Synthesis and evaluation of [11C]PBD150, a radiolabeled glutaminyl cyclase inhibitor for the potential detection of Alzheimer's disease prior to amyloid β aggregation

Article abstract of DOI:10.1039/c5md00148j

The phenol of 1-(3-(1H-imidazol-1-yl)propyl)-3-(4-hydroxy-3-methoxyphenyl)thiourea was selectively carbon-11 labelled to generate [¹¹C]PBD150 in 7.3% yield from [¹¹C]methyl triflate (non-decay corrected; radiochemical purity ≥95%, sp

Full text of DOI:10.1039/c5md00148j

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11
Synthesis and evaluation of [ C]PBD150, a
radiolabeled glutaminyl cyclase inhibitor for the
potential detection of Alzheimer's disease prior
to amyloid β aggregation†
Cite this: DOI: 10.1039/c5md00148j
a
a
a
a
a
Allen F. Brooks, Isaac M. Jackson, Xia Shao, George W. Kropog, Phillip Sherman,
a
ab
Carole A. Quesada and Peter J. H. Scott*
The phenol of 1-IJ3-IJ1H-imidazol-1-yl)propyl)-3-IJ4-hydroxy-3-methoxyphenyl)thiourea was selectively
1
1
11
Received 10th April 2015,
Accepted 21st April 2015
carbon-11 labelled to generate [ C]PBD150 in 7.3% yield from [ C]methyl triflate (non-decay corrected;
−1
11
radiochemical purity ≥95%, specific activity = 5.7 Ci μmol , n = 5). Evaluation of [ C]PBD150 by small ani-
mal PET imaging (mouse and rat) determined it does not permeate the blood brain barrier, indicating previ-
ously described therapeutic effect in transgenic mice was likely not the result of inhibiting central nervous
system glutaminyl cyclase.
DOI: 10.1039/c5md00148j
www.rsc.org/medchemcomm
7
,8
Glutaminyl cyclase (QC) is an enzyme primarily expressed
in the brain with a large amount of expression in the
hippocampus and cortex. QC is responsible for the formation
of pyroglutamate (pGlu) at the N-terminus of several neuro-
spectrometry of plaques, incubation with pGlu aminopepti-
9
dase to verify the presence of pGlu modification, and a
sandwich enzyme-linked immunosorbent assay to quantify
1
0
amount of pGlu modified Aβ. The prion like role played by
pGlu-Aβ species in the formation of aggregates in AD that are
the result of upregulated QC lead us to hypothesize that a QC
inhibitor amenable to radiolabeling would provide a means
to detect AD by positron emission tomography (PET) imaging
prior to the accumulation of senile plaques.
1
peptides including amyloid β (Aβ). This modification is
important for the biological function of those neuropeptides,
and importantly the pGlu modification stabilizes the peptide
2
against proteolytic decomposition. Changes in QC expression
between Alzheimer's disease (AD) patients and healthy control
subjects have not been fully analyzed and absolutely quanti-
fied yet. However, a post-mortem study of human neocortical
brain samples found AD patients had upregulated levels of
QC mRNA compared to patients without dementia, while
immunohistochemistry suggests 20–30% higher expression of
Many inhibitors of QC have been prepared and screened
1
1–14
in vitro.
Radiolabeled examples have been claimed in a
recent patent; however, no QC radiotracers have been
reported in the literature to date. Interestingly, PBD150
(shown in Fig. 1), was not covered in the patent despite
having good affinity for the enzyme (K = 60 nM for human
1
5
1
1
3
QC in all cortical layers of AD patients compared to controls.
i
This increase in QC leads to the formation of pGlu-Aβ pep-
QC and K = 173 nM for murine QC) and being the only agent
i
tides that are more neurotoxic and resistant to removal than
normal Aβ. They act similar to a prion, providing a template
thus far to have been demonstrated in vitro and in vivo to
3
a,11
reduce the formation of Aβ aggregates.
