Synthesis and in vitro characterization of trans- and cis-[ 18F]-4-methylbenzyl 4-[(pyrimidin-2-ylamino)methyl]-3- fluoropiperidine-1-carboxylates as new potential PET radiotracer candidates for the NR2B subtype N-methyl-d-aspartate receptor

Article abstract of DOI:10.1016/j.ejmech.2012.04.011

Diastereoisomeric compounds [¹⁸F]cis- and [¹⁸F]trans- 4-methylbenzyl 4-[(pyrimidin-2-ylamino)methyl]-3-fluoro-piperidine-1- carboxylates were successfully synthesized as new subtype-selective PET radiotracers for imaging the NR2B sub

Full text of DOI:10.1016/j.ejmech.2012.04.011

European Journal of Medicinal Chemistry 53 (2012) 408e415
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Preliminary communication
Synthesis and in vitro characterization of trans- and cis-[18F]-4-methylbenzyl
4-[(pyrimidin-2-ylamino)methyl]-3-fluoropiperidine-1-carboxylates as new
potential PET radiotracer candidates for the NR2B subtype
N-methyl-
D
-aspartate receptor
,
,
,
,
,b
Radouane Koudih a b, Gwénaëlle Gilbert a b, Martine Dhilly a b, Ahmed Abbas a b, Louisa Barré a
,
,
,b,
*
Danièle Debruyne a b, Franck Sobrio a
a CEA, I2BM, LDM-TEP, Centre Cyceron, F-14074 Caen, France
b UMR CNRS 6301, ISTCT, Université de Caen Basse-Normandie, Centre Cyceron, BP 5229, F-14074 Caen, France
a r t i c l e i n f o
a b s t r a c t
Diastereoisomeric compounds [18F]cis- and [18F]trans-4-methylbenzyl 4-[(pyrimidin-2-ylamino)methyl]-
3-fluoro-piperidine-1-carboxylates were successfully synthesized as new subtype-selective PET radio-
tracers for imaging the NR2B subunit containing NMDA receptors. Rat brain section autoradiographies
demonstrated a high specific binding in NR2B/NMDA receptor rich regions for both radioligands. The
measured logD7.4 values as well as Bmax/Kd ratios indicated that both radiotracers possess the adequate
properties required for PET radiotracers.
Article history:
Received 21 February 2012
Received in revised form
3 April 2012
Accepted 7 April 2012
Available online 19 April 2012
Ó 2012 Elsevier Masson SAS. All rights reserved.
Keywords:
Fluorine-18
PET
NR2B NMDA receptors
In vitro binding
Radiotracer
Radiolabelling
1. Introduction
[3]. Among them, the NR2B subunit containing NMDAR is the focus
of increasing interest as a therapeutic target in a wide range of CNS
A functional N-methyl-
D
-aspartate receptor (NMDAR) is a het-
pathologies, including acute and chronic pain, stroke and head
trauma, drug-induced dyskinesia or dementia [4,5]. The subtype
selectivity and cellular location of the NR2B receptors within the
CNS is expected to be related to the lesser adverse effects on
locomotion and so, to the improved therapeutic index of the NR2B
selective antagonists [6]. A lot of developed bioactive compounds
were derivatives of ifenprodil, the first selective antagonist
described for the NR2B NMDAR (Fig. 1) [7e9]. Others series were
also developed like cinnamidines or benzamidines [6].
eromeric complex of NR1 and NR2 subunits that mediates rapid
excitatory neurotransmission throughout the central nervous
system (CNS) where it is highly expressed. However, the thera-
peutic use of NMDAR antagonists is restricted by side effects
including hallucinations and loss of coordination [1,2]. Heteroge-
neity of NR2 subunits results from encoding by four distinct genes
leading to NR2A-D subunits that have diverse pharmacological
properties and substantial different expression patterns in the CNS
To explore the disease states, to provide new diagnosis tool and
to validate innovative therapies, the development of positron
emission tomography (PET) tracers targeting the NR2B subtype
NMDAR is needed and is still a challenge. All evaluated radiotracers
have suffered so far from low brain penetration, fast metabolism
and/or lack of in vivo specific binding for the NR2B subtype NMDAR
[10e12].
Abbreviations: BBB, blood brain barrier; CNS, central nervous system; EOB, end
of bombardment decay-corrected; HPLC, High pressure liquid chromatography;
HRMS, High resolution mass spectroscopy; Ms, mesylate; NMDAR, N-methyl-D-
aspartate receptor; NMR, Nuclear magnetic resonance; Ns, nosylate; PET, Positron
emission tomography; ROI, region of interest; SEM, standard error of mean.
* Corresponding author. UMR CNRS 6301, ISTCT, Université de Caen Basse-
Normandie, Centre Cyceron, BP 5229, F-14074 Caen, France. Tel.: þ 33 2 31 47 02
64; fax: þ33 2 31 47 02 75.
Among the candidate molecules that possess the characteristics
for a PET radiotracer, we focused on a series of antagonists recently
E-mail address: sobrio@cyceron.fr (F. Sobrio).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2012.04.011

R. Koudih et al. / European Journal of Medicinal Chemistry 53 (2012) 408e415
409
2. Results and discussion
2.1. Chemistry
Both fluorinated diastereoisomers were synthesized as refer-
ence compounds for the identification of the radiolabeled
compounds, starting from cis- and trans-3. Following the same
route, the two mesylate labelling precursors were prepared as
described in Scheme 1 from the cis- and trans-2. The preparation of
both isomers 3 was achieved by a Wittig reaction on the N-Boc-4-
oxo-3-fluoropiperidine [17] leading to the N-Boc-4-methylene-3-
fluoropiperidine 14 which reacted with the 9-BBN to a hydro-
boration reaction leading to the cis and trans alcohol isomers 3
(Scheme 2) [18]. Each time, for the fluorinated and alcohol
compounds, the same procedures were applied to the cis- and
trans-isomers. The azides 6 and 7 were prepared by selective
conversion of the primary alcohols [19,20] 2 and 3 into mono
sulfonates 4 and 5, followed by reaction with sodium azide.
Compounds 6 and 7 were reduced by catalytic hydrogenation and
then nucleophilic substitution with 2-chloropyrimidine yielded
compounds 8 and 9. The removal of the N-Boc protecting group by
HCl gas gave the amine of the piperidine which reacted with the in
situ generated 4-methylbenzyl succimidyl carbonate leading to the
reference compounds cis-11 and trans-11 and to alcohols 10.
Reaction of 10 with mesyl chloride in the presence of silver triflate
afforded the labelling precursors cis-12 and trans-12.
Fig. 1. Structures of NR2B NMDAR antagonists.
described by Merck [13,14]. In this series, in vitro and in vivo
pharmacology of compound 1 (Fig. 1) was evaluated and presented
very interesting properties. First,
1
had
a
high affinity
(Ki ¼ 3.4 ꢀ 2.2 nM in binding on Ltk-cells and EC50 ¼ 5.6 nM in
calcium flux assay) and was selective for the NR2B subunit without
any interactions with the hERG receptor system. The T1/2 about
2.5 h in rat and rhesus is compatible with PET scan duration.
Compound 1 was described as highly brain penetrant and without
Pgp substrate activity assessed by in vivo data and in vitro assays.
