Discovery of 3-cyclopropylmethyl-7-(4-phenylpiperidin-1-yl)-8- trifluoromethyl[1,2,4]triazolo[4,3- a ]pyridine (JNJ-42153605): A positive allosteric modulator of the metabotropic glutamate 2 receptor

Article abstract of DOI:10.1021/jm3010724

Advanced leads from a series of 1,2,4-triazolo[4,3-a]pyridines with mGlu2 receptor PAM activity are reported. By modification of the analogous imidazo[1,2-a]pyridine series, the newly reported leads have improved potency, in vitro ADMET, and hERG as well

Full text of DOI:10.1021/jm3010724

Article
pubs.acs.org/jmc
Discovery of 3‑Cyclopropylmethyl-7-(4-phenylpiperidin-1-yl)-8-
trifluoromethyl[1,2,4]triazolo[4,3‑a]pyridine (JNJ-42153605): A
Positive Allosteric Modulator of the Metabotropic Glutamate 2
Receptor
Jose María Cid,*,† Gary Tresadern,‡ Juan Antonio Vega,† Ana Isabel de Lucas,† Encarnacion Matesanz,†
́
Laura Iturrino,† María Lourdes Linares,† Aranzazu Garcia,† Jose Ignacio Andres,† Gregor J. Macdonald,§
́
́
́
Daniel Oehlrich,§ Hilde Lavreysen,∥ Anton Megens,∥ Abdellah Ahnaou,∥ Wilhelmus Drinkenburg,∥
Claire Mackie,⊥ Stefan Pype,# David Gallacher,∞ and Andres A. Trabanco†
́
†Neuroscience Medicinal Chemistry and ‡Molecular Sciences, Janssen Research & Development, Janssen-Cilag S.A., Jarama 75, 45007
Toledo, Spain
§Neuroscience Medicinal Chemistry, ∥Neuroscience Biology, ⊥Discovery ADME/Tox, #Project Management Office, and ∞Center of
Excellence for Cardiovascular Safety Research & Mechanistic Pharmacology, Janssen Research & Development, Turnhoutseweg 30,
B-2340, Beerse, Belgium
S
* Supporting Information
ABSTRACT: Advanced leads from a series of 1,2,4-triazolo-
[4,3-a]pyridines with mGlu2 receptor PAM activity are
reported. By modification of the analogous imidazo[1,2-
a]pyridine series, the newly reported leads have improved
potency, in vitro ADMET, and hERG as well as good in vivo
PK profile. The optimization of the series focused on
improving metabolic stability while controlling lipophilicity
by introducing small modifications to the scaffold substituents. Analysis of this series combined with our previously reported
mGlu2 receptor PAMs showed how lipophilic ligand efficiency was improved during the course of the program. Among the best
compounds, example 20 (JNJ-42153605) showed a central in vivo efficacy by inhibition of REM sleep state at a dose of 3 mg/kg
po in the rat sleep−wake EEG paradigm, a phenomenon shown earlier to be mGlu2 mediated. In mice, compound 20 reversed
PCP-induced hyperlocomotion with an ED50 of 5.4 mg/kg sc, indicative of antipsychotic activity.
The eight mGlu receptors are classified into three groups
based upon sequence homology, pharmacological profile, and
preferential signal transduction pathway.11,12 The mGlu2
receptor, which belongs to the group II subfamily along with
mGlu3, has proven to be of particular importance in
neuropharmacology. Preferentially expressed on presynaptic
nerve terminals mGlu2 receptor negatively modulates gluta-
mate and GABA release and is widely distributed in the brain.13
High levels of mGlu2 receptors are seen in brain areas such as
prefrontal cortex, hippocampus, and amygdala where glutamate
hyperfunction may be implicated in disorders and diseases such
as anxiety and schizophrenia.14−17 It is therefore considered
that activation of group II mGlu receptors may offer anxiolytic
and/or antipsychotic effects.2,4,18
INTRODUCTION
■
Glutamate is considered the major excitatory neurotransmitter
in the mammalian central nervous system (CNS) of
vertebrates, contributing excitatory input to as many as half
of all central synapses. It plays a major role in numerous
physiological functions such as learning and memory but also
sensory perception, development of synaptic plasticity, motor
control, respiration, and regulation of cardiovascular function.
An imbalance in glutamatergic neurotransmission is believed to
be at the center of various neurological and psychiatric
diseases.1−7 Glutamate acts via activation of ionotropic or
metabotropic glutamate receptors (iGlu or mGlu). The former
includes ion channels such as NMDA, AMPA, and kainate
receptors responsible for fast excitatory transmission, whereas
the latter are a family of eight class C G-protein-coupled
receptors contributing to the fine-tuning of synaptic effi-
cacy.1,8,9 Glutamate activates mGlu receptors through binding
at an orthosteric site in the amino terminal domain. This
induces a conformational change in the receptor, resulting in
activation of the G-protein and intracellular signaling path-
ways.10
The first generation pharmacological tools were conforma-
tionally constrained analogues of glutamate acting as agonists at
both mGlu2 and mGlu3 receptors.19 Preclinical studies
performed with mGlu2/3 receptor agonists such as (+)-2-
aminobicyclo[3.1.0]hexane-2,6-dicarboxylic acid (LY354740)
Received: July 23, 2012
Published: October 16, 2012
© 2012 American Chemical Society
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Figure 1. (a) Structures of mGlu2 PAM reported molecules (1−3). (b) Evolution from pyridone series to 1,2,4-triazolopyridine series.
showed robust anxiolytic-like20 and antipsychotic-like effects in
animal models.21 Clinical data strengthen the rationale for
group II receptor intervention as a treatment for schizophrenia.
(1R,4S,5S,6S)-2-Thiabicyclo[3.1.0]hexane-4,6-dicarboxylic acid
4-[(2S)-2-amino-4-(methylthio)-1-oxobutyl]amino-2,2-dioxide
monohydrate (LY2140023), the oral prodrug of the mGlu2/3
agonist (−)-(1R,4S,5S,6S)-4-amino-2-sulfonylbicyclo[3.1.0]-
hexane-4,6-dicarboxylic acid (LY404039), showed improve-
ment of both positive and negative symptoms in schizophrenia
in a double-blind placebo-controlled study in schizophrenic
patients.22 Importantly, treatment with LY2140023 was safe
and well-tolerated. It did not affect prolactin levels nor induce
extrapyramidal symptoms or weight gain, in contrast to many of
the current atypical antipsychotic medications.23
Given the promise offered by mGlu2 receptor activation,
there is increased interest in identifying small molecules that
activate the receptor. A preferred approach is via positive
allosteric modulators (PAMs) that bind at an alternative site to
orthosteric agonists. This strategy offers many potential
benefits: avoidance of amino acid like motifs that can be
detrimental for CNS penetration, opportunity for greater
chemical diversity, potential for improved selectivity, as the
orthosteric site is highly conserved, and reduced likelihood of
receptor desensitization, as PAMs typically activate the receptor
only in the presence of glutamate and will respond to
physiological neurotransmitter fluctuations.24−26 The number
of reports on mGlu2 receptor PAM chemical series has
increased over the past years.27 Within our laboratories we have
pursued a series of isoquinolones28,29 and 1,5-pyridones30,31 (1
and 2, respectively, Figure 1a). Those compounds were
reported to be selective and moderately potent mGlu2 receptor
PAMs; however, balancing potency and metabolic stability
remained a challenge. We have very recently reported
structurally related 3-cyanopyridones (3, Figure 1a).32−34
This scaffold delivered compounds with improved potency
and druglike properties, and the latest results will soon be
reported elsewhere.
were employed to help identify and prioritize new chemical
scaffolds. An overlay model revealed shared pharmacophoric
features between reported series which were expected to be
required for alternative motifs. On the basis of this model and
methods of shape and electrostatic similarity, a novel series of
imidazopyridines35−38 was discovered. The imidazopyridine
serves as a surrogate for the pyridone core, in which the oxygen
lone pair is replaced by a nitrogen lone pair of the imidazole
ring, the ring centers of pyridone and pyridine overlap well, and
the pendent groups are distributed along a similar vector. The
series proved capable of delivering compounds with appealing
pharmacological profile. However, exploration was mainly
limited to compounds having trifluoroethyl as R1 (Figure 1b),
which was one of the few groups conferring acceptable potency
and metabolic stability. Therefore, we considered improving
metabolic stability by reducing lipophilicity with the introduc-
tion of an additional nitrogen atom, causing little disruption to
overall molecular shape and features. This delivered the new
1,2,4-triazole template, which is the subject of this report
(Figure 1b).
First, to test the viability of the imidazopyridine to
triazolopyridine swap hypothesis, an initial set of imidazopyr-
idines (4, 6, and 8) and their corresponding 1,2,4-triazolo[4,3-
a]pyridines pairs (5, 7, and 9) were prepared (Table 1). In this
set of compounds, both phenylpiperidine and cyano were
selected as the most suitable substitutions to allow complete
comparisons across the series. The reduction of the lipophilicity
in the triazolopyridines did indeed correlate with an improve-
ment of the metabolic stability of the compounds in both
human and rat species. Interestingly, the in vitro potency was
also retained. Herein we report the synthesis and structure−
activity relationship (SAR) development of novel triazolopyr-
idines39−41 as the culmination of the exploration around the
imidazopyridine series. This exploration led to the identification
of 3-cyclopropylmethyl-7-(4-phenyl-piperidin-1-yl)-8-
trifluoromethyl[1,2,4]triazolo[4,3-a]pyridine (JNJ-42153605,
20), one of the most potent and selective mGlu2 PAMs
identified in our program. Compound 20 influenced rat sleep−
wake organization by decreasing REM sleep and reversed PCP-
induced locomotor activity in mice. Hence, compound 20 is
seen as a powerful pharmacological tool to gain more insight on
These 3-cyanopyridone series were derived from HTS
performed at the start of our program. However, with a need
to expand the SAR, identify new chemotypes, and also gain
more insight on the target, molecular modeling approaches
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which was converted into the target 8-cyanotriazolopyridines by
cyclization with several acid derivatives (orthoesters, acid
chlorides, and carboxylic acids) under the reaction conditions
shown in Scheme 1.
Table 1. Comparative Data for a Representative Set of
Imidazopyridines (4, 6, 8) and Triazolopyridines (5, 7, 9)
The triazolopyridines 14−17 substituted with a chlorine
atom at position C-8 were prepared following a route similar to
that described in Scheme 1. In this case, reaction of 2,3-
dichloro-4-iodopyridine 34 with the piperidine derivatives 30
and 35 followed by nucleophilic substitution of the C-2
chlorine in 36a,b with hydrazine led to the 2-pyridylhydrazine
derivatives 37a,b (Scheme 2). Subsequent cyclization of 37a,b
with the corresponding acid derivatives resulted in the target
molecules 14−17.
Derivatives 18 and 19 were prepared by Buchwald coupling
of the corresponding amine (4139 and 42) with the 8-chloro-7-
iodotriazolopyridine 40 (Scheme 3). The synthesis of 40 was
achieved in three steps by reaction of hydrazine with 34
followed by acylation and subsequent cyclodehydration of the
formed intermediate 39.
mGlu2
mGlu2
maxa
(%)
EC
50a
E
HLM RLM
b b
compd
X
R1
clogP
(nM)
(%)
(%)
4
5
6
7
8
9
C
N
C
N
C
N
Et
4.41
3.38
4.49
3.91
4.35
3.33
501
696
155
299
125
56
225
247
254
272
259
277
74
11
74
25
24
9
82
7
Et
nPr
nPr
81
40
41
10
CH2CF3
CH2CF3
a
b
Values are the mean of at least two experiments. HLM and RLM
data refer to % of compound metabolized after incubation with human
The C-8 trifluoromethyl substituted triazolopyridines,
bearing a cyclopropylmethyl substituent in the triazole ring
(20, 24−28), were prepared as shown in Scheme 4. Chlorine
displacement in 2,4-dichloro-3-trifluoromethylpyridine 43 with
benzyl alcohol yielded 44 with complete regioselectivity.
Introduction of hydrazine in 44 to give 45 followed by
treatment with POCl3 led to the 7-chlorotriazolopyridine 46.
Nucleophilic substitution of the activated choro atom in 46
with several piperidines (30, 47, 48) and cyclic amines
(R′R″NH = tetrahydroisoquinoline, dihydroisoindoline, and
rac-3-phenylpyrrolidine) led to the target compounds 20 and
24−28.
or rat liver microsomes for 15 min at 5 μM.
the target as well as a suitable candidate for further
development.
CHEMISTRY
■
Triazolopyridines 5, 7, 9−28 were synthesized following the
routes outlined in Schemes 1−5. For the exploration of the R1
group in cyano substituted triazolopyridines (5, 7, 9−12), 3-
cyano-2−4-dibromopyridine (29) was chosen as starting
material. Reaction of 29 with 4-phenylpiperidine (30) yielded
the C-4 substitution product 31 with complete regioselectivity.
Treatment of 31 with hydrazine under microwave heating led
to the key 2-pyridylhydrazine intermediate 32 (Scheme 1),
Finally, compounds 21−23 were obtained after nucleophilic
substitution of 39 with the piperidines 30 and 35 followed by
a
Scheme 1
a
Reagents and conditions: (a) NaH, DMF, rt, 1 h; (b) NH2NH2, THF, 160 °C, 15 min, microwave; (c) EtC(OEt)3, xylenes, 180 °C, 2 h; (d) (i)
R1COCl, Et3N, CH2Cl2 or CHCl3, rt, 1−16 h; (ii) POCl3, CH3CN, 150 °C, 10 min, microwave; (e) CF3CH2CO2H, DIPEA, CCl3CN, PS-PPh3, 1,2-
dichloroethane, 150 °C, 18 min, microwave; (f) HC(OEt)3, xylenes, 180 °C, 2 h; (g) CH2O, pyrrolidine, AcOH, 80 °C, 16 h.
