Synthesis, evaluation, and radiolabeling of new potent positive allosteric modulators of the metabotropic glutamate receptor 2 as potential tracers for positron emission tomography imaging

Article abstract of DOI:10.1021/jm300912k

The synthesis and in vitro and in vivo evaluation of a new series of 7-(phenylpiperidinyl)-1,2,4-triazolo[4,3-a]pyridines, which were conveniently radiolabeled with carbon-11, as potential positron emission tomography (PET) radiotracers for in vivo imagin

Full text of DOI:10.1021/jm300912k

Article
pubs.acs.org/jmc
Synthesis, Evaluation, and Radiolabeling of New Potent Positive
Allosteric Modulators of the Metabotropic Glutamate Receptor 2 as
Potential Tracers for Positron Emission Tomography Imaging
Jose-Ignacio Andres,*,† Jesus Alcazar,† Jose María Cid,† Meri De Angelis,† Laura Iturrino,†
́
́
́
́
́
Xavier Langlois,‡ Hilde Lavreysen,‡ Andres A. Trabanco,† Sofie Celen,§ and Guy Bormans§
́
†Medicinal Chemistry, Janssen Research & Development, Janssen-Cilag S.A., C/Jarama 75, 45007 Toledo, Spain
‡Neuroscience, Janssen Research & Development, Janssen Pharmaceutica N.V., Turnhoutseweg 30, B-2340 Beerse, Belgium
§Laboratory for Radiopharmacy and IMIR, Faculty of Pharmaceutical Sciences, K.U. Leuven, Herestraat 49, B-3000 Leuven, Belgium
S
* Supporting Information
ABSTRACT: The synthesis and in vitro and in vivo evaluation
of a new series of 7-(phenylpiperidinyl)-1,2,4-triazolo[4,3-
a]pyridines, which were conveniently radiolabeled with carbon-
11, as potential positron emission tomography (PET)
radiotracers for in vivo imaging of the allosteric binding site
of the metabotropic glutamate (mGlu) receptor subtype 2 are
described. The synthesized compounds proved to be potent
and selective positive allosteric modulators (PAMs) of the
mGlu receptor 2 (mGluR2) in a [35S]GTPγS binding assay and were able to displace an mGluR2 PAM radioligand, which we
had previously developed, with IC50 values in the low nanomolar range. The most promising candidates were radiolabeled and
subjected to biodistribution studies and radiometabolite analysis in rats. Preliminary small-animal PET (μPET) studies in rats
indicated that [11C]20f binds specifically and reversibly to an mGluR2 allosteric site, strongly suggesting that it is a promising
candidate for PET imaging of mGluR2 in the brain.
been shown to be efficacious to treat anxiety disorders in
clinical trials.11
INTRODUCTION
■
Glutamate is the major excitatory neurotransmitter in the
mammalian central nervous system (CNS) and plays a major
role in numerous physiological and behavioral processes via two
different receptor classes, the ionotropic (iGlu) and the G-
protein coupled metabotropic glutamate (mGlu) receptors.1,2
There are eight known mGlu receptor subtypes, of which
subtype 2 (mGluR2), which belongs to the group II mGlu
receptors, is an attractive potential therapeutic target in
neuropharmacology. Preferentially expressed on presynaptic
nerve terminals, mGluR2 negatively modulates glutamate and
GABA release.3 Thus, it may be hypothesized that schizo-
phrenia-like symptoms arising from increased glutamate
transmission in the forebrain could be treated by stimulating
mGluR2, thereby reducing synaptic glutamate levels.4 Based on
this mechanism, normalizing glutamate levels by mixed
mGluR2/3 agonists has shown comparable efficacy as conven-
tional antipsychotic drugs for treating schizophrenia.5 Indeed,
clinical validation was achieved in a phase II study in
schizophrenic patients with LY2140023, the orally available
prodrug of the mixed mGluR2/3 orthosteric agonist LY404039,
which demonstrated improvements in both positive and
negative symptoms.6 In addition, multiple preclinical studies
have reported efficacy of mGluR2 activation in animal models
of neurologic disorders such as schizophrenia, anxiety/stress,
and depression.7−10 Furthermore, activating mGluR2/3 has
A new avenue for developing selective agents acting at mGlu
receptors is to identify compounds that act through allosteric
mechanisms, modulating the receptor by binding to a site
different from the highly conserved orthosteric glutamate
binding site. Positive allosteric modulators (PAMs) offer several
advantages over orthosteric agonists: the ligands are not based
on an amino acid structure, which is generally detrimental for
CNS penetration, they avoid the conserved mGluR orthosteric
binding site and offer improved selectivity, they may be less
liable to cause receptor desensitization, and they only act in the
presence of endogenous glutamate, thereby responding to
physiological fluctuations.12−14 The number of chemically
diverse mGluR2 PAMs reported over the last years in both
patent applications and research journals has increased
dramatically.15
Noninvasive imaging of mGluR2 using positron emission
tomography (PET) would allow quantification of the
distribution, expression, and modulation of this receptor
under physiological and pathological conditions. PET imaging
of mGluR2 would allow better understanding of the role of this
receptor under different neuropsychiatric conditions, and it
would facilitate the clinical development of mGluR2 modu-
Received: June 28, 2012
© XXXX American Chemical Society
A
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lators as potential drug candidates by giving direct insight into
the relationship between the level of receptor occupancy and
the administered dose of the candidate drug.16,17 Several PET
radiotracers have been reported for in vivo imaging of group I
mGluRs (mGluR1 and mostly mGluR5).18−21 Very recently the
first PET radioligand for imaging group II mGluRs in vivo has
been reported ([11C]CMGDE, 1).22 However, 1 (Figure1)
us as a good tool to study allosteric mGluR2 interactions and to
explore the relation between receptor binding and function.24
Overall, the PAMs tested showed similar affinity and were able
to displace [3H]2 from recombinant human receptors and
native rat receptors.24 These promising results prompted us to
start a program aiming at the identification of a potential PET
radiotracer for in vivo imaging of the mGluR2 allosteric binding
site(s). In a first attempt, several analogs of the 4-(phenyl-
piperidinyl)-2-pyridone derivative 2, containing different
substituents that could be amenable for radiolabeling with
either carbon-11 or fluorine-18, were prepared. However, none
of the synthesized derivatives could be taken into consideration
as a potential PET radiotracer candidate due to suboptimal
potency,15,23 as well as low brain uptake (unpublished results).
In a second attempt, the 1,2,4-triazolo[4,3-a]pyridine chemical
series, exemplified by compounds 3 and 4 (Figure 1) was
developed, providing compounds with increased potency.25
Previous exploration on the 4-(phenylpiperidinyl)-2-pyridones
showed that the introduction of a methoxy substituent on the
phenyl ring in the ortho-position to the piperidine moiety
would not only facilitate the production of the carbon-11
analog but could also increase the mGluR2 PAM potency.
Furthermore, the presence of one or two fluorine atoms in
different positions of the phenyl ring could impact the
compound potency, as well as the metabolic stability and
brain penetration.26
Based on these observations, a set of differently substituted 7-
[4-(2-methoxyphenyl)-1-piperidinyl]-1,2,4-triazolo[4,3-a]-
pyridines as mGluR2 PAMs that could be easily radiolabeled
with carbon-11 were synthesized. The synthesis and in vitro
activity of this new series of mGluR2 PAMs, as well as the
radiosynthesis of the most promising candidates and their
preliminary in vivo evaluation in rats, are reported in this article.
Figure 1. Structures of two mGluR2 radioligands and two 1,2,4-
triazolo[4,3-a]pyridine mGluR2 PAMs.
shows high affinity for both mGluR2 and mGluR3, and since it
binds to the orthosteric binding site, it cannot be used for
imaging studies with novel mGluR2 PAM drug candidates.
Furthermore, [11C]CMGDE is in fact a prodrug (dimethylester
of an amino acid like molecule) and its brain retention may
therefore also reflect esterase activity in the brain; this
hypothesis is supported by the fact that blocking studies
showed only 20−30% reduced brain uptake. To our knowledge,
there is no PET radiotracer disclosed for imaging selectively
mGluR2 so far.
We started a research program to develop a PET radiotracer
for in vivo imaging of mGluR2 in brain, in parallel to the
Medicinal Chemistry efforts aiming at the identification of a
suitable mGluR2 PAM for clinical development. We have
reported several structurally diverse chemotypes of mGluR2
PAMs.15 Among them, one of the most exemplified chemical
series is the 1,4-disubstituted 2-pyridones.15,23 Radiolabeled
mGluR2 PAM [3H]JNJ-40068782 ([3H]2, Figure 1) belongs to
this chemotype and was recently characterized and described by
RESULTS AND DISCUSSION
■
Chemistry and Radiochemistry. Synthesis of key
intermediates 7b−h was achieved in a fast and efficient way
using the appropriate synthetic technology depending on the
reaction (Scheme 1), following a similar approach as that
described in the literature for the synthesis of 7a.27 In the first
step, commercially available boronate 5 was coupled with
different 1-bromo-2-methoxyphenyl derivatives in parallel
under microwave irradiation28 to give intermediates 6a−h.
These bromo-methoxy-phenyl compounds were either com-
mercially available or were synthesized according to literature
precedents (see Experimental Section). Reduction of the
double bond in compounds 6a−h was effectively performed
under flow conditions using an H-Cube reactor, and
a
Scheme 1. Synthesis of the Key Intermediates 7a−h
a
Reagents and conditions: (i) Pd(PPh3)4, K2CO3 (saturated solution), 1,4-dioxane, μW, 150 °C, 10 min; (ii) H2, EtOH, Pd(OH)2/C (20%); H-
CUBE, full-H2, 80 °C, 1 mL/min; (iii) HCl (7 M in i-PrOH), MeOH, rt, 90 min.
B
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a
Scheme 2. Synthesis of Final Products 16d and 17b
a
Reagents and conditions: (i) NaH, DMF, rt, 1 h; (ii) diisopropylamine, MeCN, 110 °C, overnight; (iii) NH2NH2, EtOH or THF, μW, 160 °C, 15−
20 min; (iv) CF3CH2COCl, Et3N, CH2Cl2, rt, 1 h; (v) POCl3, MeCN, μW, 150−160 °C, 5−10 min.
a
Scheme 3. Synthesis of Final Products 20a−h and 21d,f and Synthesis of the Precursors 22b,c,f−h and 23f
a
Reagents and conditions: (i) 18 or 19, Pd(AcO)2, ( )BINAP, Cs2CO3, toluene, 125 °C, overnight; (ii) BBr3, CH2Cl2, rt, 45 min.
a
Scheme 4. Radiosynthesis of the Selected PET Tracer Candidates
a
Reagents and conditions: (i) [11C]CH3I, Cs2CO3, DMF, 90 °C, 3 min, 35−74% RCY.
subsequent deprotection of the tert-butoxycarbonyl protecting
group afforded the desired compounds 7a−h in good yields.
From the above intermediates, two sets of final compounds
could be generated. The first set is represented by compounds
containing the 3-(2,2,2-trifluoroethyl)-1,2,4-triazolo[4,3-a]-
pyridine fragment and were synthesized from intermediates
7b,d as starting material (Scheme 2). In a first step, derivatives
7b,d were N-arylated with the substituted pyridines 829 or 9 to
give derivatives 10d and 11b. Subsequent reaction with
hydrazine under microwave conditions afforded intermediates
12d and 13b, which were subjected to an acylation reaction
with CF3CH2COCl to introduce the 2,2,2-trifluoroethyl
residue, yielding 14d and 15b. Finally, cyclization using
microwave irradiation provided the target compounds 16d
and 17b. The second set of compounds, containing the 3-
(cyclopropylmethyl)-1,2,4-triazolo[4,3-a]pyridine fragment,
C
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Table 1. Functional Activity of the Synthesized mGluR2 PAMs
a
b
clogP values were calculated from Daylight software and Biobyte, Inc. of Claremont, CA. Values are means of 2−4 experiments.
was synthesized according to the strategy reported in Scheme 3.
