Synthesis, evaluation and metabolic studies of radiotracers containing a 4-(4-[18F]-fluorobenzyl)piperidin-1-yl moiety for the PET imaging of NR2B NMDA receptors

European Journal of Medicinal Chemistry 46 (20 1 1 ) 2295e2309

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European Journal of Medicinal Chemistry

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Original article

Synthesis, evaluation and metabolic studies of radiotracers containing a

4-(4-[¹⁸F]-ﬂuorobenzyl)piperidin-1-yl moiety for the PET imaging of NR2B

NMDA receptors

,

b

,

b

,

,d

Romain Labas ^{a 1}, Gwénaëlle Gilbert ^{a 1}, Olivier Nicole ^b, Martine Dhilly ^{a b}, Ahmed Abbas ^b

,

Olivier Tirel ^{a b}, Alain Buisson ^b, Joël Henry^c, Louisa Barré ^{a b}, Danièle Debruyne ^{a b}, Franck Sobrio ^a

,

,b,

*

^aCEA, I2BM, CI-NAPS, LDM-TEP, F-14074 Caen, France

^bUniversité de Caen Basse-Normandie, UMR 6232, CNRS, CI-NAPS, Centre Cyceron, BP 5229, F-14074 Caen, France

^cUniversité de Caen Basse-Normandie, UMR 100 IFREMER, Esplanade de la paix, F-14000 Caen, France

^dINSERM-EPHE-Université de Caen Basse-Normandie, Unité 923, 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

Article history:

In this study, novel speciﬁc PET radioligands containing the 4-(4-ﬂuorobenzyl)piperidine moiety and

selectively antagonistic for the NR2B subunit containing NMDA receptors were developed. Two antag-

onists, RGH-896 (1a) and 4-(4-ﬂuorobenzyl)piperidinyl-1-methyl-2-benzimidazol-5-ol (2a), belonging to

two different structural families, were radiolabeled by an aromatic nucleophilic radioﬂuorination fol-

lowed by a reduction of the para-position carbonyl function. Radiotracers [¹⁸F]1a, [¹⁸F]2a or the pattern

4-(4-[¹⁸F]-ﬂuorobenzyl)piperidine ([¹⁸F]6) demonstrated an identical in vivo behavior with high accu-

mulation of radioactivity in bone and cartilage which would suggest a radiodeﬂuorination of the

radiotracers. The identiﬁcation of metabolites from 6 by LC-MS-MS conﬁrmed the signiﬁcant degree of

deﬂuorination as a result of the in vivo hydroxylation in the benzyl ring. In conclusion, [¹⁸F]1a or [¹⁸F]2a

are not suitable for imaging the NR2B NMDA receptors due to their poor brain penetration. We also argue

for a cautious use of the radiolabeled pattern, 4-(4-[¹⁸F]-ﬂuorobenzyl)piperidine, to develop PET

radiotracers.

Received 5 January 2011

Received in revised form

25 February 2011

Accepted 7 March 2011

Available online 15 March 2011

Keywords:

NR2B

NMDA

PET

Radiotracer

Fluorine-18

Antagonist

1. Introduction

The functional NMDA receptor is a complex consisting of two

obligatory NR1 subunits and two NR2 subunits. The NR1 subunit is

The N-methyl-

D

-aspartate (NMDA) receptors are glutamate-

widely expressed within the central nervous system and exists in

eight isoforms (NR1aeh) encoded by alternative splicing of

a unique gene [1]. NR2 subunits exist in four forms produced from

distinct genes (2A, 2B, 2C and 2D) each of which shows a charac-

teristic distribution and confers different pharmacological proper-

ties to the receptor [8,9]. The NR2A subunit is expressed in all brain

regions but to a greater degree in the cerebral cortex, hippocampus

and cerebellum. NR2B expression is largely limited to forebrain

regions, including the hippocampus, cortex and striatum [5]. NR2C

subunit is expressed exclusively in the cerebellum whereas the

midbrain contains high levels of NR2D subunits. The NR2B subunit

containing NMDA receptors are the most studied and therefore the

best known subtype. The NR2B receptors have become a thera-

peutic target for stroke, neurodegenerative diseases, schizophrenia

and neuropathic pain [5,10]. Ifenprodil (Fig. 1) was the ﬁrst antag-

onist that showed selectivity for the NR2B NMDA receptor [11]. This

property improved the neuroprotective potential, and reduced

side-effects, compared to non-selective, ﬁrst generation NMDA

receptor inhibitors [3].

gated ion channels that regulate excitatory synaptic transmission in

mammals [1,2]. The channel activation of the NMDA receptors

requires the binding of two different agonists, glutamate and

glycine [3]. The important permeability to calcium ions confers on

NMDA receptors a central role in a number of physiological

processes involved in long-term potentiation, memory and cogni-

tion [4]. Moreover, over-activation of NMDA receptors has been

implicated in several neuropathological and psychiatric disorders

including neuropathic pain, epilepsy, neurodegenerative diseases

(Alzheimer’s, Parkinson’s and Huntington’s) as well as stroke and

brain trauma [5e7].

* Corresponding author. UMR 6232, CI-NAPS/LDM-TEP, Centre Cyceron, BP 5229,

F-14074 Caen cedex, France. Tel.: þ33 (0) 231470264; fax: þ33 (0) 231470275.

E-mail address: sobrio@cyceron.fr (F. Sobrio).

1

These authors have contributed equally to this work.

doi:10.1016/j.ejmech.2011.03.013

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R. Labas et al. / European Journal of Medicinal Chemistry 46 (2011) 2295e2309

Fig. 1. Structures of ifenprodil and ifenprodil-like antagonists.

The in vivo quantiﬁcation of the NR2B subunit containing NMDA

receptors by positron emission tomography (PET) could be of great

interest to understand the contribution of this complex to neuro-

logical disorders or brain pathologies [12e14]. Several NR2B

subtype selective antagonists, such as [¹¹C]EMD-95885, N-[¹¹C]-(2-

methoxy)benzyl-3-phenyl-amidines or a [¹¹C]-methylated analog

of CP101,606, have been labeled with the short-lived positron

emitter, carbon-11 (T_1/2: 20.4 min) or ﬂuorine-18 (T_1/2: 109.7 min)

and their potential to image NR2B containing NMDA receptors in

the living brain has been investigated [15e17]. In spite of promising

in vitro binding data for some compounds, an inadequate brain

penetration and/or a uniform distribution of tracer throughout

brain structures have been reported for all the radiotracers so far

developed [18]. Therefore, an effective PET radiotracer to image

NR2B containing NMDA receptors is still needed.

Since the discovery of the NMDA related actions of ifenprodil,

considerable efforts have been pursued to develop structurally-

related compounds with higher afﬁnity and selectivity for NR2B

subtype [10,19e22]. As the 4-benzylpiperidine moiety was

considered as an essential pharmacophore for the NR2B subunit

receptor site, novel antagonists containing the 4-benzylpiperidine

pattern were investigated including different substitutions on the

aromatic ring [21,22]. Among the substituted benzylpiperidine

derivatives described, the 4-(4-ﬂuorobenzyl)piperidine moiety is

present in a number of NR2B antagonists, the most known ligands

being eliprodil, besonprodil and EMD-95885 (Fig. 1). The ﬂuorine

atom on the aromatic ring led us to envisage the radiolabeling of

such structures with ﬂuorine-18 to obtain a radiotracer for PET

imaging.

Fig. 2. Recent NR2B antagonists.

2-benzimidazol-5-methylsulfamide (3a) demonstrated

afﬁnity (K_i¼ 0.99 ꢀ 0.4 nM) and selectivity for NR2B [26].

a high

From these structureeactivity relationship (SAR) studies and

pharmacological data, we have investigated the preparation of

several ligands for the NR2B receptor that belong to these structural

classes of compounds and bear the 4-(4-ﬂuorobenzyl)piperidine

moiety. The in vitro inhibitory activities were determined and two

ligands were selected to be labeled with ﬂuorine-18. The radiosyn-

thesis of [¹⁸F]1a and [¹⁸F]2a was developed using a two-step labeling

method. Their in vivo behavior, including tissue uptake, kinetics and

radiometabolism were investigated in rats. A complementary mass

spectroscopy study was undertaken to corroborate the in vivo

metabolic pathway of the 4-(4-[¹⁸F]ﬂuorobenzyl)piperidine moiety

([¹⁸F]6) observed with [¹⁸F]1a and [¹⁸F]2a.

2. Results and discussion

From a recent series of N-oxamide ligands (1aef) (Fig. 2), two

compounds (1a, 1c) bearing the 4-(4-ﬂuorobenzyl)piperidine

moiety, have been described with high afﬁnity and selectivity for

the NR2B subunit (IC₅₀¼ 6 ꢀ 1 and 5 ꢀ 1 nM, respectively for 1a

and 1c) [23]. The addition of a ﬂuorine atom in the para-position on

the benzyl ring did not induce any loss of afﬁnity compared to the

non-ﬂuorinated analogs (IC₅₀¼ 4 ꢀ 1 and 8 ꢀ 1 nM for 1b and 1d)

in binding assay with [³H]Ro-25,6981. The compounds 1aef

(excepted for 1d) presented in vivo analgesic activities in the mouse

formalin test with an efﬁcacy dose (ED₅₀) varying from 0.5 to

14 mg/kg (p.o.). The benzoxazolone derivative of this series (1a),

namely RGH-896 or radiprodil, is currently in clinical trials

[24]. From a second series of antagonists, based on the 4-(sub-

stitutedbenzyl)piperidinyl-1-methylbenzimidazol structure [25],

the 4-(4-ﬂuorobenzyl)piperidinyl-1-methyl-2-benzimidazol-5-ol

(2a) (Fig. 2) showed an excellent afﬁnity (K_i¼ 1.1 ꢀ 0.1 nM) and

selectivity for NR2B subunit containing NMDA receptors vs. human

ether-a-go-go (hERG) potassium channel (inhibitory potency,

2.1. Chemistry

The syntheses of ﬂuorinated ligands 1a, 1g, 1h, 2a and 3b and

non-ﬂuorinated analogs 2b and 3a are showed in Schemes 1 and 2.

They were prepared by synthetic pathway differing from those

described previously for the corresponding series within the objec-

tive to get a converging approach with the benzylpiperidines [23,25].

The oxalamic acids 4aec were obtained in high yield by addition of

ethyl chlorooxoacetate to the corresponding amine [27] followed by

basic hydrolysis of the ethyl ester. These acids were coupled with the

4-(4-ﬂuorobenzyl)piperidine (6) [28] using the corresponding acid

chloride or by a peptidic coupling reaction within moderate yields

(19e30%) to obtain the carboxamides 1a, 1g and 1h in sufﬁcient

quantities for the biological evaluation (Scheme 1). The benzimid-

azole compounds 2a-b and 3a-b were prepared by the addition of

the 5-substituted-1-chloromethylbenzimidazole to the adequate

benzylpiperidines (Scheme 2). The benzimidazole compounds 7a-

b and 9a-b were prepared by addition respectively of the 1-chlor-

omethyl-5-methoxybenzimidazole [29] and 1-chloromethyl-5-

nitrobenzimidazole (8) to the corresponding 4-(4-ﬂuorobenzyl)

piperidine or benzylpiperidine in 49e70% yields. Compound 8 was

obtained in one step by condensation of the 5-nitrophenylene-1,2-

IP ¼ 500 nM) and -adrenergic receptors (IC₅₀¼ 620 nM). In this

a

series, the non-ﬂuorinated analog of 2a presented interesting

pharmacokinetic properties as an activity in carrageenan-induced

mechanical hyperalgesia in rats with an ED₅₀of 3.3 mg/kg i.v. and a

plasma half-life superior to 8 h. The 4-benzylpiperidine-1-methyl-

R. Labas et al. / European Journal of Medicinal Chemistry 46 (2011) 2295e2309

2297

Scheme 1. Synthesis of ligands 1a, 1g, 1h. Reaction conditions: (i) EtOCOCOCl, sodium acetate, acetic acid, 35e90 ^ꢁC, 2 h; (ii) KOH, EtOH, H₂O, RT, 2 h; (iii) 1a, 1h: 1) (COCl)₂, DMF,

CH₂Cl₂, 0 ^ꢁC to RT, 3 h; 2) 6, TEA, CH₂Cl₂, RT, overnight; (iv) 1g: 6, HBTU, TEA, DMF, RT, 24 h.

diamine with the chloroacetic acid in hydrochloric acid in good yield

[30]. Ligands 2a and 2b were obtained after removal of the methoxy

group by hydrolysis in HBr at 130 ^ꢁC [25]. Reduction of the nitro

intermediates 9a and 9b followed by sulfonation with mesylate

chloride led to 3a and 3b.

substituted ligand 3a or even improved it as for 2b. Within the

oxalamic acid series compounds, 1a demonstrated an IC₅₀(69 nM)

similar to 2a, 3a and 3b whereas 1g and 1h presented poor

inhibitory effects (835 nM) towards Ca^2þinﬂux, lower than for the

ifenprodil. The inhibition of calcium inﬂux evoked by NMDA in cells

expressing NR1a/NR2A NMDA receptors were measured with

10

mM of ligands to determine the subunit selectivity (Table 1).

