Discovery of 8-Trifluoromethyl-3-cyclopropylmethyl-7-[(4-(2,4-difluorophenyl)-1-piperazinyl)methyl]-1,2,4-triazolo[4,3-a]pyridine (JNJ-46356479), a Selective and Orally Bioavailable mGlu2 Receptor Positive Allosteric Modulator (PAM)

Article abstract of DOI:10.1021/acs.jmedchem.6b00913

Positive allosteric modulators of the metabotropic glutamate 2 receptor have generated great interest in the past decade. There is mounting evidence of their potential as therapeutic agents in the treatment of multiple central nervous system disorders. We have previously reported substantial efforts leading to potent and selective mGlu2 PAMs. However, finding compounds with the optimal combination of in vitro potency and good druglike properties has remained elusive, in part because of the hydrophobic nature of the allosteric binding site. Herein, we report on the lead optimization process to overcome the poor solubility inherent to the advanced lead 6. Initial prototypes already showed significant improvements in solubility while retaining good functional activity but displayed new liabilities associated with metabolism and hERG inhibition. Subsequent subtle modifications efficiently addressed those issues leading to the identification of compound 27 (JNJ-46356479). This new lead represents a more balanced profile that offers a significant improvement on the druglike attributes compared to previously reported leads.

Full text of DOI:10.1021/acs.jmedchem.6b00913

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Article
Discovery of 8-Trifluoromethyl-3-cyclopropylmethyl-7-[(4-
(2,4-difluorophenyl)-1-piperazinyl)methyl]-1,2,4-triazolo[4,3-
a]pyridine (JNJ-46356479), a Selective and Orally Bioavailable
mGlu2 receptor Positive Allosteric Modulator (PAM)
José María Cid, Gary Tresadern, Juan Antonio Vega, Ana Isabel de Lucas, Alcira del Cerro,
Encarnación Matesanz, María Lourdes Linares, Aránzazu García, Laura Iturrino, Laura Pérez-
Benito, Gregor J. Macdonald, Daniel Oehlrich, Hilde Lavreysen, Luc Peeters, Marc Ceusters,
Abdellah Ahnaou, Wilhelmus Drinkenburg, Claire Mackie, Marijke Somers, and Andrés A. Trabanco
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00913 • Publication Date (Web): 31 Aug 2016
Downloaded from http://pubs.acs.org on August 31, 2016
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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
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Page 1 of 40
Journal of Medicinal Chemistry
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Discovery of 8ꢀTrifluoromethylꢀ3ꢀcyclopropylmethylꢀ7ꢀ
[(4ꢀ(2,4ꢀdifluorophenyl)ꢀ1ꢀpiperazinyl)methyl]ꢀ1,2,4ꢀ
triazolo[4,3ꢀa]pyridine (JNJꢀ46356479), a Selective and
Orally Bioavailable mGlu2 receptor Positive Allosteric
Modulator (PAM)
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Jose María Cid,a,* Gary Tresadern,a Juan Antonio Vega,a Ana Isabel de Lucas,a Alcira del Cerro,a
Encarnación Matesanz,a María Lourdes Linares,a Aránzazu García,a Laura Iturrino,a Laura Pérezꢀ
Benito,c Gregor J. Macdonald,b Daniel Oehlrich,b Hilde Lavreysen,b Luc Peeters,b Marc Ceusters,b
Abdellah Ahnaou,b Wilhelmus Drinkenburg,b Claire Mackie,b Marijke Somers,b Andrés A. Trabanco a,*
aJanssen Research & Development, a division of JanssenꢀCilag, S.A., JanssenꢀCilag, Toledo, Spain.
bJanssen Research & Development, Janssen Pharmaceutica NV, Beerse, Belgium.
cLaboratori de Medicina Computacional Unitat de Bioestadistica, Facultat de Medicina, Universitat
Autonoma de Barcelona, Bellaterra, Spain.
RECEIVED DATE (to be automatically inserted after your manuscript is accepted if required
according to the journal that you are submitting your paper to)
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ABSTRACT: Positive allosteric modulators of the metabotropic glutamate 2 receptor have generated
great interest in the last decade. There is mounting evidence of their potential as therapeutic agents in
the treatment of multiple central nervous system disorders. We have previously reported substantial
efforts leading to potent and selective mGlu2 PAMs. However, finding compounds with the optimal
combination of in vitro potency and good drugꢀlike properties has remained elusive, in part because of
the hydrophobic nature of the allosteric binding site. Herein, we report on the lead optimization process
to overcome the poor solubility inherent to the advanced lead 6. Initial prototypes already showed
significant improvements in solubility while retaining good functional activity but displayed new
liabilities associated to metabolism and hERG inhibition. Subsequent subtle modifications efficiently
addressed those issues leading to the identification of compound 27 (JNJꢀ46356479). This new lead
represents a more balanced profile that offers a significant improvement on the druglike attributes
compared to previously reported leads.
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KEYWORDS Allosteric modulators, mGlu2, mGluR2, GRM2, PAMs, metabotropic glutamate
receptor, GPCR, class C GPCR, schizophrenia, anxiety, depression, sleep wake architecture, REM
sleep, antipsychotic, homology model, molecular dynamics.
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Journal of Medicinal Chemistry
INTRODUCTION
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3
4
The metabotropic glutamate 2 (mGlu2) receptor, which belongs to the group II subfamily of
metabotropic glutamate (mGlu) receptors along with the mGlu3 receptor, has proven to be of particular
interest in neuropharmacology.1,2 It is widely distributed in the brain and is preferentially expressed on
presynaptic nerve terminals, where it negatively modulates glutamate and GABA release.3 High levels
of mGlu2 receptors can be found in brain areas such as prefrontal cortex, hippocampus and amygdala
where glutamate hyperfunction may be implicated in disorders and diseases such as anxiety,
schizophrenia4,5 and chronic pain.6,7
5
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Clinical data have become available for several mixed mGlu2/3 agonists in anxiety and schizophrenia.
Thus, (+)ꢀ2ꢀaminobicyclo[3.1.0]hexaneꢀ2,6ꢀdicarboxylic acid (1, LY354740, Figure 1)8 was found to
have anxiolytic efficacy in healthy volunteers showing activity in fearꢀpotentiated startle and panic
induction after CO2 challenge.9 (1S,2S,5R,6S)ꢀ2ꢀ[(2'S)ꢀ(2'ꢀAmino)propionyl]aminobicyclo[3.1.0]
hexaneꢀ2,6ꢀdicarboxylic acid (2, LY544344),10 a prodrug of 1, showed significant anxiolytic effects in
a clinical trial with patients suffering from generalized anxiety disorder (GAD). Treatment with 2
resulted in a significant improvement from baseline in Hamilton Anxiety and Clinical Global
Impression Scores as well as in remission rates compared to placebo. Unfortunately this trial was
discontinued in an early stage based on findings of convulsions in preclinical studies.11 In addition,
Phase IIb clinical data have been reported for [(1R,4S,5S,6S)ꢀ2ꢀthiabicyclo[3.1.0]ꢀhexaneꢀ4,6ꢀ
dicarboxylic acid,4ꢀ[(2S)ꢀ2ꢀaminoꢀ4ꢀ(methylthio)ꢀ1ꢀoxobutyl]aminoꢀ 2,2ꢀdioxide monohydrate (3,
LY2140023, pomaglumetad methionil),12 a prodrug of the mixed mGu2/3 agonist (−)ꢀ(1R,4S,5S,6S)ꢀ4ꢀ
aminoꢀ2ꢀsulfonylbicyclo[3.1.0]hexaneꢀ4,6ꢀdicarboxylic acid (LY404039),13 showing improvement of
both positive and negative symptoms in schizophrenic patients.14 However, in 2012 the originating
company announced the decision to stop the Phase III clinical trials of 3 for the treatment of patients
suffering from schizophrenia.15,16 Moreover, a very recent analysis has shown that 3 is effective in
subgroups of schizophrenic patients that are early in disease and had not been treated with atypical
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antipsychotic antagonizing 5ꢀHT2A receptors but had been treated with classical antipsychotics
antagonizing D2 receptors.17
1
2
3
4
5
6
7
8
Given the interest in mGlu2 receptor activation, attention has turned towards the search for selective
mGlu2 receptor positive allosteric modulators as an alternative approach to enhance endogenous
glutamate agonist response with little or no intrinsic activity. The fact that allosteric modulators bind at
an alternative site to orthosteric agonists offers multiple potential benefits: opportunity to access a novel
chemical space, potential for improved selectivity versus other members of the mGlu family, and lower
potential for receptor desensitization and tolerance because PAMs function only in the presence of
physiologically controlled levels of glutamate. To date, multiple series of selective mGlu2 receptor
PAMs have been described,18ꢀ20 and two of them have entered the clinic: 7ꢀmethylꢀ5ꢀ[3ꢀ(piperazinꢀ1ꢀ
9
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ylmethyl)ꢀ1,2,4ꢀoxadiazolꢀ5ꢀyl]ꢀ2ꢀ[[4ꢀ(trifluoromethoxy)phenyl]methyl]ꢀ3Hꢀisoindolꢀ1ꢀone
(4,
AZD8529)21 from AstraZeneca and 1ꢀbutylꢀ3ꢀchloroꢀ4ꢀ(4ꢀphenylꢀ1ꢀpiperidinyl)ꢀ(1H)ꢀpyridone (5, JNJꢀ
40411813, also known as ADX71149) from Janssen Pharmaceuticals, Inc. (Janssen) and Addex
Therapeutics (Addex). Compound 4 (Figure 1) advanced into phase II clinical trials in schizophrenic
patients in 200922 but it was discontinued in early 2011 due to lack of efficacy.23 Currently 4 is in the
recruiting phase for a phase II clinical trial to study smoking cessation in female smokers.24
In an exploratory phase IIa study in schizophrenia, compound 5 met the primary objectives of safety
and tolerability. Moreover, patients treated with antipsychotics who experience residual negative
symptoms were identified as the subgroup that may potentially benefit from addꢀon treatment with 5,
although this is yet to be established in a formal proofꢀofꢀconcept study.25 In addition, in a second phase
IIa study with 5 as adjunctive therapy in patients having major depressive disorder with significant
anxiety symptoms, 5 did not meet the criterion for efficacy vs. placebo.26 Despite a lack of signal on the
primary outcome measure, treatment with 5 showed efficacy signals on several anxiety measures and on
all depression measures,27 however overall the results do not suggest efficacy for 5 as an adjunctive
treatment for patients with MDD with significant anxious symptoms.
