Triazolopyridine ethers as potent, orally active mGlu2positive allosteric modulators for treating schizophrenia

Article abstract of DOI:10.1016/j.bmc.2016.11.018

Triazolopyridine ethers with mGlu₂positive allosteric modulator (PAM) activity are disclosed. The synthesis, in vitro activity, and metabolic stability data for a series of analogs is provided. The effort resulted in the discovery of a potent, selective, and brain penetrant lead molecule BMT-133218 ((+)-7m). After oral administration at 10 mg/kg, BMT-133218 demonstrated full reversal of PCP-stimulated locomotor activity and prevented MK-801-induced working memory deficits in separate mouse models. Also, reversal of impairments in executive function were observed in rat set-shifting studies at 3 and 10 mg/kg (p.o.). Extensive plasma protein binding as the result of high lipophilicity likely limited activity at lower doses. Optimized triazolopyridine ethers offer utility as mGlu₂PAMs for the treatment of schizophrenia and merit further preclinical investigation.

Full text of DOI:10.1016/j.bmc.2016.11.018

Accepted Manuscript
Triazolopyridine Ethers as Potent, Orally Active mGlu2 Positive Allosteric
Modulators for Treating Schizophrenia
Mendi A. Higgins, Lawrence R. Marcin, F. Christopher Zusi, Robert Gentles,
Min Ding, Bradley C. Pearce, Amy Easton, Walter A. Kostich, Matthew A.
Seager, Clotilde Bourin, Linda J. Bristow, Kim A. Johnson, Regina Miller, John
Hogan, Valerie Whiterock, Michael Gulianello, Meredith Ferrante, Yanling
Huang, Adam Hendricson, Andrew Alt, John E. Macor, Joanne J. Bronson
PII:
S0968-0896(16)31181-6
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.11.018
BMC 13385
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
29 August 2016
9 November 2016
11 November 2016
Please cite this article as: Higgins, M.A., Marcin, L.R., Christopher Zusi, F., Gentles, R., Ding, M., Pearce, B.C.,
Easton, A., Kostich, W.A., Seager, M.A., Bourin, C., Bristow, L.J., Johnson, K.A., Miller, R., Hogan, J., Whiterock,
V., Gulianello, M., Ferrante, M., Huang, Y., Hendricson, A., Alt, A., Macor, J.E., Bronson, J.J., Triazolopyridine
Ethers as Potent, Orally Active mGlu2 Positive Allosteric Modulators for Treating Schizophrenia, Bioorganic &
Medicinal Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bmc.2016.11.018
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Bioorganic & Medicinal Chemistry
Triazolopyridine Ethers as Potent, Orally Active mGlu2 Positive Allosteric
Modulators for Treating Schizophrenia
Mendi A. Higgins,* Lawrence R. Marcin, F. Christopher Zusi, Robert Gentles, Min Ding, Bradley C. Pearce, Amy
Easton, Walter A. Kostich, Matthew A. Seager, Clotilde Bourin, Linda J. Bristow, Kim A. Johnson, Regina Miller,
John Hogan, Valerie Whiterock, Michael Gulianello, Meredith Ferrante, Yanling Huang, Adam Hendricson, Andrew
Alt, John E. Macor, Joanne J. Bronson
Bristol-Myers Squibb, Research and Development, 5 Research Parkway, Wallingford, CT 06492-7660, USA
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
Triazolopyridine ethers with mGlu2 positive allosteric modulator (PAM) activity are disclosed.
The synthesis, in vitro activity, and metabolic stability data for a series of analogs is provided.
The effort resulted in the discovery of a potent, selective, and brain penetrant lead molecule
BMT-133218 ((+)-7m). After oral administration at 10 mg/kg, BMT-133218 demonstrated full
reversal of PCP-stimulated locomoter activity and prevented MK-801-induced working memory
deficits in separate mouse models. Also, reversal of impairments in executive function were
observed in rat set-shifting studies at 3 and 10 mg/kg (p.o.). Extensive plasma protein binding
as the result of high lipophilicity likely limited activity at lower doses. Optimized
triazolopyridine ethers offer utility as mGlu2 PAMs for the treatment of schizophrenia and merit
further preclinical investigation.
Available online
Keywords:
schizophrenia
mGlu2
metabotropic glutamate receptors
triazolopyridine
2016 Elsevier Ltd. All rights reserved.
1. Introduction
positive symptoms. Unfortunately, they provide little or no
efficacy against negative symptoms and do not improve
Schizophrenia is a complex and chronic mental health
disorder affecting nearly 1 % of the world’s population, including
approximately 3.2 million Americans.1 Consequently, U.S.
healthcare expenses related to schizophrenia treatment have been
estimated at more than $60 billion annually.2 The disease usually
manifests in early adulthood and usually continues throughout
life.3 While the name is derived from the Greek words for split
(schizo) and mind (phrene), sufferers do not have split
personalities. Instead, schizophrenia patients present a wide array
of positive and negative symptoms along with cognitive deficits
that together inflict varying degrees of social and occupational
dysfunction.3 Positive symptoms include hallucinations,
delusions, disorganized thought, and movement disorders, while
negative symptoms encompass social withdrawal, lack of
motivation, reduced pleasure, and flat affect.
neurocognitive deficits. Also, these drugs have been associated
with multiple side-effects including weight gain, type II diabetes,
and hyperlipidemia.6 Consequently, there is a clear and present
need for additional antipsychotic medications that exploit novel
modes of action.
A growing body of evidence, including early work with
phencyclidine (PCP) and ketamine,7 has implicated N-methyl-D-
aspartate (NMDA) receptor hypofunction along with excessive
glutamatergic neurotransmission in the pathophysiology of
schizophrenia.8 Consequently, metabotropic glutamate receptor 2
(mGlu2), which is highly expressed in the forebrain and acts as an
autoreceptor to diminish glutamate tone, has emerged as an
attractive drug target.9 Activation of this G-protein coupled
receptor with exogenous ligands should temper synaptic
glutamate levels and alleviate the symptoms and deficits of
schizophrenia. Indeed, ground breaking work with a dual
mGlu2/3 orthosteric agonist LY2140023 (1, Figure 1)
demonstrated reversal of PCP-induced hyperlocomotion in
animal models.9 Unfortunately, mixed results in clinical trials
eventually lead to the discontinuation of 1 in development.10
Since mGlu2 orthosteric agonists are typically challenged by a
lack of mGlu subtype selectivity, poor CNS penetration, and
potential for receptor desensitization,11 many groups have
refocused their efforts on positive allosteric modulators (PAMs)
The etiology of schizophrenia is not well understood, but it is
believed to involve a combination of genetic predisposition and
environmental triggers.4 The standard of care is focused on
relieving symptoms through pharmacological intervention, and
improving function through psychosocial therapy. Atypical
antipyschotics have been the pharmacological treatment of choice
since the early 1990s.5 These drugs work through transient
inhibition of dopamine and serotonin receptors in the central
nervous system and are reasonably effective at alleviating

of mGlu2.12 Unlike agonists, PAMS only potentiate the function
of mGlu2 receptors that are activated by endogenous glutamate.
This alternative strategy should circumvent receptor
desensitization, confer better therapeutic activity, and minimize
side-effects. Furthermore, allosteric modulators are likely to
exhibit improved CNS penetration since they would not require
the polar functionality that is presumably required for binding to
the orthosteric site in the receptor. Additionally, mGlu2
selectivity with PAMs should be possible given the likelihood for
a less conserved allosteric binding site across receptor subtypes.
