Synthesis and Initial Characterization of a Selective, Pseudo-irreversible Inhibitor of Human Butyrylcholinesterase as PET Tracer

Article abstract of DOI:10.1002/cmdc.202000942

The enzyme butyrylcholinesterase (BChE) represents a promising target for imaging probes to potentially enable early diagnosis of neurodegenerative diseases like Alzheimer's disease (AD) and to monitor disease progression in some forms of cancer. In this study, we present the design, facile synthesis, in vitro and preliminary ex vivo and in vivo evaluation of a morpholine-based, selective inhibitor of human BChE as a positron emission tomography (PET) tracer with a pseudo-irreversible binding mode. We demonstrate a novel protecting group strategy for ¹⁸F radiolabeling of carbamate precursors and show that the inhibitory potency as well as kinetic properties of our unlabeled reference compound were retained in comparison to the parent compound. In particular, the prolonged duration of enzyme inhibition of such a morpholinocarbamate motivated us to design a PET tracer, possibly enabling a precise mapping of BChE distribution.

Full text of DOI:10.1002/cmdc.202000942

Full Papers
doi.org/10.1002/cmdc.202000942
ChemMedChem
Synthesis and Initial Characterization of a Selective,
Pseudo-irreversible Inhibitor of Human
Butyrylcholinesterase as PET Tracer
+
[a]
+ [a]
[c, d]
[e]
Christian Gentzsch , Matthias Hoffmann , Yasuhiro Ohshima,
Xinyu Chen,
Naoko Nose,
[b, c, d]
[c, d, e]
[a]
Takahiro Higuchi,*
and Michael Decker*
The enzyme butyrylcholinesterase (BChE) represents a promis-
ing target for imaging probes to potentially enable early
diagnosis of neurodegenerative diseases like Alzheimer’s dis-
ease (AD) and to monitor disease progression in some forms of
cancer. In this study, we present the design, facile synthesis,
in vitro and preliminary ex vivo and in vivo evaluation of a
morpholine-based, selective inhibitor of human BChE as a
positron emission tomography (PET) tracer with a pseudo-
irreversible binding mode. We demonstrate a novel protecting
1
8
group strategy for F radiolabeling of carbamate precursors
and show that the inhibitory potency as well as kinetic
properties of our unlabeled reference compound were retained
in comparison to the parent compound. In particular, the
prolonged duration of enzyme inhibition of such a morpholino-
carbamate motivated us to design a PET tracer, possibly
enabling a precise mapping of BChE distribution.
Introduction
smoking can delay the onset and therefore contribute to a
reduced future number of affected people, as there is still no
[1]
Dementia and its most frequent form, Alzheimer’s disease (AD),
are increasingly challenging health-care systems all around the
world. The number of affected people doubled from 1990 to
chance to cure or even modify the progress of AD. Alzheimer
[
2]
published the first description of histological alterations,
namely amyloid β deposits (Aβ plaques) and neurofibrillary
tangles (NFTs), which occur during the progression of the
disease named after him and are accompanied by memory
2
016, meaning that there are up to 50 million patients right
now. On the one hand, this can be correlated with a growing
and aging world population, on the other hand, there is
evidence that avoiding risk factors like high body mass index or
[3]
deficits and cognitive decline. These pathological markers are
still used to make a definite diagnosis of AD, which, however,
[4]
can only take place post mortem. The fatal consequences of
the disease can be ascribed to a proceeding loss of cholinergic
neurons and a decline of the neurotransmitter acetylcholine,
which is metabolized by the serine hydrolase acetylcholinester-
+
+
[
a] C. Gentzsch, Dr. M. Hoffmann, Prof. Dr. M. Decker
Pharmaceutical and Medicinal Chemistry
Institute of Pharmacy and Food Chemistry
Julius-Maximilians-University of Würzburg
Am Hubland, 97074 Würzburg (Germany)
E-mail: michael.decker@uni-wuerzburg.de
b] Dr. X. Chen
[5]
ase (AChE). Apart from the N-methyl-d-aspartate receptor
antagonist memantine, all of the approved drugs on the market
to combat AD are AChE inhibitors and can ameliorate
symptoms in the early stage of the disease by preventing
further degradation of acetylcholine through enzyme hydrol-
ysis. This is far from any disease-modifying or curative treat-
ment, and, in general, the complex pathologies of AD are yet
[
Department of Nuclear Medicine
University Hospital of Augsburg
Stenglinstraße 2, 86156 Augsburg (Germany)
c] Dr. Y. Ohshima, Dr. X. Chen, Prof. Dr. T. Higuchi
Comprehensive Heart Failure Center
University Hospital of Würzburg
[
[5a,6]
Am Schwarzenberg 15, 97078 Würzburg (Germany)
E-mail: higuchi_t@ukw.de
d] Dr. Y. Ohshima, Dr. X. Chen, Prof. Dr. T. Higuchi
too little understood.
However, there is evidence that
butyrylcholinesterase (BChE) represents an attractive target for
novel imaging probes in AD diagnostics and such imaging
probes represent tool compounds to provide further insights
into BChE’s involvement in neuropathologies and consequently
for more powerful drugs to combat the disease. It was shown
that in late-stage AD, the level of AChE dramatically decreases
by approximately 90%, while BChE activity remains unaffected
[
Department of Nuclear Medicine
University Hospital of Würzburg
Oberdürrbacher Straße 6
9
7080 Würzburg (Germany)
[
e] N. Nose, Prof. Dr. T. Higuchi
Graduate School of Medicine, Dentistry and Pharmaceutical Sciences
Okayama University
2
-5-1 Shikata-cho, Kita-ku, Okayama (Japan)
[7]
+
or can be elevated by up to 30%. Furthermore, in the human
brain large amounts of BChE can be found in regions associated
with cognition and behavior, which are among the first areas
[
] These authors contributed equally to this work.
Supporting information for this article is available on the WWW under
https://doi.org/10.1002/cmdc.202000942
[8]
©
2021 The Authors. ChemMedChem published by Wiley-VCH GmbH. This is
affected by neurodegeneration. Last, there is evidence that
BChE is involved in AD pathologies by generating harmful
an open access article under the terms of the Creative Commons Attribution
Non-Commercial NoDerivs License, which permits use and distribution in
any medium, provided the original work is properly cited, the use is non-
commercial and no modifications or adaptations are made.
[9]
attributes of Aβ plaques. In up to 30% of cognitively normal
older adults, Aβ plaques appear without being colocalized with
ChemMedChem 2021, 16, 1427–1437
1427
© 2021 The Authors. ChemMedChem published by Wiley-VCH GmbH

Full Papers
doi.org/10.1002/cmdc.202000942
ChemMedChem
[
10]
[21]
BChE, which is the case in AD. Consequently, it has been
shown in several studies that there is a positive, symptomatic
effect on memory deficits and cognitive decline, when BChE is
from histochemistry. Thereby first preclinical evidence was
provided that it is possible to distinguish healthy and AD mice
by targeting BChE with imaging probes.
This approach was extended to an inhibitor-based, irrever-
sibly acting ChE inhibitor, phenyl 4-[ I]-iodophenylcarbamate
[11]
inactivated. Moreover, it is possible to reverse the effect of
injected Aβ25–35 in an AD mouse model with highly selective
1
23
1
23
BChE inhibitors exhibiting
a
pronounced neuroprotective
( I-PIP). In ex vivo autoradiography studies using brain tissue of
profile, providing first hints that a disease-modifying treatment
AD patients, cognitively normal, Aβ positive older adults and Aβ
[12]
can potentially be achieved. Besides, BChE is known to play
an important physiological role in serum metabolism by
detoxifying xenobiotics or naturally occurring bioactive com-
pounds with its ability to hydrolyze ester or carbamate
functionalities. Therefore, the active site exhibits a low substrate
negative aged humans, this tracer could discriminate AD tissue
[22]
from the other two.
However, it is not selective towards
BChE, ex vivo studies are preliminary due to a limited number of
investigated brain tissues and the in vivo assessment of the
[22]
123
tracer is still pending. Additionally, I-PIP was investigated in
a recent study for its potential ability to detect pathological
changes in multiple sclerosis (MS). In vitro autoradiography
revealed that the tracer can detect MS lesions in white matter
[13]
specificity compared to AChE. Additionally, there is evidence
that BChE contributes to other diseases, including cardiovascu-
[14]
lar pathologies, obesity or diabetes mellitus type 2. Interest-
ingly, both ChEs are known to be involved in cellular
proliferation and differentiation, which has raised attention to
[23]
by binding to BChE.
Other irreversible ChE inhibitors are organophosphates and
a respective PET tracer was designed to target central nervous
system AChE in rats, but these compounds are highly toxic
[15]
their potential role in tumorigenesis.