2.2. In vitro studies. Functional assays

With such a concentration, only 10% of calcium inﬂux was inhibited

by the antagonists 1a and 2a whereas a 22% inhibition was obtained

with ifenprodil, demonstrating a high selectivity for the NR2B

subunit.

The inhibition potency of ligands toward the NR2B NMDA

receptors was evaluated by measuring the Ca^2þinﬂux evocated by

NMDA application (at 100

mM for 30 s) in NR1a/NR2B subunit

The choice to develop the radiolabeling of two of the best

antagonists was based on the few in vivo pharmacological proper-

ties described for these series of ligands. The in vivo analgesic

efﬁciency for 1a in the mouse formalin test was 7.7 mg/kg p.o.

(ED₅₀) and a clinical trial is currently conducted with 1a under the

name radiprodil or RGH-896 [24]. The reported pharmacokinetic

data for the non-ﬂuorinated analog 2b indicated a long half-life

superior to 480 min and an in vivo efﬁcacy of 3.3 mg/kg in rats on

carrageenan-induced mechanical hyperalgesia after i.v. injection

[25] and we speculated that the ﬂuorine atom would not change

considerably these properties. For these reasons, the ligands 1a and

2a were labeled with ﬂuorine-18 to compare the in vivo comport-

ment of two different series of compounds.

transfected human embryonic kidney (HEK) cells. Tested

compounds were applied 60 s prior and during NMDA exposure

[11]. IC₅₀values were determined on the concentration-response

curves of Ca^2þinﬂux and were compared to the reference antago-

nist, ifenprodil (Table 1). The IC₅₀found for ifenprodil (177 nM) was

of the same order as described by Williams (340 nM) [11]. The

calcium inﬂux was measured on neurons by optical microscopy

leading to differences with the reported data probably due to the

sensitivity of the technique. Ligands 2a, 3a and 3b demonstrated

identical inhibitory effects on NR2B containing NMDA receptors

(IC₅₀¼ 47e57 nM), more potent than ifenprodil (Table 1). The

presence of the ﬂuorine atom in the 4-position on the benzyl (2a,

3b) did not change the inhibition value compared with the non-

Scheme 2. Synthesis of ligands 2a, 2b, 3a and 3b. Reaction conditions: (i) benzylpiperidine or 6, acetonitrile, Na₂CO₃, reﬂux, 15 h; (ii) HBr, 130 ^ꢁC, 3 h; (iii) chloroacetic acid, HCl,

reﬂux, 7 h.; (iv) benzylpiperidine or 6, Na₂CO₃, acetonitrile, 70 ^ꢁC, overnight; (v) Pd/C, H₂, MeOH, RT, 3 h; (vi) MsCl, CH₂Cl₂, TEA, RT, overnight.

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R. Labas et al. / European Journal of Medicinal Chemistry 46 (2011) 2295e2309

Table 1

Results for inhibition of calcium inﬂux evoked by NMDA into cells expressing NR1a/

NR2B or NR1a/NR2A NMDA receptors.

Ligands

IC₅₀NR2B

(nM)^a

Inhibition at

Reported IC₅₀

References

10

m

M NR2A (%)^aNR2B (nM)

Ifenprodil

177.40 ꢀ 1.28 22.27 ꢀ 2.41

69.91 ꢀ 1.21 10.14 ꢀ 1.61

340 (66 ꢀ 15^b) [11,25]

1a X ¼ O

4 ꢀ 1

[23]

1g X ¼ CH₂830.40 ꢀ 1.27 14.46 ꢀ 2.10

1h X ¼ S

839.90 ꢀ 1.39 13.22 ꢀ 2.18

47.27 ꢀ 5.31 10.48 ꢀ 2.45

209.20 ꢀ 1.61 nd^c

2a

2b

3a

3b

12^b

[26]

[25]

[26]

8.2^b

50.29 ꢀ 1.58 nd

1.4 ꢀ 0.5^b

57.70 ꢀ 1.29 nd

a

Values represents the mean ꢀ SEM.

b

c

Glu/Gly stimulated Ca^2þinﬂux in place of NMDA.

nd: not determined.

2.3. In vitro stability study

As a pre-required property, the stability of the compounds 1a

and 2a was evaluated by in vitro incubation with plasma or liver

homogenate from rats. 0.25e10 mg of ligand were in contact for

30 min at 37 ^ꢁC with the biological material (0.45 mL). The HPLC

measurement of the compound amounts extracted from plasma or

liver homogenates were reliable and quantitatively similar to these

extracted from aqueous solution. No degradation could be statis-

tically demonstrated for 1a and 2a compounds and let to suppose

no spontaneous degradation of these products in biological

supports.

Scheme 3. Synthesis of substituted benzylpiperidines. Reaction conditions: (i) 1) 9-

BBN, THF, reﬂux, 2) phenylbromide, Pd(dppf)Cl₂.CH₂Cl₂, K₂CO₃, DMF, H₂O, 65 ^ꢁC, 3 h;

(ii) HCl, AcOEt, RT, 12 h; (iii) 1-chloromethyl-5-methoxybenzimidazole, acetonitrile,

Na₂CO₃, reﬂux, 15 h; (iv) CH₂Cl₂, FeCl₃.6H₂O, RT, 3 h.

2.4. Radiochemistry

14 (Scheme 4). Complementary studies, here not shown, led us to

hypothesize the formation of a by-product corresponding to the

oxidation at the benzylic position [35].

The radiolabeling of 2a with ﬂuorine-18 was explored by the

aromatic nucleophilic substitution of a nitro group activated by an

aldehyde function at the 3-position (Scheme 4). Then, a decar-

bonylation step using Wilkinson’s catalyst would afford the

compound [¹⁸F]7a [31]. The characterization of the radiolabeled

compound was achieved by HPLC co-migration of the radioactive

compound with the corresponding non-radioactive reference

compound. The labeling precursor 16 and intermediate reference

compound 14 were synthesized following Scheme 3. The Suzuki’s

coupling reaction between 4-methylene-N-Boc-piperidine and

bromobenzenes [32] led to substituted benzylpiperidines 5, 10 and

11 in 88 to 46% yields. After Boc deprotection, addition of 2-(1-

chloromethyl)-5-methoxybenzimidazole to the corresponding

piperidine afforded reference compound 14 or intermediate 15.

Recovery of the benzaldehyde function resulted in 1,3-dithiane

removal of 15 with iron chloride [33] and afforded the labeling

precursor 16 in 65% yield. The radiolabeling by a nucleophilic

substitution with K[¹⁸F]ﬂuoride/K_2.2.2complex and K₂CO₃or

potassium oxalate [34] as base in different solvents led to an

unidentiﬁed radiolabeled compound without any presence of [¹⁸F]

In consequence, the radioﬂuorination by nucleophilic substitu-

tion of a nitro group activated by a carbonyl group in para-position,

on the benzylic position, was studied (Scheme 6). Subsequent

reduction of the benzylic carbonyl function would lead to the ﬁnal

product. The converging synthetic strategy permitted the prepa-

ration of the labeling precursor 17 and intermediate reference

compound 18 in one step by addition of the 2-(1-chloromethyl)-5-

methoxybenzimidazole to the corresponding 4-(4-nitrobenzoyl)

piperidine [36] or 4-(4-ﬂuorobenzoyl)piperidine (Scheme 5). The

nucleophilic aromatic substitution on 17 was performed using

standard conditions i.e. K[¹⁸F]ﬂuoride/K₂CO₃/K_2.2.2complex as

ﬂuorinating agent in DMSO at 140 ^ꢁC during 10 min (Scheme 6).

After a C-18 solid-phase extraction (SPE), the ﬂuorinated interme-

diate [¹⁸F]18 was obtained with a radiochemical purity more than

90% in 24 ꢀ 5% (n ¼ 15) radiochemical yield at the end of

bombardment (EOB). The use of classical reaction conditions [37]

for the reduction with triethylsilane and triﬂuoroacetic acid (TFA)

did not reduce the benzoyl [¹⁸F]18 even at 80 ^ꢁC (Table 2). Then, the

reduction was achieved using triﬂic acid and triethylsilane to lead

Scheme 4. Strategy for the radiosynthesis of [¹⁸F]2a from precursor 16.

R. Labas et al. / European Journal of Medicinal Chemistry 46 (2011) 2295e2309

2299

Scheme 5. Synthesis of precursors 17, 19 and intermediates 18, 20. Reaction conditions: (i) 1-chloromethyl-5-methoxybenzimidazole, Na₂CO₃, acetonitrile, reﬂux, 15 h; (ii) 1) 4a,

(COCl)₂, CH₂Cl₂, DMF, RT, 3 h; 2) CH₂Cl₂, TEA, RT, overnight.

to [¹⁸F]7a within 70% yield after 10 min at 105 ^ꢁC. After evaporation

of the reaction solution, the methoxy protective group was

removed using concentrated HBr at 160 ^ꢁC during 15 min. An SPE

pre-puriﬁcation on a C-18 cartridge was followed by a reversed

phase HPLC puriﬁcation to afford [¹⁸F]2a in 9.5 ꢀ 1.5% decay-cor-

rected radiochemical overall yield. The ﬁnal product [¹⁸F]2a was

reformulated by SPE to eliminate the HPLC eluent leading to an

injectable solution containing less than 10% of ethyl alcohol. The

radiochemical purity of [¹⁸F]2a was >99% and the speciﬁc radio-

a mixture of two major products corresponding to 15 ꢀ 9% (EOB) of

the radioactivity after SPE. The compound [¹⁸F]20 represented

about 40% of the radioactivity in this solution (Fig. 3A). After

evaporation of the solvent, methanesulfonic acid and triethylsilane

were added to achieve the reduction of the benzoyl [¹⁸F]20 into

[

¹⁸F]1a without reaction of the second product (Fig. 3B). A pre-

puriﬁcation on a C-18 SPE cartridge was followed by a reversed

phase HPLC puriﬁcation and reformulation. The product [¹⁸F]1a

was radiochemically pure (>99%) and the speciﬁc radioactivity was

activity was 1.7 ꢀ 1.4 GBq/

m

mol at the end of synthesis (EOS). The

1.2 ꢀ 0.3 GBq/

mmol EOS. It is to notice that the use for the reduction

of the non-ﬂuorinated acid, methanesulfonic acid, did not improve

the speciﬁc radioactivity.

total synthesis time was 135 min including formulation.

For the radiosynthesis of [¹⁸F]1a, the labeling precursor 19 and

the intermediate 20 were synthesized by addition of the acid

chloride of 4a to the corresponding 4-benzoylpiperidine in 63e64%

yield (Scheme 5). The nucleophilic substitution of the nitro group of

19 by the K[¹⁸F]ﬂuoride/K_2.2.2complex occurred with potassium

oxalate as base (Scheme 6). The radiolabeling reaction led to

For the radiosynthesis of the 4-(4-[¹⁸F]-ﬂuorobenzyl)piperidine

([¹⁸F]6) moiety, the same preparation strategy than for [¹⁸F]1a and [¹⁸F]

2a was used (Scheme 7). The radiolabeling of [¹⁸F]22 was performed

from the precursor 21 under the same labeling conditions as for [¹⁸F]18.

The methanesulfonic acid allowed the removal of the Boc protective

Scheme 6. Radiosynthesis of [¹⁸F]1a and [¹⁸F]2a. Reaction conditions: (i) K¹⁸F, K_2.2.2, K₂CO₃, DMSO, 140 ^ꢁC, 10 min; (ii) Et₃SiH, TfOH, 105 ^ꢁC, 15 min; (iii) 48% HBr, 160 ^ꢁC, 15 min; (iv)

¹⁸F, K_2.2.2, K₂C₂O₄, K₂CO₃, DMSO, 140 ^ꢁC, 15 min; (v) Et₃SiH, methanesulfonic acid, RT, 20 min.

K

2300

R. Labas et al. / European Journal of Medicinal Chemistry 46 (2011) 2295e2309

0.035 ꢀ 0.005% ID/g for [¹⁸F]2a). This poor brain uptake cannot be

Table 2

Reduction of [¹⁸F]18.

assigned to the present experimental conditions. Indeed, if a low

speciﬁc radioactivity could lead to a decrease of the signal/noise

ratio, it could be sufﬁcient to evaluate the total binding of the

radioligand in the brain and the global distribution in the whole

organism. It was demonstrated that a low speciﬁc radioactivity did

not change signiﬁcantly the pharmacokinetics of radiotracers [38].

