Synthesis and Initial Characterization of a Reversible, Selective 18F-Labeled Radiotracer for Human Butyrylcholinesterase

Article abstract of DOI:10.1007/s11307-021-01584-2

Purpose: A neuropathological hallmark of Alzheimer’s disease (AD) is the presence of amyloid-β (Aβ) plaques in the brain, which are observed in a significant number of cognitively normal, older adults as well. In AD, butyrylcholinesterase (BChE) becomes associated with A_β aggregates, making it a promising target for imaging probes to support diagnosis of AD. In this study, we present the synthesis, radiochemistry, in vitro and preliminary ex and in vivo investigations of a selective, reversible BChE inhibitor as PET-tracer for evaluation as an AD diagnostic. Procedures: Radiolabeling of the inhibitor was achieved by fluorination of a respective tosylated precursor using K[¹⁸F]. IC₅₀ values of the fluorinated compound were obtained in a colorimetric assay using recombinant, human (h) BChE. Dissociation constants were determined by measuring hBChE activity in the presence of different concentrations of inhibitor. Results: Radiofluorination of the tosylate precursor gave the desired radiotracer in an average radiochemical yield of 20 ± 3?%. Identity and > 95.5?% radiochemical purity were confirmed by HPLC and TLC autoradiography. The inhibitory potency determined in Ellman’s assay gave an IC₅₀ value of 118.3 ± 19.6?nM. Dissociation constants measured in kinetic experiments revealed lower affinity of the inhibitor for binding to the acylated enzyme (K₂ = 68.0?nM) in comparison to the free enzyme (K₁ = 32.9?nM). Conclusions: The reversibly acting, selective radiotracer is synthetically easily accessible and retains promising activity and binding potential on hBChE. Radiosynthesis with ¹⁸F labeling of tosylates was feasible in a reasonable time frame and good radiochemical yield.

Full text of DOI:10.1007/s11307-021-01584-2

Mol Imaging Biol (2021)
DOI: 10.1007/s11307-021-01584-2
* The Author(s), 2021. This article is an open access publication
RESEARCH ARTICLE
Synthesis and Initial Characterization
18
of a Reversible, Selective F-Labeled
Radiotracer for Human Butyrylcholinesterase
Christian Gentzsch,1 Xinyu Chen,2,3,4 Philipp Spatz,1,3 Urban Košak,5 Damijan Knez,5
1
Naoko Nose,6 Stanislav Gobec,5 Takahiro Higuchi,3,4,6 Michael Decker
1Pharmaceutical and Medicinal Chemistry, Institute of Pharmacy and Food Chemistry, Julius-Maximilians-University of Würzburg, Am
Hubland, 97074, Würzburg, Germany
2Department of Nuclear Medicine, University Hospital of Augsburg, Stenglinstraße 2, 86156, Augsburg, Germany
3Department of Nuclear Medicine, University Hospital of Würzburg, Oberdürrbacher Straße 6, 97080, Würzburg, Germany
4Comprehensive Heart Failure Center, University Hospital of Würzburg, Am Schwarzenberg 15, 97078, Würzburg, Germany
5Chair of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Ljubljana, Aškerčeva cesta 7, SI-1000, Ljubljana, Slovenia
6Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, 2-5-1 Shikata-cho, Kita-ku, Okayama, Japan
Abstract
Purpose: A neuropathological hallmark of Alzheimer’s disease (AD) is the presence of amyloid-β
(Aβ) plaques in the brain, which are observed in a significant number of cognitively normal, older
adults as well. In AD, butyrylcholinesterase (BChE) becomes associated with Aβ aggregates,
making it a promising target for imaging probes to support diagnosis of AD. In this study, we
present the synthesis, radiochemistry, in vitro and preliminary ex and in vivo investigations of a
selective, reversible BChE inhibitor as PET-tracer for evaluation as an AD diagnostic.
Procedures: Radiolabeling of the inhibitor was achieved by fluorination of a respective tosylated
precursor using K[18F]. IC50 values of the fluorinated compound were obtained in a colorimetric
assay using recombinant, human (h) BChE. Dissociation constants were determined by
measuring hBChE activity in the presence of different concentrations of inhibitor.