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a
Scheme 2
a
Reagents and conditions: (a) DIPEA, CH3CN, 110 °C, 16 h; (b) NH2NH2, EtOH, 160 °C, 20 min, microwave; (c) (i) R1COCl, Et3N, CH2Cl2, rt,
1 h; (ii) POCl3, CH3CN, 150 °C, 10 min, microwave; (d) Et(OEt)3, xylenes, 180 °C, 2 h; (e) (i) CF3CH2CO2H, Et3N, CH2Cl2, rt, 1 h; (ii)
CCl3CN, PS-Ph3P, DIPEA, 1,2-dichloroethane, 100 °C, 16 h.
a
Scheme 3
a
Reagents and conditions: (a) NH2NH2, 1,4-dioxane, 70 °C, 16 h; (b) CyPrCOCl, Et3N, CH2Cl2, rt, 1 h; (c) 160 °C, 180 min; (d) Pd(AcO)2,
BINAP, Cs2CO3, toluene, 95 °C, 16 h.
introduction of the hydrazine and standard cyclization
processes with the corresponding acid derivatives.
Interestingly, α-branching to the triazole ring was detrimental
for mGlu2 PAM activity as shown with compound 11 (1230
nM). More diverse R1 substitutions were explored as
exemplified by the ether (12) and amine (13) analogues.
These compounds, although metabolically stable, displayed
decreased activity (468 nM for 12 and 1120 nM for 13). This
suggests that polar groups are not well tolerated for activity in
the R1 region, which is in agreement with the moderate activity
observed for compound 5 which has a less lipophilic R1 alkyl
chain. Of note, the cyanotriazolopyridines 5 and 9 showed
weak inhibition of the hERG channel at 3 μM in a patch clamp
assay, 20% and 33%, respectively.
RESULTS AND DISCUSSION
■
Representative compounds illustrating the SAR for this
triazolopyridine series along with functional activity, metabolic
stability, and hERG data are shown in Table 2. The
triazolopyridine exploration began with the elaboration of the
R1 substitution. For these variations the cyano and phenyl-
piperidine substituents at R2 and R3 were left constant (Table 2,
compounds 5, 7, 9−13). Initial SAR showed that the R1
substituent has an important effect on the potency (EC50 42).
Lengthening the alkyl chain from an ethyl to n-propyl led to a
more than 2-fold increase in potency as exemplified with
compounds 5 (696 nM) and 7 (299 nM). However, the
increased size of the alkyl chain was detrimental for the
metabolic stability in rat liver microsomes (RLMs) which
increased from 7% (5) to 40% metabolized (7). A more
balanced profile was found for compounds 9 and 10 which
combined better metabolic stability with increased potency.
Next, our exploration focused on the R2 substituent and was
guided by SAR previously established for the imidazopyridine
series.35 In that case chlorine appeared to be beneficial for
potency with an approximate 2-fold increase compared to the
cyano group. The increase in activity correlated with the 0.9 log
unit increase in lipophilicity by moving from cyano to chloro.
This same approach was attempted in the triazolopyridine
series; however, in this case the effect of the chlorine on the
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a
piperidine. Thus, compound 17, having a 4-fluoro-4-phenyl-
piperidine R2 substituent, showed improvement of the hERG
profile (17, 42% vs 14, 71%). This 4,4-disubstitution of the
piperidine ring also led to a 2-fold decrease in potency and
slightly higher metabolic turnover. Nevertheless 17 was
considered to have an overall better profile than 14. The
same trends were found with the 4-phenyl-4-trifluoromethylpi-
peridine analogue 18, which also showed a remarkable
reduction of hERG inhibition when compared to the
corresponding pair 15 (18, 42% vs 15, 99%). Although a
similar 2-fold decrease in functional activity was found for 18,
its metabolic profile was similar to that of 15. Replacement of
the piperidine R2 substituent by a piperazine led to a 9-fold
decrease in mGlu2 PAM activity (19, 944 nM vs 15, 102 nM).
A breakthrough was achieved by the identification of the CF3
group as an optimal replacement for the R2 cyano and chloro
substituents. A significant increase in potency was found over
the R2 cyano and chloro subclasses (20 vs 10 or 15 and 21 vs 5
and 16). In particular, compound 20 was found to be the most
potent mGlu2 PAM identified so far, with an EC50 of 17 nM
and Emax of 285%.43 This increase in potency promoted by the
R2 CF3 substituent was consistent across several R1 and R3
variations.39−41 In addition the positive effect of the CF3 group
was accompanied by acceptable metabolic stability and reduced
hERG inhibition.
Fine tuning around the structure of 20 did not result in
compounds with an improved profile. For instance, shorter
(21) and longer (22) R1 alkyl groups were tolerated in terms of
potency and metabolic stability with the more lipophilic 22
being more potent than 21 (21, 116 nM; 22, 24 nM).
However, lengthening the alkyl chain had a detrimental effect
on hERG inhibition (20, 56% vs 22, 91%). Opposite the
finding in the R3 chloro subclass (17), the introduction of
fluorine at the C-4 position of the piperidine did not result in
an improved hERG profile for the trifluoromethyltriazolopyr-
idine 23 (23, 84% vs 20, 57%). Fused variations of the 4-
phenylpiperidine, such as 24 and 25, turned out to be
detrimental for activity (149 and 400 nM, respectively) as
well as for metabolic stability. Ring contraction of the
piperidine into pyrrolidine 26 led to an 80-fold drop in
mGlu2 PAM potency together with worse metabolic stability.
Finally, SAR on the aryl substituent of the piperidine ring in 20
Scheme 4
a
Reagents and conditions: (a) BnOH, NaH, DMF, rt, 1 h; (b)
NH2NH2, 1,4-dioxane, 160 °C, 30 min, microwave; (c) (i) CyPrCOCl,
Et3N, CH2Cl2, rt, 1 h; (ii) POCl3, CH3CN, 150 °C, 5 min, microwave;
(d) amine, DIPEA, CH3CN, 90−180 °C, 10 min to 24 h.
potency was not as consistent as for the imidazopyridine series.
While the chloro analogue 16 showed a 2-fold increase
compared to its cyano pair 5 (386 nM vs 696 nM, respectively),
other chloro analogues (14, 15) displayed potency comparable
to that of their equivalent cyano versions (9, 10).
Unfortunately, chloro analogues were prone to be more
metabolically unstable than cyano analogues. Furthermore,
chlorine seemed to be detrimental for hERG liability (16, 43%
vs 5, 20% or 14, 71% vs 9, 33%), potentially a consequence of
the increased lipophilicity.
Interestingly, the increase in hERG inhibition caused by the
chlorine could be mitigated in some analogues by the
introduction of additional groups at the 4-position of the 4-
a
Scheme 5
a
Reagents and conditions: (a) 30, NaH, DMF, rt, 1 h or 35, DIPEA, CH3CN, 110 °C, 4 h; (b) NH2NH2, THF, 160 °C, 45 min, microwave; (c) (i)
R1COCl, Et3N, CH2Cl2, rt, 1 h; (ii) POCl3, MeCN, 150 °C, 10 min; (d) (i) CyPrCOCl, Et3N, CH2Cl2, rt, 1 h; (ii) CCl3CN, PS-Ph3P, PS-DIPEA,
1,2-dichloroethane, 150 °C, 10 min, microwave.
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Table 2. Functional Activity, Metabolic Stability, and hERG Data of Representative mGlu2 Receptor PAMs 5, 7, 9−28
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Table 2. continued
a
b
c
Values are the mean of at least two experiments. HLM and RLM data refer to % of compound metabolized after 15 min at 5 μM. Experiments
were performed using HEK293 cells stably transfected with the hERG potassium channel. Whole-cell currents were recorded with an automated
patch-clamp system (PatchXpress 7000A). Three concentrations of compound were tested (0.1, 0.3, and 3 μM; n = 3) and compared to vehicle
d
(0.1% DMSO; n = 3). Data are presented as % inhibition at the highest concentration tested (3 μM). Not tested.
Figure 2. (a) mGlu2 PAM pEC50 activity versus clogP (Biobyte). (b) mGlu2 PAM pEC50 activity versus log D at pH 7.4 (ACD, version 12.0).
Molecules are color-coded by order of registration in our internal database, the earliest compounds being blue and later compounds red. Plots
combine data generated from several previously reported series: pyridones, isoquinolones, imidazopyridines, and triazolopyridines reported here.
Inactive compounds for which exact pEC50 could not be determined because of concentration limits were removed from the analysis.
is illustrated with compounds 27 and 28, showing that
increased lipophilicity is well tolerated for activity (27, 10
nM) while detrimental for hERG inhibition (91%). Increased
polarity such as in 28 led to compounds with decreased activity
(28, 519 nM). In summary, a good understanding of the SAR
and the effects of lipophilicity led to compounds with optimal
in vitro profile combining potency, metabolic stability in both
human and rat species, and acceptable preliminary cardiovas-
cular profile.
As discussed here and in recent articles describing other
series,32,35,36 we have sought to balance primary in vitro activity
and lipophilicity. The detrimental effects of increased lip-
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Figure 3. (a) Plot of LLE versus MW. (b) Plot of LELP versus MW. Molecules are color-coded by order of registration in our internal database, the
earliest compounds being blue and later compounds red. Molecule 20 is highlighted in green. Plots combine data generated from several previously
reported series: pyridones, isoquinolones, imidazopyridines, and triazolopyridines reported here. Inactive compounds for which exact pEC50 could
not be determined because of concentration limits were removed from the analysis. To account for basic compounds, ACDLabs, version 12.0, log D
at pH 7.4 was used for LLE and LELP calculations.
Table 3. Effect of CN, Cl, and CF3 Substitution on Brain and Plasma Levels after 1 h of a Single Dose at 10 mg/kg in the Swiss
Mouse
a
b
c
Data are expressed as the geometric mean values of at least two runs. clogP calculated with Biobyte software. Compound dosed po in 0.5%
d
Methocel suspension. Compound dosed sc in 1 mg/mL in 20% HP-β-CD + HCl solution at pH 4.
ophilicity on many ADMET parameters44 and druglikeness are
well-known.45 Figure 2 shows mGlu2 PAM pEC50 activity
versus calculated log P and log D (pH 7.4) for molecules from
multiple chemical series pursued within our mGlu2 PAM
program. The plots include examples from previously reported
pyridone, isoquinolone, and imidazopyridine series as well as
the triazolopyridines reported herein. Such plots provide an
interesting way to analyze compounds; for instance, those in
the top left quadrant would be preferred because they are most
active while having lower clogP. The molecules in Figure 2 are
color-coded according to the rank order in which they were
registered in our database, reflecting the progression of the
project over time: blue being the oldest and red the newest
registered compounds. As might be expected for small-
molecule target interactions, clogP shows a weak correlation
with in vitro activity, with a Pearson correlation coefficient of
0.36 (r2 = 0.13). This highlights the difficulty that was faced
throughout the program to maintain lipophilicity in check. It
can be seen that potency improved during the course of the
program, with later compounds (red) generally having higher
activity compared to earlier examples (blue). The Pearson
correlation coefficient between the mGlu2 PAM pEC50 and
compound registration rank order was 0.51. This is reflected in
the SAR in Table 2, which shows more potent examples than
previously reported, for instance, compound 20. It appears that
later molecules, those colored red, have higher clogP,
suggesting that improved potency arose at the cost of increased
lipophilicity. However, among the more recent compounds
there was a large set of intentionally designed basic molecules.
Therefore, for this data set the calculated log D at pH 7.4 is a
better measure for lipophilicity (Figure 2b). In this case, for
compounds that were registered later log D values were lower,
suggesting reduced apparent lipophilicity and a more optimal
potency lipophilicity balance.
Parameters such as lipophilic ligand efficiency (LLE, pEC50
minus log D)45 and lipophilicity-corrected ligand efficiency
(LELP, ratio log D/LE)46 have emerged as useful tools for lead
optimization to address and monitor the lipophilicity and
potency balance.47 We examined how these parameters evolved
for molecules synthesized during the project (Figure 3).
Interestingly, both LLE and LELP improved over the course
of the program with the newer molecules (red) tending to have
higher LLE and lower LELP values. LLE values greater than 5
are typically preferred, and some of the later molecules reach
such levels, with compound 20 having an LLE of 5.2.48 LELP
values, which are more suitable for optimization of fragments,
are preferred to be lower. Although there are limitations for
molecules with log D < 1, it can be seen that there were many
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a
Table 4. PK Data for Compounds 17, 18, 20, and 21 in Rat after Oral and Intravenous Administration
b
b
b
b
b
b
c
compd
Cl (mL min−1 kg−1
)
T1/2 (h)
Vdss (L/kg)
Tmax (h)
AUC0−inf (ng·h/mL)
Cmax (ng/mL)
F (%)
B/P
17
18
20
21
18
77
35
53
2.6
2.1
2.7
1.9
1.9
5.1
1.4
1.4
1.7
1.3
0.5
0.7
5987
1159
1804
1009
807
289
482
63
53
36
35
1.4
1.6
1.4
1.3
359
a
b
Study in male Sprague−Dawley rat dosed at 10 mg/kg po and 2.5 mg/kg iv, formulated in 20% HP-β-CD at pH 4. Values are the mean of three
c
animals. Ratios calculated after 1 h of a single dose at 10 mg/kg sc in 1 mg/mL in 20% HP-β-CD + HCl solution at pH 4 in the Swiss mouse.