For example, the
malignancy grade of tumors in sporadic breast cancer is
positively correlated with the total number of alterations in the
[24]
nerve agents. However, due to the lack of results concerning
irreversibly acting and selective BChE PET tracers as potential
in vivo AD diagnostics, we continued our previous studies, in
which we had investigated enzyme kinetics and binding,
radiolabeling and preliminary ex vivo brain tissue binding of
pseudo-irreversible, selective BChE inhibitors with high
[16]
BChE gene. Furthermore, a biphasic alteration of BChE level
was observed in prostate cancer, meaning a downregulation in
the early stage and a subsequent upregulation in the late-stage
of the disease, which is additionally correlated with recurrence
[17]
and reduction of disease-free survival. Consequently, there is
the need for imaging probes selectively targeting BChE not
only as potential AD diagnostics to support a diagnosis
delimiting other forms of dementia, but also to shed new light
on the pathologies of other diseases with related altered BChE
activity, expression levels and occurrence. For this reason,
positron emission tomography (PET) radiotracers have been
developed to gain information about the spatial and temporal
distribution of an injected, radiolabeled compound. Ideally, the
tracer should address the target of choice with high affinity and
[12,19,25]
affinity.
Starting point of our investigations was heptylcar-
bamate 1a (Figure 1) bearing a tetracyclic carrier scaffold. Since
this compound turned out to be a highly potent carbamate-
based BChE inhibitor with pronounced selectivity over AChE,
we performed kinetic studies to gain information about the
complex binding mode of this class of BChE inhibitors (Fig-
ure 1). In the first binding step of a carbamate-based BChE
inhibitor [CÀ X] to enzyme [E] the complex [ECÀ X] forms
spontaneously, which may either dissociate or result in a
transfer of the carbamate group C onto the enzyme E, which is
subsequently inhibited by means of a covalent bond. Under the
presumption that the formation of [EC-X], characterized by the
[18]
selectivity. In case of BChE, one can design either an inhibitor-
or substrate-based compound for radiolabeling, while the
former can be classified in reversibly or irreversibly acting
inhibitors. Even though substrate analogs as BChE PET tracers
can usually pass the blood–brain barrier (BBB), their hydrolysis
rate is often too high to achieve an accurate estimation of the
kinetic rate constant k , is considerably faster than the chemical
1
reaction of carbamate transfer, the affinity of carbamate-based
BChE inhibitors can be characterized by equilibrium constant Kc.
Dissociation of [EC-X] is kinetically characterized by the rate
[
6a,19]
11
actual enzyme distribution in the brain.
1- C-Methyl-4-
1
1
18
piperidinyl-n-butyrate
( C-MP4B)
and
N-[ F]
constant k . The carbamate transfer from the tetracyclic carrier
2
fluoroethylpiperidin-4-ylmethyl butyrate are examples of such
tracers with a pronounced rate of hydrolysis in brain and
scaffold [X] onto the enzyme [E] is a nucleophilic substitution
reaction, in which the serine in the catalytically active site of
BChE attacks the activated carbon of the carbamate group,
while the carrier scaffold X is released as free alcohol. This
chemical reaction is described by the kinetic rate constant k3
and results in the formation of the carbamylated enzyme E-C,
that is, the catalytic site in the enzyme is blocked by a covalent
bond. Due to the inherent chemical instability of carbamate
bonds, the enzyme is not inhibited in an irreversible fashion,
but enzyme activity is reconstituted by hydrolysis of the
carbamate, that is, a second chemical reaction characterized
through rate constant k and half-life t of the carbamylated
1
1
plasma. The biodistribution of C-MP4B in the brains of AD
patients most widely represented the neuroanatomy of BChE
known from post mortem studies of human brain, apart from a
much higher uptake in the striatum than in the cortex. Further
studies are required to fully characterize the diagnostic value of
[6a,20]
123
both tracers.
N-Methylpiperidin-4-yl 4-[ I]iodobenzoate
was consequently developed to overcome these problems and
indeed it exhibited slower hydrolysis and retained capability of
BBB penetration. It was possible to demonstrate increased brain
retention in an AD mouse model compared to wild type and
single-photon emission computed tomography (SPECT) studies
revealed that radioactivity accumulated in cortical and sub-
cortical brain areas in accordance with BChE activity known
4
1/2
[12,25–26]
state.
For the reason of their complex binding mode,
carbamate-based PET tracers on the one hand need to be
designed particularly carefully with respect to their kinetic
ChemMedChem 2021, 16, 1427–1437
www.chemmedchem.org
1428
© 2021 The Authors. ChemMedChem published by Wiley-VCH GmbH

Full Papers
doi.org/10.1002/cmdc.202000942
ChemMedChem
Figure 1. Design of radiotracer 2 based on potent, selective BChE inhibitors 1a, 1b and 1c with (pseudo-)irreversible binding mode. The mechanism of
[12,19]
enzyme-inactivation is presented below.
1
1
behavior, but on the other hand offer great opportunities in
their application. The first aspect to be considered is the speed
heptyl chain (compound 1b, Figure 1) or introducing C as the
carbamate carbonyl group ([ C]-1a, Figure 1).
1
1
[19]
of carbamylation (k ), that is, the speed at which the enzyme is
Even though the affinity of the fluorinated compound was
not significantly altered, a harsh and complicated radiolabeling
procedure of four steps and a short half-life of enzyme
reactivation forced us to develop a new generation of
carbamates with heterocyclic moieties at the end of alkylene
chains of different lengths to examine their influence on
inhibition times. Thus, we found out that a morpholine
substitution at the end of a hexylene chain substantially
increases the time of enzyme inactivation and beneficially
increases water solubility of this highly lipophilic class of
3
deactivated and is, hence, radiolabeled through transfer of a
carbamate group comprising a positron emitting radionuclide.
Nevertheless, the carbamylation rate requires precise tuning: If
the rate is too high, the tracer may bind to BChE circulating in
the blood immediately hampering the suitability for BChE
imaging in the CNS. PET scans thus obtained only reflect the
blood flow and highlight highly vascularized areas of the body,
such as brain, liver, heart, kidneys, lung and the like, that is,
high brain uptakes may be found although not reflecting true
BChE distribution throughout the body. If the carbamylation
rate is too slow, however, the tracer may be metabolized before
binding to BChE occurs to a considerable extent, also rendering
the tracer inappropriate. Only if the carbamylation speed is at
an appropriate level, a molecular scan representing the true
distribution of BChE throughout the body and CNS can be
obtained. Once the enzyme is labeled with the radionuclide,
however, slow hydrolysis and long half-life time of the
carbamylated enzyme state result in a prolonged retention of
[12]
compounds. Consequently, in this study, we present a second
1
8
generation BChE-selective F-PET tracer with significantly
prolonged duration of action, which most widely retains its
inhibitory potency. Additionally, we demonstrate a new radio-
labeling strategy for carbamates leading to improved radio-
chemical yields. Last, we show preliminary ex vivo and in vivo
results of the tracer.
radioactivity in the area of interest allowing for increased time Results and Discussion
of observation and highly flexible study design. Therefore, the
ultimate aim for the successful design of a carbamate-based
PET tracer for BChE imaging is the provision of a radiolabeled
carbamate that is stably transferred to BChE to a considerable
extent within a short period of time, such as 5 minutes, but
remains bound to BChE for a preferably long time.
For the design of an advanced, pseudo-irreversible PET radio-
tracer, we chose highly selective and potent, carbamate-based
BChE inhibitor 1c (Figure 1, IC =49.3 nM, residual activity on
5
0
hAChE at 10 μM=26.1�1.0%) as scaffold, mainly due to its
pronounced decelerated speed of enzyme regeneration (1c: t
1/
[12,19]
Due to the scarcity of highly selective and nanomolar BChE
inhibitors, especially with pseudo-irreversible binding mode
=16.50 h, 1a: t1/2 =1.12 h, 1b: t1/2 =0.86 h).
Our aim was to
2
take advantage of a preferably long carbamoylated enzyme,
since this would presumably result in representative, precise
in vivo distribution of BChEs by PET imaging, when the radio-
1
8
meeting the demanding requirements, we developed a first F-
11
or C-labeled PET tracer by attaching fluorine at the end of the
ChemMedChem 2021, 16, 1427–1437
www.chemmedchem.org
1429
© 2021 The Authors. ChemMedChem published by Wiley-VCH GmbH

Full Papers
doi.org/10.1002/cmdc.202000942
ChemMedChem
nuclide is part of the transferred carbamate moiety (Figure 1).
Previously described structure-activity relationships revealed
that a hexylene-sized linker between the carbamate and
morpholine is best for both retaining inhibitory potency and a
nolino[1,2-b]quinazolin-10-ol carrier scaffold to yield the non-
radioactive compound 2 as a racemic mixture, which was used
for in vitro evaluation of hAChE and hBChE binding properties
and as a reference compound for analysis and purification after
[12]
18
prolonged duration of action. In contrast, the unsubstituted
carbamate bearing a heptyl chain attached at the carbamate
nitrogen exhibited a much shorter half-life of enzyme inhibition
F-labeling. Synthesis of the carrier scaffold has been described
[31]
18
before in detail. Because radiolabeling with F requires a
precursor bearing a suitable leaving group to be replaced by
the radionuclide, we developed a similar synthesis as presented
in Scheme 1 leading to a respective tosylated compound 14
(Scheme 2). N-benzylaminoethanol 3 was reacted with epichlor-
ohydrin, followed by an acid-mediated ether condensation
giving 4-benzyl-2-(chloromethyl)morpholine in satisfying
[25]
(t1/2 =1.12 h).