Results: Radiofluorination of the tosylate precursor gave the desired radiotracer in an average
radiochemical yield of 20 3 %. Identity and 9 95.5 % radiochemical purity were confirmed by
HPLC and TLC autoradiography. The inhibitory potency determined in Ellman’s assay gave an
IC50 value of 118.3 19.6 nM. Dissociation constants measured in kinetic experiments revealed
lower affinity of the inhibitor for binding to the acylated enzyme (K2 = 68.0 nM) in comparison to
the free enzyme (K1 = 32.9 nM).
Conclusions: The reversibly acting, selective radiotracer is synthetically easily accessible and
retains promising activity and binding potential on hBChE. Radiosynthesis with 18F labeling of
tosylates was feasible in a reasonable time frame and good radiochemical yield.
Key words: Alzheimer’s disease, Enzyme inhibitor, Positron emission tomography, Biodistribu-
tion, Quaternization
Correspondence to: Takahiro Higuchi; e-mail: higuchi_t@ukw.de, Michael
Decker; e-mail: michael.decker@uni-wuerzburg.de

Gentzsch C. et al.: Reversible Butyrylcholinesterase PET Tracer
concentration and biodistribution [22]. ChE PET tracers
have been developed, with the majority of them targeting
Introduction
Alzheimer’s disease (AD) is an incurable, progressive
neurodegenerative disorder and the most frequent cause of
dementia with high prevalence and a long asymptomatic
phase [1, 2]. The number of dementia-diseased individuals is
expected to further increase, due to a growing average age
and demographic change, which is hard to overcome for
health care systems and society as a whole [3]. The
definitive diagnosis of AD should no longer be solely based
on typical, clinical symptoms, since they are not conclusive
enough and begin many years after initial neuropathological
alterations [4–6]. At this point, a successful disease-
modifying or even preventive therapy is already impossible.
Due to this fact, there is an urgent need for biological targets
and biomarkers of an early disease state to help not only for
an early, conclusive diagnosis with respective imaging
probes but also to shed new light in the sequences and
significances of the various neuropathological processes
involved [7]. Three of the four FDA-approved anti-AD
medications temporarily ameliorate AD symptoms by
inhibiting cholinesterases (ChEs), whereby donepezil and
galantamine are selective acetylcholinesterase (AChE) inhib-
itors and rivastigmine additionally binds to the isoenzyme
butyrylcholinesterase (BChE). AChE inhibition prevents
degradation of the neurotransmitter acetylcholine, levels of
which are decreasing through cell death of cholinergic
neurons [8–10]. However, advanced progression of AD
involves a rapid decrease of AChE levels in the brain of ~
90 %, which is accompanied in later-stage AD by elevated
BChE expression. AChE and BChE exhibit very different
localisations, biochemical features, and physiological func-
tions [11]; however, BChE can take over the hydrolytic
function of AChE [12–16]. These findings make BChE an
attractive target for the development of more potent drugs
for combatting AD [8–10]. Established pathological AD
hallmarks are the abnormal metabolism of amyloid-β (Aβ),
accompanied by hyperphosphorylated tau (τ) proteins,
oxidative stress, and alterations concerning microglia cells
in nervous tissue [17]. Aβ peptide can impair neurovascular
homeostasis by its cerebrovascular effects, causing endothe-
lial dysfunction [18]. Interestingly, Aβ deposits develop in
nearly 30 % of aged adults, who do not show pathologically
deteriorated cognition or memory deficits [19]. However, in
AD patients, there is an increased expression of BChE along
with Aβ plaques, especially in the cerebral cortex [20, 21].
In this outer layer of cerebrum, there is usually no
considerable amount of BChE in healthy people, suggesting
that the enzyme plays a potential key role in generating the
detrimental attributes of Aβ. This finding has been supported
in a BChE knockout mouse model, where distinctly fewer
fibrillar Aβ plaques have been detected [15]. In this context,
it is highly promising to apply suitable radiotracers in
positron emission tomography (PET) studies, since this
imaging technique exhibits excellent sensitivity and can
provide an accurate estimation of the radiotracers´ in vivo
peripheral or brain AChE [23]. These compounds can be
classified in two main categories, substrate-based and
ligand-based tracers. The latter can be reversible or
irreversible inhibitors. Substrate-based tracers, which usually
have a high blood-brain barrier (BBB) permeability, can
have intrinsic drawbacks due to quick hydrolysis and
delivery limitations into the tissue [24]. In consequence,
they may not reflect the regional enzyme distribution, but
rather plasma delivery rate of the tracer [25]. Irreversible
inhibitor-derived tracers can have a complex mechanism of
enzyme inactivation and it is often complicated to accurately
determine in vivo kinetics and distribution [24, 26].