PBD150 by methylation with [ C]MeOTf or [ C]MeI through
a phenol precursor is not straightforward as PBD150 contains
Radiolabeling
4
11
11
for misfolding and as
a
seed for aggregation with
5
unmodified Aβ. The pGlu modified variants of Aβ have been
shown to be present in the plaques of AD brains by several
different analytical methods: immunohistochemistry and
1
6
a thiourea, which can readily undergo methylation instead,
6
,7
immunocytochemistry for distinct Aβ species,
mass
a
Division of Nuclear Medicine, Department of Radiology, The University of
Michigan Medical School, 2276 Medical Science I Building, Ann Arbor,
Michigan 48109, USA. E-mail: pjhscott@umich.edu; Fax: +1(734)615 2557;
Tel: +1(734)615 1756
b
The Interdepartmental Program in Medicinal Chemistry, The University of
Michigan, 428 Church St., Ann Arbor, Michigan 48109, USA
†
Electronic supplementary information (ESI) available: Procedures for
3
chemistry, radiochemistry, rodent PET imaging and metabolism experiments, as
well as spectral data. See DOI: 10.1039/c5md00148j
Fig. 1 Structure of PBD150 and properties: a , b from Pubchem
11
database, c from ChemBioOffice, d
.
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alternate product 6 was readily achievable by 2 different ana-
lytical HPLC methods (see ESI† for representative HPLC
traces).
1
1
Initial attempts to radiolabel PBD150 by [ C]methylation
were investigated using tetrabutylammonium hydroxide
(
TBA-OH) as a base (Table 1). No product was observed with
the preformed tetrabutylammonium phenoxide of 5 using
either typical reactor set up with DMF as a solvent in a
TRACERLab FXC-Pro (entries 1 and 2) or ethanolic loop chem-
2
0
11
11
Scheme
1 Synthesis of desmethyl precursor of PBD150 5 and
istry (entry 3) with either [ C]MeOTf or [ C]MeI. In addi-
tion, the subsequent HPLC-UV analysis showed that no pre-
cursor remained in these reactions. Based on those results,
we decided that a non-nucleophilic base could be required to
avoid the decomposition of the precursor. We therefore
alternate methylation reference standard 6.
1
7
making an undesired alternate product. In an example
pertinent to PET radiochemistry, Gilissen and co-workers
had problems introducing ethyl tosylate groups into methyl-
thioureas for subsequent F-fluorination, obtaining unwanted
cyclized by-products instead. To circumvent this issue, the
investigators first generated 2-ij F]fluoroethylamine, and then
introduced the thiourea functionality. Despite these issues, as
PBD150 has shown a therapeutic effect when administered
orally to transgenic mice, we resolved to develop a method to
carbon-11 label a phenol in the presence of a thiourea so that
we could evaluate [ C]PBD150 as a PET agent for the detection
of AD prior to senile plaque burden.
To radiolabel PBD150 the desmethyl precursor, 1-IJ3-IJ1H-
imidazol-1-yl)propyl)-3-IJ4-hydroxy-3-methoxyphenyl)thiourea
1
1
attempted carbon-11 labelling with NaH using [ C]MeI
1
1
(
entry 4) or [ C]MeOTf (entry 5). These conditions resulted in
1
8
predominantly alternate radiolabelled product 6, confirmed by
comparison of the radio-HPLC with the known retention
times of the 2 reference standards (see ESI†). The use of a
non-nucleophilic base had avoided precursor decomposition
1
8
1
8
1
1
but NaH favoured the alternate methylation product [ C]6,
3
a
so we next investigated the use of K CO , another non-
2
3
nucleophilic base that has a lower conjugate pK
a
. Initial
attempts using standard conditions ([ C]MeOTf in
-pentanone, entry 6) proved ineffective as precursor 5 was
insoluble. Ultimately, treatment of the precursor in DMF with
0 μL of a satd. K CO solution dried over Na SO prior to
labelling with [ C]MeOTf in a reactor (entry 7) was found to
1
1
1
1
3
1
2
3
2
4
(5), was prepared in 4 steps. The synthesis of the desmethyl
1
1
precursor, as shown in Scheme 1, was accomplished by
acylation of the hydroxyl of 4-nitroguaiacol and subsequent
reduction of the nitro of intermediate 2 to give aniline 3.
