N-(4-[18F]-fluoropyridin-2-yl)-N-{2-[4-(2-methoxyphenyl) piperazin-1-yl]ethyl}carboxamides as analogs of WAY100635. New PET tracers of serotonin 5-HT1A receptors

Article abstract of DOI:10.1016/j.ejmech.2014.07.096

N-(4-[¹⁸F]-Fluoropyridin-2-yl)-N-{2-[4-(2-methoxyphenyl) piperazin-1-yl]ethyl}-carboxamides were prepared by labeling their 4-nitropyridin-2-yl precursors through nitro substitution by the ¹⁸F anion. In vitro and in vivo tests showed

Full text of DOI:10.1016/j.ejmech.2014.07.096

European Journal of Medicinal Chemistry 85 (2014) 795e806
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
N-(4-[18F]-fluoropyridin-2-yl)-N-{2-[4-(2-methoxyphenyl)
piperazin-1-yl]ethyl}carboxamides as analogs of WAY100635.
New PET tracers of serotonin 5-HT1A receptors
a
a
a
a, *, 1
ꢀ
ꢀ
Gonzalo García , Valentina Abet , Ramon Alajarín , Julio Alvarez-Builla
,
b
b
b
~
Mercedes Delgado , Luis García-García , Pablo Bascunana-Almarcha ,
Carmen Pena-Salcedo , James Kelly , Miguel A. Pozo
c
c
b, c, **, 1
~
a
ꢀ
ꢀ
ꢀ
Departamento de Química Organica, Universidad de Alcala, Alcala de Henares, Madrid 28871, Spain
b CAI Cartografía Cerebral, Instituto Pluridisciplinar UCM, Paseo Juan XXIII, 1, Madrid 28040, Spain
c
ꢀ
ꢀ
Instituto Tecnologico PET, Calle Manuel Bartolome Cossío 10, Madrid 28040, Spain
a r t i c l e i n f o
a b s t r a c t
N-(4-[18F]-Fluoropyridin-2-yl)-N-{2-[4-(2-methoxyphenyl)piperazin-1-yl]ethyl}-carboxamides
were
Article history:
prepared by labeling their 4-nitropyridin-2-yl precursors through nitro substitution by the 18F anion.
In vitro and in vivo tests showed that the cyclohexanecarboxamide derivative is a reversible, selective and
high affinity 5-HT1A receptor antagonist (IC50 ¼ 0.29 nM, ki ¼ 0.18 nM) with high brain uptake, slow brain
clearance and stability to defluorination when compared with conventional standards. This PET radio-
ligand is a promising candidate for an improved in vivo quantification of 5-HT1A receptors in neuro-
psychiatric disorders.
Received 28 March 2014
Received in revised form
24 July 2014
Accepted 25 July 2014
Available online 26 July 2014
Keywords:
5-HT1A
© 2014 Published by Elsevier Masson SAS.
Positron emission tomography
Fluorine-18
Radioligand
N-(4-[18F]-fluoropyridin-2-yl)-N-{2-[4-(2-
methoxyphenyl)piperazin-1-yl]ethyl}
carboxamides
1. Introduction
Abbreviations: PET, positron emission tomography; 5-HT1A, receptor 1A of 5-
hydroxytryptamine; 5-HT, 5-hydroxytryptamine; WAY100635, N-{2-[4-(2-
methoxyphenyl)piperazin-1-yl]ethyl}-N-(pyridin-2-yl)cyclohexanecarboxamide;
MPPF, 4-fluoro-N-{2-[1-(2-methoxyphenyl)-piperazin-1-yl]ethyl}-N-(pyridin-2-yl)
benzamide; kD, apparent equilibrium dissociation constant; WAY100634, N-{2-[4-
(2-methoxyphenyl)piperazin-1-yl]ethyl}pyridin-2-amine; 5-HT2B, receptor 2BA of
5-hydroxytryptamine; D4,4, receptor 4,4 of dopamine; SPECT, single-photon emis-
sion computed tomography; EtOH, ethyl alcohol; MW, microwaves; dba, tris(di-
Molecular imaging techniques such as PET (Positron Emission
Tomography) are useful tools for translational neuroscience from
animal models to man. Observations carried out by this technique
have suggested that there is a relationship between the serotonin
5-HT1A receptor and human cognition by the determination of al-
terations in receptor binding in patients through the imaging of
receptor densities [1]. The neurotransmitter serotonin (5-
hydroxytryptamine, 5-HT) and its receptors have been shown to
be involved in the pathophysiology of psychiatric diseases such as
schizophrenia [2], depression [3], mood disorders [4e7], and
neurodegenerative processes such as Alzheimer's and Parkinson's
diseases [8,9]. The 5-HT1A receptor has therefore been proposed as
a target for the development of cognitive enhancers in the treat-
ment of numerous cognitive dysfunction-related disorders.
benzylideneacetone)dipalladium(0);
BINAP,
2,20-bis(diphenylphosphino)-1,10-
binaphthyl; IPrHCl, 1,3-bis(2,4,6-trimethylphenyl)imidazolium chloride; t-BuONa,
sodium butoxide; CsOAc, cesium acetate; DMSO, dimethyl sulfoxide; Chx, cyclo-
hexyl; 1-Ad, adamant-1-yl; 2-py, pyridin-2-yl; THF, tetrahydrofuran; DMF, N,N-
dimethylformamide; HPLC, high-performance liquid chromatography; Ph, phenyl;
IC50, concentration producing 50% inhibition; ki, inhibition constant; EOB, end-of-
bombardment; 8-OH-DPAT, 2-(dipropylamino)-8-hydroxytetraline; BBB, bloode-
brain barrier; BP, binding potential; ID, injected dose.
* Corresponding author.
** Corresponding author. CAI Cartografía Cerebral, Instituto Pluridisciplinar UCM,
Paseo Juan XXIII, 1, Madrid 28040, Spain.
An array of PET tracers has been developed for the imaging of 5-
HT1A receptors (Fig. 1) [1,10e12]. Two antagonists have been prin-
cipally studied in human diseases, namely [carbonyl-11C]
ꢀ
E-mail addresses: julio.alvarez@uah.es (J. Alvarez-Builla), pozo@med.ucm.es
(M.A. Pozo).
1
These authors contributed equally.
http://dx.doi.org/10.1016/j.ejmech.2014.07.096
0223-5234/© 2014 Published by Elsevier Masson SAS.

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G. García et al. / European Journal of Medicinal Chemistry 85 (2014) 795e806
Fig. 1. Evolution of structures of PET radiotracers of 5-HT1A receptors, key metabolite WAY100634 and radiolabeling methods: (a) carbonyl labeling; (b) bulky acyl group; (c)
fluorine labeling on the cyclohexane ring; (d) fluorine labeling on the aromatic acyl group; (e) halogen on the pyridine C6 atom; (f) fluorine labeling on the pyridine C6 atom; (g)
iodine labeling on the bulky acyl group.
WAY100635 and [18F]MPPF [13,14]. WAY100635 is potent, selective
and shows high affinity (kD ¼ 0.2 nM) [15]. Recent in vitro studies
showed that WAY100635 and its major metabolite WAY100634
also have affinity for other receptors such as 5-HT2B and D4,4 [16],
although this finding has not subsequently been confirmed [17]. In
addition, radiolabeling with carbon-11, which has a very short half
life, is a challenge to radiochemical yields, purities and specific
activities [18]. [18F]MPPF is a selective antagonist (kD ¼ 0.34 nM)
with a longer half-life, but it shows low brain uptake compared to
[carbonyl-11C]WAY100635 (0.05% and 0.45% ID/g at 30 min in rats,
respectively) and rapid metabolism in vivo [14,19]. In spite of its
drawbacks, [carbonyl-11C]WAY100635 remains the ‘gold standard’
for PET imaging of the 5-HT1A receptor [20,21]. However, [18F]MPPF
has the advantage of being simpler to synthesize and the longer
half-life of 18F allows for distribution to remote facilities that do not
have an in-house cyclotron. The synthesis of this compound is
based on the displacement of a nitro group on a benzene ring by a
weak nucleophile such as the 18F anion, a process that suffers from
low efficiency. However, the efficiency can be substantially
improved by changing the position of the leaving group e the nitro
group in this case e on the molecule.