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Journal of Medicinal Chemistry
To search for a more potent, efficacious and structurally differentiated backꢀup candidate to pyridone
1
2
5, a scaffoldꢀhopping drugꢀdesign strategy was carried out. Computational techniques based on 3D
shape and electrostatic similarity led to the identification of a series of imidazopyridines28ꢀ31 which
evolved into the recently disclosed 1,2,4ꢀtriazolopyridines.32,33 Structure activity relationship (SAR)
studies resulted in the lead compound 3ꢀcyclopropylmethylꢀ7ꢀ(4ꢀphenylꢀpiperidinꢀ1ꢀyl)ꢀ8ꢀ
trifluoromethyl[1,2,4]triazolo[4,3a] pyridine (6, JNJꢀ42153605)32 as a potent and selective mGlu2
receptor PAM with an acceptable pharmacokinetic profile in rodent and nonꢀrodent species (Figure 1).
Compound 6 showed centrally mediated in vivo effectivity in models sensitive to mGlu2 receptor
modulation such as sleepꢀwake electro encephalogram (swꢀEEG) in rats,34 showing suppressed REM
sleep during the first four hours after oral administration of a 3 mg/kg dose. However, 6 showed limited
solubility in water (< 0.004 mg/mL) and pH 4 buffer containing 20% HPꢀβꢀcyclodextrin (0.995
mg/mL), which could hinder its further development. These deficiencies were initially predicted by our
kinetic solubility assay, where 6 presented solubility values lower than 0.8 ꢀM at pH 4 and 7. We
hypothesized that placing a methylene spacer between the piperidine ring and the triazole core (A,
Figure 1) would generate a basic center and hence improve aqueous solubility. This strategy delivered a
new potent, selective, soluble, orally bioavailable triazolopyridine class, the subject of this report. An
extensive profiling of the most advanced lead 8ꢀtrifluoromethylꢀ3ꢀcyclopropylmethylꢀ7ꢀ[(4ꢀ(2,4ꢀ
3
4
5
6
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difluorophenyl)ꢀ1ꢀpiperazinyl)methyl]ꢀ1,2,4ꢀtriazolo[4,3ꢀa]pyridine
(27,
JNJꢀ46356479)
which
overcomes deficiencies associated with 6 is also discussed.
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Page 6 of 40
O
S
H
H
O
1
2
3
4
H
H
HO2C
HO2C
CO2H
CO2H
NH
HO2C
H
O
H
O
CO2H
NH2
NH
S
5
6
7
NH2
2, LY544344
NH2
1, LY354740
3, LY2140023
8
9
O
O
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Cl
N
N
N
O
N
OCF3
N
N
5, JNJ-40411813
4, AZD8529
HN
mGlu2 PAM EC50 = 195 nM
mGlu2 PAM EMAX (%) = 110
mGlu2 PAM EC50 = 147 nM
mGlu2 PAM EMAX (%) = 273
mGlu2 PAM EC50 = 17 nM
N
N
mGlu2 PAM EMAX (%) = 285
F3C
N
N
N
N
in vivo active:
sw-EEG at 3 mg/kg p.o.
F3C
N
N
PCP-LMA at 5.4 mg/kg s.c.
this work
Solubility:
R1
R2
A
<0.004 mg/mL in water
6, JNJ-42153605
0.995 mg/mL 20% HP-β-CD, pH = 4
Figure 1. Representative mGlu2 receptor orthosteric agonists and PAMs. Previous key leads 5 and 6 are
shown, along with schematic A describing the subject of this study with NR1R2 groups shown in Table
1.
As shown in Scheme 1, the synthesis of Cꢀ7 aminomethylene triazolopyridine derivatives 6ꢀ23 were
prepared using two alternative procedures. The key intermediate 3 was obtained from the 7ꢀ
chlorotriazolopyridine 1,32 followed by Suzuki cross coupling to introduce a vinyl group and subsequent
oxidative rupture of the double bond in 2 to afford the aldehyde intermediate 3. Final compounds 6ꢀ23
were prepared either by reductive amination with NaBH(OAc)3 or by a twoꢀstep synthesis starting with
a reduction of the carbonyl function in 3 with NaBH4, followed by reaction of 4 with mesyl chloride
leading to intermediate 5 which was then transformed into the corresponding target compounds by
nucleophilic substitution reaction with the corresponding amines.
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Journal of Medicinal Chemistry
N
N
N
(3)
N
N
N
1
2
3
4
N
N
(8)
F3C
N
F3C
O
c
a
F3C
b
N
F3C
Cl
N
N
N
(7)
R1
R2
8
9
7
12-29
5
6
7
8
d
f
9
10
11
12
13
14
15
16
17
N
N
N
N
F3C
F3C
e
N
N
OH
OMs
10
11
Scheme 1. Reagents and conditions: (a) 4,4,5,5ꢀtetramethylꢀ2ꢀvinylꢀ1,3,2ꢀdioxoborolane, Pd(PPh3)4,
1,4ꢀdioxane, aq. NaHCO3, 100 ºC, 18 h, 94 %; (b) OsO4, NaIO4, 1,4ꢀdioxane/H2O, rt, 2 h, 60 %; (c)
amine, NaBH(OAc)3, AcOH, DCE, rt, 16h, 18 – 64%; (d) NaBH4, MeOH, 0 ºC, 1 h, 81 %; (e) MsCl,
NEt3, CH2Cl2, rt, 2 h, 84 % ; (f) amine, DIPEA, CH3CN, 80ꢀ90 ºC, 1 ꢀ 4 h, 6 – 60 %.
18
19
20
21
22
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27
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32
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Representative compounds of this Cꢀ7 aminomethylene triazolopyridine series (12ꢀ29) are shown in
Table 1 along with mGlu2 PAM functional activity, kinetic solubility, human and rat metabolic stability
and hERG patchꢀclamp data. In general, the introduction of the methylene spacer between the
triazolopyridine core and the cyclic amine was beneficial for the kinetic solubility of the molecules
while not being detrimental for their functional activity. However this improved solubility was
accompanied by diminished metabolic stability in rat liver microsomes (RLM) and enhanced hERG
channel inhibition. These trends were observed for the vast majority of compounds prepared within this
series. As such the identification of a lead combining all the desired properties was not initially straight
forward.