Examples of substrate-type tracers specific for BChE include
N-methylpiperidin-4-yl 4-[123I]iodobenzoate ([123I]MP4Bz),
N-[18F]fluoroethylpiperidin-4-ylmethyl butyrate, and 1-
[11C]-methyl-4-piperidinyl n-butyrate ([11C]MP4B) [16, 27,
28]. [11C]MP4B enters the brain, but no enhanced activity
can be seen in regions where BChE-associated plaques
typically show up in AD, which is likely the same for N-
[18F]fluoroethylpiperidin-4-ylmethyl butyrate. In preliminary
in vivo studies, the tracer showed high initial uptake in rat
cerebral cortex after intravenous injection, but the hydrolysis
rate of these ester is presumably still too high [28].
[
123I]MP4Bz was applied in single-photon emission com-
puted tomography (SPECT) studies, in which it was possible
to distinguish cerebral cortical BChE activity in wild-type
mice from an AD model [16]. This represents an important
proof of concept, which demonstrates that BChE can indeed
serve as a promising biomarker in AD diagnostics [15, 21].
This could also be shown for the pseudoirreversible BChE
inhibitor phenyl 4-123I-iodophenylcarbamate (123I-PIP),
which accumulated specifically in Aβ plaques with ChE
activity in human AD brain tissue [29]. Additionally, this
tracer has been investigated very recently as a potential
diagnostic and treatment monitoring tool for BChE activity
changes in multiple sclerosis showing promising preliminary
results [30]. Regarding irreversible and highly selective
inhibitors of BChE as radiotracers, carbamate-based inhib-
itors were investigated that transfer the radiolabeled moiety
onto the enzyme, where it is covalently bound. Ex vivo
autoradiography on mice brain tissue and kinetic investiga-
tions proved such covalent transfer [24]. Furthermore,
investigations into the influence of the carbamate structure
that is transferred to BChE were made by altering spacer
lengths and attached heterocyclic moieties. This resulted in
sets of inhibitors with short, medium and long duration of
action and pronounced neuroprotectivity in an AD mouse
model, showing also the therapeutic potential of such
inhibitors [31]. Altogether, these results support BChE not
only as a promising biomarker for an early diagnosis of AD
but also as an attractive target to be addressed by imaging
probes in order to shed the light on the complex neuropa-
thology of AD. Herein, we report on synthesis, in vitro
evaluation, radiolabeling, ex vivo autoradiography, and

Gentzsch C. et al.: Reversible Butyrylcholinesterase PET Tracer
Fig. 1. a Design of the radiotracer. A potent, selective, and reversibly acting BChE inhibitor served as parent compound [32]. b
Parent ligand in the BChE binding pocket [32].
preliminary in vivo PET studies of an 18F-labeled BChE
selective tracer, based on the structure of a potent, selective
BChE inhibitor with reversible binding mode (Fig. 1a, b)
[32]. Our intention was to utilize the promising properties of
the parent compound for a suitable radiotracer, and these
properties include the high affinity and selectivity towards
the target enzyme, accompanied by pronounced lipophilicity
and a relatively low molecular weight, which is favorable for
blood-brain barrier (BBB) penetration [33]. Additionally, we
chose a reversible inhibitor to overcome the intrinsic
problems of substrate-type and irreversible radiotracers
mentioned before, possibly enabling a more precise mapping
of BChE distribution. We decided to replace the methoxy
group on the ethylene-side chain with fluorine, because this
moiety points out of the binding pocket as observed in the
resolved crystal structure of the enzyme in complex with the
parent inhibitor [32]. We assumed that this modification
would retain most of inhibitory potency. We chose to
incorporate 18F as radioisotope to take advantage of a long
half-life and high positron yields in combination with a good
spatial resolution due to relatively low positron energies in
comparison to other radioisotopes commonly used for PET
studies (e.g., 18F: 0.65 MeV, 68Ga: 1.90 MeV) [34, 35].