Our efforts in the field began with a functional screen of our
proprietary compound deck. Over 1 million compounds were
assayed for their ability to inhibit forskolin stimulated cAMP
production in HEK293 cells stably-expressing human mGlu2.
The substituted triazolopyridine (TZP) 5 (EC50 = 2.7 uM) was
identified from this campaign as an attractive small molecule hit
with low micromolar activity (Figure 2). In the course of
optimizing the mGlu2 activity of compound 5, a patent
application from Janssen had published disclosing 8-amino-
substituted TZPs with mGlu2 PAM activity,21 including a highly
potent phenylpiperidine analog 6 (later identified as JNJ-
42153605 with a reported mGlu2 PAM EC50 = 17 nM).22 While
this application narrowed novel options to continue working in
this space, it also reinforced our desire to exploit untapped
opportunities for this privileged scaffold. During our attempts at
hit optimization of 5, we prepared the triazolopyridine ether 7a as
a synthetic intermediate. Interestingly, compound 7a showed
potent mGlu2 activity (EC50 = 28 nM, Figure 2) and became the
inspiration of a focused drug discovery effort. The following
report will disclose mGlu2 PAM structure activity relationships
(SARs) for various TZP ether analogs as well as encouraging
data in preclinical rodent models of schizophrenia for an
advanced lead.
Figure 1. Clinical mGlu2 agents.
Over the past 13 years, a large variety of structurally distinct
scaffolds with mGlu2 PAM activity have been disclosed and
reviewed in the literature.13 Two drugs and one PET tracer have
been studied in clinical trials. AZD8529 (2, Figure 1) was active
in seven preclinical models that were used to predict
antipsychotic activity.14 Nevertheless, clinical development of
AZD8529 for schizophrenia was abandoned after Phase II studies
due to lack of efficacy. In retrospect, the investigators have
suggested that an inability to assess target engagement, variable
drug exposure, and dose limiting preclinical toxicology findings
contributed to the unfavorable outcome.14,15 Another mGlu2
PAM, JNJ-40411813 (3), has demonstrated robust antipsychotic
effects in preclinical models and was favorably differentiated
from an agonist profile.16,17 An exploratory study in stably
treated patients with schizophrenia suggested that individuals
with prominent negative symptoms may have benefited from
treatment with JNJ-40411813; however, no additional effect
against positive symptoms was reported.18 As with AZD8529,
the investigators have noted that without the capability to
confirm mGlu2 target engagement in the clinic, it was uncertain
whether sufficient receptor occupancy was achieved.16
Furthermore, high-dose administration was associated with
decreased tolerability. A PET imaging agent, [11C]JNJ-42491293
(4) has demonstrated specific and reversible binding to mGlu2 in
rat brain and displacement with a mGlu2 PAM.19 In clinical
studies, the PET tracer exhibited radioactivity uptake and
washout in striatum and cerebellum, consistent with the
distribution of mGlu2 in human brain.20 It has not been reported
if PET tracer 4 shows clinical utility for determining receptor
occupancy of mGlu2 PAMs 2 or 3. Since neither AZD8529 nor
JNJ-40411813 were able to unambiguously test the glutamate
hypothesis for schizophrenia in clinic trials, there is continued
motivation to discover novel mGlu2 PAMs with increased
potency and greater tolerability, which can be developed in
combination with a useful PET tracer.
Figure 2. Triazolopyridine-based mGlu2 PAMs.
2. Chemistry
As outlined in Scheme 1, a variety of 8-alkyloxy substituted
TZP ethers 9a-n were prepared via copper mediated coupling of
primary alcohols with 8-chloro-3-(cyclopropylmethyl)-7-iodo-
[1,2,4]-triazolo[4,3-a]pyridine 8.22 The yield for this method was
typically low (2-10 % yield) due to poor chemoselectivity
between halogen atoms. Nonetheless, the procedure was useful
for providing small quantities of the desired products for in vitro
testing. The choice of 8-chloro and 3-(cyclopropylmethyl)
substitution of the TZP core was driven by SAR reported for
these substituents with structurally related mGlu2 PAMs.21,22
Scheme 1. Reagents and conditions: (a) ROH (7 equiv), copper(I) iodide (2
equiv), 1,10-phenanthroline (1.2 equiv), cesium carbonate (1.8 equiv),
toluene, 100 ºC, 17-24 h, 2-10 % yield. For the structures of compounds 9a-
n, refer to Table 1.
Additional TZP ethers bearing a C-8 trifluoromethyl group
were made using alternative methods (Scheme 2). For this set of
analogs, focus was given to the hydrophobic ether side chains

that had substitution at likely metabolic softspots and/or
possessed some element of conformational restriction.
Compounds 7a, b, e-m were prepared in 4 steps and good overall
yield as shown in Scheme 2. Chlorine displacement in 2,4-
dichloro-3-trifluoromethyl pyridine 1022 with commercially
available or readily obtained alcohols afforded ethers 11 with
complete regioselectivity. Hydrazine displacement of the second
chlorine atom provided hydrazine adducts 12. Acylation with
cyclopropylacetyl chloride followed by mild cyclodehydration
using the Burgess reagent afforded the final products in good
overall yield. Chiral separation of racemic 7m using supercritical
fluid chromotography (SFC) provided the pure enantiomers (+)-
7m and (-)-7m. The absolute configuration of (+)-7m was
assigned 1S,2S through single crystal X-ray analysis using heavy
atom techniques.23
A more convergent palladium catalyzed etherification strategy
was used to prepare TZP ethers 7c and 7d from intermediate 18
(Scheme 4). The use of JOSIPHOS as a ligand for palladium
was critical to the success of this coupling procedure. It should
be noted that an excess of primary alcohol was necessary to
achieve yields of up to 20 %. Also, secondary alcohols were
unreactive under these conditions.
Scheme 4. Reagents and conditions: (a) ROH (5 equiv), JOSIPHOS (0.2
equiv), Cs2CO3 (2 equiv), allylpalladium (II) chloride dimer (0.1 equiv),
toluene, 135 ºC, 3 h, 13-20 % yield. For the structures of compounds 7c and
7 d, refer to Table 2.
3. Results
3.1. In Vitro Characterization
The mGlu2 functional activity for 8-chloro TZP ethers 9a-n is
detailed in Table 1. The simple benzyl ether 9a exhibited modest,
yet encouraging, mGlu2 functional activity (EC50 = 180 nM).
Single chlorine substitution of the benzylic ring at the ortho-,
meta-, and para-positions provided a 3-16 fold improvement in
activity as evidenced by compounds 9b-d. The ortho and meta
isomers 9c and 9d were both more potent (EC50 = 17 nM and 11
nM, respectively) than the para isomer (EC50 = 60 nM).
A
slightly larger increase in potency was observed with the
corresponding isomeric biphenyls 9e-g. In contrast to the chloro
analogs, the para substitution pattern of 9e was preferred (EC50
=
Scheme 2. Reagents and conditions: (a) ROH, NaH, DMF, 0 ºC, 1 h, ~70%;
(b) NH2NH2, dioxane, 95 ºC, 1 h, quantitative yield; (c) cyclopropylacetyl
chloride, TEA, CH2Cl2, 0 ºC, 1 h; (d) Burgess reagent, dioxane/acetonitrile,
85 ºC, 12 h, 9-65 % (over two steps). For the structures of compounds 7a, b,
e-m, refer to Table 2.