By way of example, 3ꢀ(cyclopropylmethyl)ꢀ7ꢀ[(4ꢀphenylpiperidinꢀ1ꢀyl)methyl]ꢀ8ꢀ(trifluoromethyl)ꢀ
[1,2,4]triazolo[4,3ꢀa]pyridine (12, also known as JNJꢀ46281222)35, the direct analogue of 6, showed
much improved solubility in the kinetic solubility assay at pH 4 and 7 and displayed equipotent activity
(12: EC50 = 21 nM vs 6: EC50 = 17 nM) in the mGlu2 receptor functional assay. Unfortunately 12 was
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Page 8 of 40
found to be unstable in RLM (84% metabolized after 15 minutes incubation at 5ꢀM) and showed strong
1
2
inhibition (83%) of the hERG channel at 3 ꢀM. The introduction of additional substituents at position
Cꢀ4 of the piperidine ring (13ꢀ15) was detrimental for mGlu2 PAM potency and did not result in
improved metabolic stability in RLM. Shifting of the phenyl group from position Cꢀ4 to position Cꢀ3 of
the piperidine ring (16) did not improve the overall profile of the molecule. Of note is that an additional
methyl substituent at Cꢀ3 position (17) gave a remarkable increase in potency, with a mGlu2 PAM EC50
∼3 nM; however both 16 and 17 showed poor metabolic stability in HLM and RLM, which halted
further exploration of the 3ꢀphenylpiperidine moiety. The introduction of halogen atoms in the 4ꢀphenyl
substituent of the piperidine ring (18ꢀ20) led to a slight increase in potency, but these analogues still
showed high metabolism in RLM and strong inhibition of the hERG channel. Reduction of metabolism
and diminished hERG channel inhibition was also observed for 21 which contains the 2,4ꢀ
difluorophenyl substituent attached to the C4ꢀposition of the piperidine ring. Compounds 22 and 23,
containing a spacer between the 2,4ꢀdifluorophenyl and piperidine ring, were nearly 4ꢀfold less potent
than 21 and showed higher metabolic instability in both HLM and RLM. The 4ꢀ(2,4ꢀ
difluorophenyl)cyclohexylamine derivatives 24 and 25 were also found to be either metabolically
unstable (24) or show a hERG liability (25). Replacement of the piperidine ring by a piperazine was
detrimental for activity and thus compounds 26 and 27 were ∼2.6ꢀ and ∼5ꢀfold less potent than their
corresponding piperidine counterparts 18 and 19. Compound 27 also combined good kinetic solubility
and microsomal stability, and moreover it only showed 30 % inhibition of the hERG channel at 3 ꢀM
concentration. Although replacement of the aromatic fluorine atoms in 27 by the more lipophilic
chlorine atoms in 28 led to a potency increase (28: EC50 = 17 nM vs 27: EC50 = 78 nM) the inhibition of
the hERG channel was also increased for the latter compound (28: hERG = 86 % inh.). Good primary
activity was also found for the homopiperazine analogue (29) of compound 27, however, this potency
increase came together with high metabolic instability in HLM and RLM. The calculated basicity (pKa)
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
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49
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Journal of Medicinal Chemistry
was highest for molecules such as 25, 27 and 28 which had better HLM and RLM. However, this alone
is insufficient because 26 and 29 have similarly higher basicity but poor metabolic stability.
1
2
3
4
5
6
7
8
9
Table 1. mGlu2 PAM functional activity, kinetic solubility, human and rat metabolic stability and
hERG patchꢀclamp data of representative Cꢀ7 aminomethylene triazolopyridine mGlu2 receptor PAMs
12ꢀ29.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
N
N
F3C
R
N
12-29
hERG
(% inh.
@ 3
Kin sol
(ꢀM)
pH4/pH7c
mGlu2
EC50
(nM)b
mGlu2
Emax
(%)b
pKa
(calc.)a
HLM
(%)d
RLM
(%)d
Compd
R
ꢀM)
< 0.8 / <
0.8
ꢀꢀ
ꢀꢀ
1.1
147
273
23
53
51
5
< 0.8 / <
0.8
3.1
3.7
17
21
285
261
23
11
25
84
57
87
6
N
> 100 / 3
90 / 2
12
N
F
F3C
O
2.4
2.4
2.6
132
58
260
276
249
24
35
69
78
98
98
70
13
14
15
N
93 / 13
n.t.e
n.t.e
N
26
n.t. e/ n.t.e
5.7
5.9
65
240
278
91 / 45
51
97
98
99
n.t.e
n.t.e
16
17
N
N
2.6
n.t. e/ n.t.e
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F
N
N
N
1
2
3
4
5
6
7
8
3.5
3.5
3.5
15
13.5
9.7
353
328
330
> 100 / 2
n.t. e/ n.t.e
n.t. e/ n.t.e
8
13
5
85
90
75
86
18
19
20
Cl
F
93
88
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
F
F
N
3.5
4.4
4.7
15
57
57
255
272
332
n.t. e/ <0.6
> 100 / 7
> 100 / 3
6
9
69
63
21
22
23
F
F
F
N
25
67
57
96
O
F
N
n.t.e
O
F
F
6.5
6.5
38
83
279
264
81 / 31.
81
33
84
34
n.t.e
83
24
25
F
N
H
F
n.t. e/ n.t.e
F
F
N
H
N
5.3
5.5
26
78
366
256
n.t. e/ n.t.e
95 / 17
55
7
99
29
n.t.e
30
N
F
26
27
F
N
N
F
Cl
N
N
4.2
5.8
17
22
262
255
71 / 1
1
9
86
28
29
Cl
F
n.t. e/ n.t.e
91
99
n.t.e
N
F
N
a Most basic pKa calculated with ACS Labs v2014. b Values are means of at least two experiments and within Confidence
Interval > 95%. c Compound concentrations were measured by UPLC/UV using a three point reference calibration and are
d
means of at least two experiments. HLM and RLM data refer to % of compound metabolized after 15 min at 5 ꢀM
concentration e n.t. Not tested.
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Table 2. mGlu2 receptor PAM [35S]ꢀGTPγS functional activity of Glutamate, 27, 5 and 6 on wild type
1
2
3
and mutant receptors.
4
5
6
7
8
9
mGlu2 WT a
(Transient b)
EC50 (nM)
mGlu2
L639A
EC50 (nM)
mGlu2
mGlu2
mGlu2
N735D
EC50 (nM)
mGlu2
W773A
EC50 (nM)
mGlu2
F776A EC50
(nM)
Compd
F643A EC50 L732A EC50
(nM)
(nM)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Glutamate c
5010
65
6310
1350
n.t. d
n.t. d
10000
2000
385
3980
77
6310
1630
27.0
10000
2570
5010
265
5
6
4.8
46.4
>10000
23.6
n.t. d
27
45.7
6610
977
1410
2340
Values are means of at least two experiments. See experimental Methods for more details. WT wild type receptor. b All
data including WT and mutants are for transiently transfected CHOꢀK1 cells, in contrast to stably transfected cells reported
in Table 1. c Glutamate agonist EC50 (glutamateꢀinduced [35S]GTPγS binding). d n.t. Not tested
a
On the basis of these results, compound 27 displayed the best combination of mGlu2 PAM activity,
good microsomal stability and low hERG liability within this series. It was pursued further with
extensive characterization and comparison with the previous leads.
To further understand the in vitro SAR and binding at mGlu2 receptors, we performed binding
displacement, mutagenesis and computational molecular modeling on the key leads. Radioligand
binding studies using [3H]ꢀ12 as an allosteric mGlu2 receptor radioligand35 showed that 27, 5 and 6 fully
displaced [3H]ꢀ12 with an Ki of 150, 180 and 15 nM respectively, suggesting these molecules share a
common allosteric binding site. Experimental mutagenesis was performed analogous to previous
work33,36 (Table 2). Molecules 27, 5 and 6 were affected by mutation of similar amino acids, such as
F6433.36a.40c, N7355.47a.47.c W7736.48a.50c implying the lead molecules make comparable receptor
interactions and likely share a common allosteric binding site.
Molecules 27 and 6 were docked into an mGlu2 receptor homology model revealing a similar
plausible binding mode that were consistent with the binding and mutagenesis experiments; see Figure
2A and Figure S1. They align in a parallel orientation to the alphaꢀhelical segments of the 7ꢀ
transmembrane domain with the cyclopropylmethyl triazolopyridine being oriented toward the
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intracellular side of the receptor and interacting with a hydrophobic cluster formed by L6393.32a.36c
,
1
2
3
4
5
6
7
8
9
F6433.36a.40c, L7325.43a.44c, and W7736.48a.50c, some of which were important in the mutagenesis study. A
triazolopyridine nitrogen of the ligand core accepts a hydrogen bond acceptor from N7355.47a.47.c and
satisfies an important pharmacophoric feature of mGlu2 receptor PAMs.28 The N735D mutation
reduced activity suggesting this amino acid provides a hydrogen bond donor to the ligand. The main
difference occurs towards the extracellular side, whereby the flexibility of 27 permits a more ‘bent’
orientation compared to the more linear orientation of 6. This region of the receptor is more open, which
is consistent with the structural diversity that is permitted on the Cꢀ7 substituents of the
triazolopyridines (Table 1). Allosteric modulators of mGlu receptors typically form few Hꢀbonds, and
instead bind with steric and hydrophobic complementarity in a narrow binding site.37 It is plausible that
the increased potency of 6 compared to 27 comes from a combination of electrostatic and
conformational differences between the piperidine and piperazine rings with the former showing higher
activity for comparable analogues. Alternatively, the increased basicity of 27 compared to 6 (calculated
pKa of 5.5 and 3.1 respectively) may incur an extra desolvation cost contributing to its lower activity.
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26
27
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The stability and behavior of the binding mode for 27 was studied with molecular dynamics (MD) for
a total of 3 (1+2 replicas) 1 microsecond simulations, in the presence of ligand, membrane, solvent and
Gꢀprotein. The receptor was stable throughout the simulations showing little structural movement,
Figure 2(B). The key hydrogen bond between N7355.47a.47.c and the ligand was maintained, Figure 2(C).