Numerous modifications to the structures of WAY100635 and
MPPF have been reported as part of the development of new PET
and SPECT (Single-Photon Emission Computed Tomography)
radiolabeled ligands for 5-HT1A with improved specificity [22],
in vivo kinetics [22e26] and metabolic stability [27e33] (Fig. 1).
In spite of the improved parameters, the aforementioned tracers
also suffer from poor radiosynthetic yields [22,23,34], low brain
uptake [33], extensive in vivo defluorination [32,35] and hydro-
lysis of the amide bond [36,37]. There is therefore a need for new
5-HT1A radiotracers that combine an optimal radiosynthesis
process with improved pharmacological profiles and imaging
properties.
We report here a series of novel analogs of WAY100634 and
MPPF labeled with fluorine-18 for the quantification of 5-HT1A re-
ceptors by PET imaging. These tracers all bear a [18F]fluorine label
on the pyridine C4 atom, which a priori would allow an improve-
ment in radiolabeling efficiency, and they possess different
aliphatic (cyclohexyl, adamant-1-yl), aromatic (phenyl) and heter-
oaromatic (pyridine-2-yl) radicals at the acyl group. The chemical
synthesis of the precursors is described and we report the first
radiosynthesis of these derivatives. The results of in vitro and in vivo
assays of binding specificity and kinetics are also reported. One
analog, the 4-[18F]fluoropyridine derivative of WAY100635, was
found to be a potent 5-HT antagonist and it showed good selectivity
for 5-HT1A receptors, higher brain uptake and slower washout than
[
18F]MPPF, a commonly used fluorine-18 PET tracer for 5-HT1A
.
2. Chemistry
The synthetic route for the precursors was designed in order
to build molecules with a 4-nitropyridin-2-yl moiety on which
the fluorination step would subsequently be carried out (Scheme
1). Commercially available piperazine 1 was transformed into 2
and 3 in 98% yield using modifications of previously reported
methods [38]. 2-Chloro-4-nitropyridine-1-oxide (4) was used to
link amine 3 by nucleophilic substitution to give pyridine N-ox-
ide 5. Compound 5 was the only reaction product but it was
obtained in low yield (31%) in spite of the harsh conditions
applied (1.5 equiv of 4, EtOH, 100 ꢀC, 1 h, microwaves). The yield
was not improved by carrying out the reaction in the presence of
a
stoichiometric
amount
of
tertiary
amine

G. García et al. / European Journal of Medicinal Chemistry 85 (2014) 795e806
797
and 21%, respectively) and disubstitution product 8 (8%) was also
formed.
i
N-Oxide 5 was deoxygenated with PCl3 to yield pyridine 7
quantitatively. Subsequent acylation of 7 gave precursors 9. For the
benzoyl (9a) and cyclohexanecarbonyl (9b) derivatives, acylation
was carried out at room temperature in dichloromethane (Method
A, 95% and 92% yield, respectively). For the bulky 1-
adamantanecarbonyl derivative (9c) it was necessary to use
toluene under reflux (Method B, 60%). The picolinoyl derivative
(9d) was obtained from picolinoyl chloride, prepared in situ using
oxalyl chloride in dichloromethane, with heating in toluene
(Method C, 70%). All of the desired precursors 9aed were suc-
cessfully prepared in low overall yields (20e30%) from commer-
cially available 1.
1
2
ii
3
iv
iii
v
Cold fluorination experiments were performed in order to
obtain standard samples of compounds 10 for the characterization
of their radiolabeled counterparts and to provide samples for
binding assays. Experiments were carried out by conventional and
microwave-accelerated heating. The preparation of 4-
fluoropyridine derivatives 10 was optimized by microwave-
assisted synthesis (9aec, 100 W; 9d, 140 ꢀC) using an excess of
CsF (9a, b, d, 5 equiv; 9c, 10 equiv) in DMSO (9aec, 0.02 M; 9d,
0.004 M) for 5 min (10b, 52%; 10c, 70%), 3 min (10d, 62%) and 2 min
(10a, 40%). The microwave-assisted procedure was up to 2-times
faster and the optimal power for 10aec was 100 Watts, although
a larger excess of fluoride was necessary for 10c in order to achieve
good conversion. For 10d, temperature-controlled rather than
power-controlled conditions were required to obtain good yields.
The reaction was also optimized by classical heating (180 ꢀC) using
5 equivalents of CsF in DMSO (9aed, 0.02 M) for 10 min (10b, 59%),
5
7
vi
a: R = Ph
b: R = Chx
c: R = 1-Ad
d: R = 2-Py
vii
9
viii
18
10
[
18F]10
8
Scheme 1. Synthesis of precursors, cold derivatives and [18F]-labeled derivatives. Re-
agents and conditions: (i) Bromoacetonitrile, K2CO3, cat NaI, acetone, reflux; (ii) LiAlH4,
THF, reflux; (iii) 2-chloro-4-nitropyridine-1-oxide (4), EtOH, MW, 100 ꢀC; (iv) 2-chloro-
4-nitropyridine (6), t-BuONa, Pd2(dba)3, BINAP, toluene, 80 ꢀC; (v) PCl3, CH2Cl2, rt; (vi)
Method A (9a, 9b): RCOCl, CH2Cl2, rt; Method B (9c): RCOCl, toluene, reflux; Method C
(9d): a. RCOOH, oxalyl chloride, cat DMF, CH2Cl2, rt; b. 7, toluene, 80 ꢀC; (vii) CsF,
DMSO, 180 ꢀC; (viii) [kryptofix 2.2.2. K][18F]F, DMSO, 180e200 ꢀC.
10a
7
11
12
b
R = Ph
(ethyldiisopropylamine) vs 3. When the reaction was carried out
under reflux, poorer results were obtained (4 h, 23%; 20 h, 17%)
even though a 2-fold excess of amine 3 was used (6 h, 26%).
Microwave-assisted conditions accelerated the process 6-fold
(1 h MW vs 6 h reflux) but only a slight improvement in the
yield was achieved (31% MW vs 26% reflux). These results were
unexpected since the reaction of 2-bromo-4-nitropyridine-1-
oxide was previously reported to give moderate to good yields
on using methylamine (3 equiv, 73%), aniline and cyclohexyl-
amine (2 equiv, 56% and 69%, respectively) [39,40]. Steric hin-
9a-d
a
drance caused by the bulky substituent at the
b-carbon to the
nucleophilic amine is the most likely explanation for the low
yield obtained. Pd-catalyzed CeN cross-coupling reactions were
also unsuccessfully evaluated. Although this reaction has not
been reported to date for N-oxides, we tested the reactivity of N-
7
10a-d
oxide
4 by conventional heating but found that the yield
diminished. The reaction of 4 with 3 catalyzed by CuI or
Pd2(dba)3, in the presence of BINAP or IPrHCl as ligands and t-
BuONa or CsOAc as bases, gave lower yields of 5 when the pro-
cess was carried out in DMSO (90 ꢀC, 9%), toluene (80 ꢀC, 21%) or
1,4-dioxane (100 ꢀC, 14%) for 24 h [41e43]. The CeN cross-
coupling reaction with 2-chloro-4-nitropyridine (6) using Buch-
wald and Nolan procedures [41,43] gave only low yields of 7 (4%
11
Scheme 2. Major by-products formed during cold fluorination of precursors 9aed and
compound 12 formed in the presence of water. Reagents and conditions: (a) CsF
(5 equiv), DMSO, 180 ꢀC, 3e10 min; (b) CsF (5 equiv), H2O (2.8 equiv), DMSO, 180 ꢀC,
5 min.