The plasma concentration of 1 for the occupancy of 50% of the NR2B
NMDAR was evaluated by an ex vivo binding assay and was found to
be 230 nM. Compound 1 was active in two animal pain models and
in one preclinical Parkinson’s disease model demonstrating that
compound 1 was CNS active and therefore crossed the blood brain
barrier (BBB) [13]. Merck developed a fluorinated analogue of 1,
namely MK-0657 (Fig. 1) [14,15], which presented similar proper-
ties as an IC50 ¼ 3.6 nM and selectivity vs. NR2A. In vivo, an effective
plasma concentration of 400 nM in a Parkinson’s primate model
was needed to occupy 50% of the NR2B receptor [16]. The phar-
macological properties of MK-0657 and of its non-fluorinated
analogue 1 and the presence of a fluorine atom on the alipha-
tic piperidine ring led us to consider its radiolabelling with
fluorine-18. To study further the influence of the fluorine orienta-
tion on the pharmacological proprieties of the molecule, we
investigated the radiosynthesis of both diastereoisomers cis and
trans-11 (Scheme 1). The in vitro pharmacological evaluation
was achieved to compare the two radiotracers and to evaluate
their capability to satisfy the necessary prerequisites for a radio-
tracer for PET imaging.
2.2. Radiochemistry
The radiolabelling was achieved by a one-step nucleophilic
substitution reaction of the precursors cis-12 and trans-12 by 18F
complex with Kryptofix 2.2.2 and K2CO3 as shown in Scheme 3.
After azeotropic drying with acetonitrile of the [18F]-fluoride
complex, the radiofluorination occurred on mesylate precursor
(11 m
mol) in acetonitrile (1 mL) at 90 ꢁC for 20 min. The identities of
the labelled compounds were confirmed by co-injection of their
corresponding fluorine-19 references cis-11 or trans-11 using
analytical HPLC. The reaction of the trans-12 precursor led to the
formation of the [18F]cis-11 with an inversion of configuration
corresponding to the classical SN2 substitution mechanism.
Following the same way, the reaction of the precursor cis-12 gave
the [18F]trans-11 compound. The [18F]-fluorine incorporation yield
was measured from radio-TLC integration and was about 31e45%
for both radiotracers. The radiofluorination occurred with the
elimination side-reaction which led to the alkene 13. This elimi-
nation was so prevalent that the synthetic pathway was not
Scheme 1. Reagents: (a) MsCl, pyridine, 0 ꢁC or NsCl, pyridine, ꢂ50 ꢁC, (b) NaN3, DMF, (c) i) Pd/C, H2, methanol, ii) 2-chloropyrimidine, DIPEA, amyl alcohol, (d) i) HCl gas, ethyl
acetate, ii) DSC, 4-MeBnOH, CH2Cl2, acetonitrile, DMF, DIPEA then piperidine, (e) MsCl, AgOTf, pyridine, 0 ꢁC.

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R. Koudih et al. / European Journal of Medicinal Chemistry 53 (2012) 408e415
cis-11 was shown in the forebrain regions. The highest density was
measured in the hippocampus and cortex (especially with [18F]cis-
11), the intermediate in striatum and thalamus, and the lowest in
olfactory tubercle (Fig. 2A). No specific binding sites were detect-
able in the cerebellum for [18F]trans-11 as well as for [18F]cis-11. As
previously reported for the [3H]CP-101,606 [21], another NR2B
subunit selective antagonist, the binding was the strongest in the
dentate gyrus within the hippocampus and the outer layers of the
neocortex were more labelled into the cortex (Fig. 2A). The
maximal densities of [18F]trans-11 and [18F]cis-11 binding sites
measured in the different brain structures (Table 1) agreed with the
mean Bmax value obtained from scatchard analysis of the binding of
racemic [3H]CP 101,606 (Bmax ¼ 1.6 pmol mg proteinꢂ1) [24] or of
the [3H] Ro 25-6981 (Bmax ¼ 1.4 ꢀ 0.2 pmol mg proteinꢂ1) [25]
obtained using rat forebrain membranes. The Kd values were
lower than 10 nM for both radiotracers (Table 1) assuming
a favourable binding affinity in accordance with the IC50 value
reported for the MK-0657 (3.6 nM) [16]. The Kd values was of the
same order as [3H] Ro 25-6981 (Kd ¼ 2.3 nM) [25] and [3H]CP
101,606 (Kd ¼ 10 nM) [24] both measured on rat forebrain
membranes. The Kd values calculated here on specific brain struc-
tures ranged from 1.9 to 7.1 nM for [18F]trans-11 and were lower
compared to 5.9e15.2 nM for [18F]cis-11, suggesting that the [18F]
trans-11 affinity was about two fold higher than for the cis isomer.
The Bmax/Kd ratio that is considered to be one of the best criteria to
select a radiotracer [26] is also promising for both compounds since
the ratios were superior to 10, with values (17-37) slightly higher
for [18F]trans-11.
Scheme 2. Reagents: (a) nBuLi, CH3PPh3Br, THF, ꢂ78 ꢁC to RT, (b) i) 9-BBN, THF, reflux,
ii) NaOH 3N, H2O2, 0 ꢁC.
successful in fluorine-19 chemistry to obtain the reference
compounds cis- and trans-11. The trace amount of fluoride-18
compared to the precursor concentration allowed this reaction by
a kinetic effect. The purification was achieved by reversed phase
HPLC followed by a reformulation by solid phase extraction to
obtain a radioactive concentrated saline/ethanol (9/1) solution for
i.v. injection. The radiosynthesis was automated on a commercial
TRACERlab FX F-N module (GE Healthcare) and the radiochemical
yields (n ¼ 8) were 18 ꢀ 6% end of bombardment decay-corrected
(EOB) for [18F]trans-11 for 89 min of synthesis time and 13 ꢀ 5%
(EOB) for [18F]cis-11 produced within 100 min. 400e750 MBq of the
radiotracers were produced and the specific radioactivities were
236 ꢀ 45 and 170 ꢀ 80 GBq
m
molꢂ1 for [18F]trans-11 and [18F]cis-
11, respectively. The radiochemical purities were more than 98%.
The logD7.4 was measured by the shake flask method by radio-
active counting of each phase. The logD7.4 was 2.80 ꢀ 0.02 for [18F]
cis-11 and 2.66 ꢀ 0.09 for the [18F]trans-11. Both values are between
2 and 3.5 which are the best values to predict a good bloodebrain
barrier passage [27]. The weak difference between the two
stereoisomers could be due to the fluorine position (equatorial or
axial) influencing the pKa value of the amine of the piperidine about
0.9 units [25].
2.3. In vitro characterization
In order to assess the subunit selectivity and specificity of these
new radiotracers and to know the distribution pattern of the
radiolabelled compounds, autoradiographic in vitro binding
experiments were performed on rat brain slices [21e23]. The
radioactive binding (0.15e48 nM) of [18F]cis-11 and [18F]trans-11
3. Conclusion
was strongly inhibited by Ro 25e6981 (10
mM), a specific and
selective NR2B NMDAR antagonist, leading to a low non-specific
binding measured in all brain regions (Fig. 2). The percentage of
non-specific binding to total binding varied according to the region
of interest (ROI). At the [18F]cis-11 concentration of 8 nM, the
specific binding represented 76% in the thalamus and up to 91% in
the hippocampus and was higher than for the trans isomer (62%
and 84%, respectively) (Fig. 3). The ratio of structure vs. cerebellum,
a region devoid of NR2B subunit, ranged from 6.5 for the thalamus
to 12.1 for the hippocampus for the [18F]cis-11 compound and was
greater than for [18F]trans-11 (4.1 and 6.4, respectively). An
extremely high specific localization of both [18F]trans-11 or [18F]
The diastereoisomeric reference compounds cis- and trans-11
and the labelling precursors cis- and trans-12 were prepared in six
or seven steps. The nucleophilic radiofluorinations of both [18F]cis-
and [18F]trans-11 from the mesylate precursors trans- and cis-12
respectively, were successfully achieved and adapted to an auto-
mated synthesis module. The two potential radiotracers appeared
to be promising candidates as PET ligands for studying NMDA/
NR2B subunit receptor with equivalent in vitro properties.
