Preparation of a first 18F-labeled agonist for M1 muscarinic acetylcholine receptors

Article abstract of DOI:10.3390/molecules25122880

M₁ muscarinic acetylcholine receptors (mAChRs) are abundant in postsynaptic nerve terminals of all forebrain regions and have been implicated in the cognitive decline associated with Alzheimer’s disease and other CNS pathologies. Consequently,

Full text of DOI:10.3390/molecules25122880

molecules
Article
Preparation of a First 18F-Labeled Agonist for M1
Muscarinic Acetylcholine Receptors
Boris D. Zlatopolskiy 1,2,3 , Felix Neumaier 4 , Till Rüngeler 2 , Birte Drewes 1
,
Niklas Kolks 1,2 and Bernd Neumaier 1,2,3,
*
1
Institute of Neuroscience and Medicine, Nuclear Chemistry (INM-5), Forschungszentrum Jülich GmbH,
52428 Jülich, Germany; boris.zlatopolskiy@uk-koeln.de (B.D.Z.); b.drewes@fz-juelich.de (B.D.);
niklas.kolks@uk-koeln.de (N.K.)
Institute of Radiochemistry and Experimental Molecular Imaging, University Clinic Cologne,
50931 Cologne, Germany; till@ruengeler.eu
Max Planck Institute for Metabolism Research, 50931 Cologne, Germany
Institute of Neurophysiology, University Hospital Cologne, Robert-Koch Str. 39, 50931 Cologne, Germany;
felix@neumaier-net.de
2
3
4
*
Correspondence: b.neumaier@fz-juelich.de; Tel.: +49-2461-61-4141
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 22 April 2020; Accepted: 17 June 2020; Published: 23 June 2020
Abstract: M1 muscarinic acetylcholine receptors (mAChRs) are abundant in postsynaptic nerve
terminals of all forebrain regions and have been implicated in the cognitive decline associated with
Alzheimer’s disease and other CNS pathologies. Consequently, major efforts have been spent in
the development of subtype-selective positron emission tomography (PET) tracers for mAChRs
resulting in the development of several 11C-labeled probes. However, protocols for the preparation of
18F-labeled mAChR-ligands have not been published so far. Here, we describe a straightforward
procedure for the preparation of an 18F-labeled M1 mAChR agonist and its corresponding pinacol
boronate radiolabeling precursor and the non-radioactive reference compound. The target compounds
were prepared from commercially available aryl fluorides and Boc protected 4-aminopiperidine using
a convergent reaction protocol. The radiolabeling precursor was prepared by a modification of the
Miyaura reaction and labeled via the alcohol-enhanced Cu-mediated radiofluorination. The developed
procedure afforded the radiotracer in a non-decay-corrected radiochemical yield of 17
±
3% (n = 3)
and in excellent radiochemical purity (>99%) on a preparative scale. Taken together, we developed
a straightforward protocol for the preparation of an 18F-labeled M1 mAChR agonist that is amenable
for automation and thus provides an important step towards the routine production of a 18F-labeled
M1 selective PET tracer for experimental and diagnostic applications.
Keywords: radiolabeling; fluorine-18; positron emission tomography; acetylcholine receptor agonist;
Cu-mediated radiofluorination
1. Introduction
Muscarinic acetylcholine receptors (mAChRs) in the central and peripheral nervous system
participate in autonomic, cognitive, and motor function. To date, five subtypes (M1–M5) have been
identified, all of which share a highly conserved ligand-binding site for acetylcholine. M1 mAChRs
are the most prevalent subtype in the CNS and abundant in postsynaptic nerve terminals of
all forebrain regions [
cognition and that loss of cholinergic function contributes to the cognitive decline associated
with Alzheimer’s disease, schizophrenia, and other neurological and psychiatric disorders [ ].
1,2]. Substantial evidence suggests that they are critically involved in
3–5
These findings have spurred interest into M1 receptors as a target for experimental or clinical
applications. In this context, functional imaging techniques and especially positron emission
Molecules 2020, 25, 2880; doi:10.3390/molecules25122880
www.mdpi.com/journal/molecules

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tomography (PET) have an enormous potential for in vivo assessment of target engagement [
6].
A lot of effort spent in the development of PET tracers for mAChRs led to the discovery of
several subtype selective 11C-labeled radioligands [
7,8]. However, to the best of our knowledge
M1 receptor subtype selective 18F-labeled mAChR ligands have not been reported so far.
Herewith we disclose the preparation of the radiofluorinated allosteric M1-specific mAChR agonist,
5-[18F]fluoro-6-methyl-1-(1-(tetrahydro-2H-pyran-4-yl)piperidin-4-yl)-1,3-dihydro-2H-benz[d]imida-
zole-2-one ([18F]
1), discovered by Budzik et al. [9] (Scheme 1). Ligand 1 has been shown to possess
excellent pharmacokinetic properties and robust pro-cognitive activity in animal models [9], making
[18F]1 a potentially useful probe for in vivo tracer studies.
Scheme 1. Preparation of intermediates 6a and
et al. [ ]. Conditions: ( ) KNO3, H2SO4, 16 h, r.t., 64% and 72% for 4a and 4b, respectively;
) 4-amino-N-Boc-piperidine, DIEA, DMF, 70–80 ◦C, 38 h, 63% and 72% for 5a and 5b, respectively;
) Raney nickel, N2H4 ) triphosgene,
H2O, EtOH, 45 ◦C, 2 h, 70% and 29% for 6a and 6b, respectively; (
) TFA/CH2Cl2, 1 h or HCl/EtOAc, r.t., 1 h, then ( ) NaBH3CN, Bu4NBr,
8 according to the modified protocol of Budzik
9
a
(
(
b
c
·
d
Et3N, THF, 45 ◦C, 2 h, 94%; (
e
f
Et3N, DMF, r.t., 48 h, 6%. r.t.—room temperature.