Multiple amino acids around the ligand such as F6433.36a.40c showed only small fluctuations in their
position, Figure 2(D). In contrast, W7736.48a.50c made a large inward movement (seen in all three
simulations) in synergy with the cyclopropylmethyl of the ligand to form a hydrogen bond with
Y6473.39a.43c. The tryptophan is in a conserved motif (WLAFxPI) in transmembrane (TM) 6 in mGlu
receptors, suggesting functional importance. Also, the 6.48a position is part of the TM6 transmission
switch important for activation in class A GPCRs. Ligand induced movement of this amino acid may be
a source of functional activity of mGlu2 PAMs. Compared to the MD simulations performed for 6,35
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Journal of Medicinal Chemistry
here we have run the simulations for a further 500 ns, reaching a full microsecond. The results reveal
consistent behavior adding further evidence to the behavior of PAMs of this chemical series. Overall,
the docking and MD simulations confirmed a stable binding mode agreeing with experimental data for
27 and 6.
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5
6
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Figure 2. MD simulation of the “active” state of mGlu2 receptor in complex with the PAM 27. (A)
Proposed docking binding mode of 27 used as input for MD simulations. (B) Rootꢀmeanꢀsquare
deviations (RMSD) during MD simulations on protein αꢀcarbons after superposition of the αꢀcarbons of
the 7ꢀtransmembrane mGlu2 receptor. The three separate simulations are shown as grey, blue and red
solid lines. (C) Evolution of the hydrogen bond between the nitrogen acceptor of the triazolopyridine
scaffold and the amino acid N7355.47a.47.c (D) Snapshots of 27 and selected amino acids at different time
steps of the MD simulation, showing the static nature of some sidechains compared to the inward
movement of the W7736.48a.50c
.
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1
2
3
4
5
6
7
8
9
Compound 27 was further characterized in vitro and in vivo to gain a deeper understanding of the
solubility and metabolism. Thus, improved solubility in the kinetic screen was confirmed in a
thermodynamic assay (Table 3) where 27 showed significantly higher solubility at pH 7.4 and more
significantly at pH 2 consistent with the presence of a protonatable basic center (pKa = 5). That
improvement also may impact protein binding which, along with membrane permeability significantly
decreased compared to compounds such as 5 and 6. Moreover, the increased solubility of 27 translated
into a significant improvement in several in vivo PK parameters in both rat and dog (Tables 4 and 5).
Thus, 27 was rapidly absorbed and showed almost complete absorption. Unlike 5 and 6, compound 27
showed lower clearance and longer elimination halfꢀlife after intravenous (i.v.) administration resulting
in higher bioavailability and ultimately higher distribution to the brain (B/P = 2.1).
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11
12
13
14
15
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23
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26
27
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Table 3. Physicoꢀchemical properties, permeability and rat plasma protein binding for compounds 27, 5
and 6.
Permeability
rat fu plasma/braind
(% free)
Solubility in H2O; pH
7.4 / 2 (mg/mL)
Compd
LogPa / pKaa
(Cacoꢀ2 monolayers;
PappAꢀ>B x 10 cm sꢀ1)
5
6
4.6 / <2
4.4 / 4.1
4.2 / 5
<0.004 / 0.001
<0.004 / 0.078
0.05 / 2.5
12b
12c
22c
0.69/0.38
0.4/0.06
5.6/1.21
27
a Experimental values. b In vitro passive permeability using Cacoꢀ2 monolayers. c In vitro passive permeability using LLCꢀ
PK1 cells. Permeability experiments were conducted at a single concentration (1 ꢁM) in a transwell system with an
incubation of 120 min. The apical to basolateral (AtoB) transport in the presence of the Pꢀgp inhibitor GF120918 was
d
measured and permeation rates (Apparent Permeability) of the test compounds (Papp x10ꢀ6 cm/sec) were calculated.
Unbound plasma and brain concentrations determined by rapid equilibrium dialysis at a concentration of 5 ꢁM.
Finally, 27 showed low cytochrome P450 inhibition potential for the main isoenzymes tested (3A4,
2C9, 2D6, 1A2, 2C19 and 2C8 all IC50 > 20 μM) and no genotoxic potential.
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Journal of Medicinal Chemistry
1
2
3
4
5
6
Table 4. Rat PKa data and in vivo activity in swꢀEEG for compounds 27. 5 and 6.
7
8
9
Cl
AUC0ꢀinf
(ng.h/mL)b
Cmax
(ng/mL)b
swꢀEEG
LAD/LACe
Compd
T1/2 (h)b
Tmax (h)
F (%)
B/Pd
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(mL min/ kg)
23 ± 1b
31, 37c
16 ± 3b
2.3 ± 0.5
2.7 ± 0.2
3 ± 0.6
0.5 ± 0b
0.5c
2250 ± 417
1804 ± 415
938 ± 300
482 ± 151
980 ± 237
31
36
1
3 / 249
3 / 65
5
6
1.4
5 ± 3b
11314 ± 4286
100
2.1
3 / 247
27
a
Study in male SpragueꢀDawley rats dosed at 10 mg/kg p.o. and 2.5 mg/kg i.v., formulated in 20% HPꢀβꢀCD + HCl
solution at pH 4. b Values are the mean of three animals ± SD. Individual values. Ratios calculated after 1 h of a single
c
d
dose at 10 mg/kg sc in 20% HPꢀ
β
ꢀCD + HCl solution at pH 4 in the Swiss mouse. e Compound administered orally. LAD
stands for lowest active dose expressed in mg/kg. LAC stands for lowest active concentration in plasma, estimated on the
basis of Cmax expressed in ng/mL.
Table 5. Dog PKa data for compounds 27, 5 and 6.
Cl
AUC0ꢀinf
Cmax
Compd
T1/2 (h)
Tmax (h)b
F (%)
(mL min/kg)
(ng.h/mL)
(ng/mL)
23 ± 0.8b
29 ± 0.3b
4, 6c
3.7 ± 0.3b
1.1, 1.8c
7, 9c
0.5 ± 0
0.5 ± 0
0.5 ± 0
743 ± 152b
531, 957c
373 ± 88b
218, 472c
459, 1400c
20 ± 4b
18, 33c
18, 44c
5
6
3959, 9864c
27
a Study in male Beagle dogs dosed at 5 mg/kg p.o. and 1 mg/kg i.v., formulated in 20% HPꢀ
mean of three animals ± SD. c Individual values (two dogs)
β
ꢀCD + HCl solution at pH 4. b
In order to rule out possible offꢀtarget interactions compound 27 was tested for positive allosteric
modulation, agonistic or antagonistic activity at the other mGlu receptor subtypes. 27 acts as a weak
mGlu3 receptor PAM (60ꢀfold EC50 ratio compared to mGlu2 PAM activity), and is >85ꢀfold selective
for mGlu2 versus other mGlu receptor subtypes. Overall, 27 showed no or negligible affinity at any of
the targets in the CEREP38 panel of receptors (>100ꢀfold selective for mGlu2).
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As in previously reported mGlu2 receptor PAM series, the in vivo pharmacological activity of
compound 27 was investigated in the sleepꢀwakeꢀEEG paradigm.39 This is a highly sensitive readꢀout of
a compound’s central functional activity that may provide additional insight into its potential therapeutic
application. It has been previously shown that systemic administration of an mGlu2/3 receptor agonist
suppresses REM sleep in rats.40 Internal efforts confirmed these results and have furthermore shown that
this effect is mGlu2 receptorꢀmediated, i.e. absent in mGlu2 KO mice.41
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After acute p.o. administration of a 3 mg/kg dose, compound 27 significantly decreased the amount of
REM sleep in rats during the first 4 h without clear effects on other sleep−wake stages (see Figure 3,
bottom right panel, blue bar). The active dose of 3 mg/kg p.o. corresponded to a peak of plasma
concentration of 247 ng/mL, which is very comparable to those of 5 (249 ng/mL) and ∼ 4ꢀfold higher
than those of 6 (65 ng/mL).
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Journal of Medicinal Chemistry
Figure 3. Effect of single oral doses of 1, 3 and 10 mg/kg of 27 or vehicle (20% CD + 1HCl) on
sleep−wake organization in rats during 20 consecutive hours after administration (line graphs). Mean
percentage of occurrence per 30 min period is indicated for each sleep−wake state. Shaded area
indicates dark period. Small bar charts indicate average amounts of vigilance states in minutes (±SEM,
n = 8 for each group) during the first 4 h postꢀadministration. Dots on the lines of the graph indicate p <
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0.05 versus vehicle (Wilcoxon−Mann−Whitney rank sum);
Wilcoxon−Mann−Whitney rank sum tests compared to vehicle values.
*
indicates
p
<
0.05:
CONCLUSIONS
Herein, we report the further optimization of 6, the most potent and selective mGlu2 PAM lead
compound previously reported from our labs which had an acceptable pharmacokinetic profile in rodent
and nonꢀrodent species. Despite its promising pharmacological features, we were concerned that poor
solubility and suboptimal PK could pose a challenge for achieving sufficient exposure in the clinic,
thereby hindering its further development. Using 6 as a starting point, improvement of the solubility was
achieved by the introduction of a methylene spacer between the piperidine ring and the triazole core.