Table 5. Overview of Compound 20
physicochemical properties
in vitro
MW = 440
exptl pKa = 4.1
exptl log P = 4.4 at pH 7
mGlu2 PAM EC50 = 17 nM; Emax = 67%
mGlu2 Ago EC50 = 270 nM; Emax = 285%
mGlu2 PAM binding57 IC50 = 15 nM
selectivity (mGlu1,3−8) of >100-fold vs mGlu2 PAM
selectivity (CEREP)49 of >50-fold vs mGlu2 PAM
solubility of <0.004 mg/mL in water and 0.995 mg/mL 20% HP-β-CD at pH 4
plasma protein binding (%) of 99.82 (h), 99.65 (r)
ADME profile
a
metabolism (%) of 23 (HLM), 25 (RLM)
CYP450 inhibition of 1A2, 2D6, 3A4 (>30 μM), 2C9 (11 μM), 2C19 (20 μM)
permeability (LLC-PK1 cells, ABCB1) AB/BA Papp × 10 cm s−1 = 12/6
Cl = 35 mL min−1 kg−1; Vdss = 1.4 L/kg; Tmax = 0.5 h; T1/2 = 2.7 h
AUC0−inf = 1804 ng·h/mL; Cmax = 482 ng/mL; F = 35%; B/P = 1.4
Cl = 29 mL min−1 kg−1; Vdss = 1.3 L/kg; Tmax = 0.5 h; T1/2 = 0.8−1.1 h
AUC0−inf = 531−957 ng·h/mL; Cmax = 218−472 ng/mL; F = 18−33%
LAD = 3 mg/kg (free plasma concn of 0.14 ng/mL)
rat PK: 2.5 mg/kg iv, 10 mg/kg po
dog PK: 1 mg/kg iv, 2.5 mg/kg po
swEEG rat po
PCP-LMA mice sc
CV safety
ED50 = 5.4 mg/kg (free plasma concn of 2.6 ng/mL)
b
d
hERG PC (% inhat 3 μM) = 57%; NOEL = 0.1 μM; margin = 285
d
rabbit purkinje fiber; NOEL = 0.075 μM; margin = 214
d
anesthetized guinea pig; NOEL = 9000 ng/mL; margin = 225
d
anesthetized dog; NOEL = 6625 ng/mL; margin = 165
genotoxicity
Ames TA98:58 clean up to 125 mg/mL
c
in vitro micronucleus: clean up to 150 μg/mL
a
b
HLM and RLM data refer to % of compound metabolized after 15 min at 5 μM. Experiments were performed using HEK293 cells stably
transfected with the hERG potassium channel. Whole-cell currents were recorded with an automated patch-clamp system (PatchXpress7000A).
Three concentrations of compound were tested (0.1, 0.3, and 3 μM; n = 3) and compared with vehicle (0.1% DMSO; n = 3). Data are presented as
c
percent inhibition at the highest concentration tested (3 μM). Study conducted in human lymphoblastoid TK6 cell line in the presence or absence
d
of metabolic activation (rat liver S9). Based on efficacy free concentration in sw-EEG model (0.14 ng/mL).
compounds with preferred values less than 10; for instance,
compound 20 had LELP of 9.8. The retrospective analysis of
these parameters showing their improvement throughout lead
optimization suggests that they can be useful for guiding
medicinal chemistry decision making.
We next investigated the ability of a representative set of
compounds to cross the blood−brain barrier (BBB). Plasma
and brain levels measured 1 h after dosing at 10 mg/kg are
listed in Table 3. The cyano subclass (5) was much less brain
penetrant (B/P = 0.2) than chloro or CF3 subclasses (6.5- to 9-
fold higher B/P ratios for 14, 15, and 20). This improvement
for the chloro and CF3 subseries may well be explained by a
reduction of the TPSA and/or higher lipophilicity as shown in
Table 3.
To ascertain the druglike properties of the series, further in
vivo PK work was conducted with the most promising
compounds (17, 18, 20, and 21). Data for relevant PK
parameters are shown in Table 4. The most optimal PK data
were obtained for compounds 17 and 20 with moderate to
good plasma exposures after oral (po) administration (AUC of
5987 and 1804 ng·h/mL, respectively) combined with
acceptable intravenous (iv) PK parameters, resulting in
moderate to good bioavailability (F). In contrast, compounds
18 and 21 had a less favorable PK, with higher clearance (Cl)
after iv dosing and shorter half-lives (T1/2) resulting in more
limited plasma exposures.
On the basis of these results, compound 20 showed the best
overall profile among all analogues prepared, combining an
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Figure 4. Effects of oral administration of 20 (3 mg/kg) or vehicle (20% CD + 2H2T) on sleep−wake organization in rats during 20 consecutive
hours. Mean percentage of occurrence per minute is indicated for each sleep−wake state. Small bar charts indicate amounts of vigilance states in min
(plus SEM) during the first 2 h postadministration: ROL, REM sleep onset latency; ∗, p < 0.05; Wilcoxon−Mann−Whitney rank sum tests
compared with vehicle values.
excellent mGlu2 PAM activity (15 nM) with acceptable PK
profile and brain penetration. In addition, 20 was assessed for
its selectivity for the mGlu2 receptor (data shown in Table 5)
and was found to not have agonist or antagonist activity toward
other mGlu receptor subtypes up to 30 μM (>50-fold vs
mGluR2) and also showed no or negligible affinity or activity at
any of the targets in the CEREP49panel of receptors (>100-fold
selectivity for mGlu2 receptor). Therefore, 20 is a selective
mGlu2 receptor PAM. Altogether, compound 20 represented
suitable candidate to progress to further studies including in
vivo pharmacology.
The link between sleep abnormalities and neurological and
psychiatric disorders is widely reported. For instance,
disturbances in rapid eye movement (REM) sleep are
frequently seen in patients with major depressive disorder,
and many clinically effective antidepressants produce common
changes in sleep pattern such as reduced REM sleep and REM
sleep onset latency. Glutamate concentrations fluctuate across
different vigilance states, suggesting that glutamate in the
forebrain may play a role in the sleep process.50 Given the
relationship between the mechanism of sleep and glutamate
signaling, sleep−wake neurophysiological measures can offer a
highly reliable, sensitive, and translational index of brain activity
and target engagement. We have previously shown that mGlu2
receptor activation with the agonist LY354740 and PAM 3′-
([[2-cyclopentyl-6,7-dimethyl-1-oxo-2,3-dihydro-1H-inden-5-
yl]oxy]methyl)biphenyl-4-carboxylic acid (BINA) elicited
common changes in the rodent's sleep−wake pattern (i.e.,
selectively suppress REM sleep), which supports an important
role for the mGlu2 receptor in the regulation of REM sleep51
and that combining both compounds produces synergistic
effects. Subsequently we reported similar results for examples
from our 1,4-pyridone and imidazopyridine series.32,36 In
addition, reports from groups elsewhere confirm that mGlu2
activation by agonists or allosteric potentiators has consistent
and robust effects seen in sleep−wake EEG.52−54 Therefore, we
have evaluated the central activity of 20 in the sw-EEG
paradigm, and the present results were in accordance with our
earlier findings. Accordingly, 20 at a dose of 3 mg/kg (po) was
centrally active and suppressed REM sleep during the first 2 h
after administration without clear effects on the other sleep−
wake stages (Figure 4, bottom right panel). In addition, 20
prolonged REM sleep onset latency (Figure 4, bottom right
panel) whereas no indications of sleep fragmentation were
revealed by examination of the total number of transitions from
sleep states toward waking (data not shown). On the basis of
plasma protein binding (data shown in Table 5), the active
dose of 3 mg/kg po corresponded to an approximate free
concentration in plasma of 0.14 ng/mL.55
Compound 20 was further evaluated for its effect on
phencyclidine-induced hyperlocomotion (PCP-LMA) in mice.
The PCP-LMA model is based on the evidence that the
noncompetitive NMDA receptor channel blocker phencycli-
dine (PCP) is capable of evoking schizophrenia-like symptoms
in healthy subjects and worsens psychosis in schizophrenic
patients,56 and hence, PCP is widely used in animal models of
psychosis. In rodents, PCP causes an increase in motor activity.
As this induced behavior may be linked to increased glutamate
release, it is speculated that mGlu2 receptor activation may
block these specific PCP-mediated effects. Therefore, this
represents a useful model of psychosis and serves as the basis
for the “glutamate hypothesis of schizophrenia”. We have
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administered 20 in mice pretreated with PCP and evaluated its
effect on PCP-induced hyperlocomotion. As shown in Figure 5,
CONCLUSION
■
In summary, optimization of the previously reported
imidazopyridine series led to the discovery of a novel series
of 1,2,4-triazolo[4,3-a]pyridines. Lowering lipophilicity of the
central core by the introduction of an additional nitrogen was
key to delivering compounds with better metabolic stability
while the potency remained comparable. These promising
results were followed by sequential optimizations of hERG and
PK profiles that led to compound 20 as the best
triazolopyridine derivative identified. 20 turned out to be
among the most potent mGlu2 receptor PAMs prepared so far,
displaying very high in vitro potency of 17 nM and good
selectivity for the mGlu2 receptor (>50-fold). Compound 20
showed an acceptable PK profile in rodent and non-rodent
species. Likewise, it displayed good in vivo activity in models
sensitive to mGlu2 modulation such as sw-EEG and PCP-LMA,
both at doses remarkably better compared to leads previously
reported in our program. Furthermore, there were no safety
concerns identified that would hamper the progression of the
compound. Collectively, compound 20 is considered a very
attractive pharmacological agent to advance our knowledge of
how mGlu2 receptor modulation may reduce the severity of
diseases where disruption of glutamatergic transmission is
pronounced and as an excellent candidate to progress toward
clinical settings.
Figure 5. Effect of 20 on PCP-induced locomotor activity in mice. The
gray horizontal bar represents the activity level measured in solvent
treated control mice not challenged with PCP.
compound 20 dose-dependently and significantly attenuated
the increase in locomotor activity induced by PCP (5 mg/kg
sc) with an ED50 of 5.4 mg/kg sc which corresponded to an
approximate free concentration in plasma of 2.6 ng/mL.55
As shown in Table 5, 20 was extensively characterized for its
ADMET and safety profile. Compound 20 has a low aqueous
solubility (<0.004 mg/mL in water) which increases at pH ≈ 4
and in complexation with cyclodextrines (0.995 mg/mL 20%
HP-β-CD at pH 4).
EXPERIMENTAL SECTION
■
Chemistry. 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 of 230−400 (Merck)
under standard techniques. Microwave assisted reactions were
performed in a single-mode reactor, Biotage Initiator Sixty microwave
reactor (Biotage), or in a multimode reactor, MicroSYNTH Labstation
(Milestone, Inc.). Nuclear magnetic resonance (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 (LCMS) was performed on either a
HP 1100 HPLC system (Agilent Technologies) or Advanced
Chromatography Technologies system composed of a quaternary or
binary pump with degasser, an autosampler, a column oven, a diode
array detector (DAD), and a column as specified in the respective
methods below. Flow from the column was split to a MS spectrometer.
The MS detector was configured with either an electrospray ionization
source or an ESCI dual ionization source (electrospray combined with
atmospheric pressure chemical ionization). Nitrogen was used as the
nebulizer gas. Data acquisition was performed with MassLynx-
Openlynx software or with Chemsation-Agilent data browser software.
More detailed information about the different LCMS methods
employed can be found in the Supporting Information. Melting
point values are peak values and were obtained with experimental
uncertainties that are commonly associated with this analytical
method. Melting points were determined in open capillary tubes on
a Mettler FP62 apparatus with a temperature gradient of 10 °C/min.
Maximum temperature was 300 °C.
Compound 20 showed a rapid rate of absorption from the
gastrointestinal tract, reaching the maximal concentration after
0.5 h. Clearance in vivo was moderate to high in both rat and
dog (35 and 29 mL min−1 kg−1, respectively). Elimination half-
lives are on the shorter side across the species, being 2.7 h in rat
and 0.8−1.1 h in dog. Volume of distribution is slightly higher
than total body water, indicating distribution outside the
plasma, which was confirmed in a rat tissue distribution study
(data not shown). In vitro studies (LLC-PK1-MDR1) showed
high permeability with no indication for P-glycoprotein (P-gp)
efflux. Bioavailability is low to moderate across the species
(35% in rat and 18−33% in dog). For a highly permeable
compound, the low to moderate bioavailability might be related
to an in vivo solubility/dissolution rate limiting factor that
could reduce the extent of absorption.
Safety characterization of 20 did not reveal relevant alerts.
Thus, compound 20 showed a low potential to inhibit the
metabolism of co-medication (IC50 for five major CYP450 of
>10 μM; Table 5) and no genotoxic alerts based on both the
Ames and the micronucleus tests.
Compound 20 displayed good safety margins in various
cardiovascular studies. It did not affect IKr in hERG-transfected
HEK293 cells at 0.1 μM and did not induce electro-
physiological effects in the isolated rabbit Purkinje fiber at
≤0.075 μM. This was more than 200-fold the estimated free
efficacious plasma exposure of 0.14 ng/mL (taking into
consideration the high plasma protein binding, i.e., 0.35%
free). The no-effect levels (NOEL) in vivo (anesthetized guinea
pigs and dogs, 9000 and 6625 ng/mL, respectively) are more
than 160-fold the efficacious plasma exposure. On the basis of
the currently available preclinical safety and efficacy data, the
good safety margins attained support for further studies with
compound 20.
Purities of all new compounds were determined by analytical 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.
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1
3-Ethyl-7-(4-phenylpiperidin-1-yl)[1,2,4]triazolo[4,3-a]-
pyridine-8-carbonitrile (5). A solution of 32 (0.05 g, 0.17 mmol)
and EtC(OEt)3 (0.46 mL, 2.56 mmol) in xylenes (1 mL) was heated
in a sealed tube at 180 °C for 2 h. After the mixture was cooled to
room temperature, the volatiles were evaporated in vacuo. The residue
thus obtained was precipitated by treatment with Et2O. The resulting
solid was filtered off and dried in the vacuum oven (50 °C) to give 5
CH2Cl2, 0/100 to 3/97) to give 10 (0.14 g, 15%). H NMR (500
MHz, CDCl3) δ 0.27−0.37 (m, 2 H), 0.56−0.69 (m, 2 H), 1.08−1.18
(m, 1 H), 1.85−1.98 (m, 2 H), 2.07 (br d, J = 11.6 Hz, 2 H), 2.85 (tt, J
= 12.1, 3.5 Hz, 1 H), 3.00 (d, J = 6.6 Hz, 2 H), 3.39 (td, J = 12.8, 1.9
Hz, 2 H), 4.31 (br d, J = 13.3 Hz, 2 H), 6.61 (d, J = 7.8 Hz, 1 H),
7.22−7.27 (m, 3 H), 7.30−7.38 (m, 2 H), 7.85 (d, J = 7.8 Hz, 1 H).