This is also obviously illustrated by the use of tritiated radioligands,

with speciﬁc radioactivity lower than those obtained in this work,

for ex vivo or in vivo receptor occupancy studies [39]. In the present

work, the radioactivity measured in the brain was highly over the

detection limits and the low brain/blood ratio may be quietly

related to a poor blood brain barrier crossing. These weak brain

penetrations were not expected since their calculated log D_{(pH ¼ 7.4)}

were optimal (2.5 and 3.0 for 1a and 2a) for a passive brain pene-

Entry

Acid

Quantity

L)

Et₃SiH

L)

T (^ꢁC)

Time

(min)

[

¹⁸F]7a (%)^a

(

m

(m

1

2

3

4

5

6

7

TFA

TfOH

500

400

300

100

200

400

300

600

800

80

55

RT

105

20

10

0

22

32

55

71

a

Radiochemical conversions were determined by HPLC analysis.

group and addition of triethylsilane permitted the reduction into

product [¹⁸F]6. HPLC puriﬁcation and formulation using SPE gave pure

[

¹⁸F]6 with a radiochemical overall yield of 12% (EOB) in 125 min.

tration with values comprised between 2 and 3.5 [40]. The mPET

images for both radiotracers showed a high uptake in liver and

radioactivity in bone, skull and cartilage (Fig. 4). The radioactive

level for each radiotracer in bone and cartilage increased regularly

during the entire exam and the clavicle articulation represented the

highest standardized uptake value (SUV) at 120 min (Fig. 4). These

in vivo results could correspond to a typical skeletal uptake of

ﬂuorine-18 due to a deﬂuorination process. Biodistribution in

peripheral organs demonstrated a high uptake in liver at 120 min

and high radioactive concentration in kidney and in urine for [¹⁸F]

1a and [¹⁸F]2a (Table 3). These data indicated a rapid urinary

excretion of the products and suggested a hepatic metabolism.

2.5. Pharmacological evaluation

After intravenous injection of [¹⁸F]1a or [¹⁸F]2a in rats,

acquired time activity curves showed a very weak passage of the

radiotracers into the brain during the 120 min of the exam (Fig. 4).

mPET

The ex vivo biodistribution study in rat at the end of mPET exam

conﬁrmed the low brain uptakes of [¹⁸F]1a or [¹⁸F]2a (0.054%

injected dose/g (ID/g) for [¹⁸F]1a and 0.035%ID/g for [¹⁸F]2a). The

microdissection of rat brains at this time did not show any signif-

icant differences between cerebral structures with high or low

densities of NR2B subunits as hippocampus and cerebellum

(0.064 ꢀ 0.005 vs. 0.046 ꢀ 0.004% ID/g for [¹⁸F]1a, 0.037 ꢀ 0.002 vs.

Fig. 3. HPLC radiochromatograms of the reaction mixture after [¹⁸F]ﬂuorination of [¹⁸F]20 (A) and reduction into [¹⁸F]1a (B).

R. Labas et al. / European Journal of Medicinal Chemistry 46 (2011) 2295e2309

2301

The evaluation of the in vivo radiometabolism in rat of [¹⁸F]1a

and [¹⁸F]2a was achieved by radio thin layer chromatography

(radioTLC) analysis from the intravenous blood sampling (Fig. 5).

The radiolabeled parent compound was also identiﬁed by radi-

oHPLC analysis. For both radiotracers, the radioTLC showed the

presence of the labeled parent compound and the apparition of

very polar compounds at the TLC origin. [¹⁸F]1a presented a slow

degradation with the presence of a second polar radiometabolite

and a half-life about 50 min (Fig. 5A). The half-life of [¹⁸F]2a was

about 35 min in plasma (Fig. 5B). The parent compounds were

detected in plasma during all the experimental time and therefore,

we could conﬁrm that [¹⁸F]1a and [¹⁸F]2a did not cross the blood

brain barrier.

In order to broaden the use of the 4-[¹⁸F]ﬂuorobenzyl-4-piper-

idine pattern for other radiotracers development, we decided to

explore more in details the pharmacokinetic behavior and meta-

bolic pathway of the 4-[¹⁸F]ﬂuorobenzyl-4-piperidine itself, [¹⁸F]6.

First, in vitro assays conﬁrmed that compound 6 added to samples

of blood or liver tissue maintained at 37 ^ꢁC during 30 min was not

spontaneously altered as for 1a and 2a. This in vitro stability agreed

with the fact that ﬂuoro-substituted aromatic compounds are

known to not undergo or even avoid undesirable metabolic trans-

formation at the carbon bearing the halogen atom [41,42]. Indeed,

several radiotracers labeled with [¹⁸F]-ﬂuorine at the 4-position on

a phenyl cycle, as [¹⁸F]-FBFPA [43] or [¹⁸F]-G379 [44], did not reveal

any deﬂuorination. Widely used radiopharmaceuticals possessing

the benzoyl carbonylated analog moiety 4-[¹⁸F]-ﬂuorobenzoyl-4-

piperidine as [¹⁸F]-setoperone or [¹⁸F]-altanserin were extensively

studied and no radiodeﬂuorination was observed [40,45e47].

Secondly, the in vivo behavior of [¹⁸F]6 was studied. The compound

[

¹⁸F]6 demonstrated a similar high uptake in liver (2.307 ꢀ 0.020%

ID/g) and a more important uptake in kidney (1.206 ꢀ 0.061% ID/g)

and in urine (10.1 ꢀ 2.3% injected dose/mL) than for [¹⁸F]1a and

[

¹⁸F]2a.

mPET acquired time activity curves in bone, jaw, skull or

cartilage revealed an identical proﬁle to the two other radiotracers

(Fig. 4C), typical of a radiodeﬂuorination process. The radio-

metabolism study of [¹⁸F]6 indicated a fast degradation with

a plasmatic half-life of 10 min (Fig. 5C). The extremely polar

compound at the origin of the radioTLC, specially under elution

conditions for [¹⁸F]6, could correspond to [¹⁸F]-ﬂuoride ion, as

suspected for [¹⁸F]1a and [¹⁸F]1b. From these data, we concluded to

a similar radiometabolism for [¹⁸F]6 and [¹⁸F]1a and/or [¹⁸F]2a,

corresponding to a radiodeﬂuorination.

Table 3

Ex vivo biodistribution in rats of [¹⁸F]1a, [¹⁸F]2a and [¹⁸F]6 as percentage of injected

dose/g of tissue (ꢀSEM) in rats at the end of

mPET scan.

Tissues

[

¹⁸F]1a^a

[

¹⁸F]2a^a

[

¹⁸F]6^b

Thymus

Lung

Heart

Pancreas

Liver

Spleen

Kidney

Urine

Bladder

Intestine

Muscle

Fat

Brain

Bone

0.057 ꢀ 0.006

0.132 ꢀ 0.016

0.076 ꢀ 0.008

0.080 ꢀ 0.014

1.365 ꢀ 0.137

0.169 ꢀ 0.045

0.377 ꢀ 0.016

3.237 ꢀ 0.646

0.175 ꢀ 0.029

0.107 ꢀ 0.016

0.069 ꢀ 0.009

0.056 ꢀ 0.007

0.054 ꢀ 0.005

0.397 ꢀ 0.106

0.776 ꢀ 0.155

0.225 ꢀ 0.005

0.445 ꢀ 0.018

0.068 ꢀ 0.003

0.391 ꢀ 0.039

2.255 ꢀ 0.082

0.305 ꢀ 0.014

0.384 ꢀ 0.009

1.327 ꢀ 0.282

0.373 ꢀ 0.029

0.188 ꢀ 0.008

0.044 ꢀ 0.003

0.098 ꢀ 0.006

0.035 ꢀ 0.001

1.576 ꢀ 0.070

nd^c

0.187 ꢀ 0.027

0.507 ꢀ 0.071

0.135 ꢀ 0.016

0.285 ꢀ 0.071

2.307 ꢀ 0.020

0.322 ꢀ 0.057

1.206 ꢀ 0.061

10.125 ꢀ 2.346

0.542 ꢀ 0.068

0.902 ꢀ 0.063

0.145 ꢀ 0.010

0.052 ꢀ 0.000

0.395 ꢀ 0.035

0.750 ꢀ 0.307

2.312 ꢀ 0.253

Fig. 4. Time course of radioactivity in rat tissues determined by

¹⁸F]1a (A), [¹⁸F]2a (B) or [¹⁸F]6 (C): A, skull; -, articulation;

brain (n ¼ 3 except for liver and plasma, n ¼ 1).

m

PET after injection of

[

6, liver; B, plasma; >,

Cartilage

a

Radioactivity measured 120 min after injection of the radiotracer.

Radioactivity measured 90 min after injection of [¹⁸F]6.

Not determined.

b

c

2302

R. Labas et al. / European Journal of Medicinal Chemistry 46 (2011) 2295e2309

Scheme 7. Radiosynthesis of [¹⁸F]6. Reaction conditions: (i) K¹⁸F, K_2.2.2, K₂CO₃, DMSO,

140 ^ꢁC, 10 min; (ii) Et₃SiH, methanesulfonic acid, RT, 10 min.

To complete our ﬁndings and to assess that the deﬂuoration was

subject to in vivo metabolic pathways, a study was conducted by LC-

MS/MS to explore the structure of metabolites coming from 6. The

cold compound 6 was administered (10 mg/kg) to rats and the

analyses were achieved on plasma and urine samples collected 1 h

after i.v. injection. The analysis was realized on the additional peaks

obtained by comparison of the full-scan MS of samples from treated

rat with sample from control rat. The unmetabolized parent

compound 6 was not found in urine and plasma samples. The masses

correspondingtothe differentpotential metabolic pathways of phase

I and II i.e. hydroxylation, N-oxidation, glucuronidation, O-methyla-

tion, glutathione addition, sulfation and combinations of them, were

searched in the full-scan MS peaks [48]. Protonated molecular ion

mass was found at m/z 386.30 and 384.37. The MS/MS generated

product ions accounted for cleavage at the bond resulting in loss of

neutral species such as H₂O, CO₂, acetic acid, etc. The mass m/z 386

was hypothesized to be due to the presence of a hydroxyl and

a glucuronide (Glu) adduct to 6. The MS/MS ion spectrum of m/z 386

showed a fragmentation at m/z 326 (MH-CH₃COOH)^þand the major

fragment ion m/z 210 (MH-Glu)^þcorresponded to the hydroxylation

of 6. The presence of fragment ions for the m/z 386 ion at m/z125 (m/z

109 þ O) and m/z 151 (m/z 135 þ O) is consistent with a hydroxylation

on the aromatic benzyl moiety (Scheme 8) and could be compared to

the MS/MS spectra of 6 (m/z 194 (M þ H)^þ, 109 and 135). The

protonated molecular ion m/z 384.37 was suggested from the frag-

ment pattern to be the product of the corresponding metabolite m/z

386 in which a hydroxyl group was bonded to the benzyl cycle by

displacing the ﬂuoride ion (Scheme 8). In MS/MS analysis, frag-

mentation was similar to the precedent compound with m/z 324

(MH-CH₃COOH)^þand the major fragment ion m/z 208 (MH-Glu)^þ.

Characterization of hydroxylated metabolites provided indirect

evidence for [¹⁸F]-ﬂuoride ion formation as a radiometabolite of [¹⁸F]

6 and corroborated the uptake in bone, skull and cartilage. The

presence of glucuronide metabolite implied a metabolism pathway

by a phase I hydroxylation by cytochrome P450 and a phase II glu-

curonidation by uridine diphosphate-glucuronyl transferase both

mainly located in liver where the highest uptake was observed. We

supposed a glucuronidation on the hydroxyl group rather than on the

amine of the piperidine since the N-glucuronidation is considered as

secondary and the N-glucuronide are less stable than O-analogs.

These ﬁndings could be compared with the metabolism describedfor

the traxoprodil which presented some structural similarity with our

compounds. In this case, hydroxylation at the 4-position and di-

hydroxylation at the 4- and 3-positions occurred on the 4-phenyl-

piperidine moiety followed by glucuronidation [49].

Fig. 5. Time course of plasma radioactive concentration (SUV) in rats after injection of

[

¹⁸F]1a (A), [¹⁸F]2a (B) or [¹⁸F]6 (C): , total radioactivity; A, [¹⁸F]parent compound;

6

,, polar compound including [¹⁸F]ﬂuoride; B, unknown radiometabolite.