This delivered a new Cꢀ7 aminomethylene triazolopyridine class, that showed significantly increased
solubility while retaining the in vitro mGlu2 PAM potency. However, that improvement turned out to be
detrimental for the metabolism and hERG affinity, which was subsequently addressed by optimization
of the phenylpiperidine moiety. These efforts brought us to the discovery of 27, a compound with
balanced activity vs solubility. Experimental binding, mutagenesis and computational modelling suggest
that 27 shares a similar binding site and binding mode as previous leads, but with increased flexibility
due to the methylene spacer. Furthermore, 27 exhibited improved drugꢀlike properties with a better PK
profile as reflected by higher oral exposures, lower clearance and higher bioavailability in rodent and
nonꢀrodent species. All this combined with improvements in central penetration and higher free
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fractions positions 27 as a more suitable clinical candidate to explore a wider exposure window in
eventual therapeutic indications.
1
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3
4
5
6
7
8
Therefore, compound 27 is considered an 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.
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ASSOCIATED CONTENT
Biology. Membrane Preparation. CHO cells expressing the human mGlu2 receptor were grown
until they were 80% confluent, washed in iceꢀcold phosphate buffered saline, and stored at −20 °C until
membrane preparation. After thawing, cells were suspended in 50 mM TrisꢀHCl, pH 7.4, and collected
through centrifugation for 10 min at 23 500g at 4 °C. Cells were lysed in 5 mM hypotonic TrisꢀHCl, pH
7.4, and after recentrifugation for 20 min at 30 000g at 4 °C, the pellet was homogenized with an Ultra
Turrax homogenizer in 50 mM Trisꢀ HCl, pH 7.4. Protein concentrations were measured by the BioꢀRad
protein assay using bovine serum albumin as standard.
[35S]GTPγS Binding Assay. For [35S]GTPγS measurements, compound and glutamate were diluted in
buffer containing 10 mM HEPES acid, 10 mM HEPES salt, pH 7.4, containing 100 mM NaCl, 3 mM
MgCl2, and 10 ꢁM GDP. Membranes were thawed on ice and diluted in the same buffer, supplemented
with 14 ꢁg/mL saponin (final assay concentration of 2 ꢁg/mL saponin). Final assay mixtures contained
7 ꢁg of membrane protein and were preincubated with compound alone (determination of agonist
effects) or together with an EC20 concentration (4 ꢁM) 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 Unifilterꢀ96 GF/ B filter plates
(Packard) using a 96ꢀwell Packard filtermate harvester. Filters were washed six times with iceꢀcold 10
mM NaH2PO4/10 mM Na2HPO4, pH 7.4, and filterꢀbound radioactivity was counted in a microplate
scintillation and luminescence counter from Packard.
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Data Analysis. Data were processed using an internal software interface and were calculated as the
percent of the control agonist challenge. Sigmoid concentration−response curves plotting these
percentages versus the log concentration of the test compound were analyzed using nonlinear regression
analysis. The EC50 is the concentration of a compound that causes a halfꢀmaximal potentiation of the
glutamate response. The pEC50 values are calculated as the –log EC50 (wherein EC50 is expressed in mol
L−1).
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Sleep−Wake EEG: Animals, Drug Treatment, and Experimental 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 lightsꢀon 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 on the basis of the combination of
dynamics of five EEG frequency domains, integrated EMG, EOG, and body activity level: active wake
(AW); passive wake (PW); intermediate stage (preꢀREM transients); rapid eye movement sleep (REM);
light nonꢀREM sleep (lSWS); deep nonꢀREM sleep (dSWS). Different sleep−wake parameters were
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investigated over 20 h postꢀadministration, and time spent in each vigilance state, sleep parameters,
latencies for first REM sleep period and the number of transitions between states were determined.
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, followed by a
Wilcoxon−Mann−Whitney rank sum test of comparisons with the control group.
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Mutagenesis experiments. The methodology is identical to as described in the work of Farinha et
al.36 In brief, cDNA encoding human nonꢀmutated and mutated mGlu2 receptors was synthesized by
GeneArt® (Life Technologies, Carlsbad, CA, USA) and subcloned to the mammalian expression vector
pcDNA3.1(+) (Life Technologies). Plasmid DNA was amplified through Escherichia coli
transformation and used for transient transfection in CHOꢀK1 cells. All transfections were performed
using the cationic lipid transfection reagent LipofectamineTM (Life Technologies). The cells were
maintained in DMEM supplemented with 10% heatꢀinactivated FBS, penicillin G, streptomycin
sulphate, pyruvic acid and Lꢀglutamine. Cells were washed, thawed and treated as described above for
membrane preparation. Also, the [35S]GTPγS assay was performed as described above. Proper mutant
receptor expression and glutamate agonistic activity was previously tested.36
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 fourꢀparameter logistic
equation Y = Bottom + (Top − Bottom)/(1 + 10(log EC50−X)Hill slope) allowing determination of EC50
values. The EC50 is the concentration of a compound that causes a halfꢀmaximal 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 halfꢀ
maximal effect is then calculated as EC50. The pEC50 values are calculated as the −log EC50 (wherein
EC50 is expressed in mol L−1).
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Journal of Medicinal Chemistry
Molecular modeling. An active state mGlu2 receptor model was built into which the compound was
docked using GlideXP and the best binding pose was subjected to molecular dynamics simulations. The
mGlu2 receptor model was built using a combination of structural templates. The 7ꢀtransmembrane and
loop sections of the human mGlu2 receptor (Uniprot code Q14416) model were based on the crystal
structures of the human mGlu5 receptor (PDB ID 4OO9)42 and mGlu1 receptor (PDB ID 4OR2)43. To
account for the open conformation of transmembrane helix 6 which is expected in an active state
conformation, this helix was modelled based on the active structure of the β2AR (PDB ID 3SN6)44. The
structure was prepared for further work within Maestro45, for instance to fix missing sidechains/atoms,
assigning protein protonation states with PROPKA, optimize the hydrogen bonding network, etc. The
ligand was prepared for docking using the LigPrep tool and conformers of 27 were calculated with
ConfGen. The 3D conformers were docked into the receptor model using Glide XP with the grid
centered on the crystallized ligand in the mGlu1 receptor. The Glide XP scoring function was used, and
sampling was increased through modifying a number of parameters within Glide: expanded sampling
was turned on, and 100 initial poses were passed to postꢀdocking minimisation. All other docking
parameters were set to the defaults. Docking solutions were then visually inspected.
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Molecular dynamics simulations were performed with the GROMACS v 5.0.5 simulation package.46
The ligandꢀreceptor complexes were embedded in a preꢀequilibrated box (10x10x19 nm) containing a
lipid bilayer (297 molecules of POPC) with explicit solvent (∼47.000 water) and 0.15 M concentration
of Na+ and Clꢀ (∼490 ions).47 The model system was energy minimized and subsequently subjected to a
5 steps MD equilibration. The first step was a 10ns equilibration with the whole system fixed except
hydrogens. Then a 5 ns equilibration was performed in which the loops of the protein were released
from restraints, using the dssp program48 to calculate secondary structure. Finally, three steps of 2 ns
each were performed, the restraints in the ligand and the protein were consecutively relaxed (100ꢀ50ꢀ10
Kcal). After these restraints were released, the final 1000 ns production simulation was performed. An
additional two replicas of 1000 ns were also performed using different initial velocities. All simulations
were run at a constant temperature of 300 K using separate vꢀrescale thermostats for ProteinꢀLigand,
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lipid and water and ions.49 A time step of 2fs was used for the integration of equation of motion. All
bonds and angles were kept frozen using LINCS algorithm.50 LennardꢀJones interactions were
computed using a cutoff of 10 Å, and the electrostatic interactions were treated using PME51 with the
same realꢀspace cutoff. The AMBER99SDꢀILDN force field52 was used for the protein, the parameters
described by Berger and coꢀworkers for lipids,53 and the general Amber force field (GAFF) and HF/6ꢀ
31G*ꢀderived RESP atomic charges for the ligand.54 This combination of protein and lipid parameters
has recently been validated.55
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PatchꢀClamp 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% heatꢀinactivated fetal bovine serum, 1% Lꢀglutamine− 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 Lꢀ
glutamine−penicillin−streptomycin. For use in the automated patchꢀclamp system PatchXpress 7000A
(Axon Instruments), cells were harvested to obtain cell suspension of single cells. The extracellular
solution contained the following (mM): 150 NaCl, 4 KCl, 1 MgCl2, 1.8 CaCl2, 10 HEPES, 5 glucose
(pH 7.4 with NaOH). The pipette solution contained the following (mM): 120 KCl, 10 HEPES, 5
EGTA, 4 ATPꢀMg2+, 2 MgCl2, 0.5 CaCl2 (pH 7.2 with KOH). Patchꢀclamp experiments were performed
in the voltage clamp mode, and wholeꢀcell currents were recorded with an automated patchꢀclamp 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. The 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 wholeꢀcell 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
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Journal of Medicinal Chemistry
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.