Materials and Methods
Chemistry
The non-radioactive “cold” inhibitor 4 and precursor 5 for
radiolabeling were synthesized as shown in Fig. 2. Briefly,
the piperidine ring of building block 1 was benzylated under
Leuckart-Wallach conditions [36]. Subsequent demethyla-
tion of the methoxy group in compound 2 applying BF3 ·
Et2O in propane-1-thiol [37] gave the central building block,

Gentzsch C. et al.: Reversible Butyrylcholinesterase PET Tracer
Fig. 2. Synthesis of precursor 5 and cold, reversible inhibitor of hBChE 4. Reaction conditions and yields: (i) benzaldehyde,
formic acid, 6 h, 180 °C, 44 %; (ii) BF3 · Et2O, propane-1-thiol, 50 °C, 60 h, 60 %; (iii) DAST, CH2Cl2, − 10 °C → RT, 24 %; (iv) 1.
TosCl, NEt3, CH2Cl2, RT, 18 h, 98 %, 2. p-TosOH, MeOH; (v) benzoyl chloride, K2CO3, THF/H2O, 0 °C – RT, 20 h, 88 %; (vi)
ethanolamine, HBTU, NEt3, DMF, RT, 18 h, 88 %; (vii) TBDMS-Cl, imidazole, DMF, RT, 24 h, 69 %; (viii) 1. LiAlH4, THF, reflux,
1 h; 2.naphthalene-2-sulfonyl chloride, DIPEA, CH2Cl2, 0 °C – RT, 54 % (two steps).
the alcohol 3, which can also be obtained from commercially
available nipecotic acid (Fig. 2) [38]. Cold compound 4 was
obtained by fluorination of 3 with diethylaminosulfur
trifluoride (DAST) [39]. For radiolabeling with K[18F], a
tosylate-leaving group was chosen, which was introduced by
reacting 3 with p-toluenesulfonyl chloride (TosCl). We
chose this synthetic strategy to utilize the advantage of
having one central building block for generating both the
precursor 5 for radiolabeling and the cold reference
compound 4.
were performed by measuring progress curves of product
formation with ~ 50 μM BTCI as substrate. BTCI hydrolysis
by hBChE was measured in the absence and presence of
inhibitor 4 at three different concentrations (namely 40 nM,
80 nM, and 160 nM, respectively). The experiments were
carried out at 25 °C in 25-mM phosphate buffer (pH = 7.0)
according to the method of Ellman [40]. The concentration
of purified hBChE, which was always the final addition to
the assay probes, was approx. 1 nM. The hydrolysis of
46 μM BTCI was followed until completion in the presence
of 1 mM DTNB (cf. ESM for further experimental details).
In Vitro Studies
Radiochemistry
The inhibitory potency of compound 4 (Fig. 2) on hBChE
was determined in the colorimetric Ellman’s assay [40]. The
incubation time of the compound stock solution (100 % in
dimethylsulfoxide, DMSO) with Ellman’s reagent (5,5′-
dithiobis-(2-nitrobenzoic acid), DTNB) and recombinant
hBChE was 5 min. The final concentrations were 370 μM
of DTNB and 1 nM of the enzyme in a 0.1-M phosphate
buffer at pH = 8.0. Reactions were started by addition of
butyrylthiocholine iodide (BTCI) in a concentration of
500 μM with a final DMSO content of 1 %. IC50 values
were determined by plotting residual enzyme activities
against seven respective inhibitor concentrations. Tacrine
served as a positive control (cf. ESM for further experimen-
tal details).
Radiofluorination of tosylate precursor 5 (Fig. 2) was
performed in an established procedure. 18F-Fluoride was
separated from 18O-water by an anion-exchange cartridge,
which was eluted with 300 μl of 66-mM K2CO3
aqueous solution into a v-vial containing 15 mg of
Kryptofix222 in 500-μl acetonitrile. The mixture was dried
azeotropically at 120 °C, which was repeated twice using
dry acetonitrile (500 μl each time). Then, 1 mg of the
precursor 5 (Fig. 2) in 400 μl of dry acetonitrile was added
to the mixture and reacted for 10 min at 110 °C. After
cooling to room temperature, the reaction mixture was
neutralized by adding 5 % acetic acid (300 μl). The labeled
compound was purified by semi-preparative, reversed
phase HPLC (cf. ESM for further experimental details).