3. Conclusion

Several ﬂuorinated ligands were synthesized and their antag-

onist effects for the NR2B NMDA receptor were measured by an in

R. Labas et al. / European Journal of Medicinal Chemistry 46 (2011) 2295e2309

2303

Scheme 8. LC-MS/MS fragmentation of 6, m/z 386 and m/z 384.

vitro functional assay. Two NR2B NMDA antagonists from two

different series of compounds and presenting high afﬁnity and

selectivity vs. NR2A, [¹⁸F]1a and [¹⁸F]2a, were labeled by a multi-

step radiosynthesis. The syntheses of the reference compounds

and of the different labeling precursors were optimized with the

converging synthesis used by coupling the different methyl-

benzimidazole or oxalamic acid with the adequate 4-substituted

piperidines. The radioﬂuorination occurred by nucleophilic subs-

titution followed by the reduction of the benzoyl function with

triethylsilane in strong organic acid. After injection of [¹⁸F]1a or

and are uncorrected. NMR spectroscopy was performed on a Bruker

DRX 400 operating at 400 MHz 100.6 MHz and 376.4 MHz for

respectively ¹H, ¹³C and ¹⁹F NMR spectra, or on a Bruker DPX 250

operating at 250 MHz (¹H NMR), 62.9 MHz (¹³C NMR) and 235 MHz

(

¹⁹F NMR). Chemical shifts are reported as parts per million (

d)

using tetra-methylsilane (TMS) as the internal standard or by

reference to the proton resonances resulting from incomplete

deuteration of the NMR solvent. Elemental analyses were deter-

mined on a ThermoQuest analyzer CHNS and were within ꢀ0.4% of

the calculated values. Mass spectra (MS) were performed on

a Varian GCMS Saturn 2000 spectrometer by electronic impact (EI)

or on a Waters Q-TOF micro spectrometer by electrospray ioniza-

tion (ESI). High-resolution mass spectra (HRMS) were obtained on

a Waters Q-TOF micro spectrometer by ESI. Relative intensities are

given in brackets. Thin Layer Chromatographies (TLC) were run on

pre-coated aluminum plates of silica gel 60F₂₅₄(Merck) and Rf were

established using an UV-lamp at 254 nm or by ninhydrin or

phosphomolybdic acid hydrate spray reagent. Liquid chromatog-

raphies were performed on 40e63 mesh silica gel 60 (Merck)

columns. Radioactive TLCs were measured using an Instant

Imager^ÒPackard apparatus. High Performance Liquid Chromatog-

raphy (HPLC) was carried out on a Waters 600 pump and controller,

a Waters 717 plus autosampler and a Waters 996 photodiode array

[

¹⁸F]2a in rats,

mPET scans showed low brain uptake and high bone

uptakes. The accumulation of radioactivity in bones, skull and

articulations was supposed to be due to an in vivo radiodeﬂu-

orination on the 4-(4-[¹⁸F]ﬂuorobenzyl)piperidine moiety inde-

pendently of the piperidinyl substituent. This in vivo behavior was

assessed by injection of [¹⁸F]6. Ex vivo LC-MS/MS studies on

plasma and urine samples after injection of the non-radioactive 6

to rats indicated a rapid metabolism by hydroxylations on the

phenyl ring followed by glucuronidation. The metabolic conver-

sion of [¹⁸F]6 in a glucuronoconjugate having lost its ﬂuorine atom

conﬁrmed the now expected in vivo deﬂuorination of compounds

that include in their structures the 4-(4-[¹⁸F]ﬂuorobenzyl)piperi-

dine moiety as we observed for [¹⁸F]1a and [¹⁸F]2a. The poor brain

penetration and radiodeﬂuorination processes of [¹⁸F]1a and [¹⁸F]

2a harm the pursuit of their evaluation as radiotracers. The

development of radiolabeled probes for NR2B NMDA receptor

imaging remains important and could be achieved with ligands of

different structural series.

detector (

l

¼ 210e380 nm) coupled with a NaI probe radioactive

detector (Novelec, Meylan, France). HPLC puriﬁcation of the

radiotracers were performed using a Waters 501 pump, a LC

spectrophotometer (

l

¼ 254 nm) coupled with a GeigereMuller

probe radioactive detector (SDS, Paris, France) and a Valco Vici

injector. Radioactivity quantities were determined with dose cali-

brator (Capintec R15C). The radio-TLC was revealed with a Packard

Instant Imager^Ò(Perkin Elmer, Courtaboeuf, France).

4. Experimental

4.1. General methods

4.2. Synthesis of NR2B NMDAR antagonists

All reagents were purchased from Fluka or SigmaeAldrich

(Saint-Quentin Fallavier, France) and were used without further

puriﬁcation. Anhydrous THF, DMF and acetonitrile were obtained

from a Mbraun SPS-800 solvent delivery system. HPLC quality

solvents were purchased from Merck (France) or SDS (Peypin,

France). [¹⁸F]-ﬂuoride was produced using a Cyclone 18/9 (IBA)

cyclotron at the Cyceron PET Center, via the ¹⁸O(p,n)¹⁸F nuclear

reaction. Melting points were determined in open capillary tubes

using a Barnstead Electrothermal IA 9100 melting point apparatus

4.2.1. 2-Acetamido-N-(2-oxo-3H-1,3-benzoxazol-6-yl)acetic acid

(4a)

6-Amino-3H-1,3-benzoxazol-2-one (700 mg, 4.66 mmol) and

sodium acetate (497 mg, 6.05 mmol) were dissolved in acetic acid

(20 mL) at 35 ^ꢁC. Ethyl chlorooxoacetate (677

mL, 6.05 mmol) was

added dropwise and the reaction mixture was stirred at 35 ^ꢁC for

1 h and 45 min at 90 ^ꢁC then water (50 mL) was added to the

solution. The precipitate was ﬁltered, washed with water

2304

R. Labas et al. / European Journal of Medicinal Chemistry 46 (2011) 2295e2309

(2 ꢂ 30 mL) and dried overnight under vacuum in presence of P₂O₅

to afford the intermediate ethyl 2-acetamido-N-(2-oxo-3H-1,3-

benzoxazol-6-yl)acetate (857 mg, 74%) as a white solid. Mp 185 ^ꢁC.

NMR (DMSO-d₆) d 170.1,162.2, 156.8, 133.3, 132.9,123.7, 119.6,114.8,

111.7. HRMS (m/z): [M ꢃ H]^ꢃcalcd for C₉H₅N₂O₄S, 236.9970; found,

236.9967.

Anal. (C₁₁H₁₀N₂O₅.0.1H₂O) C, H, N. ¹H NMR (DMSO-d₆)

d 11.64 (s,

1H), 10.85 (s, 1H), 7.73 (d, J₄¼ 4.0 Hz, 1H), 7.51 (dd, J₃¼ 8.0 Hz,

4.2.4. N-[4-(4-Fluorobenzyl)piperidin-1-yl]-N⁰-(2-oxo-3H-1,3-

benzoxazol-6-yl)oxamide (1a)

J₄¼ 4.0 Hz,1H), 7.07 (d, J₃¼ 8.0 Hz,1H), 4.30 (q, J₃¼ 8.0 Hz, 2H),1.31

(t, J₃¼ 8.0 Hz, 3H). ¹³C NMR (DMSO-d₆)

d

160.6, 155.3, 154.5, 143.0,

Under nitrogen atmosphere, 4a (100 mg, 0.45 mmol) was

132.1, 127.2, 116.2, 109.5, 102.8, 62.4, 13.9. HRMS (m/z): [M þ H]^þ

partially dissolved in dry CH₂Cl₂(3 mL) and anhydrous DMF (20

m

L)

calcd for C₁₁H₁₁N₂O₅, 251.0668; found, 251.0657.

at 0 ^ꢁC. Oxalyl chloride (50

mL, 0.49 mmol) was added dropwise and

A solution of potassium hydroxide (101 mg, 1.80 mmol) in water

(400 mL) was added to a suspension of ethyl 2-acetamido-N-(2-oxo-

the reaction mixture was stirred at 0 ^ꢁC for 10 min and 3 h at room

temperature. Solvents were evaporated and the resulting yellow

solid was dried under reduced pressure, then partially dissolved in

dry CH₂Cl₂(3 mL) at 0 ^ꢁC. A solution of 6 hydrochloride salt

3H-1,3-benzoxazol-6-yl)acetate obtained above (150 mg,

0.60 mmol) in ethanol (3 mL) and the reaction mixture was stirred

at room temperature for 2 h. Water (5 mL) was added to the

mixture and the resulting homogenous solution was acidiﬁed to

pH ¼ 1 with concentrated HCl. The precipitate was ﬁltered and

dried overnight under vacuum in presence of P₂O₅to give 4a

(115 mg, 87%) as a white solid. Mp 248 ^ꢁC. Anal. (C₉H₆N₂O₅) C, H, N.

(103 mg, 0.45 mmol) and TEA (253 mL, 1.80 mmol) in dry CH₂Cl₂

(1 mL) was added dropwise and the reaction mixture was stirred

for 10 min at 0 ^ꢁC and overnight at room temperature. The reaction

was quenched with a solution of 10% NaHCO₃(20 mL) then CH₂Cl₂

(20 mL) was added. The layers were separated and the aqueous

layer was extracted with CH₂Cl₂(3 ꢂ 20 mL). The combined organic

layers were dried over MgSO₄, ﬁltered, concentrated and puriﬁed

by chromatography on silica gel with CH₂Cl₂/MeOH/NH₄OH

(98:2:0.1 to 95:5:0.1) to afford 1a (45 mg, 25%) as a white solid. Mp

¹H NMR (DMSO-d₆)

d

11.63 (s, 1H), 10.78 (s, 1H), 7.76 (d, J₄¼ 1.6 Hz,

1H), 7.54 (dd, J₃¼ 8.4 Hz, J₄¼ 1.6 Hz,1H), 7.06 (d, J₃¼ 8.4 Hz,1H). ¹³

C

NMR (DMSO-d₆) d 162.1, 156.6, 154.6, 143.1, 132.4, 127.0, 116.1, 109.6,

102.6. HRMS (m/z): [M ꢃ H]^ꢃcalcd for C₉H₅N₂O₅, 221.0198; found,

221.0209.

223e225 ^ꢁC. Anal. (C₂₁H₂₀FN₃O₄) C, H, N. ¹H NMR (CDCl₃)

d 9.33 (s,

1H), 8.96 (s, 1H), 7.81 (d, J₄¼ 1.9 Hz, 1H), 7.22 (dd, J₃¼ 8.5 Hz,

J₄¼1.9 Hz,1H), 7.14e6.91 (m, 5H), 5.16e4.96 (m,1H), 4.64e4.46 (m,

1H), 3.17e2.94 (m, 1H), 2.82e2.62 (m, 1H), 2.54 (d, J₃¼ 6.7 Hz, 2H),

4.2.2. 2-Acetamido-N-(2-oxo-1,3-dihydroindol-5-yl)acetic acid

(4b)

The intermediate ethyl 2-acetamido-N-(2-oxo-1,3-dihy-

droindol-5-yl)acetate was obtained as a white solid (532 mg, 79%)

from 5-amino-1,3-dihydroindol-2-one (400 mg, 2.70 mmol)

following the same procedure as described for 4a. Mp 245e249 ^ꢁC.

1.94e1.68 (m, 3H), 1.46e1.13 (m, 2H). ¹³C NMR (CDCl₃)

d 160.5,

159.0, 157.5 (d, J₁¼ 242 Hz), 155.6, 144.1, 135.4, 132.7, 130.5 (d,

J₃¼ 8.2 Hz), 126.3, 116.0, 115.4 (d, J₂¼ 20.8 Hz), 109.9, 103.4, 47.0,

44.4, 42.1, 38.3, 33.0, 31.9. ¹⁹F NMR (CDCl₃)

d

ꢃ117.12. HRMS (m/z):

Anal. (C₁₂H₁₂N₂O₄.0.3H₂O) C, H, N. ¹H NMR (DMSO-d₆)

d

10.65 (s,

[M þ H]^þcalcd for C₂₁H₂₁FN₃O_4,398.1516; found, 398.1524.

1H), 10.37 (s, 1H), 7.62 (d, J₄¼ 4.0 Hz, 1H), 7.51 (dd, J₃¼ 8.0 Hz,

J₄¼ 4.0 Hz, 1H), 6.78 (d, J₃¼ 8.0 Hz, 1H), 4.29 (q, J₃¼ 8.0 Hz, 2H),

4.2.5. N-[4-(4-Fluorobenzyl)piperidin-1-yl]-N⁰-(2-oxo-1,3-

dihydroindol-5-yl)oxamide (1g)

3.49 (s, 2H), 1.31 (t, J₃¼ 8.0 Hz, 3H). ¹³C NMR (DMSO-d₆)

d 176.3,

160.9, 155.2, 140.6, 131.4, 126.2, 120.0, 117.5, 108.9, 62.3, 36.0, 13.9.

HRMS (m/z): [M þ H]^þcalcd for C₁₂H₁₃N₂O₄, 249.0875; found,

249.0887.