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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 singleꢀmode 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 DPXꢀ400 or a Bruker AVꢀ500 spectrometer (Bruker AG)
with standard pulse sequences, operating at 400 and 500 MHz, respectively, using CDCl3 and DMSOꢀd6
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).
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Liquid chromatography combined with mass spectrometry (LCMS) was performed using either an HP
1100 HPLC system (Agilent Technologies, Waldbronn, Germany) or an Acquity UPLC® system
(Waters®, Milford, MA, USA) comprising an LC pump (quaternary or binary) with degasser, an
autosampler, a column oven, a diode array detector (DAD), and a column as specified in the respective
methods. Flow from the column was brought to the MS spectrometer. The MS detector (either SQD or
TOF) was configured with an atmospheric pressure ion source. Nitrogen was used as the nebulizer gas.
Data acquisition was performed with MassLynxꢀOpenlynx software or with ChemsationꢀAgilent 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
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were determined in open capillary tubes either on a Mettler FP62 (A) or a Mettler FP 81HT / FP90 (B)
apparatus with a temperature gradient of 10 °C/min. Maximum temperature was 300 °C.
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Purities of all new compounds were determined by analytical reverse phase RP HPLC or RP UPLC
coupled to a mass spectrometry detector, using the area percentage method on the UV trace scanning
from 200 to 450 nm, and compounds were found to have ≥95% purity unless otherwise specified.
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7ꢀVinylꢀ3ꢀcyclopropylmethylꢀ8ꢀtrifluoromethyl[1,2,4]triazolo[4,3ꢀa]pyridine (8). A suspension of
7 (1.65 g, 5.986 mmol), vinylboronic acid pinacol ester (1.218 ml, 7.183 mmol), Pd(PPh3)4 (0.346, 0.3
mmol) and NaHCO3 (aqueous sat. solution, 12.5 mL) in 1,4ꢀdioxane (64.5 mL) was heated at 150 ºC
under microwave irradiation for 13 min. After cooling, the resulting reaction mixture was diluted with
EtOAc/water and filtered through a pad of diatomaceous earth. The filtrate was washed with water and
NaCl (aqueous sat. solution) and extracted with EtOAc. The organic layer was separated, dried
(Na2SO4) and concentrated in vacuo. The residue was purified again by column chromatography (silica;
EtOAc in DCM 0/100 to 40/60). The desired fractions were collected and concentrated in vacuo to give
8 (1.34 g, 83.7%). LCMS: m/z 268 [M + H]+, tR = 2.27 min. 1H NMR (500 MHz, CDCl3) δ ppm 0.29 ꢀ
0.40 (m, 2 H), 0.57 ꢀ 0.69 (m, 2 H), 1.14 ꢀ 1.23 (m, 1 H), 3.10 (d, J=6.6 Hz, 2 H), 5.72 (d, J=11.0 Hz, 1
H), 5.94 (d, J=17.3 Hz, 1 H), 7.09 (d, J=7.2 Hz, 1 H), 7.18 ꢀ 7.28 (m, 1 H), 8.07 (d, J=7.2 Hz, 1 H)
7ꢀCarboxaldehydeꢀ3ꢀcyclopropylmethylꢀ8ꢀtrifluoromethyl[1,2,4]triazolo[4,3ꢀa]pyridine (9). A
solution of 8 (6.24 g, 21.014 mmol), sodium periodate (13.484 g, 63.041 mmol), osmium tetroxide
(2.5% in tertꢀbutanol, 10.873 mL, 0.841 mmol) in water (55 mL) and 1,4ꢀdioxane (221 mL) was stirred
at rt for 2 h. The resulting reaction mixture was diluted with EtOAc/water and filtered through a pad of
diatomaceous earth. The filtrate was extracted with EtOAc. The organic layer was separated, dried
(Na2SO4) and concentrated in vacuo. The solid residue was washed with Et2O, filtered and dried in
vacuo to give 9 (3.84 g, 67.9%). LCMS: m/z 270 [M + H]+, tR = 1.62 min. 1H NMR (500 MHz, CDCl3)
δ ppm 0.32 ꢀ 0.43 (m, 2 H), 0.61 ꢀ 0.73 (m, 2 H), 1.16 ꢀ 1.29 (m, 1 H), 3.17 (d, J=6.9 Hz, 2 H), 7.49 (d,
J=7.2 Hz, 1 H), 8.21 (d, J=7.2 Hz, 1 H), 10.54 (d, J=1.2 Hz, 1 H).
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7ꢀHydroxymethylꢀ3ꢀcyclopropylmethylꢀ8ꢀtrifluoromethyl[1,2,4]triazolo[4,3ꢀa]pyridine (10). To a
solution of 9 (1.73 g, 6.426 mmol) in MeOH (58 mL) stirred at 0º C, was added portionwise sodium
borohydride (0.243, 6.426 mmol). The resulting mixture was stirred at rt for 1 h. The resulting mixture
was concentrated in vacuo. The residue was treated with water and NaCl (aqueous sat. solution) and
extracted with EtOAc. The organic layer was separated and concentrated in vacuo. The residue was
purified by column chromatography (silica; MeOH/NH3 in DCM 0/100 to 5/95). The desired fractions
were collected and concentrated in vacuo to give 10 (1.015 g, 58%) as a brown syrup. LCMS: m/z 272
[M + H]+, tR = 0.53 min. 1H NMR (400 MHz, CDCl3) δ ppm 0.15 ꢀ 0.36 (m, 2 H), 0.46 ꢀ 0.64 (m, 2 H),
1.04 ꢀ 1.19 (m, 1 H), 3.01 (d, J=6.7 Hz, 2 H), 5.00 (br s, 2 H), 6.26 (br s, 1 H), 7.63 (d, J=7.2 Hz, 1 H)
8.12 (d, J=7.4 Hz, 1 H).
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7ꢀ(Methylsulfonyloxy)methylꢀ3ꢀcyclopropylmethylꢀ8ꢀtrifluoromethyl[1,2,4]triazolo[4,3ꢀ
a]pyridine (11). To a solution of 10 (1.341 g, 9.678 mmol) and Et3N (0.778 mL, 5.612 mmol) in DCM
(42 mL) stirred at 0ºC, was added dropwise methylsulfonyl chloride (0.749 mL, 9.678 mmol) and
stirred at rt for 2 h. The resulting mixture was treated with NaHCO3 (aqueous sat. solution) and
extracted with DCM. The organic layer was separated and concentrated in vacuo to give 11 (2.6 g,
87%). LCMS: m/z 350 [M + H]+, tR = 0.74 min. 1H NMR (400 MHz, CDCl3) δ ppm 0.28 ꢀ 0.41 (m, 2
H), 0.57 ꢀ 0.70 (m, 2 H), 1.14 ꢀ 1.25 (m, 1 H), 3.13 (d, J=6.7 Hz, 2 H), 3.16 (s, 3 H), 5.49 (br d, J=1.6
Hz, 2 H), 7.17 (d, J=7.2 Hz, 1 H), 8.30 (d, J=7.2 Hz, 1 H).
8ꢀTrifluoromethylꢀ3ꢀcyclopropylmethylꢀ7ꢀ[(4ꢀphenylꢀ1ꢀpiperidinyl)methyl]ꢀ1,2,4ꢀtriazolo[4,3ꢀ
a]pyridine (12). 5 (0.4 g, 0.9 mmol) was added to a stirred solution of phenylpiperidine (0.174 mg, 1.08
mmol) and DIPEA (0.233 mL, 1.35 mmol) in CH3CN (10 mL). The resulting mixture was heated in a
sealed tube at 100 ºC for 4 h. The resulting mixture was concentrated in vacuo. The residue was purified
by column chromatography (silica gel; DCM/EtOAc from 100/0 to 50/50 as eluent). The desired
fractions were collected and concentrated in vacuo. The residue thus obtained was triturated with DIPE
to yield final compound 12 (0.272 g, 77%) as a white solid. LCMS: m/z 415 [M + H]+, tR = 3.46 min.
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1H NMR (500 MHz, DMSOꢀd6)
δ ppm 0.27 ꢀ 0.40 (m, 1 H), 0.45 ꢀ 0.62 (m, 2 H), 1.14 ꢀ 1.34 (m, 1 H),
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1.94 (br. d, J=13.3 Hz, 2 H), 2.15 ꢀ 2.34 (m, 2 H), 2.81 ꢀ 2.95 (m, 1 H), 3.12 (d, J=6.9 Hz, 2 H), 3.16 ꢀ
3.33 (m, 2 H), 3.54 (br. d, J=11.6 Hz, 2 H), 4.61 (br. s., 2 H), 7.17 ꢀ 7.29 (m, 3 H), 7.34 (t, J=7.5 Hz, 2
H), 7.88 (d, J=7.2 Hz, 1 H), 8.93 (d, J=7.2 Hz, 1 H), 11.22 (br. s., 1 H). M. p. 144.6 ºC (B).
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8ꢀTrifluoromethylꢀ3ꢀcyclopropylmethylꢀ7ꢀ[(4ꢀphenylꢀ4ꢀfluoroꢀ1ꢀpiperidinyl)methyl]ꢀ1,2,4ꢀ
triazolo[4,3ꢀa]pyridine (13). To a solution of 4ꢀfluoroꢀ4ꢀphenylpiperidine hydrochloride [C.A.S.