2. Results and Discussion
2.1. Preparation of the Reference Compound 1 and Pinacol Boronate Precursor 8 for Radiolabeling
Initially the modified procedure of Budzik et al. [ ] was applied in order to prepare the
non-radioactive ligand and the intermediate (Scheme 1). This synthetic route consists of nitration of
9
1
8
the respective 2-fluoro-5-halotoluene followed by the selective nucleophilic substitution of the fluorine
substituent in ortho-position to the nitro group with 4-N-Boc-aminopiperidine, reduction of the nitro
group, cyclization to the benzimidazolone, N-Boc deprotection and, finally, reductive alkylation of the
resulting intermediate with 4-tetrahydropyranone. The nitration step was performed as described
by Kher et al. [10], except that the reaction time was increased from 30 min to 16 h, affording known
4a and 4b in yields of 64% and 72%, respectively. The subsequent steps furnished the corresponding
o-phenylendiamines, 6a and 6b, in 70% and 29% yield, respectively. Cyclization of 6b using triphosgene
instead of the originally proposed 1,10-carbonyldiimidazole, afforded benzimidazolone
7 in almost
quantitative yield. Unexpectedly, deprotection of this intermediate by HCl in EtOAc or trifluoroacetic
acid (neat or in CH2Cl2) gave rise to a number of by-products. Reductive alkylation of the respective
crude amine afforded 8 in only 6% yield.

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In order to circumvent the problems mentioned above, 1-(tetrahydro-2H-pyran-
4-yl)piperidine-4-amine (10) was prepared by reductive alkylation of 4-N-Boc-aminopiperidine
(9) with tetrahydro-4H-pyran-4-one using NaBH3CN as reductant and Bu4NBr as phase transfer
catalyst followed by N-Boc deprotection and was allowed to react with 4a or 4b (Scheme 2) affording
the corresponding o-nitroanilines 11a and 11b in good yields. Reduction of the nitro group in
11a using Raney nickel and hydrazine hydrate proceeded smoothly, furnishing the corresponding
o-phenylenediamine intermediate in 63% yield. In contrast, application of the same procedure to the
bromo-substituted nitroaniline 11b afforded only traces of the product. Among several reductants
tested, powdered Zn/NH4Cl in a mixture of EtOH and EtOAc according to the modified procedure of
Tsukinoki and Tsuzuki [11] enabled the preparation of the desired intermediate in an excellent (>90%)
yield. Subsequent cyclization of the prepared o-phenylenediamines with triphosgene afforded ligand
and substituted imidazolone 8 in 14% and 33% yield over four steps, respectively.
1
Scheme 2. Convergent synthesis of ligand
4H-pyran-4-one, NaBH3CN, Bu4NBr, Et3N, DMF, 36 h, 57%; (
4a or 4b, DIEA, DMF, 70 ◦C, 16 h, 58% and 99% (crude) for 11a and 11b, respectively; (
nickel, N2H4 ) triphosgene,
H2O, EtOH, 45 ◦C, 2 h, 63% or 11b, NH4Cl, Zn, EtOH/EtOAc, 16 h, 90%; (
Et3N, THF, 45 ◦C, 2 h, 60% and 52% for 1 and 8, respectively.
1
and intermediate
) HCl/EtOAc, 1 h, then NaOH, 93%;
11a, Raney
8. Conditions: (a) tetrahydro-
b
(c)
d)
·
e
The boronic acid pinacol ester
2 was prepared in 48% yield by the Miyaura reaction, using
a modification of the protocol described by Ishiyama et al. [12] (Scheme 3). The Pd content determined
by ICP/MS amounted to 1.2 ± 0.1 ppm.
Scheme 3. Preparation of the precursor for radiolabeling
2 by Miyaura cross-coupling reaction.
Conditions: B2Pin2, Pd(dppf)Cl2, KOAc, 1,4-dioxane, 110 ◦C, 3 h, 48%.
2.2. Preparation of [18F]1
Radiolabeling of the Bpin ester
Cu-mediated alcohol-enhanced radiofluorination [13
onto an anion exchange resin and eluted with a solution of Et4NHCO3 in MeOH. After evaporation of
MeOH, [18F]Et4NF/Et4NHCO3 was taken up in a solution of
and Cu(py)4(OTf)2 in 2:1 DMA/nBuOH
and the reaction mixture was heated at 110 ◦C for 10 min under Ar or air to afford the 18F-labeled
ligand [18F]
in excellent (>90%) radiochemical conversions (RCCs). The crude tracer obtained after
concentration of the reaction mixture under reduced pressure was purified by HPLC and formulated
as a ready-to-use solution. On a preparative scale [18F]
was produced in a non-decay corrected
radiochemical yield (n.d.c RCY) of 17 3% (n = 3) and in excellent radiochemical purity (>99%) within
2
was performed according to the modified protocol for
–
17] (Scheme 4). [18F]Fluoride was loaded
2
1
1
±
90–100 min (Figure 1). Besides the molar activity, which is dependent on the activity amount, the carrier
amount per batch was measured (please refer to [18] for further discussion). The latter amounted

Molecules 2020, 25, 2880
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to 25.2 nmol/batch and the molar activity to 30.8 GBq/
µ
mol (measured for 770 MBq [18F]
1; refer to
the Supplementary Materials for more details). The Cu content, measured by ICP/MS, amounted to
3.4 0.1 g/batch and was below any level of concern according to the ICH Guideline of Elemental
±
µ
Impurities (Q3D) [19].
O
O
HN
HN
a)
N
N
O
B
O
N
18F
2
N
18
O
1
[ F]
O
Scheme 4. Preparation of [18F]
1
via alcohol-enhanced Cu-mediated radiofluorination. Conditions: (
, Cu(py)4(OTf)2,
3% (n = 3)]; HPLC purification; formulation
a)
elution of 18F– in the form of [18F]Et4NF with Et4NHCO3 in MeOH; removal of MeOH;
9
2:1 DMA/nBuOH, 110 ◦C, 10 min, air or argon [RCC = 96
(n.d.c RCY = 13–19%).