However, the contrast between structures and cerebellum was
higher for [18F]cis-11 than for [18F]trans-11. Even if [18F]cis-11 pre-
sented a higher specific binding than [18F]trans-11, the Bmax/Kd ratio
criterion was slightly higher for the trans isomer than for the cis
one. Moreover, the found logD7.4 reinforced the hypothesis based
on the use of MK-0657 in clinical trial that those radiotracers
should cross the BBB. All the criteria as defined by Pike [27] are
present to achieve high contrast brain images of the NR2B subunit
NMDAR.
4. Experimental section
4.1. Chemistry. Materials and reagents
Reagents and solvents used, unless stated otherwise, were of
commercially available reagents grade quality and were used
without further purification. Nuclear magnetic resonance spectra
1H, 13C and 19F NMR were recorded on a Bruker DRX 400, at
Scheme 3. Radiolabelling of [18F]-11.

R. Koudih et al. / European Journal of Medicinal Chemistry 53 (2012) 408e415
411
Fig. 2. Autoradiograms of rat brain sagittal sections after in vitro binding of [18F]cis-11 (8 nM) alone (A) and with co-incubation with Ro 25-6981 (10
mM) (B). Cb: cerebellum; Cx:
cortex; Hip: hippocampus; ST: striatum; OT: olfactory tubercle; Th: thalamus.
400 MHz, 100.6 MHz and 376.4 MHz respectively. 1H NMR spectra
were referenced internally on CDCl3
1H: 7.26), 13C NMR spectra
were referenced internally on CDCl3
13C: 77.20) and 19F NMR
spectra were referenced internally on CFCl3
19F: 0.00). Coupling
a Waters 996 photodiode arrays detector (210e380 nm) coupled
with a NaI probe radioactive detector (Novelec, France). Purity was
determined by HPLC on an analytical column CC 250/4 Nucleodur
C-18 Pyramid (excepted for 3: C-18 Gravity), MachereyeNagel,
(
d
(
d
(d
constants (J) are expressed in hertz (Hz) and coupling patterns are
abbreviated as: s (singlet), bs (broad singlet), d (doublet), t (triplet),
q (quadruplet), m (mutiplet), t (triplet), dd (doublet of doublet),
dddd (doublet of doublet of doublet of doublet) and dt (doublet of
triplet). Flash chromatography purifications were performed on
MachereyeNagel silica gel 60 M (0.04e0.063 mm). Thin Layer
Chromatography (TLC) was run on pre-coated aluminium plates of
silica gel 60F254 (Merck) and Rf were established using a UV-lamp
at 254 nm or by ninhydrin or phosphomolybdic acid hydrate
spray reagent. Radioactive TLC was measured using an Instant
ImagerÒ Packard apparatus. Melting points were determined on
a Barnstead Electrothermal IA 9100 melting point apparatus and
are uncorrected. HPLC experiments were performed using a Waters
600 pump and controller, a Waters 717 plus autosampler and
5 mm; 1 mL/min at 235 nm and the purities of compounds were
found to be at least 98%. [18F]-fluoride was produced by the
18O(p,n)18F nuclear reaction using the Cyclone 18/9 (IBA) cyclotron
at the Cyceron PET Centre. Irradiation occurred on target filled with
18O-enriched water (97%, Eurisotop).
4.2. Chemical synthesis
4.2.1. tert-Butyl 1-[3-fluoro-4-methylenepiperidin-1-yl]carboxylate
(14)
A solution of n-butyllithium in THF (2.5 M, 11.9 mL, 19.0 mmol)
was slowly added at ꢂ78 C to a solution of methyl triphenylphos-
phonium bromide (4.71 g, 20 mmol) in THF (40 mL) and the
mixture was stirred at ꢂ78 ꢁC for 2 h. A solution of tert-butyl 1-(4-
oxo-3-fluoropiperidin-1-yl) carboxylate (3.45 g, 15.8 mmol) in THF
(10 mL) was added drop wise and the mixture was allowed to warm
at room temperature and stirred overnight. The reaction mixture
was quenched with water (40 mL) and extracted with heptanes
(3 ꢃ 50 mL). The phosphine was removed by filtration and the
combined organic layers were dried over MgSO4, filtered, concen-
trated under reduced pressure and purified by chromatography on
silica gel (heptanes:ethyl acetate, 95:5) to afford 14 as colourless oil
(2.22 g, 65%). 1H NMR (CDCl3):
J ¼ 48 Hz, 1H), 3.68e3.21 (m, 4H), 2.44e2.37 (m, 1H), 2.13e2.07 (m,
d 5.02 (s, 1H), 4.91 (s, 1H), 4.75 (d,
1H), 1.40 (s, 9H). 13C NMR (CDCl3):
d
154.8, 142.8 (d, J ¼ 16 Hz), 111.5,
88.0 (d, J ¼ 176 Hz), 80.1, 49.3 (d, J ¼ 52 Hz), 44.3, 31.3, 28.3. 19F NMR
(CDCl3):
d
ꢂ182.8 (d, J ¼ 372 Hz). HRMS m/z calcd. for C11H19FNO2
216.1400, found: 216.1398.
4.2.2. (ꢀ)-cis-tert-Butyl 1-[3-fluoro-4-(hydroxymethyl)piperidin-
1-yl]carboxylate and (ꢀ)-cis-tert-butyl 1-[3-fluoro-4-
(hydroxymethyl)piperidin-1-yl]carboxylate (cis-3 and trans-3)
A solution of 9-BBN (0.5 M in THF, 44.65 mL, 22.32 mmol)
was added under nitrogen atmosphere to a solution of tert-butyl
1-(3-fluoro-4-methylenepiperidin-1-yl) carboxylate 14 (3.2 g,
14.88 mmol) in THF (55 mL). The resulting solution was refluxed for
2 h. After cooling to 0 ꢁC, NaOH 3 N (25 mL) was added and the
Fig. 3. Radioligands concentration in brain structures after in vitro binding of [18F]-11
(8 nM) alone and with co-incubation with Ro 25-6981 (10 mM) on rat brain sections.
Table 1
Bmax, Kd and Bmax/Kd ratio for [18F]trans-11 and [18F]cis-11 in rich NR2B NMDAR rat brain structures.
a
Brain structure
Bmax ꢀ SEM (pmol mg proteinꢂ1
)
Kd ꢀ SEM (nM)
Bmax/Kd
18F]cis-11
[
18F]cis-11
[
18F]trans-11
[
18F]cis-11
[
18F]trans-11
[
[
18F]trans-11
Cortex
Hippocampus
Striatum
Thalamus
Olfactory tubercle
1.50 ꢀ 0.17
1.65 ꢀ 0.14
1.41 ꢀ 0.13
1.12 ꢀ 0.05
1.05 ꢀ 0.05
0.77 ꢀ 0.10
1.58 ꢀ 0.18
1.05 ꢀ 0.23
0.86 ꢀ 0.05
0.69 ꢀ 0.13
9.8 ꢀ 0.9
9.1 ꢀ 2.1
9.3 ꢀ 0.6
3.2 ꢀ 0.6
7.1 ꢀ 1.7
5.1 ꢀ 1.1
5.4 ꢀ 1.1
1.9 ꢀ 0.2
15 ꢀ 6
19 ꢀ 2
15 ꢀ 1
8 ꢀ 1
25 ꢀ 2
22 ꢀ 1
21 ꢀ 1
17 ꢀ 3
37 ꢀ 3
15.2 ꢀ 1.7
5.9 ꢀ 0.5
18 ꢀ 1
a
Assuming 10% proteins per gram of tissue.