1056382ꢀ25ꢀ2] (0.096 g, 0.446 mmol) in DCE stirred at rt (2.14 mL) was added 3 (0.1 g, 0.371 mmol)
and the resulting mixture was stirred at rt overnight. Then, acetic acid (0.037 mL) was added and stirred
at rt for 4 h. Then, sodium triacetoxyꢀborohydride (0.87 g, 0.409 mmol) was added and stirred at rt
overnight. The reaction mixture was neutralized with Na2CO3 (aqueous sat. solution) and extracted with
DCM. The organic layer was dried (Na2SO4) and concentrated in vacuo. The crude product thus
obtained was purified by column chromatography (silica gel; DCM/EtOAc from 100/0 to 50/50 as
eluent). The desired fractions were collected and concentrated in vacuo. The residue obtained was
triturated with Et2O to give 13 (0.029 g, 18%). LCMS: m/z 433 [M + H]+, tR = 3.26 min. 1H NMR (500
MHz, CDCl3)
δ ppm 0.31 ꢀ 0.41 (m, 2 H), 0.59 ꢀ 0.69 (m, 2 H), 1.14 ꢀ 1.24 (m, 1 H), 2.02 (br t, J=11.7
Hz, 2 H), 2.05 ꢀ 2.13 (m, 1 H), 2.17 (td, J=13.2, 4.8 Hz, 1 H), 2.65 (br t, J=11.0 Hz, 2 H), 2.71 ꢀ 2.81
(m, 2 H), 3.11 (d, J=6.9 Hz, 2 H), 3.82 (d, J=1.4 Hz, 2 H), 7.29 ꢀ 7.34 (m, 1 H), 7.36 ꢀ 7.46 (m, 4 H),
7.39 (d, J=7.2 Hz, 1 H), 8.06 (d, J=7.2 Hz, 1 H). M. p. 179.2 °C (B).
8ꢀTrifluoromethylꢀ3ꢀcyclopropylmethylꢀ7ꢀ[(4ꢀphenylꢀ4ꢀtrifluoromethylꢀ1ꢀpiperidinyl)methyl]ꢀ
1,2,4ꢀtriazolo[4,3ꢀa]pyridine (14). Starting from 9 (0.1 g, 0.371 mmol) and 4ꢀtrifluoroꢀ4ꢀ
phenylpiperidine hydrochloride [C.A.S. 1254981ꢀ45ꢀ7] (0.102 g, 0.446 mmol) and following the
procedure described for 13, compound 14 was obtained as a solid (0.073 g, 39%). LCMS: m/z 483 [M +
H]+, tR = 2.75 min. 1H NMR (500 MHz, CDCl3) δ ppm 0.30 ꢀ 0.41 (m, 2 H), 0.59 ꢀ 0.70 (m, 2 H), 1.15 ꢀ
1.24 (m, 1 H), 2.14 ꢀ 2.28 (m, 4 H), 2.67 ꢀ 2.77 (m, 1 H), 3.10 (d, J=6.6 Hz, 2 H), 3.60 (br s, 2 H), 7.34 ꢀ
7.42 (m, 2 H), 7.42 ꢀ 7.47 (m, 4 H), 8.05 (d, J=7.2 Hz, 1 H). M. p. 128.0 °C (B).
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8ꢀTrifluoromethylꢀ3ꢀcyclopropylmethylꢀ7ꢀ[(4ꢀphenylꢀ4ꢀmethoxyꢀ1ꢀpiperidinyl)methyl]ꢀ1,2,4ꢀ
triazolo[4,3ꢀa]pyridine (15). Starting from 11 (0.4 g, 0.9 mmol) and 4ꢀmethoxyꢀ4ꢀphenylpiperidine
hydrochloride [C.A.S. 83949ꢀ38ꢀ6] (0.072 mg, 0.378 mmol) and following the procedure described for
12, compound 15 was obtained as a white solid (0.084 g, 60%). LCMS: m/z 445 [M + H]+, tR = 3.16
min. 1H NMR (400 MHz, CDCl3) δ ppm 0.28 ꢀ 0.41 (m, 2 H), 0.56 ꢀ 0.70 (m, 2 H), 1.15 ꢀ 1.23 (m, 1 H),
1.59 (br s, 1 H) 1.92 ꢀ 2.11 (m, 4 H), 2.55 ꢀ 2.71 (m, 4 H), 2.97 (s, 3 H), 3.10 (d, J=6.7 Hz, 2 H), 3.79 (d,
J=1.6 Hz, 2 H), 7.27 ꢀ 7.32 (m, 1 H), 7.33 ꢀ 7.43 (m, 4 H), 7.44 (d, J=7.4 Hz, 1 H), 8.04 (d, J=7.2 Hz, 1
H). M. p. 143.1 °C (B).
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3ꢀ(Cyclopropylmethyl)ꢀ7ꢀ[(3ꢀphenylꢀ1ꢀpiperidinyl)methyl]ꢀ8ꢀ(trifluoromethyl)ꢀ1,2,4ꢀ
triazolo[4,3ꢀa]pyridine (16). Starting from 11 (0.123 g, 0.315 mmol) and 3ꢀphenylpiperidine (0.063 g,
0.394 mmol), and following the procedure described for 12, compound 16 was obtained as a white solid
(0.011 g, 9%). LCMS: m/z 415 [M + H]+, tR = 3.45 min. 1H NMR (500 MHz, CDCl3)
δ ppm 0.29 ꢀ 0.40
(m, 2 H), 0.58 ꢀ 0.69 (m, 2 H), 1.14 ꢀ 1.24 (m, 1 H), 1.50 (qd, J=12.5, 4.2 Hz, 1 H), 1.69 ꢀ 1.77 (m, 1 H),
1.78 ꢀ 1.86 (m, 1 H), 1.91 ꢀ 2.02 (m, 1 H), 2.18 (td, J=11.5, 2.7 Hz, 1 H), 2.24 (t, J=11.0 Hz, 1 H), 2.77 ꢀ
2.87 (m, 2 H), 2.88 ꢀ 2.95 (m, 1 H), 3.09 (d, J=6.6 Hz, 2 H), 3.68 ꢀ 3.84 (m, 2 H), 7.16 ꢀ 7.24 (m, 3 H),
7.27 ꢀ 7.33 (m, 2 H), 7.46 (d, J=7.2 Hz, 1 H), 8.05 (d, J=7.2 Hz, 1 H).
3ꢀ(Cyclopropylmethyl)ꢀ7ꢀ[(3ꢀmethylꢀ3ꢀphenylꢀ1ꢀpiperidinyl)methyl]ꢀ8ꢀ(trifluoromethyl)ꢀ1,2,4ꢀ
triazolo[4,3ꢀa]pyridine (17). Starting from 9 (0.085 g, 0.316 mmol) and (RS)ꢀ3ꢀmethylꢀ3ꢀ
phenylpiperidine [(C.A.S. 19735ꢀ13ꢀ8), 0.075 g, 0.428 mmol] and following the procedure described for
13, compound 17 was obtained as a white solid (0.07 g, 45.5 %). LCMS: m/z 429 [M + H]+, tR = 3.45
min. 1H NMR (400 MHz, CDCl3)
δ ppm 0.29 ꢀ 0.41 (m, 2 H), 0.57 ꢀ 0.70 (m, 2 H), 1.14 ꢀ 1.22 (m, 1 H),
1.22 ꢀ 1.27 (m, 1 H), 1.30 (br. s, 3 H), 1.53 ꢀ 1.62 (m, 1 H), 1.67 ꢀ 1.82 (m, 1 H), 1.88 ꢀ 2.06 (m, 1 H),
2.32 ꢀ 2.45 (m, 1 H), 2.51 (br. d, J=10.9 Hz, 2 H), 2.78 (br. d, J=9.7 Hz, 1 H), 3.03 ꢀ 3.16 (m, 2 H), 3.67
ꢀ 3.80 (m, 2 H), 7.16 ꢀ 7.24 (m, 1 H), 7.27 ꢀ 7.40 (m, 5 H), 8.00 (d, J=7.4 Hz, 1 H). M. p. 148.5 °C (A).