Tracer identity and sufficient radiochemical purity were
Kinetic studies to measure dissociation constants K1 and
K2 of inhibitor 4 for binding to the free and acylated enzyme

Gentzsch C. et al.: Reversible Butyrylcholinesterase PET Tracer
confirmed by radio-thin layer chromatography (TLC). The
radiotracer was diluted with saline to the required
concentration for further investigation.
reaction on the other hand. Since building block 1 is not
commercially available, we synthesized alcohol 3 additionally
out of low cost and easy to handle nipecotic acid 6 (Fig. 2) [36,
37]. In the first step, the piperidine nitrogen was benzoylated in
very good yields applying benzoyl chloride. Next, the
carboxylic acid of compound 7 was activated with 3-
[bis(dimethylamino)methyliumyl]-3H-benzotriazol-1-oxide
hexafluorophosphate (HBTU) and coupled to 2-aminoethanol
in very good yields. Subsequently, compound 8 was protected
at its hydroxyl group with tert-butyldimethylsilyl chloride
(TBDMS-Cl) in good yields. Finally, both amide groups were
reduced with lithium aluminum hydride and the crude product
was used directly in a one-pot-two-steps manner to be coupled
naphthalene-2-sulfonyl chloride at the reduced secondary
amine function. It was found that the TBDMS group is cleaved
under the applied conditions and alcohol 3 (Fig. 2) was
obtained in satisfying yields.
Preliminary Ex Vivo Tissue Binding Assay and
In Vivo PET Imaging
Animal protocols were approved by the local Animal Care
and Use Committee and conducted according to the Guide
for the Care and Use of Laboratory Animals. One C57BL/
6N mouse from Charles River was used for ex vivo tissue
binding studies. Horizontal slices with 20-μm thickness were
made and separated into two series for either control or
blocking group. The frozen mice brain slices were incubated
in a buffer at pH = 8.0 (150-mM NaCl, 5-mM EDTA, 50-
mM Na2HPO4) containing 18F-labeled compound 4 (A =
1.44 MBq) with or without ethopropazine hydrochloride
(60 μM) as blocking agent. After incubating for 30 min at
25 °C, the slices were rinsed five times in PBS buffer (1 min
each time). After drying at room temperature, the slices were
exposed to a phosphor imaging plate (Fuji SR-type image
plate, Fujifilm Corporation, Tokyo, Japan). The images were
obtained using a digital autoradiographic system (Typhoon
FLA 7000). For in vivo PET imaging, a healthy male Wistar
rat, which was anesthetized and maintained with isoflurane,
was scanned using a micro-PET system (FOCUS, Siemens).
Directly after the injection of tracer 4 (6.3 MBq), a 60-min
dynamic imaging protocol was started. The obtained PET
images were analyzed with the public domain tool AMIDE
imaging software (A Medical Imaging Data Examiner,
version 1.01) and used to generate time-activity curves of
regions of interest.
In Vitro Studies
Subsequently, compound 4 was tested in an Ellman’s assay
for its inhibitory potency against hBChE [40]. We deter-
mined a low submicromolar value (IC50 = 118.3 19.6 nM),
meaning a drop in inhibitory potency compared to the parent
methoxy derivative (Fig. 1a, b; IC50 = 4.9 0.3 nM) [32].
This can be explained by means of the BChE crystal
structure in complex with the inhibitor. Although the
methoxy-ethylene moiety as a whole rather points out of
the binding pocket, the missing methoxy-oxygen has been
described to act as an additional H-bond acceptor with a
structural water and Asn68 (Fig. 1b). This is still a good
compromise, since the structure-activity relationships
revealed that other positions to introduce fluorine in the
molecule would presumably lead to a more drastic decline of
activity (cf. Table 1). Still, this class of compounds exhibits
high selectivity over hAChE, which had been described also
for several derivatives thereof [32].
Results
Chemistry
To gain in-depth insight into the binding potential of
inhibitor 4, the effects of different concentrations of the
compound on hBChE activity were studied by measuring
progressive curves of product formation at approximately
50 μM of BTCI (Fig. 4a). The analysis of these progressive
curves revealed good agreement between the experimental
curves (Fig. 4a, in blue) and a theoretical model (Fig. 4a, in
red) that defined a mixed reaction mechanism with binding
of the inhibitor to both the free and the acylated enzyme
(Fig. 4b). The dissociation constants revealed that the
binding affinity of compound 4 to the acylated enzyme
(K2 = 68.0 nM) is lower than to the free enzyme (K1 =
32.9 nM).