412
R. Koudih et al. / European Journal of Medicinal Chemistry 53 (2012) 408e415
mixture was stirred for 5 min. Then, H2O2 (25 mL) was added and
the stirring was hold for 10 min. The reaction was quenched with
water (50 mL) and the mixture was extracted with CH2Cl2
(3 ꢃ 50 mL). The combined organic layers were dried over MgSO4
and concentrated under reduced pressure. Purification by silica gel
chromatography (CH2Cl2:AcOEt, 9:1) permitted to obtain cis-3
(1.7 g, 49%) and trans-3 (1.5 g, 43%) as colourless oils. TLC
(CH2Cl2:MeOH, 95:5) Rf(trans-3) ¼ 0.23, Rf(cis-3) ¼ 0.23. Cis-3: 1H
(3 ꢃ 20 mL). The combined organic layers were dried over MgSO4,
concentrated under reduced pressure and purified by chroma-
tography on silica gel (CH2Cl2:AcOEt, 9:1) gave cis-5 as a white
solid (1.25 g, 85%). Mp 81e83 ꢁC. 1H NMR (CDCl3):
d 4.70 (d,
J ¼ 48 Hz, 1H), 4.38e4.06 (m, 4H), 2.97 (s, 3H), 2.78e2.64 (m, 2H),
2.08e1.95 (m, 1H), 1.58e1.47 (m, 2H), 1.40 (s, 9H). 13C NMR
(CDCl3):
d
155.0, 84.6 (d, J ¼ 176 Hz), 80.1, 69.4 (d, J ¼ 3.9 Hz), 47.6,
42.6, 39.4 (d, J ¼ 19.8 Hz), 37.3, 28.4, 22.1. 19F NMR (CDCl3):
NMR (CDCl3):
d
4.74 (d, J ¼ 48 Hz,1H), 4.36e4.04 (m, 2H), 3.67e3.62
d L204.1. HRMS m/z calcd. for C12H22FNO5SNa 334.1100, found:
(m, 1H), 3.56e3.52 (m, 1H), 2.82e2.59 (m, 2H), 1.76e1.54 (m, 3H),
1.39 (s, 9H). 13C NMR (CDCl3) 155.2, 86.1 (d, J ¼ 176 Hz), 79.8, 63.2 (d,
J ¼ 3.3 Hz), 49.0, 42.6, 41.8 (d, J ¼ 19.8 Hz), 28.4, 22.4. 19F NMR
334.1106.
4.2.6. (ꢀ)-trans-tert-Butyl 1-{3-fluoro-4-[(methylsulfonyl)
oxymethyl]piperidin-1-yl}carboxylate (trans-5)
Following the procedure for the preparation of cis-5, the
compound trans-5 was obtained from trans-3 (1.169 mg,
5.02 mmol) as a white solid (1.38 g, 88%). Mp: 63e65 ꢁC. 1H NMR
(CDCl3):
d
ꢂ203.4. HRMS m/z calcd. for C11H21FNO3 234.1505,
found: 234.1517. HPLC purity: H2O:MeCN, 60:40, tR ¼ 8.8 min.
Trans-3: 1H NMR (CDCl3)
d
(ppm): 4.39e4.21 (m, 2H), 3.97e3.95 (m,
1H), 3.70 (dd, J ¼ 4.3 Hz, J ¼ 10.8 Hz, 1H), 3.60 (dd, J ¼ 5.2 Hz,
J ¼ 10.8 Hz, 1H), 2.68e2.62 (m, 2H), 1.77e1.69 (m, 2H), 1.38e1.25
(CDCl3):
d 4.45e4.31 (m, 4H), 4.12 (bs, 1H), 3.07 (s, 3H), 2.77e2.71
(m, 10H). 13C NMR (CDCl3):
d
154.5, 88.0 (d, J ¼ 176 Hz), 80.2, 63.3
(m, 2H), 2.07e2.04 (m, 1H), 1.93e1.89 (m, 1H), 1.55e1.50 (m, 9H).
(d, J ¼ 2.2 Hz), 48.0, 44.0 (d, J ¼ 16 Hz), 41.0, 28.5, 26.2. 19F NMR
13C NMR (CDCl3):
d
154.3, 86.6 (d, J ¼ 178.8 Hz), 80.4, 69.0, 47.6,
(CDCl3):
d
ꢂ186.8 (d, J ¼ 49 Hz). HRMS m/z calcd. for C11H21 F NO3
42.6, 41.9 (d, J ¼ 17.0 Hz), 37.2, 28.3, 26.3. 19F NMR (CDCl3):
ꢂ187.9
d
234.1505, found: 234.1510. HPLC purity: H2O:MeCN, 60:40,
(d, J ¼ 45.1 Hz). HRMS m/z calcd. for C12H23FNO5S 312.1281, found:
312.1288. Anal. calcd for C12H22FNO5S: C 46.29, H 7.12, N 4.50,
found: C 46.79, H 7.38, N 4.33.
tR ¼ 10.7 min.
4.2.3. (ꢀ)-cis-tert-Butyl 1-{4-{[(4-nitrophenyl)sulfonyl]oxymethyl}
piperidin-3-ol-1-yl}carboxylate (cis-4)
4.2.7. (ꢀ)-cis-tert-Butyl 1-[4-(azidomethyl)piperidin-3-ol-1-yl]
carboxylate (cis-6)
To
piperidin-3-ol-1-yl]carboxylate (cis-2) (760 mg, 3.28 mmol),
Et3N (540 L, 3.6 mmol) and catalytic amount of 4-
a
solution of (ꢀ)-cis-tert-butyl 1-[4-(hydroxymethyl)
To a solution of cis-4 (1.17 g, 2.81 mmol), NH4Cl (296 mg,
5.6 mmol) in DMF (30 mL), was added NaN3 (364 mg, 5.6 mmol)
under nitrogen atmosphere. The reaction mixture was heated to
80 ꢁC for 20 h. The reaction mixture was diluted with water (20 mL)
and extracted with EtOAc (3 ꢃ 30 mL). The combined organic layers
were dried over MgSO4 and concentrated under reduced pressure.
Purification by silica gel chromatography (CH2Cl2:MeOH, 98:2)
gave cis-6 as yellow solid (690 mg, 96%). Mp 48e50 ꢁC. 1H NMR
m
dimethylaminopyridine in CH2Cl2 (24 mL), was added portion
wise 4-nitrobenzenesulfonyl chloride (797 mg, 3.6 mmol) at ꢂ50 ꢁC
under nitrogen atmosphere. After 3 h, the reaction mixture was
warmed up to room temperature and was stirred further overnight.
Then, the reaction mixture was diluted with water (10 mL) and
extracted with EtOAc (3 ꢃ 10 mL). The combined organic layers
were dried over MgSO4 and concentrated under reduced pressure.