6.8 nM). The naphthalene 9h and tetrahydronapthalene 9i
derivatives also maintained high levels of functional activity
(EC50 = 29 nM and 18 nM, respectively). Incorporation of polar
basic functionality in the form of a pyridine, pyrimidine, or
isoquinoline was not tolerated as demonstrated by compounds 9j-
m. Interestingly, the homologated phenethylether 9n also gained
Obviating the need for chiral separation, cyclopropylmethyl
ether (+)-7m was also synthesized using ((1S,2S)-2-(4-
chlorophenyl)cyclopropyl)methanol (17, Scheme 3). The
homochiral alcohol 17 was made from (E)-3-(4-
chlorophenyl)acrylic acid (14) via diastereroselective palladium–
catalyzed cyclopropanation of the (+)-camphorsultam acrylamide
15, followed by lithium aluminum hydride reduction (Scheme 3).
The diastereoselectivity of the cyclopropanation event was
greater than 95% diastereomeric excess (de) and was further
enriched to greater than 99 % de after silica gel column
chromatography of intermediate 16. An X-ray crystal structure
of 16 confirmed the stereochemistry of the newly formed
cyclopropane as 1S,2S.24 Alcohol 17 was converted to (+)-7m via
the methods outlined in Scheme 2.
~6 fold improvement in activity over the benzyl ether 9a.
A
significant issue across the series was moderate to rapid in vitro
metabolism in human, rat, and mouse liver microsomes (HLM,
RLM, and MLM, respectively). Percent of drug remaining after
a 10 minute incubation with RLM and MLM was typically low.
Biotransformation studies of representative analogs with liver
microsomes and freshly prepared rat hepatocytes revealed that O-
dealkylation of the benzyl ether in our analogs was the major
route of metabolism. Glutathione displacement of the 8-
chlorosubstitutent was also observed and considered to be a
significant liability. In order to curb metabolism and avoid
reactive metabolite formation, subsequent efforts to build
additional SAR was carried out with the C-8 trifluoromethyl
group on the TZP core.
Table 1
Functional mGlu2 potency and metabolic stability of TZP ethers 9a-n.
N
N
Cl
N
R
O
mGlu2 PAM EC50
nM (Glu max) a
,
HLM, RLM, MLM
(% remaining)b
cmpd
R
Scheme 3. Reagents and conditions: (a) thionyl chloride, DCM, reflux, 24 h,
quantitative yield; (b) (3aR,6R,7aS)-8,8-dimethylhexahydro-1H-3a,6-
methanobenzo[c]isothiazole 2,2-dioxide, TEA, DCM, 0 ºC, 82 % yield; (c)
N-methyl-N'-nitro-N-nitrosoguanidine, Pd(OAc)2, -10 ºC, DCM, 73 % yield;
(d) LAH, THF, -40 ºC, 1 h, 99 % yield.
benzyl
180(100%)
60(94%)
17(97%)
11(99%)
74, 39, 32
79, 52, 47
77, 34, 52
65, 15, 16
9a
9b
9c
9d
4-chlorobenzyl
3-chlorobenzyl
2-chlorobenzyl

6.8(95%)
13(95%)
20(95%)
83, 50, 55
13, 0.1, 0.2
45, 0.1, 0.2
N
N
9e
9f
F3C
Ph
Ph
N
R
O
mGlu2 PAM EC50, HLM, RLM, MLM
Ph
9g
cmpd
R
nM (Glu max) a
28(92%)
(% remaining)b
80, 10, 24
benzyl
7a
29(96%)
18(96%)
80, 27, 3
9h
9i
6.1(95%)
10(97%)
72, 47, 22
40, 8, 12
7b
7c
F
F
49, 15, 0.3
F
>15,000(nd)
93, 48, 37
9j
F
Cl
F
N
11(97%)
5.5(94%)
16(94%)
90, 79, 49
91, 79, 47
83, 57, 55
7d
7e
7f
>3,000(nd)
720(95%)
80, 83, 75
57, 18, 30
9k
9l
N
N
N
N
230(97%)
32(101%)
44, 3, 2
9m
9n
Cl
13(92%)
1.8(95%)
77, 63, -
7g
7h
73, 13, 4
a EC50 values represent functional activity for inhibition of forskolin
stimulated cAMP production in HEK293 cells stably-expressing human
mGlu2. Values are mean of ≥ 2 experiments. bIn vitro metabolic stability
in human, rat, and mouse liver microsomes. Results are expressed as %
remaining after 10 min incubation.
15, 0.8, 21
0.4(94%)
0.5(98%)
0.4(100%)
85, 75, 83
72, 36, 27
83, 69, 60
7i
7j
In vitro data for C-8 trifluoromethyl TZP ethers 7a-m is
detailed in Table 2. The benzyl ether 7a was 6 fold more potent
than the corresponding C-8 chloro analog 9a, but microsomal
stability was still poor in RLM and MLM. While in vitro
potency of variously substituted phenethylethers 7b-h was
encouraging (EC50 = 1.8-16 nM), microsomal stability was
highly dependent on the nature of substitution. Neither the
geminal difluoro analog 7b nor -methyl compound 7c showed
differentiated metabolism. Dimethyl analogs 7d-e and cycloalkyl
derivatives 7f-h demonstrated a significant improvement in
percent remaining after 10 min incubation with liver microsomes.
The most promising category of compounds was the ring
constrained ethers 7i-m. Phenylcyclohexyl and phenylcyclobutyl
ethers 7i and 7j both displayed subnanomolar mGlu2 PAM
activity (EC50 = 0.4-0.5 nM). Metabolic stability of the
cyclohexane 7i was significantly better than the cyclobutane 7j,
but improvements with the latter were realized with 4-
fluorophenyl substitution (compound 7k). The racemic trans-
methylcyclopropanes (±)-7l and (±)-7m exhibited excellent
mGlu2 activity (EC50 = 1.3-1.7 nM). Metabolic stability of the 4-
chloro isomer (±)-7m in rodent liver microsomes was marginally
better than the 4-fluoro congener (±)-7l. This prompted
characterization of the separated enantiomers (+)-7m and (-)-7m.
The dextrorotatory isomer (+)-7m, with an absolute configuration
of 1S,2S, showed 3-4 fold greater mGlu2 activity than the 1R,2R
enantiomer (-)-7m. In contrast to prior results with 8-chloro TZP
analogs, reactive metabolites were not observed in
biotransformation studies with the 8-trifluoromethyl TZP (+)-7m.
Given the encouraging in vitro data for (+)-7m (also referred to
7k
1.7(96%)
1.3(96%)
1.4(99%)
4.0(96%)
79, 53, 46
76, 83, 60
83, 84, 74
92, 79, 52
7l
F
7m
Cl
Cl
Cl
(+)-7m
(-)-7m
a EC50 values represent functional activity for inhibition of forskolin
stimulated cAMP production in HEK293 cells stably-expressing
human mGlu2. Values are mean of ≥ 2 experiments. bIn vitro
metabolic stability in human, rat, and mouse liver microsomes.
Results are expressed as % remaining after after 10 min incubation.