This obvious influence of the morpholine
moiety on the duration of enzyme inhibition has been well
investigated using ChE inhibitors carrying either a morpholine
[27]
or dimethylmorpholine moiety at the end of alkylene chain.
The carrier X is responsible for generating selectivity over AChE
[12]
[29]
and contributes to BChE binding. Considering the design of
yield. After that, the chlorine atom was replaced by a hydroxy
1
8
the radiotracer, we chose to introduce F in the compound,
because it has advantageous properties over other radio-
isotopes commonly used for nuclear imaging techniques like
group in a nucleophilic substitution reaction with excellent
[30]
yield,
and the benzyl protecting group was subsequently
removed by catalytic hydrogenation to yield morpholine-2-yl
methanol 9 quantitatively (Scheme 2). Compound 9 was N-
alkylated with 2-(6-bromohexyl)isoindoline-1,3-dione in satisfy-
ing yields for an upcoming hydrazinolysis (Gabriel synthesis).
Then the hydroxy group on the morpholine moiety was
activated by tosylation with 4-toluenesulfonyl chloride (TosCl)
in good yield. The resulting tosylate 10 was subjected to a
hydrazinolysis, and the amine 11 was subsequently activated
with p-nitrophenyl chloroformate and transferred onto 13-
methyl-5,8,13,13a-tetrahydro-6H-isoquinolino[1,2-b]quinazolin-
10-ol to give the desired tosyl carbamate 12 as precursor again
in excellent yields (Scheme 2). As carbamates can easily
6
8
PET. These include a long half-life (t =110 min, t1/2 ( Ga)=
6
1
/2
6
8
8 min), high positron yields (96.86%, Ga: 89.14%) and a good
spatial resolution, which is a result of relatively low positron
6
8
[28]
energies (0.65 MeV, Ga: 1.90 MeV). Consequently, we aimed
at the synthesis of the cold, fluorinated inhibitor 2 at first to
investigate its binding properties and kinetics. Moreover, this
compound was essential as reference during radiolabeling. Our
18
first investigated radiotracer [ F]-1b (Figure 1) required an
inconvenient radiolabeling procedure of four steps and the
respective cold reference had an even shorter half-life of
enzyme reactivation (t1/2 =0.86 h) than heptyl carbamate 1a (t1/
1
8
=
1.12 h). The synthetic procedure is shown in Scheme 1. The
decompose under the harsh conditions of F labeling, it is
2
[
19]
synthesis started from N-benzylaminoethanol 3, which was
reacted with epifluorohydrin and cyclized in an acid-mediated
intramolecular ether formation to give 4-benzylated 2-
necessary to employ a protecting group strategy.
We
adopted a facile method by introducing a methoxymethyl ether
(MOM) group at the carbamate nitrogen, without using highly
[29]
[32]
fluoromethylmorpholine 4 in satisfying yield (Scheme 1).
Subsequent deprotection of the benzylamine by catalytic
cancerogenic methoxymethyl chloride.
In the first step, a
chloromethyl group was attached in situ at the carbamate
yielding compound 13. The reaction mixture was subsequently
quenched with methanol to give the desired MOM-protected
precursor in very good yields (Scheme 2). However, this
inevitable modification of our precursor forced us to perform an
additional deprotection step after radiolabeling, which might
[30]
hydrogenation, and Gabriel synthesis with 2-(6-bromohexyl)
isoindoline-1,3-dione, were followed by a hydrazinolysis, which
gave hexylamine 7 in very good yields. The resulting amine was
activated with p-nitrophenyl chloroformate and subsequently
transferred onto the 13-methyl-5,8,13,13a-tetrahydro-6H-isoqui-
1
8
Scheme 1. Synthesis of nonradioactive compound 2 for in vitro ChE testing and as reference compound for HPLC purification after F labeling. i) 1.
epifluorohydrine, RT, 3 h; 2. H
2
SO
4
, 140°C, 1 h, 67%; ii) H
2
, Pd/C, MeOH, RT, 1 h, quant.; iii) 2-(6-bromohexyl)isoindoline-1,3-dione, NEt
3
, DMF, 105°C, 4 h, 63%;
iv) H
4
N
2
·H
2
O, EtOH, 85°C, 4 h, 77%; v) 1. p-nitrophenyl chloroformate, NEt
3 2 2
, CH Cl , 2 h, RT, 67%; 2. 13-methyl-5,8,13,13a-tetrahydro-6H-isoquinolino[1,2-b]
2 2
quinazolin-10-ol, NaH, CH Cl , 1 h, RT, 75%.
ChemMedChem 2021, 16, 1427–1437
www.chemmedchem.org
1430
© 2021 The Authors. ChemMedChem published by Wiley-VCH GmbH

Full Papers
doi.org/10.1002/cmdc.202000942
ChemMedChem
1
8
18
Scheme 2. Synthesis of MOM-protected, tosylated precursor 14 for F labeling to [ F]-2. i) 1. epichlorohydrine, RT, 3 h; 2. H
2
SO
4
, 140°C, 1 h, 67% over two
steps; 3. H
2
O, CHONH
2
, 145°C, 20 h, 93%; ii) H
2
, Pd/C, MeOH, RT, 1 h, quant.; iii) 1. 2-(6-bromohexyl)isoindoline-1,3-dione, N,N-diisopropylethylamine (DIPEA),
DMF, 100°C, 20 h, 66%; 2. TosCl, NEt
, CH
Cl
, RT, 20 h, 71%; iv) H
N
2
·H
O, EtOH, 80°C, 1.5 h, 88%; v) 1. p-nitrophenyl chloroformate, NEt
, CH
Cl
2 2
Cl , 3 h, RT, 55%;
3
2
2
4
2
3
2
. 13-methyl-5,8,13,13a-tetrahydro-6H-isoquinolino[1,2-b]quinazolin-10-ol, NaH, CH
MeOH, RT, 1 h, 85% (two steps); viii) 1. [ F]KF, K222, MeCN, 110°C, 10 min; 2. 6 M HClaq, 90°C, 5 min.
2
Cl
2
, 2 h, RT, 94%; vi) TMSÀ Cl, para-formaldehyde, CH
2
2
, RT, 18 h; vii)
1
8
C 3 4
Table 1. Inhibitory potencies and kinetic parameters of heptylcarbamate 1a, and cold reference compound 2. K , k and k are presented with SEM (standard
error of the mean) for triplicates of the respective experiments (n=3).
Cmpd.
IC50 (hBChE [nM])
IC50 (hAChE [nM])
K
c
� SEM (hBChE [nM])
[12,19]
[12,19]
[12,19]
1a
1b
1c
2
6.4[
3800
28.5�17.2
24.3�19.3
19]
[19]
[19]
5.2
3600
[12]
[12]
[12]
49.3
>100000
202�55
66.6
>100000
199.9�160.4
k
3
�SEM (hBChE [minÀ 1])
k
4
�SEM (hBChE [hÀ 1])
t1/2 [h]
[
[
[
12,19]
[12,19]
[12,19]
[19]
1a
1b
1c
2
0.66�0.32
0.78�0.48
0.16�0.02
0.42�0.25
0.62�0.04
0.81�0.04
1.12
0.86
16.50
16.13
19]
[19]
12]
[12]
[12]
0.042�0.004
0.043�0.003
be disadvantageous due to additional time investment for
reaction and work-up. Even though the introduction of a
fluoromethyl group might represent just a minor modification
on the parent BChE inhibitor 1c (Figure 1), we fully charac-
terized cold reference compound 2 for its retained inhibitory
potency in colorimetric Ellman’s assay and performed an
measured by adding the synthetic substrate of each ChE
(acetylthiocholine for AChE and butyrylthiocholine for BChE).
After reaction of the hydrolyzed thiols with DTNB and their
photometric quantification, enzyme activity was calculated. The
evaluation in kinetic studies revealed that the pseudo-irrever-
sible binding of carbamate 2 is comparable to that of
compound 1c. K and k representing kinetic values for the
[12,19, 33]
additional thorough kinetic study to compare the results.
c
3
Thus, we have proven that both the binding properties and
kinetic characteristics are in excellent accordance with the
parent compound 1c and greatly exceed the properties of
heptylcarbamate 1a (Table 1). The colorimetric assay (Ellman’s
assay) was performed by incubation of a solution of 5,5’-
dithiobis-(2-nitrobenzoic acid) (DTNB) and the respective hu-
man ChE with the inhibitor. After 20 min, enzyme activity was
carbamoylation step appear to be in the suitable range to allow
for molecular scans reflecting the true BChE distribution among
the whole body. The decarbamoylation step is kinetically
characterized by k and the half-life of enzyme reactivation, t1/2.