The purification by silica gel chromatography (CH2Cl2:MeOH, 99:1)
led to cis-4 as a yellow solid (1.26 g, 93%). mp 117e119 ꢁC. 1H NMR
(CDCl3):
d
4.16e4.06 (m, 2H), 3.86 (s, 1H), 3.38 (dd, J ¼ 8 Hz,
J ¼ 12.4 Hz, 1H), 3.25 (dd, J ¼ 6.4 Hz, J ¼ 12.4 Hz, 1H), 2.78 (d,
J ¼ 14 Hz, 1H), 2.65 (t, J ¼ 12.4 Hz, J ¼ 12.4 Hz, 1H), 1.66e1.63 (m,
1H), 1.53 (dddd, J ¼ 12.8 Hz, J ¼ 12.8 Hz, J ¼ 12.8 Hz, J ¼ 4.4 Hz, 1H),
(CDCl3):
d
8.33 (d, J ¼ 8 Hz, 2H), 8.04 (d, J ¼ 8 Hz, 2H), 4.14e4.05 (m,
3H), 3.96e3.92 (m, 1H), 3.82 (s, 1H), 2.78e2.74 (m, 1H), 2.66e2.60
1.40e1.37 (m, 10H). 13C NMR (CDCl3):
d 156.0, 80.1, 65.2, 53.4, 50.1,
(m, 1H), 1.92e1.88 (m, 1H), 1.55e1.37 (m, 11H). 13C NMR (CDCl3):
43.7, 40.3, 28.4, 23.6. HRMS m/z calcd. for C11H21N4O3 257.1614,
found: 257.1620. HPLC purity: H2O:MeCN, 60:40, tR ¼ 11.1 min
(220 nm).
d
154.5, 150.8, 141.6, 129.3, 124.5, 80.3, 72.3, 64.0, 49.9, 43.2, 39.9,
28.3, 22.0. HRMS m/z calcd. for C17H25N2O8S 417.1332, found:
417.1324. Anal. calcd for C17H24N2O8S: C 49.03, H 5.81, N 6.73, S 7.70,
found: C 49.15, H 6.03, N 7.11, S 8.03.
4.2.8. (ꢀ)trans-tert-Butyl 1-[4-(azidomethyl)piperidin-3-ol-1-yl]
carboxylate (trans-6)
4.2.4. (ꢀ)-trans-tert-Butyl 1-{4-{[(4-nitrophenyl)sulfonyl]
oxymethyl}piperidin-3-ol-1-yl}carboxylate (trans-4)
Following the procedure for the preparation of cis-4,
compounds trans-4 was obtained from (ꢀ)-trans-tert-butyl 1-[4-
(hydroxymethyl)piperidin-3-ol-1-yl]carboxylate (trans-2) (1.03 g,
4.45 mmol) as yellow crystal (1.2 g, 65%). Mp 120e123 ꢁC. 1H NMR
Following the procedure for the preparation of cis-6, the
compound trans-6 was obtained from trans-4 (1.2 g, 2.88 mmol) as
a yellow oil (670 mg, 90%). 1H NMR (CDCl3):
d 4.17e4.15 (m, 1H),
3.98 (bs, 1H), 3.50e3.33 (m, 3H), 2.60 (dd, J ¼ 12.4 Hz, J ¼ 12.4 Hz,
1H), 2.45 (t, J ¼ 12 Hz, J ¼ 12 Hz, 1H), 1.70e1.64 (m, 1H), 1.59e1.54
(m, 1H), 1.39 (s, 9H), 1.29e1.20 (m, 1H). 13C NMR (CDCl3):
d 154.6,
(CDCl3):
d
8.34 (d, J ¼ 8 Hz, 2H), 8.04 (d, J ¼ 8 Hz, 2H), 4.27e4.0 (m,
80.1, 68.5, 53.8, 49.9, 43.4, 43.3, 28.4, 27.8. HRMS m/z calcd. for
C11H21N4O3 257.1614, found: 257.1618.
4H), 3.38 (dt, J ¼ 5 Hz, J ¼ 10 Hz, 1H), 2.60e2.55 (m, 1H), 2.45e2.42
(m, 1H), 1.67e1.65 (m, 2H), 1.37e1.30 (m, 10H). 13C NMR (CDCl3):
d
154.5, 150.8, 141.6, 129.2, 124.5, 80.2, 72.0, 66.5, 49.9, 43.2, 43.2,
4.2.9. (ꢀ)-cis-tert-Butyl 1-[3-fluoro-4-(azidomethyl)piperidin-1-yl]
carboxylate (cis-7)
28.3, 26.8. HRMS m/z calcd. for C17H25N2O8S 417.1332, found:
417.1325.
Following the procedure for the preparation of cis-6, the
compound cis-7 was obtained from cis-5 (1.24 g, 3.98 mmol) as
4.2.5. (ꢀ)-cis-tert-Butyl 1-{3-fluoro-4-[(methylsulfonyl)oxymethyl]
piperidin-1-yl}carboxylate (cis-5)
To a solution of cis-3 (1.1 g, 4.7 mmol), Et3N (1 mL, 7.21 mmol)
in CH2Cl2 (35 mL), methanesulfonyl chloride (550
was added at room temperature under nitrogen atmosphere. The
reaction mixture was stirred overnight at room temperature,
then diluted with water (20 mL) and extracted with CH2Cl2
a yellow oil (880 mg, 86%). 1H NMR (CDCl3):
d
4.74 (d, J ¼ 47.6 Hz,
1H), 4.47e4.18 (m, 2H), 3.45 (dd, J ¼ 8.6 Hz, J ¼ 12.1 Hz, 1H), 3.32
(dd, J ¼ 6.4 Hz, J ¼ 12.1 Hz, 1H), 2.98e2.73 (m, 2H), 1.86e1.56 (m,
m
L, 7.2 mmol)
3H), 1.49 (s, 9H). 13C NMR (CDCl3):
d
155.1, 85.5 (d, J ¼ 176 Hz), 80.0,
52.5 (d, J ¼ 3.4 Hz), 47.4, 42.9, 39.7 (d, J ¼ 20.2 Hz), 28.3, 23.6. 19F
NMR (CDCl3):
d
ꢂ203.85. HRMS m/z calcd. for C11H20N4O2F
259.1570, found: 259.1580.

R. Koudih et al. / European Journal of Medicinal Chemistry 53 (2012) 408e415
413
4.2.10. (ꢀ)-trans-tert-Butyl 1-[3-fluoro-4-(azidomethyl)piperidin-
1-yl]carboxylate (trans-7)
4.33e4.17 (m, 2H), 3.9 (bs, 1H), 3.63e3.42 (m, 2H), 2.73e2.63 (m,
2H), 1.92e1.78 (m, 2H), 1.38 (s, 9H), 1.33e1.25 (m, 1H). 13C NMR
2
Following the procedure for the preparation of cis-6, the
compound trans-7 was obtained from trans-5 (1.33 g, 4.27 mmol) as
(CDCl3):
d
162.5, 158.0, 154.4, 110.7, 90.1 (d, JCeF ¼ 176 Hz), 80.1,
47.0, 43.1 (d, J ¼ 2.2 Hz), 43, 42.2 (d, J ¼ 16.6 Hz), 28.3, 27.2. 19F NMR
a yellow oil (1.04 g, 94%). 1H NMR (CDCl3):
d
4.40e4.22 (m, 2H), 4.10
(CDCl3):
d
ꢂ185.8 (d, J ¼ 48.8 Hz). HRMS m/z calcd. for C15H24FN4O2
(bs, 1H), 3.62 (dd, J ¼ 3.4 Hz, J ¼ 12.2 Hz, 1H), 3.44 (dd, J ¼ 6.4 Hz,
311.1883, found: 311.1873. Anal. calcd for C15H23FN4O2: C 58.05, H
7.47, N 18.05, found: C 58.39, H 8.08, N 17.79. HPLC purity:
H2O:MeCN, 60:40, tR ¼ 12.7 min.