In alignment with its robust functional activity, BMT-133218
demonstrated potent binding affinity (Ki = 1.0 nM) for the
allosteric site of mGlu2 in a competition binding assay using the
PAM radioligand [3H]-JNJ-40068782 (Table 3).25 BMT-133218
also exhibited > 600-fold functional selectivity for the reduction
of cAMP levels in cells overexpressing human mGlu2 versus
human mGlu3. To more definitively determine that BMT-133218
functioned as a PAM at mGlu2, it was evaluated in a humanized
as BMT-133218), we embarked on
characterization of this interesting lead.
a
more in-depth
Table 2
Functional mGlu2 potency and metabolic stability of TZP ethers 7a-m.

monkey mGlu2 Gqi5 FLIPR assay. When glutamate dose
response curves were run in the presence of increasing
concentrations of BMT-133218, a “left-shift” of greater than 18-
fold was observed for the EC50 of glutamate, Figure 3.26 When
the FLIPR assay was carried out in agonist mode (no added
glutamate), BMT-133218 displayed a low level of mGlu2 agonist
activity (mGlu2 agonist EC50 = 530 nM).
aCalculated using Chem Axon software. bDetermined through application
of published HPLC methods. cThermodynamic solubility of crystalline
material in 50 mM phosphate buffer.
Pharmacokinetic data for BMT-133218 in male Sprague-
Dawley rats is summarized in Table 5. After oral administration
at 3 mg/kg, BMT-133218 displayed a plasma Cmax of 4.1 M at
2.7 hours post dose and an AUC0-24 of 39 M*h. A brain/plasma
ratio of 0.38 was observed 1 hour post dose in a separate rat brain
uptake experiment. Brain uptake in C57B1/6 mice (10 mg/kg,
p.o.) was very similar (b/pmouse = 0.46) to the rat. Despite
apparent challenges in physiochemical properties, it was
anticipated that good pharmacokinetics and high target potency
observed with BMT-133218 would be sufficient to drive
pharmacodynamic effects in animal models of schizophrenia.
Table 3
In vitro characterization of (+)-7m (BMT-133218).
N
N
F3C
O
Cl
Table 5
cmpd
mGlu2 mGlu3 PAM
mGlu2
mGlu2
mGlu2 FLIPR
PK and brain uptake data for compound (+)-7m (BMT-133218) after oral
administration at 3 mg/kg in Sprague-Dawley Rats.
PAM
EC50 (nM)a
binding FLIPR PAM agonist
EC50 (nM)a
Ki (nM)b
1.0 ± 0.3
fold-shiftc
> 18
EC50 (nM)c
530 ± 220 e
(+)-7m 1.4 ± 1.1
a
890 ± 230 d
cmpd
Cmax
Tmax
(h) a
AUC0-24 plasma
(M*h) a (M)b
brain
brain/
(M)a
(M) b
plasmab
Inhibition of forskolin-stimulated cAMP production in HEK293 cells
stably-expressing human mGlu2 or human mGlu3. Values are mean of ≥ 2
experiments. bRadioligand competition experiment using HEK293 cell
membrane homogenates and the PAM radioligand [3H]-JNJ-40068782.
cHumanized monkey mGlu2 FLIPR assay. dmGlu3 max = 76%. emGlu2
max = 85%.
(+)-7m 4.1 ± 0.2 2.7 ± 0.6 39.4 ± 0.4 3.6 ± 0.5 1.4 ± 0.2
0.38
aValues are the mean of three animals. bPlasma and brain concentrations
determined after 1 h of a single dose at 3 mg/kg. c10% DMSO/18% TPGS/
72% water
3.3. In Vivo Efficacy
BMT-133218 was tested for its ability to inhibit
phencyclidine-induced hyperlocomotion (PCP-LMA) in mice.
This model is widely used as an in vivo screen for novel
antipsychotics due to its sensitivity to many typical and atypical
antipsychotics. This assay is also well-suited for testing mGlu2-
based mechanisms, as PCP stimulates an excess of glutamate
release coincident with locomotor hyperactivity. Mice treated
with BMT-133218 dosed at 10 mg/kg (p.o.) demonstrated a
statistically significant reduction in locomotor activity after PCP
administration when compared to animals that received PCP
alone (Figure 4). BMT-133218 dosed at 3 mg/kg (p.o.) showed a
non-significant trend for reversal. The positive control,
risperidone, was fully effective in reducing hyperlocomotion in
the same study. Plasma and brain drug concentrations from
satellite animals dosed with BMT-133218 at 10 mg/kg (p.o.) and
harvested at a time corresponding to the peak PCP response (1
hour) were 15.4 uM and 6.8 uM, respectively. The mice dosed at
3 mg/kg had proportionally lower exposures. Brain to plasma
ratios were similar across dose groups (b/p = 0.35-0.44) and
consistent with prior brain uptake experiments in mice. The
calculated free plasma drug concentrations at 10 and 3 mg/kg
were 76 and 21 nM, respectively.
Figure 3. Humanized monkey mGlu2 FLIPR glutamate shift assay results for
(+)-7m (BMT-133218), n = 4.
3.2. Physiochemical Properties and Pharmacokinetics
A summary of physiochemical properties for BMT-133218 is
presented in Table 4. The mass of 421 Daltons is slightly above
the average of 357 Daltons for all marketed neuroscience drugs.27
The calculated LogP and experimentally determined HPLC
LogD values of 4.4 and 4.7, respectively, suggested lipophilic
character approaching the high end of ideal drug properties. The
consequence of high lipophilicity was reflected in extensive
protein binding (> 99.4 %) across species and low aqueous
solubility at neutral pH (~2 ug/mL @ pH 6.5).
Table 4
Physiochemical properties of (+)-7m (BMT-133218).
cmpd
MW
cLogPa exptl. plasma protein binding aqueous solub.
(Daltons) value LogDb
value
hum/rat/mouse
(% free)
@ pH 6.5
(g/mL)
(+)-7m
421
4.4
4.7
0.2/0.6/0.5
2

cmpd
dose behavioral plasma
(mg/kg)a efficacy
brain
brain/
plasma plasma (nM)c
free drug
(M)b
(M) b
cmpd
dose efficacy
(mg/kg)
plasma
brain
brain/ free plasma
(M)a
(M) a
plasma
(nM)
3
No
4.2 ± 0.3 1.5 ± 0.4 0.35
15.4 ± 0.3 6.8 ± 0.4 0.44
21
76
(+)-7m
(+)-7m
1
3
No
No
0.84 ± 0.18 0.24 ± 0.05 0.29
2.2 ± 0.3 0.63 ± 0.9 0.35
4
10
Yes
(+)-7m
(+)-7m
(+)-7m
aDrug samples were prepared in 10% DMSO/18% TPGS/72% water and
dosed orally 30 minutes prior to PCP treatment (3.2 mg/kg, s.c.).
Risperidone was prepared in 0.3% tartaric acid. bValues are the mean of
three animals from satellite group. cAssuming 99.5% protein binding in
mouse plasma.
11
41
10
Yes
8.2 ± 1.0
4.1 ± 1.0
0.44
aValues are the mean of three animals from satellite groups. Assuming
c
99.5% protein binding in mouse plasma.
Figure 4. In vivo dose response of (+)-7m (BMT-133218) at 3 and 10 mg/kg
(p.o.) in PCP-stimulated locomotion studies using C57B1/6J male mice.