4
[12,19, 34]
Dilution experiments (Figure 2)
revealed a time-depend-
ent plot of enzyme activity characterized by a first-order
ChemMedChem 2021, 16, 1427–1437
www.chemmedchem.org
1431
© 2021 The Authors. ChemMedChem published by Wiley-VCH GmbH

Full Papers
doi.org/10.1002/cmdc.202000942
ChemMedChem
In the next step, we characterized the radiotracer’s in vitro
properties in protein binding assays on hBChE. First, the time
course of binding was investigated by incubating a mixture of
1
8
hBChE and radiotracer [ F]-2 in PBS for 5, 10 and 20 min at
3
2
7°C. A maximum of tracer-bound hBChE was reached after
0 min (Figure 3). This demonstrates the specificity of tracer
binding to hBChE and the maximum after 20 min is in good
agreement with previous kinetic studies of compounds 1a, 1b
and 1c (Figure 1), where equilibrium constant K and rate
C
[
12,19,25]
constant of carbamoylation k were determined.
Addition-
3
ally, we studied the blocking of tracer binding in vitro by the
known reversible BChE-selective inhibitor ethopropazine and
[35]
nonselective, reversible ChE inhibitor tacrine.
Therefore, a
solution of hBChE in PBS was preincubated with ethopropazine
hydrochloride (60 μM) or tacrine (1 μM), respectively. Subse-
1
8
quently, [ F]-2 was added to each of the inhibitors and
incubated for 20 min at 37°C (cf. Figure S1 in the Supporting
Information). Next, we investigated the binding properties of
1
8
[
F]-2 to mice brain tissue using autoradiography. After
incubation of healthy mouse brain slices with the tracer,
autoradiography images exhibited high accumulation of radio-
activity in relevant brain areas (Figure 4), proving a good
1
8
[36]
binding of [ F]-2 to brain tissue. In parallel, we performed
blocking experiments on mice brain tissue preincubated with
ethopropazine hydrochloride, a selective inhibitor of BChE that
prevents binding of other BChE inhibitors and substrates due to
Figure 2. Recovery of hBChE activity after pseudo-irreversible inhibition with
compounds 1a, 1b, 1c, and 2 and subsequent dilution. Normalized enzyme
activity (1=full activity) was measured in triplicate for each time point
(mean�SD).
[35a]
its blocking of the active site.
Consequently, we found a
1
8
significantly decreased binding of [ F]-2 to the preincubated
tissue, which demonstrates the specificity of the tracer towards
BChE binding in areas with high BChE activity like white matter
bundles, thalamus, upper brainstem and cerebral cortex. It
has to be mentioned that a higher level of displaceable binding
exponential curve and the related Equation (1), out of which t1/2
and k were calculated (Figure 2).
4
[
36]
À
�
À k4t
A ¼ 1 À e
ꢀ ð1 À A Þ þ A
(1)
0
0
1
8
was observed in autoradiography experiments with [ F]-1b,
which is likely due to its higher binding affinity (IC =
In summary, we found that our cold inhibitor 2 shows a
very minor decrease in its inhibitory potency compared to
parent compound 1c (Table 1 and Figure 2), but still exhibits
binding affinity in the two-digit nanomolar range. Importantly,
50
[19]
5.2 nM). Nevertheless, the complicated radiolabeling proce-
dure and unfavorable kinetic parameters of this tracer are
1
8
disadvantageous compared to [ F]-2. Due to the successful
it retains long duration of action considering the half-life (t1/2
=
1
6.13 h) and rate constant k (Table 1, Figure 2), which met our
4
expectations. Altogether, the pronounced selectivity over
hAChE, the sufficiently fast transfer of the carbamate onto the
enzyme and long binding represent promising attributes for a
suitable BChE PET tracer. Consequently, we used precursor 14
1
8
(
Scheme 2) for F radiolabeling to generate the respective
radiotracer. In the first step, we adopted an established
1
8
procedure for F labeling of tosylates by nucleophilic substitu-
1
8
tion. [ F]KF was reacted with the precursor at elevated temper-
atures in the presence of a [2.2.2.]cryptand (K2.2.2). Finally, the
MOM group was deprotected with 6 M hydrochloric acid.
Purification of the tracer was achieved by semi-preparative
high-performance liquid chromatography (HPLC). Thus, we
obtained the tracer in a time frame of 180 min and a
radiochemical yield of 13% after decay correction (specific
activity a=191 GBq/mmol). Autoradiography of a thin-layer
chromatogram (TLC) indicated sufficient purity (95.3%) of our
tracer for subsequent preliminary ex vivo and in vivo experi-
ments.
1
8
Figure 3. Time-dependent in vitro hBChE binding assay with radiotracer [ F]-
. Enzyme-bound radioactivity was measured in triplicate after 5, 10 and
2
20 min (mean�SD). % Dose: the percentage of binding dose according to
overall radioactivity used, U: the enzyme unit for BChE added in each vial.
ChemMedChem 2021, 16, 1427–1437
www.chemmedchem.org
1432
© 2021 The Authors. ChemMedChem published by Wiley-VCH GmbH

Full Papers
doi.org/10.1002/cmdc.202000942
ChemMedChem
tracer reaches heart, lung and liver with increased activity, a
potential application is given in prognosis, diagnosis and
monitoring disease state and progress or malignancy grade in
certain types of cancer, which can be associated with down-
[38]
and/or upregulation of BChE. In particular, BChE expression
has been found to decrease significantly in large-cell- and
[39]
squamous-cell lung carcinoma. Therefore, an application of
the tracer in diseases monitoring and prognosis in lung cancer
might be conceivable. Further studies in animal models are
warranted based on the current initial evaluation to prove its
feasibility.
Figure 4. Autoradiography of mice brain slices incubated with radiotracer
18
[
F]-2. Left: without and right: with ethopropazine hydrochloride. Bright
Conclusion
areas represent high binding of the tracer. Tracer intensity [arbitrary units] is
shown as a percentage of the maximum tracer activity.
1
8
In this study, we present design and synthesis of an F-labeled
radiotracer for PET imaging of BChE. A potent and selective
BChE inhibitor with pseudo-irreversible binding mode served as
parent compound due to its long duration of action. We
modified the carbamate moiety by attaching a fluoromethyl
group to the morpholine moiety, which is responsible for the
prolonged enzyme inhibition. We present a facile synthesis of
both cold reference compound and a MOM-protected tosylate
radiolabeling and promising in vitro and ex vivo results, we felt
motivated to initially evaluate the radiotracers in vivo character-
istics in PET studies. Therefore, a solution of the tracer (5%
ethanol in saline) was injected via tail vein to a healthy male
Wistar rat, which was directly scanned in a small animal PET
system to obtain dynamic images and generate time-activity
curves of regions of interest (Figure 5). From both dynamic
images and time activity curves only limited brain uptake of the
tracer could be observed, which could indicate limited BBB
penetration. After initial blood-pool circulation and a rapid
enrichment in heart and lung, the tracer accumulates in liver
tissue. BChE occurs in the above-mentioned organs in an
1
8
precursor, which is very suitable for F-radiolabeling in good
radiochemical yields. Thus, we provide access to a new method
to radiolabel sensitive carbamates. Colorimetric in vitro studies
revealed that the compound widely retains the inhibitory
potency of the parent compound, and kinetic investigations
exhibited that the half-life of enzyme reactivation due to slow
hydrolysis of the carbamate is in excellent accordance with the
parent compound as well. Radiolabeling of the tosylate
precursor was achieved in two steps through an established
procedure with subsequent MOM deprotection in a reasonable
time frame and good radiochemical yields. Subsequently, we
investigated the tracer’s properties in protein binding studies
and found that the time course of binding was in accordance
with our expectations. Consequently, we applied the radiotracer
in preliminary ex vivo autoradiography studies on mice brain
slices, where we could observe good binding to brain tissue.
Additionally, we demonstrated the specificity of binding to
BChE in blocking experiments with the selective BChE inhibitor
ethopropazine. Finally, we present a preliminary in vivo PET
study on a healthy male rat, where we found only limited brain
uptake. The tracer rapidly addressed heart and lung, accom-
panied by long-term accumulation in liver tissue - organs with
BChE expression. Future studies will build upon these results
and elucidate the precise biodistribution of the tracer to clarify
the remaining question of its limited brain uptake, which is
likely due to the inability to penetrate the blood-brain-barrier
efficiently or extrusion by P-glycoprotein. A potential applica-
tion of this tracer in diagnosis and monitoring of diseases with
altered BChE activity in the addressed organs, such as certain
types of lung cancer, is conceivable.
[37]
elevated manner. It is noteworthy that parent compound 1c
and closely related compounds) exhibited pronounced activity
(
[12]
in vivo. Duration of action at hBChE in vitro (t1/2) correlates
well with neuroprotective effects in vivo. However, since the
Figure 5. Preliminary in vivo evaluation of the tracer. Top: Dynamic coronal
18
PET images after tail vein injection of a solution of [ F]-2 to a male Wistar
rat and subsequent microPET scanning over 40 min. Bottom: Time–activity
curves of regions of interest obtained from the above images (A.U=arbitrary
unit).