J ¼ 12.2 Hz, 1H), 2.76e2.69 (m, 2H), 1.89e1.85 (m, 2H), 1.50e1.39
(m, 10H). 13C NMR (CDCl3):
d
154.3, 87.8 (d, J ¼ 176 Hz), 80.3, 52.3
(d, J ¼ 2.3 Hz), 46.9, 42.2, 42.0, 28.4, 27.3. 19F NMR (CDCl3):
d L187.7
(d, J ¼ 46 Hz). HRMS m/z calcd. for C11H20N4O2F 259.1570, found:
4.2.15. (ꢀ)-cis-4-Methylbenzyl 1-[4-(pyrimidin-2-ylamino)
259.1562. HPLC purity: H2O:MeCN, 55:45, tR ¼ 13.18 min (220 nm).
methylpiperidin-3-ol-1-yl]carboxylate (cis-10)
HCl gas was bubbled through a solution of cis-8 (560 mg,
1.81 mmol) in EtOAc (3 mL) for about 45 min. The mixture was
then concentrated under reduced pressure and the crude amine
hydrochloride was dissolved in dry DMF (5 mL). DIPEA (710 mg,
5.43 mmol) was added and the resulting solution was
stirred for 30 min. To a solution of N,N0-disuccinimidyl carbonate
(926 mg, 3.62 mmol) in dry acetonitrile (10 mL), was added 4-
methylbenzyl alcohol (441 mg, 3.54 mmol) in dry CH2Cl2
(10 mL) and a catalytic amount of DMAP. The reaction mixture
was stirred overnight at room temperature. The solution was then
added to the amine hydrochloride previous prepared and stirred
for two days at room temperature. The reaction mixture was
diluted with water (15 mL) and extracted with EtOAc (4 ꢃ 15 mL).
The combined organic layers were dried over MgSO4, concen-
trated under reduced pressure and purified by chromatography
on silica gel (CH2Cl2:MeOH, 98:2) led to cis-10 as a yellow oil
4.2.11. (ꢀ)-cis-tert-Butyl 1-[4-(pyrimidin-2-ylamino)
methylpiperidin-3-ol-1-yl]carboxylate (cis-8)
A mixture of cis-6 (690 mg, 2.69 mmol) and 10% palladium on
carbon (200 mg) in methanol (40 mL) was stirred under H2
atmosphere (1.2 bars) at room temperature for 7 h. The reaction
mixture was filtered off then concentrated to give the intermediate
amine as a yellow oil (610 mg, 99%). The intermediate amine
(305 mg,1.32 mmol) was dissolved in tert-amyl alcohol (4 mL), then
2-chloropyrimidine (226 mg, 1.98 mmol) and DIPEA (2 mL) were
added under nitrogen atmosphere. The reaction mixture was stir-
red 48 h at 90 ꢁC, then diluted with water (10 mL) and extracted
with EtOAc (3 ꢃ 10 mL). The combined organic layers were dried
over MgSO4 and concentrated under reduced pressure. Purification
by silica gel chromatography (CH2Cl2:MeOH, 99:1) afforded cis-8 as
yellow crystals (210 mg, 51%). Mp 125e127 ꢁC. 1H NMR (CDCl3):
d
8.18 (d, J ¼ 4.8 Hz, 2H), 6.46 (m, 1H), 4.20e4.10 (m, 2H), 3.70 (bs,
(466 mg, 72%). 1H NMR (CDCl3):
d
8.15 (d, J ¼ 4.8 Hz, 2H), 7.17 (d,
1H), 3.57e3.49 (m, 1H), 3.14e3.10 (m, 1H), 2.73e2.63 (m, 2H),
J ¼ 8 Hz, 2H), 7.06 (d, J ¼ 8 Hz, 2H), 6.44 (t, J ¼ 4.8 Hz, 1H), 5.83
(bs, 1H), 5.15e4.98 (m, 2H), 4.30e4.20 (m, 2H), 3.73e3.66 (m, 1H),
3.53e3.47 (m,1H), 3.12e3.11 (m, 1H), 2.76e2.69 (m, 2H), 2.25 (s,
1.65e1.59 (m, 2H), 1.39 (s, 9H), 1.28e1.26 (m, 1H). 13C NMR (CDCl3):
d
162.7, 158.1, 154.6, 110.5, 79.4, 63.8, 49.6, 43.3, 43.2, 41.5, 28.4,
23.8. HRMS m/z calcd. for C15H25N4O3 309.1927, found: 309.1924.
Anal. calcd for C15H24N4O3.0.2H2O: C 57.74, H 7.88, N 17.95, found: C
57.34, H 8.30, N 17.57. HPLC purity: H2O:MeCN, 70:30, tR ¼ 9.8 min.
3H), 1.65e1.64 (m, 2H), 1.27 (bs, 1H). 13C NMR (CDCl3):
d 162.7,
158.1, 156.2, 137.6, 134.0, 129.1, 128.0, 110.6, 67.0, 63.6, 49.7, 43.8,
43.2, 41.5, 23.7, 21.2. HRMS m/z calcd. for C19H25N4O3 357.1927,
found: 357.1928.
4.2.12. (ꢀ)-trans-tert-Butyl 1-[4-(pyrimidin-2-ylamino)
methylpiperidin-3-ol-1-yl]carboxylate (trans-8)
Following the procedure for the preparation of cis-8, the
compound trans-8 was obtained from trans-6 (670 mg, 2.6 mmol)
as yellow crystals (495 mg, 61%). Mp 114e116 ꢁC. 1H NMR (CDCl3):
4.2.16. (ꢀ)-trans-4-Methylbenzyl 1-[4-(pyrimidin-2-ylamino)
methylpiperidin-3-ol-1-yl]carboxylate (trans-10)
Following the procedure for the preparation of cis-10, the
compound trans-10 was obtained from trans-8 (250 mg,
d
8.18 (d, J ¼ 4.8 Hz, 2H), 6.50 (t, J ¼ 4.8 Hz, 2H), 4.17e3.94 (m, 3H),
3.19e3.05 (m, 2H), 2.59e2.46 (m, 2H), 1.55e1.39 (m, 12H). 13C NMR
(CDCl3): 162.8, 158.1, 154.6, 110.7, 79.7, 67.1, 49.1, 45.4, 43.2, 43.0,
0.81 mmol) as a yellow oil (160 mg, 55%). 1H NMR (CDCl3):
d 8.30 (d,
J ¼ 4.8 Hz, 2H), 7.27 (d, J ¼ 8 Hz, 2H), 7.18 (d, J ¼ 8 Hz, 2H), 6.60 (t,
J ¼ 4.8 Hz, 1H), 5.62 (bs, 1H), 5.1 (bs, 2H), 4.15e4.08 (m, 3H), 3.28
(bs, 1H), 3.19e3.15 (m, 1H), 2.78e2.67 (m, 2H), 2.38 (s, 3H),
d
28.4, 27.9. HRMS m/z calcd. for C15H25N4O3 309.1927, found:
309.1919. HPLC purity: H2O:MeCN, 60:40, tR ¼ 5.1 min.
1.63e1.58 (m, 3H). 13C NMR (CDCl3):
d 162.8, 158.1, 155.2, 137.8,
133.6, 129.1, 128.1, 110.9, 67.1, 66.9, 49.2, 45.5, 43.7, 42.8, 27.8, 23.2.