1
1
be the best method for generating [ C]PBD150. These condi-
tions were based upon institutional knowledge for
radiolabeling phenols (unpublished results), and the use of
1
9
The aniline was then converted to thioisocynate 4 with thio-
phosgene. The product was used directly in the next reaction
with 1-IJ3-aminopropyl)imidazole to form thiourea 5 in 41%
yield from 3. The synthesis of desmethyl precursor (5) was
completed in 4 steps and an overall yield of 28% from 1. To
confirm the identity of the radiotracer, it was also necessary
to synthesize unlabelled reference standards. Authentic
PBD150 was synthesized as previously described. Alternate
methylation product 6 was also prepared in 11% yield by
treating 5 with MeI. Baseline separation of PBD150 and
11
the more efficient methylating agent is essential. Use of [ C]
11
MeI in the presence of K
PBD150 over [ C]6, but in low yield (entry 8).
The radiolabeling of [ C]PBD150 with [ C]MeOTf using
the optimum conditions was then automated in
2
CO
3
also favours generation of [ C]
1
1
1
1
11
a
TRACERLab FXC-Pro system. The product was purified by
reverse phase HPLC and the ethanolic buffer with product
1
1
(
2 mL) was added to saline (8 mL) to prepare the dose for
injection. The synthesis (Scheme 2) resulted in 66.1 mCi of
1
1
formulated product (7.3% yield from [ C]MeOTf, non-decay
corrected; radiochemical purity ≥95%, specific activity =
−
1
1
1
Table 1 Strategies for the radiosynthesis of [ C]PBD150
5.7 Ci μmol , n = 5). The doses produced were determined
by standard quality control techniques (see ESI†) to be appro-
priate for preclinical studies.
a
Entry Solvent
Base
Methylating agent RCC (PBD150 : 6)
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
b
11
1
2
3
4
5
6
7
8
DMF
DMF
EtOH loop
DMF
DMF
3-Pentanone
DMF
DMF
TBAOH
TBAOH
TBAOH
NaH
[
[
[
[
[
[
[
[
C]MeOTf
C]MeI
C]MeOTf
C]MeI
C]MeOTf
C]MeOTf
C]MeOTf
C]MeI
0% (0 : 0)b
The imaging capability of [ C]PBD150 was investigated
0% (0 : 0)
through small animal PET imaging with Sprague Dawley rats
b
0% (0 : 0)
10% (28 : 72)
1% (50 : 50)
0% (0 : 0)
12% (95 : 5)
1% (75 : 25)
1
1
(n = 2; Fig. 2 and ESI†). [ C]PBD150 was administered i.v. via
NaH
tail vein injection and dynamic PET scans were acquired for
c
K CO
2
K CO
2
K CO
2
3
3
3
60 min post-injection of the radiotracer. Analysis of summed
frame images showed no brain uptake and early frames
showed no first pass uptake in the brain. The fact that
PBD150 had previously demonstrated a therapeutic effect on
AD progression in transgenic mice made the lack of blood–
a
%
Radiochemical conversions (RCC) represent combined
11 11 11
conversion to [ C]PBD150 and [ C]6. RCC are based upon [ C]
MeOTf and are uncorrected. Product ratios in parentheses were
determined by analytical HPLC. Precursor decomposed. Precursor
insoluble.
