600 – 2100 mCi/µmol). Notably
however, RCP (>90%) and chemical purity were lower for the NCA product (see
SI for further details of both CA and NCA syntheses).
N
N
1) [11C]CO2
O
MgBr
N
Cl
*
2) Phthaloyl
dichloride or
oxalyl chloride
N
10
9
Ph
N
O
H
Precursor (11)
DTBP
(0.5-1% RCY based
on [11C]CO2)
[11C]CO2
N
N
O
N
Precursor 11
N
OH
*
N
HATU, DIPEA
DMF
O
12
*
O
[11C]Ibrutinib
(2-5% RCY
based on [11C]CO2)
(3)
Scheme 3. Approaches to [11C]Ibrutinib (3)
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In an initial baseline PET study in a female CD-1 mouse, NCA-
[11C]ibrutinib showed significant uptake and retention in spleen, liver and
intestines (Figure 5), consistent with the known distribution and excretion of
ibrutinib. 43 Further investigations of the biodistribution and metabolism of
[11C]ibrutinib using normal and tumor bearing rodents, as well as nonhuman
primates, are underway and will be reported in due course.
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Liver
Intestines
Spleen
Figure 5. Summed coronal mouse PET image with [11C]ibrutinib (3) (0 – 60 min
after tail-vein injection of the radiotracer)
In summary we have demonstrated the synthesis of complex PET
radiotracers using different building blocks all accessed by [11C]CO2 fixation
chemistry. This work demonstrates that the ability to rapidly trap [11C]CO2 and
incorporate it into building blocks using a number of different approaches allows
quite sophisticated downstream chemistry to take place in spite of the 20 min
half-life of carbon-11. For example, we demonstrate that intramolecular
cyclizations, intermolecular cycloaddition reactions and peptide coupling
reactions are all possible. Each process is fully automated through new
modifications to a standard radiochemical synthesis module, and the
radiochemical syntheses provide isolated and formulated doses of radiotracers,
which are appropriate for pre-clinical in vivo imaging in rodents and non-human
primates. To date, radiochemical yields are somewhat low but reflect the
challenging chemistry being conducted with short-lived carbon-11. Notably,
yields could be improved by adding carrier [12C]CO2 to the reactions rather than
using NCA-[11C]CO2, which we attribute to competing side reactions that become
significant when working with NCA carbon-11. This is a current limitation of using
this new chemistry with very high specific activity carbon-11, and should be taken
into consideration when considering [11C]CO2 fixation approaches for
radiolabeling. Exploring the scope of [11C]CO2 fixation reactions is ongoing in our
laboratory, including efforts to determine the minimal amount of [12C]CO2 that can
be added to minimize side reactions, and maximize specific activities of the
resulting radiotracers. Nevertheless, the new reactions reported herein have
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enabled access to 3 new radiotracers inaccessible by other carbon-11
radiochemistry in amounts suitable for initial pre-clinical evaluation. [11C]QZ does
not cross the BBB and so is unsuitable for imaging glutaminyl cyclase in the
CNS. Contrastingly, initial results with [11C]tideglusib and [11C]ibrutinib are
promising and suggest that additional research with both radiotracers is
warranted.
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EXPERIMENTAL PROCEDURES
Chemistry
General considerations. All solvents and reagents were commercially available
and used without further purification unless otherwise stated. NMR spectra were
recorded with a Varian 400 MHz instrument at room temperature with
tetramethylsilane (TMS) as an internal standard. Mass spectra were performed
on an Agilent Q-TOF HPLC-MS or VG (Micromass) 70-250-S Magnetic sector
mass spectrometer employing the electrospray ionization (ESI) method or
electron ionization (EI) method. High performance liquid chromatography (HPLC)
was performed using a Shimadzu LC-2010A HT system equipped with a Bioscan
B-FC-1000 radiation detector. All procedures including anhydrous solvents were
performed with rigorously dried glassware under inert atmosphere.
Precursors. Precursors 6 and 8 were commercially available (Aldrich); precursor
11 was obtained from Bristol-Myers Squibb and used as received; precursor 5
was synthesized as described below.