Locomotor activity is expressed as the mean of total distance travelled (n=
14-16/group) during the 2 h post-PCP administration. Data were analyzed by
one-way ANOVA followed by Dunnet’s test; *** p < 0.001 versus Veh/PCP
treatment. Risperidone was used as a positive control.
Figure 5. Dose response of BMT-133218 or (+)-7m in two-trial Y-maze
experiment. Results from the free-choice trial are presented as the mean %
time spent in the novel arm ± SEM (n = 14-16/treatment group) and were
analyzed by one-way ANOVA followed by Dunnett’s test; ** P<0.01, ***
P<0.001 versus Veh/Mk-801 treatment. PNU-282987 (987) was used as a
positive control.
The effects of BMT-133218 on two-trial Y-maze performance
in MK-801 treated mice are shown in Figure 5. The Y-maze
assay is commonly used to evaluate novel chemical entities for
their effects on working memory. The test measures a rodent’s
preference to investigate a new arm of the maze during a free
choice trial rather than returning to one that was previously
visited during the forced choice trial, indicating recognition of
the previously visited arm. MK-801 is a nonselective NMDA
receptor antagonist that imparts cognitive deficits in this model.
In the study, mice treated with vehicle showed a strong
preference for the novel arm. In contrast, mice treated with MK-
801 (0.05 mkg/kg, s.c.) spent an equivalent amount of time in
each arm. Animals treated with BMT-133218 at 10 mg/kg (p.o.),
30 minutes prior to MK-801 administration and 60 min prior to
the forced choice trial, demonstrated a significant increase in
time spent in the novel arm. Animals treated with 3 mg/kg
BMT-133218 showed a trend toward reversing MK-801-induced
cognitive deficits, but the results did not reach statistical
significance. The effective dose of 10 mg/kg showed comparable
activity to the positive control, the 7 nACh receptor full agonist
PNU-282987.28 Exposure data for BMT-133218 from satellite
animals revealed dose-proportional drug concentrations in brain
and plasma, and an average unbound drug plasma level of 41 nM
for animals receiving the active dose of 10 mg/kg.
BMT-133218 was also evaluated at oral doses of 1, 3, and 10
mg/kg for its ability to reverse MK-801 deficits in the rat maze-
based set-shifting paradigm (Figure 6).29 The induced impairment
in attentional set-shifting parallels deficits observed on tests of
executive function in schizophrenia patients and can serve as a
useful preclinical tool in rats for measuring prefrontal cortical
function. In this assay, results are expressed as the number of
trials to reach performance criterion during the set-shift task.
Rats receiving MK-801 (0.04 mg/kg, i.p.) 35 minutes prior to the
set-shift task required significantly more trials to reach criterion
compared to rats that only received vehicle (saline) injection.
Conversely, rats that received the well characterized 7 nAch
receptor partial agonist SSR180771,30 immediately prior to MK-
801 treatment, demonstrated a significantly reduced number of
trials to reach performance criterion compared to the MK-801
only treated group. BMS-133218 demonstrated a statistically
significant reduction in trials at oral doses of 3 and 10 mg/kg, but
not at 1 mg/kg. Brain and plasma samples from BMT-133218
treated satellite animals were used to project drug exposure
during the set-shift experiment. Again, drug exposure was dose-
proportional and brain-to-plasma ratios were in close alignment
with other experiments. Animals receiving the active dose of 3
and 10 mg/kg of BMT-133218 had free drug plasma
concentrations of 230 and 610 nM at 1 hour after oral
administration, respectively.

and that substitution of the aryl ring and its incorporation into a
bicyclic ring system was well tolerated. Polarity in the form of a
basic heteroarene decreased activity. These observations, along
with the knowledge that the C-8 chlorine substitution on the TZP
was a metabolic liability, guided the design of second generation
analogs toward more hydrophobic and ring constrained
triazolopyridine ethers bearing a C-8 trifluoromethyl group,
which are exemplified in Table 2. Ultimately, aryl-substituted
cyclohexyl, cyclobutyl, and trans-cyclopropylmethyl ethers all
displayed excellent mGlu2 PAM activity with good metabolic
stability. Halogen substitution of the pendent aryl group
generally improved metabolic stability. The homochiral trans-
cyclopropylmethyl ether (+)-7m (also referred to as BMT-
133218) was selected as an attractive candidate for further
characterization. It showed potent mGlu2 PAM functional
activity, high selectivity for modulation of mGlu2 versus mGlu3,
and demonstrated potent binding to the allosteric site of mGlu2 in
a radioligand displacement assay. Furthermore, increasing
concentrations of BMT-133218 displayed a left-shift for the EC50
of glutamate, which is fully consistent with PAM mode of action.
While BMT-133218 did exhibit a low level of agonist activity in
the absence of glutamate, a similar observation has been reported
for other mGlu2 PAMs, including JNJ-42153605.22
cmpd
dose efficacy
(mg/kg)
plasma
brain
brain/
free drug
(M)a
(M) a
plasma plasma (nM)
1
3
No
Yes
Yes
1.1 ± 0.7
3.8 ± 0.2
10 ± 2
0.7 ± 0.5
1.5 ± 0.3
5.8 ± 0.4
0.66
0.40
0.59
63
(+)-7m
(+)-7m
(+)-7m
230
610
10
aValues are the mean of three animals from satellite group. cAssuming
99.4% protein binding in rat plasma.
Figure 6. Effect of (+)-7m (BMT-133218) on set-shifting performance in
rat. Results are expressed as the number of trials to meet criterion (n = 8) and
were analyzed by one way ANOVA followed by Dunnett’s test; * p<0.05, **
p<0.01, *** p<0.001 versus Veh/MK-801. SSR180,771 was used as a
positive control. Saline was used as the vehicle.
4. Molecular Modeling
BMT-133218 and JNJ-42153605 only differ in substitution at
the C7 position of the triazolopyridine core. To better understand
the
shared
pharmacophore
of
the
corresponding
cyclopropylmethyl ether and phenylpiperidine substituents, a
computational study was performed.31 JNJ-42153605 was
conformationally sampled to find the global minimum and a set
of local minima within 3.5 kcal/mol. The same analysis was
done with BMS-133218. The two molecules with all of their
minima were overlayed on the central heterocycle. The global
minimum of JNJ-42153605 adopts an extended conformation and
is shown in yellow in Figure 7. In contrast, the global minimum
of BMT-133218, shown in purple, is collapsed on itself and gains
intramolecular hydrophobic contacts and -stacking interactions.
The striking difference in global minima can be attributed to the
presence of 2 additional rotatable bonds in the ether side chain of
BMT-133218, allowing increased conformational flexibility.
Nevertheless, a low energy conformer of BMT-133218, shown in
blue, that is within 2.7 kcal/mol of the global minimum overlays
nicely with the global minimum of JNJ-42153605. The energy
cost for BMT-133218 to adopt this extended conformation may
be partially off-set via interaction with the hydrophobic mGluR2
allosteric binding pocket.
Figure 7. Computational modeling showing overlays of the global minimum
for JNJ-42153605 (yellow), BMT-133218 (purple), and a low energy
conformer (+ 2.7 kcal/mol) of BMT-133218 (blue).