Under nitrogen atmosphere, compound 4b (100 mg, 0.52 mmol)

was dissolved in DMF (8 mL), then TEA (123

mL, 0.87 mmol), 6

hydrochloride salt(100 mg, 0.44 mmol) and HBTU (199 mg,

0.52 mmol) were added. The solution was stirred for 24 h at room

temperature and the solvent was evaporated. 10% NaHCO₃(80 mL)

and CH₂Cl₂(50 mL) were added. The layers were separated and the

aqueous layer was extracted with CH₂Cl₂(3 ꢂ 50 mL). The

combined organic layers were dried over MgSO₄, ﬁltered, concen-

trated and puriﬁed by chromatography on silica gel with CH₂Cl₂/

MeOH/NH₄OH (96:4:0.1) to afford 1g (32 mg, 19%) as a yellow solid.

Mp 186 ^ꢁC. Anal. (C₂₂H₂₂FN₃O₃.0.6H₂O) C, H, N. ¹H NMR (CDCl₃)

Compound 4b was obtained as a red solid (323 mg, 81%) from

the intermediate ethyl 2-acetamido-N-(2-oxo-1,3-dihydroindol-5-

yl)acetate (450 mg, 1.81 mmol) following the same procedure as

described for 4a. Mp 246 ^ꢁC. Anal. (C₁₀H₈N₂O₄.0.1H₂O) C, H, N. ¹H

NMR (DMSO-d₆)

d

10.59 (s, 1H), 10.36 (s, 1H), 7.64 (d, J₄¼ 4.0 Hz,

1H), 7.55 (dd, J₃¼ 8.0 Hz, J₄¼ 4.0 Hz, 1H), 6.77 (d, J₃¼ 8.0 Hz, 1H),

3.48 (s, 2H). ¹³C NMR (DMSO-d₆)

d 176.4, 162.3, 156.4, 140.5, 131.7,

126.1, 119.8, 117.4, 108.9, 36.0. HRMS (m/z): [M ꢃ H]^ꢃcalcd for

C₁₀H₇N₂O_4,219.0406; found, 219.0395.

d

9.19 (s, 1H), 8.32 (s, 1H), 7.61 (d, J₄¼ 2.1 Hz, 1H), 7.33 (dd,

J₃¼ 8.4 Hz, J₄¼ 2.1 Hz, 1H), 7.15e7.04 (m, 2H), 7.03e6.91 (m, 2H),

6.84 (d, J₃¼ 8.4 Hz, 1H), 5.13e4.99 (m, 1H), 4.62e4.49 (m, 1H), 3.54

(s, 2H), 3.12e2.98 (m, 1H), 2.76e2.64 (m, 1H), 2.53 (d, J₃¼ 6.8 Hz,

4.2.3. 2-Acetamido-N-(2-oxo-3H-1,3-benzothiazol-6-yl)acetic acid

(4c)

The intermediate ethyl 2-acetamido-N-(2-oxo-3H-1,3-benzo-

thiazol-6-yl)acetate was obtained as a white solid (1.02 g, 91%) from

6-amino-3H-1,3-benzothiazol-2-one (700 mg, 4.21 mmol) following

the same procedure as described for 4a. Mp 239 ^ꢁC. Anal.

2H), 1.93e1.68 (m, 3H), 1.47e1.16 (m, 2H). ¹³C NMR (CDCl₃)

d 177.2,

162.8, 161.6 (d, J₁¼ 244.0 Hz), 159.0, 139.6, 135.5 (d, J₄¼ 3.2 Hz),

132.1,130.5 (d, J₃¼ 8.2 Hz),126.2, 120.0,117.5, 115.2 (d, J₂¼ 20.7 Hz),

109.8, 47.0, 44.4, 42.1, 38.3, 36.7, 33.0, 31.9. ¹⁹F NMR (CDCl₃)

(C₁₁H₁₀N₂O₄S.0.2H₂O) C, H, N. ¹H NMR (DMSO-d₆)

d

11.90 (s, 1H),

d

ꢃ117.16. HRMS (m/z): [M þ H]^þcalcd for C₂₂H₂₃FN₃O_3,396.1723;

10.83 (s,1H), 7.95 (d, J₄¼ 2.0 Hz,1H), 7.60 (dd, J₃¼ 8.0 Hz, J₄¼ 2.0 Hz,

found, 396.1739.

1H), 7.10 (d, J₃¼ 8.0 Hz, 1H), 4.30 (q, J₃¼ 8.0 Hz, 2H), 1.31 (t,

J₃¼ 8.0 Hz, 3H). ¹³C NMR (DMSO-d₆)

d

169.9, 160.7, 155.4, 133.3,

4.2.6. N-[4-(4-Fluorobenzyl)piperidin-1-yl]-N⁰-(2-oxo-3H-1,3-

benzothiazol-6-yl)oxamide (1h)

132.6, 123.6, 119.6, 114.9, 111.6, 62.4, 13.9. HRMS (m/z): [M þ H]^þ

calcd for C₁₁H₁₁N₂O₄S, 267.0440; found, 267.0428.

Compound 1h was prepared from 4c (100 mg, 0.42 mmol)

following the same procedure as described for 1a. Puriﬁcation

using CH₂Cl₂/MeOH/NH₄OH (99:1:0.1 to 95:5:0.1) afforded 1h

(51 mg, 30%) as a white solid. Mp 218 ^ꢁC. Anal. (C₂₁H₂₀FN₃O₃S.H₂O)

Compound 4c was obtained as a white solid (626 mg, 87%)

from

ethyl

2-acetamido-N-(2-oxo-3H-1,3-benzothiazol-6-yl)

acetate (800 mg, 3.0 mmol) following the same procedure as

described for 4a. Mp 242 ^ꢁC. Anal. (C₉H₆N₂O₄S.0.5H₂O) C, H, N. ¹H

C, H, N, S. ¹H NMR (CD₃OD)

d

7.85 (d, J₄¼ 2.2 Hz, 1H), 7.43 (dd,

NMR (DMSO-d₆)

d

: 11.92 (s, 1H), 10.75 (s, 1H), 7.95 (d, J₄¼ 2.0 Hz,

J₃¼ 8.8 Hz, J₄¼ 2.2 Hz, 1H), 7.21e7.14 (m, 2H), 7.10 (d, J₃¼ 8.8 Hz,

1H), 7.60 (dd, J₃¼ 6.0 Hz, J₄¼ 2.0 Hz,1H), 7.10 (d, J₃¼ 6.0 Hz,1H). ¹³

C

1H), 7.03e6.94 (m, 2H), 4.48e4.40 (m, 1H), 4.03e3.94 (m, 1H),

R. Labas et al. / European Journal of Medicinal Chemistry 46 (2011) 2295e2309

2305

3.18e3.08 (m, 1H), 2.80e2.70 (m, 1H), 2.57 (d, J₃¼ 7.2 Hz, 2H),

1.93e1.80 (m, 1H), 1.79e1.67 (m, 2H), 1.36e1.16 (m, 2H). ¹³C NMR

(DMSO-d₆)

(m, 2H). ¹³C NMR (CDCl₃)

d

187.5 (d, J₃¼ 6.9 Hz), 163.5 (d,

J₁¼ 257.3 Hz), 154.9, 137.1 (d, J₃¼ 8.8 Hz), 136.9 (d, J₄¼ 3.7 Hz),

d

169.9, 162.4, 162.2, 160.7 (d, J₁¼ 241 Hz), 136.0 (d,

128.6 (d, J₃¼ 1.9 Hz), 123.9 (d, J₂¼ 8.9 Hz), 116.4 (d, J₂¼ 20.8 Hz),

J₄¼ 2.82 Hz), 133.0, 132.7, 130.8 (d, J₃¼ 7.7 Hz), 123.7, 118.7, 114.8 (d,

79.5, 43.6, 42.2, 38.1, 31.9, 28.6. ¹⁹F NMR (CDCl₃)

d

: ꢃ126.14. HRMS

J₂¼ 20.9 Hz), 114.1, 111.6, 45.8, 41.0, 40.8, 37.3, 31.8, 30.9. ¹⁹F NMR

(m/z): [M þ Na]^þcalcd for C₁₈H₂₄FNO₃Na, 344.1638; found:

(CD₃OD)

414.1288; found, 414.1283.

d

ꢃ120.11. HRMS (m/z): [M þ H]^þcalcd for C₂₁H₂₁FN₃O₃S,

344.1648.

4.3.2. tert-Butyl 1-[4-(3-[1,3-dithian-2-yl]-4-nitrobenzyl)piperidin-

4.2.7. 2-[4-(4-Fluorobenzyl)piperidin-1-yl]methyl-5-

1-yl]carboxylate (11)

nitrobenzimidazole (9b)

To

a

solution of 5-bromo-2-nitrobenzaldehyde (10 g,

Under nitrogen atmosphere, 8 (317 mg, 1.5 mmol) and 6

(290 mg, 1.5 mmol) were dissolved in dry acetonitrile (5 mL) and

sodium carbonate (477 mg, 4.5 mmol) was added to the solution.

The reaction mixture was heated at 70 ^ꢁC for 15 h, cooled down to

room temperature, quenched with water (40 mL) and extracted

with EtOAc (3 ꢂ 50 mL). The combined organic layers were dried

over MgSO₄, ﬁltered, concentrated and puriﬁed by chromatography

on silica gel with CH₂Cl₂/MeOH/NH₄OH (98:2:0.1) to afford 9b

43.5 mmol) and BF₃.OEt₂(6.61 mL, 52.2 mmol) in toluene (30 mL)

was added under nitrogen atmosphere 1,3-propanedithiol

(5.24 mL, 52.2 mmol) and the solution was stirred overnight at

room temperature. The reaction mixture was quenched by adding

water (150 mL) and extracted with dichloromethane (3 ꢂ 100 mL).

The combined organic layers were washed with 1 M NaOH

(2 ꢂ 200 mL) and water (2 ꢂ 200 mL), dried over MgSO₄, ﬁltered

and concentrated under vacuum to give a yellow solid. Recrystal-

lization from propan-2-ol afforded 2-(5-bromo-2-nitro-phenyl)-

[1,3]-dithiane (10.3 g, 74%) as yellow needles. Mp 115.5 ^ꢁC. Anal.

(350 mg, 63%). ¹H NMR (CD₃OD)

d 8.42 (s, 1H), 8.19e8.13 (m, 1H),

7.62 (d, J ¼ 14.4 Hz, 1H), 7.15e7.10 (m, 2H), 7.10e6.90 (m, 2H), 3.80

(s, 2H), 2.96e2.87 (m, 2H), 2.54e2.50 (m, 2H), 2.18e2.08 (m, 2H),

(C₁₀H₁₀BrNO₂S₂) C, H, N. ¹H NMR (CDCl₃)

d

8.03 (d, J₄¼ 2.0 Hz, 1H),

1.69e1.25 (m, 5H). ¹³C NMR (CD₃OD)

d

164.7, 160.8, 158.7, 144.8,

7.76 (d, J₃¼ 8.4 Hz,1H), 7.56 (dd, J₃¼ 8.4 Hz, J₄¼ 2.0 Hz, 1H), 5.87 (s,

1H), 3.11 (ddd, J₂¼ 14.4 Hz, J₃¼ 14.4 Hz, J₃¼ 2.4 Hz, 2H), 2.93 (ddd,

J₂¼ 14.4 Hz, J₃¼ 4.4 Hz, J₃¼ 3.2 Hz, 2H), 2.20 (dtt, J₂¼ 14.4 Hz,

J₃¼ 4.4 Hz, J₃¼ 2.4 Hz, 1H), 2.95 (dtt, J₂¼ 14.4 Hz, J₃¼ 14.4 Hz,

141.4 (d, J₁¼ 224 Hz), 137.5 (d, J₄¼ 3.1 Hz), 131.6 (d, J₃¼ 8.2 Hz),

119.2, 115.7 (d, J₂¼ 20.7 Hz), 115.3, 112.9, 57.1, 55.1, 43.0, 38.8, 32.1.

HRMS (m/z): [M þ H]^þcalcd for C₂₀H₂₂FN₄O_2,369.1727; found,

369.1725.

J₃¼ 3.2 Hz, 1H). ¹³C NMR (CDCl₃)

d 146.6, 135.7, 134.0, 132.4, 128.6,

126.4, 45.4, 32.3, 25.0.

4.2.8. N-(2-([4-(4-Fluorobenzyl)piperidin-1-yl]methyl)

benzimidazol-5-yl)methanesulfonamide (3b)

The intermediate 2-[4-(4-ﬂuorobenzyl)piperidin-1-yl]methyl-

5-aminobenzimidazole was prepared from 9b (320 mg, 0.87 mmol)

as a white solid (285 mg, 97%) following the same procedure as

described for 9a without chromatography puriﬁcation. ¹H NMR

Compound 11 was prepared from 2-(5-bromo-2-nitrophenyl)-

1,3-dithiane (1 g, 3.12 mmol) following the same procedure as

described for 5. Puriﬁcation using pentanes/EtOAc (96:4 to 85:15)

afforded 11 (630 mg, 46%) as a yellow solid. Mp 136 ^ꢁC. Anal.