N-(3-(1H-imidazol-1-yl)propyl)-2-aminobenzamide (5). Isatoic anhydride 4
(0.300 g, 2.4 mmol) was dissolved in THF (14.4 mL, 0.17 M) and 1-(3-
aminopropyl)imidazole (0.286 mL, 2.4 mmol) was added. The solution was
stirred overnight at room temperature, at which point solvent was removed under
vacuum. The resulting product was extracted with DCM and the organic layer
was washed with 5 mL saturated sodium bicarbonate solution, 2X with water,
dried (MgSO4), and concentrated in vacuo. The product was purified by silica gel
chromatography (DCM/MeOH gradient). After purification, product 5 was
1
collected as an oil (0.23 g, 39% yield); Rf 0.17 (9:1 DCM:MeOH); H NMR (400
MHz d4-MeOD): 7.68 (1H, s), 7.40 (1H, d, J=7.8), 7.17-7.14 (2H, m), 6.95 (1H,
br), 6.72 (1H, d, J=8.1), 6.60 (1H, t, J=7.5), 4.08 (2H, t, J=7.0), 3.31 (2H, t,
J=6.8), 2.05 (2H, quin, J=6.9); HRMS: calculated for C13H16N4O: 244.1397,
found: 245.1394 ([M+H]+).
Reference Standards. The identity of all 11C-labeled radiotracers was confirmed
by co-injection onto HPLC with the corresponding unlabeled reference standard.
Details on commercial suppliers and/or synthesis of reference standards is
described in the supporting information (SI).
Radiochemistry
General Considerations. Standard reagents and solvents were commercially
available and used without further purification, unless otherwise noted: sodium
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chloride, 0.9% USP and sterile water for Injection, USP were purchased from
Hospira; Dehydrated Alcohol for Injection, USP was obtained from Akorn Inc.;
Ascorbic Acid for Injection, USP was acquired from Bioniche Pharma; molecular
sieves were purchased from Alltech; and HPLC columns were acquired from
Phenomenex. Other synthesis components were obtained as follows: sterile
filters were acquired from Millipore; C18 Sep-Paks were purchased from Waters
Corporation; 10 cc sterile vials were obtained from HollisterStier. Sep-Paks were
flushed with 10 mL of ethanol followed by 10 mL of sterile water prior to use.
[11C]CO2 was produced with a General Electric (GE) PETTrace cyclotron. High
purity nitrogen containing 0.5% O2 was irradiated with 16.5 MeV to generate
[11C]CO2, which was delivered to a GE TRACERlab FXC-Pro synthesis module via
stainless steel lines. Radiochemical syntheses were carried out using GE
TRACERLab FXC-Pro synthesis modules housed in Comecer hot-cells. Doses
produced were assessed via standard quality control techniques (see below) and
were appropriate for rodent studies.
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[11C]QZ (1): A solution of precursor 5 (4 mg) and BEMP (5 µL) dissolved in 0.1
mL acetonitrile was sparged for 4 min with [11C]CO2 (300 mCi) that had been
diluted with 1 mL of 12CO2 at 7 mL/min (Ar used for transfer). A solution of POCl3
(0.4 µL in 0.1 mL of acetonitrile) was added and the resulting mixture was stirred
for 4 minutes. Then 1 mL of HPLC mobile phase was added and used to transfer
the reaction mixture to the HPLC loop. The product was purified by reverse
phase HPLC (column: Luna C18 semi-prep, 150 x 10 mm, 10 µ; mobile phase:
25% EtOH, 10 mM NaH2PO4, pH 8.0, 0.50 g/L of L-ascorbic acid; flow rate: 4
mL/min) and the product peak (tR ~ 9 – 10 min) was collected and diluted with
USP saline to achieve a 10% ethanol solution suitable for injection. Yield was 2
mCi (1% non-corrected isolated and formulated RCY based upon [11C]CO2; RCP
= 100%; pH = 6.5).
[11C]Tideglusib (2). A solution of naphthalen-1-amine (6) (0.72 mg, 5 µmol) and
BEMP (5 µL, 17.5 µmol) dissolved in 0.2 mL acetonitrile was sparged for 6 min
12
with [11C]CO2 (800 mCi) that had been diluted with 0.5 mL of CO2 at 5 mL/min
(He used for transfer). After this time the reaction mixture was transferred to a
vial containing POCl3 (10 µL, 100 µmol) in 100 µL THF (inhibitor-free) and
allowed to react for 2 min to generate [11C]7. The solution was then transferred
to a vial containing benzylisothiocyanate (8) (3.7 µL, 25 µmol) and N-
chlorosuccinimide (13.4 mg, 100 µmol) in 200 µL THF (inhibitor-free) with 20 mL
air and allowed to react for 5 min. The crude reaction mixture was diluted with
600 µL of 1:1 ethanol: water and transferred (using 60 mL air) into an 8 mL sterile
vial. Analysis of the reaction mixture revealed 14.6 mCi of [11C]titdeglusib, which
equated to 2% non-decay corrected RCY based upon [11C]CO2. Total synthesis
time was 60 minutes from end of beam. Efforts to purify and reformulate
[11C]tideglusib were complicated by radioloysis, but were eventually
accomplished using ethanolic solvent systems (see SI for detailed information).