Intermediates 7a−h were coupled with intermediates 1830 or
1931 under Buchwald conditions, providing the target
compounds 20a−h and 21d,f. Demethylation of selected
compounds was performed in the presence of boron
tribromide, to give derivatives 22b,c,f−h and 23f, which were
used as precursors to prepare the appropriate radiolabeled
analogues. In view of the in vitro pharmacological results (see
D
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Pharmacology section), derivatives 20b,c,f−h and 21f were
selected as potential PET radioligands for imaging of mGluR2.
Their radiolabeled analogues [11C]20b,c,f−h and [11C]21f
were prepared by methylation of the hydroxyl precursors
22b,c,f−h and 23f with [11C]MeI in the presence of cesium
carbonate in DMF (Scheme 4). The crude radiolabeling
mixtures were purified using reverse phase-HPLC (RP-HPLC),
affording [11C]20b,c,f−h and [11C]21f in 35%−74% radio-
chemical yields (relative to starting radioactivity of [11C]MeI,
nondecay corrected). The radiochemical purity of all six tracers
was examined using RP-HPLC and was superior to 96%. The
good radiochemical yield and purity of the employed
radiochemistry procedure thus could allow easy translation to
the production of clinical doses. The identity of the radiotracers
was confirmed using analytical HPLC after coinjection with
their nonradioactive analogues and comparison of the retention
time of the observed peaks in the radiometric and UV channel,
respectively.
[35S]GTPγS data (Table 2). Furthermore, the binding data
using the radioligand [3H]2 suggested that the 7-(phenyl-
Table 2. In-Vitro Binding of mGluR2 PAMs Derivatives 20b,
a
20c, 20f−h, and 21f
Pharmacology. We evaluated the effect of all target
compounds on [35S]GTPγS binding to the human mGluR2
receptor induced by an EC20-equivalent concentration of
glutamate (see Experimental Section) (Table 1). All new
derivatives proved to be among the most potent mGluR2
PAMs reported so far, with EC50’s ranging from 4.1 to 37 nM,
and Emax values ranging from 256% to 528%. It was not obvious
to identify structure−activity (SAR) trends within this series of
compounds. In general, comparing the 8-chloro-3-(cyclo-
propylmethyl)-substituted derivatives, it seemed that the
introduction of different fluorine atoms on the methoxy-
substituted-phenyl ring increased functional potency, although
there were some exceptions (37 nM for 20d and 26 nM for
20e, compared to 20 nM for the nonfluorinated derivative
20a). On the other hand, the presence of a fluorine atom in the
other position of the phenyl ring that is adjacent to the
piperidinyl moiety seemed to increase potency in the
[35S]GTPγS assay, as was observed for 20h and 21f.
Lipophilicity did not seem to play a crucial role in activity
either, as could be deduced from the comparison of clogP
values of the target compounds (see Table 1). Usually,
moderate lipophilicity in the clogP range of 2.5−3.5 is
considered optimal for adequate brain penetration without an
excessive level of nonspecific binding.32−34 Hence, at first sight
most of the synthesized compounds, with the exception of 16d,
might have been too lipophilic for their consideration as
potential PET radiotracer candidates. However, based on
previous experience searching for the identification of potential
PET radiotracers for imaging PDE10A, we did not discard any
compounds at this stage.35,36 The 8-cyano- derivative 16d was
one of the first compounds that we synthesized, and we decided
to check its ability to cross the blood−brain barrier (BBB). Due
to its insolubility, brain and plasma levels were studied after a
10 mg/kg oral dose in rats as a suspension, showing that brain
levels were below the quantification limits at the four measured
time points, 0.5, 1, 2, and 4 h after administration. Plasma levels
were also very low, with the mean maximal concentration being
12.6 ng/mL after 2 h. The disappointing results of 16d led to
deprioritization of this 8-cyano-substituted subseries.
a
Experimental details of the in vitro binding protocol are described in
b
the Experimental Section. Values are means of two or three
experiments.
piperidinyl)-1,2,4-triazolo[4,3-a]pyridines and the 4-(phenyl-
piperidinyl)-2-pyridones may bind to the same or a mutually
exclusive allosteric site of the mGluR2. These results were very
promising for our goal to identify a suitable PET radiotracer
from this chemical series.
We decided to select one of these compounds as a
representative example to evaluate their potential to cross the
BBB, assuming the hypothesis that the structural similarity
between all derivatives would warrant comparable behavior in
terms of brain penetration. We chose compound 20b for a fast
study in rats to measure brain and plasma levels after a 0.63
mg/kg intravenous (iv) dose at four different time points. This
experiment showed a very quick brain uptake with maximum
concentration after ∼5 min (316 ng/g) followed by a gradual
decline up to 1 h (∼50 ng/g). The mean brain/plasma ratio
was around 1.1 (Figure 2). These results indicated that the
compound showed relatively good brain penetration and some
brain retention, and thus, this chemical series deserved to be
further explored as potential PET tracers.
The most potent compounds in the functional assay, 20b,
20c, 20f-h, and 21f, were also subjected to in vitro binding
studies using radioligand [3H]2. These experiments showed
that all compounds tested displaced the radioligand with IC50
values in the low nanomolar range and that, in general, there
was a nice correlation between the binding and the functional
E
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radiotracer;32 however, if dissociation from plasma protein
occurs fast enough, high plasma protein binding will not be
detrimental for adequate brain uptake. Indeed, our previous
experience in the PDE10 PET program in which we identified a
potential radioligand with plasma protein binding >99.5%
prompted us to go ahead with the evaluation of the most
promising compounds.35 Also there are different reports in the
literature of other successful PET tracers that have a high log D
and high protein binding but still show good brain
penetration.32 Compounds 20b and 20f were profiled against
∼50 targets in a selectivity screen performed at CEREP.38
Selectivity overall was good, with the only relevant interactions
at a 10 μM concentration being dopamine-1 receptor (D1, 71%
for 20b and 76% for 20f) and the dopamine transporter (DAT,
83% for 20b and 52% for 20f). However, both compounds
were then tested in-house in a concentration−response curve to
measure their affinity, which resulted to be negligible: D1, Ki ≥
6200 nM for both compounds; DAT, Ki = 5005 nM for 20b
and >8500 nM for 20f.
Figure 2. Brain and plasma levels for compound 20b. Compound was
formulated in 20% HP-β-CD. Study was performed in two male
Sprague−Dawley rats dosed iv at 0.63 mg/kg. Brain levels are
expressed in ng/g and plasma levels in ng/mL. The graphs represent
the data as geometric mean values of the two runs.
Based on all the results shown above, compounds 20b, 20c,
20f, 20g, 20h, and 21f, were selected for radiolabeling with
carbon-11.
Biodistribution Studies. All six carbon-11 labeled
triazolopyridines ([11C]20b,c,f--h and [11C]21f) were evaluated
in vivo in male healthy Wistar rats by tissue distribution studies
at 2, 30, and 60 min postinjection (pi). Table 3 shows the brain
uptake of the six tracers at 2 min post-tracer-injection. All
tracers produced a similar moderate brain uptake, average
0.71% of the injected dose (ID), with on average 0.52% ID in
the cerebrum and 0.16% ID in the cerebellum. Table 4 shows
The mGluR2 selectivity of the final compounds listed in
Table 2 was determined using functional receptor assays. Thus,
they were tested for agonist and antagonist activity in
fluorescent Ca2+ assays using HEK293 cells expressing human
mGluR1, mGluR3, mGluR5, mGluR7, and mGluR8. Effects on
the human mGluR4 and rat mGluR6, expressed in L929 or
CHO cells, were assessed in [35S]-GTPγS functional assays. All
compounds displayed mGluR2 selectivity >350−500 fold over
the other mGluRs, with the exception of the following
compounds, whose selectivity over the mGluR3 receptor was
somewhat lower: 20b, ∼40 fold; 20g, ∼70-fold.
We identified the main metabolites formed from the
metabolic turnover of some derivatives after incubation with
human and rat liver microsomes.37 In both species two major
metabolites were observed, one of them being the correspond-
ing O-demethylated derivatives, and the others being M+O
oxidative metabolites in which oxidation occurred somewhere
on the 3-alkyl-1,2,4-triazolo[4,3-a]pyridine fragment. The
nonradioactive phenolic derivative formed by O-[11C]-
demethylation is thus an expected metabolite for a PET tracer
containing a [11C]methoxyphenyl group. As discussed later, the
radiometabolite analysis of some of the radiolabeled derivatives
showed that only the parent compounds were indeed
responsible for brain retention. All the synthesized compounds
showed very high plasma protein binding with free fractions
lower than 1% in both human and rat plasma, with the
exception of 16d (free fraction 4% and 4.7% in rat and human
plasma, respectively). In theory, those levels of plasma protein
binding could be considered too high for a suitable PET
Table 4. Concentration of [11C]20f in Different Rat Brain
Regions, Total Brain, and Blood at 2, 30, and 60 min Post-
tracer-injection
a
SUV
2 min
30 min
60 min
striatum
1.22 0.24
0.90 0.12
1.46 0.28
1.32 0.26
1.59 0.34
1.39 0.24
0.61 0.09
2.14 0.38
1.49 0.33
1.77 0.39
1.96 0.30
2.62 0.42
2.13 0.33
0.39 0.12
1.72 0.19
0.73 0.61
1.28 0.24
1.11 0.60
1.75 0.36
1.18 0.55
0.28 0.00
hippocampus
cortex
cerebrum
cerebellum
total brain
blood
a
Calculated as (radioactivity in cpm in organ/weight of organ in
grams)/(total cpm recovered/body weight rat in grams). Data are
expressed as mean SD; n = 3 per time point.
the standardized uptake values (SUVs) of the different studied
brain regions, the total brain, and the blood for [11C]20f. Table
5 presents the relative retention in the studied brain regions for
the six tracers. Similar concentrations in brain and blood were
Table 3. Brain Uptake of [11C]20b,c,f−h and [11C]21f in Normal Rats
a
%ID at 2 min pi
[11C]20b
[11C]20c
[11C]20f
[11C]20g
[11C]20h
[11C]21f
total brain
cerebrum
cerebellum
0.58 0.03
0.45 0.03
0.10 0.00
0.65 0.08
0.45 0.07
0.17 0.01
0.88 0.16
0.69 0.15
0.17 0.04
0.64 0.13
0.46 0.11
0.15 0.05
0.75 0.15
0.53 0.10
0.18 0.04
0.75 0.04
0.54 0.05
0.17 0.01
a
Percentage of injected dose calculated as counts per minute (cpm) in organ/total cpm recovered. Data are expressed as mean SD; n = 3 per time
point.