LCMS: m/z 358 [M + H]+, tR = 3.88 min.
1
(0.042 g, 74%). H NMR (500 MHz, CDCl3) δ 1.31 (t, J = 7.5 Hz, 3
3-tert-Butyl-7-(4-phenyl-1-piperidin-1-yl)-1,2,4-triazolo[4,3-
a]pyridine-8-carbonitrile (11). To a solution of 32 (0.3 g, 1.02
mmol) in dry CH2Cl2 (2 mL) cooled at 0 °C were added Et3N (0.14
mL, 1.02 mmol) and 2,2-dimethylpropionyl chloride (0.15 g, 1.23
mmol). The resulting reaction mixture was gradually warmed to room
temperature and further stirred for 16 h. NaHCO3 (aqueous saturated
solution) was added, and the resulting solution was then extracted with
CH2Cl2. The organic layer was separated, dried over MgSO4, and
concentrated in vacuo. The residue thus obtained was dissolved in 1,2-
dichloroethane (2 mL), and POCl3 (0.094 mL, 1.01 mmol) was added.
The resulting mixture was heated at 150 °C for 5 min under
microwave irradiation. After cooling to room temperature, the mixture
was diluted with CH2Cl2 and washed with NaHCO3 (aqueous
saturated solution). The organic layer was separated, dried over
Na2SO4, and concentrated in vacuo. The residue thus obtained was
purified by column chromatography (silica gel, EtAcO in CH2Cl2, 0/
H), 1.77 (qd, J = 12.6, 3.8 Hz, 2 H), 1.95 (br d, J = 11.3 Hz, 2 H),
2.86−2.95 (m, 1 H), 3.00 (q, J = 7.5 Hz, 2 H), 3.34−3.42 (m, 2 H),
4.32 (br d, J = 13.3 Hz, 2 H), 6.96 (d, J = 7.8 Hz, 1 H), 7.19−7.25 (m,
1 H), 7.26−7.37 (m, 4 H), 8.34 (d, J = 8.1 Hz, 1 H). LCMS: m/z 332
[M + H]+, tR = 3.46 min.
7-(4-Phenylpiperidin-1-yl)-3-propyl[1,2,4]triazolo[4,3-a]-
pyridine-8-carbonitrile (7). To a solution of 32 (0.2 g, 0.68 mmol)
in dry CH2Cl2 (2 mL) cooled at 0 °C were added Et3N (0.14 mL, 1.02
mmol) and butanoyl chloride (0.087 g, 0.82 mmol). The resulting
reaction mixture was allowed to gradually warm to room temperature
and then further stirred for 1 h. NaHCO3 (aqueous saturated solution)
was added, and the resulting solution was then extracted with CH2Cl2.
The organic layer was separated, dried over MgSO4, and the volatiles
were evaporated in vacuo. The residue thus obtained was dissolved in
1,2-dichloroethane (2 mL), and POCl3 (0.035 mL, 0.371 mmol) was
added. The resulting mixture was heated at 150 °C for 5 min under
microwave irradiation. After cooling to room temperature, the mixture
was diluted with CH2Cl2 and washed with NaHCO3 (aqueous
saturated solution). The organic layer was separated, dried over
Na2SO4, and concentrated in vacuo. The residue thus obtained was
purified by column chromatography (silica gel, EtAcO in CH2Cl2, 0/
100 to 40/50) to give 7 (0.039 g, 17%). Mp > 300 °C. 1H NMR (400
MHz, CDCl3) δ 1.05 (t, J = 7.4 Hz, 3 H), 1.81−1.98 (m, 4 H), 2.07
(br d, J = 12.0 Hz, 2 H), 2.84 (tt, J = 12.1, 3.5 Hz, 1 H), 2.97 (t, J = 7.5
Hz, 2 H), 3.38 (br t, J = 12.0 Hz, 2 H), 4.30 (br d, J = 13.2 Hz, 2 H),
6.60 (d, J = 7.9 Hz, 1 H), 7.20−7.29 (m, 3 H), 7.30−7.38 (m, 2 H),
7.75 (d, J = 7.9 Hz, 1 H). LCMS: m/z 346 [M + H]+, tR = 3.03 min.
7-(4-Phenylpiperidin-1-yl)-3-(2,2,2-trifluoroethyl)[1,2,4]-
triazolo[4,3-a]pyridine-8-carbonitrile (9). To a suspension of 32
(0.3 g, 1.02 mmol) in dry CH2Cl2 (10 mL) at room temperature were
added diisopropylethylamine (0.36 mL, 2.045 mmol), polymer
supported PPh3 (1.43 g, 3.07 mmol, loading 2.15 mmol/g),
CF3CO2H (0.09 mL, 1.02 mmol), and trichloroacetonitrile (0.21
mL, 2.04 mmol) in 1,2-dichloroethane (10 mL). The resulting reaction
mixture was heated at 150 °C under microwave irradiation for 18 min.
After cooling to room temperature, the reaction mixture was filtered
through a Celite pad and solids were washed thoroughly with CH2Cl2
and MeOH. The combined filtrates were concentrated in vacuo and
the residue thus obtained was purified by column chromatography
(silica gel, MeOH in CH2Cl2, 0/100 to 5/100) to give 9 (100 mg,
1
100 to 20/80) to give 11 (0.11 g, 31%) as a solid. Mp > 300 °C. H
NMR (400 MHz, CDCl3) δ 1.55 (s, 9 H), 1.90 (qd, J = 12.3, 3.5 Hz, 2
H), 2.06 (dd, J = 12.7, 1.8 Hz, 2 H), 2.84 (tt, J = 12.1, 3.7 Hz, 1 H),
3.37 (td, J = 12.8, 2.3 Hz, 2 H), 4.31 (dt, J = 13.6, 2.1 Hz, 2 H), 6.57
(d, J = 7.9 Hz, 1 H), 7.20−7.28 (m, 3 H), 7.29−7.38 (m, 2 H), 8.05
(d, J = 8.1 Hz, 1 H). LCMS: m/z 360 [M + H]+, tR = 3.18 min.
3-Methoxymethyl-7-(4-phenyl-1-piperidin-1-yl)-1,2,4-
triazolo[4,3-a]pyridine-8-carbonitrile (12). To a solution of 32
(0.2 g, 0.68 mmol) in dry CH2Cl2 (2 mL) cooled at 0 °C were added
Et3N (0.19 mL, 1.36 mmol) and methoxyacetyl chloride (0.074 g, 0.68
mmol). The resulting reaction mixture was gradually warmed to room
temperature and further stirred for 1 h. NaHCO3 (aqueous saturated
solution) was added, and the resulting solution was then extracted with
CH2Cl2. The organic layer was separated, dried over MgSO4, and
concentrated in vacuo. The residue thus obtained was dissolved in 1,2-
dichloroethane (2 mL), and POCl3 (0.035 mL, 0.37 mmol) was added.
The resulting mixture was heated at 150 °C for 5 min under
microwave irradiation. After cooling to room temperature, the mixture
was diluted with CH2Cl2 and washed with NaHCO3 (aqueous
saturated solution). The organic layer was separated, dried over
Na2SO4, and concentrated in vacuo. The residue thus obtained was
purified by column chromatography (silica gel, MeOH−NH3 in
1
CH2Cl2 0/100 to 10/90) to give 12 (0.075 g, 32%). H NMR (500
MHz, CDCl3) δ 1.78 (qd, J = 12.6, 3.6 Hz, 2 H), 1.95 (br d, J = 11.0
Hz, 2 H), 2.92 (tt, J = 12.0, 3.5 Hz, 1 H), 3.29 (s, 3 H), 3.39−3.47 (m,
2 H), 4.35 (br d, J = 13.3 Hz, 2 H), 4.86 (s, 2 H), 7.03 (d, J = 7.8 Hz, 1
H), 7.19−7.25 (m, 1 H), 7.26−7.36 (m, 4 H), 8.33 (d, J = 8.1 Hz, 1
H). LCMS: m/z 348 [M + H]+, tR = 3.62 min.
1
25%). Mp > 300 °C. H NMR (500 MHz, CDCl3) δ 1.92 (qd, J =
12.7, 3.8 Hz, 2 H), 2.09 (br d, J = 12.4 Hz, 2 H), 2.87 (tt, J = 12.1, 3.5
Hz, 1 H), 3.42 (br t, J = 12.7, 12.7 Hz, 2 H), 3.98 (q, J = 9.8 Hz, 2 H),
4.36 (br d, J = 13.3 Hz, 2 H), 6.70 (d, J = 7.8 Hz, 1 H), 7.21−7.26 (m,
3 H), 7.31−7.38 (m, 2 H), 7.86 (d, J = 7.8 Hz, 1 H). LCMS: m/z 386
[M + H]+, tR = 3.82 min.
7-(4-Phenylpiperidin-1-yl)-3-pyrrolidin-1-ylmethyl[1,2,4]-
triazolo[4,3-a]pyridine-8-carbonitrile (13). To a solution of 33
(0.15 g, 0.49 mmol) in acetic acid (2 mL) were added pyrrolidine
(0.63 g, 0.89 mmol) and paraformaldehyde (0.46 mL, 2.03 mmol).
The resulting mixture was stirred at 80 °C for 16 h. After the mixture
was cooled to room temperature, water was added. The resulting
aqueous solution was extracted with CH2Cl2. The organic layer was
separated, dried over MgSO4, and the volatiles were evaporated in
vacuo. The residue thus obtained was purified by column
chromatography (silica gel, MeOH in CH2Cl2 0/100 to 3/97) to
give 13 as a white solid (0.084 g, 44%). 1H NMR (400 MHz, DMSO-
d6) δ 1.68 (quin, J = 3.1 Hz, 4 H), 1.77 (qd, J = 12.6, 3.8 Hz, 2 H),
1.94 (dd, J = 12.5, 1.8 Hz, 2 H), 2.46 (br s, 4 H), 2.91 (tt, J = 12.0, 3.6
Hz, 1 H), 3.37−3.46 (m, 2 H), 4.04 (s, 2 H), 4.32 (br d, J = 13.4 Hz, 2
H), 6.98 (d, J = 7.9 Hz, 1 H), 7.19−7.24 (m, 1 H), 7.25−7.36 (m, 4
H), 8.36 (d, J = 8.1 Hz, 1 H). LCMS: m/z 387 [M + H]+, tR = 4.09
min.
3-Cyclopropylmethyl-7-(4-phenyl-1-piperidin-1-yl)-1,2,4-
triazolo[4,3-a]pyridine-8-carbonitrile (10). To a solution of 32
(0.8 g, 2.73 mmol) in dry CHCl3 (14 mL) cooled at 0 °C were added
Et3N (0.61 mL, 4.36 mmol) and cyclopropylacetyl chloride (0.48 g,
4.09 mmol). The resulting reaction mixture was gradually warmed to
room temperature and further stirred for 1 h. Then NaHCO3
(aqueous saturated solution) was added, and the resulting solution
was then extracted with CH2Cl2. The organic layer was separated,
dried over MgSO4, and concentrated in vacuo. The resulting residue
was dissolved in 1,2-dichloroethane (10 mL), and POCl3 (0.4 mL,
4.26 mmol) was added. The mixture was heated at 150 °C for 5 min
under microwave irradiation. After cooling to room temperature, the
mixture was diluted with CH2Cl2 and washed with NaHCO3 (aqueous
saturated solution). The organic layer was separated, dried over
Na2SO4, and concentrated in vacuo. The residue thus obtained was
purified by column chromatography (silica gel, MeOH−NH3 in
8-Chloro-7-(4-phenylpiperidin-1-yl)-3-(2,2,2-trifluoroethyl)-
1,2,4-triazolo[4,3-a]pyridine (14). To a solution of 37a (0.45 g,
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1.47 mmol) in dry CH2Cl2 (2 mL) were added Et3N (0.31 mL, 2.23
mmol), 3,3,3-trifluoropropionyl chloride (0.22 mL, 1.78 mmol), and
the resulting mixture was stirred at room temperature for 1 h. Then
the volatiles were evaporated in vacuo. The residue thus obtained was
dissolved in 1,2-dichloroethane (5 mL), and POCl3 (0.13 mL, 1.45
mmol) was added at room temperature. The resulting mixture was
heated at 150 °C for 5 min under microwave irradiation. After cooling
to room temperature, the mixture was diluted with CH2Cl2 and
washed with NaHCO3 (aqueous saturated solution). The organic layer
was separated, dried over Na2SO4, and concentrated in vacuo. The
residue thus obtained was purified by column chromatography (silica
gel EtOAc in CH2Cl2, 0/100 to 10/90) to give 14 as a white solid
supercritical fluid purification (pyridine 20 mm; mobile phase, isocratic
83% CO2, 17% MeOH) to give 17 as a white solid (2.36 g, 31%). Mp
> 300 °C. H NMR (500 MHz, CDCl3) δ 2.11−2.22 (m, 2 H), 2.30
1
(td, J = 13.2, 4.8 Hz, 1 H), 2.41 (td, J = 13.3, 4.9 Hz, 1 H), 3.39 (td, J =
12.2, 2.0 Hz, 2 H), 3.53−3.64 (m, 2 H), 4.04 (q, J = 9.9 Hz, 2 H), 6.90
(d, J = 7.3 Hz, 1 H), 7.31−7.38 (m, 1 H), 7.38−7.50 (m, 4 H), 7.91
(d, J = 7.7 Hz, 1 H). LCMS: m/z 413 [M + H]+, tR = 1.05 min.