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[11C]Ibrutinib (3). [11CO2] (~3 Ci diluted with 0.5 mL of CO2) was bubbled into
vinylmagnesium bromide (200 µL of 1.0 M Grignard diluted with 400 µL of THF)
at 7 mL/min flow rate for 4 minutes. 250 µL of HCl solution (100 µL of 4.0 M
HCl/dioxane diluted with 800 µL of DMF) was then added and the reaction stirred
for an additional 1 min. A solution of precursor 11 (3.9 mg) and DIPEA (25 µL) in
DMF (150 µL), and a solution of (1-[bis(dimethylamino)methylene]-1H-1,2,3-
triazolo[4,5-b]pyridinium 3- oxide hexafluorophosphate) (HATU, 6.5 mg) in DMF
(150 µL) were then added and the reaction mixture was stirred at room
temperature for 10 min. The reaction was then diluted with 1 mL of HPLC mobile
phase and purified by semi-preparative HPLC (column: Prodigy ODS 250x10
mm; mobile phase: 30 mM NH4OAc in 45% MeCN/H2O; flow rate: 4 mL/min).
The product peak (tR ~11 – 12 min) was collected into 50 mL of water, and
loaded onto a C18 Sep-Pak (Waters, 1cc). The final dose was eluted by 0.5 mL
of ethanol followed by 4.5 mL of injectable saline. Total synthesis time was 70
minutes from end of beam and gave 150 mCi of isolated and formulated product
(5% non-decay corrected RCY based upon [11C]CO2; RCP >99%; specific activity
= 55 mCi/µmol; pH = 6.0). No-carrier-added syntheses were also attempted and
further details are provided in the SI).
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Quality Control: Radiochemical purity of products was assessed using
Shimadzu LC-2010A HT system equipped with UV-Vis and radioactivity
detectors as follows:
[11C]QZ. Column: Phenomenex Luna C18, 150x4.6 mm; mobile phase: 10 mM
NH4OAc in 15 % MeCN; flow rate: 2.0 mL/min; wavelength: 254 nm; room
temperature; product tR = 9.9 min.
[11C]Tideglusib. Column: Phenomenex Luna phenyl hexyl, 5 micron,
150x4.6mm; mobile phase: 10 mM KH2PO4 in 70% EtOH, pH 4.6; flow rate: 1
mL/min; wavelength: 254 nm; product tR = 7.2 min.
[11C]Ibrutinib. Column: Kinetex XB-C18 150x4.6 mm; mobile phase: 30 mM
NH4OAc in 45% MeCN; flow rate: 1.0 mL/min; wavelength: 254 nm; product tR =
6.9 min.
ACKNOWLEDGEMENTS
PJHS acknowledges US DOE/NIBIB (DE-SC0012484), NIH (T32-EB005172)
and the Alzheimer’s Association (NIRP-14-305669) for financial support. Ibrutinib
precursor and reference standard were generously provided by Bristol-Myers
Squibb.
Supporting Information Available: Experimental procedures for the synthesis
of precursors, reference standards and radiotracers, spectral/chromatographic
data for all new compounds synthesized, semi-preparative and analytical radio-
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HPLC for all radiotracers, and details of pre-clinical PET imaging studies. This
material is available free of charge via the Internet at http://pubs.acs.org.
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TABLE OF CONTENTS GRAPHIC
9
N
10
11
12
13
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N
Ph
O
O
N
S
O
*
*
N
HN
N
O
[
11C]QZ
N
[
11C]Tideglusib
[11C]CO2
Fixation Chemistry
N
N
N
N
PhO
*
O
[
11C]Ibrutinib
LITERATURE CITED
(1) Ametamey, S.M., Honer, M., and Schubiger, P.A. (2008) Molecular imaging
with PET. Chem. Rev.,108, 1501–1516.
(2) Pither, R. (2003) PET and the Role of In Vivo molecular Imaging in
Personalized Medicine. Expert Rev. Mol. Diagn., 3, 703–713.