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G. García et al. / European Journal of Medicinal Chemistry 85 (2014) 795e806
5 min (10a, 43%; 10c, 95%) and 3 min (10d, 58%). Both conventional
and microwave-assisted syntheses gave deacylated starting mate-
rial 7 (ꢁ10%) and deacylated reaction product 11 (ꢁ7%) as major by-
products (Scheme 2). Since the solvent employed was rigorously
dried, amide bond hydrolysis by water was ruled out. In an effort to
shed light on this aspect, compound 9a was reacted under classical
conditions, as described above, in the presence of water (2.8 equiv)
and a ratio of 89:4:3:4 was found for 10a:7:11:12 by HPLC (See
Scheme 2). Compound 12 is therefore formed by the displacement
of either the nitro group or fluoro-substituent by water. Fluoride
ion-mediated hydrolysis is probably the origin of deacylated by-
products 7 and 11, as reported for phosphoroselenoic esters and
amides [44]. In addition, the strong electron-withdrawing effect of
3.2. Radiolabeling
The radiolabeling of precursors 9 was accomplished by nucle-
ophilic aromatic substitution of the nitro group by [18F]fluoride in
DMSO at 180e200 ꢀC (Scheme 1) [48]. The compounds were pu-
rified by semi-preparative HPLC and the structures of labeled
compounds [18F]10 were confirmed by comparison with the cold
standards. Compound [18F]10a was radiolabeled in 25.7 8% yield
(corrected to EOB), while compounds [18F]10bed were synthe-
sized in 27.8 8%, 25.9 5% and 22.7 4% yields, respectively.
Specific activities were in the range 25e500 GBq/mmol. Under the
same conditions [18F]MPPF was radiosynthesized in 23.8
yield.
9%
both the 4-nitro group and the
a-azine-nitrogen make the 4-
The radiolabeling of pyridine moieties with [18F]fluoride ions
has previously been undertaken at the C6 carbon [49,50]. However,
it has been shown that analogs of WAY100635 labeled at this po-
sition are rapidly metabolized [31]. In principle, labeling at the C4
position of the pyridine ring should be less favorable for metabo-
lization and this hypothesis has been borne out by previously re-
ported labeling studies [51]. It was found that fluorination at C4 is
more favorable than labeling in the benzenecarboxamide moiety
([18F]10a: 25.7% vs [18F]MPPF: 23.8%), although the effect was small.
Our radiolabeling procedure represents a significant improvement
on the one reported for 6-[18F]FWAY (~2% decay-corrected yield,
Fig. 1) [31].
nitropyridin-2-yl-amide anion a good leaving group and this
potentially assists the fluorolysis. Attempts have previously been
made to identify impurities formed during the radiolabeling of [18F]
MPPF but, as the structures of the labeled by-products could not be
assigned [45], it was not possible to confirm that this fluorolysis
had taken place during the radiolabeling.
3. Results and discussion
3.1. Receptor binding in vitro
3.1.1. 5-HT1A receptor binding
3.3. In vitro brain autoradiography
Radioligand binding experiments carried out with 10b, which
showed better imaging results both in vitro and in vivo (see below),
were performed in triplicate using the 5-HT1A agonist [3H]8-OH-
DPAT [46]. Inhibition curves were measured and the concentrations
that produced 50% inhibition (IC50) were derived from non-linear
regression curve fitting and apparent equilibrium inhibition con-
stant (ki) values were calculated according to the ChengePrusoff
equation [47]. Compound 10b (IC50: 0.35 nM; ki: 0.22 nM) is an
even better 5-HT1A receptor antagonist than its defluorinated
analog WAY-100635 (IC50: 0.91 nM; ki: 0.39 nM) [32,33].
The binding patterns of [18F]10a (not shown) and [18F]10b
(Fig. 2) were identical to those obtained with [18F]MPPF and [3H]8-
OH-DPAT ligands and correspond to the known 5-HT1A receptor rat
brain distribution [52]. Furthermore, in both cases the specific
3.1.2. Receptor binding profile
The selectivity of 10b for the 5-HT1A receptor over a panel of
seven relevant receptors was determined at a concentration of
0.55 nM and the results are shown in Table 1. Compound 10b
inhibited the 5-HT1A receptor between 2- to 20-fold better than
other receptors tested. The lowest selectivity was found for adre-
noreceptor a1D
.
Table 1
Inhibition of relevant receptors by 10b at 0.55 nM (n ¼ 3).
Receptor
Reference compound
Inhibition (%)
Name
IC50 (nM)
ki (nM)
5-HT1A
5-HT1D
5-HT2B
5-HT7
a1A-A
a1B-A
a1D-A
D4
[3H]8-OH-DPAT
[3H]serotonin
[3H]mesulergine
[3H]serotonin
[3H]WB-4101
[3H]prazosin
0.29
3.8
0.18
1.3
37
6
0.49
0.38
0.46
0.22
0.14
91
0.27
0.14
0.23
0.06
0.06
35
ꢂ21a
6
2
4
21
8
[3H]prazosin
[3H]clozapine
a
Low to moderate negative values have no real meaning and are attributable to
variability of the signal around the control level. High negative values (ꢃ50%)
sometimes obtained with high concentrations of test compounds are generally
attributable to non-specific effects of the test compounds in the assays. On rare
occasion they could suggest an allosteric effect of the test compound.
Fig. 2. In vitro autoradiograms of coronal brain sections of rat incubated with [18F]10b,
[
HIP, hippocampus; OC, occipital cortex; EC, entorhinal cortex; RN, raphe nucleus.
18F]MPPF and [18F]8-OH-DPAT. SEPT, septum; PC, parietal cortex; TC; temporal cortex;

G. García et al. / European Journal of Medicinal Chemistry 85 (2014) 795e806
799
Fig. 3. Time-activity curves for [18F]10a, [18F]10b and [18F]MPPF. PET timeeactivity curves in different structures measured after injecting the different tracers. Data are expressed as
the mean (n ¼ 8) of the average radioactivity (KBq/cc) bound in different structures (prefrontal cortex, septum, hippocampus and cerebellum) at different time points. Legend:
hippocampus (squares), prefrontal cortex (diamonds), septum (circles) and cerebellum (triangles).
signal was abolished when the tracer was incubated in the presence
reports that WAY100635 also shows some in vitro affinity for the 5-
HT2B and dopamine D4,4 receptors [16] and the similarity in
structure between these derivatives and WAY100635, it is possible
that non-target interactions with the dopamine system could
explain the lack of specificity of these compounds for 5-HT1A
receptors.
of 1 mM WAY100635, a well characterized 5-HT1A antagonist. These
results indicate that [18F]10a and [18F]10b show high selectivity for
5-HT1A receptors. However, compounds [18F]10c and [18F]10d
showed high accumulation in the brain regions with poor 5-HT1A
receptor density, such as the striatum and the thalamus. Given the

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G. García et al. / European Journal of Medicinal Chemistry 85 (2014) 795e806
3.4. In vivo brain distribution studies (dynamic PET)
The brain distribution of the radiolabeled compounds was
evaluated by dynamic PET. While compound [18F]10a crossed the
blood brain barrier (BBB), it showed quick washout, as a low signal
was detected in the brain at 15 min post-injection. Furthermore,
uptake in the cerebellum was higher than in the hippocampus and
cortex at 15 min post-injection (Fig. 3). The rest of the compounds
showed good brain retention, but only [18F]10b showed a similar
binding distribution to that observed in animals administered with
[
[
18F]MPPF. As in the in vitro experiments, compounds [18F]10c and
18F]10d bound highly to the striatum, indicating poor selectivity
for 5-HT1A receptors. On the basis of its favorable kinetics and
selectivity for 5-HT1A receptors, [18F]10b was selected for additional
in vivo evaluation.
The dynamic PET images revealed rapid accumulation of [18F]
10b in the brain following intravenous injection. The timeeactivity
curves showed a high uptake in 5-HT1A receptor-rich structures
such as the hippocampus, septum and prefrontal cortex a few mi-
nutes after injection (Fig. 3). Weak binding was observed in the
cerebellum, a region known to have low expression of 5-HT1A re-
ceptors. The average timeeactivity curve for [18F]10b obtained from
8 rats is displayed in Fig. 3. Tracer uptake remains almost constant
from 10 to 40 min post-injection in contrast to the [18F]MPPF
timeeactivity curve, which shows a gradual decrease over time.
One possible explanation for the rapid brain washout of [18F]10a
is that it is a substrate with high affinity for P-glycoprotein. Indeed,
several 5-HT1A receptor radioligands with similar structures, based
on an arylpiperazinyl-alkyl backbone, including [18F]MPPF, are
known to act as substrates for P-glycoprotein at the rat BBB [53,54].
Metabolic instability may be another contributing factor, as a low
uptake of activity in the skull, consistent with in vivo defluorination,
was observed when a 45 min dynamic PET scan was performed.