8-Chloro-3-cyclopropylmethyl-7-(4-phenyl-4-trifluorome-
thylpiperidin-1-yl)[1,2,4]triazolo[4,3-a]pyridine (18). To a
stirred solution of 40 (1 g, 3 mmol) in toluene (10 mL) were
added 4-trifluoromethyl-4-phenylpiperidine 41 (0.962 mL, 4.2 mmol),
Pd(OAc)2 (0.034 g, 0.15 mmol), Cs2CO3 (1.46 g, 4.5 mmol), and
BINAP (0.14 g, 0.225 mmol), and the reaction mixture was heated at
95 °C for 16 h in a sealed tube. After cooling to room temperature, the
mixture was concentrated in vacuo and the resulting residue was
suspended in water and extracted with CH2Cl2. The organic layer was
separated, dried (Na2SO4), concentrated in vacuo, and purified by
column chromatography (silica gel, MeOH in CH2Cl21, 0/100 to 10/
90) to give 18 as a solid (0.5 g, 39%). Mp 276.7 °C. H NMR (500
MHz, CDCl3) δ 0.24−0.34 (m, 2 H), 0.53−0.64 (m, 2 H), 1.07−1.19
(m, 1 H), 2.38−2.48 (m, 2 H), 2.64 (d, J = 12.7 Hz, 2 H), 2.91 (t, J =
11.8 Hz, 2 H), 3.02 (d, J = 6.6 Hz, 2 H), 3.51 (br d, J = 12.1 Hz, 2 H),
6.57 (d, J = 7.5 Hz, 1 H), 7.36−7.42 (m, 1 H), 7.46 (t, J = 7.7 Hz, 2
H), 7.49−7.53 (m, 2 H), 7.76 (d, J = 7.2 Hz, 1 H). LCMS: m/z 435
[M + H]+, tR = 2.43 min.
1
(0.15 g, 26%). Mp 228.6 °C. H NMR (500 MHz, CDCl3) δ 1.94−
2.06 (m, 4 H), 2.66−2.78 (m, 1 H), 3.00−3.11 (m, 2 H), 3.77 (br d, J
= 12.1 Hz, 2 H), 4.02 (q, J = 9.8 Hz, 2 H), 6.85 (d, J = 7.2 Hz, 1 H),
7.21−7.31 (m, 3 H), 7.31−7.38 (m, 2 H), 7.86 (d, J = 7.5 Hz, 1 H).
LCMS: m/z 395 [M + H]+, tR = 3.66 min.
8-Chloro-3-cyclopropylmethyl-7-(4-phenylpiperidin-1-yl)-
[1,2,4]triazolo[4,3-a]pyridine (15). To a solution of 37a (0.15 g,
0.49 mmol) in dry CH2Cl2 (4 mL) were added Et3N (0.12 mL, 0.89
mmol) and cyclopropylacetyl chloride (0.059 mL, 0.49 mmol). The
resulting mixture was stirred at room temperature for 1 h. Then the
volatiles were evaporated in vacuo. The residue thus obtained was
dissolved in 1,2-dichloroethane (5 mL), and POCl3 (0.13 mL, 1.45
mmol) was added. The reaction mixture was heated at 150 °C for 5
min under microwave irradiation. After cooling to room temperature,
the mixture was diluted with CH2Cl2 and washed with NaHCO3
(aqueous saturated solution). The organic layer was separated, dried
over Na2SO4, and concentrated in vacuo. The residue thus obtained
was purified by column chromatography (silica gel, CH2Cl2) to give 15
8-Chloro-3-cyclopropylmethyl-7-(4-phenylpiperazin-1-yl)-
1,2,4-triazolo[4,3-a]pyridine (19). To a stirred solution of 40 (0.3
g, 0.9 mmol) in toluene (4 mL) were added 4-phenylpiperazine 42
(0.18 mL, 1.69 mmol), Pd(OAc)2 (0.010 g, 0.045 mmol), Cs2CO3
(0.73 g, 2.24 mmol), and BINAP (0.042 g, 0.068 mmol), and the
reaction mixture was heated at 95 °C for 16 h in a sealed tube. After
cooling to room temperature, the mixture was concentrated in vacuo
and the resulting residue was suspended in water and extracted with
CH2Cl2. The organic layer was separated, dried (Na2SO4),
concentrated in vacuo, and purified by column chromatography (silica
gel, EtOAc in CH2Cl2, 0/100 to 40/60). The desired fractions were
collected, and the volatiles were evaporated in vacuo. The residue thus
obtained was precipitated by treatment with diisopropyl ether to give
1
as a white solid (0.069 g, 36%). Mp 194.1 °C. H NMR (500 MHz,
CDCl3) δ 0.26−0.39 (m, 2 H), 0.55−0.68 (m, 2 H), 1.10−1.23 (m, 1
H), 1.93−2.07 (m, 4 H), 2.65−2.77 (m, 1 H), 2.95−3.04 (m, 2 H),
3.05 (d, J = 6.7 Hz, 2 H), 3.72 (br d, J = 12.0 Hz, 2 H), 6.77 (d, J = 7.4
Hz, 1 H), 7.20−7.32 (m, 3 H), 7.32−7.39 (m, 2 H), 7.84 (d, J = 7.4
Hz, 1 H). LCMS: m/z 367 [M + H]+, tR = 3.69 min.
8-Chloro-3-ethyl-7-(4-phenylpiperidin-1-yl)[1,2,4]triazolo-
[4,3-a]pyridine (16). A solution of 37a (0.1 g, 0.33 mmol) and
EtC(OEt)3 (0.058 mL, 0.33 mmol) in xylene (2 mL) was heated in a
sealed tube at 180 °C for 2 h. After the mixture was cooled to room
temperature, the volatiles were evaporated in vacuo. The residue thus
obtained was purified by column chromatography (silica gel, MeOH−
NH3 in CH2Cl2, 0/100 to 3/97) to give 16 as a white solid (0.056 g,
50%). Mp 227.7 °C. 1H NMR (500 MHz, DMSO-d6) δ 1.34 (t, J = 7.5
Hz, 3 H), 1.84 (qd, J = 12.1, 3.8 Hz, 2 H), 1.91 (br d, J = 10.4 Hz, 2
H), 2.73 (tt, J = 11.9, 3.8 Hz, 1 H), 2.98−3.09 (m, 4 H), 3.62 (br d, J =
12.1 Hz, 2 H), 6.98 (d, J = 7.5 Hz, 1 H), 7.19−7.26 (m, 1 H), 7.28−
7.37 (m, 4 H), 8.32 (d, J = 7.2 Hz, 1 H). LCMS: m/z 341 [M + H]+, tR
= 4.34 min.
8-Chloro-7-(4-fluoro-4-phenylpiperidin-1-yl)-3-(2,2,2-tri-
fluoroethyl)-1,2,4-triazolo[4,3-a]pyridine (17). To a solution of
37b (6 g, 18.7 mmol) in dry CH2Cl2 (30 mL) cooled at 0 °C were
added Et3N (4.69 mL, 33.67 mmol) and 3,3,3-trifluoropropionyl
chloride (3.08 mL, 29.93 mmol). The reaction mixture was allowed to
warm to room temperature and then further stirred for 1 h. Then
NaHCO3 (aqueous saturated solution) was added, and the resulting
solution was then extracted with CH2Cl2. The organic layer was
separated, dried over MgSO4, and concentrated in vacuo. The residue
thus obtained was dissolved in 1,2-dichloroethane (180 mL), and
polymer supported diisopropylethylamine (13.87 g, 54.09 mmol,
loading 3.9 mmol/g), polymer supported PPh3 (25.04 g, 45.07 mmol,
loading 1.8 mmol/g) and trichloroacetonitrile (2.17 mL 21.64 mmol)
were added. The resulting mixture was heated at 150 °C for 10 min
under microwave irradiation. After cooling to room temperature, the
mixture was filtered through a Celite pad and the solids were washed
thoroughly with CH2Cl2 and MeOH. The combined filtrates were
concentrated in vacuo, and the residue thus obtained was purified by
column chromatography (silica gel, EtOAc in CH2Cl2, 0/100 to 40/
60). The desired fractions were collected, and the volatiles were
evaporated in vacuo. The resulting residue was purified by preparative
1
compound 19 as a solid (0.2 g, 60%). Mp > 300 °C. H NMR (400
MHz, CDCl3) δ 0.27−0.39 (m, 2 H), 0.55−0.69 (m, 2 H), 1.12−1.22
(m, 1 H), 3.06 (d, J = 6.7 Hz, 2 H), 3.33−3.48 (m, 8 H), 6.77 (d, J =
7.4 Hz, 1 H), 6.92 (t, J = 7.3 Hz, 1 H), 7.00 (d, J = 8.1 Hz, 2 H), 7.31
(t, J = 8.1 Hz, 2 H), 7.88 (d, J = 7.4 Hz, 1 H). LCMS: m/z 368 [M +
H]+, tR = 2.79 min.
3-Cyclopropylmethyl-7-(4-phenylpiperidin-1-yl)-8-
trifluoromethyl[1,2,4]triazolo[4,3-a]pyridine (20). A mixture of
46 (4 g, 14.51 mmol), 4-phenylpiperidine 30 (3.3 g, 17.41 mmol), and
diisopropyethylamine (5.05 mL, 29.02 mmol) in CH3CN (8.5 mL)
was heated at 180 °C for 20 min under microwave irradiation. After
cooling to room temperature, the mixture was concentrated in vacuo.
The residue thus obtained was purified by column chromatography
(silica gel, MeOH−NH3 in CH2Cl2, 0/100 to 10/90) to give 20 as a
1
solid (2.8 g, 48%). Mp 211.3 °C. H NMR (500 MHz, CDCl3) δ
0.28−0.39 (m, 2 H), 0.57−0.72 (m, 2 H), 1.06−1.22 (m, 1 H), 1.78−
2.11 (m, 4 H), 2.72 (tt, J = 11.5, 4.4 Hz, 1 H), 3.04 (d, J = 6.6 Hz, 2
H), 3.18 (td, J = 12.1, 2.0 Hz, 2 H), 3.62 (br d, J = 12.4 Hz, 2 H), 6.76
(d, J = 7.5 Hz, 1 H), 7.21−7.28 (m, 3 H), 7.34 (t, J = 7.7 Hz, 2 H),
7.92 (d, J = 7.8 Hz, 1 H). LCMS: m/z 401 [M + H]+, tR = 4.78 min.
3-Ethyl-7-(4-phenylpiperidin-1-yl)-8-trifluoromethyl-1,2,4-
triazolo[4,3-a]pyridine (21). To a solution of 50a (2.6 g, 7.73
mmol) in dry CH2Cl2 (70 mL) at 0 °C were added Et3N (1.9 mL,
13.91 mmol) and propanoyl chloride (0.83 mL, 9.28 mmol). The
resulting reaction mixture was stirred at room temperature for 10 min.
The mixture was then concentrated in vacuo. The residue thus
obtained was dissolved in 1,2-dichloroethane (5 mL), and POCl3 (0.7
mL, 1.15 mmol) was added. The resulting mixture was heated at 150
°C for 5 min under microwave irradiation. After cooling to room
temperature, the mixture was diluted with CH2Cl2 and washed with
NaHCO3 (aqueous saturated solution). The organic layer was
separated, dried over Na2SO4, and concentrated in vacuo. The crude
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product was purified by column chromatography (silica gel, CH2Cl2)
to give 21 (0.14 g, 50%). H NMR (500 MHz, CDCl3) δ 1.46 (t, J =
cooling to room temperature, the mixture was concentrated in vacuo.
The crude product was purified by column chromatography (silica gel,
MeOH−NH3 in CH2Cl2, 0/100 to 1/99) to give 25 as a solid (0.13 g,
50%). Mp > 300 °C. 1H NMR (500 MHz, CDCl3) δ 0.28−0.37 (m, 2
H), 0.56−0.66 (m, 2 H), 1.10−1.18 (m, 1 H), 3.02 (d, J = 6.6 Hz, 2
H), 5.00 (s, 4 H), 6.80 (d, J = 7.8 Hz, 1 H), 7.30−7.37 (m, 4 H), 7.85
(d, J = 7.8 Hz, 1 H). LCMS: m/z 359 [M + H]+, tR = 2.66 min.
rac-3-Cyclopropylmethyl-7-(3-phenylpyrrolidin-1-yl)-8-
trifluoromethyl[1,2,4]triazolo[4,3-a]pyridine (26). A mixture of
46 (0.2 g, 0.73 mmol), rac-3-phenylpyrrolidine (0.21 g, 1.45 mmol),
and diisopropylethylamine (0.76 mL, 4.35 mmol) in CH3CN (10 mL)
was heated at 180 °C for 20 min under microwave irradiation. After
cooling, the mixture was concentrated in vacuo. The crude product
was purified by column chromatography (silica gel, MeOH−NH3 in
CH2Cl2, 0/100 to 1/99) to give 26 as a solid (0.092 g, 32%). Mp 178.3
1
7.5 Hz, 3 H), 1.87−2.01 (m, 4 H), 2.72 (tt, J = 11.5, 4.3 Hz, 1 H), 3.06
(q, J = 7.5 Hz, 2 H), 3.18 (td, J = 12.4, 2.3 Hz, 2 H), 3.62 (br d, J =
12.7 Hz, 2 H), 6.77 (d, J = 7.5 Hz, 1 H), 7.21−7.28 (m, 3 H), 7.31−
7.37 (m, 2 H), 7.81 (d, J = 7.5 Hz, 1 H). LCMS: m/z 375 [M + H]+, tR
= 4.4 min.