(C₂₁H₃₀N₂O₄S₂) C, H, N. ¹H NMR (CDCl₃)

d

7.84 (d, J₃¼ 13.3 Hz, 1H),

7.62 (d, J₄¼ 3.0 Hz, 1H), 7.18 (dd, J₃¼ 13.3 Hz, J₄¼ 3.0 Hz, 1H), 5.93

(s, 1H), 4.18e3.96 (m, 2H), 3.19e3.06 (m, 2H), 2.96e2.87 (m, 2H)

2.69e2.55 (m, 4H), 2.24e2.15 (m, 1H), 2.00e1.87 (m, 1H), 1.73e1.53

(CD₃OD)

2.86 (m, 2H), 2.45 (m, 2H), 2.03 (m, 2H), 1.58e1.15 (m, 5H). ¹³C NMR

(CD₃OD)

164.6, 160.8, 151.4, 144.6, 137.5 (d, J₄¼ 3.1 Hz), 136.8 (d,

J₁¼ 309 Hz), 131.6 (d, J₃¼ 8.1 Hz), 117.0, 115.7 (d, J₂¼ 21.4 Hz), 114.2,

d 7.30 (m,1H), 7.09e6.88 (m, 6H), 6.72 (m,1H), 3.45 (s, 2H),

d

(m, 3H), 1.45 (s, 9H), 1.24e1.07 (m, 2H). ¹³C NMR (CDCl₃)

d

154.9,

147.2, 145.8, 133.7, 131.4, 129.8, 125.1, 79.5, 46.2, 44.0, 42.9, 38.1,

100.4, 56.9, 54.8, 43.0, 38.7, 32.8. ¹⁹F NMR (CD₃OD)

d

ꢃ119.87. HRMS

32.5, 32.0, 28.6, 25.2. HRMS (m/z): [M

C₂₁H₃₀N₂O₄S₂Na, 461.1545; found : 461.1524.

þ

Na]^þcalcd for

(m/z): [M þ H]^þcalcd for C₂₀H₂₄FN₄, 339.1985; found: 339.1978.

Compound 3b was prepared from the amino intermediate

(225 mg, 0.66 mmol) as a white solid (132 mg, 48%) following the

same procedure as described for 3a as a white solid. Mp 237.5 ^ꢁC.

4.3.3. 4-[3-(1,3-Dithian-2-yl)-4-nitrobenzyl]piperidine

hydrochloride (13)

Anal. (C₂₁H₂₅FN₄O₂S.2HCl) C, H, N. ¹H NMR (CD₃OD)

d

7.73 (d,

To a solution of 11 (216.5 mg, 0.49 mmol) in CH₂Cl₂(15 mL) was

added TFA (1.45 mL, 19.8 mmol) and the solution was stirred at

room temperature for 3 h. The reaction was diluted with CH₂Cl₂

(15 mL) and the organic layer was washed with 6 N HCl (2 ꢂ 20 mL),

dried over MgSO₄and concentrated under vacuum to afford 13 as

the hydrochloride salt (184 mg, 99%) as a brown solid. Mp 188 ^ꢁC.

J₃¼ 8.7 Hz, 1H), 7.65 (s, 1H), 7.33 (d, J₃¼ 8.7 Hz, 1H), 7.16 (m, 2H),

6.96 (dd, J₃¼ 8.7 Hz, J₃¼ 8.7 Hz, 2H), 4.73 (s, 2H), 3.63 (m, 2H), 3.17

(m, 2H), 2.98 (s, 3H), 2.56 (m, 2H), 1.65 (m, 3H), 1.55 (m, 2H). ¹³C

NMR (CD₃OD)

d

161.9, 159.5, 138.7 (d, J₁¼ 403 Hz), 135.2, 134.9 (d,

J₄¼ 3.1 Hz), 132.3, 130.3 (d, J₃¼ 7.5 Hz), 119.9, 115.8, 114.5 (d,

J₂¼ 20.1 Hz), 106.4, 53.1, 50.7, 40.0, 37.9, 34.5, 28.5. ¹⁹F NMR

Anal. (C₁₆H₂₂N₂O₂S₂.HCl) C, H, N. ¹H NMR (CDCl₃)

d 7.84 (d,

(CD₃OD)

417.1761; found : 417.1761.

d

ꢃ118.97. HRMS (m/z): [M þ H]^þcalcd for C₂₁H₂₆FN₄O₂S,

J₃¼ 8.20 Hz, 1H), 7.64 (s, 1H), 7.19 (d, J₃¼ 8.20 Hz, 1H), 5.90 (s, 1H),

3.91 (bs, 2H), 3.58e3.32 (m, 2H), 3.18e3.07 (m, 2H), 2.98e2.73 (m,

4H), 2.69 (d, J₃¼ 3.60 Hz, 2H), 2.26e2.12 (m,1H), 2.03e1.60 (m, 6H).

4.3. Synthesis of precursors and reference compounds for

radiolabeling

¹³C NMR (CDCl₃)

d 146.2, 145.7, 134.1, 131.4, 129.7, 125.3, 46.0, 44.2,

42.0, 36.0, 32.5, 28.6, 25.1. HRMS (m/z): [M þ H]^þcalcd for

C₁₆H₂₃N₂O₂S₂, 339.1201; found, 339.1192.

4.3.1. tert-Butyl 1-[4-(3-formyl-4-ﬂuorobenzyl)piperidin-1-yl]

carboxylate (10)

4.3.4. 2-Fluoro-5-[1-(5-methoxybenzimidazol-2-ylmethyl)

Compound 10 was prepared from 5-bromo-2-ﬂuo-

robenzaldehyde (252 mg, 1.24 mmol) following the same proce-

dure as described for 5. Puriﬁcation using pentanes/EtOAc (95:5 to

90:10) afforded 10 as a yellow solid (165 mg, 50%). Mp 95e97 ^ꢁC.

piperidin-4-ylmethyl]-benzaldehyde (14)

To a solution of compound 10 (500 mg, 1.56 mmol) in EtOAc

(5 mL) was added 3 N HCl (5 mL) and the mixture was stirred at

room temperature overnight. The layers were separated, the

aqueous layer was alkalinized to pH 11 with 5 N NaOH and

extracted with CH₂Cl₂(3 ꢂ 15 mL). The organic layers were

combined, dried over MgSO₄, ﬁltered and concentrated to afford

the free amine 12 as a white solid (262 mg). Compound 14 was

Anal. (C₁₈H₂₄FNO₃) C, H, N. ¹H NMR (CDCl₃)

d 10.34 (s, 1H), 7.61 (dd,

J₄¼ 2.3 Hz, J₄¼ 6.6 Hz, 1H), 7.35 (ddd, J₃¼ 8.5 Hz, J₄¼ 2.3 Hz,

J₄¼ 5.1 Hz, 1H), 7.08 (dd, J₃¼ 8.5 Hz, J₃¼ 10.0 Hz, 1H), 4.14e3.96 (m,

2H), 2.70e2.48 (m, 4H), 1.70e1.47 (m, 3H), 1.43 (s, 9H), 1.22e1.01

2306

R. Labas et al. / European Journal of Medicinal Chemistry 46 (2011) 2295e2309

prepared from 12 (100 mg, 0.45 mmol) following the same proce-

dure as described for 7a. Puriﬁcation using CH₂Cl₂/MeOH/NH₄OH

(98:2:0.1) afforded 14 (115 mg, 67%) as a white solid. Mp 103 ^ꢁC.

Anal. (C₂₂H₂₄FN₃O₂.H₂O) C, H, N. ¹H NMR (CD₃OD) with aldehyde as

d

8.30 (d, J₃¼ 8.8 Hz, 2H), 8.06 (d, J₃¼ 8.8 Hz, 2H), 7.46 (d,

J₃¼ 8.8 Hz, 1H), 7.04 (bs, 1H), 6.87 (dd, J₃¼ 8.8 Hz, J₄¼ 2.4 Hz, 1H),

3.83 (s, 5H), 3.33e3.24 (m, 1H), 3.06e2.94 (m, 2H), 2.39e2.27 (m,

2H), 1.93e1.78 (m, 4H). ¹³C NMR (CD₃OD)

d 201.6, 160.6, 152.0,

gem-diol form

d

7.72 (d, J₃¼ 9.0 Hz, 1H), 7.35 (dd, J₄¼ 1.6 Hz,

142.5, 141.2, 135.4, 130.9, 129.2, 125.1, 118.3, 116.9, 97.3, 56.7, 53.6,

52.0, 41.7, 27.2. HRMS (m/z): [M þ H]^þcalcd for C₂₁H₂₃N₄O₄,

395.1719; found, 395.1719.

J₄¼ 6.8 Hz, 1H), 7.31 (d, J₄¼ 2.0 Hz, 1H), 7.22 (dd, J₃¼ 9.0 Hz,

J₄¼ 2.0 Hz, 1H), 7.18 (m, 1H), 7.0 (dd, J₃¼ 8.8 Hz, J₃¼ 10.0 Hz, 1H),

5.55 (s, 1H), 4.88 (s, 2H), 3.90 (s, 3H), 3.69e3.56 (m, 2H), 3.25e3.10

(m, 2H), 2.62 (d, J₃¼ 6.4 Hz, 2H), 1.98e1.82 (m, 3H), 1.69e1.51 (m,

4.3.8. [1-(5-Methoxybenzimidazol)-2-ylmethylpiperidin-4-yl]-(4-

ﬂuorophenyl)methanone (18)

2H). ¹³C NMR (CD₃OD) with aldehyde as gem-diol form

d 160.9,

160.5 (d, J₁¼ 246.5 Hz), 159.3, 141.7, 136.1 (d, J₄¼ 3.8 Hz), 132.1 (d,

J₃¼ 7.8 Hz), 129.6 (d, J₃¼ 3.8 Hz), 128.7, 126.5 (d, J₂¼ 13.2 Hz), 118.3,

116.3 (d, J₂¼ 21.4 Hz), 113.8, 100.0, 97.1, 56.5, 54.5, 51.2, 41.8, 36.2,

Compound 18 was prepared following the same procedure as

described for 7a. Puriﬁcation using CH₂Cl₂/MeOH/NH₄OH (98:2:0.1

to 97:3:0.1) afforded 18 (175 mg, 28%) as a white solid. Mp 190 ^ꢁC.

30.0. ¹⁹F NMR (CD₃OD)

d

ꢃ124.99. HRMS (m/z): [M þ H]^þcalcd for

¹H NMR (CDCl₃)

d

: 8.02e7.93 (m, 2H), 7.47 (d, J₃¼ 8.8 Hz, 1H),

C₂₂H₂₅FN₃O₂, 382.1931; found, 383.1929.

7.18e7.10 (m, 2H), 7.05 (bs,1H), 7.88 (dd, J₃¼ 8.8 Hz, J₄¼ 2.4 Hz,1H),

3.86 (s, 2H), 3.84 (s, 3H), 3.37e3.24 (m, 1H), 3.09e2.95 (m, 2H),

4.3.5. 2-(4-(3-[1,3-Dithian-2-yl]-4-nitrobenzyl)piperidin-1-

ylmethyl)-5-methoxybenzimidazole (15)

Compound 15 was prepared from 13 (342 mg, 0.91 mmol)

following the same procedure as described for 7a. Puriﬁcation

using CH₂Cl₂/MeOH/NH₄OH (98:2:0.1) afforded 15 (217 mg, 48%) as

a yellow solid. Mp 142 ^ꢁC. Anal. (C₂₅H₃₀N₄O₃S₂.H₂O) C,H,N,S. ¹H

2.46e2.29 (m, 2H), 1.98e1.78 (m, 4H). ¹³C NMR (CDCl₃)

d 201.0,

165.7 (d, J₁¼ 254.7 Hz), 156.3, 151.7, 132.2 (d, J₄¼ 3.1 Hz), 130.9,

130.8 (d, J₃¼ 9.4 Hz), 116.0, 115.7 (d, J₂¼ 21.4 Hz), 111.8, 97.6, 56.7,

55.8, 53.4, 43.0, 28.7. ¹⁹F NMR (CDCl₃)

d

ꢃ106.97. HRMS (m/z):

[M þ H]^þcalcd for C₂₁H₂₃FN₃O₂, 368.1774; found, 368.1762.