Tracers [18F]10b, [18F]10c and [18F]10d did not show significant
accumulation of activity in bone, although intense signal uptake in
striatal tissue during the dynamic scan did reinforce the lack of
specificity by [18F]10c and [18F]10d for 5-HT1A receptors observed
in vitro. Washout of [18F]10b from brain regions was quite slow,
with the slope of the timeeactivity curve being small from 20 min
after injection (Fig. 3), but its uptake in known 5-HT1A receptor-rich
regions was similar to that of [18F]MPPF. The uptake ratios of [18F]
10b and [18F]MPPF in several brain regions vs the cerebellum at
45 min is shown in Table 2.
Fig. 4. Representative BP parametric map for [18F]10b. Coronal view (top panel) and
transversal view (bottom panel).
Uptake ratios for [18F]10b and [18F]MPPF are similar in the hip-
pocampus, but [18F]10b shows a higher ratio in the cortex. The
hippocampus-to-cerebellum ratio reaches a maximum between 10
and 12 min for [18F]MPPF (reaching a value of 2.44 0.07), at which
time the corresponding ratio for [18F]10b is 2.34 0.09. While the
ratio for [18F]MPPF begins to diminish after 12 min, the ratio for
The BP values for [18F]10b are significantly higher than those found
for [18F]MPPF (Table 3), with the hippocampus showing the highest
BP value (1.412 0.077 vs 0.872 0.035).
The BP values of [18F]10b taken from the parametric maps ob-
tainedin5-HT1A-richbrainregionsaresignificantlyhigherthanthose
found for [18F]MPPF (Table 3). The highest values were observed in
the hippocampus and the prefrontal cortex and these were 1.62 and
1.71 times higher (P < 0.01), respectively, than the values reported for
[
18F]10b slowly increases before reaching a maximum value of
2.53 0.13 at 18 min. This ratio exceeds the values reported for new
5-HT1A tracers [54].
The binding potential (BP) parametric maps for [18F]10b ob-
tained by using the simplified reference tissue model (Fig. 4)
enabled the BP values in several regions of interest to be calculated.
[
18F]MPPF. The binding potential of [18F]10b in the cerebellum was
elevated with respect to that of [18F]MPPF, possibly reflecting the fact
that [18F]10b binds the few 5-HT1A receptors in this region with
higheraffinity. Collectivelytheseresults demonstrate thatthe affinity
of [18F]10b is higher than that of [18F]MPPF for the 5-HT1A receptor.
Table 2
Uptake ratios at 45 min for [18F]10b and [18F]MPPF in selected brain regions vs
cerebellum.
Brain region
[
18F]10b (n ¼ 8)
[
18F]MPPF (n ¼ 8)
3.5. In vivo competition studies
Hippocampus
Septum
Cortex
1.67 0.13
1.15 0.24
2.04 0.15(*)
1.48 0.06
1.28 0.03
1.60 0.05
The specificity of [18F]10b for 5-HT1A receptors was assessed by
carrying out dynamic PET studies with [18F]10b in adult rats in the
presence of 8-OH-DPAT, WAY100635, or with cold 10b as
Data are presented as a ratio SD of uptake (kBq/cc) in 5-HT1A receptor-rich brain
regions vs cerebellum at 45 min after injection. (*) P < 0.05 (non-paired t-test).

G. García et al. / European Journal of Medicinal Chemistry 85 (2014) 795e806
801
Table 3
18F]10b BP values in several brain regions of interest vs [18F]MPPF.
Table 5
[
Average tissue distribution vs time (in %ID) of [18F]10b.
Brain region
[
18F]10b BP (n ¼ 10)
[
18F]MPPF BP (n ¼ 8)
Tissue
Time (min)
15
Prefrontal cortex
Septum
Caudate putamen
Amygdala
Hippocampus
Thalamus
Cerebellum
Brain stem
1.039 0.082
0.675 0.026
0.470 0.028
0.784 0.051
1.412 0.077
0.373 0.029
0.234 0.017
0.225 0.046
0.664 0.035
0.608 0.036
0.677 0.039
0.386 0.025
0.477 0.023
0.872 0.035
0.404 0.027
0.084 0.004
0.118 0.013
0.362 0.016
30
60a
120
Kidneys
Heart
Spleen
Lungs
0.814 0.187 0.463 0.068 0.285 0.074 0.133 0.012
0.325 0.038 0.153 0.013 0.076 0.012 0.032 0.001
0.303 0.032 0.168 0.003 0.103 0.013 0.049 0.007
0.344 0.057 0.212 0.005 0.118 0.001 0.044 0.008
Muscle (soleus) 0.160 0.005 0.094 0.002 0.061 0.10 0.031 0.000
Retroper. fat 0.327 0.043 0.335 0.042 0.321 0.037 0.271 0.005
Small intestine 7.559 3.478 13.188 0.085 1.443 0.374 0.680 0.201
Liver
Stomach
Whole brain
Whole brain
1.337 0.176 0.716 0.208 0.589 0.002 0.326 0.025
0.567 0.041 0.247 0.081 0.239 0.018 0.174 0.072
0.412 0.051 0.380 0.023 0.303 0.022 0.075 0.021
displacers. Cold 10b was found to induce a dose-dependent
Large intestine 0.243 0.027 0.160 0.004 0.214 0.012 0.845 0.605
decrease in the [18F]MPPF binding potential. The BP values for
[
18F]10b when simultaneously administered with one of the three
aforementioned cold ligands are shown in Table 4. The binding of
[
13.2% ID/g at 30 min), while elevated concentrations were also
observed in the kidneys and liver. These distributions probably
reflect the involvement of these organs in the metabolism and
excretion of the compound. Total activity in the small intestine
continued to accumulate up to 30 min after injection, but in other
tissues the concentration peaked at 15 min post-injection. The
relativelyconstant activity in the brain up to 60 min provides further
evidence of the slow washout of the radiolabeled compound.
18F]10b was almost completely reversed when it was co-
administered with WAY100635 or cold 10b. The BP of [18F]10b in
the hippocampus and prefrontal cortex in these displacement ex-
periments was reduced by 96.25% and 95.38% (P < 0.01), respec-
tively, after administration of the cold ligand 10b (0.5 mg/kg). The
co-administration of the selective antagonist WAY100635 (1 mg/
kg) with [18F]10b decreased hippocampal and cortex binding by
99.15% and 95.77% (P < 0.01), and the hippocampus/cerebellum and
prefrontal cortex/cerebellum ratios from 6.03 to 0.04 and 4.44 to
0.15, respectively. These results are consistent with the reversible
binding of [18F]10b to 5-HT1A receptors.
4. Conclusions
Four new 4-[18F]fluoropyridine-labeled derivatives of
WAY100635 bearing benzoyl ([18F]10b), cyclohexanecarbonyl ([18F]
10b), adamantane-1-carbonyl ([18F]10c) and pyridine-2-carbonyl
([18F]10d) groups were radiosynthesized by nucleophilic displace-
ment of the nitro group of 4-nitropyridine precursors. Yields in the
range 22.7%e27.8% (corrected to EOB) were obtained and [18F]10b
was obtained in the highest yield after a synthesis time of 105 min.
The nitro precursors were prepared in modest yields (20e30%) by a
new approach involving a pyridine-N-oxide intermediate. Cold
fluorination experiments showed that amide fluorolysis is the main
pathway to impurities.
In the case of co-administration with 8-OH-DPAT at a dose of
1 mg/kg, the decreases were 61.05% and 61.02% (P < 0.01) in the
hippocampus and frontal cortex, respectively. The smaller de-
creases observed in these cases are probably due to the lower af-
finity of 8-OH-DPAT for 5-HT1A receptors in comparison to
WAY100635, as demonstrated in pharmacological studies [55,56].
3.6. Biodistribution in rats
The uptake and washout kinetics of [18F]10b in rat were evalu-
ated by carrying out a biodistribution experiment in normal male
rats. Compound [18F]10b displayed a high initial brain uptake
(0.90% ID/g) at just 1 min post-injection. The radioactivity in the
brain displayed a moderate washout rate (0.34% ID/g at 30 min)
with a brain 1 min/brain 30 min ratio of 1.7.