With knowledge that BMT-133218 displayed good oral PK
and micromolar brain drug levels at deliverable doses in rodents,
this molecule was subjected to further characterization in
multiple preclinical models relevant to positive and cognitive
symptoms of schizophrenia. Toward this end, BMT-133218
reversed a PCP-stimulated increase in locomotion in mice at 10
mg/kg (p.o.). It also alleviated MK-801 induced cognitive
deficits in both the two trial Y-maze performance in mice at 10
mg/kg (p.o.) and attentional set-shifting in rats in a dose
dependent fashion at 3 and 10 mg/kg (p.o.). To our knowledge,
this is the first report that an mGlu2 PAM alleviates cognitive
deficits in working memory and attention in acute NMDA
antagonist models of CIAS. Reversal of cognitive impairment
was also detected in a neonatal PCP model (unpublished
observations), an effect not detected with mGlu2 agonists. All
together, these experiments suggest that the TZP ether BMT-
133218 has potential utility to alleviate the positive symptoms
and improve cognitive deficits of schizophrenia. For all
experiments a rudimentary pharmacodynamic/pharmacokinetic
relationship could be assembled using exposure data from
satellite animals. This analysis suggests that free plasma drug
5. Discussion and Conclusions
BMT-133218 resulted from optimization of the mGlu2
functional activity and in vitro metabolic stability of our novel
triazolopyridine ethers. Guided by literature reports for
triazolopyridine amines, initial efforts were focused on preparing
and testing various arylalkyl ethers. The data presented in Table 1
suggested that an alkyl chain length of 2-3 bonds was optimal,

concentrations of at least 30 times the functional mGlu2 EC50 of
1.4 nM were necessary to achieve in vivo activity. While this
value seems high, several factors have to be taken into
consideration. First, brain concentrations of BMT-133218 were
typically less than half of the drug plasma concentrations.
Secondly, protein binding data in plasma may not accurately
reflect protein binding in brain tissue. Indeed, exploratory studies
using rat brain homogenates suggested that BMT-133218 may be
as much as 99.9% protein bound in brain tissue (data not shown).
Lastly, exposure data used to calculate the free drug plasma
levels for the in vivo experiments only came from a single time
point at t = 1 h, and behavioral observations were often collected
over a period of hours. Regardless of the analysis, it remains
evident that high concentrations of BMT-133218 in plasma (≥
3.8 M at t = 1 h) were required to drive activity in preclinical
models. The need for high active exposures of BMT-133218
created concern for the continued progression of this lead
molecule. Areas for improvement included formulation/delivery
of a poorly soluble compound, off-target activity, and drug-drug
interactions. Extensive profiling and safety characterization of
BMT-133218 revealed significant inhibition of the hERG ion
channel (patch clamp hERG IC50 = 0.38 uM) and submicromolar
inhibition of cytochrome P450 2C19 (rCYP2C19 IC50 = 0.62
uM). While the hERG liability could not be fully assessed
without knowledge of unbound plasma concentrations at the
projected human dose (an exercise that was not undertaken), the
inhibition of CYP2C19 was a potential risk for drug-drug
interactions. Many, if not all, of the known issues with BMT-
133218, including extensive protein binding, inactivity at lower
oral doses, poor solubility, hERG, and CYP inhibition were
likely intertwined and a result of the highly lipophilic character
of this molecule. It was our conclusion that improvements in the
PK/PD relationship and reducing liabilities for TZP ethers must
involve a reduction in lipophilicity. Not surprisingly, balancing
lipophilicity and the mGlu2 PAM activity of related small
molecules has been a recognized challenge.22
conditions typically used for reverse phase preparative HPLC are
as follows: column = Sunfire PrepC18 OBD 10 m, 50 x 250
mm column; mobile phase A = 90% MeOH/10% water with
0.1% TFA; mobile phase B = 90% MeOH/10% water with 0.1%
TFA; linear gradient 10-100 % B over 34 min, then a 20 minute
hold at 100% B; flow = 50 mL/min. Low-resolution mass
spectrometry (MS) data were obtained using ES ionization mode
(positive).
6.1 General synthetic procedure for final products 9a-n
Examples 9 a-n were prepared in parallel fashion according to
the following method.
A stock solution of 8-chloro-3-
(cyclopropylmethyl)-7-iodo-[1,2,4]triazolo[4,3-a]pyridine22 (0.9
M in toluene, 1 mL, 0.090 mmol) was added to a 2.0-5.0 mL
microwave vial charged with the corresponding commercially
available alcohol (0.648 mmol), cesium carbonate (50.0 mg,
0.153 mmol), 1,10-phenanthroline (20.0 mg, 0.111 mmol), and
20.0 mg of copper(I) iodide (20.0 mg, 0.105 mmol). The vials
were sealed and heated to 100 °C for 17 hours. The reaction
mixtures were cooled to rt, diluted with 0.5 mL of acetonitrile,
and then filtered. The filtrates were concentrated in vacuo at 34
°C for 1 hour. Each sample was diluted with 0.5 mL of DMF (0.5
mL) and purified using reverse phase preparative LC-MS to
afford the title product.
6.1.2
8-Chloro-7-(benzyloxy)-3-(cyclopropylmethyl)-
[1,2,4]triazolo[4,3-a]pyridine (9a). 9% yield as a colorless oil;
1H NMR (500MHz, METHANOL-d4)  ppm 8.49 (d, J=7.6 Hz,
1H), 7.54 - 7.50 (m, 2H), 7.46 - 7.41 (m, 2H), 7.40 - 7.36 (m,
2H), 5.49 (s, 2H), 3.09 (d, J=6.9 Hz, 2H), 1.33 - 1.24 (m, 1H),
0.69 - 0.64 (m, 2H), 0.39 - 0.35 (m, 2H); LC–MS (M+H)+ 314.2.
6.1.3 8-chloro-7-((4-chlorobenzyl)oxy)-3-(cyclopropylmethyl)-
[1,2,4]triazolo[4,3-a]pyridine (9b). 12% yield as a white solid;
1H NMR (500MHz, METHANOL-d4)  8.67 (d, J=7.6 Hz, 1H),
7.56 (d, J=7.6 Hz, 1H), 7.54 - 7.49 (m, 2H), 7.48 - 7.41 (m, 2H),
5.53 (s, 2H), 3.11 (d, J=6.9 Hz, 2H), 1.36 - 1.25 (m, 1H), 0.73 -
0.66 (m, 2H), 0.42 - 0.36 (m, 2H); LC–MS (M+H)+ 348.4.
In summary, the synthesis, in vitro mGlu2 PAM activity, and
metabolic stability data for a series of triazolopyridine (TZP)
ether analogs was provided. The effort resulted in the discovery
of a potent, selective, and brain penetrant lead molecule BMT-
133218 ((+)-7m) with activity in several rodent models that
recapitulate the symptoms and cognitive deficits of
schizophrenia. Inactivity at lower doses was likely the result of
high lipophilicity. Optimized triazolopyridine ethers may offer
utility as mGlu2 PAMs for the treatment of schizophrenia. Our
continued efforts toward this end will be disclosed in due course.