F
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Table 5. Wash-out from Different Rat Brain Regions
Table 6. Relative Percentages of Intact Tracer and
a
Calculated as 2 min to 30 min Ratio for [11C]20b,c,f−h and
Radiometabolites in Rat Plasma at 30 min Post-injection of
a
[11C]21f
[11C]20b, [11C]20f, [11C]20g, and [11C]20h
[11C]
20b
[11C]
20c
[11C]
20f
[11C]
20g
[11C]
20h
[11C]
21f
mean SD
[11C]20f
[11C]20b
[11C]20g
[11C]20h
striatum
0.63
0.66
0.88
0.83
0.63
0.79
0.59
0.63
0.71
0.60
0.71
0.72
0.57
0.60
0.82
0.67
0.61
0.65
0.99
1.19
0.84
0.84
0.95
0.93
0.67
0.71
0.66
0.72
0.77
0.88
0.86
1.01
1.04
0.98
0.99
1.08
polar metabolites 59.0 7.1
30.3 5.1
69.7 5.1
54.5 2.1
45.5 2.1
69.2 7.0
30.8 7.0
hippocampus
cortex
intact tracer
41.0 7.1
a
Results are presented as mean SD; n = 3 for [11C]20f, n = 2 for
cerebrum
cerebellum
total brain
[11C]20b, [11C]20g, and [11C]20h.
radioactivity in plasma, present as intact tracer at 30 min post-
tracer-injection. The reconstructed radiochromatograms of a
perfused rat cerebrum and cerebellum analysis for [11C]20f are
also shown in the Supporting Information. The peak
corresponding to the intact tracer eluted at ∼12 min. An
overview of the results from the perfused rat brain radio-
metabolite analysis for [11C]20b, [11C]20f, [11C]20g, and [11C]
20h is presented in Table 7. Results were very similar for the
four studied tracers. The fraction of apolar radiometabolites
detected in brain was negligible. The percentage of polar
radiometabolites detected in brain was very small. On average,
about 92% of the recovered radioactivity was present as intact
tracer in cerebrum and 95% in cerebellum. Future small-animal
PET (μPET) imaging studies will reveal whether this small
percentage of radiometabolites in brain needs to be taken into
account for determination of quantitative parameters.
a
Calculated from the 2 and 30 min SUV values.
observed for [11C]20b, [11C]20c, [11C]20g, [11C]20h, and
[11C]21f (data not shown). For all studied brain regions, the
radioactivity concentration increased from 2 to 30 min pi and
the 2-to-30 min wash-out ratios were ≤1 for all tracers in all
brain regions (except for [11C]20g in the hippocampus 2/30
ratio = 1.19). This accumulation of radioactivity in all studied
brain regions is in accordance with the reported intracerebral
distribution of mGluR2.39 For all brain regions the radioactivity
concentration at 60 min pi was lower compared to the 30 min
time point, indicating dissociation of the tracers from their
binding site. Since radioactivity was spread throughout the
brain, no reference region was available to correct for
nonspecific binding in kinetic modeling using a reference
tissue model, thus requiring determination of the arterial input
function in order to obtain quantitative data. The complete
tissue distribution study of [11C]20f is presented in the
Supporting Information. At 2 min pi, 4.3% ID was present in
blood, and this cleared to 2.0% by 60 min pi. The tracer was
cleared mainly by the hepatobiliary system, as there was in total
35.7% of ID present in liver and intestines 60 min after
injection of the radiotracer. Because of its extensive plasma
protein binding, the urinary excretion of the tracer was minimal
with only 2.4% ID present in the urinary system at 60 min pi. A
similar clearance pattern was observed for [11C]20b, [11C]20c,
[11C]20g, and [11C]20h (data not shown).
Radiometabolite Analysis in Rat Plasma and Perfused Rat
Brain. In order to use these PET radioligands for accurate
measurements of target density and target occupancy in drug
dose occupancy studies, knowledge of radioligand metabolism
is required, especially the extent to which radioactive
metabolites are present in plasma and their ability to penetrate
the brain and contribute to specific and/or nonspecific binding.
Therefore, the metabolic stability of [11C]20b, [11C]20f, [11C]
20g, and [11C]20h was studied in normal rats by determination
of the relative amounts of parent tracer and radiometabolites in
plasma and brain at 30 min pi of the tracers.
Preliminary μPET Studies. [11C]20f was further evaluated in
vivo in normal Wistar rats using μPET imaging (Figure 3). As
was also observed in the biodistribution studies, baseline
imaging (Figure 3A,B) showed tracer accumulation in all
studied brain regions. The maximum radioactivity concen-
tration (SUV 1.7) in brain was reached at 12 min pi, remained
constant until 27 min, and was followed by slow wash-out. After
injection of JNJ42153605, a potent PAM discovered by our
Janssen R&D team with high affinity and selectivity for
mGluR2,40 a clear displacement of the radioactivity in all brain
regions was observed (Figure 3C,D), demonstrating that the
binding of [11C]20f to the mGluR2 allosteric site is reversible
and specific.
CONCLUSIONS
■
In summary, we have synthesized a series of 7-(phenyl-
piperidinyl)-1,2,4-triazolo[4,3-a]pyridines as potent and selec-
tive mGluR2 PAMs, which could be radiolabeled with carbon-
11 for their evaluation as potential PET radioligands for in vivo
imaging of the mGluR2 allosteric binding site. From this
investigation we identified several promising candidates that
were selected in view of their good potency and selectivity, as
well as for their potential to cross the BBB, and they were
radiolabeled with [11C]MeI. Biodistribution studies, plasma and
brain radiometabolite analysis, and a preliminary μPET baseline
experiment in rats showed that [11C]20f was the most
promising PET radioligand candidate for in vivo imaging of
mGluR2, due to its good brain uptake and retention and its
relatively small fraction of radiometabolites in plasma and brain.
The μPET displacement experiment with a potent and selective
PAM discovered by our research team demonstrated that the
tracer binding to mGluR2 was specific and reversible. However,
due to the widespread distribution of mGluR2 in the brain, no
reference region is available for quantification of kinetic
parameters and estimation of receptor occupancy. Therefore,
extended μPET imaging studies with [11C]20f are ongoing to
The reconstructed radiochromatogram of a rat plasma
analysis of [11C]20f is included as Supporting Information.
From co-injection of the plasma with the authentic reference
compound we could identify the peak eluting at ∼11 min as the
intact tracer. The radioactivity corresponding to the more
lipophilic fractions eluting after the intact tracer was negligible,
indicating the absence of lipophilic radiometabolites, which, if
present, could penetrate the BBB. Unidentified polar radio-
metabolite(s) were eluting from 3 to 6 min. Similar results were
found for [11C]20b, [11C]20g, and [11C]20h. An overview of
the results of the plasma radiometabolite analysis is presented
in Table 6. Of the four 11C-labeled tracers, [11C]20f was found
to have the highest fraction (70
5%) of the recovered
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Table 7. Relative Percentages of Intact Tracer and Radiometabolites in Perfused Rat Cerebrum and Cerebellum at 30 min Post-
a
injection of [11C]20b, [11C]20f, [11C]20g, and [11C]20h
[11C]20b
[11C]20f
[11C]20g
[11C]20h
cbr
cbll
cbr
cbll
cbr
cbll
cbr
cbll
polar metabolite
intact tracer
7.6
7.3
9.7 0.3
4.1 1.5
6.9
3.6
7.1
4.5
92.4
92.7
90.3 0.3
95.5 1.3
93.1
96.4
92.9
95.5
a
Results are presented as mean SD; n = 2 for [11C]20f, n = 1 for [11C]20b, [11C]20g, [11C]20h. cbr = cerebrum, cbll = cerebellum.
Figure 3. μPET images and TACs for [11C]20f in rat striatum, cerebellum, cortex, hippocampus, thalamus, and total brain. (A) Untreated baseline
scan. (B) Transversal image corresponding to a baseline scan: averaged image (12−28 min after pi) of a representative rat injected with 41 MBq of
[11C]20f. (C) Chase experiment: JNJ42153605, 1 mg/kg, was injected iv (arrow) 30 min pi. (D) Transversal images corresponding to the chase
experiment: averaged image (12−28 min pi) before chase injection (left) and averaged image (68−88 min pi) after chase injection (right). max =
maximum; min = minimum.
(NMR) spectra were recorded with either a Bruker DPX-400 or a
Bruker AV-500 spectrometer (Bruker AG) with standard pulse
sequences, operating at 400 and 500 MHz, respectively, using
CDCl3 and DMSO-d6 as solvents. Chemical shifts (δ) are reported
in parts per million (ppm) downfield from tetramethylsilane (δ = 0).
Coupling constants are reported in hertz. Splitting patterns are defined
by s (singlet), d (doublet), dd (double doublet), t (triplet), q
(quartet), quin (quintet), sex (sextet), sep (septet), or m (multiplet).
Liquid chromatography combined with mass spectrometry (LC-MS)
was performed either on a HP 1100 HPLC system (Agilent
Technologies) or Advanced Chromatography Technologies, com-
posed of a quaternary or binary pump with a degasser, an autosampler,
a column oven, a diode-array detector (DAD), and a column, as
specified in the respective methods. Flow from the column was split to
a MS spectrometer. The MS detector was configured either with an
electrospray ionization source or an ES-CI dual ionization source
further evaluate the kinetics of this tracer and its potential for
human brain imaging, the results of which will be reported
elsewhere.
EXPERIMENTAL SECTION
■
Chemistry and Radiochemistry. Materials and General
Methods. Unless otherwise noted, all reagents and solvents were
obtained from commercial suppliers and used without further
purification. Thin layer chromatography (TLC) was carried out on
silica gel 60 F254 plates (Merck). Flash column chromatography was
performed on silica gel, particle size 60 Å, mesh = 230−400 (Merck)
under standard techniques. Microwave assisted reactions were
performed in a single-mode reactor, a Biotage Initiator Sixty
microwave reactor (Biotage), or in a multimode reactor, a Micro-
SYNTH Labstation (Milestone, Inc.). Nuclear magnetic resonance
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(electrospray combined with atmospheric pressure chemical ioniza-
tion). Nitrogen was used as the nebulizer gas. Data acquisition was
performed with MassLynx-Openlynx software or with Chemsation-
Agilent Data Browser software. Detailed information about the
different LCMS methods employed can be found in the Supporting
Information. Retention time (tR) is expressed in minutes. Gas
chromatography combined with mass spectrometry (GC-MS) was
performed using a 6890 Series gas chromatograph (Agilent
Technologies) system comprising a 7683 Series injector and
autosampler, a column oven, and a J&W HP-5MS, coupled to a
5973N MSD mass selective detector (single quadrupole, Agilent
Technologies). The MS detector was configured with an electronic
impact ionization source/chemical ionization source (EI/CI). EI low-
resolution mass spectra were acquired by scanning from 50 to 550 at a
rate of 14.29 scans/s. The source temperature was maintained at 230
°C. Helium was used as the nebulizer gas. Data acquisition was
performed with Chemstation-Open Action software. Melting point
(mp) values are peak values, were obtained with experimental
uncertainties that are commonly associated with this analytical
method, and were determined in open capillary tubes on a Mettler
FP62 apparatus with a temperature gradient of 10 °C/min. The
maximum temperature was 300 °C. The melting point was read from a
digital display.
The purities of all new compounds were determined by analytical
reverse phase RP-HPLC using the area percentage method on the UV
trace recorded at a wavelength of 254 nm, and compounds were found
to have ≥95% purity unless otherwise specified. For carbon-11 labeled
compounds, HPLC analysis was performed on a LaChrom Elite HPLC
system (Hitachi, Darmstadt, Germany). The HPLC eluate after
passage through the UV detector was led over a shielded 3-in. NaI(Tl)
scintillation detector connected to a multichannel analyzer (Gabi box,
Raytest, Straubenhardt Germany). The output signal was recorded and
analyzed using a GINA Star data acquisition system (Raytest,
Straubenhardt, Germany).