Labeled compounds [18F]10aed were evaluated as 5-HT1A radi-
oligands in vitro and in vivo. Compounds [18F]10a and [18F]10b
showed good in vitro distribution to 5-HT1A-rich regions while [18F]
10c and [18F]10d, in contrast, showed poor selectivity for the 5-HT1A
receptor, with considerable uptake observed in 5-HT1A-poor regions
such as the striatum and the cerebellum. [18F]10a was found to un-
dergo rapid brain clearance and evidence for the loss of fluorine was
found in vivo. [18F]10b was shown to reversibly and selectively bind
5-HT1A receptors with high affinity. This compound has a low rate of
brain clearance and evidence for radiodefluorination was not found.
Compound 10b was found to be a potent and selective 5-HT1A re-
ceptor antagonist by in vitro binding experiments.
It can be seen from the results in Table 5 that [18F]10b was
simultaneously taken up in several other organs. The distribution of
the radiotracer in the liver and kidneys was initially high (1.34 and
0.81% ID/g at 15 min, respectively) and the tracer was subsequently
washed out at a moderate rate (0.72 and 0.46% ID/g at 30 min).
However, radioactivity was observed to accumulatewithin the small
intestine over time (the uptake increased from 7.6% ID/g at 15 min to
On the basis of its high affinity and good selectivity for 5-HT1A
receptors, [18F]10b was identified as a promising candidate for PET
imaging of the serotonergic pathway. It is envisioned that its slow
brain washout makes [18F]10b suitable for the static study and
quantification of 5-HT1A receptor densities as part of the in vivo
study of neuropsychiatric disorders.
Table 4
BP values for [18F]10b when simultaneously administered with three cold ligands.
Brain region
[
18F]10b
8-OH-DPAT
(1 mg/kg)
10b
(0.5 mg/
kg)
WAY100635
(1 mg/kg)
Prefrontal cortex
Septum
Caudate
0.405 0.089
0.183 0.061
0.195 0.043
0.048
0.001
0.018
0.044
0.014
0.022
5. Experimental section
putamen
Amygdala
5.1. Chemistry
0.359 0.071
0.550 0.103
0.123 0.029
0.184 0.036
0.207 0.079
0.354 0.053
0.106
0.053
0.021
0.244
0.086
0.186
0.063
0.012
0.010
0.286
0.080
0.261
Hippocampus
Thalamus
Cerebellum
Brain stem
Whole brain
Solvents (HPLC quality, Scharlau) were dried in a Solvent Purifi-
cation System (MBraun) by passage through a pre-activated alumina
column or were purchased with anhydrous quality. Reagents were
purchased from SigmaeAldrich and they were used as received.

802
G. García et al. / European Journal of Medicinal Chemistry 85 (2014) 795e806
Reactions involving moisture-sensitive compounds wereperformed
under an atmosphere of dry argon. Microwave-assisted reactions
were carried out in a 5 mL vial using an Initiator 2.5 synthesizer
(Biotage). The reactions were monitored by thin-layer chromatog-
raphy (TLC) on silica-coated aluminum sheets (Alugram silica gel 60
(5H, m, HAr), 7.06e6.92 (4H, m, HAr), 4.39 (2H, t, J ¼ 6.2 Hz,
CH2NCO), 3.87 (3H, s, OCH3), 2.93 (4H, br. s, 2ꢄ CH2N), 2.83 (2H, t,
J ¼ 6.2 Hz, CH2N), 2.67 (4H, m, 2ꢄ CH2N) ppm. 13C NMR (50 MHz,
CDCl3):
d 170.9, 158.5, 153.8, 151.9, 149.8, 140.9, 135.1, 130.6, 128.2
(ꢄ2), 122.7, 120.7, 117.8, 114.3, 112.2, 110.9, 56.6, 55.1, 53.1, 50.4,
45.5 ppm. Anal. (C25H28N5O4) theoretical: C, 69.42; H, 6.10; N, 15.14.
Found: C, 69.74; H, 6.21; N, 14.83. IR (nmax, NaCl): 2941, 2818, 1661,
F254). The compounds were visualized by UV light (254 nm). Puri-
fication by column chromatography was undertaken with Merck
silica gel (0.030e0.075 mm) and the solvents were used as received
(Scharlau). Infrared spectra (IR, NaCl windows) were recorded on a
1574, 1534, 1500, 1465, 1355, 1304, 1240, 1141, 1022, 911, 733 cme1
.
HRMS (ESI-TOF): Calculated for C25H28N5O4 [MþH]þ: 462.2141.
PerkineElmer FTIR 1725X instrument. The frequencies (
n
) of the
Found: 462.2145.
more intense bands are given in cmꢂ1. Nuclear magnetic resonance
spectra (1H and 13C NMR) were recorded using Varian Gemini 200
(200 and 50 MHz, respectively), Varian Mercury-VX-300 MHz (300
and 75 MHZ, respectively) and Varian-UNITYPLUS-500 (500 and
5.2.2. N-{2-[4-(2-methoxyphenyl)piperazin-1-yl]ethyl}-N-(4-
nitropyridin-2-yl)-cyclohexanecarboxamide (9b)
Yield: 601 mg, 92% (method A). 1H NMR (200 MHz, CDCl3):
125 MHz, respectively) instruments. Chemical shifts (d) are given in
d
8.68 (1H, d, J ¼ 5.5 Hz, H6py), 8.31 (1H, dd, J ¼ 1.7 Hz, J ¼ 5.5 Hz,
ppm and are referenced to the residual signal of the non-deuterated
solvent. Coupling constants (J) are given in Hz. Elemental analyses
(C, H, N) were carried out on a LECO CHNSO-932 elemental analyzer
and the results were within 0.4% of the theoretical values. All
compounds were analyzed by tandem HPLC-MS. HPLC was per-
H5py), 7.82 (1H, d, J ¼ 1.7 Hz, H3py), 7.02e6.80 (4H, m, HAr), 4.07 (2H,
t, J ¼ 6.4 Hz, CH2NCO), 3.82 (3H, s, OCH3), 2.97 (4H, br. s, 2ꢄ CH2N),
2.65 (6H, m, 3ꢄ CH2N), 2.44 (1H, br. dt, Hchx), 1.90e1.00 (10H, m,
Hchx) ppm. 13C NMR (50 MHz, CDCl3):
d 176.4, 154.3, 151.7, 149.7,
140.5, 122.5, 120.5, 117.7, 113.8, 112.9, 110.6, 65.5, 54.9, 53.0, 50.0,
44.7, 42.4, 30.5, 29.2, 25.1 ppm. Anal. (C25H34N5O4) theoretical: C,
formed on a C18, 3
using a gradient of MeOH/water-4% formic acid at a flow rate of
1.0 mL/min. Products were detected at
mm column (Luna, Phenomenex, 3 ꢄ 100 mm)
64.08; H, 7.31; N, 14.95. Found: C, 64.23; H, 7.40; N, 14.77. IR (nmax
,
l
¼ 254 nm. Mass spectra
NaCl): 2932, 2853, 1670, 1536, 1500, 1451, 1388, 1355, 1241, 1146,
1027, 737. HRMS (ESI-TOF): Calculated for C25H34N5O4 [MþH]þ:
468.2611. Found: 468.2612.
were recorded on a HewlettePackard 5988A mass spectrometer.
High-resolution mass spectra were recorded on an Agilent 6210 LC/
MS TOF mass spectrometer.