6.1.4 8-chloro-7-((3-chlorobenzyl)oxy)-3-(cyclopropylmethyl)-
[1,2,4]triazolo[4,3-a]pyridine (9c). 31% yield as a colorless oil;
1H NMR (500 MHz, DMSO-d6)  8.51 (d, J=7.6 Hz, 1H), 7.58
(s, 1H), 7.52 - 7.39 (m, 3H), 7.23 (d, J=7.6 Hz, 1H), 5.46 (s, 2H),
3.04 (d, J=7.0 Hz, 2H), 1.19 (s, 1H), 0.62 - 0.45 (m, 2H), 0.28 (d,
J=5.2 Hz, 2H); LC–MS (M+H)+ 348.1.
6.1.5 8-chloro-7-((2-chlorobenzyl)oxy)-3-(cyclopropylmethyl)-
[1,2,4]triazolo[4,3-a]pyridine (9d). 19% yield as a colorless oil;
1H NMR (500 MHz, DMSO-d6)  8.53 (d, J=7.6 Hz, 1H), 7.71 -
7.64 (m, 1H), 7.60 - 7.53 (m, 1H), 7.48 - 7.40 (m, 2H), 7.29 (d,
J=7.6 Hz, 1H), 5.48 (s, 2H), 3.05 (d, J=7.0 Hz, 2H), 1.26 - 1.13
(m, 1H), 0.56 - 0.48 (m, 2H), 0.32 - 0.25 (m, 2H); LC–MS
(M+H)+ 348.4.
6. Chemistry
Unless otherwise noted, all reagents and materials were
obtained from commercial suppliers and used without further
purification. Reactions involving air- or moisture-sensitive
reagents were performed under a nitrogen or argon atmosphere.
Silica gel chromatography was performed using prepacked silica
gel cartridges (Biotage or ISCO). NMR spectra were recorded
with Bruker spectrometers. Chemical shifts are reported in parts
per million (ppm, d units). All final compounds were purified to
95% purity unless otherwise noted as determined by LCMS
(LCAP at 215 nm) obtained on an Agilent 1100 or 1200
spectrometer using one of the following methods: Method A:
column = XBridge C18, 19 x 200 mm, 5-μm particles; mobile
phase A = 5:95 acetonitrile : water with 10-mM ammonium
acetate; mobile phase B = 95/5 acetonitrile : water with 10-mM
ammonium acetate; gradient = 30-70% B over 20 minutes, then a
7-minute hold at 100% B; flow = 20 mL/min. Standard
6.1.6
7-([1,1'-biphenyl]-4-ylmethoxy)-8-chloro-3-
(cyclopropylmethyl)-[1,2,4]triazolo[4,3-a]pyridine (9e). 22%
yield as a colorless oil; 1H NMR (500MHz, DMSO-d6)  8.50
(d, J=7.6 Hz, 1H), 7.76 - 7.67 (m, 4H), 7.59 (d, J=7.9 Hz, 2H),
7.48 (t, J=7.6 Hz, 2H), 7.42 - 7.36 (m, 1H), 7.28 (d, J=7.6 Hz,
1H), 5.50 (s, 2H), 3.04 (d, J=7.0 Hz, 2H), 1.19 (br. s., 1H), 0.55 -
0.48 (m, 2H), 0.28 (q, J=4.8 Hz, 2H); LC–MS (M+H)+ 390.4.
6.1.7
7-([1,1'-biphenyl]-3-ylmethoxy)-8-chloro-3-
(cyclopropylmethyl)-[1,2,4]triazolo[4,3-a]pyridine (9f). 20%
1
yield as a colorless oil; H NMR (500MHz, DMSO-d6)  8.55 -
8.38 (m, 1H), 7.77 - 7.59 (m, 3H), 7.59 - 7.44 (m, 5H), 7.39 (br.
s., 1H), 7.33 - 7.24 (m, 1H), 5.51 (br. s., 2H), 3.06 - 2.93 (m, 2H),

1.16 (br. s., 1H), 0.50 (d, J=5.2 Hz, 2H), 0.26 (br. s., 2H); LC–
6.2.1. (4-(benzyloxy)-2-chloro-3-(trifluoromethyl)pyridine. A
solution of benzyl alcohol (commercially available, 1.201 mL,
11.55 mmol) was added to a mixture of 60% sodium hydride
dispersion in mineral oil (0.484 g, 12.10 mmol) and DMF (30
mL) in an ice bath at 0 °C. The mixture was allowed to stir at 0
MS (M+H)+ 390.4.
6.1.8
7-([1,1'-biphenyl]-2-ylmethoxy)-8-chloro-3-
(cyclopropylmethyl)-[1,2,4]triazolo[4,3-a]pyridine (9g). 41%
yield as a colorless oil; 1H NMR (500MHz, DMSO-d6)  8.42
(d, J=7.3 Hz, 1H), 7.66 (d, J=7.0 Hz, 1H), 7.50 - 7.36 (m, 8H),
7.00 (d, J=7.3 Hz, 1H), 5.23 (s, 2H), 3.00 (br. s., 2H), 1.16 (br. s.,
1H), 0.50 (d, J=7.3 Hz, 2H), 0.26 (br. s., 2H); LC–MS (M+H)+
390.4.
°C for 30 min, then
a
solution of 2,4-dichloro-3-
(trifluoromethyl)pyridine30 (2.376 g, 11 mmol) in DMF (3 mL)
was quickly added. The resulting mixture was stirred at 0 °C for
1 hour, then quenched by the addition of water. The aqueous
mixture was extracted with ethyl acetate. The combined organic
layers were washed with brine, dried over magnesium sulfate,
filtered, and concentrated in vacuo. The residue was purified
using silica gel column chromatography (5:1 hexanes/ethyl
6.1.9
8-chloro-3-(cyclopropylmethyl)-7-(naphthalen-2-
ylmethoxy)-[1,2,4]triazolo[4,3-a]pyridine (9h). 17% yield as a
1
colorless oil; H NMR (500MHz, DMSO-d6)  8.49 (d, J=7.6 Hz,
acetate)
to
afford
4-(benzyloxy)-2-chloro-3-
1H), 8.02 - 7.93 (m, 4H), 7.63 (dd, J=8.4, 1.7 Hz, 1H), 7.57 -
7.53 (m, 2H), 7.30 (d, J=7.6 Hz, 1H), 5.62 (s, 2H), 3.02 (d, J=6.7
Hz, 2H), 1.20 - 1.14 (m, 1H), 0.53 - 0.48 (m, 2H), 0.30 - 0.24 (m,
2H); LC–MS (M+H)+ 364.2.
(trifluoromethyl)pyridine (1.59 g, 50 % yield) as a white solid.
1H NMR (400MHz, CDCl3)  8.36 (d, J=5.9 Hz, 1H), 7.47 - 7.37
(m, 6H), 6.95 (d, J=5.6 Hz, 1H), 5.28 (s, 2H); LC–MS (M+H)+
288.1. CDCl3
6.1.10
8-chloro-3-(cyclopropylmethyl)-7-((1,2,3,4-
6.2.2.
4-(benzyloxy)-2-hydrazinyl-3-
tetrahydronaphthalen-2-yl)oxy)-[1,2,4]triazolo[4,3-a]pyridine
(9i). 3% yield as a colorless oil; 1H NMR (500MHz,
METHANOL-d4)  8.37 (d, J=7.3 Hz, 1H), 7.24 (d, J=7.6 Hz,
1H), 7.15 - 7.05 (m, 4H), 5.13 (br. s., 1H), 3.26 (dd, J=16.5, 4.6
Hz, 1H), 3.16 - 3.01 (m, 4H), 2.95 - 2.84 (m, 1H), 2.25 - 2.10 (m,
2H), 1.32 - 1.22 (m, 1H), 0.69 - 0.59 (m, 2H), 0.40 - 0.31 (m,
2H); LC–MS (M+H)+ 354.3.