3-Butyl-7-(4-phenylpiperidin-1-yl)-8-trifluoromethyl-1,2,4-
triazolo[4,3-a]pyridine (22). To a solution of 50a (0.35 g, 1.04
mmol) in dry CH2Cl2 (10 mL) was added Et3N (0.25 mL, 1.87 mmol)
and pentanoyl chloride (0.13 mL, 1.04 mmol). The resulting reaction
mixture was stirred at room temperature for 10 min. The mixture was
then concentrated in vacuo. The residue thus obtained was dissolved
in 1,2-dichloroethane (5 mL), and POCl3 (0.15 mL, 1.57 mmol) was
added. The resulting mixture was heated at 150 °C for 5 min under
microwave irradiation. After cooling to room temperature, the mixture
was diluted with CH2Cl2 and washed with NaHCO3 (aqueous
saturated solution). The organic layer was separated, dried over
Na2SO4, and concentrated in vacuo. The residue thus obtained was
purified by column chromatography (silica gel, MeOH−NH3 in
CH2Cl2, 0/100 to 3/97) to give 22 as a solid (0.16 g, 38%). Mp 221.7
1
°C. H NMR (500 MHz, CDCl3) δ 0.26−0.37 (m, 2 H), 0.56−0.64
(m, 2 H), 1.08−1.18 (m, 1 H), 2.10−2.21 (m, 1 H), 2.36−2.45 (m, 1
H), 2.94−3.06 (m, 2 H), 3.44−3.53 (m, 1 H), 3.65−3.73 (m, 2 H),
3.76−3.84 (m, 1 H), 3.84−3.91 (m, 1 H), 6.68 (d, J = 7.8 Hz, 1 H),
7.23−7.32 (m, 3 H), 7.33−7.39 (m, 2 H), 7.79 (d, J = 7.8 Hz, 1 H).
LCMS: m/z 387 [M + H]+, tR = 2.18 min.
1
°C. H NMR (500 MHz, CDCl3) δ 0.97 (t, J = 7.3 Hz, 3 H), 1.42−
3-Cyclopropylmethyl-7-[4-(2,4-difluorophenyl)piperidin-1-
yl]-8-trifluoromethyl[1,2,4]triazolo[4,3-a]pyridine (27). A mix-
ture of 46 (0.06 g, 0.22 mmol), 4-(2,4-difluorophenyl)piperidine 27
(0.061 g, 0.261 mmol), and diisopropylethylamine (0.076 mL, 0.43
mmol) in CH3CN (2 mL) was heated at 180 °C for 15 min under
microwave irradiation. After the mixture was cooled to room
temperature, the volatiles were evaporated in vacuo. The residue
thus obtained was purified by column chromatography (silica gel,
EtOAc in CH2Cl2, 0/100 to 30/70) to give 27 as a solid (0.041 g,
43%). Mp > 300 °C. 1H NMR (500 MHz, CDCl3) δ 0.28−0.39 (m, 2
H), 0.57−0.68 (m, 2 H), 1.11−1.19 (m, 1 H), 1.86−1.97 (m, 4 H),
2.97−3.08 (m, 1 H), 3.05 (d, J = 6.6 Hz, 2 H), 3.13−3.24 (m, 2 H),
3.60 (d, J = 12.1 Hz, 2 H), 6.77 (d, J = 7.5 Hz, 1 H), 6.81 (ddd, J =
10.7, 8.7, 2.6 Hz, 1 H), 6.87 (td, J = 8.3, 2.2 Hz, 1 H), 7.22 (td, J = 8.5,
6.6 Hz, 1 H), 7.94 (d, J = 7.5 Hz, 1 H). LCMS: m/z 437 [M + H]+, tR
= 2.55 min.
1.53 (m, 2 H), 1.78−1.87 (m, 2 H), 1.87−2.02 (m, 4 H), 2.65−2.78
(m, 1 H), 2.99−3.08 (m, 2 H), 3.12−3.24 (m, 2 H), 3.61 (br d, J =
12.5 Hz, 2 H), 6.76 (d, J = 7.6 Hz, 1 H), 7.20−7.30 (m, 3 H), 7.30−
7.39 (m, 2 H), 7.81 (d, J = 7.9 Hz, 1 H). LCMS: m/z 403 [M + H]+, tR
= 3.96 min.
3-Cyclopropylmethyl-7-(4-fluoro-4-phenylpiperidin-1-yl)-8-
trifluoromethyl-1,2,4-triazolo[4,3-a]pyridine (23). To a solution
of 50b (0.29 g, 0.82 mmol) in dry CH2Cl2 (10 mL) were added Et3N
(0.2 mL, 1.47 mmol) and cyclopropylacetyl chloride (0.12 g, 0.98
mmol). The resulting reaction mixture was stirred at room
temperature for 10 min. The mixture was then concentrated in
vacuo. The residue was dissolved in 1,2-dichloroethane (10 mL), and
polymer supported diisopropylethylamine (0.65 g, 2.54 mmol, loading
3.9 mmol/g), polymer supported PPh3 (1.77 g, 2.12 mmol, loading 1.8
mmol/g), and trichloroacetonitrile (0.1 mL, 1.02 mmol) were added.
The resulting mixture was heated at 150 °C for 10 min under
microwave irradiation. After cooling to room temperature, the mixture
was filtered through a Celite pad and the solids were washed
thoroughly with CH2Cl2 and MeOH. The combined filtrates were
concentrated in vacuo and the residue thus obtained was purified by
column chromatography (silica gel, EtOAc in CH2Cl2, 0/100 to 40/
60) to give a residue that was purified by supercritical fluid purification
(pyridine 20 mm; mobile phase, isocratic 85% CO2, 15% MeOH) to
give 23 as a solid (0.1 g, 29%). 1H NMR (500 MHz, CDCl3) δ 0.27−
0.41 (m, 2 H), 0.57−0.70 (m, 2 H), 1.11−1.22 (m, 1 H), 2.08−2.19
(m, 2 H), 2.20−2.31 (m, 1 H), 2.36 (td, J = 13.2, 5.1 Hz, 1 H), 3.06
(d, J = 6.5 Hz, 2 H), 3.33−3.43 (m, 2 H), 3.43−3.55 (m, 2 H), 6.83
(d, J = 7.6 Hz, 1 H), 7.31−7.37 (m, 1 H), 7.38−7.48 (m, 4 H), 7.98
(d, J = 7.6 Hz, 1 H). LCMS: m/z 419 [M + H]+, tR = 1.09 min.
2-(3-Cyclopropylmethyl-8-trifluoromethyl[1,2,4]triazolo-
[4,3-a]pyridin-7-yl)-1,2,3,4-tetrahydroisoquinoline (24). A mix-
ture of 46 (0.25 g, 0.91 mmol), 1,2,3,4-tetrahydroisoquinoline (0.14
mL, 1.09 mmol), and diisopropylethylamine (0.31 mL, 1.81 mmol) in
CH3CN (5 mL) was heated at 180 °C for 20 min under microwave
irradiation. After cooling to room temperature, the mixture was
concentrated in vacuo. The crude product was purified by column
chromatography (silica gel, MeOH−NH3 in CH2Cl2, 0/100 to 1/99)
to give 24 as a solid (0.12 g, 36%). Mp > 300 °C. 1H NMR (400 MHz,
CDCl3) δ 0.26−0.38 (m, 2 H), 0.54−0.67 (m, 2 H), 1.07−1.17 (m, 1
H), 2.99−3.05 (m, 2 H), 3.03 (d, J = 6.7 Hz, 2 H), 3.68 (t, J = 5.9 Hz,
2 H), 4.52 (s, 2 H), 6.79 (d, J = 7.9 Hz, 1 H), 7.08−7.14 (m, 1 H),
7.14−7.25 (m, 3 H), 7.89 (d, J = 7.6 Hz, 1 H). LCMS: m/z 373 [M +
H]+, tR = 4.2 min.
1′-(3-Cyclopropylmethyl-8-trifluoromethyl[1,2,4]triazolo-
[4,3-a]pyridin-7-yl)-1′,2′,3′,4′,5′,6′-hexahydro[3,4′]bipyridinyl
(28). A mixture of 46 (0.22 g, 0.8 mmol), 4-(3-pyridyl)piperidine
(0.14 g, 0.84 mmol), and diisopropylethylamine (0.17 mL, 0.96 mmol)
in CH3CN (5 mL) was heated at 90 °C for 1 day. After the mixture
was cooled to room temperature, the volatiles were evaporated in
vacuo. The residue thus obtained was purified by column
chromatography (silica gel, EtOAc in CH2Cl2, 0/100 to 30/70) to
1
give 28 as a solid (0.11 g, 35%). Mp 201.9 °C. H NMR (400 MHz,
CDCl3) δ 0.27−0.40 (m, 2 H), 0.55−0.69 (m, 2 H), 1.09−1.22 (m, 1
H), 1.86−2.03 (m, 4 H), 2.70−2.81 (m, 1 H), 3.05 (d, J = 6.7 Hz, 2
H), 3.12−3.24 (m, 2 H), 3.61 (br d, J = 12.5 Hz, 2 H), 6.77 (d, J = 7.6
Hz, 1 H), 7.28 (dd, J = 8.1, 4.9 Hz, 1 H), 7.58 (dt, J = 8.0, 1.9 Hz, 1
H), 7.95 (d, J = 7.6 Hz, 1 H), 8.50 (dd, J = 4.7, 1.5 Hz, 1 H), 8.54 (d, J
= 2.1 Hz, 1 H). LCMS: m/z 402 [M + H]+, tR = 1.58 min.
2-Bromo-3-cyano-4-(4-phenylpiperidin-1-yl)pyridine (31).
To a suspension of NaH (0.15 g, 3.82 mmol, 60% in mineral oil) in
DMF (20 mL) at 0 °C was added portionwise 4-phenylpiperidine 30
(0.62 g, 3.82 mmol). The resulting mixture was stirred at 0 °C for 5
min, and then 2,4-dibromo-3-cyanopyridine 29 (1 g, 3.016 mmol) was
added. The resulting reaction mixture was stirred for 1 h. The reaction
mixture was then quenched with NH4Cl (aqueous saturated solution)
and extracted with Et2O. The organic layer was separated, dried over
Na2SO4, and the volatiles were evaporated in vacuo. The residue thus
obtained was purified by column chromatography (silica gel, MeOH−
1
NH3 in CH2Cl2, 0/100 to 1/99) to give 31 (0.98 g, 75%). H NMR
(500 MHz, CDCl3) δ 1.91 (qd, J = 12.7, 3.6 Hz, 2 H), 2.03 (br d, J =
12.4 Hz, 2 H), 2.80 (tt, J = 12.1, 3.6 Hz, 1 H), 3.19 (br t, J = 12.1 Hz, 2
H), 4.15 (br d, J = 13.0 Hz, 2 H), 6.76 (d, J = 6.1 Hz, 1 H), 7.21−7.28
(m, 3 H), 7.30−7.37 (m, 2 H), 8.10 (d, J = 6.1 Hz, 1 H). LCMS: m/z
342 [M + H]+, tR = 4.48 min.
3-Cyclopropylmethyl-7-(1,3-dihydroisoindol-2-yl)-8-
trifluoromethyl[1,2,4]triazolo[4,3-a]pyridine (25). A mixture of
46 (0.2 g, 0.73 mmol), isoindoline (0.17 g, 1.45 mmol), and
diisopropylethylamine (0.76 mL, 4.35 mmol) in CH3CN (10 mL) was
heated at 180 °C for 20 min under microwave irradiation. After
[3-Cyano-4-(4-phenylpiperidin-1-yl)pyridin-2-yl]hydrazine
(32). To a solution of 31 (0.5 g, 1.46 mmol) in THF (4 mL) at room
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temperature was added hydrazine monohydrate (0.37 g, 7.30 mmol),
and the mixture was heated at 160 °C for 15 min under microwave
irradiation. The reaction mixture was then cooled to room
temperature, and the volatiles were evaporated in vacuo. The residue
thus obtained was purified by column chromatography (silica gel,
MeOH−NH3 in CH2Cl2, 0/100 to 1/99) to give 32 as a white solid
1 H), 7.26−7.36 (m, 5 H), 7.93 (d, J = 5.5 Hz, 1 H). LCMS: m/z 303
[M + H]+, tR = 4.13 min.
(3′-Chloro-4-fluoro-4-phenyl-3,4,5,6-tetrahydro-2H-[1,4′]-
bipyridinyl-2′-yl)hydrazine (37b). To a suspension of compound
36b (0.97 g, 2.97 mmol) in EtOH (6 mL) was added hydrazine
monohydrate (2.88 mL, 59.41 mmol). The reaction mixture was
heated under microwave irradiation at 160 °C for 20 min. After the
mixture was cooled to room temperature, the volatiles were evaporated
in vacuo and the residue thus obtained was taken up in CH2Cl2. The
resulting solution was washed with NaHCO3 (aqueous saturated
solution). The organic layer was separated, dried over MgSO4, and
concentrated in vacuo. The residue thus obtained was precipitated by
treatment with Et2O. The resulting solid was filtered off and dried in
the vacuum oven (50 °C) to give 37b as a white solid (0.8 g, 84%). 1H
NMR (400 MHz, DMSO-d6) δ 2.04 (dd, J = 12.3, 11.3 Hz, 2 H), 2.19
(td, J = 13.2, 4.6 Hz, 1 H), 2.29 (td, J = 13.4, 4.0 Hz, 1 H), 3.07 (t, J =
11.4 Hz, 2 H), 3.37−3.49 (m, 2 H), 4.17 (s, 2 H), 6.53 (d, J = 5.8 Hz,
1 H), 7.31−7.39 (m, 2 H), 7.42 (t, J = 7.5 Hz, 2 H), 7.46−7.53 (m, 2
H), 7.95 (d, J = 5.5 Hz, 1 H). LCMS: m/z 321 [M + H]+, tR = 3.37
min.
(3-Chloro-4-iodopyridin-2-yl)hydrazine (38). To a solution of
2,3-dichloro-4-iodopyridine 34 (8 g, 29.21 mmol) in 1,4-dioxane (450
mL) was added hydrazine monohydrate (14.17 mL, 175.25 mmol).
The reaction mixture was heated in a sealed tube at 70 °C for 16 h.