The fluorinated, reversible hBChE inhibitor 4 (Fig. 2) was
synthesized in three steps with satisfying yields [36, 37, 39].
We applied the DAST fluorination method for alcohol 3 due to
the short reaction times, mild conditions, and uncomplicated
workup, despite lower yields (24 %). This compound was only
required in minor amounts as reference during radiolabeling
and for in vitro assays. The respective precursor for 18F-
labeling was synthesized using the same building block,
alcohol 3, in almost quantitative yields. However, precursor 5
exhibited an instability problem, when present as free base. It
was resolved by turning 5 into its tosylate salt. During purity
control by liquid chromatography/mass spectrometry (LCMS),
we found a slow side reaction due to intramolecular ring
closure by nucleophilic attack of the piperidine nitrogen
(Fig. 3). This can be explained with the excellent leaving
group quality of tosylate, which is on the one hand required for
a facile radiolabeling under preferably mild conditions, but
makes the compound sensitive for this quaternization side
Radiochemistry
Next, precursor 5 (Fig. 2) was subjected to 18F-radio-
labeling. We obtained [18F]-labeled tracer 4 in

Gentzsch C. et al.: Reversible Butyrylcholinesterase PET Tracer
Fig. 3. Quaternization of precursor 5 when present as free base.
approximately 120 min with an average radiochemical yield
of 20 3 % (decay-corrected, n = 2) without reaction
condition optimization. The identity of the tracer and its
radiochemical purity (≥ 95.3 %) were confirmed by TLC
autoradiography. In a preceding 18F radiolabeling approach
of precursor 5 applying established conditions as described
before (cf. “Materials and Methods”), we were able to verify
tracer identity by its Rf value on radio-TLC. TLC autoradi-
ography indicated a progress of fluorination by more than
33 % (Fig. 5c). Furthermore, the retention time of the
radiotracer (γ detection, Fig. 5b) corresponded to that of the
cold compound on HPLC (UV detection, Fig. 5a).
Preliminary Ex Vivo Tissue Binding and In Vivo
PET Imaging
In the ex vivo autoradiography study, we found accumu-
lation of high radioactivity in most of the brain area,
especially high intensity at the cortex, where BChE
activity is elevated even more during AD progression
[41]. Ethopropazine hydrochloride, a selective and re-
versible inhibitor of BChE [42], successfully reduced
tracer uptake, suggesting specific binding of the tracer to
BChE in the brain tissue (Fig. 6a). Unfortunately,
dynamic PET images in a healthy rat indicated low
tracer retention in brain (Fig. 6b, c).
Table 1 Structures and inhibitory potencies of cold, reversible hBChE inhibitor 4 (Fig. 2) in contrast to respective derivatives with substituted benzyl group,
naphthalene group, and altered alkyl chain length on sulfonamidic nitrogen [32]. aSEM standard error of means, bRA residual activity
%RAb SEM
at 10 M on
mAChE
IC50 SEMa
(nM) on
R
R1
R2
hBChE
-(CH2)2-F
118.3 19.6
~ 53000
-
-(CH2)2-OMe
-(CH2)3-OMe
-
14.4 0.8
95% 5%
-(CH2)2-OMe
156 33
95% 7%

Gentzsch C. et al.: Reversible Butyrylcholinesterase PET Tracer
Fig. 4. a Time courses of product formation in the reactions between BTCI (~ 46 μM) and purified hBChE (1 nM) in the absence
(curve a) and presence of compound 4 (b, 40 nM; c, 80 nM; d, 160 nM). b Scheme for the inhibition of BTCI hydrolysis by
hBChE in the presence of DTNB by compound 4. E, free enzyme; EA, acylated intermediate; S, substrate (BTCI); P, all of the
stoichiometrically released products (thiocholine-TNB, TNB−); and I, compound 4 (inhibitor). The symbols for the constants are
kcat (k1), catalytic constant for BTCI turnover; Km (k0), Michaelis constant; Kp = k3/k2, inhibition constant for binding of the
product thiocholine-TNB; K1 = k5/k4 and K2 = k7/k6, dissociation constants for binding of the compound to the free and acylated
enzyme, respectively.