4-(3,6-Difluoro-2-methoxyphenyl)-3,6-dihydro-2H-pyridine-1-
carboxylic Acid tert-Butyl Ester (6f). Step 1: to a solution of 2,5-
difluorophenol (2.0 g, 15.37 mmol) and isopropylamine (1.61 mL,
15.37 mmol) in dry THF (40 mL) was added NBS (3.01 g, 16.19
mmol) portionwise at −40 °C. The reaction mixture was stirred at that
temperature for 30 min and then allowed to reach rt. The resulting
mixture was diluted with HCl (1 N in H2O) and Et2O, the organic
layer was separated and dried (Na2SO4), and the solvent was
evaporated in vacuo to yield intermediate 2-bromo-3,6-difluorophenol
(3.23 g, 51% pure) that was used as such in the next reaction step. 1H
NMR (500 MHz, CDCl3) δ 5.71 (br s, 1 H), 6.69 (ddd, J = 9.2, 7.7,
4.2 Hz, 1 H), 7.05 (td, J = 9.5, 4.9 Hz, 1 H).
Step 2: to a solution of 2-bromo-3,6-difluorophenol (3.23 g, 15.45
mmol) in dry MeCN (25 mL) were added K2CO3 (6.4 g, 46.36 mmol)
and MeI (2.88 mL, 46.36 mmol), and the resulting mixture was heated
under microwave irradiation at 150 °C for 10 min. Then the mixture
was diluted with DCM and filtered off, and the filtrate was evaporated
in vacuo to yield crude 2-bromo-1,4-difluoro-3-methoxybenzene that
was used without further purification in the next reaction step.
C7H5BrF2O3. GCMS: Rt 6.67, m/z 223 [M]+ (major product).
Step 3: 2-bromo-1,4-difluoro-3-methoxybenzene obtained from the
previous step (crude material) was added to a stirred mixture of 3,6-
dihydro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1(2H)-pyridi-
necarboxylic acid, 1,1-dimethylethyl ester (5, 1.26 g, 4.08 mmol),
Pd(PPh3)4 (0.07 g, 0.06 mmol), and a saturated aqueous solution of
K2CO3 (3.5 mL) in 1,4-dioxane (7 mL). The reaction mixture was
heated under microwave irradiation at 150 °C for 10 min. After
cooling, the mixture was diluted with water and extracted with Et2O.
The organic phase was separated and dried (Na2SO4), and the solvent
was evaporated in vacuo. The crude product was purified by column
chromatography (silica gel; EtOAc in heptane 10/90 to 20/80). The
desired fractions were collected and concentrated in vacuo to give a
residue that was triturated with Et2O to yield compound 6f as a sticky
oil (0.23 g, 22%). C17H21F2NO3. LCMS: Rt 3.25, m/z 326 [(M +
H)]+. 1H NMR (400 MHz, CDCl3) δ 1.50 (s, 9 H), 2.38 (br s, 2 H),
3.63 (br s, 2 H), 3.87 (d, J = 1.6 Hz, 3 H), 4.06 (br s, 2 H), 5.72 (br s,
1 H), 6.74 (td, J = 8.9, 3.9 Hz, 1 H), 6.95 (ddd, J = 10.6, 9.2, 5.1 Hz, 1
H).
In a similar manner, compounds 6b-e,g,h were also synthesized,
whereas 6a is described in the literature.25
tert-Butyl 4-(5-Fluoro-2-methoxyphenyl)-3,6-dihydropyridine-
1(2H)-carboxylate (6b). Starting from 2-bromo-4-fluoroanisole (2.28
g, 11.12 mmol) and 5 (2.86 g, 9.26 mmol), compound 6b (3.4 g,
quant yield) was obtained as colorless oil. C17H22FNO3. LCMS: Rt
3.25, m/z 308 [(M + H)]+.
tert-Butyl 4-(2-Fluoro-6-methoxyphenyl)-3,6-dihydropyridine-
1(2H)-carboxylate (6c). Starting from 2-bromo-3-fluoroanisole (2.2
g, 10.73 mmol) and 5 (3.01 g, 9.75 mmol), compound 6c (2.02 g,
67%) was obtained as a colorless oil that solidified on standing.
C17H22FNO3. LCMS: Rt 3.25, m/z 208 [(M − Boc) + H]+.
tert-Butyl 4-(3-Fluoro-2-methoxyphenyl)-3,6-dihydropyridine-
1(2H)-carboxylate (6d). Starting from 2-bromo-6-fluoroanisole
(0.974 mL, 7.31 mmol) and 5 (2.37 g, 7.68 mmol), compound 6d
(1.13 g, 50%) was obtained as a colorless oil. C17H22FNO3. LCMS: Rt
3.60, m/z 308 [(M + H)]+. 1H NMR (400 MHz, CDCl3) δ 1.49 (s, 9
H), 2.47 (br s, 2 H), 3.62 (br t, J = 5.7 Hz, 2 H), 3.89 (s, 3 H), 4.06
(br d, J = 2.5 Hz, 2 H), 5.96 (br s, 1 H), 6.92 (dd, J = 9.0, 8.6 Hz, 1 H),
7.04−7.16 (m, 2 H).
tert-Butyl 4-(3,4-Difluoro-2-methoxyphenyl)-3,6-dihydropyridine-
1(2H)-carboxylate (6e). Step 1: to a solution of 2,3-difluorophenol
(0.50 g, 3.84 mmol) and isopropylamine (0.40 mL, 3.84 mmol) in dry
DCM (20 mL) was added NBS (3.01 g, 16.19 mmol) portionwise at
−10 °C. The mixture was stirred at that temperature for 30 min and
then allowed to reach rt. The reaction mixture was then diluted with
HCl (1 N in H2O), the organic layer was separated and dried
(Na2SO4), and the solvent evaporated in vacuo. The crude compound
was purified by chromatography (silica gel, EtOAc in heptane 0:100 to
20:80). The desired fractions were collected and the solvent
evaporated in vacuo to yield 6-bromo-2,3-difluorophenol (0.63 g,
1
78%). C6H3BrF2O. GCMS: Rt 6.86, m/z 209 [M]+. H NMR (400
MHz, CDCl3) δ 6.36 (td, J = 9.4, 7.6 Hz, 1 H), 6.50 (br s, 1 H), 7.12
(ddd, J = 8.8, 6.1, 2.4 Hz, 1 H).
Step 2: 6-bromo-2,3-difluorophenol (0.63 g, 2.99 mmol), MeI
(0.281 mL, 4.51 mmol), and K2CO3 (0.623 g, 4.51 mmol) in MeCN
87.5 mL) were heated at 150 °C for 10 min in a microwave oven.
Then DCM was added, the solid was filtered off, and the filtrate
solvent was evaporated under vacuum, affording 1-bromo-3,4-difluoro-
2-methoxybenzene (0.62 g, 92%) as a sticky oil that was used as such
in the next reaction step. C7H35BrF2O. GCMS: Rt 6.54, m/z 222
[M]+. 1H NMR (500 MHz, CDCl3) δ 4.00 (d, J = 1.44 Hz, 3 H), 6.83
(td, J = 9.25, 7.51 Hz, 1 H), 7.23−7.29 (m, 1 H).
Step 3: starting from 1-bromo-3,4-difluoro-2-methoxybenzene (0.86
g, 3.83 mmol) and 5 (0.22 g, 0.19 mmol), compound 6e was obtained
(0.79 g, 63%) as a colorless oil. C17H21F2NO3. LCMS: Rt 3.31, m/z
206 [(M − Boc) + H]+. 1H NMR (500 MHz, CDCl3) δ 1.50 (s, 9 H),
2.45 (br s, 2 H), 3.59 (br s, 2 H), 3.90 (d, J = 1.4 Hz, 3 H), 4.04 (br s,
2 H), 5.76 (br s, 1 H), 6.79−6.88 (m, 2 H).
tert-Butyl 4-(2,3-Difluoro-6-methoxyphenyl)-3,6-dihydropyridine-
1(2H)-carboxylate (6g). Starting from 2-bromo-3,4-difluoroanisole
(0.18 g, 0.81 mmol) and 5 (0.25 g, 0.81 mmol), compound 6g (0.5 g,
quant yield) was obtained as a colorless oil. C17H21F2NO3. LCMS: Rt
3.17, m/z 206 [(M − Boc) + H]+.
tert-Butyl 4-(2,4-Difluoro-6-methoxyphenyl)-3,6-dihydropyridine-
1(2H)-carboxylate (6h). Step 1: methyl iodide (0.60 mL, 9.57 mmol)
was added to a solution of 2-bromo-3,5-difluorophenol (1.0 g, 4.78
mmol) and K2CO3 (1.32 g, 9.57 mmol) in DMF (10 mL). The
mixture was stirred under microwave irradiation at 150 °C for 10 min,
and after cooling to rt it was then treated with H2O and extracted with
EtOAc. The organic layer was separated, dried (Na2SO4), and filtered,
and the solvent was evaporated in vacuo to give 2-bromo-1,5-difluoro-
3-methoxybenzene (1.05 g, quant yield) as an oil that solidified upon
standing. The compound was used as such in the next reaction step.
C7H5BrF2O. GCMS: Rt 2.40, m/z 222 [M]+. Step 2: starting from 2-
bromo-1,5-difluoro-3-methoxybenzene (1.05 g, 4.78 mmol) and 5 (1.5
g, 4.78 mmol), compound 6h (1.13 g, 73%) was obtained as a colorless
oil that solidified on standing. C17H21F2NO3. LCMS: Rt 3.87, m/z 311
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[(M − Me) + H]+. 1H NMR (500 MHz, CDCl3) δ 1.50 (s, 9 H), 2.32
(br s, 2 H), 3.60 (br s, 2 H), 3.78 (s, 3 H), 4.04 (br s, 2 H), 5.63 (br s,
1 H), 6.40−6.48 (m, 2 H).
reaction mixture was quenched with water and extracted with DCM.
The organic layer was separated, dried (Na2SO4), and filtered, and the
solvent was evaporated. The solid compound obtained was then
washed with diisopropyl ether to afford compound 10d that was used
as such in the next reaction step (0.943 g, 74%). C18H17BrFN3O.
LCMS: Rt 3.34, m/z 390 [(M + H)]+.
4-(3,6-Difluoro-2-methoxyphenyl)piperidine (7f). A solution of 6f
(0.23 g, 0.71 mmol) in EtOH (15 mL) was hydrogenated in a H-Cube
reactor (1 mL/min, Pd(OH)2 20% cartridge, full H2 mode, 80 °C).
The solvent was evaporated in vacuo. The crude material obtained was
used in the next reaction step without further purification.
2,3-Dichloro-4-[4-(5-fluoro-2-methoxyphenyl)piperidin-1-yl]-
pyridine (11b). To a suspension of 7b (0.79 g, 3.78 mmol) and 2,3-
dichloro-4-iodopyridine (9, 0.87 g, 3.15 mmol) in MeCN (8 mL) was
added diisopropylethylamine (1.37 mL, 7.89 mmol). The mixture was
heated at 110 °C overnight. Then the solvent was evaporated and the
crude mixture was purified by column chromatography (silica gel,
DCM in heptane 80/20), and the desired fractions were collected and
concentrated in vacuo, yielding compound 11b (0.55 g, 48.5%) as a
white solid. C17H17ClFN2O. LCMS: Rt 3.30, m/z 355 [(M + H)]+.
4-[4-(3-Fluoro-2-methoxyphenyl)piperidin-1-yl]-2-hydrazinopyri-
dine-3-carbonitrile (12d). A mixture of 10d (0.94 g, 2.41 mmol) and
hydrazine (0.59 mL, 12.1 mmol) in THF (10 mL) was heated in a
microwave oven for 15 min at 160 °C. Then the solvent was
evaporated to dryness, the residue was taken up with a solution of NH3
in MeOH and then treated with Na2CO3 (aqueous saturated solution),
and the mixture was extracted with DCM. The organic layer was
separated, dried (Na2SO4), and filtered, and the solvent was
evaporated. The solid material obtained was then washed with Et2O,
yielding compound 12d that was used without further purification in
the next reaction step (0.62 g, 75%). C20H20FN5O. LCMS: Rt 3.02, m/
z 342 [(M + H)]+.