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Journal of Medicinal Chemistry
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8ꢀTrifluoromethylꢀ3ꢀcyclopropylmethylꢀ7ꢀ[(4ꢀ(2ꢀfluorophenyl)ꢀ1ꢀpiperidinyl)methyl]ꢀ1,2,4ꢀ
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triazolo[4,3ꢀa]pyridine (18). Starting from 5 (0.140 g, 0.315 mmol) and 4ꢀ(2ꢀfluorophenyl)piperidine
(0.075 g, 0.378 mmol), and following the procedure described for 12, compound 18 was obtained (0.065
g, 49%). LCMS: m/z 433 [M + H]+, tR = 3.49 min. 1H NMR (500 MHz, CDCl3) δ ppm 0.30 ꢀ 0.41 (m, 2
H), 0.58 ꢀ 0.69 (m, 2 H), 1.15 ꢀ 1.24 (m, 1 H), 1.75 ꢀ 1.90 (m, 4 H), 2.26 ꢀ 2.36 (m, 2 H), 2.84 ꢀ 2.99 (m,
3 H), 3.10 (d, J=6.6 Hz, 2 H), 3.77 (d, J=1.4 Hz, 2 H), 6.97 ꢀ 7.06 (m, 1 H), 7.07 ꢀ 7.14 (m, 1 H), 7.15 ꢀ
7.22 (m, 1 H), 7.22 ꢀ 7.27 (m, 1 H), 7.47 (d, J=7.2 Hz, 1 H), 8.06 (d, J=7.2 Hz, 1 H). M. p. 151 °C (B).
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8ꢀTrifluoromethylꢀ3ꢀcyclopropylmethylꢀ7ꢀ[(4ꢀ(2ꢀchlorophenyl)ꢀ1ꢀpiperidinyl)methyl]ꢀ1,2,4ꢀ
triazolo[4,3ꢀa]pyridine (19). Starting from 11 (0.140 g, 0.315 mmol) and 4ꢀ(2ꢀchlorophenyl)piperidine
(0.087 g, 0.378 mmol), and following the procedure described for 12, compound 19 was obtained as a
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white solid (0.041 g, 29%). LCMS: m/z 449 [M + H]+, tR = 3.76 min. H NMR (500 MHz, CDCl3) δ
ppm 0.31 ꢀ 0.40 (m, 2 H), 0.58 ꢀ 0.69 (m, 2 H), 1.15 ꢀ 1.24 (m, 1 H), 1.74 (qd, J=12.1, 3.2 Hz, 2 H), 1.88
(br d, J=12.7 Hz, 2 H), 2.34 (td, J=11.8, 2.0 Hz, 2 H), 2.94 (br d, J=11.6 Hz, 2 H), 3.07 (tt, J=12.1, 3.5
Hz, 1 H), 3.10 (d, J=6.6 Hz, 2 H), 3.78 (br d, J=1.7 Hz, 2 H), 7.15 (td, J=7.8, 1.7 Hz, 1 H), 7.23 (dd,
J=8.1, 0.9 Hz, 1 H), 7.29 (dd, J=7.8, 1.7 Hz, 1 H), 7.36 (dd, J=8.1, 1.2 Hz, 1 H), 7.46 (d, J=7.5 Hz, 1
H), 8.05 (d, J=7.2 Hz, 1 H).
8ꢀTrifluoromethylꢀ3ꢀcyclopropylmethylꢀ7ꢀ[(4ꢀ(2,6ꢀdifluorophenyl)ꢀ1ꢀpiperidinyl)methyl]ꢀ1,2,4ꢀ
triazolo[4,3ꢀa]pyridine (20). Starting from 11 (0.123 g, 0.316 mmol)
and 4ꢀ(2,6ꢀ
difluorophenyl)piperidine (0.097 g, 0.395 mmol), and following the procedure described for 12,
compound 20 was obtained as a white solid (0.049 g, 35%). LCMS: m/z 451 [M + H]+, tR = 3.55 min.
1H NMR (500 MHz, CDCl3) δ ppm 0.30 ꢀ 0.41 (m, 2 H), 0.58 ꢀ 0.70 (m, 2 H), 1.14 ꢀ 1.24 (m, 1 H), 1.72
(br d, J=13.3 Hz, 2 H), 2.12 ꢀ 2.23 (m, 2 H), 2.23 ꢀ 2.32 (m, 2 H), 2.91 (br d, J=11.0 Hz, 2 H), 3.03 (tt,
J=12.1, 3.5 Hz, 1 H), 3.11 (d, J=6.9 Hz, 2 H), 3.77 (d, J=1.4 Hz, 2 H), 6.84 (t, J=8.2 Hz, 2 H), 7.09 ꢀ
7.18 (m, 1 H), 7.53 (d, J=7.2 Hz, 1 H), 8.07 (d, J=7.2 Hz, 1 H). M. p. 186.2 °C (B).
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Page 29 of 40
Journal of Medicinal Chemistry
8ꢀTrifluoromethylꢀ3ꢀcyclopropylmethylꢀ7ꢀ[(4ꢀ(2,4ꢀdifluorophenyl)ꢀ1ꢀpiperidinyl)methyl]ꢀ1,2,4ꢀ
triazolo[4,3ꢀa]pyridine (21). Starting from 9 (0.1 g, 0.371 mmol) and 4ꢀ(2,4ꢀdifluorophenyl)piperidine
[C.A.S. 291289ꢀ50ꢀ4] (0.088 g, 0.446 mmol) and following the procedure described for 13, compound
21 was obtained (0.099 g, 59%). LCMS: m/z 451 [M + H]+, tR = 3.58 min. 1H NMR (500 MHz, CDCl3)
δ ppm 0.30 ꢀ 0.41 (m, 2 H), 0.58 ꢀ 0.69 (m, 2 H), 1.15 ꢀ 1.24 (m, 1 H), 1.72 ꢀ 1.87 (m, 4 H), 2.30 (td,
J=11.6, 2.9 Hz, 2 H), 2.80 ꢀ 2.89 (m, 1 H), 2.92 (br d, J=11.6 Hz, 2 H), 3.10 (d, J=6.6 Hz, 2 H), 3.77 (d,
J=1.7 Hz, 2 H), 6.78 (ddd, J=11.0, 9.0, 2.3 Hz, 1 H), 6.83 (td, J=8.2, 2.0 Hz, 1 H), 7.16 ꢀ 7.22 (m, 1 H),
7.45 (d, J=7.2 Hz, 1 H), 8.06 (d, J=7.2 Hz, 1 H). M. p. 153.4 °C (B).
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8ꢀTrifluoromethylꢀ3ꢀcyclopropylmethylꢀ7ꢀ[(4ꢀ(2,4ꢀdifluorophenoxy)ꢀ1ꢀpiperidinyl)methyl]ꢀ1,2,4ꢀ
triazolo[4,3ꢀa]pyridine (22). Starting from 11 (0.140 g, 0.315 mmol)
and 4ꢀ(2,4ꢀ
difluorophenoxy)piperidine [C.A.S. 367501ꢀ08ꢀ4] (0.081 g, 0.378 mmol), and following the procedure
described for 12, compound 22 was obtained as a white solid (0.009 g, 6%). LCMS: m/z 467 [M + H]+,
tR = 3.28 min. 1H NMR (400 MHz, CDCl3) δ ppm 0.29 ꢀ 0.41 (m, 2 H), 0.57 ꢀ 0.70 (m, 2 H), 1.14 ꢀ 1.25
(m, 1 H), 1.79 ꢀ 1.91 (m, 2 H), 1.94 ꢀ 2.05 (m, 2 H), 2.30 ꢀ 2.41 (m, 2 H), 2.74 ꢀ 2.83 (m, 2 H), 3.10 (d,
J=6.7 Hz, 2 H), 3.75 (br d, J=1.6 Hz, 2 H), 4.18 ꢀ 4.27 (m, 1 H), 6.74 ꢀ 6.82 (m, 1 H), 6.82 ꢀ 6.90 (m, 1
H), 6.92 ꢀ 7.01 (m, 1 H), 7.42 (d, J=7.2 Hz, 1 H), 8.05 (d, J=7.2 Hz, 1 H).
8ꢀTrifluoromethylꢀ3ꢀcyclopropylmethylꢀ7ꢀ[(4ꢀ(2,4ꢀdifluorobenzyloxy)ꢀ1ꢀpiperidinyl)methyl]ꢀ
1,2,4ꢀtriazolo[4,3ꢀa]pyridine (23). Starting from 11 (0.140 g, 0.315 mmol)
and 4ꢀ(2,4ꢀ
difluorobenzyloxy)piperidine [C.A.S. 1121592ꢀ61ꢀ7] (0.095 g, 0.378 mmol), and following the
procedure described for 12, compound 23 was obtained (0.054 g, 36%). LCMS: m/z 481 [M + H]+, tR =
3.32 min. 1H NMR (500 MHz, CDCl3) δ ppm 0.30 ꢀ 0.40 (m, 2 H), 0.58 ꢀ 0.69 (m, 2 H), 1.12 ꢀ 1.23 (m,
1 H), 1.64 ꢀ 1.74 (m, 2 H), 1.89 ꢀ 2.01 (m, 2 H), 2.23 ꢀ 2.33 (m, 2 H), 2.70 ꢀ 2.79 (m, 2 H), 3.10 (d,
J=6.6 Hz, 2 H), 3.43 ꢀ 3.51 (m, 1 H), 3.72 (d, J=1.7 Hz, 2 H), 4.55 (s, 2 H), 6.80 (td, J=9.5, 2.3 Hz, 1 H)
6.88 (td, J=8.2, 2.0 Hz, 1 H), 7.37 ꢀ 7.44 (m, 2 H), 8.04 (d, J=7.2 Hz, 1 H). M. p. 126.0 °C (B).
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