The Supplementary data section includes experimental pro-
cedures and characterization data for compounds 2, 3, 5, 7 and 8,
radiochromatograms for crude radiotracers [18F]10aed and addi-
tional in vivo brain distribution data (dynamic PET): PET/CT images
of [18F]10aed and [18F]MPPF; Time-activity curves for [18F]10c and
5.2.3. N-{2-[4-(2-methoxyphenyl)piperazin-1-yl]ethyl}-N-(4-
nitropyridin-2-yl)adamantane-1-carboxamide (9c)
Yield: 467 mg, 60% (method B). 1H NMR (500 MHz, CDCl3):
d
8.66 (1H, d, J ¼ 5.4 Hz, H6py), 8.11 (1H, d, J ¼ 1.9 Hz, H3py), 7.86 (1H,
dd, J ¼ 1.9 Hz, J ¼ 5.4 Hz, H5py), 6.96 (1H, m, HAr), 6.88 (2H, t,
J ¼ 4.1 Hz, H4Ar and H5Ar), 6.82 (1H, d, J ¼ 7.9 Hz, H6Ar), 3.92 (2H, t,
J ¼ 7.9 Hz, CH2NCO), 3.82 (3H, s, OCH3), 2.98 (4H, m, 2ꢄ CH2N), 2.62
(6H, m, 3ꢄ CH2N), 1.88 (4H, m, HAd), 1.76 (2H, d, J ¼ 2.8 Hz, HAd), 1.60
(3H, d, J ¼ 11.7 Hz, HAd), 1.52 (3H, d, J ¼ 11.7 Hz, HAd) ppm. 13C NMR
[
18F]10d. The synthesis of precursors 9aed, 10aed and the radio-
synthesis of [18F]10aed are described below.
5.2. General procedures for the synthesis of 9aed
(125 MHz, CDCl3):
d 180.6, 159.9, 155.1, 152.2, 150.2, 141.1, 122.9,
Method A: In a 25 mL round-bottomed flask a solution of pyr-
idylamine 7 (500 mg, 1.40 mmol) and Et3N (0.24 mL, 1.68 mmol) in
anhydrous dichloromethane (15 mL) was prepared. The corre-
sponding acyl chloride (1.54 mmol) was added. The mixture was
stirred for 24 h at room temperature and the solvent was evapo-
rated. The residue was purified by silica gel flash chromatography
using ethyl acetate/hexane (1:1) as eluent. Amides 9 were obtained
as yellowish oils.
Method B: In a 25 mL round-bottomed flask a solution of pyr-
idylamine 7 (357 mg, 1.00 mmol) and NEt3 (0.21 mL, 1.50 mmol)
was prepared in anhydrous toluene (15 mL). The acyl chloride
(1.50 mmol) was added. The mixture was stirred for 24 h at 110 ꢀC
and the solvent was evaporated. The residue was purified by silica
gel column flash chromatography using ethyl acetate as eluent.
Amides 9 were obtained as yellowish oils.
Method C: Oxalyl chloride (0.11 mL, 1.3 mmol) and a catalytic
amount of dry DMF were added to a solution of the acid (1.2 mmol)
in dry dichloromethane (5 mL) at 0 ꢀC. The reaction mixture was
stirred for 2 h at room temperature. This solution was added
dropwise to a solution of the amine 7 (357 mg, 1 mmol) and NEt3
(0.18 mL, 1.3 mmol) in dry toluene (10 mL). The mixture was heated
for 24 h at 80 ꢀC and then purified by silica gel flash chromatog-
raphy using ethyl acetate as eluent. Amides 9 were obtained as
yellowish oils.
121.0, 118.1, 114.8, 114.0, 111.1, 56.6, 55.3, 53.5, 50.4, 48.6, 44.6, 40.1,
36.3, 28.2 ppm. Anal. (C29H38N5O4) theoretical: C, 66.9; H, 7.36; N,
13.45. Found: C, 67.23; H, 7.27; N, 13.5. IR (nmax, NaCl): 1674, 1534,
1500, 1411, 1355, 1240, 1147, 1023, 737 cme1. HRMS (ESI-TOF):
Calculated for C29H38N5O4 [MþH]þ: 520.2924. Found: 520.2930.
5.2.4. N-{2-[4-(2-hydroxyphenyl)piperazin-1-yl]ethyl}-N-(3-
nitrophenyl)pyridine-2-carboxamide (9d)
Yield: 323 mg, 70% (method C). 1H NMR (300 MHz, CDCl3):
d
8.50 (1H, d, J ¼ 5.3 Hz, H6py), 8.22 (1H, d, J ¼ 3.9 Hz, H3py), 7.88
(2H, m, H5py þ H6py), 7.77 (1H, td, J ¼ 1.9 Hz, J ¼ 7.9 Hz, H4py), 7.69
(1H, dd, J ¼ 1.9 Hz, J ¼ 5.6 Hz, H3py), 7.23 (1H, m, H5py), 6.95e6.84
(4H, m, HAr), 4.32 (2H, t, J ¼ 6.2 Hz, CH2NCO), 3.81 (3H, s, OCH3),
2.87 (4H, m, 2ꢄ CH2N), 2.79 (2H, t, J ¼ 6.3 Hz, CH2N), 2.61 (4H, m,
2ꢄ CH2N) ppm. 13C NMR (75 MHz, CDCl3):
d 168.9, 159.0, 154.4,
152.8, 152.1, 149.7, 147.8, 141.0, 137.1, 125.1, 124.9, 122.9, 120.9, 118.0,
113.7, 112.6, 111.0, 56.7, 55.2, 53.3, 50.5, 46.1 ppm. Anal.
(C24H27N6O4) theoretical: C, 62.19; H, 5.87; N, 18.13. Found: C,
62.24; H, 6.12; N, 17.77. IR (nmax, NaCl): 2818, 1661, 1574, 1532, 1500,
1463, 1354, 1240, 1143, 1024, 912, 745 cme1. HRMS (ESI-TOF):
Calculated for C24H27N6O4 [MþH]þ: 463.2094. Found: 463.2098.
5.3. General procedure for the synthesis of 10aed
5.2.1. N-{2-[4-(2-methoxyphenyl)piperazin-1-yl]ethyl}-N-(4-
nitropyridin-2-yl)benzamide (9a)
Amide 9 (25 mg, 0.054 mmol) was added to a vial containing CsF
in dry DMSO (3 mL). The mixture was placed in a Biotage® Initiator
synthesizer at 100 W for the time indicated in each case. Water
(5 mL) was added and the organic phase was extracted with ethyl
Yield: 614 mg, 95% (method A); 1H NMR (200 MHz, CDCl3):
d
8.65 (1H, d, J ¼ 5.5 Hz, H6py), 7.72 (2H, m, H5py þ HAr), 7.50e7.26

G. García et al. / European Journal of Medicinal Chemistry 85 (2014) 795e806
803
acetate (3 ꢄ 5 mL), dried over MgSO4, filtered and evaporated. The
residue was purified by silica gel flash chromatography.
extracted with ethyl acetate (3 ꢄ 5 mL), dried over MgSO4, filtered
and evaporated. The residue was purified by silica gel column flash
chromatography using ethyl acetate as the eluent. Yield: 23 mg,
5.3.1. N-{2-[4-(2-methoxyphenyl)piperazin-1-yl]ethyl}-N-(4-
nitropyridin-2-yl)benzamide (10a)
yellow oil, 62%. 1H NMR (200 MHz, CDCl3):
d
8.29 (1H, d, J ¼ 4.7 Hz,
H3py), 8.21 (1H, dd, J ¼ 5.5 Hz, J ¼ 9.1 Hz, H6py), 7.73 (2H, m,
H5py þ H6py), 7.21 (1H, m, H4py), 6.98e6.73 (6H, m, Hpy þ HAr), 4.62
(2H, t, J ¼ 6.7 Hz, CH2NCO), 3.82 (3H, s, OCH3), 2.93 (4H, m,
2ꢄ CH2N), 2.77 (2H, t, J ¼ 6.4 Hz, CH2N), 2.63 (4H, m, 2ꢄ CH2N) ppm.