3-Chloro-4-[4-(5-fluoro-2-methoxyphenyl)piperidin-1-yl]-2-hy-
drazinopyridine (13b). Compound 13b was synthesized following a
similar procedure as described for 12d. Starting from 11b (0.55 g, 1.53
mmol), compound 13b was obtained (0.52 g, 92%) as a yellow oil.
C17H20ClFN4O. LCMS: Rt 2.95, m/z 351 [(M + H)]+.
N′-{3-Cyano-4-[4-(3-fluoro-2-methoxyphenyl)piperidin-1-yl]-
pyridin-2-yl}-3,3,3-trifluoropropanehydrazide (14d). To a solution of
compound 12d (0.10 g, 0.293 mmol) and Et3N (0.061 mL, 0.44
mmol) in DCM was added 3,3,3-trifluoropropionyl chloride (0.044
mL, 0.352 mmol) portionwise at 0 °C. The reaction was stirred for 10
min at rt. Then Na2CO3 (aqueous saturated solution) was added; the
organic layer was separated, dried (Na2SO4), and filtered; and the
solvent was evaporated in vacuum to give compound 14d, which was
used as such in the next reaction step (0.13 g, quant yield).
1
C17H23F2NO3. LCMS: Rt 3.29, m/z 655 [(MM + H)]+. H NMR
(400 MHz, CDCl3) δ 1.49 (s, 9 H), 1.61 (br d, J = 13.2 Hz, 2 H), 2.05
(qd, J = 12.6, 3.5 Hz, 2 H), 2.77 (br t, J = 11.3, 11.3 Hz, 2 H), 3.19 (tt,
J = 12.4, 3.5 Hz, 1 H), 3.91 (d, J = 1.8 Hz, 3 H), 4.23 (br s, 2 H), 6.70
(td, J = 9.6, 3.9 Hz, 1 H), 6.90 (ddd, J = 10.8, 9.1, 4.9 Hz, 1 H). The
residue was dissolved in MeOH, HCl (7 M in iPrOH, 2 mL) was
added, and the mixture was stirred at rt for 1.5 h. Then, the reaction
mixture was diluted with Na2CO3 (aqueous saturated solution) and
extracted with DCM. The organic phase was separated and dried
(Na2SO4), and the solvent was evaporated in vacuo to yield compound
7f (0.12 g, 85%) as a white solid. C12H15F2NO. LCMS: Rt 1.19, m/z
1
228 [(M + H)]+. H NMR (400 MHz, CDCl3) δ 1.85 (br d, J = 13.9
Hz, 2 H), 2.65 (qd, J = 13.3, 3.7 Hz, 2 H), 2.98 (td, J = 13.1, 2.5 Hz, 2
H), 3.27 (tt, J = 12.4, 3.2 Hz, 1 H), 3.63 (br d, J = 12.5 Hz, 2 H), 4.01
(d, J = 2.5 Hz, 3 H), 6.71 (td, J = 9.4, 3.8 Hz, 1 H), 6.94 (ddd, J = 11.0,
9.2, 5.2 Hz, 1 H), 7.27 (br s, 1 H).
In a similar manner, compounds 7b−e,g,h were also synthesized.
4-(5-Fluoro-2-methoxyphenyl)piperidine(7b). Starting from 6b
(3.4 g, 7.41 mmol) after reduction of the double bond and
deprotection of the N-Boc protecting group, compound 7b (0.76 g,
44% yield) was obtained as white solid. C12H16FNO. LCMS: Rt 1.85,
m/z 210 [(M + H)]+.
4-(2-Fluoro-6-methoxyphenyl)piperidine (7c). Starting from 6c (2
g, 6.5 mmol) after reduction of the double bond and deprotection of
the N-Boc protecting group, compound 7c (1.0 g, 73% yield) was
obtained as a white solid. C12H16FNO. LCMS: Rt 0.55, m/z 210 [(M
+ H)]+.
4-(3-Fluoro-2-methoxyphenyl)piperidine Hydrochloride (7d).
Starting from 6d (1.13 g, 3.67 mmol), the double bond was reduced
as previously described. The crude material was then dissolved in HCl
(7 M in iPrOH, 8.5 mL), and the reaction mixture was stirred for 1.5
h. The solvent was then evaporated to give 7d as the hydrochloride
salt (0.9 g, quant yield) as a white solid. C12H16FNO. LCMS: Rt 0.74,
m/z 210 [(M + H)]+. 1H NMR (400 MHz, DMSO-d6) δ 1.81 (qd, J =
12.5, 4.2 Hz, 2 H), 1.90 (br d, J = 11.8 Hz, 2 H), 2.79 (tt, J = 11.8, 3.8
Hz, 1 H), 2.87−3.02 (m, 2 H), 3.32 (br d, J = 12.9 Hz, 2 H), 3.81 (s, 3
H), 6.99 (dd, J = 8.6, 1.8 Hz, 1 H), 7.06 (dd, J = 12.9, 2.1 Hz, 1 H),
7.12 (t, J = 8.8 Hz, 1 H), 8.96 (br s, 1 H), 9.06 (br s, 1 H).
4-(3,4-Difluoro-2-methoxyphenyl)piperidine (7e). Starting from
6e (0.54 g, 1.66 mmol) after reduction of the double bond and
deprotection of the N-Boc protecting group, derivative 7e (0.38 g,
quant yield) was obtained as a white solid. C12H15F2NO. LCMS: Rt
1.35, m/z 228 [(M + H)]+.
4-(2,3-Difluoro-6-methoxyphenyl)piperidine (7g). Starting from
6g (0.13 g, 0.41 mmol) after reduction of the double bond and
deprotection of the N-Boc protecting group, compound 7g (0.09 g,
quant yield) was obtained as white solid. C12H15F2NO. LCMS: Rt
0.85, m/z 228 [(M + H)]+.
4-(2,4-Difluoro-6-methoxyphenyl)piperidine (7h). Starting from
compound 6h (0.726 g, 2.23 mmol) after reduction of the double
bond and deprotection of the N-Boc protecting group, compound 7h
(0.525 g, quant yield) was obtained as a yellow oil. C12H15F2NO.
LCMS: Rt 0.67, m/z 228 [(M + H)]+. 1H NMR (500 MHz, CDCl3) δ
1.60 (br d, J = 13.3 Hz, 2 H), 2.08−2.21 (m, 2 H), 2.53 (br s, 1 H),
2.71−2.80 (m, 2 H), 3.13−3.26 (m, 3 H), 3.81 (s, 3 H), 6.34−6.46
(m, 2 H).
́
́
3,3,3-Trifluoropropionic Acid N-[3-Chloro-4-(5-fluoro-2-methox-
́
́
yphenyl)-3,4,5,6,tetrahydro-2H-[1,4]bipyridinyl-2-yl]hydrazide
(15b). Compound 15b was synthesized following a similar procedure
as described for 14d. Starting from 13b (0.53 g, 1.51 mmol),
compound 15b was obtained (0.35 g, 54%) as a yellow oil.
C20H21ClF4N4O2. LCMS: Rt 2.99, m/z 461 [(M + H)]+.
7-[4-(3-Fluoro-2-methoxyphenyl)piperidin-1-yl]-3-(2,2,2-
trifluoroethyl)[1,2,4]triazolo[4,3-a]pyridine-8-carbonitrile (16d). A
solution of 14d (0.133 g, 0.295 mmol) and POCl3 (0.055 mL, 0.59
mmol) in MeCN was heated in a microwave oven for 5 min at 150 °C.
Then Na2CO3 (aqueous saturated solution) and DCM were added.
The organic layer was separated and dried (Na2SO4), and the solvent
was evaporated. The residue was purified by column chromatography
(silica gel, EtOAc in DCM 10/90 to 80/20). The solid compound
obtained was then washed with diisoproyl ether to yield compound
16d as a sticky solid (0.073 g, 57%). C21H19ClF4N5O. LCMS: Rt 4.19,
m/z 434 [(M + H)]+. 1H NMR (500 MHz, DMSO-d6) δ 1.80 (qd, J =
12.4, 3.2 Hz, 2 H), 1.87 (br d, J = 10.7 Hz, 2 H), 3.29 (tt, J = 11.8, 3.8
Hz, 1 H), 3.39−3.48 (m, 2 H), 3.87 (d, J = 1.2 Hz, 3 H), 4.37 (br d, J
= 12.7 Hz, 2 H), 4.40 (q, J = 10.7 Hz, 2 H), 7.04−7.16 (m, 4 H), 8.52
(d, J = 7.8 Hz, 1 H).
8-Chloro-7-[4-(5-fluoro-2-methoxyphenyl)-1-piperidinyl]-3-
(2,2,2-trifluoroethyl)-1,2,4-triazolo[4,3-a]pyridine (17b). Compound
17b was synthesized following a similar procedure as described for
16d. Starting from 15b (0.35 g, 0.77 mmol), compound 17b was
obtained as a white solid (0.11 g, 33%); mp 259 °C. C20H19ClF4N4O.
LCMS: Rt 3.87, m/z 443 [(M + H)]+. 1H NMR (500 MHz, CDCl3) δ
1.89 (qd, J = 12.4, 3.8 Hz, 2 H), 1.94−2.00 (m, 2 H), 3.08 (td, J =
11.8, 2.3 Hz, 2 H), 3.15 (tt, J = 11.9, 3.4 Hz, 1 H), 3.73−3.79 (m, 2
H), 3.83 (s, 3 H), 4.02 (q, J = 9.8 Hz, 2 H), 6.80 (dd, J = 9.0, 4.6 Hz, 1
2-Bromo-4-[4-(3-fluoro-2-methoxyphenyl)piperidin-1-yl]pyridine-
3-carbonitrile (10d). To a suspension of NaH (60% in mineral oil,
0.13 g, 3.27 mmol) in DMF (10 mL) was added 7d at 0 °C. The
reaction mixture was stirred at 0 °C for 5 min, and 2,4-
dibromopyridine-3-carbonitrile (8,28 0.86 g, 3.27 mmol) was added.
The reaction was allowed to reach rt and stirred for 1 h. Then the
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Journal of Medicinal Chemistry
Article
H), 6.85 (d, J = 7.5 Hz, 1 H), 6.86−6.91 (m, 1 H), 6.97 (dd, J = 9.5,
3.2 Hz, 1 H), 7.86 (d, J = 7.2 Hz, 1 H).
compound 20f. Starting from intermediates 18 (0.10 g, 0.30 mmol)
and 7d (0.075 g, 0.36 mmol), the title compound 20d was obtained as
an off-white solid (0.025 g, 19.5%); mp 168.3 °C. C22H24ClFN4O.
LCMS: Rt 3.40, m/z 415 [(M + H)]+. 1H NMR (400 MHz, CDCl3) δ
0.26−0.39 (m, 2 H), 0.54−0.68 (m, 2 H), 1.11−1.23 (m, 1 H), 1.86−
2.04 (m, 4 H), 2.98−3.10 (m, 4 H), 3.11−3.21 (m, 1 H), 3.67−3.75
(m, 2 H), 3.95 (d, J = 1.8 Hz, 3 H), 6.77 (d, J = 7.4 Hz, 1 H), 6.94−
7.09 (m, 3 H), 7.85 (d, J = 7.4 Hz, 1 H).