(3) Matthews, P.M.; Rabiner, E.A.; Passchier, J.; Gunn, R.N. (2011) Positron
Emission Tomography Molecular Imaging for Drug Development. Br. J. Clin.
Pharmacol., 73, 175–186.
11
18
(4) Miller, P. W., Long, N. J., Vilar, R., Gee, A. D. (2008) Synthesis of C, F,
15O, and N Radiolabels for Positron Emission Tomography. Angew. Chem. Int.
13
Ed., 47, 8998-9033.
(5) Scott, P. J. H. (2009) Methods for the Incorporation of Carbon-11 To
Generate Radiopharmaceuticals for PET Imaging. Angew. Chem. Int. Ed., 48,
6001-6004.
(6) Antoni, G. (2015). Development of Carbon-11 Labelled PET Tracers –
Radiochemical and Technological Challenges in a Historic Perspective. J. Label.
Compd. Radiopharm., 58, 65–72.
(7) Baumann, M., and Baxendale, I. R. (2013). An Overview of the Synthetic
Routes to the Best-selling Drugs Containing 6-membered Heterocycles. Beilstein
J. Org. Chem. 9, 2265–2319.
(8) Runkle, A. C., Shao, X., Tluczek, L. J. M., Henderson, B. D., Hockley, B. G.,
and Scott, P. J. H. (2011) Automated Production of [11C]Acetate and
[11C]Palmitate using a Modified GE Tracerlab FXC-Pro. Appl. Radiat. Isot., 69, 691-
698.
(9) Riss, P.J., Lu, S., Telu, S., Aigbirhio, F.I., and Pike, V. W. (2012) CuI-
Catalyzed 11C Carboxylation of Boronic Acid Esters: a Rapid and Convenient
ACS Paragon Plus Environment

Page 15 of 18
Bioconjugate Chemistry
1
2
3
4
5
6
11
Entry to C-Labeled Carboxylic Acids, Esters, and Amides. Angew. Chem. Int.
Ed., 51, 2698-2702.
7
8
9
(10) Rotstein, B.H., Hooker, J.M., Woo, J., Collier, T.L., Brady, T.J., Liang, S.H.,
and Vasdev, N. (2014) Synthesis of [11C]Bexarotene by Cu-Mediated
[11C]Carbon Dioxide Fixation and Preliminary PET Imaging. ACS Med. Chem.
Lett., 5, 668-672.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(11) Rotstein, B.H., Liang, S.H., Holland, J.P., Collier, T.L., Hooker, J.M., Wilson,
A.A., and Vasdev N. (2013) [11C]CO2 Fixation: a Renaissance in PET
Radiochemistry. Chem. Commun., 49, 5621-5629.
(12) Haji Dheere, A.K., Bongarzone, S., Taddei, C., Yan, R., and Gee, A.D.
11
(2015) Synthesis of C-Labelled Symmetrical Ureas via the Rapid Incorporation
of [11C]CO2 into Aliphatic Amines. Synlett, 26, 2257-2260.
(13) Haji Dheere, A.K., Yusuf, N., and Gee, A. (2013) Rapid and Efficient
Synthesis of [11C]Ureas via the Incorporation of [11C]CO2 into Aliphatic and
Aromatic Amines. Chem. Commun., 49, 8193-8195.
(14) Hicks, J.W., Parkes, J., Tong, J., Houle, S., Vasdev, N., and Wilson, A.A.
(2014) Radiosynthesis and Ex Vivo Evaluation of [11C-Carbonyl]carbamate- and
Urea-based Monoacylglycerol Lipase Inhibitors. Nucl. Med. Biol., 41, 688-694.
(15) Hicks, J.W., Parkes, J., Sadovski, O., Tong, J., Houle, S., Vasdev, N., and
Wilson, A.A. (2013) Synthesis and Preclinical Evaluation of [11C-Carbonyl]PF-
04457845 for Neuroimaging of Fatty Acid Amide Hydrolase. Nucl. Med. Biol., 40,
740-746.
(16) Hicks, J.W., Wilson, A.A., Rubie, E.A., Woodgett, J.R., Houle, S., and
Vasdev, N. (2012) Towards the Preparation of Radiolabeled 1-Aryl-3-benzyl
ureas: Radiosynthesis of [11C-Carbonyl] AR-A014418 by [11C]CO2 Fixation.
Bioorg. Med. Chem. Lett., 22, 2099-2101.
(17) Wilson, A.A., Garcia, A., Houle, S., Sadovski, O., and Vasdev, N. (2011).