After the mixture was cooled to room temperature, NH4OH (32%
aqueous solution) was added and the resulting mixture was
concentrated in vacuo. The resulting residue was taken up in EtOH,
and the suspension thus obtained was heated to reflux. The remaining
solid was filtered off while hot, and the filtrate was cooled to room
temperature. The precipitate newly formed was filtered off and then
the filtrate concentrated in vacuo to give 38 as a white solid (2.67 g,
52%). 1H NMR (400 MHz, DMSO-d6) δ 3.90 (br s, 3 H), 7.10 (d, J =
5.1 Hz, 1 H), 7.70 (d, J = 5.1 Hz, 1 H), 7.84 (br s, 1 H). LCMS: m/z
270 [M + H]+, tR = 1.13 min.
1
(0.38 g, 89%). H NMR (400 MHz, DMSO-d6) δ 1.71 (qd, J = 12.6,
3.7 Hz, 2 H), 1.88 (dd, J = 12.0, 1.4 Hz, 2 H), 2.78 (tt, J = 12.0, 3.7 Hz,
1 H), 3.07 (td, J = 12.5, 2.1 Hz, 2 H), 3.97 (br d, J = 12.9 Hz, 2 H),
4.28 (s, 2 H), 6.34 (d, J = 6.2 Hz, 1 H), 7.17−7.24 (m, 1 H), 7.24−
7.35 (m, 4 H), 7.77 (s, 1 H), 7.96 (d, J = 6.0 Hz, 1 H). LCMS: m/z
294 [M + H]+, tR = 3.62 min.
7-(4-Phenylpiperidin-1-yl)[1,2,4]triazolo[4,3-a]pyridine-8-
carbonitrile (33). A mixture of 32 (1 g, 3.41 mmol) and HC(OEt)3
(7.58 g, 51.13 mmol) in xylenes (25 mL) was heated in a sealed tube
at 180 °C for 2 h. The reaction mixture was cooled to room
temperature and then concentrated in vacuo. The residue thus
obtained was treated with Et2O and the resulting solid was filtered off
1
and dried in the vacuum oven (50 °C) to give 33 (0.93 g, 90%). H
NMR (400 MHz, DMSO-d6) δ 1.77 (qd, J = 12.6, 3.6 Hz, 2 H), 1.89−
1.99 (m, 2 H), 2.91 (tt, J = 12.1, 3.7 Hz, 1 H), 3.25−3.45 (m, 2 H),
4.32 (br d, J = 13.4 Hz, 2 H), 7.00 (d, J = 7.9 Hz, 1 H), 7.19−7.25 (m,
1 H), 7.25−7.36 (m, 4 H), 8.45 (d, J = 7.9 Hz, 1 H), 8.98 (s, 1 H).
LCMS: m/z 304 [M + H]+, tR = 3.11 min.
2′,3′-Dichloro-4-phenyl-3,4,5,6-tetrahydro-2H-[1,4′]-
bipyridinyl (36a). A mixture of 2,3-dichloro-4-iodopyridine 34 (4 g,
14.61 mmol), 4-phenylpiperidine 30 (3.53 g, 21.91 mmol), and
diisopropylethylamine (5.09 mL, 29.21 mmol) in CH3CN (150 mL)
was heated in a sealed tube at 110 °C for 16 h. The mixture was then
cooled to room temperature, and then NaHCO3 (aqueous saturated
solution) was added. The resulting mixture was extracted with EtOAc.
The organic layer was separated, dried (Na2SO4), and the volatiles
were evaporated in vacuo. The residue thus obtained was purified by
column chromatography (silica gel, MeOH−NH3 in CH2Cl2, 0/100 to
1/99). The desired fractions were collected and concentrated in vacuo
Cyclopropylacetic Acid N′-(3-Chloro-4-iodopyridin-2-yl)-
hydrazide (39). To a solution of 38 (0.73 g, 2.71 mmol) in dry
CH2Cl2 (8 mL), cooled at 0 °C, were added Et3N (0.56 mL, 4.06
mmol) and cyclopropylacetyl chloride (0.38 g, 3.25 mmol). The
reaction mixture was stirred at room temperature for 16 h, and then
NaHCO3 (aqueous saturated solution) was added. The resulting
solution was extracted with CH2Cl2. The organic layer was separated,
dried over Na2SO4, and concentrated in vacuo to give 39 (0.94 g,
99%). 1H NMR (400 MHz, CDCl3) δ 0.27−0.40 (m, 2 H), 0.56−0.70
(m, 2 H), 1.11−1.23 (m, 1 H), 1.68 (s, 2 H), 3.07 (d, J = 6.7 Hz, 2 H),
7.17 (d, J = 7.2 Hz, 1 H), 7.69 (d, J = 7.2 Hz, 1 H). LCMS: m/z 352
[M + H]+, tR = 1.82 min.
1
to give 36a as a white solid (2.32 g, 52%). H NMR (400 MHz,
DMSO-d6) δ 1.80 (qd, J = 12.3, 3.7 Hz, 2 H), 1.90 (dd, J = 12.3, 2.3
Hz, 2 H), 2.75 (tt, J = 12.0, 3.8 Hz, 1 H), 2.93 (td, J = 12.1, 2.1 Hz, 2
H), 3.68 (br d, J = 12.5 Hz, 2 H), 7.14 (d, J = 5.5 Hz, 1 H), 7.18−7.24
(m, 1 H), 7.26−7.35 (m, 4 H), 8.16 (d, J = 5.5 Hz, 1 H). LCMS: m/z
307 [M + H]+, tR = 3.46 min.
2′,3′-Dichloro-4-fluoro-4-phenyl-3,4,5,6-tetrahydro-2H-
[1,4′]bipyridinyl (36b). A mixture of 2,3-dichloro-4-iodopyridine 34
(2 g, 7.30 mmol), 4-fluoro-4-phenylpiperidine hydrochloride 35 (2.05
g, 9.49 mmol), and diisopropylethylamine (5.05 mL, 29.21 mmol) in
CH3CN (10 mL) was heated in a sealed tube at 110 °C for 16 h. The
mixture was cooled to room temperature, and then NaHCO3 (aqueous
saturated solution) was added. The resulting mixture was extracted
with EtOAc. The organic layer was separated, dried over Na2SO4, and
the volatiles were evaporated in vacuo. The residue thus obtained was
purified by column chromatography (silica gel, CH2Cl2 in heptane 25/
75 to 75/25) to give 36b as a white solid (0.88 g, 37%). 1H NMR (500
MHz, CDCl3) δ 2.08−2.20 (m, 2 H), 2.27 (ddd, J = 13.6, 13.0, 4.6 Hz,
1 H), 2.35 (td, J = 13.3, 4.9 Hz, 1 H), 3.25 (td, J = 12.3, 2.0 Hz, 2 H),
3.53−3.63 (m, 2 H), 6.90 (d, J = 5.5 Hz, 1 H), 7.31−7.37 (m, 1 H),
7.38−7.49 (m, 4 H), 8.14 (d, J = 5.5 Hz, 1 H). LCMS: m/z 325 [M +
H]+, tR = 3.32 min.
(3′-Chloro-4-phenyl-3,4,5,6-tetrahydro-2H-[1,4′]bipyridinyl-
2′-yl)hydrazine (37a). To a suspension of 36a (0.25 g, 0.81 mmol)
in 1,4-dioxane (3 mL) at room temperature was added hydrazine
monohydrate (0.79 mL, 16.27 mmol). The reaction mixture was
heated at 160 °C for 20 min under microwave irradiation. After the
mixture was cooled to room temperature, the solvent was evaporated
in vacuo, and the residue thus obtained was taken up in CH2Cl2. The
resulting solution was washed with NaHCO3 (aqueous saturated
solution). The organic layer was separated, dried over MgSO4, and
concentrated in vacuo to give 37a (0.24 g, 99%). 1H NMR (400 MHz,
DMSO-d6) δ 1.79 (qd, J = 12.3, 3.7 Hz, 2 H), 1.84−1.91 (m, 2 H),
2.63−2.75 (m, 1 H), 2.81 (td, J = 11.8, 2.1 Hz, 2 H), 3.54 (br d, J =
12.0 Hz, 2 H), 4.16 (s, 2 H), 6.48 (d, J = 5.5 Hz, 1 H), 7.18−7.24 (m,
8-Chloro-3-cyclopropylmethyl-7-iodo[1,2,4]triazolo[4,3-a]-
pyridine (40). Compound 39 (0.74 g, 2.39 mmol) was heated at 160
°C for 3 h. After the mixture was cooled to room temperature, the
brown gum thus obtained was triturated with diisopropyl ether to give
a solid that was filtered off and dried in the vacuum oven (50 °C),
1
yielding 40 (0.74 g, 93%). H NMR (400 MHz, CDCl3) δ 0.26−0.41
(m, 2 H), 0.56−0.70 (m, 2 H), 1.12−1.24 (m, 1 H), 3.07 (d, J = 6.7
Hz, 2 H), 7.18 (d, J = 7.2 Hz, 1 H), 7.69 (d, J = 7.2 Hz, 1 H). LCMS:
m/z 334 [M + H]+, tR = 1.5 min.
4-Benzyloxy-2-chloro-3-trifluoromethylpyridine (44). To a
suspension of NaH (0.49 g, 12.73 mmol, 60% in mineral oil) in DMF
(50 mL) cooled at 0 °C was added benzyl alcohol (1.26 mL 12.2
mmol). The resulting mixture was stirred for 2 min, and then 43 (2.5
g, 11.57 mmol) was added. The mixture was gradually warmed to
room temperature and further stirred for 1 h. The reaction mixture
was quenched with water and extracted with Et2O. The organic layer
was separated, dried over Na2SO4, and concentrated in vacuo. The
residue thus obtained was purified by column chromatography (silica
1
gel, heptane in CH2Cl20/100 to 50/50) to give 44 (1.1 g, 33%). H
NMR (500 MHz, CDCl3) δ 5.25 (s, 2 H), 6.93 (d, J = 5.8 Hz, 1 H),
7.33−7.39 (m, 2 H), 7.38−7.44 (m, 3 H), 8.33 (d, J = 5.8 Hz, 1 H).
LCMS: m/z 288 [M + H]+, tR = 2.57 min.
(4-Benzyloxy-3-trifluoromethylpyridin-2-yl)hydrazine (45).
To a suspension of 44 (1.09 g, 3.79 mmol) in 1,4-dioxane (9 mL),
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was added hydrazine monohydrate (3.68 mL, 75.78 mmol). The
reaction mixture was heated at 160 °C for 30 min under microwave
irradiation. After the mixture was cooled to room temperature, the
volatiles were evaporated in vacuo. The residue thus obtained was
dissolved in CH2Cl2 and washed with NaHCO3 (aqueous saturated
solution). The organic layer was separated, dried over Na2SO4, and
(4-Fluoro-4-phenyl-3′-trifluoromethyl-3,4,5,6-tetrahydro-
2H-[1,4′]bipyridinyl-2′-yl)hydrazine (50b). To a suspension of
compound 49b (0.53 g, 1.15 mmol) in THF (10 mL) was added
hydrazine monohydrate (0.224 mL, 4.61 mmol). The reaction mixture
was heated at 160 °C for 45 min under microwave irradiation. After
the mixture was cooled to room temperature, the volatiles were
evaporated in vacuo. The residue thus obtained was precipitated by
1
concentrated in vacuo to give 45 as a white solid (0.89 g, 83%). H
1
treatment with Et2O to give 50b as a white solid (0.28 g, 69%). H
NMR (500 MHz, CDCl3) δ 4.01 (br s, 2 H), 5.18 (s, 2 H), 6.39 (d, J =
5.8 Hz, 1 H), 6.53 (br s, 1 H), 7.31−7.41 (m, 3 H), 7.39 (d, J = 4.6 Hz,
2 H), 8.15 (d, J = 5.8 Hz, 1 H). LCMS: m/z 284 [M + H]+, tR = 1.88
min.
NMR (500 MHz, CDCl3) δ 2.03−2.12 (m, 2 H), 2.16−2.25 (m, 1 H),
2.25−2.34 (m, 1 H), 3.22−3.32 (m, 4 H), 4.04 (br s, 2 H), 6.26 (br s,
1 H), 6.49 (d, J = 5.8 Hz, 1 H), 7.30−7.38 (m, 1 H), 7.38−7.46 (m, 4
H), 8.12 (d, J = 5.5 Hz, 1 H). LCMS: m/z 355 [M + H]+, tR = 2.50
min.
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
23500g at 4 °C. Cells were lysed in 5 mM hypotonic Tris-HCl, pH 7.4,
and after recentrifugation for 20 min at 30000g 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.
7-Chloro-3-cyclopropylmethyl-8-trifluoromethyl[1,2,4]-
triazolo[4,3-a]pyridine (46). A mixture of 45 (1.14 g, 1.87 mmol)
and POCl3 (0.35 mL, 3.74 mmol) in 1,2-dichloroethane (10 mL) was
heated at 150 °C under microwave irradiation for 10 min. After
cooling to room temperature, the resulting reaction mixture was
diluted with CH2Cl2 and washed with NaHCO3 (aqueous saturated
solution). The organic layer was separated, dried over Na2SO4, and the
volatiles were evaporated in vacuo. The residue thus obtained was
purified by column chromatography (silica gel, MeOH−NH3 in
CH2Cl2, 0/100 to 20/80) to give 46 as a white solid (0.26 g, 51%). Mp
184.5 °C. 1H NMR (400 MHz, CDCl3) δ 0.28−0.40 (m, 2 H), 0.58−
0.71 (m, 2 H), 1.11−1.24 (m, 1 H), 3.10 (d, J = 6.7 Hz, 2 H), 6.92 (d,
J = 7.4 Hz, 1 H), 8.05 (d, J = 7.4 Hz, 1 H). LCMS: m/z 276 [M + H]+,
tR = 1.79 min.
[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 concentration 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 (PerkinElmer) using a Unifilter-96 Harvester
(PerkinElmer). Filters were washed 3 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.