3
a
b
c
brain barrier (BBB) permeability surprising. To investigate
if efflux by P-glycoprotein (Pgp) transporter is an issue with
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In summary, a method for the carbon-11 methylation of a
phenol in the presence of thiourea has been developed and
1
1
applied to the synthesis of [ C]PBD150: a QC inhibitor dem-
onstrated in vitro and in vivo to decrease the formation of
pGlu-Aβ. Preclinical rodent PET imaging experiments with
Scheme 2 The development of a method to selectively label the
phenol over the thiourea was required to produce [ C]PBD150.
11
1
1
[
C]PBD150 revealed a lack of brain uptake. Further experi-
ments demonstrated that the lack of brain uptake is not due
to metabolism, and nor is it due to efflux by Pgp. The reasons
for the lack of BBB permeability are unclear at this time, but
could simply be due to the polarity of the compound. Never-
theless, this work does suggest that the therapeutic effect
observed in transgenic mice in prior literature was not due to
the inhibition of CNS QC, but could be due to inhibition of
peripheral QC or changes to the ADME of PBD150 in the
transgenic mice studied due to long term treatment. Inhibi-
tion of QC remains a promising target for the treatment of
AD and early detection of AD prior to senile plaque forma-
tion. A BBB-permeable QC inhibitor would represent a step
forward in the development of a therapeutic agent and could
serve as a companion diagnostic when developed as a PET
radioligand. To that end, we continue our work to identify a
BBB-permeable QC inhibitor. In addition, this work demon-
strates that a more detailed understanding of QC inhibition
is essential prior to clinical translation.
2
1,22
PBD150 (given previous examples observed
) cyclosporin A
blocking experiments were performed. Animals were treated
with cyclosporin A (50 mg kg ) 60 min prior to injection of
C]PBD150. The imaging data obtained showed no brain
uptake as before with baseline scan data. In the work with
transgenic mice, PBD150 had been administered orally in the
−
1
1
1
[
3
a
food of the mice for several months. To examine if brain
uptake was facilitated by a higher concentration of PBD150
in the blood, an experiment was conducted where [ C]
PBD150 was co-administered, via tail vein injection, with an
equivalent amount of unlabelled PBD150 to the oral dose
reported previously (12 mg total; ~37 mg kg ). The PET
imaging data again showed no brain uptake (Fig. 2A), compa-
rable to previous experiments. As the therapeutic studies
were conducted in mice, we also repeated imaging in a
female CD-1 mouse to investigate any differences in brain
uptake between species. However, there was also no brain
uptake of the radiotracer in the mouse brain (see ESI† for rat
and mouse imaging data), suggesting that the poor brain
uptake is not due to a species difference.
For each experiment a region of interest was drawn
around the brain and standardized uptake values (SUV) were
calculated and charted versus time. The analysis showed no
difference between the baseline, cyclosporin A treatment and
co-administered experiments (Fig. 2B). To investigate whether
the imaging results were due to metabolic instability, metab-
olism of PBD150 was examined by incubation with rat liver
microsomes. The LC-MS/MS data analysis at microsome incu-
bation time points out to 60 min (the length of the rodent
PET imaging experiments) demonstrated the molecule had a
half-life >60 min, with 78% of authentic PBD150 remaining
at 60 min, indicating that the lack of brain uptake is likely
not due to rapid metabolism of the radiotracer.
1
1
−
1 3a
3
a
Acknowledgements
We acknowledge the NIH IJT32-EB005172), Alzheimer's Associ-
ation (NIRP-14-305669) and the Michigan Research Commu-
nity/Undergraduate Research Opportunity Program for finan-
cial support, and also thank the Pharmacokinetics Core at the
University of Michigan for conducting metabolism studies.
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Fig. 2 Rodent PET data: A, representative image from [ C]PBD150
co-administration PET scan (summed image 0–60 min post-injection
of the radiotracer); B, time-radioactivity curves for baseline, cyclo-
sporin A-treated and PBD150 co-administration scans.
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