ChemMedChem 2021, 16, 1427–1437
www.chemmedchem.org
1433
© 2021 The Authors. ChemMedChem published by Wiley-VCH GmbH

Full Papers
doi.org/10.1002/cmdc.202000942
ChemMedChem
1
3
Experimental Section
3.18–3.12 (m, 1H), 3.10–2.99 ppm (m, 1H); C NMR (101 MHz,
CD OD): δ=168.2 (1 C), 84.8 (1 C), 83.1 (1 C), 73.6 (0.5 C), 73.4
0.5 C), 64.9 (1 C), 43.8 ppm (1 C); MS (ESI): [M+H] calcd for
3
+
(
Chemistry
C H FNO=120.08; found 120.15.
5
10
Reagents and solvents were obtained from commercial suppliers in
reagent grade and were used without further purification unless
stated otherwise. Dichloromethane as solvent was distilled from
CaH2 under argon. The reaction progress was controlled with
analytical thin-layer chromatography (TLC) on precoated silica gel
GF254 plates (Macherey Nagel, Düren, Germany). Compounds were
detected under UV light (254 and 366 nm) or through staining with
2
(
1
-{6-[2-(Fluoromethyl)morpholino]hexyl}isoindoline-1,3-dione (6): 2-
Fluoromethyl)morpholine hydroformiate (5; 1001 mg, 6.06 mmol,
equiv.) was dissolved in dry dimethylformamide (10 mL) and
triethylamine (3 mL) was added. Then 2-(6-bromohexyl)isoindoline-
,3-dione (2 g, 6,45 mmol, 1.06 equiv.) was added and the resulting
1
solution was heated to 105°C for 4 h. The mixture was allowed to
cool down to room temperature and water was added. The
aqueous layer was extracted with ethyl acetate (3x). Then the
combined organic layers were washed with water and brine, dried
over sodium sulphate, filtered, and the solvent was evaporated
under reduced pressure. The residue was purified with flash column
chromatography (CH Cl /MeOH 98:2, R =0.33) to yield 2-(6-[2-
iodine, KMnO , or Ehrlich’s reagent. Crude products after work-up
4
were purified by manual flash column chromatography. Silica gel
(particle size of 40–63 μM; VWR chemicals, Leuven, Belgium) was
used as the stationary phase and mixtures from petroleum ether/
ethyl acetate or dichloromethane/methanol as eluent systems. The
pure compounds were submitted for nuclear magnetic resonance
spectra on a AV-400 NMR instrument (Bruker) in deuterated
solvents (DMSO-d , CDCl , CD Cl , CD OD). Chemical shifts are
2
2
f
(fluoromethyl)morpholino]hexyl}isoindoline-1,3-dione (6; 1.334 g,
1
3
.83 mmol, 63%) as colorless oil. H NMR (400 MHz, CDCl ): δ=
3
6
3
2
2
3
7
.80–7.74 (m, 2H), 7.66–7.61 (m, 2H), 4.41–4.36 (m, 1H), 4.29–4.24
expressed in ppm relative to DMSO-d , CDCl , CD Cl , or CD OD
6
3
2
2
3
1
13
(m, 1H), 3.83 (ddd, J=11.3, 3.3, 1.7 Hz, 1H), 3.66–3.50 (m, 4H), 2.74–
.67 (m, 1H), 2.61 (dd, J=11.5, 1.9 Hz, 1H), 2.34–2.22 (m, 2H), 2.05
td, J=11.4, 3.3 Hz, 1H), 1.94–1.82 (m, 1H), 1.69–1.56 (m, 3H), 1.50–
(
2.50/7.26/5.32/3.31 for H; 39.5/77.2/53.5/49.0 for C). Purity of
2
(
compounds was controlled with analytical HPLC on a Shimadzu
system equipped with a DGU-20A3R controller, LC20AB liquid
chromatograph, and SPD-20 A UV/vis detector (Shimadzu): Sta-
tionary phase: Synergi 4 U fusion RP (Synergi, Aschaffenburg,
Germany) column; mobile phase: water+0.1% formic acid (phase
A) and methanol+0.1% formic acid (phase B) with a flow of
1
3
1
1
7
3
.36 (m, 3H), 1.37–1.24 ppm (m, 4H); C NMR (101 MHz, CDCl ): δ=
3
68.4 (1 C), 133.9 (2 C), 132.2 (1 C), 123.3 (2 C), 84.9 (1 C), 83.1 (1 C),
4.3 (0.5 C), 74.1 (0.5 C), 66.8 (1 C), 58.8 (1 C), 54.0 (1 C), 52.9 (1 C),
7.9 (1 C), 28.5 (1 C), 27.0 (1 C), 26.7 (1 C), 26.4 ppm (1 C); MS (ESI):
+
[M+H] calcd for C H N O =349.19, found 349.15.
1
9
26
2
4
1.0 mL/min. Method: conc. B, gradient 5!90% from 0 to 8 min,
90% isocratic from 8 to 13 min, gradient 90!5% from 13 to
15 min, 5% isocratic from 15 to 18 min. Compounds were detected
6
2
1
-[2-(Fluoromethyl)morpholino]hexan-1-amine (7): To a solution of
-{6-[2-(fluoromethyl)morpholino]hexyl}isoindoline-1,3-dione (6;
.2 g, 3.44 mmol, 1 equiv.) in ethanol (50 mL), hydrazine hydrate
at λ=254 nm, and target compounds were ꢁ95% pure.
(
1 mL, >5 equiv.) was added. The solution was heated to 85°C for
4
-Benzyl-2-(fluoromethyl)morpholine (4): In a flask, 2-(benzylamino)
4
h. Then the mixture was allowed to cool down to room
ethan-1-ol (3; 1.866 mL, 1.988 g, 13.15 mmol, 1 equiv.) and epifluor-
ohydrin (1 g, 13.15 mmol, 1 equiv.) were mixed together and stirred
for 3 h at room temperature. Then concentrated H SO (5 mL) was
temperature and the precipitate was filtered off. The filtrate was
evaporated to dryness under reduced pressure. The residue was
triturated with CH Cl , filtered, and the filtrate was evaporated
2
4
2
2
added carefully and the mixture was heated to 140°C for 1 h.
Afterwards, the reaction mixture was allowed to cool down to room
temperature and was poured onto ice. The aqueous layer was
basified with aqueous sodium hydroxide (10 M) and extracted with
three portions of ethyl acetate. The combined organic layers were
washed with water and brine, dried over sodium sulphate, filtered,
and evaporated to dryness under reduced pressure. The residue
was purified with flash column chromatography (petroleum ether/
ethyl acetate 3:1, Rf =0.35) to yield 4-benzyl-2-(fluoroethyl)
under reduced pressure to yield 6-[2-(fluoromethyl)morpholino]
hexan-1-amine (7; 577 mg, 2.64 mmol, 77%) as colorless oil without
further purification. H NMR (400 MHz, CDCl ): δ=4.48–4.42 (m, 1H),
1
3
3
.95–3.86 (m, 1H), 3.72–3.64 (m, 1H), 3.64–3.58 (m, 1H), 2.80–2.74
(m, 1H), 2.71–2.63 (m, 3H), 2.37–2.29 (m, 2H), 2.12 (td, J=11.5,
3
4
7
.4 Hz, 1H), 2.01–1.89 (m, 1H), 1.55–1.41 (m, 4H), 1.39–1.27 ppm (m,
1
3
H); C NMR (101 MHz, CDCl ): δ=85.0 (1 C), 83.3 (1 C), 74.4 (0.5 C),
3
4.2 (0.5 C), 67.0 (1 C), 59.1 (1 C), 54.2 (1 C), 54.1 (1 C), 53.1 (1 C),
1
42.2 (1 C), 33.7 (1 C), 27.4 (1 C), 26.9 (1 C), 26.6 ppm (1 C); MS (ESI):
morpholine (4; 1.31 g, 6.26 mmol, 48%) as colorless oil. H NMR
+
[M+H] calcd for C H FN OH=219.19; found 219.20.
11 23 2
(
400 MHz, CDCl ): δ=7.29–7.14 (m, 5H), 4.41–4.31 (m, 1H), 4.29–
3
4
.21 (m, 1H), 3.89–3.58 (m, 3H), 3.53–3.35 (m, 2H), 2.74–2.55 (m, 2H),
13
4
a
-Nitrophenyl-{6-[2-(fluoromethyl)morpholino]hexyl}-carbamate: To
solution of 6-[2-(fluoromethyl)morpholino]hexan-1-amine (7;
2
.14 (td, J=11.4, 3.3 Hz, 1H), 2.04–1.86 ppm (m, 1H); C NMR
(101 MHz, CDCl ): δ=137.7 (1 C), 129.3 (2 C), 128.5 (2 C), 127.4 (1 C),
3
500 mg, 2.29 mmol) in dry dichloromethane (10 mL), triethylamine
0.5 mL) and 4-nitrophenylchloroformiate (508 mg, 2.52 mmol)
8
5.0 (1 C), 83.3 (1 C), 74.5 (0.5 C), 74.3 (0.5 C), 66.9 (1 C), 63.5 (1 C),
(
+
53.9 (1 C), 53.8 (1 C), 53.0 ppm (1 C); MS (ESI): [M+H] calcd for
were added. The resulting solution was stirred for 2 h at room
temperature. Then the solvent was evaporated under reduced
pressure and the residue was purified by flash column chromatog-
raphy (CH Cl /MeOH 99:1, R =0.11) to yield 4-nitrophenyl {6-[2-
C H NOF=210.13, found 210.10.