±
Figure 1. HPLC traces of the purified and formulated radiotracer [18F]
1
, and the 19F-reference
compound
1
. Blue trace: [18F]
1
(radioactivity channel); green trace: [18F]
1
(UV channel, = 210 nm);
λ
red trace: 19F-reference compound (UV channel, λ = 210 nm).
3. Materials and Methods
3.1. General
Chemicals and solvents were purchased from Sigma-Aldrich (Steinheim, Germany), Merck KGaA
(Darmstadt, Germany), OxChem (Wood Dale, IL, USA), VWR International (Radnor, PA, USA) and
Alfa Aesar (Haverell, MA, USA) and used without further purification.

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3.2. Nuclear Magnetic Resonance Spectroscopy (NMR)
Unless otherwise stated, all NMR-Spectra were measured in CDCl3. 1H-NMR spectra were
obtained with a Bruker DPX Avance 300 (Bruker, Rheinstetten, Germany). 1H chemical shifts are
reported in ppm relative to residual peaks of deuterated solvents. The observed signal multiplicities are
characterized as follows: s = singlet, d = doublet, t = triplet, m = multiplet and q = quartet.
Coupling constants are reported in Hertz (Hz). 13C-NMR spectra [additional APT (Attached
Proton Test)]: Bruker DPX Avance 300 (75 MHz). 13C chemical shifts are reported in ppm relative
to residual peaks of deuterated solvents. 1H-, 13C- and 19F-NMR spectra are provided in the
Supplementary Materials.
3.3. Mass Spectroscopy
Mass spectra (MS) were measured with a LTQ Orbitrap XL (Thermo Fisher Scientific Inc.,
Bremen, Germany).
3.4. Chemistry
All reactions were carried out with magnetic stirring. Air or moisture sensitive reagents were
handled under argon using either a glovebox or a Schlenk line. Organic extracts were dried over
anhydrous MgSO4. Solutions were concentrated under reduced pressure at 40–50 ◦C using a rotary
evaporator (Bruker, Rheinstetten, Germany).
Column chromatography was performed with silica gel (w/Ca, 0.1–0.3%), 60Å, 230–400 mesh
particle size from Sigma-Aldrich GmbH (Steinheim, Germany). Solvent proportions are indicated in
a volume/volume ratio.
Thin layer chromatography (TLC) was performed using aluminium sheets coated with silica
gel 60 F254 (Merck KGaA, Darmstadt, Germany). Chromatograms were inspected under UV light
(λ = 254 nm) and stained with molybdophosphoric acid (10% in ethanol), ninhydrin (0.2% in ethanol)
or Dragendorff reagent.
3.4.1. 2,5-Difluoro-4-nitrotoluene (4a)
KNO3 (3.2 g, 31.2 mmol) was added to an ice-cold solution of 2,5-difluorotoluene (3a) (4 g,
31.2 mmol) in concentrated H2SO4 (15 mL), the mixture was allowed to warm to ambient temperature
◦
and stirred at 28 C overnight. The reaction mixture was then poured over ice and the resulting
suspension was extracted with EtOAc (3
reduced pressure and the residue was purified by column chromatography (EtOAc/hexane = 1:15
×
50 mL). The combined organic layers were concentrated under
)
to afford the title compound 4a
[
9
]. Yield: 3.45 g, 20 mmol (64%). Appearance: yellow crystalline
1
solid [
9
,
10]. H-NMR: 7.78 (dd, J = 8.4, 6.3 Hz, 1H), 7.16 (dd, J = 10.9, 6.1 Hz, 1H), 2.39 (d, J = 1.8 Hz, 3H).
3.4.2. tert-Butyl 4-[(4-fluoro-5-methyl-2-nitrophenyl)amino] piperidine-1-carboxylate (5a)
Diisopropylethylamine (0.87 mL, 0.65 g, 5 mmol, 1 eq) was added to a solution of 4a (0.87 g,
5 mmol, 1 eq) and 4-amino-1-N-Boc-piperidine (1 g, 5 mmol, 1 eq) in DMF (8 mL) at 40 ◦C and the
resulting solution was stirred at 80 ◦C for 38 h. After removal of the solvent under reduced pressure
and addition of water, the resulting mixture was extracted with CH2Cl2 (3
organic fractions were dried and concentrated under reduced pressure. The residue was purified by
×
40 mL). The combined
column chromatography (EtOAc/hexane = 1:15) to afford the title compound 5a
[
9
]. Yield: 1.12 g,
1
3.16 mmol (63%). Appearance: orange-red solid. H-NMR: 8.02 (br, 1H), 7.84 (d, J = 10.0 Hz, 1H),
6.67 (d, J = 6.3 Hz, 1H), 4.21–3.85 (m, 2H), 3.79–3.55 (m, 1H), 3.08 (t, J = 11.1 Hz, 2H), 2.32 (s, 3H),
2.15–1.97 (m, 2H), 1.63–1.52 (m, 2H), 1.49 (s, 9H). 13C-NMR: 169.52, 152.27 (d, J = 353.3 Hz), 153.07,
141.35, 115.32 (d, J = 3.0 Hz), 111.70 (d, J = 27.0 Hz), 79.91, 49.40, 42.09, 36.47, 31.78, 28.41.

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3.4.3. tert-Butyl 4-([2-amino-4-fluoro-5-methylphenyl]amino)piperidine-1-carboxylate
Hydrazine hydrate (700
µL, 680 mg, 14 mmol, 10 eq) was slowly added to a suspension
of Raney nickel (0.7 mL, 50% aq.
suspension) in a solution of 5a (0.5 g, 1.4 mmol,
1 eq) in EtOH (30 mL). The mixture was stirred at 45 ◦C for 2 h and filtered over Celite.