Synthesis and Application of Isocyanates Radiolabeled with Carbon-11. Chem.
Eur. J., 17, 259-264.
(18) Hooker, J.M., Reibel, A.T., Hill, S.M., Schueller, M.J., and Fowler, J.F.
(2009). One-Pot, Direct Incorporation of [11C]CO2 into Carbamates. Angew.
Chem. Int. Ed., 48, 3482-3485.
(19) Wilson, A.A., Hicks, J.W., Sadovski, O., Parkes, J., Tong, J., Houle, S.,
Fowler, C.J., and Vasdev, N. (2013) Radiosynthesis and Evaluation of [11C-
Carbonyl]-Labeled Carbamates as Fatty Acid Amide Hydrolase Radiotracers for
Positron Emission Tomography. J. Med. Chem., 56, 201-209.
(20) Vasdev, N., Sadovski, O., Garcia, A., Dollé, F., Meyer, J., Houle, S., and
Wilson, A.A. (2011) Radiosynthesis of [11C]SL25.1188 via [11C]CO2 Fixation for
Imaging Monoamine Oxidase B. J. Label. Compd. Radiopharm., 54, 678–680.
(21) Wilson, A.A., Garcia, A., Houle, S., and Vasdev, N. (2010) Direct Fixation of
[11C]-CO2 by Amines: Formation of [11C-Carbonyl]-methylcarbamates. Org.
Biomol. Chem., 8, 428–432.
(22) Miller, P.W. and Bender, D. (2012) [11C]Carbon Disulfide: A Versatile
Reagent for PET Radiolabelling. Chem. Eur. J., 18, 433-436.
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(23) Tran, P-T., Hoang, V-H., Thorat, S.A., Kim, S. E., Ann, J., Chang, Y.J., Nam,
D.W., Song, H., Mook-Jung, I., Lee, J., and Lee, J. (2013) Structure-activity
Relationship of Human Glutaminyl Cyclase Inhibitors having an N-(5-methyl-1H-
imidazol-1-yl)propyl Thiourea Template. Bioorg. Med. Chem., 21, 3821-3830.
(24) Domínguez, J.M., Fuertes, A., Orozco, L., del Monte-Millán, M., Delgado, E.,
and Medina, M. (2012). Evidence for Irreversible Inhibition of Glycogen Synthase
Kinase-3β by Tideglusib. J. Biol. Chem., 287, 893-904.
10
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47
48
49
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56
57
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59
60
(25) Davids, M.S., and Brown, J.R. (2014) Ibrutinib: a First in Class Covalent
Inhibitor of Bruton’s Tyrosine Kinase. Future Oncol. 10, 957-967.
(26) Shao, X., Hoareau, R., Runkle, A. C., Tluczek, L. J. M., Hockley, B. G.,
Henderson, B. D., and Scott, P. J. H. (2011) Highlighting the versatility of the
Tracerlab synthesis modules. Part 2: fully automated production of [11C]-labeled
radiopharmaceuticals using a Tracerlab FXC-Pro. J. Label. Compd. Radiopharm.
54, 819-838.
(27) Schilling, S., Zeitschel, U., Hoffmann, T., Heiser, U., Francke, M., Kehlen,
A., Holzer, M., Hutter-Paier, B., Prokesch, M., Windisch, M. et al. (2008)
Glutaminyl Cyclase Inhibition Attenuates Pyroglutamate Aβ and Alzheimer's
Disease-like Pathology. Nat. Med., 14, 1106–1111.
(28) Morawski, M., Schilling, S., Kreuzberger, M., Waniek, A., Jäger, C., Koch,
B., Cynis, H., Kehlen, A., Arendt, T., Hartlage-Rübsamen, M. et al. (2014)
Glutaminyl Cyclase in Human Cortex: Correlation with (pGlu)-amyloid-β Load
and Cognitive Decline in Alzheimer's Disease. J. Alzheimer's Dis., 39, 385–400.
( 29 ) Brooks, A.F., Jackson, I.M., Shao, X., Kropog, G.W., Sherman, P.,
Quesada, C.A., and Scott, P.J.H. (2015) Synthesis and Evaluation of
[11C]PBD150, a Radiolabeled Glutaminyl Cyclase Inhibitor for the Potential
Detection of Alzheimer's Disease Prior to Amyloid β Aggregation. Med. Chem.
Commun., 6, 1065-1068.