1
2
16
2-(Fluoromethyl)morpholine hydroformiate (5): A solution of 4-
2
2
f
benzyl-2-(fluoromethyl)morpholine (4; 1.3 g, 6.21 mmol, 1 equiv.) in
MeOH (30 mL) was prepared and formic acid (0.5 mL) was added.
No precipitation was observed. Then palladium on activated
charcoal (10 wt/%, 130 mg) was added and the atmosphere was
replaced with hydrogen. The mixture was stirred for 1 h at room
temperature. After that, the catalyst was filtered off by a pad of
celite, which was washed with methanol. The filtrate was evapo-
rated to dryness in vacuo to yield 2-(fluoromethyl)morpholine
hydroformiate (5; 1.025 g, 6.21 mmol, quant.) as colorless oil.
NMR (400 MHz, CD OD): δ=8.37 (s, 1H), 4.57–4.49 (m, 1H), 4.44–
4
(
6
fluoromethyl)morpholino]hexyl}carbamate (588 mg, 1.53 mmol,
7%) as yellow oil. MS (ESI): [M+H] calcd for C H FN O =
18 26 3 5
+
3
84.20, found 384.05.
Due to high instability of the title compound only LCMS data was
measured, before directly reacting it in the next step.
1
3-Methyl-5,8,13,13a-tetrahydro-6H-isoquinolino[1,2-b]quinazolin-
1
10-yl-{6-[2-(fluoromethyl)morpholino]-hexyl}carbamate (2): To a sol-
ution of 4-nitrophenyl [6-(2-(fluoromethyl)morpholino)hexyl]
carbamate (86 mg, 0.23 mmol, 1.2 equiv.) and 13-methyl-5,8,13,13a-
H
3
.37 (m, 1H), 4.15–4.06 (m, 1H), 4.04–3.90 (m, 1H), 3.90–3.80 (m, 1H),
.39–3.32 (m, 1H), 3.29–3.26 (m, 1H), 3.25 (dt, J=2.5, 1.3 Hz, 1H),
tetrahydro-6H-isoquinolino[1,2-b]quinazolin-10-ol
(50 mg,
3
ChemMedChem 2021, 16, 1427–1437
www.chemmedchem.org
1434
© 2021 The Authors. ChemMedChem published by Wiley-VCH GmbH

Full Papers
doi.org/10.1002/cmdc.202000942
ChemMedChem
0
.19 mmol, 1 equiv.), sodium hydride (60% suspension in paraffin
instead of the compound solution. 10% of ethanol did not reduce
enzyme activity. The enzyme activity in percent of maximum
activity was plotted against the logarithmic inhibitor concentration,
from which IC50 values were calculated with the software GraphPad
Prism 5.
oil, 9 mg, 0.23 mmol, 1 equiv.) was added. The resulting mixture
was stirred for 1 h at room temperature. Then water was added,
and the aqueous layer was extracted with three portions of
dichloromethane. The combined organic layers were washed with
water, saturated aqueous sodium hydrogen carbonate solution and
brine, dried over sodium sulphate, filtered, and evaporated to
dryness under reduced pressure. The residue was purified by flash
column chromatography (CH Cl /MeOH 98:2, R =0.14) to yield 13-
Kinetic studies: For determination of K and k values, the same
c
3
general setup was used (900 μL of the assay buffer, 30 μL of DTNB
solution (10 mM, 0.3 mM in the assay), and 30 μL of hBChE solution
2
2
f
(
2.5 units/mL) were mixed at room temperature). Then, 30 μL of
methyl-5,8,13,13a-tetrahydro-6H-isoquinolino[1,2-b]quinazolin-10-yl
6-[2-(fluoromethyl)morpholino]hexyl}carbamate (2; 72 mg, 0.14
inhibitor 2 (Scheme 1) solutions with different concentrations were
{
1
added, but enzyme activity was determined after 1, 2, 4, 6, 10, 15,
μmol, 75%) as colorless oil. H NMR (400 MHz, CD Cl ): δ=7.38–7.32
2
2
2
0, 30, and 40 min of incubation by addition of 6 μL of BTC solution
(m, 1H), 7.22–7.15 (m, 2H), 7.14–7.09 (m, 1H), 6.89–6.81 (m, 2H),
(75 mM, 452 μM in the assay). The absorbance at λ=412 nm was
6
3
3
2
1
1
.74–6.68 (m, 1H), 4.77 (s, 1H), 4.42–4.35 (m, 1H), 4.30–4.22 (m, 1H),
.94 (d, J=15.6 Hz, 1H), 3.89–3.79 (m, 2H), 3.78–3.58 (m, 2H), 3.24–
.11 (m, 3H), 3.10–2.98 (m, 1H), 2.83–2.60 (m, 4H), 2.51 (s, 3H), 2.34–
.26 (m, 2H), 2.06 (td, J=11.4, 3.3 Hz, 1H), 1.94–1.82 (m, 1H), 1.55–
measured after 2.5 min. All concentrations were measured three
times, at least five different concentrations were measured for at
least seven time points. The obtained enzyme activities in percent
of maximum enzyme activity were plotted in a time-dependent
manner and fitted to Equation (2) to determine the rate constant
kobs using the software GraphPad Prism 5.
13
.39 (m, 4H), 1.37–1.26 ppm (m, 4H); C NMR (101 MHz, CD Cl ): δ=
2
2
55.5 (1 C), 146.3 (1 C), 144.9 (1 C), 136.7 (1 C), 134.5 (1 C), 129.1
(1 C), 128.9 (1 C), 127.7 (1 C), 126.1 (1 C), 120.7 (1 C), 120.5 (1 C),
1
6
5
20.0 (1 C), 85.5 (1 C), 83.8 (1 C), 76.8 (1 C), 74.8 (0.5 C), 74.6 (0.5 C),
7.0 (1 C), 56.5 (1 C), 54.38 (1 C), 54.33 (1 C), 54.26 (1 C), 53.8 (1 C),
A ¼ A
ꢀ eÀ kobsꢀt þ A
(2)
0
1
3.4 (1 C), 48.3 (1 C), 41.5 (1 C), 38.2 (1 C), 30.2 (1 C), 29.0 (1 C), 27.4
+
(
1 C), 27.0 (1 C), 26.8 ppm (1 C); MS (ESI): [M+H] calcd for
A: enzyme activity at time t, A : enzyme activity at time t=0, 100%;
0
A : enzyme activity at infinite time.
1
C H FN O =511.31, found 511.15; HPLC: t =6.74 min, 97.5%
2
9
39
4
3
R
purity.
The obtained inverse rate constants k were plotted against the
reciprocal concentration [I] , and k was calculated from the y-
intercept of the resulting curve, and Kc from the slope of the
resulting linearization according to Equation (3) using the software
GraphPad Prism 5.
obs
À 1
Experimental procedures for the synthesis of MOM-protected
precursor 14 (Scheme 2) are described in the Supporting Informa-
tion.
3
Enzyme inhibition
1
Kc
1
1
¼
ꢀ
þ
(
3)
hAChE (EC 3.1.1.7, from human erythrocytes), DTNB (Ellman’s
reagent), ATC and BTC iodides were purchased from Sigma–Aldrich.
hBChE (E.C. 3.1.1.8) was kindly donated by Dr. Oksana Lockridge,
Nebraska Medical Centre. For the assays, the inhibitors were
dissolved in ethanol (absolute, reag. Ph. Eur.) to give a concen-
tration of 3.33 mM (100 μM in the assay) and stepwise diluted to
kobs k3 ½Iꢂ k3
[
I]: concentration of BChE inhibitor 2 (Scheme 1).
For the measurement of decarbamylation kinetics, the enzyme was
incubated with inhibitor 2 to carbamylate >85% of the enzyme.
After 1 h, the solution was diluted 1000-fold so that no enzyme was
carbamylated anymore. The enzyme activity was measured at
several (at least 8) time points, as described above. For the
determination of full enzyme activity, a batch of the enzyme was
treated with ethanol instead of inhibitor solution and diluted 1000-
fold in the same manner. The enzyme activity in percent was
plotted against time after dilution to give first-order rate constant
3
.33 nM (0.1 nM in the assay). Buffer was prepared from 3.12 g of
potassium dihydrogen phosphate in 500 mL of doubly distilled
water. After the potassium dihydrogen phosphate was dissolved,
the pH value was adjusted to pH 8.0 with 0.1 M sodium hydroxide
solution. Both enzymes were dissolved in assay buffer and diluted
to 2.5 units/mL. The solutions were stabilized with 1 mg/mL of
bovine serum albumin (Sigma–Aldrich) and were stored at 7°C until
usage. DTNB was dissolved in buffer at 10 mM (0.3 mM in the
assay). The substrates ATC and BTC were prepared with a
concentration of 75 mM (452 μM in the assay) in assay buffer and
kept frozen until usage. The absorbance of probes was measured
with a Shimadzu UVmini-1240 spectrometer at 412 nm.
k according to Equation (1) using the software GraphPad Prism 5.