After removal of volatiles under reduced pressure and addition of saturated NaHCO3 (30 mL),
the resulting emulsion was extracted with CH2Cl2 (3
were dried and concentrated under reduced pressure. The residue was purified by column
×
30 mL). The combined organic layers
chromatography (EtOAc/hexane = 1:1) followed by crystallization from hexane to afford the
title compound tert-butyl-4-([2-amine-4-fluoro-5-methylphenyl]amine)piperidine-1-carboxylate [9].
Yield: 0.32 g, 0.98 mmol (70%). Appearance: brown solid. 1H-NMR: 6.47 (d, J = 7.5 Hz, 1H),
6.42 (d, J = 10.5 Hz, 1H, H-60), 4.10 (d, J = 7.2 Hz, 1H), 4.03 (d, J = 11.8 Hz, 2H), 3.45 (t, J = 13.4 Hz, 2H),
3.25 (ddd, J = 13.8, 9.9, 3.7 Hz, 1H), 2.91 (t, J = 11.4 Hz, 2H), 2.15 (s, 3H), 1.97 (dd, J = 9.1, 3.7 Hz, 2H),
1.47 (s, 9H), 1.41–1.29 (m, 2H). 13C-NMR: 157.29, 154.49 (d, J = 48.0 Hz), 135.75 (d, J = 9.8 Hz), 130.64,
118.46 (d, J = 5.7 Hz), 103.66 (d, J = 26.1 Hz), 79.54, 51.24, 32.58, 28.44, 22.65, 14.06.
3.4.4. 2-Bromo-5-fluoro-4-nitrotoluene (4b)
KNO3 (2 g, 20 mmol, 1 eq) was added to an ice-cold solution of 3b (3.78 g, 20 mmol, 1 eq) in
concentrated H2SO4 (15 mL). The mixture was allowed to reach ambient temperature and stirred
overnight. Afterwards, the reaction mixture was poured over ice and extracted with EtOAc (3
The combined organic fractions were dried, concentrated under reduced pressure and the remaining
×
50 mL).
brown-red oil purified by column chromatography (CH2Cl2/MeOH = 9:1) to afford the title compound
1
4b
[
10]. Yield: 4.25 g, 14.4 mmol (72%). Appearance: yellow oil. H-NMR: 8.14 (d, J = 7.1 Hz, 1H),
7.18 (d, J = 11.4 Hz, 1H), 2.44 (s, 3H).
3.4.5. tert-Butyl 4-[(4-bromo-5-methyl-2-nitrophenyl)amino]piperidine-1-carboxylate (5b)
DIEA (0.87 mL, 0.65 g, 5 mmol, 1 eq) was added to a solution of 4b (1.5 g, 5 mmol, 1 eq) and
4-amino-1-N-Boc-piperidine (1 g, 5 mmol, 1 eq) in DMF (8 mL) at 40 ◦C. The resulting mixture was
stirred at 80 ◦C for 38 h and concentrated under reduced pressure. H2O (40 mL) was added to the
oily residue and the resulting suspension was extracted with CH2Cl2 (3
organic fractions were dried and concentrated under reduced pressure. The residue was purified by
×
30 mL). The combined
column chromatography (EtOAc/hexane = 1:15) to afford the title compound. Yield: 1.6 g, 3.6 mmol
1
(72%). Appearance: orange solid. H-NMR: (300 MHz, CDCl3)
δ
(ppm) = 8.35 (s, 1H), 8.06 (br, 1H),
6.74 (s, 1H), 4.02 (d, J = 13.6 Hz, 2H), 3.75–3.57 (m, 1H), 3.21–3.02 (m, 2H), 2.41 (s, 3H), 2.15–1.97 (m,
2H), 1.61–1.45 (m, 2H), 1.47 (s, 9H). 13C-NMR: 154.65, 147.22, 143.26, 130.65, 129.94, 115.06, 109.92,
79.98, 49.33, 42.11, 31.71, 28.45, 23.87.
3.4.6. tert-Butyl 4-[(2-amino-4-bromo-5-methylphenyl)amino]piperidine-1-carboxylate (6b)
Hydrazine hydrate (0.94 mL, 0.97 g, 18.83 mmol, 3 eq) was slowly added to a suspension of Raney
nickel (6 mL; 50% suspension in H2O) in a solution of 5b (2.6 g, 6.28 mmol, 1 eq) in ethanol (30 mL).
The mixture was stirred at 45 ◦C for 2 h and filtered over Celite. After removal of volatiles under
reduced pressure and addition of saturated NaHCO3 (30 mL), the resulting emulsion was extracted
with CH2Cl2 (3
×
30 mL). The combined organic layers were dried and concentrated under reduced
pressure. The residue was purified by column chromatography (EtOAc/hexane = 1:1) followed by
crystallization from hexane to afford the title compound. Yield: 0.71 g, 1.85 mmol (29%). Appearance:
1
orange solid. H-NMR: 6.91 (s, 1H), 6.52 (s, 1H), 4.05 (d, J = 12.1 Hz, 2H), 3.36 (td, J = 9.9, 4.9 Hz, 1H),
3.25 (s, 2H), 2.97 (t, J = 11.4 Hz, 2H), 2.30 (s, 3H), 2.03 (d, J = 10.5 Hz, 2H), 1.49 (s, 9H), 1.41 (dd, J = 17.3
,
7.3 Hz, 2H), 1.30 (dd, J = 12.3, 3.7 Hz, 1H); NH2-Group is unobservable. 13C-NMR: 135.57, 133.82, 129.18,
120.47, 115.20, 79.61, 50.15, 42.41, 31.71, 28.44, 22.30. MS: m/z: [M + H]+ calcd: 384.1; found: 384.1.