NMR (DMSO-d₆)

d

7.86 (d, J₃¼ 8.4 Hz, 1H), 7.74 (d, J₃¼ 8.8 Hz, 1H),

4.3.9. N-[4-(4-Nitrobenzoyl)piperidin-1-yl]-N⁰-(2-oxo-3H-1,3-

benzoxazol-6-yl)oxamide (19)

7.58 (d, J₄¼ 1.6 Hz, 1H), 7.38 (dd, J₃¼ 8.4 Hz, J₄¼ 1.6 Hz, 1H), 7.28 (d,

J₄¼ 2.0 Hz, 1H), 7.15 (dd, J₃¼ 8.8 Hz, J₄¼ 2.0 Hz, 1H), 5.77 (s, 1H),

4.74 (s, 2H), 3.84 (s, 3H), 3.63e3.51 (m, 2H), 3.26e3.05 (m, 4H),

2.93e2.82 (m, 2H), 2.69 (d, J₃¼ 6.0 Hz, 2H), 2.13e2.06 (m, 1H),

19 was prepared following the same procedure as described for

1a using 4-(4-nitrobenzoyl)-piperidine hydrochloride (200 mg,

0.74 mmol) to afford 19 (207 mg, 64%) as a yellow solid. Mp 274 ^ꢁC.

1.92e1.65 (m, 4H), 1.56e1.38 (m, 2H). ¹³C NMR (DMSO-d₆)

d

158.6,

Anal. (C₂₁H₁₈N₄O₇) C, H, N. ¹H NMR (DMSO-d₆)

d 11.61 (s, 1H), 10.81

147.0, 145.5, 141.4, 134.0, 133.1, 130.5, 130.1, 127.7, 123.7, 116.5, 116.0,

96.8, 55.3, 53.2, 44.6, 41.6, 36.8, 31.2, 31.0, 24.5. HRMS (m/z):

[M þ H]^þcalcd for C₂₅H₃₁N₄O₃S₂, 499.1838; found, 499.1818.

(s, 1H), 8.35 (d, J₃¼ 8.2 Hz, 2H), 8.23 (d, J₃¼ 8.2 Hz, 2H), 7.68 (s, 1H),

7.34 (d, J₃¼ 8.4 Hz, 1H), 7.06 (d, J₃¼ 8.4 Hz, 1H), 4.42e4.29 (m, 1H),

3.95e3.76 (m, 2H), 3.41e3.23 (m, 1H), 3.08e2.91 (m, 1H), 2.02e1.81

(m, 2H), 1.67e1.40 (m, 2H). ¹³C NMR (DMSO-d₆)

d 201.0, 162.5,

4.3.6. 5-[1-(5-Methoxybenzimidazol-2-ylmethyl)piperidin-4-

ylmethyl]-2-nitrobenzaldehyde (16)

162.0, 154.5, 150.0, 143.1, 140.2, 132.5, 129.7, 126.7, 124.0, 115.4,

109.7, 102.2, 45.1, 42.8, 39, 28.3, 27.3. HRMS (m/z): [M þ H]^þcalcd

for C₂₁H₁₉N₄O₇, 439.1254; found, 439.1271.

Iron (III) chloride hexahydrate (887 mg, 3.28 mmol) was added

to a stirred solution of compound 15 (273 mg, 0.546 mmol) in

CH₂Cl₂(10 mL). The reaction mixture was stirred vigorously for 3 h

at room temperature, quenched with saturated solution of NaHCO₃

(10 mL), diluted with water (15 mL) and extracted with CH₂Cl₂

(3 ꢂ 30 mL). The combined organic layers were dried over MgSO₄,

ﬁltered, concentrated and puriﬁed by chromatography on silica gel

with CH₂Cl₂/MeOH/NH₄OH (98:2:0.1 to 95:5:0.1) to afford 16

(145 mg, 65%) as a yellow solid. Mp 99 ^ꢁC. Anal. (C₂₂H₂₄N₄O₄) C, H,

4.3.10. N-[4-(4-Fluorobenzoyl)piperidin-1-yl]-N⁰-(2-oxo-3H-1,3-

benzoxazol-6-yl)oxamide (20)

20 was prepared following the same procedure as described for

1a using 4-(4-ﬂuorobenzoyl)piperidine hydrochloride (150 mg,

0.62 mmol) to afford (160 mg, 63%) of compound as a white solid.

Mp 243 ^ꢁC. Anal. (C₂₁H₁₈FN₃O₅.1.5H₂O) C, H, N. ¹H NMR (DMSO-d₆)

d

11.60 (s, 1H), 10.81 (s, 1H), 8.14e8.07 (m, 2H), 7.68 (d, J₄¼ 2.0 Hz,

N. ¹H NMR (CDCl₃)

d

10.42 (s, 1H), 8.11 (d, J₃¼ 8.3 Hz, 1H), 7.69 (m,

1H), 7.42e7.30 (m, 2H), 7.06 (d, J₃¼ 8.4 Hz, 1H), 4.41e4.28 (m, 1H),

3.90e3.73 (m, 2H), 3.40e3.24 (m, 1H), 3.04e2.90 (m, 1H), 1.96e1.78

2H), 7.55 (dd, J₃¼ 8.3 Hz, J₄¼ 1.8 Hz, 1H), 7.45 (bs, 1H), 7.28 (m, 1H),

7.20 (d, J₄¼ 2.1 Hz, 1H), 5.04 (s, 2H), 3.91 (s, 3H), 3.63e3.19 (m, 4H),

(m, 2H), 1.67e1.44 (m, 2H). ¹³C NMR (DMSO-d₆)

d 200.2, 162.4,

2.79 (d, J₃¼ 6.8 Hz, 2H), 2.17e1.85 (m, 3H), 1.80e1.55 (m, 2H). ¹³

C

161.9, 160.3 (d, J₁¼ 240.3 Hz), 154.2, 143.1, 132.5, 132.0, 131.3 (d,

NMR (DMSO-d₆)

d 189.9, 148.3, 146.0, 138.0, 134.4, 132.5, 131.6,

J₃¼ 9.4 Hz), 127.4, 116.0, 115.5 (d, J₂¼ 22.0 Hz), 109.6, 102.1, 45.1,

130.1, 125.4, 121.6, 120.4, 115.6, 107.9, 95.7, 56.3, 53.8, 49.5, 41.3,

34.4, 29.0. HRMS (m/z): [M þ H]^þcalcd for C₂₂H₂₅N₄O₄, 409.1876;

found, 409.1866.

42.0, 36.4, 28.4, 27.7. ¹⁹F NMR (DMSO-d₆)

d

ꢃ106.33. HRMS (m/z):

[M þ H]^þcalcd for C₂₁H₁₉FN₃O₅, 412.1309; found, 412.1317.

4.3.11. tert-Butyl-1-[4-(4-nitrobenzoyl)piperidin-1-yl]carboxylate

(21)

Di-tert-butyl dicarbonate (230 mg, 1.05 mmol) in solution in

DMF (2 mL) was added to a solution of 4-(4-nitrobenzoyl)

4.3.7. [1-(5-Methoxybenzimidazol-2-ylmethyl)piperidin-4-yl]-(4-

nitrophenyl)methanone (17)

Under nitrogen atmosphere, compound 4-(4-nitrobenzoyl)

piperidine (150 mg, 0.55 mmol) and 1-chloromethyl-5-methox-

ybenzimidazole (129 mg, 0.55 mmol) were dissolved in dry

acetonitrile (25 mL) and sodium carbonate (235 mg, 2.22 mmol)

was added. The reaction mixture was heated at 45 ^ꢁC for 72 h,

cooled to room temperature, quenched with water (50 mL) and

extracted with CH₂Cl₂(3 ꢂ 50 mL). The combined organic layers

were dried over MgSO₄, ﬁltered, concentrated and puriﬁed by

chromatography on silica gel EtOAc/MeOH/NH₄OH (98:2:0.1) to

afford the labeling precursor 17 (168 mg, 77%) as a yellow solid. Mp

150e152 ^ꢁC. Anal. (C₂₁H₂₂N₄O₄.0.5H₂O) C, H, N. ¹H NMR (CDCl₃)

piperidine (380 mg, 1 mmol) and TEA (272 mL, 2 mmol) in DMF

(3 mL) and stirred for 3 days at room temperature. After evapo-

ration of DMF under vacuum, water was added (5 mL) and

extraction was achieved with diethyl ether (2 ꢂ 25 mL). The

organic layer were combined and dried over MgSO₄, ﬁltered and

concentrated and puriﬁed by chromatography on silica gel with

CH₂Cl₂/MeOH/NH₄OH (98:2:0.1) to afford (247 mg, 74%) of 21 as

a light yellow solid. Mp 141 ^ꢁC. Anal. (C₁₇H₂₂N₂O₅) C, H, N. ¹H

NMR (CDCl₃)

d

8.24 (d, J₂¼ 8.9 Hz, 2H), 8.02 (d, J₂¼ 8.9 Hz, 2H),

4.12e4.07 (m, 2H), 3.37e3.30 (m, 1H), 2.85 (t, J₂¼ 12.1 Hz, 2H),

R. Labas et al. / European Journal of Medicinal Chemistry 46 (2011) 2295e2309

2307

1.81e1.77 (m, 2H), 1.65e1.61 (m, 2H), 1.39 (s, 9H). ¹³C NMR

(CDCl₃) 200.6, 154.7, 150.3, 140.5, 129.3, 124.0, 79.8, 44.1, 43.1,

puriﬁcation occurred on a HPLC column (

m

Bondapack Waters,

d

300

ꢂ

7.8 mm) at 3

mL min^ꢃ1with H₂O/acetonitrile/TFA

28.45, 28.2. HRMS (m/z): [M

335.1607; found, 335.1592.

þ

H]^þcalcd for C₁₇H₂₃N₂O₅,

(60:40:0.1) as eluent and the collected fraction containing [¹⁸F]1a

(t_R¼ 26 min) was diluted with water (1 vol). The solution was

passed through two SPE C₁₈cartridges (Chromaﬁx Shorty 20 mg,

4.4. Radiosynthesis

MachereyeNagel). [¹⁸F]1a was eluted with EtOH (200

m

L) and

diluted with saline (>2 mL). Radiochemical purity was assayed by

4.4.1. Radiosynthesis of [¹⁸F]2a

analytical HPLC using a Luna C18 column (Phenomenex,

Cyclotron-produced [¹⁸F]ﬂuoride ion in [¹⁸O]water was passed

4.6 ꢂ 250 mm, 1 mL min^ꢃ1) with H₂O/acetonitrile/TFA as eluent

through an exchange resin (QMA light, Waters, ABX) eluted with

(70:30:0.1) and radio-TLC on silica gel (Merck; CH₂Cl₂/MeOH/

aqueous K₂CO₃(300

m

L, 2 mg mL^ꢃ1) and acetonitrile (300

mL). The

NH₄OH (90:10:0.1)). The radiochemical purities of [

¹⁸F]1a

aqueous [¹⁸F]ﬂuoride solution was collected into a conical Reac-

(t_R¼ 18.5 min) were greater than 98%. Speciﬁc activity was

tivial^Òcontaining Kryptoﬁx 2.2.2. (22 mg) and K₂CO₃(7 mg) dis-

measured from the puriﬁcation chromatogram.

solved in a water:acetonitrile mixture (600 mL, 1:1, v:v). The water

was removed azeotropically with acetonitrile (3 ꢂ 1 mL) under

4.4.3. Radiosynthesis of [¹⁸F]6

a stream of nitrogen at 105 ^ꢁC. A solution of the labeling precursor

[

¹⁸F]22 was prepared following the same procedure as described

17 (5 mg, 12.7 _þ

m

mol) dissolved in DMSO (500

m

L) was added to the

for [¹⁸F]18 and [¹⁸F]6 was obtained following the procedure

described for [¹⁸F]1a. The HPLC puriﬁcation of [¹⁸F]6 (t_R¼ 17 min)

was achieved using a C18 XTerra column (250 ꢂ 10 mm, Waters,

4 mL min^ꢃ1) using H₂O/acetonitrile/TFA (80:20:0.1) as eluent.

Radiochemical purity was assayed by analytical HPLC using

a Nucleodur C18 column (C18 Gravity, Phenomenex, 4.6 ꢂ 250 mm,

1 mL min^ꢃ1) with H₂O/acetonitrile/TFA as eluent (75:25:0.1) and

radio-TLC on silica gel (Merck; CH₂Cl₂/MeOH/cyclohexane/NH₄OH

(68:15:15:2)). The radiochemical purities of [¹⁸F]6 (t_R¼ 8.9 min)

was greater than 98%.

dried [K/K₂₂₂

]

¹⁸F^ꢃcomplex and the sealed reaction vial was

heated at 140 ^ꢁC for 15 min. The reaction mixture was diluted with

water (6 mL) and passed through a SPE C₁₈cartridge (Waters). The

cartridge was washed with water (6 mL) and eluted by acetonitrile

or dichloromethane (2 mL) in a conical Reactivial^Òyielding 24 ꢀ 5%

(n ¼ 15) of the labeled compound [¹⁸F]18 with a radiochemical

purity greater than 90%. After evaporation of the solvent followed

by azeotropic distillation of residual water with acetonitrile

(2 ꢂ 1 mL), triﬂic acid (300

Then triethylsilane (800 L) was added dropwise to the solution

and the mixture was vigorously stirred at 105 ^ꢁC for 5 min affording

¹⁸F]7a. The reactants were evaporated under nitrogen ﬂow at

105 ^ꢁC for 5 min. HBr (500 L, 48%) was added and heated at 160 ^ꢁC

mL) was added to the reaction vessel.

m

4.5. HEK293 cells culture and plasmid transfection

[

m

CDNAs encoding the full length sequences of NR1-1a and NR2B-

GFP were a generous gift from Dr. Stefano Vicini, (Georgetown

University Medical Center, Washington). Human embryonic kidney

293 cells (HEK 293) were grown in Dulbecco’s medium (Invitrogen)

for 15 min leading to [¹⁸F]2a. NaOH solution (6 mL, 6 N) was added

and the solution was passed through a SPE C₁₈cartridge (Waters).