8-Chloro-3-(cyclopropylmethyl)-7-[4-(3,4-difluoro-2-methoxy-
phenyl)-1-piperidinyl]-1,2,4-triazolo[4,3-a]pyridine (20e). Com-
pound 20e was synthesized following the same approach described
for compound 20f. Starting from intermediates 18 (0.15 g, 0.45 mmol)
and 7e (0.12 g, 0.54 mmol), compound 20e was obtained as an off-
white solid (0.042 g, 21%); mp 173.5 °C. C22H23ClF2N4O. LCMS: Rt
3.56, m/z 433 [(M + H)]+. 1H NMR (400 MHz, CDCl3) δ 0.26−0.39
(m, 2 H), 0.54−0.68 (m, 2 H), 1.11−1.23 (m, 1 H), 1.85−2.00 (m, 4
H), 2.97−3.13 (m, 5 H), 3.70 (br d, J = 11.8 Hz, 2 H), 4.00 (d, J = 2.1
Hz, 3 H), 6.75 (d, J = 7.4 Hz, 1 H), 6.83−6.92 (m, 1 H), 6.93−6.99
(m, 1 H), 7.84 (d, J = 7.4 Hz, 1 H).
8-Chloro-3-(cyclopropylmethyl)-7-[4-(2,3-difluoro-6-methoxy-
phenyl)-1-piperidinyl]-1,2,4-triazolo[4,3-a]pyridine (20g). Com-
pound 20g was synthesized following the same procedure described
for compound 20f. Starting from intermediates 18 (0.10 g, 0.30 mmol)
and 7g (0.08 g, 0.36 mmol), compound 20g was obtained as an off-
white solid (0.04 g, 27.6%); mp 208.5 °C. C22H23ClF2N4O. LCMS: Rt
3.48, m/z 433 [(M + H)]+. 1H NMR (400 MHz, CDCl3) δ 0.26−0.39
(m, 2 H), 0.54−0.68 (m, 2 H), 1.11−1.22 (m, 1 H), 1.71−1.80 (m, 2
H), 2.45 (qd, J = 12.4, 3.4 Hz, 2 H), 3.00 (br t, J = 11.4, 2 H), 3.05 (d,
J = 6.7 Hz, 2 H), 3.30 (tt, J = 12.4, 3.5 Hz, 1 H), 3.67−3.75 (m, 2 H),
3.83 (s, 3 H), 6.52−6.61 (m, 1 H), 6.76 (d, J = 7.6 Hz, 1 H), 6.98 (q, J
= 9.2 Hz, 1 H), 7.83 (d, J = 7.6 Hz, 1 H).
8-Chloro-3-(cyclopropylmethyl)-7-[4-(3,6-difluoro-2-methoxy-
phenyl)-1-piperidinyl]-1,2,4-triazolo[4,3-a]pyridine (20f). To a mix-
ture of 8-chloro-3-(cyclopropylmethyl)-7-iodo[1,2,4]triazolo[4,3-a]-
pyridine29 (18, 0.25 g, 0.75 mmol) and 7f (0.22 g, 0.97 mmol) in
toluene (2.5 mL) were added Pd(OAc)2 (0.008 g, 0.04 mmol),
( )BINAP (0.046 g, 0.07 mmol), and Cs2CO3 (0.37 g, 1.12 mmol).
The reaction mixture was heated at 125 °C overnight. Then DCM was
added, the solid was filtered off, the filtrate solvent evaporated in vacuo,
and the crude material purified by column chromatography (MeOH in
DCM 0/100 to 5/95). The desired fractions were collected, the
solvent was evaporated in vacuo, and the solid material obtained was
then washed with Et2O to yield compound 20f as an off-white solid
(0.19 g, 59.2%); mp 163.2 °C. C22H23ClF2N4O. LCMS: Rt 2.43, m/z
443 [(M + H)]+. 1H NMR (500 MHz, CDCl3) δ 0.20−0.38 (m, 2 H),
0.47−0.67 (m, 2 H), 1.13−1.20 (m, 1 H), 1.78 (br d, J = 12.4 Hz, 2
H), 2.41 (qd, J = 12.5, 2.7 Hz, 2 H), 3.01 (t, J = 12.1 Hz, 2 H), 3.05 (d,
J = 6.9 Hz, 2 H), 3.25 (tt, J = 12.5, 3.4 Hz, 1 H), 3.72 (br d, J = 11.8
Hz, 2 H), 3.95 (d, J = 1.7 Hz, 3 H), 6.74 (td, J = 9.2, 4.0 Hz, 1 H), 6.76
(d, J = 7.5 Hz, 1 H), 6.93 (ddd, J = 10.5, 9.2, 5.1 Hz, 1 H), 7.84 (d, J =
7.5 Hz, 1 H).
8-Chloro-3-(cyclopropylmethyl)-7-[4-(2-methoxyphenyl)-1-piper-
idinyl]-1,2,4-triazolo[4,3-a]pyridine (20a). Compound 20a was
synthesized following the same procedure as described for compound
20f. Starting from intermediates 18 (0.15 g, 0.45 mmol) and 4-(2-
methoxyphenyl)piperidine26 (7a, 0.1 g, 0.54 mmol), compound 20a
was obtained as a foam (0.056 g, 29.5%). C22H25ClN4O. LCMS: Rt
3.38, m/z 397 [(M + H)]+ 1H NMR (400 MHz, CDCl3) δ 0.25−0.39
(m, 2 H), 0.54−0.67 (m, 2 H), 1.10−1.23 (m, 1 H), 1.87−2.03 (m, 4
H), 3.00−3.09 (m, 4 H), 3.11−3.21 (m, 1 H), 3.71 (br d, J = 12.5 Hz,
2 H), 3.86 (s, 3 H), 6.77 (d, J = 7.4 Hz, 1 H), 6.89 (br d, J = 8.1 Hz, 1
H), 6.97 (br t, J = 7.4, 7.4 Hz, 1 H), 7.19−7.24 (m, 1 H), 7.25−7.29
(m, 1 H), 7.84 (d, J = 7.6 Hz, 1 H).
8-Chloro-3-(cyclopropylmethyl)-7-[4-(2,4-difluoro-6-methoxy-
phenyl)-1-piperidinyl]-1,2,4-triazolo[4,3-a]pyridine (20h). Com-
pound 20h was synthesized following the same procedure described
for 20f. Starting from intermediate 18 (0.10 g, 0.30 mmol) and 7h
(0.08 g, 0.36 mmol), compound 20h was obtained as an off-white solid
(0.05 g, 38%); mp 162.2 °C. C22H23ClF2N4O. LCMS: Rt 3.6, m/z 433
8-Chloro-3-(cyclopropylmethyl)-7-[4-(5-fluoro-2-methoxyphen-
yl)-1-piperidinyl]-1,2,4-triazolo[4,3-a]pyridine (20b). A suspension of
compounds 18 (0.10 g, 0.30 mmol), 7b (0.13 g, 0.60 mmol), and
NaHCO3 (0.061 g, 0.75 mmol) in MeCN (1 mL) was heated in a
pressure tube (Q-Tube) at 180 °C overnight. Then the mixture was
diluted with DCM and HCl (2 N in H2O), the organic layer separated
and dried (Na2SO4), and the solvent evaporated in vacuo. The crude
material was purified by column chromatography (EtOAc in DCM 0/
100 to 100/0), the desired fractions were collected, and the solvent
was evaporated in vacuo. The solid compound obtained was then
washed with diisopropyl ether to yield compound 20b as an off-white
solid (0.06 g, 49%); mp >300 °C. C22H24ClFN4O. LCMS: Rt 3.43, m/
1
[(M + H)]+. H NMR (500 MHz, CDCl3) δ 0.28−0.38 (m, 2 H),
0.55−0.67 (m, 2 H), 1.12−1.21 (m, 1 H), 1.68−1.76 (m, 2 H), 2.40
(qd, J = 12.3, 3.3 Hz, 2 H), 2.98 (br t, J = 11.7 Hz, 2 H), 3.05 (d, J =
6.6 Hz, 2 H), 3.22 (tt, J = 12.4, 3.6 Hz, 1 H), 3.70 (br d, J = 11.8 Hz, 2
H), 3.84 (s, 3 H), 6.39−6.47 (m, 2 H), 6.76 (d, J = 7.5 Hz, 1 H), 7.83
(d, J = 7.5 Hz, 1 H).
3-(Cyclopropylmethyl)-7-[4-(3,6-difluoro-2-methoxyphenyl)-1-pi-
peridinyl]-8-(trifluoromethyl)-1,2,4-triazolo[4,3-a]pyridine (21f). A
mixture of 7-chloro-3-(cyclopropylmethyl)-8-(trifluoromethyl)[1,2,4]-
triazolo[4,3-a]pyridine30 (19, 0.30 g, 1.09 mmol), 7f (0.37 g, 1.63
mmol), and diisopropylamine (0.38 mL, 2.18 mmol) in MeCN (3
mL) was heated under microwave irradiation at 190 °C for 20 min.
Then the solvent was evaporated, and the crude residue was purified
by column chromatography (EtOAc in DCM 0/100 to 100/0). The
desired fractions were collected, and the solvent was evaporated in
vacuo. The solid compound obtained was then washed with
diisopropyl ether to yield compound 21f as an off-white solid (0.25
g, 48.2%); mp 180.7 °C. C23H23F5N4O. LCMS: Rt 3.56, m/z 467 [(M
1
z 415 [(M + H)]+. H NMR (500 MHz, CDCl3) δ 0.27−0.38 (m, 2
H), 0.55−0.67 (m, 2 H), 1.13−1.20 (m, 1 H), 1.89 (qd, J = 12.1, 3.8
Hz, 2 H), 1.93−1.99 (m, 2 H), 3.00−3.07 (m, 2 H), 3.05 (d, J = 6.6
Hz, 2 H), 3.14 (tt, J = 11.7, 3.6 Hz, 1 H), 3.71 (br d, J = 11.8 Hz, 2 H),
3.83 (s, 3 H), 6.76 (d, J = 7.5 Hz, 1 H), 6.80 (dd, J = 9.0, 4.6 Hz, 1 H),
6.86−6.92 (m, 1 H), 6.97 (dd, J = 9.5, 3.2 Hz, 1 H), 7.84 (d, J = 7.5
Hz, 1 H).
8-Chloro-3-(cyclopropylmethyl)-7-[4-(2-fluoro-6-methoxyphen-
yl)-1-piperidinyl]-1,2,4-triazolo[4,3-a]pyridine (20c). Compound 20c
was synthesized following a similar procedure to that described for its
analogue 20b, changing the heating system from pressure tube to
microwave irradiation (230 °C, 30 min). Thus, starting from
intermediate 18 (0.10 g, 0.30 mmol) and intermediate 7c (0.094 g,
0.45 mmol), the title compound 20c was obtained as a foam (0.05 g,
1
+ H)]+. H NMR (500 MHz, CDCl3) δ 0.28−0.38 (m, 2 H), 0.57−
0.67 (m, 2 H), 1.11−1.20 (m, 1 H), 1.75 (dd, J = 12.1, 1.7 Hz, 2 H),
2.35 (qd, J = 12.4, 3.2 Hz, 2 H), 3.04 (d, J = 6.6 Hz, 2 H), 3.18 (br t, J
= 12.4 Hz, 2 H), 3.27 (tt, J = 12.4, 3.6 Hz, 1 H), 3.62 (br d, J = 12.7
Hz, 2 H), 3.94 (d, J = 2.0 Hz, 3 H), 6.72 (ddd, J = 9.8, 9.3, 4.1 Hz, 1
H), 6.75 (d, J = 7.5 Hz, 1 H), 6.93 (ddd, J = 10.8, 9.2, 4.9 Hz, 1 H),
7.91 (d, J = 7.5 Hz, 1 H).