Discussion
elimination or carbonium-ion type rearrangements [39].
Tosylation of alcohol 3 proceeded almost quantitatively
under standard conditions [44]; however, the product has to
be turned into its tosylate salt to prevent slow, but continuous
quaternization (Fig. 3). In our additional synthetic approach
towards central building block 3 (Fig. 2), we found that both
amide groups of TBDMS-protected compound 9 can be
reduced by lithium aluminum hydride and the crude product
can directly be coupled to naphthalene-2-sulfonyl chloride to
obtain deprotected alcohol 3. This abbreviates the synthetic
procedure by one additional TBDMS-deprotection step.
Next, we measured the inhibitory potency of our cold,
reversible hBChE inhibitor in a colorimetric Ellman’s assay
and determined an IC50 value of 118.3 19.6 nM, meaning a
significant drop of inhibitory activity compared to the parent
compound (Fig. 1a, IC50 = 4.9 0.3 nM). However, the
structure-activity relationships for this class of compounds
revealed that altering the N-alkyl moieties of the sulfon-
amide nitrogen led to the lowest changes in inhibitory
In our synthetic approach towards cold, reversible hBChE
inhibitor 4, and the respective precursor 5 for 18F labeling
(Fig. 2), we were able to perform several optimizations of
reaction conditions. Yields of the Leuckart-Wallach benzyla-
tion in the first step were significantly increased, when
benzaldehyde was freshly distilled before the reaction to
remove benzoic acid, which is formed due to slow oxidation
of benzaldehyde in the presence of air [36, 43]. In the next
step, we achieved demethylation of a methoxy group with
boron trifluoride etherate in propane-1-thiol. However, at
first, this step required long reaction times (6–7 days), even
though it was described that increased amounts of BF3 ∙ Et2O
can accelerate conversion [37]. We found that slightly
elevated temperatures (up to 50 °C) significantly decreased
reaction times (60 h). Fluorination of the alcohol 3 with
DAST gave the desired product 4; however, attempts to
increase the yields failed. This is likely due to the side
reactions that can appear when DAST is applied, namely

Gentzsch C. et al.: Reversible Butyrylcholinesterase PET Tracer
Fig. 5. HPLC – and radio-TLC analysis after first 18F radiolabeling of precursor 5. a HPLC chromatogram of reference
compound 4. The UV absorption at 220 nm was detected. b HPLC chromatogram of the purified radiotracer. γ-ray emission
produced by the β+-(positron-) decay of 18F and subsequent annihilation with electrons was detected. The retention time (Rt) is
in good agreement with the reference (cold compound: Rt = 9.273 min; radiotracer: Rt = 9.902 min). c Radio-TLC analysis of
labeling progress. The Rf values of cold compound (cpd. 4) and the respective spot of the tracer in the reaction mixture (r. m.)
are in good agreement. TLC autoradiography indicated a process of fluorination by more than 33 %.
potency, while substituted naphthalene or benzyl groups
significantly decreased inhibitory potency (Table 1 and Fig.
1b) [32]. As an example, we synthesized a respective
derivative with a fluoromethyl group in para position of
the benzyl ring, which turned out to be almost inactive as
hBChE inhibitor (Table 1). Even though replacement of the
methoxy group with a fluorine atom reduced inhibitory
potency more than expected considering the previously
established structure-activity relationships, the submicromo-
lar IC50 value in combination with the high selectivity ratio
over AChE for this class of compounds still represent
promising attributes for a suitable radiotracer [33]. Addi-
tionally, kinetic experiments revealed a good binding
potential of compound 4 (Fig. 2) to hBChE. The dissociation
constant K1, representing the binding affinity to the free
enzyme, was generally lower than K2, the respective
constant for binding to the acylated enzyme (K1 = 32.9 nM,
K2 = 68.0 nM, cf. Fig. 4).
limiting factor for time-consuming preparations of radio-
tracers [34]. In a precedent radiolabeling approach, we could
already determine a facile progress of fluorination by TLC
autoradiography. The tracer identity could be confirmed
both by radio-TLC and HPLC retention times (Fig. 5). Since
harsh radiolabeling conditions can lead to decomposition of
unstable functional groups and unexpected side reactions of
the respective precursor, our promising radiolabeling results
motivated us to perform preliminary ex vivo and in vivo
investigations.