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3.4.7. tert-Butyl 4-(5-bromo-6-methyl-2-oxo-2,3-dihydro-1H-benz[d]imidazol-1-yl)-piperidine-1-
carboxylate (7)
Et3N (1.15 mL, 0.83 g, 8.25 mmol, 2.01 eq) was added dropwise to a solution of 6b (1.58 g,
4.1 mmol, 1 eq) and triphosgene (0.41 g, 1.37 mmol, 0.33 eq) in THF (10 mL). The reaction mixture was
stirred at 45 ◦C for 2 h. After removal of volatiles under reduced pressure and addition of saturated
NaHCO3 (30 mL), the resulting emulsion was extracted with EtOAc (3
fraction was dried and concentrated under reduced pressure. The residue was purified by column
×
30 mL). The combined organic
chromatography (EtOAc/hexane = 1:1) to afford the known title compound
7 [20], which was directly
used for the next step. Yield: 1.59 g, 3.87 mmol (94%). Appearance: white solid. MS: m/z: [2M + H]+2
calcd: 409.6; found: 410.1.
3.4.8. Deprotection of 7
Procedure A
An excess of 4 m HCl in EtOAc was added to a solution of
7 (0.27 g, 0.66 mmol) in CH2Cl2 (2 mL)
and the resulting mixture was stirred at ambient temperature for 1 h. After removal of volatiles
under reduced pressure and addition of 0.1 m NaOH (50 mL), the resulting mixture was extracted
with CH2Cl2 (3
×
30 mL). The combined organic fraction was dried and concentrated under reduced
pressure to afford the crude amine hydrochloride, which was used for the next step without any
purification and characterization. Yield: 0.18 g, 0.6 mmol (90% crude). Appearance: white solid.
Procedure B
TFA (30 mL) was slowly added to a solution of
7 (1.55 g, 3.78 mmol) in CH2Cl2 (30 mL) and
the reaction mixture was stirred at ambient temperature for 1 h. After concentration under reduced
pressure and addition of saturated NaHCO3 (30 mL) the resulting mixture was extracted with CH2Cl2
(3
×
30 mL). The combined organic fraction was dried and concentrated under reduced pressure to
afford the crude amine trifluoroacetate, which was used for the next step without any purification and
characterization. Yield: 1.44 g, ≤3.78 mmol (100% crude). Appearance: white solid.
3.4.9. 5-Bromo-6-methyl-1-[1-(tetrahydro-2H-pyran-4-yl)piperidin-4-yl]-1,3-dihydro-2H-
benz[d]imidazol-2-one (8). Protocol A
NaBH3CN (1.54 g, 24.5 mmol, 7 eq) was added to a solution of
7 (1.08 g, 3.5 mmol, 1 eq),
tetrahydro-4H-pyran-4-one (2.45 g, 24.5 mmol, 7 eq), Bu4NBr (0.79 g, 2.45 mmol, 0.7 eq) and Et3N
(1.46 mL, 1.06 g, 10.5 mmol, 3 eq) in CH2Cl2 (80 mL) and the reaction mixture was stirred for 24 h.
After concentration under reduced pressure and addition of 0.1 n NaOH (50 mL), the resulting
emulsion was extracted with CH2Cl2 (3
and concentrated under reduced pressure. The residue was purified by column chromatography
CH2Cl2/MeOH = 9:1) to afford the title compound. Yield: 83 mg, 0.21 mmol (6%). Appearance: white
solid. 1H-NMR [(CD3)2SO + TFA; mixture of
×
30 mL). The combined organic fraction was dried
(
8
and 8·TFA]: 7.22 (s, 1H), 7.16 (s, 1H), 4.57–4.51 (m,
1H), 4.00–3.78 (m, 2H), 3.63–3.61 (m, 2H), 3.61–3.51 (m, 1H), 3.49–3.38 (m, 2H), 3.25–3.19 (m, 2H),
2.72–2.63 (m, 2H), 2.36 (s, 3H), 1.98 (m, 4H), 1.76–1.171 (m, 2H). 13C-NMR [(CD3)2SO + TFA; mixture of
8
and 8·TFA]: 154.05, 153.93; 129.28, 129.24; 128.99, 128.41; 128.26, 115.39, 112.50, 110.86, 65.99, 62.08,
48.34, 47.54, 27.41, 26.26, 22.88; TFA: 158.86 (q, J = 37.4 Hz, CO2H), 115.76 (q, J = 289.9 Hz, CF3) 120.07,
117.20, 114.32, 112.50, MS: m/z: [2 M + MeOH + H]+2 calcd: 396.6; found: 396.1.
3.4.10. tert-Butyl [1-(tetrahydro-2H-pyran-4-yl)piperidin-4-yl]carbamate
NaBH3CN (0.16 g, 2.5 mmol, 1 eq) was added to a solution of 4-N-Boc-aminopiperidine (0.5 g,
2.5 mmol, 1 eq), tetrahydropyran-4-one (0.3 g, 3 mmol, 1.2 eq), acetic acid (0.23 mL, 0.24 g, 4 mmol,
1.6 eq), and Bu4NBr (97 mg, 0.3 mmol, 0.12 eq) in CH2Cl2 (20 mL) and the reaction mixture was stirred
for 2 days. Thereafter, the mixture was washed with saturated K2CO3 (10 mL), the organic layer was

Molecules 2020, 25, 2880
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dried and concentrated under reduced pressure. The residue was purified by column chromatography
(CH2Cl2/MeOH = 9:1) to afford the title compound. Yield: 0.34 g, 1.44 mmol (57%). Appearance:
1
white solid. H-NMR: 4.47 (s, 1H), 4.02 (d, J = 10.7 Hz, 2H), 3.44 (s, 1H), 3.37 (dd, J = 11.7, 9.7 Hz, 2H),
2.95 (d, J = 12.4 Hz, 2H), 2.50 (d, J = 11.1 Hz, 1H), 2.29 (t, J = 10.6 Hz, 2H), 1.98 (d, J = 10.9 Hz, 2H),
1.79 (d, J = 9.9 Hz, 2H), 1.71–1.57 (m, 2H), 1.55 (d, J = 7.1 Hz, 2H), 1.45 (s, 9H). 13C-NMR: 144.39, 67.49,
61.23, 48.03, 32.57, 29.30, 28.44. MS: m/z: [M + H]+ calcd: 285.2 found: 285.2.