All experiments were carried out in triplicates.
4
Radiochemistry
Solvents and chemicals were purchased from Aldrich and directly
IC50 determination: The assay was carried out at room temperature
1
8
À
(
25°C). Thereby, 900 μL of the buffer, 30 μL of DTNB solution
(
used without further purification. [ F]F was produced by a
cyclotron (GE Medical Systems, Uppsala) at the Department of
Nuclear Medicine of the University Hospital of Würzburg. Enriched
10 mM, 0.3 mM in the assay) and 30 μL of enzyme solution (hAChE
or hBChE, 2.5 units/mL) were mixed in a cuvette. The incubation
was started directly after the addition of 30 μL of inhibitor 2
1
8
18
À
[
O]H O was irradiated with protons to produce [ F]F and
2
radiofluorination was carried out manually. HPLC was used for
purification and analyses of radioactive products (Shimadzu system
equipped with UV detector, λ=220 and 254 nm, and γ-detector).
Purified radiotracer 2 (Scheme 1) was diluted with either PBS or
saline to the corresponding concentration for further evaluation.
(
Scheme 1) solutions with different concentrations. The solutions
were mixed well by manual stirring. After 20 min incubation, 6 μL
of substrate solution (ATC or BTC; 75 mM, 452 μM in the assay)
were added. The mixture was left for 2.5 min to allow substrate
hydrolysis, and the absorbance was measured at λ=412 nm,
whereas enzyme activity was determined three times for every
concentration with at least seven different concentrations. A blank
value was determined by replacing the enzyme solution with
buffer; the compound solution was replaced with ethanol. The
maximum enzyme activity was determined with 30 μL of ethanol
1
1
2
3-Methyl-5,8,13,13a-tetrahydro-6H-isoquinolino[1,2-b] quinazolin-
1
8
1
8
0-yl-{6-[2-([ F]-fluoromethyl)morpholino]-hexyl}carbamate ([ F]-
18
À
18
): [ F]F was separated from [ O]H O by an anion exchange
2
cartridge (Sep-Pak QMA Cartridge) and eluted with 0.5 mL of
ChemMedChem 2021, 16, 1427–1437
www.chemmedchem.org
1435
© 2021 The Authors. ChemMedChem published by Wiley-VCH GmbH

Full Papers
doi.org/10.1002/cmdc.202000942
ChemMedChem
2
5 mM potassium carbonate and 50 mM Kryptofix222 solution in
Center) for providing hBChE. Open access funding enabled and
organized by Projekt DEAL.
MeCN/H O (6:2) into a V-vial. The solution was dried at 120°C
2
under a nitrogen flow, which was repeated twice with 500 μL of
anhydrous MeCN. Labeling was carried out using 1.3 mg of
precursor 14 (Scheme 2) dissolved in 0.4 mL of anhydrous MeCN at
Conflict of Interest
1
10°C for 20 min, followed by addition of 100 μL of 6 M
hydrochloric acid with further 5 min heating at 90°C. After addition
of 500 μL of 1 M aqueous sodium hydrogencarbonate solution and
The authors declare no conflict of interest.
1
0 mL water to quench the reaction, the crude product was
trapped on a Sep-Pak light C18, which was washed with 5 mL of
water and eluted with 0.5 mL of ethanol. The crude radiotracer was
purified via semi-preparative HPLC. Column: 10×100 mm C18
Keywords: carbamate · enzyme kinetics · fluorine-18 · positron
emission tomography · radiotracers
Phenomenex Onyx Monolithic, Mobile phase: Phase A: H O, Phase
2
B: MeCN, 0–20 min, 20%!80% B, 20–26 min, 95% B, 28–30 min
20% B, Flow rate: 1.5 ml/min. The collected fraction was diluted
with 10 mL water and trapped on a Sep-Pak light C18, which was
dried with 5 mL air and eluted with 0.3 mL ethanol. The total
radiolabeling procedure was feasible in 180 min in a radiochemical
yield of 13% (corrected for decay). Sufficient radiochemical purity
was measured by TLC autoradiography (95.3%).
[
1] a) E. Nichols, C. E. I. Szoeke, S. E. Vollset, N. Abbasi, F. Abd-Allah, et al.,
Lancet Neurol. 2019, 18, 88–106; b) M. Crous-Bou, C. Minguillón, N.
Gramunt, J. Molinuevo, Alzheimer’s Res. Ther. 2017, 9, 71; c) Alzheimer’s
Dementia 2019, 15, 321–387.
[2] A. Alzheimer, Neurologisches Centralblatt 1906, 25, 1134.
[
[
[
3] R. Katzman, Arch. Neurol. 1976, 33, 217–218.
4] R. Sengoku, Neuropathology 2020, 40, 22–29.
5] a) P. Davies, A. J. F. Maloney, Lancet 1976, 308, 1403; b) E. Perry, R. Perry,
G. Blessed, B. Tomlinson, Lancet 1977, 309, 189; c) J. Coyle, D. Price, M.
DeLong, Science 1983, 219, 1184–1190; d) R. Bartus, R. Dean, B. Beer, A.
Lippa, Science 1982, 217, 408–414.
Experimental procedures for protein binding assays with tracer
1
8
[
F]-2 (time course and blocking of binding) are described in the
Supporting Information.
[
6] a) S. Darvesh, Curr. Alzheimer Res. 2016, 13, 1–5; b) S. D. Rountree, W.
Chan, V. N. Pavlik, E. J. Darby, S. Siddiqui, R. S. Doody, Alzheimer’s Res.
Ther. 2009, 1, 7.
Tissue binding and in vivo PET imaging
Ex vivo tissue binding studies were carried out with one C57BL/6 N
mouse from Charles River. Two series of horizontal brain slices with
[
[
[
7] N. H. Greig, T. Utsuki, Q. Yu, X. Zhu, H. W. Holloway, et al., Curr. Med. Res.
Opin. 2001, 17, 159–165.
8] a) S. Darvesh, D. L. Grantham, D. A. Hopkins, J. Comp. Neurol. 1998, 393,
3
20 μm thickness were prepared for either control or blocking group.
A buffer (150 mM NaCl, 5 mM EDTA, 50 mM Na HPO , pH 8.0)
2
4
74–390; b) S. Darvesh, D. A. Hopkins, J. Comp. Neurol. 2003, 463, 25–43.
9] a) A. L. Guillozet, J. F. Smiley, D. C. Mash, M. M. Mesulam, Ann. Neurol.
997, 42, 909–918; b) M. Mesulam, C. Geula, Ann. Neurol. 1994, 36, 722–
727.
18
containing [ F]-2 (A=1.44 MBq) with or without ethopropazine
hydrochloride (60 μM) as blocking agent was prepared for
incubation with the mice brain slices. Slices were incubated for
1
3
0 min at 25°C and then rinsed five times in PBS buffer (1 min each
[10] I. R. Macdonald, S. P. Maxwell, G. A. Reid, M. K. Cash, D. R. DeBay, et al., J.
time). The slices were dried at room temperature and exposed to a
phosphor imaging plate (GE healthcare, BAS IP MS 2025 E, Munich,
Germany). Images were produced with a digital autoradiographic
system (Typhoon FLA 7000).
Alzheimer’s Dis. 2017, 58, 491–505.
[
11] a) M. M. Mesulam, A. Guillozet, P. Shaw, A. Levey, E. G. Duysen, et al.,
Neuroscience 2002, 110, 627–639; b) J. Hartmann, C. Kiewert, E. G.
Duysen, O. Lockridge, N. H. Greig, et al., J. Neurochem. 2007, 100, 1421–
1429; c) Y. Furukawa-Hibi, T. Alkam, A. Nitta, A. Matsuyama, H.
Mizoguchi, et al., Behav. Brain Res. 2011, 225, 222–229; d) N. H. Greig, T.
Utsuki, D. K. Ingram, Y. Wang, G. Pepeu, et al., Proc. Natl. Acad. Sci. USA
All applicable international, national, and/or institutional guidelines
for the care and use of animals were followed. Animal protocols
were approved by the local Animal Care and Use Committee
2
005, 102, 17213–17218; e) T. Maurice, M. Strehaiano, N. Siméon, C.
Bertrand, A. Chatonnet, Behav. Brain Res. 2015, 296, 351–360; f) R. S.
Naik, J. Hartmann, C. Kiewert, E. G. Duysen, O. Lockridge, et al., J. Pharm.
Pharm. Sci. 2009, 12, 79–85; g) T. Darreh-Shori, S. Brimijoin, A. Kadir, O.
Almkvist, A. Nordberg, Neurobiol. Dis. 2006, 24, 326–333.