The ex vivo autoradiography study demonstrated good
binding of our tracer to BChE rich areas in mice brain
tissues. We observed high intensities in cortex, which is in
good agreement with the known BChE distribution in mice
brain [45–47]. After preincubating the tissue with ethopro-
pazine hydrochloride, a selective inhibitor of BChE [42], we
found a significant decrease of binding (Fig. 6a). This
finding met our expectations and provides further evidence
of the pronounced selectivity over AChE for this compound.
Nevertheless, a significant amount of tracer remains bound
to the tissue despite blocking. Possible reasons are non-
specific binding due to the lipophilicity of the tracer and/or
potential off-target effects.
Subsequently, we performed 18F-radiolabeling of the
tosylate precursor 5 by nucleophilic substitution. Applying
an established procedure with some variations on the
technical details led to a reasonable radiochemical yield.
Importantly, the whole process including labeling, tracer
identification, and purification was feasible in approximately
120 min, considering the half-life of 18F (1.8288 h) as a
However, in our first approach to utilize the tracer for
in vivo PET studies, we found only low brain uptake after

Gentzsch C. et al.: Reversible Butyrylcholinesterase PET Tracer
Conclusion
In our study, we present design and evaluation of a novel
18F-PET radiotracer selectively targeting BChE with revers-
ible mode of binding. Precursor and the respective cold
compound are now synthetically easily accessible. Inhibitory
potency of the cold compound was decreased compared to
the parent compound. Therefore, future studies could
additionally focus on 11C labeling at the methoxy group to
completely retain inhibitory potency of the radiotracer.
Radiolabeling was achieved in a reasonable time frame and
a good radiochemical yield applying a standard procedure.
Preliminary ex vivo autoradiography on mice brain slices
preincubated with the tracer revealed its good binding to
brain tissue and blocking studies with ethopropazine
hydrochloride demonstrated its selectivity towards BChE.
However, future studies should focus on determining
possible reasons for the significant compound retention
despite blocking with respect to potential off-target effects
and non-specific binding. Our preliminary in vivo PET study
showed only limited brain uptake of the tracer after tail vein
injection. After the initial blood pool circulation, the tracer
was accumulated in liver and kidneys and excreted fast into
intestine and urine. The tracer’s ability to pass the
BBB might be restricted. On the other hand, the parent
compound, which served as design schedule for us,
demonstrated neither low passive permeability nor active
efflux. Due to these facts, the tracer will be applied in future
studies towards its precise biodistribution to clarify the
reason of its limited brain uptake.
Acknowledgments The authors are thankful to Dr. Jure Stojan (Medical
faculty, University of Ljubljana, Slovenia) for helpful discussions regarding
kinetic analysis and progress-curve analysis.
Funding Open Access funding enabled and organized by Projekt DEAL.
This work was supported by the German Research Council (Deutsche
Forschungsgemeinschaft DFG grants DE 1546/6-3 for M. Decker and HI
1789/3-3 Prof. T. Higuchi) and the Slovenian Research Agency (core
financing P1-0208 and projects Z1-9195 granted to UK and NC-0009 to S.
Gobec).
Fig. 6. a Autoradiographic image of tissue binding assay
with healthy mice brain slices incubated with [18F]-labeled
BChE – tracer 4 without (left) and with (right) ethopropazine.
Purple areas represent low binding; bright yellow areas
represent high binding. b Dynamic PET images of sagittal
sections after administration of radiotracer 4 in a healthy rat.
Dark green areas represent high tracer accumulation; light
green areas represent low accumulation. c Time-activity
curves in each organ derived from the PET imaging.
Compliance with Ethical Standards 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 and conducted according to the Guide for the Care and Use of
Laboratory Animals.
administering the compound via tail vein to a male rat (Fig.
6b, c). This can reflect the compounds limited
BBB permeability, since a moderate brain-to-plasma ratio
(0.44 vs. Donepezil = 6.3) had already been described for the
parent, methoxy compound (Fig. 1a) after in vivo blood
plasma-brain distribution studies [32]. On the other hand,
this compound had been investigated in permeability
measurements using Caco-2 cells, where it exhibited neither
low passive permeability nor active efflux by membrane
transport proteins like P-glycoprotein or breast cancer
resistance protein [32].
Conflict of Interest
The authors declare that they have no conflict of interest.
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