Using CsF (41 mg, 0.27 mmol) and heating for 2.5 min. Ethyl
acetate/hexane (4:1) was used as the eluent. Yield: 10 mg, 40%. 1H
NMR (500 MHz, CDCl3):
d
8.33 (1H, dd, J ¼ 5.7 Hz, J ¼ 8.7 Hz, H6py),
7.72 (2H, m, H5py þ HAr), 7.35e7.30 (3H, m, HAr), 7.25e7.22 (2H, m,
HAr), 6.96 (1H, td, J ¼ 2.1 Hz, J ¼ 6.0 Hz), 6.90e6.85 (2H, m, HAr), 6.82
(1H, dd, J ¼ 1.1 Hz, J ¼ 8.1 Hz), 7.87 (1H, td, J ¼ 2.1 Hz, J ¼ 5.7 Hz),
6.58 (1H, dd, J ¼ 2.1 Hz, J ¼ 10.2 Hz, H3py), 4.27 (2H, t, J ¼ 6.2 Hz,
CH2NCO), 3.82 (3H, s, OCH3), 2.96 (4H, br. s, CH2N), 2.64 (2H, br s,
13C NMR (75 MHz, CDCl3):
d
168.9 (d, 1J ¼ 261.1 Hz, C4py), 168.8,
158.6 (d, 3J ¼ 10.5 Hz, C6py), 153.5, 152.1, 149.9 (d, 2J ¼ 13.8 Hz, C2py),
148.0, 141.1, 136.8, 124.7, 124.3, 122.8, 120.8, 118.0, 111.0, 109.4 (d,
2J ¼ 15.0 Hz, C5py), 108.5 (d, 2J ¼ 19.8 Hz, C3py), 56.3, 55.2, 53.3, 50.5,
45.8 ppm. Anal. (C24H27N5O2F) theoretical: C, 66.04; H, 6.23; N,
16.04. Found: C, 66.27; H, 6.31; N, 15.90. IR (nmax, NaCl): 2939, 2818,
1660, 1583, 1500, 1385, 1240, 1138, 1025, 856, 747 cme1. HRMS (ESI-
CH2N), 2.62 (4H, m, 2ꢄ CH2N) ppm. 13C NMR (125 MHz, CDCl3):
1
d
171.1, 170.8, 168.6 (d, JCeF ¼ 261.7 Hz, C4py), 152.2, 150.3 (d,
3JCeF ¼ 8.3 Hz, C6py), 141.3, 135.8, 130.5, 128.3, 122.8, 120.9, 118.1,
111.2, 110.0 (d, 2JCeF ¼ 20.1 Hz, C5py), 109.0 (d, 2JCeF ¼ 16.6 Hz, C3py),
60.4, 56.5, 55.3, 50.6 ppm. Anal. (C25H28N4O2F) theoretical: C,
TOF): Calculated for
436.2146.
C
24H27N5O2F [MþH]þ: 436.2146. Found
68.95; H, 6.48; N, 12.86. Found: C, 69.26; H, 6.66; N, 12.43. IR (nmax
,
5.4. Radiolabeling
NaCl): 2941, 2818, 1661, 1574, 1534, 1500, 1465, 1355, 1304, 1240,
1141, 1022, 911, 733 cme1
.
HRMS (ESI-TOF): Calculated for
5.4.1. General procedure for the radiosynthesis of compounds [18F]
10a and [18F]10ced
C
25H28N4O2F [MþH]þ: 435.2196. Found 435.2196.
No carrier added [18F]Fꢂ (20.6e89.5 GBq) was transferred in a
solution of H128O (3.6 mL; Rotem Industries) to a TRACERlab MX
synthesizer (GE Healthcare) loaded with a modified cassette for
FDG synthesis (Rotem Industries). The [18F]Fꢂ ions were trapped on
a QMA light cartridge and eluted with a solution of kryptofix K222
5.3.2. N-(4-fluoropyridin-2-yl)-N-{2-[4-(2-methoxyphenyl)
piperazin-1-yl]ethyl}-cyclohexanecarboxamide (10b)
Using CsF (41 mg, 0.27 mmol) and heating for 2.5 min. Ethyl
acetate/hexane (4:1) was used as the eluent. Yield: 12 mg, 52%. 1H
NMR (200 MHz, CDCl3):
d
8.42 (1H, dd, J ¼ 3.0 Hz, J ¼ 14.5 Hz,
(22 mg) and K2CO3 (7 mg) in H2O/MeCN (500 mL/500 mL). The so-
H6pyr), 7.19 (1H, dd, J ¼ 8.7 Hz, J ¼ 14.5 Hz, H5pyr), 6.97e6.80 (5H, m,
HAr þ Hpyr), 3.97 (2H, t, J ¼ 6.6 Hz, CH2NCO), 3.82 (3H, s, OCH3), 2.98
(4H, br s, 2ꢄ CH2N), 2.63e2.56 (6H, m, 3ꢄ CH2N), 2.44 (1H, br. dt,
lution was dried azeotropically (four cycles at 95 ꢀC) and a solution
of the precursor compound (9a, 9ced; 10e15 mg) dissolved in
DMSO (2.5 mL) was added to the reactor. Fluorination was under-
taken at 180 ꢀC over 5e10 min and the crude mixture was passed
through a pre-activated Sep-Pak C18 column. The column was
washed with H2O and eluted with MeOH (1.5 mL). The eluate was
collected and manually injected into an HPLC (semi-preparative,
H
d
chx), 1.73e1.07 (10H, m, Hchx) ppm. 13C NMR (75 MHz, CDCl3):
176.5, 169.4 (d, 1JCeF ¼ 263.0 Hz, C4py), 158.3 (d, 3J ¼ 10.5 Hz, C6py),
152.3, 150.8 (d, 3J ¼ 8.0 Hz, C2py), 141.2, 122.9, 120.9, 118.2, 111.2,
110.1 (d, 2J ¼ 16.7 Hz, C5py), 109.6 (d, 2J ¼ 19.2 Hz, C3py), 56.5, 55.4,
53.4, 50.5, 45.1, 42.6, 29.6, 25.6 ppm. Anal. (C25H34N4O2F) theoret-
ical: C, 68.00; H, 7.76; N, 12.69. Found: C, 68.32; H, 7.97; N, 12.4. IR
reversed phase column; Spherisorb ODS C18, 250 ꢄ 10 mm, 5
mm,
80 Å; H2O/MeOH/THF ¼ 65:22:13, acidified to pH 5e6; flow rate of
4 mL/min). The purified fraction, identified by comparison with the
cold reference, was collected and formulated for injection by
dilution with H2O (20e40 mL), passage through a pre-activated
Sep-Pak C18 cartridge and elution of the retained compound with
EtOH (2 mL).
(
nmax, NaCl): 2930, 2816, 1667, 1581, 1501, 1450, 1240, 1135, 1028,
822, 748 cme1
. HRMS (ESI-TOF) Calculated for C25H34N4O2F
[MþH]þ: 441.2666. Found 441.2678.
5.3.3. N-(4-fluoropyridin-2-yl)-N-{2-[4-(2-methoxyphenyl)
piperazin-1-yl]ethyl}-adamantane-1-carboxamide (10c)
Using CsF (82 mg, 0.54 mmol) and heating for 2.0 min. Ethyl
5.4.1.1. [18F]-N-(4-fluoropyridin-2-yl)-N-{2-[4-(2-methoxyphenyl)
piperazin-1-yl]ethyl}-benzamide ([18F]10a). Retention time in HPLC:
21 min. The purified compound was isolated and reformulated for
injection in 25.7% radiochemical yield (~23 GBq; decay corrected to
EOB) and the sample was analyzed by analytical HPLC system
(analytical, reversed phase column; ZORBAX Eclipse Plus C18,
acetate was used as the eluent. Yield: 19 mg, 70%. 1H NMR
(300 MHz, CDCl3):
d 8.41 (1H, m, H6py), 7.07 (1H, dd,
J ¼ 1.9 Hz J ¼ 9.5 Hz, H5py), 7.00e6.94 (2H, m, H5py þ HAr),
6.90e6.87 (2H, m, HAr), 6.82 (1H, d, J ¼ 7.6 Hz, HAr), 3.82 (5H, m,
CH2NCO þ OCH3), 3.00 (4H, m, 2ꢄ CH2N), 2.60 (6H, m, 3ꢄ CH2N),
1.85e1.48 (15H, m, HAd) ppm. 13C NMR (75 MHz, CDCl3):
d
179.5,
150 ꢄ 4.6 mm, 5
m
m) to determine its radiochemical purity (>99%)
169.5 (d, 1J ¼ 263.6 Hz, C4pr),159.6 (d, 3J ¼ 9.9 Hz, C6py),152.2,150.4,
(d, 3J ¼ 8.1 Hz, C2py), 141.2, 122.8, 120.9, 118.1, 110.7 (d, 2J ¼ 16.7 Hz,
C5py), 110.2 (d, 2J ¼ 18.0 Hz, C2py), 55.9, 55.3, 53.4, 50.5, 48.8, 44.3,
39.9, 36.4, 28.3 ppm. Anal. (C29H38N4O2F) theoretical: C, 70.56; H,
7.76; N, 11.35. Found: C, 70.87; H, 7.81; N, 11.08. IR (nmax, NaCl):
and its specific activity (72e450 GBq/
m
mol).