(
approval no. 15063) and conducted according to the Guide for the
Care and Use of Laboratory Animals. A healthy, male Wistar rat was
anaesthetized and maintained with isoflurane for in vivo PET
imaging using a micro PET system (FOCUS, Siemens, Erlangen,
Germany). A 60-min dynamic imaging protocol was started directly
[12] M. Hoffmann, C. Stiller, E. Endres, M. Scheiner, S. Gunesch, et al., J. Med.
Chem. 2019, 62, 9116–9140.
[13] a) O. Lockridge, Pharmacol. Ther. 2015, 148, 34–46; b) Y. Ashani, Drug
Dev. Res. 2000, 50, 298–308; c) B. Li, M. Sedlacek, I. Manoharan, R.
Boopathy, E. G. Duysen, et al., Biochem. Pharmacol. 2005, 70, 1673–
18
after the injection of a solution of [ F]-2 (5% ethanol in saline,
6.3 MBq). Analysis of the obtained PET images was performed with
the public domain tool AMIDE imaging software (A Medical
Imaging Data Examiner, version 1.01) and time-activity curves of
regions of interest were generated.
1684.
[
14] a) B. Li, E. G. Duysen, O. Lockridge, Chem.-Biol. Interact. 2008, 175, 88–
91; b) T. Iwasaki, M. Yoneda, A. Nakajima, Y. Terauchi, Intern. Med. J.
2
007, 46, 1633–1639; c) K. K. Sato, T. Hayashi, I. Maeda, H. Koh, N. Harita,
et al., Clin. Endocrinol. (Oxf) 2014, 80, 362–367; d) M. Stojanov, A.
Stefanović, G. Džingalašević, S. Mandić-Radić, M. Prostran, Clin. Biochem.
2011, 44, 623–626; e) V. M. Alcântara, E. A. Chautard-Freire-Maia, M.
Scartezini, M. S. J. Cerci, K. Braun-Prado, et al., Scand. J. Clin. Lab. Invest.
Acknowledgements
2
002, 62, 399–404.
This work was supported by the German Research Council
[
15] a) P. G. Layer, Cell. Mol. Neurobiol. 1991, 11, 7–33; b) R. B. Aziz-Aloya, M.
Sternfeld, H. Soreq, Progress in Brain Research, Vol. 98 (Ed.: A. C. Cuello),
Elsevier, 1993, pp. 147–153; c) P. G. Layer, E. Willbold, Prog. Histochem.
Cytochem. 1994, 29, III–92.
16] C. C. Bernardi, E. d. S. F. Ribeiro, I. J. Cavalli, E. A. Chautard-Freire-Maia,
R. L. R. Souza, Cancer Genet. Cytogenet. 2010, 197, 158–165.
17] Y. Gu, M. J. Chow, A. Kapoor, W. Mei, Y. Jiang, et al., Transl. Oncol. 2018,
(Deutsche Forschungsgemeinschaft DFG; grants DE 1546/6-3 for
M.D. and HI 1789/3-3 for T.H.). M.H. was supported by the German
Academic Scholarship Foundation (“Studienstiftung des deutschen
Volkes”) with a PhD scholarship. C. G. and M. H. were supported
by the MuTaLig COST Action (active participation at the 3rd
Working Group Meeting, Paris). We gratefully acknowledge
Professor Oksana Lockridge (University of Nebraska Medical
[
[
[
11, 1012–1022.
18] S. P. McCluskey, C. Plisson, E. A. Rabiner, O. Howes, Eur. J. Nucl. Med.
Mol. Imaging 2020, 47, 451–489.
ChemMedChem 2021, 16, 1427–1437
www.chemmedchem.org
1436
© 2021 The Authors. ChemMedChem published by Wiley-VCH GmbH

Full Papers
doi.org/10.1002/cmdc.202000942
ChemMedChem
[
[
19] E. Sawatzky, E. Al-Momani, R. Kobayashi, T. Higuchi, S. Samnick, et al.,
[29] a) S. K. Fehler, S. Maschauer, S. B. Höfling, A. L. Bartuschat, N. Tscham-
mer, et al., Chem. Eur. J. 2014, 20, 370–375; b) S. Kato, T. Morie, K. Hino,
T. Kon, S. Naruto, et al., J. Med. Chem. 1990, 33, 1406–1413.
[30] J. A. H. Lainton, M. C. Allen, M. Burton, S. Cameron, T. R. G. Edwards,
et al., J. Comb. Chem. 2003, 5, 400–407.
ChemMedChem 2016, 11, 1540–1550.
20] a) A. Roivainen, J. Rinne, J. Virta, T. Järvenpää, S. Salomäki, et al., J. Nucl.
Med. 2004, 45, 2032–2039; b) T. Kikuchi, M.-R. Zhang, N. Ikota, K.
Fukushi, T. Okamura, et al., Bioorg. Med. Chem. Lett. 2004, 14, 1927–
1
930.
[31] E. Sawatzky, J. Bukowczan, M. Decker, Tetrahedron Lett. 2014, 55, 2973–
[
21] a) D. R. DeBay, G. A. Reid, I. R. Pottie, E. Martin, C. V. Bowen, et al.,
Alzheimers Dement. (N Y) 2017, 3, 166–176; b) I. R. Macdonald, G. A.
Reid, E. E. Joy, I. R. Pottie, G. Matte, et al., Mol. Imaging Biol. 2011, 13,
2976.
[32] D. M. Barnes, J. Barkalow, D. J. Plata, Org. Lett. 2009, 11, 273–275.
[33] G. L. Ellman, K. D. Courtney, V. Andres, R. M. Featherstone, Biochem.
Pharmacol. 1961, 7, 88–95.
1
250–1261.
22] I. R. Macdonald, G. A. Reid, I. R. Pottie, E. Martin, S. Darvesh, J. Nucl. Med.
016, 57, 297–302.
[
[
[
[
[
[34] C. Bartolucci, J. Stojan, Q.-s. Yu, N. H. Greig, D. Lamba, Biochem. J. 2012,
2
444, 269–277.
23] M. W. D. Thorne, M. K. Cash, G. A. Reid, D. E. Burley, D. Luke, I. R. Pottie,
S. Darvesh, Mol. Imaging Biol. 2021, 23, 127–138.
24] S. L. James, S. K. Ahmed, S. Murphy, M. R. Braden, Y. Belabassi, et al., ACS
Chem. Neurosci. 2014, 5, 519–524.
[35] a) W. J. Meuling, M. J. Jongen, J. J. van Hemmen, Am. J. Ind. Med. 1992,
22, 231–241; b) M. Ahmed, J. B. T. Rocha, M. Corrêa, C. M. Mazzanti, R. F.
Zanin, et al., Chem.-Biol. Interact. 2006, 162, 165–171.
[36] G. A. Reid, S. Darvesh, Neuroscience 2015, 298, 424–435.
[37] M. Uhlén, L. Fagerberg, B. M. Hallström, C. Lindskog, P. Oksvold, et al.,
Science 2015, 347, 1260419 (http://www.proteinatlas.org).
[38] L. Santarpia, I. Grandone, F. Contaldo, F. Pasanisi, J. Cachexia Sarcopenia
Muscle 2013, 4, 31–39.
25] F. H. Darras, B. Kling, J. Heilmann, M. Decker, ACS Med. Chem. Lett. 2012,
3
, 914–919.
26] a) E. Sawatzky, S. Wehle, B. Kling, J. Wendrich, G. Bringmann, et al., J.
Med. Chem. 2016, 59, 2067–2082; b) G. Huang, B. Kling, F. H. Darras, J.
Heilmann, M. Decker, Eur. J. Med. Chem. 2014, 81, 15–21.
[39] P. Martínez-Moreno, S. Nieto-Cerón, J. Torres-Lanzas, F. Ruiz-Espejo, I.
Tovar-Zapata, et al., Carcinogenesis 2005, 27, 429–436.
[
27] a) C. Bartolucci, E. Perola, L. Cellai, M. Brufani, D. Lamba, Biochemistry
1
999, 38, 5714–5719; b) E. Perola, L. Cellai, D. Lamba, L. Filocamo, M.
Brufani, Biochim. Biophys. Acta Protein Struct. Mol. Enzymol. 1997, 1343,
1–50; c) A. Rampa, L. Piazzi, F. Belluti, S. Gobbi, A. Bisi, et al., J. Med.
4
Chem. 2001, 44, 3810–3820; d) A. Rampa, M. Bartolini, A. Bisi, F. Belluti,
S. Gobbi, et al., ACS Med. Chem. Lett. 2012, 3, 182–186.
28] a) A. Sanchez-Crespo, Appl. Radiat. Isot. 2013, 76, 55–62; b) C. Kesch, C.
Kratochwil, W. Mier, K. Kopka, F. L. Giesel, J. Nucl. Med. 2017, 58, 687–
[
Manuscript received: December 7, 2020
Revised manuscript received: January 19, 2021
Version of record online: March 1, 2021
688.
ChemMedChem 2021, 16, 1427–1437
www.chemmedchem.org
1437
© 2021 The Authors. ChemMedChem published by Wiley-VCH GmbH