3.4.11. 1-(Tetrahydro-2H-pyran-4-yl)piperidine-4-amine (10)
A solution of anhydrous HCl in EtOAc was prepared by addition of AcCl (2.25 mL, 2.48 g, 31.6 mmol,
4.9 eq) to an ice-cold solution of MeOH (1.34 mL, 1.06 g, 33 mmol, 5.2 eq) in EtOAc (8.25 mL). The resulting
solution was added to a solution of tert-butyl [1-(tetrahydro-2H-pyran-4-yl)piperidin-4-yl]carbamate
(1.83 g, 6.4 mmol, 1 eq) in CH2Cl2 (5 mL) and the mixture was stirred for 2 h. Volatiles were removed
under reduced pressure, the residue was taken up in 0.1 m NaOH (50 mL) and the resulting mixture
was extracted with CH2Cl2 (3
under reduced pressure to afford the title compound, which was directly used for the next step. Yield:
1.1 g, 5.96 mmol (93%). Appearance: yellow solid. H-NMR (CD3OD + TFA; mixture of 10
×
30 mL). The combined organic fraction was dried and concentrated
1
·TFA and
10·
2 TFA): 4.10–4.04 (m, 2H), 3.75–3.72 (m, 2H), 3.59–3.41 (m, 4H), 3.25–3.19 (m, 2H), 2.36–2.34 (m, 2H),
2.19–2.05 (m, 4H), 1.91–1.81 (m, 2H). MS: m/z: [M + H]+ calcd: 185.2; found: 185.4.
3.4.12. N-(4-Fluoro-5-methyl-2-nitrophenyl)-1-(tetrahydro-2H-pyran-4-yl)piperidine-4-amine (11a)
A solution of 10 (0.184 g, 1 mmol, 1 eq), 4a (0.173 g, 1 mmol, 1 eq) and DIEA (0.175 mL, 0.13 g,
5.8 mmol, 5.8 eq) in DMF (5 mL) was stirred at 70 ◦C for 16 h. After removal of volatiles under reduced
pressure and addition of H2O (30 mL), the resulting mixture was extracted with CH2Cl2 (3
×
10 mL).
The combined organic fraction was dried and concentrated under reduced pressure. The residue
was purified by column chromatography (CHCl3/acetone = 1:5) to afford the title compound which
was directly used for the next step without any characterization. Yield: 0.19 g, 0.58 mmol (58%).
Appearance: red-orange solid.
3.4.13. 4-Fluoro-5-methyl-N-(1-(tetrahydro-2H-pyran-4-yl)piperidin-4-yl)benzene-1,2-diamine
Hydrazine hydrate (56 µL, 61 mg, 0.95 mmol, 1.7 eq) was slowly added to a suspension of
Raney nickel (0.5 mL 50% suspension in H2O) in a solution of 11a (0.19 g, 0.56 mmol, 1 eq) in EtOH
(5 mL). The reaction mixture was stirred at 45 ◦C for 2 h and filtered over Celite. After removal of
volatiles under reduced pressure saturated NaHCO3 (20 mL) was added and the resulting mixture was
extracted with CH2Cl2 (3
×
30 mL). The combined organic fraction was dried and concentrated under
reduced pressure. The residue was purified by column chromatography (MeOH/CH2Cl2 = 1:6) and
recrystallized from hexane to afford the title compound which was used for the next step without any
characterization. Yield: 0.11 g, 0.35 mmol (63%). Appearance: brown solid.
3.4.14. 5-Fluoro-6-methyl-1-[1-(tetrahydro-2H-pyran-4-yl)piperidin-4-yl]-1,3-dihydro-2H-benz[d]-
imidazole-2-one (1)
Et3N (98
µL, 71 mg 0.7 mmol,
2
eq) was added dropwise to
a
solution
of 4-fluoro-5-methyl-N-(1-(tetrahydro-2H-pyran-4-yl)piperidin-4-yl)benzene-1,2-diamine (0.1 g,
0.35 mmol, 1 eq) and triphosgene (35 mg, 0.12mmol, 0.34 eq) in THF (5 mL). The mixture was
stirred at 45 ◦C for 2 h. After removal of volatiles under reduced pressure and addition of saturated
NaHCO3 (10 mL), the mixture was extracted with CH2Cl2 (3
fraction was dried and concentrated under reduced pressure. The residue was purified by column
×
10 mL). The combined organic
chromatography (MeOH/CH2Cl2 = 1:6) and recrystallized from CH2Cl2/hexane to afford
1
[
9
]. Yield:
1
70 mg, 0.2 mmol (60%). Appearance: white solid. H-NMR: 9.90 (s, 1H), 7.07 (d, J = 4.6 Hz, 1H), 6.83 (d,
J = 9.1 Hz, 1H), 4.35 (s, 1H), 4.08 (d, J = 9.2 Hz, 2H), 3.43 (t, J = 11.4 Hz, 2H), 3.15 (d, J = 4.1 Hz, 2H),
2.69–2.51 (m, 1H), 2.50–2.21 (m, 4H), 2.31 (s, 3H), 1.98–1.49 (m, 6H). 19F-NMR: −124.79 (s).