1
38.5%). C22H24ClFN4O. LCMS: Rt 4.72, m/z 415 [(M + H)]+. H
NMR (500 MHz, CDCl3) δ 0.28−0.38 (m, 2 H), 0.56−0.66 (m, 2 H),
1.13−1.22 (m, 1 H), 1.71−1.78 (m, 2 H), 2.45 (qd, J = 12.3, 3.2 Hz, 2
H), 3.01 (br t, J = 11.8 Hz, 2 H), 3.05 (d, J = 6.6 Hz, 2 H), 3.31 (tt, J =
12.3, 3.5 Hz, 1 H), 3.72 (br d, J = 11.8 Hz, 2 H), 3.85 (s, 3 H), 6.66−
6.72 (m, 2 H), 6.77 (d, J = 7.5 Hz, 1 H), 7.15 (td, J = 8.3, 6.5 Hz, 1 H),
7.83 (d, J = 7.5 Hz, 1 H).
8-Chloro-3-(cyclopropylmethyl)-7-[4-(3-fluoro-2-methoxyphen-
yl)-1-piperidinyl]-1,2,4-triazolo[4,3-a]pyridine (20d). Compound 20d
was synthesized following the same procedure described for
8-Chloro-3-(cyclopropylmethyl)-7-[4-(3-fluoro-2-methoxyphen-
yl)-1-piperidinyl]-1,2,4-triazolo[4,3-a]pyridine (21d). Compound 21d
was synthesized following the same procedure described for
compound 21f. Starting from intermediate 19 (0.10 g, 0.36 mmol)
and 7d (0.09 g, 0.44 mmol), compound 21d was obtained as an off-
white solid (0.045 g, 27.6%); mp 195.7 °C. C22H24F4N4O. LCMS: Rt
3.57, m/z 449 [(M + H)]+. 1H NMR (500 MHz, CDCl3) δ 0.28−0.39
(m, 2 H), 0.56−0.68 (m, 2 H), 1.08−1.20 (m, 1 H), 1.82−1.97 (m, 4
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H), 3.05 (d, J = 6.6 Hz, 2 H), 3.10−3.18 (m, 1 H), 3.18−3.28 (m, 2
H), 3.60 (br d, J = 13.0 Hz, 2 H), 3.95 (d, J = 1.7 Hz, 3 H), 6.77 (d, J =
7.8 Hz, 1 H), 6.92−7.07 (m, 3 H), 7.93 (d, J = 7.8 Hz, 1 H).
2-[1-[8-Chloro-3-(cyclopropylmethyl)-1,2,4-triazolo[4,3-a]-
pyridin-7-yl]-4-piperidinyl]-3,6-difluorophenol (22f). To a solution of
compound 20f (0.05 g, 0.116) in DCM (0.5 mL) was added BBr3
(0.231 mL, 0.231 mmol) dropwise at 0 °C. The reaction was stirred
for 45 min at rt. The excess BBr3 was quenched dropwise with MeOH
(1 mL) at 0 °C, and then Na2CO3 (saturated aqueous solution) was
added (to pH ∼ 7). The organic layer was separated, dried (Na2SO4),
and filtered, and the solvent was evaporated in vacuo. The residue was
purified by column chromatography (silica gel, MeOH in DCM 0/100
to 6/94), the desired fractions were collected, and the solvent was
evaporated in vacuo. The compound obtained was then treated with
MeCN and then purified again by chromatography (same eluent as
before) and then treated with Et2O to yield finally the title compound
22f (0.018 g, 38%) as a white solid; mp >300 °C. C21H21ClF2N4O.
6.51−6.61 (m, 1 H), 6.96 (d, J = 7.5 Hz, 1 H), 8.37 (d, J = 7.5 Hz, 1
H), 10.44 (br s, 1 H).
2-[1-[3-(Cyclopropylmethyl)-8-(trifluoromethyl)-1,2,4-triazolo-
[4,3-a]pyridin-7-yl]-4-piperidinyl]-3,6-difluorophenol (23f). Com-
pound 23f was synthesized following the same procedure reported
for 22f. Starting from compound 21f (0.15 g, 0.32 mmol) after
deprotection with BBr3, compound 23f was obtained (0.01 g, 8.9%) as
a white solid; mp >300 °C. C22H21F5N4O. LCMS: Rt 2.92, m/z 453
[(M + H)]+. 1H NMR (500 MHz, DMSO-d6) δ 0.21−0.35 (m, 2 H),
0.42−0.59 (m, 2 H), 1.11−1.21 (m, 1 H), 1.67 (br d, J = 11.0 Hz, 2
H), 2.15−2.34 (m, 2 H), 3.00 (d, J = 6.9 Hz, 2 H), 3.17 (br t, J = 12.1
Hz, 2 H), 3.53 (br d, J = 12.4 Hz, 2 H), 6.60 (td, J = 9.5, 3.3 Hz, 1 H),
7.00 (d, J = 7.8 Hz, 1 H), 7.05 (td, J = 9.6, 5.1 Hz, 1 H), 8.47 (d, J =
7.5 Hz, 1 H), 9.96 (br s, 1 H).
Radiochemistry. Carbon-11 was produced via a [14N(p,α)11C]
nuclear reaction. The target gas, which is a mixture of N2 (95%) and
H2 (5%) was irradiated using 18-MeV protons at a beam current of 25
μA. The irradiation was done for about 30 min to yield [11C]methane
([11C]CH4). [11C]CH4 was then reacted with vaporous I2 at 650 °C to
convert it to [11C]methyl iodide ([11C]MeI). The resulting volatile
[11C]MeI was bubbled with a flow of helium through a solution of
radiolabeling precursor 22b (for [11C]20b), 22c (for [11C]20c), 22f
(for [11C]20f), 22g (for [11C]20g), 22h (for [11C]20h), 23f (for [11C]
21f) (0.2 mg), and Cs2CO3 (1−3 mg) in anhydrous DMF (0.2 mL).
When the amount of radioactivity in the reaction vial had stabilized,
the reaction mixture was heated at 90 °C for 3 min. After dilution with
water (0.7 mL), the crude reaction mixture was injected onto an
HPLC system consisting of a semipreparative XBridge column (C18, 5
μm; 4.6 mm × 150 mm; Waters, Milford, MA, USA) that was eluted
with a mixture of 0.05 M sodium acetate buffer (pH 5.5) and EtOH
(50:50 v/v) at a flow rate of 1 mL/min. The radiolabeled product was
collected between 12 and 16 min (small difference in retention time
for the different tracers). The collected peak corresponding to the
desired radioligand was then diluted with saline (Mini Plasco, Braun,
Melsungen, Germany) to obtain a final ethanol concentration of 10%,
and the solution was sterile filtered through 0.22 μm membrane filter
(Millex-GV, Millipore, Ireland). Quality control was performed on an
analytical HPLC system consisting of an XBridge C18 3.5 μm column
(3 mm × 100 mm; Waters) eluted with a mixture of 0.05 M NaOAc
buffer (pH 5.5) and MeCN (55:45 v/v) at a flow rate of 0.8 mL/min
(Rt = 4−7 min, small difference in retention time for the different
tracers). UV detection was performed at 254 nm. The six carbon-11
labeled tracers were synthesized with a radiochemical yield of 35−74%
(relative to starting radioactivity of [11C]MeI, nondecay corrected, n >
3 for each tracer). The radiochemical purity as examined using the
above-described analytical HPLC system was >96%, and the average
specific radioactivity was found to be in the range 54−239 GBq/μmol
at end of synthesis (EOS) (n > 3 for each tracer).
1
LCMS: Rt 2.02, m/z 419 [(M + H)]+. H NMR (500 MHz, DMSO-
d6) δ 0.21−0.35 (m, 2 H), 0.45−0.56 (m, 2 H), 1.11−1.22 (m, 1 H),
1.70 (br d, J = 10.7 Hz, 2 H), 2.24−2.40 (m, 2 H), 2.98 (br t, J = 11.8
Hz, 2 H), 3.02 (d, J = 6.9 Hz, 2 H), 3.24 (tt, J = 12.4, 3.3 Hz, 1 H),
3.61 (br d, J = 11.8 Hz, 2 H), 6.63 (td, J = 9.8, 3.9 Hz, 1 H), 6.97 (d, J
= 7.5 Hz, 1 H), 7.07 (td, J = 9.7, 4.9 Hz, 1 H), 8.38 (d, J = 7.2 Hz, 1
H), 9.99 (br s, 1 H).
2-[1-[8-Chloro-3-(cyclopropylmethyl)-1,2,4-triazolo[4,3-a]-
pyridin-7-yl]-4-piperidinyl]-4-fluorophenol (22b). Compound 22b
was synthesized following the same procedure described for
compound 22f. Starting from compound 20b (0.238 g, 0.57 mmol),
the title compound 22b was obtained as an off-white solid (0.065 g,
28.2%); mp 256 °C. C21H22ClFN4O. LCMS: Rt 3.04, m/z 401 [(M +
H)]+ 1H NMR (400 MHz, DMSO-d6) δ 0.21−0.33 (m, 2 H), 0.44−
0.57 (m, 2 H), 1.09−1.22 (m, 1 H), 1.72−1.83 (m, 2 H), 1.81−1.96
(m, 2 H), 2.89−3.13 (m, 5 H), 3.61 (br d, J = 11.8 Hz, 2 H), 6.73−
6.91 (m, 2 H), 6.91−6.99 (m, 1 H), 6.98 (d, J = 7.6 Hz, 1 H), 8.38 (d,
J = 7.4 Hz, 1 H), 9.40 (s, 1 H).
2-[1-[8-Chloro-3-(cyclopropylmethyl)-1,2,4-triazolo[4,3-a]-
pyridin-7-yl]-4-piperidinyl]-3-fluorophenol (22c). Compound 22c
was synthesized following the same procedure described for
compound 22f. Starting from compound 20c (0.124 g, 0.29 mmol),
the title compound 22c was obtained as an off-white solid (0.034 g,
28.3%); mp 242.6 °C C21H22ClFN4O. LCMS: Rt 2.76, m/z 401 [(M +
1
H)]+. H NMR (500 MHz, DMSO-d6) δ 0.21−0.34 (m, 2 H), 0.45−
0.56 (m, 2 H), 1.05−1.21 (m, 1 H), 1.67 (br d, J = 10.7 Hz, 2 H),
2.25−2.35 (m, 2 H), 2.97 (br t, J = 11.7 Hz, 2 H), 3.02 (d, J = 6.6 Hz,
2 H), 3.22 (tt, J = 12.3, 3.3 Hz, 1 H), 3.60 (br d, J = 11.8 Hz, 2 H),
6.56 (dd, J = 10.4, 8.7 Hz, 1 H), 6.67 (d, J = 8.1 Hz, 1 H), 6.97 (d, J =
7.2 Hz, 1 H), 6.99−7.07 (m, 1 H), 8.39 (d, J = 7.5 Hz, 1 H), 9.96 (br s,
1 H).
Biology. Membrane Preparation. CHO cells expressing the
human mGlu2 receptor were grown until 80% confluence, washed
in ice-cold phosphate-buffered saline, and stored at −20 °C until
membrane preparation. After thawing, cells were suspended in 50 mM
Tris-HCl, pH 7.4, and collected through centrifugation for 10 min at
23,500g at 4 °C. Cells were lysed in 5 mM hypotonic Tris-HCl, pH
7.4, and after recentrifugation for 20 min at 30,000g at 4 °C, the pellet
was homogenized with an Ultra Turrax homogenizer in 50 mM Tris-
HCl, pH 7.4. Protein concentrations were measured by the Bio-Rad
protein assay using bovine serum albumin as standard. [35S]GTPγS
binding assay. For [35S]GTPγS measurements, compound and
glutamate were diluted in buffer containing 10 mM HEPES acid, 10
mM HEPES salt, pH 7.4, containing 100 mM NaCl, 3 mM MgCl2, and
10 μM GDP. Membranes were thawed on ice and diluted in the same
buffer, supplemented with 14 μg/mL saponin (final assay concen-
tration of 2 μg/mL saponin). Final assay mixtures contained 7 μg of
membrane protein and were preincubated with compound alone
(determination of agonist effects) or together with an EC20
concentration (4 μM) of glutamate (determination of PAM effects)
for 30 min at 30 °C. [35S]GTPγS was added at a final concentration of
0.1 nM and incubated for another 30 min at 30 °C. Reactions were
terminated by rapid filtration through Unifilter-96 GF/B filter plates
2-[1-[8-Chloro-3-(cyclopropylmethyl)-1,2,4-triazolo[4,3-a]-
pyridin-7-yl]-4-piperidinyl]-3,4-difluorophenol (22g). Compound
22g was synthesized following the same synthetic procedure described
for compound 22f. Starting from 20g (0.142 g, 0.33 mmol), the title
compound 22g was obtained (0.016 g, 11.6%) as an off-white solid;
mp >300 °C. C21H21ClF2N4O. LCMS: Rt 2.85, m/z 419 [(M + H)]+.