5.4.1.2. [18F]-N-(4-Fluoropyridin-2-yl)-N-{2-[4-(2-methoxyphenyl)
piperazin-1-yl]ethyl}-adamantane-1-carboxamide
([18F]10c).
Retention time in HPLC: 13 min. The purified compound was iso-
lated and reformulated for injection in 25.9% radiochemical yield
(~20 GBq; decay corrected to EOB) and the sample was analyzed by
analytical HPLC (analytical, reversed phase column; ZORBAX
2095, 2245, 1644, 1580, 1500, 1451, 1240, 1142, 1026, 911, 731 cme1
.
HRMS (ESI-TOF): Calculated for C29H38N4O2F [MþH]þ: 493.2999.
Found 493.3003.
Eclipse Plus C18, 150 ꢄ 4.6 mm, 5
m
m) to determine its radio-
5.3.4. N-(4-fluoropyridin-2-yl)-N-{2-[4-(2-methoxyphenyl)
piperazin-1-yl]ethyl}pyridine-2-carboxamide (10d)
Amide 9d (40 mg, 0.086 mmol) was added to a vial containing
CsF (66 mg, 0.42 mmol) in dry DMSO (2 mL). The vial was placed
into a Biotage® Initiator microwave oven and heated for 3 min at
140 ꢀC. Water (5 mL) was added and the organic phase was
chemical purity (>99%) and its specific activity (80e250 GBq/
mmol).
5.4.1.3. [18F]-N-(4-Fluoropyridin-2-yl)-N-{2-[4-(2-methoxyphenyl)
piperazin-1-yl]ethyl}-pyridine-2-carboxamide
([18F]10d).
Retention time in HPLC: 12 min. The purified compound was iso-
lated and reformulated for injection in 22.7% radiochemical yield

804
G. García et al. / European Journal of Medicinal Chemistry 85 (2014) 795e806
(~18 GBq; decay corrected to EOB) and the sample was analyzed by
analytical HPLC (analytical, reversed phase column; ZORBAX
5.7. [18F]10b dynamic PET
Eclipse Plus C18, 150 ꢄ 4.6 mm, 5
m
m) to determine its radio-
These studies were carried out to examine the pharmacokinetics
and binding behavior of the tracer when evaluated in vivo, in order
to confirm that the compound crosses the bloodebrain barrier and
binds to 5-HT1A receptors. The protocol used was almost identical
to that described for [18F]MPPF displacement, with the principal
difference being that [18F]10b was used instead of [18F]MPPF.
chemical purity (>99%) and its specific activity (25e60 GBq/
mmol).
5.4.2. [18F]-N-(4-Fluoropyridin-2-yl)-N-{2-[4-(2-methoxyphenyl)
piperazin-1-yl]ethyl}-cyclohexanecarboxamide ([18F]10b)
The radiolabeled compound [18F]10b was prepared by nucleo-
philic aromatic substitution of the nitro precursor 9b with 18Fꢂ
using a Synthera® (IBA Molecular) automated radiosynthesizer
loaded with an integrated fluidic processor (IFP; ABX Bio-
chemicals). In a typical procedure, no carrier added [18F]Fꢂ (79 GBq)
produced by proton bombardment of H128O (3.6 mL; Rotem In-
dustries) was transferred to the synthesizer and the anions were
retained in a QMA light cartridge. The [18F]Fꢂ was eluted to the
reactor with a solution of K2CO3 (7 mg) and K222 (22 mg) in H2O/
5.8. In vivo displacement of [18F]10b
To assess the specificity of [18F]10b for 5-HT1A receptors, animal
PET studies with [18F]10b were performed in the presence of two
well-characterized 5-HT1A ligands, namely 8-OH-DPAT and WAY-
100635, as well as with cold 10b. The protocol was again similar
to that outlined for [18F]MPPF displacement, with the difference
that [18F]10b was used as the tracer instead of [18F]MPPF.
MeCN (500 mL/500 m
L) and dried at 100 ꢀC and variable pressure
over 5 min. A solution of 11 mg of compound 9b in DMSO (1.2 mL)
was reacted with the Kþ18Fꢂ at 180 ꢀC over 8 min. The reaction
mixture was cooled and diluted with H2O (4.5 mL). The solution
was subsequently passed through a pre-activated C-18 Sep-Pak™
column. After eluting with methanol (1.5 mL), the crude prepara-
5.9. Biodistribution in rats
t
The biodistribution of compound [18F]10b was evaluated by
injecting it (approx. 500
mCi (18.5 MBq), i.v. via tail vein) into
tion was purified by automated HPLC (semi-preparative, reversed
SpragueeDawley male rats weighing approximately 200 g. The
animals were killed by decapitation at 15, 30, 60, and 120 min after
injection of the radiotracer. In order to determine the brain uptake
another set of rats were killed at just 1 min after injection of the
tracer. The organs and tissues were immediately removed and
dissected on an ice-cooled glass plate. After dissection, the tissues
were submerged and washed in saline solution (0.9% NaCl) to
remove adhering blood and the samples were weighed and placed
in counting vials. The radioactivity of each sample was measured in
a gamma counter (Cobra II Auto-Gamma, Packard; energy window
450e570 KeV, 1 min/vial). The counts per minute (CPM) were
corrected by means of a standard calibration curve. Finally, the
calibrated activities were expressed as a percentage of injected
dose per gram of tissue (%ID/g), taking into account the radioactive
decay from the injection until the measurement of the vial.
phase column; Luna C18(2), 250 ꢄ 10 mm, 5
mm, 100 Å), with a
mobile phase consisting of H2O (65%)/MeOH (22%)/THF (13%),
acidified to pH 5.5 (flow rate of 4 mL/min). The typical retention
time of [18F]10b was 70 min. The compound was formulated for
in vivo injection by dilution of the HPLC solution with H2O (40 mL),
retention of the compound in a pre-activated tC-18 Sep-Pak™
column, and elution with ethanol (0.2e1.0 mL). The purified and
formulated compound was injected into an analytical HPLC system
(analytical, reversed phase column; ZORBAX Eclipse Plus C18,
150 ꢄ 4.6 mm, 5
and its specific activity at the time of product release
(110e510 GBq/ mol). The radiochemical yield, corrected to end-of-
mm) to determine its radiochemical purity (>99%)
m
bombardment (EOB), was 27.8%.
5.5. [18F]10b in vitro brain autoradiography
Acknowledgments
Coronal rat brain slices (40
m
m thickness placed on glass slides)
were incubated with [18F]10b (1
mCi/mL in 50 mM TriseHCl pH 7.5,
ꢀ
The authors acknowledge the Instituto Tecnologico PET (CENIT-
20 min). After washing to remove the non-incorporated radioac-
tivity, the slides were dried and apposed on Agfa Curix RP-2 plus
films during 1 h. After exposure, the films were manually devel-
oped and the images were digitally captured. The binding pattern of
20081013, CDTI UAH-17/2009), Comunidad de Madrid (Projects S-
BIO-0170-2006 and S2011/BMD-2349), Ministerio de Economía y
Competitividad (Projects CTQ-2008/05027/BQU and SAF2009-
09020) and Universidad de Alcala (Projects UAH2011/EXP-034 and
UAH GC2012/001) for financial support. The collaboration of Ms.
[
18F]10b was identical to those obtained with ligands [18F]MPPF and
ꢀ
[3H]8-OH-DPAT, which correspond to the known 5-HT1A receptor
rat brain distribution. Furthermore, the specific signal was abol-
ished when the positron emitter tracer was incubated in the
presence of a high concentration of cold (non-radioactive) 8-OH-
DPAT, WAY-100635 or 10b.
ꢀ
ꢀ
Mary Brennan in some steps of the synthesis and Ruben Fernandez
de la Rosa in PET recordings is also acknowledged.
Appendix A. Supplementary data
5.6. [18F]MPPF displacement
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.07.096.
Male SpragueeDawley rats (n ¼ 13) weighing approximately
200 g were dynamically scanned (25 frames, 45 min) in a PET-CT
small animal device (Albira ARS, Oncovision) immediately after
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