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3.4.15. N-(4-Bromo-5-methyl-2-nitrophenyl)-1-(tetrahydro-2H-pyran-4-yl)piperidine-4-amine (11b)
A solution of 10 (1.07 g, 5.8 mmol, 1 eq), 4b (1.36 g, 5.8 mmol, 1 eq) and DIEA (1 mL, 0.74 g,
5.8 mmol, 1 eq) in DMF (15 mL) was stirred at 70 ◦C for 16 h. After removal of volatiles under reduced
pressure, the residue was taken up with H2O (50 mL), and the mixture was extracted with CH2Cl2
(3
×
10 mL). The combined organic fraction was dried and concentrated under reduced pressure
affording the title compound (1.5 g, 66%). 1H-NMR (CDCl3): 8.35 (s, 1H),8.10 (t, J = 10.6 Hz, 1H),
6.74 (s, 1H), 4.07–4.04 (m, 2H), 3.57–3.55 (m, 1H), 3.43–3.38 (m, 2H), 2.93–2.91 (m, 2H), 2.59–2.45 (m,
3H), 2.41 (s, 3H), 2.19–2.05 (m, 2H), 1.79–1.68 (m, 6H). 13C-NMR (CDCl3): 147.06, 143.5, 130.60, 129.85,
115.13, 109.62, 67.54, 61.08, 49.12, 47.17, 31.93, 29.38, 23.82. MS: m/z: [M + H]+ calcd: 398.1; found: 398.1.
3.4.16. 4-Bromo-5-methyl-N-(1-(tetrahydro-2H-pyran-4-yl)piperidin-4-yl)benzene-1,2-diamine
Zn powder (2.66 g, 40.7 mmol, 10.7 eq) was added to a stirred solution of crude 11b (1.5 g, max.
3.8 mmol, 1 eq) and NH4Cl (2.18 g, 3.8 mmol, 1 eq) in a mixture of EtOAc/EtOH 1:1 (50 mL). The reaction
mixture was stirred overnight, then diluted with EtOAc (100 mL) and filtered over Celite. The filtrate
was concentrated under reduced pressure. The residue was purified by column chromatography
(MeOH/CH2Cl2, 1:6) followed by recrystallization from hexane to afford the title compound. Yield:
1
1.26 g, 3.42 mmol (58% over two steps). Appearance: brown solid. H-NMR: 6.90 (s, 1H), 6.51 (s, 1H),
4.05 (d, J = 8.7 Hz, 2H), 3.40 (t, J = 10.9 Hz, 2H), 3.25 (s, 2H), 2.96 (d, J = 11.6 Hz, 2H), 2.52 (s, 1H),
2.45–2.32 (m, 2H), 2.29 (s, 3H), 2.09 (d, J = 12.0 Hz, 2H), 1.81 (s, 1H), 1.76 (s, 2H), 1.69–1.34 (m, 4H).
13C-NMR: 135.96, 133.65, 129.18, 120.37, 119.87, 115.00, 67.65, 61.12, 50.22, 47.94, 32.83, 29.50, 22.39.
MS: m/z: [M + H]+ calcd: 368.1; found: 368.1.
3.4.17. 5-Bromo-6-methyl-1-[1-(tetrahydro-2H-pyran-4-yl)piperidin-4-yl]-1,3-dihydro-2H-
benz[d]imidazol-2-one (8). Protocol B
Et3N (0.75
µL, 0.55 g, 5.4 mmol, 2 eq) was added dropwise to a solution of
4-bromo-5-methyl-N-(1-(tetrahydro-2H-pyran-4-yl)piperidin-4-yl)benzene-1,2-diamine (1 g, 2.7 mmol,
1 eq) and triphosgene (0.27 g, 0.9 mmol, 0.33 eq) in THF (60 mL). The mixture was stirred at 45 ◦C for
2 h. After removal of volatiles under reduced pressure and addition of saturated NaHCO3 (60 mL),
the resulting mixture was extracted with CH2Cl2 (3
×
60 mL). The combined organic fraction was
dried and concentrated under reduced pressure to 5–7 mL. Hexane (50–70 mL) was added and the
precipitated solid was filtered off furnishing the title compound, which was directly used for the next
step. Yield: 0.56 g, 1.40 mmol (52%).
3.4.18. 6-Methyl-1-[1-(tetrahydro-2H-pyran-4-yl)piperidin-4-yl]-5-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)-1,3-dihydro-2H-benz[d]imidazol-2-one (2)
A suspension KOAc (0.39 g, 3.9 mmol, 3 eq) in a solution of
(47.5 mg, 65 mol, 0.05 eq) and bis(pinacolato)diboron (0.41 g, 1.63 mmol, 1.25 eq) in anhydrous
dioxane (6 mL) was stirred at 110 ◦C for 3 h. After removal of the volatiles under reduced pressure and
addition of 0.1 n NaOH (5 mL), the mixture was extracted with CH2Cl2 (3 20 mL). The combined
8 (0.51 g, 1.3 mmol, 1 eq), Pd(dppf)Cl2
µ
×
organic fraction was dried and concentrated under reduced pressure. The residue was purified by
column chromatography (MeOH/CH2Cl2 = 1:10) and recrystallized from CH2Cl2/hexane to afford the
1
title compound. Yield: 0.28 g, 0.62 mmol (48%). Appearance: white solid. H-NMR: 10.04 (s, 1H),
7.53 (d, J = 19.9 Hz, 1H), 7.12 (s, 1H), 4.37 (s, 1H), 4.09 (d, J = 9.5 Hz, 2H), 3.43 (t, J = 11.4 Hz, 2H),
3.16 (s, 2H), 2.60 (s, 3H), 2.69–2.25 (m, 2H), 2.05–1.55 (m, 6H), 1.33 (s, 12H). 13C-NMR: 155.44, 125.42,
116.74, 113.08, 111.02, 109.18, 83.35, 67.63, 48.83, 29.70, 24.89, 22.35; C-Bpin was not observed. MS: m/z:
[M + H]+ calcd: 442.3; found: 442.3.

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3.5. Radiochemistry
3.5.1. General
[18F]Fluoride was produced by the 18O (p,n) 18F reaction by bombardment of enriched [18O] water
with 17 MeV protons at the BC1710 cyclotron (The Japan Steel Works, Tokyo, Japan) of the INM-5
(Forschungszentrum Jülich).