(30) Buchholz, M., Heiser, U., Schilling, S., Niestroj, A.J., Zunkel, K., and Demuth
H.U. (2006) The First Potent Inhibitors for Human Glutaminyl Cyclase: Synthesis
and Structure-activity Relationship. J. Med. Chem., 49, 664-677.
(31) Woodget, J. R. (1990) Molecular Cloning and Expression of Glycogen
Synthase Kinase-3/Factor A. EMBO J., 9, 2431−2438.
(32) Hooper, C., Killick, R., and Lovestone, S. (2008) The GSK3 Hypothesis of
Alzheimer’s Disease. J. Neurochem. 104, 1433−1439.
(33) Avila, J., Wandosell, F., and Hernańdez, F. (2010) Role of Glycogen
Synthase Kinase-3 in Alzheimer’s Disease Pathogenesis and Glycogen
Synthase Kinase-3 Inhibitors. Expert Rev. Neurother. 10, 703−710.
(34) Cole, E.L., Shao, X., Sherman, P., Quesada, C., Fawaz, M.V. Desmond,
T.J., and Scott, P.J.H. (2014) Synthesis and evaluation of [11C]PyrATP-1, a Novel
Radiotracer for PET Imaging of Glycogen Synthase Kinase-3β (GSK-3β). Nucl.
Med. Biol., 41, 507-512.
(35) Li, L., Shao, X., Ohnmacht, S.A., Ferrari, V., Hong, Y.T., Williamson, D.J.,
Fryer T.D., Quesada, C.A., Sherman, P., Riss, P.J., Scott, P.J.H., and Aigbirhio,
F.I. (2015) Synthesis and Initial In Vivo Studies with [11C]SB-216763: The First
Radiolabeled Brain Penetrative Inhibitor of GSK-3. ACS Med. Chem. Lett., 6,
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Page 17 of 18
Bioconjugate Chemistry
1
2
3
4
5
548-552.
(36) Tolosa, E., Litvan, I., Höglinger, G.U., Burn, D., Lees, A., Andrés, M.V.,
Gómez-Carrillo, B., León, T., Del Ser, T., and the TAUROS Investigators. (2014)
A Phase 2 Trial of the GSK-3 Inhibitor Tideglusib in Progressive Supranuclear
Palsy. Mov. Disord., 29, 470-478.
(37) Lovestone, S., Boada, M., Dubois, B., Huell, M., Rinne, J.O., Huppertz, H-J.,
Calero, M., Andres, M.V., Gomez-Carrillo, B., Leon, T., and del Ser, T. (2015) A
Phase II Trial of Tideglusib in Alzheimer’s Disease. Journal of Alzheimer's
Disease, 45, 75-88.
(38) Medina Padilla, M., Alonso Cascon, M., Dorronsoro Diaz, I., Martinez Gil, A.,
Panizo Del Pliego, G., Fuertes Huerta, A., Perez Puerto, M.J. (2005) GSK-3
Inhibitors. US20050222220.
(39) Nasim, S., and Crooks, P.A. (2009) N-Chlorosuccinimide is a Convenient
Oxidant for the Synthesis of 2,4-Disubstituted 1,2,4-Thiadiazolidine-3,5-diones.
Tetrahedron Lett., 50, 257-259.
(40) Domínguez, J. M., Fuertes, A., Orozco, L., del Monte-Millán, M., Delgado,
E., and Medina M. (2012) Evidence for Irreversible Inhibition of Glycogen
Synthase Kinase-3β by Tideglusib. J. Biol. Chem., 287, 893-904.
(41) Van der Veldt, A.A.M., Lubberink, M., Mathijssen, R.H.J., Loos, W.J.,
Herder, G.J.M., Greuter, H.N., Comans, E.F.I., Rutten, H.B., Eriksson, J.,
Windhorst, A.D., Hendrikse, N.H., Postmus, P.E., Smit, E.F., and Lammertsma,
A.A. (2013) Toward Prediction of Efficacy of Chemotherapy: a Proof of Concept
Study in Lung Cancer Patients using [11C]Docetaxel and Positron Emission
Tomography. Clin. Cancer Res., 19, 4163–4173.
6
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43
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45
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49
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51
52
53
54
55
56
57
58
59
60
(42) Mishani, E., Levitzki, A., Ortu, G., Ben-David, I., and Rozen, Y. (2005)
Radiolabeled Anilinoquinazolines and their use in Radioimaging and
Radiotherapy. PCT Int. Appl., WO 2005023315.
(43) http://www.imbruvica.com; accessed March 2016.
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