HRMS m/z calcd. for C19H25N4O3 357.1927, found: 357.1920. HPLC
purity: H2O:MeCN, 60:40, tR ¼ 9.4 min.
4.2.13. (ꢀ)-cis-tert-Butyl 1-[3-fluoro-4-(pyrimidin-2-ylamino)
methylpiperidin-1-yl]carboxylate (cis-9)
Following the procedure for the preparation of cis-8, the
compound cis-9 was obtained from cis-7 (818 mg, 3.17 mmol) as
a yellow solid (457 mg, 46%). Mp 131e133 ꢁC. 1H NMR (CDCl3):
4.2.17. (ꢀ)-cis-4-Methylbenzyl 1-[3-fluoro-4-(pyrimidin-2-
ylamino)methylpiperidin-1-yl]carboxylate (cis-11)
d
8.20 (d, J ¼ 4.7 Hz, 2H), 6.46 (t, J ¼ 4.8 Hz,1H), 5.76 (bs,1H), 4.67 (d,
Following the procedure for the preparation of cis-10, the
compound cis-11 was obtained from cis-9 (160 mg, 0.516 mmol) as
J ¼ 48 Hz, 1H), 4.33e4.18 (m, 2H), 3.43e3.31 (m, 2H), 2.79e2.64 (m,
2H), 2.09e1.84 (m, 1H), 1.56e1.50 (m, 2H), 1.39 (s, 9H). 13C NMR
a yellow solid (65 mg, 35%). mp 131e133 ꢁC. 1H NMR (CDCl3):
d 8.18
2
(CDCl3):
d
162.5, 158.1, 155.2, 110.7, 86.6 (d, JCeF ¼ 176 Hz), 79.8,
(d, J ¼ 4.8 Hz, 2H), 7.17 (d, J ¼ 8.4 Hz, 2H), 7.08 (d, J ¼ 7.8 Hz, 2H),
6.45 (t, J ¼ 4.8 Hz, 1H), 5.77 (bs, 1H), 5.02e5.01 (m, 2H), 4.79e4.17
(m, 3H), 3.44e3.29 (m, 2H), 2.85e2.67 (m, 2H), 2.27 (s, 3H),
48.2, 42.9, 42.7 (d, J ¼ 3.3 Hz), 38.7 (d, J ¼ 19.9 Hz), 28.4, 23.7. 19F
NMR (CDCl3):
d
ꢂ203.2 (d, J ¼ 30 Hz). HPLC purity: H2O:MeCN,
60:40, tR ¼ 15.4 min.
2.02e1.89 (m, 1H), 1.54 (bs, 2H). 19F NMR (CDCl3):
m/z calcd. for C19H24FN4O2: 359.1883, found: 359.1887. HPLC purity:
H2O:MeCN, 55:45, tR ¼ 12.6 min.
d
ꢂ203.1. HRMS
4.2.14. (ꢀ)-trans-tert-Butyl 1-[3-fluoro-4-(pyrimidin-2-ylamino)
methylpiperidin-1-yl]carboxylate (trans-9)
Following the procedure for the preparation of cis-8, the
compound trans-9 was obtained from trans-7 (789 mg, 3.06 mmol)
as a yellow solid (574 mg, 60%). Mp 124e126 ꢁC. 1H NMR (CDCl3):
4.2.18. (ꢀ)-trans-4-Methylbenzyl 1-[3-fluoro-4-(pyrimidin-2-
ylamino)methylpiperidin-1-yl]carboxylate (trans-11)
Following the procedure for the preparation of cis-10, the
compound trans-11 was obtained from trans-9 (164 mg, 0.52 mmol)
d
8.20 (d, J ¼ 4.7 Hz, 2H), 6.46 (t, J ¼ 4.7 Hz, 1H), 5.43 (bs, 1H),

414
R. Koudih et al. / European Journal of Medicinal Chemistry 53 (2012) 408e415
as a yellow oil (72 mg, 38%). 1H NMR (CDCl3):
d
8.18 (d, J ¼ 4.8 Hz,
4.3. Radiochemistry
2H), 7.18 (d, J ¼ 7.9 Hz, 2H), 7.09 (d, J ¼ 7.9 Hz, 2H), 6.44 (t, J ¼ 4.8 Hz,
1H), 5.5 (bs, 1H), 5.0 (s, 2H), 4.32e4.20 (m, 2H), 3.96 (bs, 1H),
3.63e3.57 (m, 1H), 3.47e3.40 (m, 1H), 2.82e2.70 (m, 2H), 2.27 (s,
4.3.1. General procedure for the radiosynthesis of [18F]-11 using the
TRACERlab FX F-N
3H), 1.93e1.79 (m, 2H), 1.26e1.23 (m, 1H). 13C NMR (CDCl3):
d
162.5,
The fluorine-18 produced by the cyclotron was trapped on an
ion exchange resin (QMA light, Waters, ABX), separated from 18O-
enriched water then eluted with a solution of potassium carbonate
and Kryptofix 2.2.2 in acetonitrile (0.3 mL) and water (0.3 mL) (vial
1). The mixture was heated to 95 ꢁC under reduced pressure under
a flow of helium. 0.6 mL of acetonitrile (vial 2) was added and the
mixture was further heated at 95 ꢁC under reduced pressure. 5 mg
158.0, 155.1, 138.0, 133.4, 129.2, 128.2, 110.7, 89.8 (d, J ¼ 178 Hz),
67.4, 47.0 (d, J ¼ 29.9 Hz), 43.0, 42.1, 42.0, 27.0, 21.2. 19F NMR
(CDCl3):
d
ꢂ185.8 (d, J ¼ 47.8 Hz). HRMS m/z calcd. for C19H24FN4O2
359.1883, found: 359.1874. Anal. calcd for C19H23FN4O2: C 63.67, H
6.47, N 15.63, found: C 63.86, H 6.85, N 15.54. HPLC purity:
H2O:MeCN, 55:45, tR ¼ 14.3 min.
(11 mmol) of the labelling precursor 12 in 1 mL of acetonitrile (vial
4.2.19. (ꢀ)-cis-4-Methylbenzyl 1-[4-(pyrimidin-2-yl)aminomethyl-
3-(methylsulfonyl)hydroxypiperidin-1-yl]carboxylate (cis-12)
3) was added to the reactor and the reaction mixture was heated at
95 ꢁC for 20 min, then diluted with 2 mL of water (vial 4). The
reaction mixture was passed through an SPE cartridge (Shorty C18
ec, 20 mg, MachereyeNagel) to separate the radiolabeled
product from reaction mixture. The cartridge was eluted with
0.5 mL of acetonitrile (vial 5) then washed with 0.5 mL of water
To a solution of cis-10 (85 mg, 0.238 mmol) in dry pyridine
(2 mL), was added silver trifluoromethanesulfonate (123 mg,
0.47 mmol) under nitrogen atmosphere at 0 ꢁC, followed by the
addition of methanesulfonyl chloride (87 mg, 1.03 mmol). The
reaction mixture was stirred at 0 ꢁC for 1 h, then further 1 h at room
temperature. The reaction mixture was diluted with water (2 mL)
and extracted with EtOAc (4 ꢃ 3 mL). The combined organic layers
were dried over MgSO4 and concentrated under reduced pressure.