Radioactivity was measured using a CRC-55tR Dose Calibrator from Capintec, Inc.
(Florham Park, Netherlands).
All radiosyntheses were carried out in 5 mL V-Vials (Wheaton) equipped with PTFE wing coated
stir bars using anhydrous DMA (Aldrich), anhydrous nBuOH and anhydrous MeOH dried over
molecular sieves (both Acros Organics, Geel, Belgium). Cu(OTf)2(py)4 was prepared according to the
literature [21] and stored under ambient conditions without any precautions.
Radiolabeling experiments were performed using AREX-9 Digital Pro hot plate equipped with
VTF digital thermometer (VELP SCIENTIFICA, Usmate Velate MB, Italy), vacuum pump Laboport
(KNF, Freiburg im Breisgau, Germany) in the customized hot cell installed by Von Gahlen Nederland
B.V (Zevenaar, Netherlands).
Radiosyntheses were carried out under synthetic air (80% N2 + 20% O2) or Ar.
Sep-Pak Plus C18 cartridges and Sep-Pak Accell Plus QMA carbonate plus light cartridges, 46 mg
sorbent per cartridge, (both Waters GmbH, Eschborn, Germany) were applied.
HPLC analyses and preparative separations were carried out using an Ultimate® 3000 HPLC
system from Thermo Fisher Scientific (Sunnyvale, CA, USA) with variable wavelength detector coupled
in series with a HERM LB 500 radio-flow monitor (Berthold Technologies, Bad Wildbad, Germany).
The unselective adsorption of 18F− onto HPLC columns was determined to be <10% in each case.
The UV and radioactivity detectors were connected in sequence, giving a time delay of 0.1–0.9 min
between the corresponding responses, depending on the flow rate. The identity of [18F]
by the co-injection of the non-radioactive reference compound.
Analytical HPLC
1 was confirmed
Column: Luna C18 (2), 250
(0.1% TFA); flow rate: 1.5 mL/min.
×
4.6 mm (Phenomenex, Aschaffenburg, Germany); eluent: 20% MeCN
Preparative HPLC
Column: Luna C18 (2), 250
(0.1% TFA); flow rate: 4 mL/min.
×
10 mm (Phenomenex, Aschaffenburg, Germany); eluent: 25% MeCN
3.5.2. Processing [18F]fluoride
Aqueous [18F]fluoride (0.02–7 GBq) was loaded onto an anion-exchange resin (QMA cartridge)
from the male side. The resin was flushed with MeOH (2 mL) and dried with 5–10 mL of air from
the male side. 18F− was slowly eluted into the reaction vial from the female side using a solution of
Et4NHCO3 (2 mg, 10 µmol) in MeOH (0.5 mL). It should be noted that flushing with MeOH was
carried out from the male side whereas drying and 18F− elution were carried out from the female
side. If the QMA cartridge had been loaded, flushed, and eluted from the female side only, sometimes
a significant amount of [18F]fluoride remained on the resin, probably because QMA light cartridges
have a single frit on the male side but four frits on the female side. MeOH was evaporated using a
flow of air at 80 ◦C within 10 min. Using this procedure, recovery of 18F− amounted to ≥85%.
3.5.3. 5-[18F]Fluoro-6-methyl-1-(1-(tetrahydro-2H-pyran-4-yl)piperidin-4-yl)-1,3-dihydro-2H-benz-
[d]-imidazol-2-one ([18F]1)
A solution of the pinacol boronate precursor 2 (7.2 mg, 16 µmol, 1 eq) and Cu(py)4(OTf)2 (13.6 mg,
20 µmol, 1.25 eq) in DMA/nBuOH 2:1 (750 µ
L) was added to [18F]Et4NF, and the resulting solution was
heated at 110 ◦C for 10 min under air or argon. For the determination of RCC, the reaction mixture was
cooled to <40 ◦C, 0.1% TFA (1 mL) was added and the mixture was stirred for 30 s. HPLC analysis

Molecules 2020, 25, 2880
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demonstrated that [18F]
1
formed in a RCC of 96
±
3% (n = 3). For isolation of [18F]
1
, the reaction mixture
was concentrated at 110 ◦C for 10 min, the residue was taken up in 25% MeCN (0.1% TFA) and the
mixture was purified by preparative HPLC. The fraction containing [18F]
1 was diluted with a 10-fold
volume of H2O and loaded onto a C18 cartridge. The cartridge was washed with 5% MeCN (10 mL),
H2O (5 mL) and dried with air (10 mL). The tracer was eluted with EtOH (1 mL). EtOH was evaporated
at 90 ◦C and the residue was taken up in saline for injection affording [18F]
1 as a ready-to-use solution
in 13–19% n.d.c RCY (Table 1).
Table 1. Preparation of [18F]1 on a preparative scale.
18F− (MBq)
[
18F]1 (MBq)
n.d.c. RCY (%)
3300
3970
1850
460
770
340
13
19
18
4. Conclusions
We developed a straightforward and efficient protocol for the manual preparation of [18F]
1,
a potentially M1 selective PET-probe for in vivo studies. The protocol is amenable to automation owing
to its simplicity, and thus provides an important step towards the routine production of 18F-labeled M1
selective PET tracers for experimental and diagnostic applications.
Supplementary Materials: The following are available online at http://www.mdpi.com/1420-3049/25/12/2880/s1,
1H-, 13C- and 19F-NMR spectra and determination of molar activity and carrier amount.
Author Contributions: B.D.Z., T.R., B.D. and N.K. performed experiments, B.N. and B.D.Z. conceived and
designed experiments, F.N., B.D.Z. and B.N. wrote the paper. All authors have read and agreed to the published
version of the manuscript.
Funding: This work was funded by the DFG (grant ZL 65/1-1).
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of all compounds are available from the authors.
©
2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).