1H NMR (500 MHz, DMSO-d6) δ 0.21−0.33 (m, 2 H), 0.44−0.56
(m, 2 H), 1.12−1.21 (m, 1 H), 1.71 (br d, J = 10.7 Hz, 2 H), 2.18−
2.36 (m, 2 H), 2.98 (br t, J = 11.7 Hz, 2 H), 3.02 (d, J = 6.6 Hz, 2 H),
3.19−3.27 (m, 1 H), 3.61 (br d, J = 11.8 Hz, 2 H), 6.60 (dd, J = 9.0,
2.9 Hz, 1 H), 6.98 (d, J = 7.5 Hz, 1 H), 7.04 (q, J = 9.5 Hz, 1 H), 8.39
(d, J = 7.5 Hz, 1 H), 10.10 (br s, 1 H).
2-[1-[8-Chloro-3-(cyclopropylmethyl)-1,2,4-triazolo[4,3-a]-
pyridin-7-yl]-4-piperidinyl]-3,5-difluorophenol (22h). Compound
22h was synthesized following the same procedure described for
22f. Starting from 20h, the title compound 22h (0.129 g, 0.29 mmol)
was obtained as an off-white solid (0.014 g, 11.2%); mp >300 °C.
1
C21H21ClF2N4O. LCMS: Rt 2.97, m/z 419 [(M + H)]+. H NMR
(500 MHz, DMSO-d6) δ 0.22−0.32 (m, 2 H), 0.45−0.56 (m, 2 H),
1.12−1.21 (m, 1 H), 1.66 (br d, J = 10.7 Hz, 2 H), 2.18−2.34 (m, 2
H), 2.96 (br t, J = 11.7 Hz, 2 H), 3.02 (d, J = 6.9 Hz, 2 H), 3.10−3.20
(m, 1 H), 3.59 (br d, J = 11.8 Hz, 2 H), 6.48 (br d, J = 10.4 Hz, 1 H),
L
dx.doi.org/10.1021/jm300912k | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
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(PerkinElmer) using a Unifilter-96 Harvester (PerkinElmer). Filters
were washed three times with ice-cold 10 mM NaH2PO4/10 mM
Na2HPO4, pH 7.4, and filter-bound radioactivity was counted in a
Topcount Microplate Scintillation and Luminescence Counter from
PerkinElmer. Binding Assay. After thawing, membranes from
hmGlu2-CHO cells were homogenized using an Ultra Turrax
homogenizer and suspended in ice-cold binding buffer containing 50
mM Tris-HCl (pH 7.4), 10 mM MgCl2, and 2 mM CaCl2.
Displacement studies were performed using 10 nM of radioligand
[3H]2, JNJ-40068782. Assay mixtures were incubated for 60 min at
room temperature in a volume of 0.5 mL containing 75 μg hmGlu2
CHO membrane protein. Nonspecific binding (about 30% of total
binding) was estimated in the presence of 10 μM JNJ-40264796, a
structurally-related molecule. Filtration was performed using Unifilter-
96 GF/C filters presoaked in 0.1% PEI and a 40-well manifold or 96-
well Brandell harvester 96. After the addition of scintillation liquid,
radioactivity on the filters was counted in a Microplate Scintillation
and Luminescence Counter or Liquid Scintillation Analyzer from
Perkin-Elmer.
Biodistribution Studies and Radiometabolite Analysis. Quantifi-
cation of radioactivity measurements in samples of biodistribution
studies and radiometabolite analysis was done using an automated
gamma counter equipped with a 3-in. NaI(Tl) well crystal coupled to a
multichannel analyzer (Wallac 1480 Wizard, Wallac, Turku, Finland).
The results were corrected for background radiation, physical decay,
and counter dead time. All animal experiments were conducted with
the approval of the institutional ethical committee for conduct of
experiments on animals. Biodistribution Studies. Biodistribution
studies were carried out in healthy male Wistar rats (body weight
200−450 g) at 2, 30, and 60 min pi (n = 3/time point). Rats were
injected with about 22 MBq of the tracer via tail vein under anesthesia
(2.5% isoflurane in O2 at 1 L/min flow rate) and sacrificed by
decapitation at the above specified time points. Blood and major
organs were collected in tared tubes and weighed. The radioactivity in
blood, organs, and other body parts was measured using an automated
gamma counter. For calculation of total radioactivity in blood, blood
mass was assumed to be 7% of the body mass. Plasma radio-
metabolite analysis. After iv administration of about 74 MBq of the
radioligand via tail vein under anesthesia (2.5% isoflurane in O2 at 1 L/
min flow rate), rats were sacrificed by decapitation at 30 min pi (n = 2
for [11C]20b, [11C]20g, [11C]20h; n = 3 for [11C]20f). Blood was
collected in heparin containing tubes (4.5 mL LH PST tubes; BD
vacutainer, BD, Franklin Lakes, NJ, USA) and stored on ice. Next, the
blood was centrifuged for 5 min at 3000 rpm to separate the plasma.
Plasma (0.5 mL) was spiked with 10 μg of the authentic
nonradioactive compound. Plasma was then analyzed with HPLC
(Chromolith C18, 3 mm × 100 mm, Merck) eluted with gradient
mixtures of 0.05 M sodium acetate (pH 5.5) (A) and MeCN (B): 0−4
min: 0% B, flow rate 0.5 mL/min; 4−9 min: linear gradient 0% B to
90% B, flow rate 1 mL/min; 9−12 min: 90% B flow rate 1 mL/min;
12−20 min: linear gradient 90% B to 0% B, flow rate of 0.5 mL/min.
After passing through an in-line UV detector (254 nm), the HPLC
eluate was collected as 1 mL fractions. The radioactivity in all fractions
was measured using an automated gamma counter. Perfused brain
radiometabolite analysis. After administration of about 74 MBq of
the radioligand via tail vein under anesthesia (2.5% isoflurane in O2 at
1 L/min flow rate), rats (n = 1 for [11C]20b, [11C]20g, [11C]20h; n =
2 for [11C]20f) were sacrificed at 30 min pi by administering an
mm, Waters) eluted with a mixture of 0.05 M sodium acetate (pH 5.5)
and MeCN (60:40 v/v) at a flow rate of 0.8 mL/min. The HPLC
eluate was collected as 1-mL fractions after passing through the UV
detector (254 nm), and the radioactivity in the fractions was measured
using an automated gamma counter.
Small-Animal PET Studies. Imaging experiments were performed
on a Focus 220 microPET scanner (Concorde Microsystems,
Knoxville, TN, USA) using male Wistar rats. During all scan sessions,
animals were kept under gas anesthesia (2.5% isoflurane in O2 at 1 L/
min flow rate). Dynamic 90-min-scans were acquired in list mode.
Acquisition data were Fourier rebinned in 27 time frames (4 × 15 s, 4
× 1 min, 5 × 3 min, 14 × 5 min) and reconstructed with Filtered Back
Projection (FBP). The images were spatially normalized to the rat
brain Paxinos coordinate system using affine transformation, allowing
the use of a predefined volumes of interest (VOIs) map. Time−activity
curves (TAC) were generated for striatum, cortex, cerebellum,
hippocampus, thalamus, and total brain for each individual scan,
using PMOD software (v 3.2, PMOD Technologies Ltd.). The
radioactivity concentration in the different brain regions was expressed
as standardized uptake value (SUV) as a function of time π of the
radiotracer by normalization for body weight of the animal and
injected dose. Rats were injected with about 40−53 MBq of high
specific activity formulation of [11C]20f via the tail vein under
isoflurane anesthesia (2.5% in O2 at 1 L/min flow rate). For the
displacement experiment, a solution of JNJ42153605 in 20% (2-
hydroxypropyl)-β-cyclodextrine with 2 equiv of hydrochloric acid was
injected iv at a dose of 1 mg/kg 30 min after radiotracer injection.
ASSOCIATED CONTENT
■
S
* Supporting Information
The different methods used for the LCMS and GCMS
characterization of the compounds described in this manu-
script; the biodistribution of [11C]20f in normal rats at 2, 30,
and 60 min post-tracer-injection; the reconstructed radio-
chromatogram corresponding to a rat plasma analysis at 30 min
postinjection of [11C]20f; and reconstructed radiochromato-
grams corresponding to a rat perfused cerebrum and
cerebellum analysis at 30 min postinjection of [11C]20f. This
material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Tel.: + 34 925 245 780. Fax: + 34 925 245 771. E-mail:
iandres@its.jnj.com.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Jose
́
Manuel Alonso, Luis Font, and Alberto Fontana
of the Analysis & Purification department in Toledo for their
skillful assistance. We also thank Ivan Sannen and Julie Cornelis
of the Laboratory for Radiopharmacy, KU Leuven, for their
skillful help with the biodistribution and radiometabolite
studies, and Ann Van Santvoort, Peter Vermaelen, and Michel
Koole of the department of Nuclear Medicine (KU Leuven) for
their assistance in the small-animal PET studies. We also thank
Heidi Szel, Ilse Biesmans, and Luc Peeters (Janssen R&D,
Neuroscience, Beerse) for their expert assistance with the in
vitro pharmacology studies. Finally, we thank ADDEX
Therapeutics for their collaboration on the mGluR2 PAM
discovery program.
́
overdose of Nembutal (CEVA Sante Animale, 200 mg/kg intra-
peritoneal). When breathing had stopped, the rats were perfused with
saline (Mini Plasco, Braun, Melsungen, Germany) until the liver
turned pale. Brain was isolated, and cerebrum and cerebellum were
separated and homogenized in 3 and 2 mL of MeCN, respectively, for
about 2 min. A volume of 1 mL of this homogenate was diluted with
an equal volume of water, and a part of this homogenate was filtered
through a 0.22 μm filter (Millipore, Bedford, USA). About 0.5 mL of
the filtrate was diluted with 0.1 mL of water and spiked with 10 μg of
authentic nonradioactive compound. The cerebrum/cerebellum
homogenate extracts were then injected onto an HPLC system
consisting of an analytical XBridge column (C18, 3.5 μM, 3 mm × 100
M
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Chem. 2011, 18, 47−68.
ABBREVIATIONS USED
■
BBB, blood-brain barrier; CNS, central nervous system; DCM,
dichloromethane; ID, injected dose; iv, intravenous; mGlu,
metabotropic glutamate; mGluR2, metabotropic glutamate
receptor 2; PAM, positive allosteric modulator; PET, positron
emission tomography; μPET, small-animal PET; pi, post
injection; RP-HPLC, reversed phase-high performance liquid
chromatography; rt, room temperature; SD, standard deviation;
SUV, standardized uptake value; TAC, time activity curve
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