Purification by silica gel chromatography (CH2Cl2:MeOH, 98:2)
gave cis-12 as yellow crystals (73 mg, 70%). Mp 107e109 ꢁC. 1H NMR
(vial 6). 200 mL of this elution solution was injected in HPLC to
facilitate the separation (column: Nucleosil 100-5 C-18 Nautilus,
250 ꢃ 10 mm, MachereyeNagel; eluent: acetonitrile:H2O 35:65
for tR (cis-11) ¼ 36 min and 37:63 for tR (trans-11) ¼ 38 min; flow
rate: 6 mL minꢂ1). The collected fraction containing the pure
radiolabeled product was diluted with 25e40 mL of water and then
passed through an SPE cartridge (Shorty C18 ec, MachereyeNagel).
The cartridge was then washed with 2 mL of water (vial 9) and
eluted with 0.2 mL of ethanol (vial 8) and 1.8 mL of saline (vial 7).
(CDCl3):
d
8.18 (d, J ¼ 4.8 Hz, 2H), 7.18 (m, 2H), 7.10 (m, 2H), 6.45 (t,
J ¼ 4.8 Hz, 1H), 5.65 (bs, 1H), 5.14e4.86 (m, 3H), 4.54e4.41 (m, 1H),
4.31e4.16 (m, 1H), 3.54e3.47 (m, 1H), 3.11 (bs, 1H), 2.97e2.66 (m,
5H), 2.27 (s, 3H), 2.11 (bs, 1H), 1.51e1.45 (m, 2H). 13C NMR (CDCl3):
d
162.1, 158.1, 156.2, 138.0, 134.0, 129.1, 128.0, 110.8, 74.9, 67.3, 47.6,
4.3.2. Quality control
43.5, 42.4, 38.7, 38.1, 23.7, 21.2. HRMS m/z calcd. for C20H27N4O5S
435.1702, found: 435.1683. Anal. calcd for C20H26N4O5S: C 55.28, H
6.03, N 12.89, found: C 54.73, H 6.18, N 12.93. HPLC purity:
H2O:MeCN, 60:40, tR ¼ 10.4 min.
The radiochemical purity of [18F]-11 was determined by HPLC
(column: Nucleodur 100/5 C-18 Pyramid, 250
ꢃ
4.6 mm,
MachereyeNagel; eluent: acetonitrile:H2O, 45:55, tR (trans-
11) ¼ 14.6 min; tR (cis-11) ¼ 12.5 min; tR (13) ¼ 13.3 min; flow rate:
1 mL minꢂ1) and was greater than 98%.
4.2.20. (ꢀ)-trans-4-Methylbenzyl 1-[4-(pyrimidin-2-ylamino)
methyl-3-(methylsulfonyl)hydroxypiperidin-1-yl]carboxylate
(trans-12)
4.4. LogD7.4 measurement
Following the procedure for the preparation of cis-12, the
compound trans-12 was obtained from trans-10 (50 mg,
The logD7.4 measurement was achieved with 10
Four tubes containing 1 mL of octanol and 1 mL of NaH3PO4 buffer
m
L of [18F]-11.
0.14 mmol) as a yellow oil (60 mg, 98%). 1H NMR (CDCl3):
d
8.18
(3M, pH ¼ 7.4) were stirred for 20 min. 10
mL of the solution con-
(d, J ¼ 4.8 Hz, 2H), 7.17 (d, J ¼ 8 Hz, 2H), 7.08 (d, J ¼ 8 Hz, 2H), 6.45
(t, J ¼ 4.8 Hz, 1H), 5.40 (bs, 1H), 5.01 (bs, 2H), 4.46e4.45 (m, 1H),
4.33e4.29 (m, 1H), 3.92 (bs, 1H), 3.69 (bs, 1H), 3.45e3.38 (m, 1H),
3.03e2.79 (m, 5H), 2.27 (s, 3H), 1.95e1.93 (m, 1H), 1.87e1.83 (m,
1H), 1.36e1.33 (m, 1H). HRMS m/z calcd. for C20H27N4O5S
435.1702, found: 435.1696. HPLC purity: H2O:MeCN, 60:40,
tR ¼ 12.9 min.
taining the radiotracer [18F]-11 were added in each tube, and the
mixture was stirred further for 40 min, then centrifuged for 5 min.
Two aliquots of 100 mL were taken out from the both phases and the
radioactivity was measured in each sample using a Perkin Elmer
(Packard B5003) radioactivity counter. LogD7.4 measurement was
obtained by calculation of the ratio of octanol phase to aqueous
phase radioactivity quantities.
4.2.21. 4-Methylbenzyl 1-[4-(pyrimidin-2-yl)aminomethyl-2,5,6-
tetrahydro-pyridin-1-yl]carboxylate (13)
4.5. [18F]cis-11 and [18F]trans-11 binding assay
To a solution of cis-12 (61 mg, 0.14 mmol) in acetonitrile (2 mL)
was added potassium fluoride (12 mg, 0.21 mmol) and K222 (40 mg,
0.21 mmol). The reaction mixture was stirred for two days at 80 ꢁC.
The reaction mixture was diluted with water (2 mL) and extracted
with EtOAc (4 ꢃ 3 mL). The combined organic layers were dried
over MgSO4 and concentrated under reduced pressure. Purification
by silica gel chromatography (CH2Cl2:MeOH, 99:1) gave 13 as
Sagittal adjacent brain sections (20 mm) were obtained from
brains of male SpragueeDawley rats (250g, Charles River, France)
and applied to glass slides with a very low non-specific capacity
(SuperfrostÒ Plus, Menzel-Glaser GmbH, Braunscheig, Germany).
Mounted tissue sections were pre-incubated (twice, 10 min, r.t.) in
50 mM TriseHCl buffer solution (pH ¼ 7.4) containing 10 mM EDTA
(ethylenediaminetetraacetic acid). The radiotracer concentration
was established by the measurement of the specific radioactivity
and of the radioactivity concentration before the beginning of the
binding experiment. The brain slices were incubated at 4 ꢁC for
90 min in 50 mM TriseHCl buffer containing increasing concen-
trations (0.15, 0.5, 1, 2, 8, 24, 48 nM) of [18F]cis-11 or [18F]trans-11.
Non-specific binding was determined by addition, to the incubation
a brown oil (32 mg, 67%). 1H NMR (CDCl3):
d
8.17 (d, J ¼ 4.8 Hz, 2H),
7.14 (d, J ¼ 8 Hz, 2H), 7.06 (d, J ¼ 8 Hz, 2H), 6.44 (t, J ¼ 4.8 Hz, 1H),
5.52 (bs, 1H), 5.46 (bs, 1H), 5.01 (s, 2H), 3.95e3.80 (m, 4H),
3.55e3.45 (m, 2H), 3.25 (s, 3H), 2.03 (bs, 2H). 13C NMR (CDCl3):
d
162.4, 158.1,155.4, 137.8, 133.8, 129.1,128.1,124.3,118.3,110.8, 67.0,
46.1, 43.3, 40.5, 26.4, 21.2. HRMS m/z calcd. for C19H23N4O2
339.1821, found: 339.1818. HPLC purity: H2O:MeCN, 60:40,
tR ¼ 24.2 min.
medium, of an excess of Ro 25-6981 (10
mM). At the end of the
incubation period, excess of radioligands were eliminated by three

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water at 4 ꢁC to remove the excess of buffer salts. Sections were
dried onto a hot plate. At the same time, spiked standards were
prepared by adding six appropriate solutions (50
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moment as rat brain sections to a storage phosphor screen in the
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4.6. Quantification and characterization of [18F]cis-11 and [18F]
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Images were captured with a CycloneÒ phosphor imaging
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Radouane Koudih and Gwénaëlle Gilbert were financed by the
Ministère de l’Enseignement Supérieur et de la Recherche. The
authors thank Olivier Tirel and Jérôme Delamare for technical help
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