The cartridge was washed with water (6 mL) and the product was

eluted with methanol (2 mL). The puriﬁcation occurred on a HPLC

column (XTerra C18, Waters, 250 ꢂ 10 mm) at 6 mL min^ꢃ1with

H₂O/acetonitrile/TEA (74:26:0.01) as eluent and the collected

fraction containing [¹⁸F]2a (t_R¼ 28 min) was diluted with water.

The solution was passed through two SPE C₁₈cartridges (Chromaﬁx

Shorty 20 mg, MachereyeNagel). [¹⁸F]2a was eluted with EtOH

supplemented with 1% L-glutamine, 1% streptomycin/penicillin/

neomycin (SigmaeAldrich) and 10% fetal bovine serum (Invi-

trogen). Exponentially growing cells were plated on 12 mm glass

coverslips (Biospace, MatTek Corporation) coated and grown at

37 ^ꢁC in a humidiﬁed atmosphere containing 5% CO2. Twenty four

hours after plating, cells were transfected with cDNA (3

Transfast Reagent (7.5 L, Promega). Co-transfections of NR1-1a

and NR2A-GFP or NR2B-GFP were done with a ratio 1:1. Addi-

tionally, upon transfection, cells were treated with D-APV (100 M)

mg) using

(200

mL) and diluted with saline (>2 mL). Speciﬁc radioactivity and

m

radiochemical purity were assayed by analytical HPLC using a Luna

C18 column (Phenomenex, 4.6 ꢂ 250 mm, 1 mL min^ꢃ1) with

acetonitrile/H₂O/TEA as eluent (50:50:0.01) and radio thin layer

chromatography (TLC) on silica gel (Merck; CH₂Cl₂/MeOH/NH₄OH

(90:10:0.1)). The radiochemical purities of [¹⁸F]2a (t_R¼ 6.6 min)

were greater than 99%.

m

to prevent cells death. Studies of the recombinant receptors were

performed 24 h after transfection.

4.6. Calcium inﬂux assay

4.4.2. Radiosynthesis of [¹⁸F]1a

HEK293 NR2B-GFP/NR1-1a or NR2A-GFP/1-1a transfected cells

[

¹⁸F]20 was prepared following a modiﬁed procedure as

were loaded for 45 min at 37 ^ꢁC with Fura-2/AM (10

mM) and 0.2%

described for [¹⁸F]18. The eluting solution for the QMA cartridge

was changed for a mixture of acetonitrile (350 L) and potassium

oxalate solution in water (350

L, 9.5 mg mL^ꢃ1). The aqueous

¹⁸F]ﬂuoride solution was collected into a conical Reactivial^Ò

containing Kryptoﬁx 2.2.2. (22 mg), potassium oxalate (14 mg)

and K₂CO₃solution (44 L, 1 mg/mL). After radioﬂuorination, the

pluronic acid (F-127, Invitrogen) and incubated for an additional

15 min at room temperature in HEPES and Bicarbonate-Buffered

Saline Solution (HBBSS) containing: NaCl (116 mM); KCl (5.4 mM);

CaCl₂(1.8 mM); MgSO₄(0.8 mM); NaH₂PO₄(1.3 mM); HEPES

(12 mM); glucose (5.5 mM); bicarbonate (25 mM) and glycine

m

[

m

(10 mM) at pH 7.45. Experiments were performed at room

SPE C₁₈cartridge (Waters) was eluted by acetonitrile (2 mL). After

evaporation of the solvent followed by azeotropic distillation of

residual water with acetonitrile (2 ꢂ 1 mL), methanesulfonic acid

temperature with continuous perfusion at 2 mL/min with a peri-

staltic pump, on the stage of a Nikon Eclipse inverted microscope

equipped with a 100 W mercury lamp and oil immersion Nikon

40ꢂ objective with a 1.4 numerical aperture. Fura-2 (excitation:

340/380 nm, emission: 510 nm) ratio images were acquired every

2 s with a digital camera (Princeton Instruments) using Metaﬂuor

6.3 (Universal Imaging Corporation). Measurements were per-

formed on HEK cell bodies by using ampliﬁcation rates that prevent

saturation of the ﬂuorescence signal and a preliminary calibration

of the Ca^2þmeasurements. Fluorescence ratios (340/380 nm) were

(300

(800

mL) was added to the reaction vessel. Then triethylsilane

m

L) was added dropwise to the solution and the mixture was

vigorously stirred using a conical magnetic stirrer at RT for

15 min affording [¹⁸F]1a. Water (3 mL) was added and the solu-

tion was passed through a SPE cartridge (C₁₈plus, Waters). The

cartridge was washed with water (5 mL) and the product was

eluted with acetonitrile (1 mL) and methanol (1 mL). The

2308

R. Labas et al. / European Journal of Medicinal Chemistry 46 (2011) 2295e2309

converted to intracellular Ca^2þconcentrations using the following

formula: [Ca^2þ]_i¼ K_d[(R ꢃ R_min)/(R_maxꢃ R)] F0/Fs, where R is the

ratio for observed 340/380 ﬂuorescence ratio, R_minis a ratio for

a Ca^2þ-free solution, R_maxis the ratio for a saturated Ca^2þsolution,

K_d¼ 135 nM (the dissociation constant for Fura-2), F0 is the

intensity of a Ca^2þ-free solution at 380 nm, and Fs is the intensity of

a saturated Ca^2þsolution at 380 nm. Ligands were applied 60 s

4.9. Radiometabolite study in plasma

Male SpragueeDawley rats (235e330 g, C.E.R.J., France) were

injected with radiotracer (3.2e15.1 MBq) under anesthesia.

Successive blood samples were taken from the femoral artery during

90 min. Plasma samples (5

Cobra for measuring the radioactivity. Another aliquot of plasma

(20 L) was diluted with acetonitrile (20 L) and vortexed. After

mL) were placed in the g-counter Packard

prior and during agonist exposure. Application of NMDA (100

mM)

m

induced a rapid and sustained increase in [Ca^2þ]_i. Following a 5 min

rinse period, increasing concentrations of ligands were applied. The

integrated area under the curve (in Arbitrary Units) for changes in

[Ca^2þ]_iwas calculated on a 2-min period after the beginning of drug

application. The two control responses were averaged and used to

calculate the percentage of inhibition induced by ligands. The IC₅₀

values were calculated with GraphPad software using a log(i-

nhibitor) vs. response model curve.

centrifugation, the liquid supernatant was deposed on a silica gel

TLC (Merck) and co-eluted with the corresponding non-radioactive

reference. Elution phase was CH₂Cl₂/MeOH/NH₄OH (90:10:0.1) for

[

¹⁸F]1a and [¹⁸F]2a, CH₂Cl₂/MeOH/cyclohexane/NH₄OH (68:15:15:2)

for 6. The composition of radioactivity in plasma, in term of

percentage of radioligand and radiometabolites was calculated from

the acquired data.

4.10. Metabolite assessment in plasma and urine after injection of 6

4.7. In vitro stability of ligands

Two male SpragueeDawley rats were placed under anesthesia

and injected through the tail vein with 6 (10 mg/kg) dissolved in

sterile water. Controls were obtained from two rats injected with

sterile water (1 mL/kg). Rats were maintained awake for 1 h and

re-anesthetized before euthanasia by decapitation. Plasma and

Rat plasma and liver homogenate samples (0.450 mmL) were

spiked (50 mL) with different amounts (5e200 mg/L) of the studied

ligands (1a, 2a and 6). Samples were deproteinized with acetoni-

trile (1 mL) immediately or after incubation at 37 ^ꢁC for 30 min.

After mixing and centrifugation, supernatant (0.9 mL) was removed

and dried with a SpeedVac system (Thermo Fisher Scientiﬁc,

Courtaboeuf, France). Residue was dissolved in mobile phase

(0.3 mL) (acetonitrile/H₂O/triethylamine, 53:47:0.1 for 1a; aceto-

nitrile/H₂O/TFA, 75:25:0.1 for 2a; acetonitrile/H₂O/TFA, 40:60:0.1

urine were collected. Acetonitrile (800

samples (400 L), mixed and centrifuged at 2000g for 10 min.

Supernatants (900 L) were evaporated in a SpeedVac apparatus

mL) was added to biological

m

(Thermo Fisher Scientiﬁc, Courtaboeuf, France) and stored

at ꢃ20 ^ꢁC until liquid chromatographyemass spectrometry

(LCeMS) analysis. Before injection (5 mL) in the Thermo electron

for 6) and an aliquot (10

system (Column Phenomenex Luna C18, 5

Column MachereyeNagel Nucleodur C18 gravity,

4.6 ꢂ 250 mm for 2a, Column Waters Bondapack C18, 10

mL) was injected in triplicate in the HPLC

corporation C18 column (0.5 ꢂ 50 mm, 5

solved in mobile phase (300 L) (acetonitrile/H₂O/acetic acid,

20:20:0.1) and brieﬂy centrifuged. Mass spectrum analysis was

performed with liquid chromatographic system Surveyor

mm), residue was dis-

m

m, 4.6 ꢂ 250 mm for 1a;

m

5

m

m,

m

a

3.9 ꢂ 300 mm for 6). Detection wavelength was 254 nm. Chro-

matogram peak area were integrated with the Millenium software

(Waters, Saint-Quentin en Yvelines, France) and compared with

water spiked samples. The effect of matrix and incubation were

statically analyzed with the SigmaStat software (Jandel Scientiﬁc,

Chicago, USA).

(Thermo Electron SAS, Les Ulis, France) coupled on-line to

a Thermoﬁnnigan Deca XP MAX orthogonal electrospray source

(Thermo Fisher Scientiﬁc, Courtaboeuf, France) operating in the

positive electrospray ionization mode. The mass spectra were

acquired during 35 ms from 100 to 1000 m/z in MS and MS/MS

mode. The capillary exit of the electrospray ion source was set at

70 V, the octopole at 3 V and the capillary temperature was at

200 ^ꢁC. The spray gas was nitrogen. MS data were acquired in scan

mode considering the positive ion signal using a Xcalibur system

and a turbo Sequest data system.

4.8. PET imaging experiments and biodistribution studies

Male SpragueeDawley rats (235e310 g; C.E.R.J., France) were

induced with isoﬂurane 5% and maintained under anesthesia with

isoﬂurane 2.5e1.5% in oxygen and nitrous oxide (30/70) for the

duration of the experiment. Rats were positioned in an Inveon^Ò

Acknowledgments

Romain Labas received a bursary from the CEA and the Conseil

Régional de Basse-Normandie. Gwénaëlle Gilbert was ﬁnanced by

the Ministère de l’Enseignement Supérieur et de la Recherche. The

authors thank Eric T. MacKenzie for the proof-reading of the

manuscript.

mPET (Siemens, Saint-Denis, France) with brain centered in the FOV

(Field Of View). A transmission scan of 10 min using a ⁵⁷Cobalt

source was performed before tracer injection to provide attenua-

tion correction. [¹⁸F]1a, [¹⁸F]2a or [¹⁸F]6 (3.2e18.6 MBq) were

injected via a catheter in the tail vein to animals (n ¼ 3 for each

radioligand excepted n ¼ 2 for [¹⁸F]6). PET dynamic images were

reconstructed from 90 or 120 min emission scans with an OSEM 2D

algorithm using the Inveon Acquisition Workplace software

(version 1.2.2.2; Siemens Medical Solution, Knoxville, USA). Decay

corrected time-activity curves (TAC) were obtained for ﬁve ROI

(Region of Interest) selected in the upper part of rat body: brain,

clavicle bone, wrist articulation, liver and muscle. Radioactivity

data were injected dose- and bodyweight- normalized by expres-

sion of SUV (Standardized Uptake Value).

Appendix. Supplementary material

Supplementary data related to this article can be found online at

doi:10.1016/j.ejmech.20 1 1 .03.013.

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Synthesis, evaluation and metabolic studies of radiotracers containing a 4-(4-[18F]-fluorobenzyl)piperidin-1-yl moiety for the PET imaging of NR2B NMDA receptors

DOI: 10.1016/j.ejmech.2011.03.013

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