2′-Chloro-4-phenyl-3′-trifluoromethyl-3,4,5,6-tetrahydro-
2H-[1,4′]bipyridinyl (49a). To a suspension of NaH (0.19 g, 4.83
mmol, 60% in mineral oil) in DMF (20 mL) cooled at 0 °C was added
4-phenylpiperidine 30 (0.84 g, 5.24 mmol). The resulting reaction
mixture was stirred at 0 °C for 10 min, and then 43 (0.87 g, 4.03
mmol) was added. The reaction mixture was allowed to warm to room
temperature and further stirred for 1 h. The mixture was then
quenched with water and extracted with Et2O. The organic layer was
separated, dried (Na2SO4), and the volatiles were evaporated in vacuo.
The residue thus obtained was purified by column chromatography
(silica gel, MeOH−NH3 in CH2Cl2, 0/100 to 2/98) to give 49a as a
1
solid (0.73 g, 53%). Mp 111.4 °C. H NMR (400 MHz, CDCl3) δ
1.84−2.00 (m, 4 H), 2.63−2.75 (m, 1 H), 3.05 (td, J = 12.0, 3.0 Hz, 2
H), 3.58 (br d, J = 12.7 Hz, 2 H), 6.88 (d, J = 5.8 Hz, 1 H), 7.19−7.29
(m, 3 H), 7.29−7.37 (m, 2 H), 8.17 (d, J = 5.8 Hz, 1 H). LCMS: m/z
341 [M + H]+, tR = 3.13 min.
Data Analysis. The concentration−response curves in the
presence of added EC25 of mGlu2 agonist glutamate to determine
positive allosteric modulation (PAM) were generated using the Prism
GraphPad software (Graph Pad Inc., San Diego, CA, U.S.). The curves
were fitted to a four-parameter logistic equation Y = Bottom + (Top −
2′-Chloro-4-fluoro-4-phenyl-3′-trifluoromethyl-3,4,5,6-tet-
rahydro-2H-[1,4′]bipyridinyl (49b). A mixture of 43 (0.4 g, 1.85
mmol), 4-fluoro-4-phenylpiperidine hydrochloride 35 (0.4 g, 1.85
mmol), and diisopropyletylamine (0.645 mL, 3.70 mmol) in CH3CN
(4 mL) was heated in a sealed tube at 110 °C for 4 h. The mixture was
cooled to room temperature, diluted with EtOAc, and washed with
NaHCO3 (aqueous saturated solution). The organic layer was
separated, dried over Na2SO4, and the volatiles were evaporated in
vacuo to give a residue that was purified by column chromatography
Bottom)/(1 + 10(log EC −X)Hill slope) allowing determination of EC50
50
values. The EC50 is the concentration of a compound that causes a
half-maximal potentiation of the glutamate response. This is calculated
by subtracting the maximal responses of glutamate in the presence of a
fully saturating concentration of a positive allosteric modulator from
the response of glutamate in the absence of a positive allosteric
modulator. The concentration producing the half-maximal effect is
then calculated as EC50. The pEC50 values are calculated as the
−log EC50 (wherein EC50 is expressed in mol L−1).
Patch Clamp Assay. Experiments were performed using HEK293
cells stably expressing the HERG potassium channel. Cells were grown
at 37 °C and 5% CO2 in culture flasks in MEM medium supplemented
with 10% heat-inactivated fetal bovine serum, 1% L-glutamine−
penicillin−streptomycin solution, 1% nonessential amino acids
(100×), 1% sodium pyruvate (100 mM), and 0.8% Geneticin (50
mg/mL). Before use the cells were subcultured in MEM medium in
the absence of 5 mL of L-glutamine−penicillin−streptomycin. For use
in the automated patch-clamp system PatchXpress 7000A (Axon
Instruments) cells were harvested to obtain cell suspension of single
cells. Extracellular solution contained the following (mM): 150 NaCl,
4 KCl, 1 MgCl2, 1.8 CaCl2, 10 HEPES, 5 glucose (pH 7.4 with
NaOH). Pipette solution contained the following (mM): 120 KCl, 10
HEPES, 5 EGTA, 4 ATP-Mg2, 2 MgCl2, 0.5 CaCl2 (pH 7.2 with
KOH). Patch-clamp experiments were performed in the voltage-clamp
mode, and whole-cell currents were recorded with an automated
1
(silica gel, CH2Cl2) to give 49b (0.53 g, 62%). H NMR (500 MHz,
CDCl3) δ 2.08−2.17 (m, 2 H), 2.23 (dt, J = 14.3, 8.8 Hz, 1 H), 2.31
(dt, J = 13.9, 8.8 Hz, 1 H), 3.40 (br d, J = 8.1 Hz, 4 H), 6.94 (d, J = 5.8
Hz, 1 H), 7.32−7.38 (m, 1 H), 7.38−7.45 (m, 4 H), 8.23 (d, J = 5.5
Hz, 1 H). LCMS: m/z 359 [M + H]+, tR = 3.98 min.
(4-Phenyl-3′-trifluoromethyl-3,4,5,6-tetrahydro-2H-[1,4′]-
bipyridinyl-2′-yl)hydrazine (50a). To a suspension of compound
49a (0.35 g, 1.03 mmol) in THF (6 mL) at room temperature was
added hydrazine monohydrate (0.2 mL, 4.11 mmol). The reaction
mixture was heated at 160 °C for 45 min under microwave irradiation.
Addition of hydrazine monohydrate (0.2 and 0.25 mL) to the reaction
mixture followed by heating at 160 °C under microwave irradiation for
45 min was repeated twice. After cooling to room temperature, the
resulting solution was concentrated in vacuo and the residue thus
obtained was precipitated by treatment with Et2O to give 50a as a
1
white solid (0.32 g, 93%). H NMR (400 MHz, DMSO-d6) δ 1.82−
1.92 (m, 2 H), 1.99 (qd, J = 12.5, 3.9 Hz, 2 H), 2.65−2.76 (m, 1 H),
2.86 (br t, J = 11.2, 2 H), 3.84 (br d, J = 12.3 Hz, 2 H), 4.90 (s, 2 H),
6.38 (d, J = 5.3 Hz, 1 H), 7.16−7.25 (m, 1 H), 7.25−7.38 (m, 5 H),
8.08 (d, J = 5.1 Hz, 1 H). LCMS: m/z 337 [M + H]+, tR = 2.94 min.
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patch-clamp assay utilizing the PatchXpress 7000A system (Axon
Instruments). Current signals were amplified and digitized by a
Multiclamp amplifier, stored, and analyzed by using the PatchXpress
and DataXpress software and Igor 5.0 (Wavemetrics). The holding
potential was −80 mV. The HERG current (K+-selective outward
current) was determined as the maximal tail current at −40 mV after a
2 s depolarization to +60 mV. Pulse cycling rate was 15 s. Before each
test pulse a short pulse (0.5 s) from the holding potential to −60 mV
was given to determine (linear) leak current. After establishment of
whole-cell configuration and a stability period, the vehicle was applied
for 5 min followed by the test substance at increasing concentrations
of 10−7, 3 × 10−7, and 3 × 10−6 M. Each concentration of the test
substance was applied twice. The effect of each concentration was
determined after 5 min as an average current of three sequential
voltage pulses. To determine the extent of block, the residual current
was compared with vehicle pretreatment.
Method for Identification of Main Metabolites. The test
compounds were incubated with human and rat liver microsomes at
0.4% final solvent concentration (0.16% DMSO and 0.24% CH3CN).
The study was conducted to identify the main metabolites formed
after 0 min (control) and 60 min of incubation time in the presence of
NADPH generating system at 37 °C. Test concentration of the
compound was 5 μM. Samples were compared to the control
incubations in which compound was added at termination. Data were
acquired on a Waters UPLC/QToF Premier mass spectrometer using
a 10 min generic UPLC method and a generic MSe method.
Complementary MS/MS experiments were performed when neces-
sary. An Acquity UPLC C18 (2.1 mm × 100 mm, 1.8 μm) column was
used. Interpretation of data was performed with Waters Metabolynx
software.
Sleep−Wake EEG. Animals, Drug Treatment, and Exper-
imental Procedure. All in vivo experimental procedures were
performed according to the applicable European Communities
Council Directive of November 24, 1986 (86/609/EEC) and
approved by the Animal Care and Use Committee of Janssen
Pharmaceutical Companies of Johnson & Johnson and by the local
ethical committee.
Male Sprague−Dawley rats (Charles River, France) weighing 250−
300 g were used. Animals were chronically implanted with electrodes
for recording the cortical electroencephalogram (EEG), electrical neck
muscle activity (EMG), and ocular movements (EOG). All animals
were housed in individually ventilated cages under environmentally
controlled conditions (ambient temperature, 22 °C; humidity, 60%)
on a 12 h light/dark cycle (lights on from 12:00 a.m. to 12:00 p.m.,
illumination intensity of ∼100 lx). The animals had free access to food
and tap water.
The effects of the tested molecule and vehicle on sleep−wake
distribution during the lights-on period were investigated in 16 rats (n
= 8 each group). Two EEG recording sessions were performed: the
first recording session started at 13:30 h and lasted 20 h following oral
administration of saline. The second recording session was performed
during the same consecutive circadian time and for the same duration
following administration of either vehicle (20% CD + 2H2T) or tested
compound.
Sleep polysomnographic variables were determined offline as
described elsewhere using a sleep stages analyzer, scoring each 2 s
epoch before averaging stages over 30 min periods. Sleep−wake state
classifications were assigned based upon combination of dynamics of
five EEG frequency domains, integrated EMG, EOG, and body activity
level: active wake (AW); passive wake (PW); intermediate stage (pre-
REM transients); rapid eye movement sleep (REM); light non-REM
sleep (lSWS); deep non-REM sleep (dSWS). Different sleep−wake
parameters were investigated over 20 h postadministration, and time
spent in each vigilance state, sleep parameters, latencies for first REM
sleep period and the number of transitions between states were
determined.
followed by a Wilcoxon−Mann−Whitney rank sum test of
comparisons with the control group.
Phencyclidine (PCP) Induced Hyperlocomotion in Mice. Male
NMRI mice (Charles River, France or Germany) were housed 5 per
cage and kept under a 12 h light/dark cycle under constant
temperature and humidity conditions. All experimental procedures
were performed in full compliance with the European legislation
governing the protection of animals used in scientific research.
Phencyclidine (PCP) was purchased from Sigma-Aldrich (France).
Nonhabituated animals were treated with the vehicle, or 20, and
immediately challenged with either PCP (5.0 mg/kg, sc) or vehicle
and individually placed into open-fields for a 30 min period. The
distance traveled by animals was measured using video tracking and
computerized analysis systems (Ethovision XT video tracking system,
version 3.1; Noldus, Wageningen, The Netherlands). Averaged activity
in solvent-treated control mice was 11096 3612 counts (mean
SD; n = 2334). The criterion for drug-induced inhibition of
hyperlocomotion was the following: total distance of < 5500 counts
(4.9% false positives). ED50 values (the dose inducing 50% responders
to this criterion) and corresponding 95% confidence limits were
determined according to the modified Spearman−Kaerber estimate,
using theoretical probabilities instead of empirical ones.59 This
modification allows the determination of the ED50 and its confidence
interval as a function of the slope of the log dose−response curve.60
ASSOCIATED CONTENT
* Supporting Information
LCMS methods for the characterization of intermediate and
final compounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
*Phone: +34 925 245767. Fax: +34 925 245771. E-mail: jcid@
its.jnj.com.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors thank collaborators from Addex Therapeutics and
the members of the purification and analysis group and the
biology and in vitro metabolism team from Janssen R&D.
ABBREVIATIONS USED
■
ADMET, absorption, distribution, metabolism, excretion, and
toxicity; AMPA, α-amino-3-hydroxy-5-methyl-4-isoxazolepro-
pionic acid; BBB, blood−brain barrier; AUC, area under the
curve; AW, active wake; CNS, central nervous system; CyPr,
cyclopropyl; DMS-IV, diagnostic statistical manual of mental
disorders volume IV; dSWS, deep non-REM sleep; EEG,
electroencephalogram; EMG, electrical neck muscle activity;
EOG, ocular movement; GABA, γ-aminobutyric acid, GPCR,
G-protein-coupled receptor; HLM, human liver microsome;
HP-β-CD, 2-hydroxylpropyl-β-cyclodextrin; HTS, high
throughput screening; imidazopyridine, imidazo[1,2-a]-
pyridine; iv, intravenous; LAD, lowest active dose; LE, ligand
efficiency; LELP, lipophilicity-corrected ligand efficiency; LLE,
lipophilic ligand efficiency; LMA, locomotor activity; mGlu2,
metabotropic glutamate 2; lSWS, light non-REM sleep; MW,
molecular weight; ND, not determined; nm, not measurable;
NMDA, N-methyl-D-aspartate; NOEL, no effect level; nt, not
tested; PAM, positive allosteric modulator; PCP, phencyclidine;
PK, pharmacokinetics; PW, passive wake; SAR, structure−
activity relationship; SEM, standard error of mean; po, oral;
REM, rapid eye movement; RLM, rat liver microsome; ROL,
Statistical Analysis. Time spent in each vigilance state (AW, PW,
lSWS, dSWS, IS, and REMS) was expressed as a percentage of the
recording period. A statistical analysis of the obtained data was carried
out by a nonparametric analysis of variance of each 30 min period,
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Griffey, K. R.; Tizzano, J. P.; Kallman, M. J.; Helton, D. R.; Schoepp,
D. D. Design, synthesis, and pharmacological characterization of
(+)-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylic acid (LY354740): a
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that the decision was made after a recently conducted independent
futility analysis concluded that HBBN, the second of Lilly’s two pivotal
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703018.
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Cluzeau, P.; Nhem, V.; Furnari, R.; Behaj, N.; Poulain, G.; Finn, T.;
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REM sleep onset latency; sc, subcutaneous; SD, standard
deviation; sw-EEG, sleep−wake electroencephalogram; TPSA,
total polar surface area; triazolopyridine, 1,2,4-triazolo[4,3-
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