(trifluoromethyl)pyridine. A mixture of 4-(benzyloxy)-2-
chloro-3-(trifluoromethyl)pyridine (from step 6.2.1, 1.2 g, 4.17
mmol), dioxane (10 mL), and hydrazine monohydrate (4.08 mL,
83 mmol) was heated together in a sealed vial for 18 h in an oil
bath at 100 °C. The reaction mixture was cooled to rt and
concentrated in vacuo. The residue was partitioned between
aqueous sodium bicarbonate and dichloromethane. The aqueous
layer was extracted with dichloromethane. The combined
organic extracts were washed with brine, dried over magnesium
sulfate, filtered, and concentrated in vacuo to afford 4-
(benzyloxy)-2-hydrazinyl-3-(trifluoromethyl)pyridine (820 mg,
69 % yield) as an off-white solid. LC–MS (M+H)+ 284.1.
6.1.11
8-chloro-3-(cyclopropylmethyl)-7-(pyridin-3-
ylmethoxy)-[1,2,4]triazolo[4,3-a]pyridine (9j). 6% yield as a
colorless oil; 1H NMR (500MHz, METHANOL-d4)  8.80 (s,
1H), 8.64 (d, J=4.3 Hz, 1H), 8.53 (d, J=7.8 Hz, 1H), 8.18 (d,
J=8.1 Hz, 1H), 7.65 (dd, J=8.1, 5.3 Hz, 1H), 7.41 (d, J=7.6 Hz,
1H), 5.57 (s, 2H), 3.11 (d, J=7.0 Hz, 2H), 1.29 (dd, J=11.7, 4.8
Hz, 1H), 0.69 - 0.64 (m, 2H), 0.40 - 0.36 (m, 2H); LC–MS
(M+H)+ 315.2.
6.2.3.
N'-(4-(benzyloxy)-3-(trifluoromethyl)pyridin-2-yl)-2-
1.0 solution of 2-
cyclopropylacetohydrazide.
A
M
cyclopropylacetyl chloride in DCM (3.99 mL, 3.99 mmol) was
added to a flask charged with a solution of 4-(benzyloxy)-2-
hydrazinyl-3-(trifluoromethyl)pyridine (from step 6.2.2., 946 mg,
3.34 mmol) and triethylamine (0.70 mL, 5.01 mmol) in DCM (30
mL) at 0 °C. After 1 h at 0 °C, the reaction was diluted with
water. The aqueous mixture was extracted with DCM. The
organic extracts were washed with brine, dried (sodium sulfate),
filtered, and concentrated in vacuo to afford N'-(4-(benzyloxy)-3-
(trifluoromethyl)pyridin-2-yl)-2-cyclopropylacetohydrazide (943
mg, 77% yield). The crude product was carried forward without
purification. 1H NMR (500MHz, CDCl3)  8.13 (d, J=5.8 Hz,
1H), 7.43 - 7.40 (m, 5H), 6.49 (d, J=5.6 Hz, 1H), 5.22 (s, 2H),
2.30 (d, J=7.2 Hz, 2H), 1.15 - 1.08 (m, 1H), 0.70 - 0.65 (m, 2H),
0.32 - 0.27 (m, 2H); LC–MS (M+H)+ 366.2.
6.1.12
8-chloro-3-(cyclopropylmethyl)-7-(pyridin-2-
ylmethoxy)-[1,2,4]triazolo[4,3-a]pyridine (9k). 62% yield as a
colorless oil; 1H NMR (500MHz, DMSO-d6)  8.52 (d, J=7.3
Hz, 1H), 7.37 - 7.22 (m, 6H), 5.37 (s, 2H), 3.04 (d, J=6.7 Hz,
2H), 1.18 (d, J=6.7 Hz, 1H), 0.54 - 0.49 (m, 2H), 0.28 (q, J=5.0
Hz, 2H); LC–MS (M+H)+ 315.2.
6.1.13
8-chloro-3-(cyclopropylmethyl)-7-((3-(pyrimidin-5-
yl)benzyl)oxy)-[1,2,4]triazolo[4,3-a]pyridine (9l). 7% yield as
a colorless oil; 1H NMR (500MHz, DMSO-d6)  9.20 (br. s.,
1H), 9.16 - 9.11 (m, 2H), 8.46 (s, 1H), 7.92 (br. s., 1H), 7.80 (br.
s., 1H), 7.60 (br. s., 3H), 5.50 (br. s., 2H), 3.00 (br. s., 2H), 1.16
(br. s., 1H), 0.49 (br. s., 2H), 0.25 (br. s., 2H); LC–MS (M+H)+
392.4.
6.2.4
7-(benzyloxy)-3-(cyclopropylmethyl)-8-
6.1.14
8-(((8-chloro-3-(cyclopropylmethyl)-
(trifluoromethyl)-[1,2,4]triazolo[4,3-a]pyridine. A mixture of
N'-(4-(benzyloxy)-3-(trifluoromethyl)pyridin-2-yl)-2-
[1,2,4]triazolo[4,3-a]pyridin-7-yl)oxy)methyl)isoquinoline
(9m). 6% yield as a colorless oil; H NMR (500MHz, DMSO-
1
cyclopropylacetohydrazide (from step 6.2.3, 20 mg, 0.55 mmol),
Burgess reagent (26.1 mg, 0.109 mmol), acetonitrile (2 mL), and
dioxane (2 mL) was heated at 85 °C in a sealed vial for 18 h.
After cooling to rt, the reaction mixture was evaporated in vacuo
and the residue was partitioned between water and ethyl acetate.
The aqueous layer was extracted with ethyl acetate, and the
combined organic layers were washed with brine, dried over
magnesium sulfate, filtered and concentrated in vacuo. The
crude product was dissolved in a minimum amount of boiling
ethyl acetate. The solution was allowed to cool to rt. After 3 h,
the crystalline solid was collected by vacuum filtration to afford
7-(benzyloxy)-3-(cyclopropylmethyl)-8-(trifluoromethyl)-
d6)  8.42 (d, J=7.3 Hz, 1H), 7.66 (d, J=7.0 Hz, 1H), 7.47 - 7.38
(m, 5H), 7.00 (d, J=7.3 Hz, 1H), 5.23 (s, 2H), 3.00 (br. s., 2H),
1.16 (br. s., 1H), 0.50 (d, J=7.3 Hz, 2H), 0.26 (br. s., 2H); LC–
MS (M+H)+ 365.4.
6.1.15
8-chloro-3-(cyclopropylmethyl)-7-phenethoxy-
[1,2,4]triazolo[4,3-a]pyridine (9n). 24% yield as a colorless oil;
1H NMR (500 MHz, DMSO-d6)  8.47 (d, J=7.6 Hz, 1H), 7.41 -
7.31 (m, 4H), 7.28 - 7.22 (m, 1H), 7.19 (d, J=7.6 Hz, 1H), 4.49 (t,
J=6.9 Hz, 2H), 3.10 (t, J=6.9 Hz, 2H), 3.04 (d, J=7.0 Hz, 2H),
1.23 - 1.12 (m, 1H), 0.54 - 0.48 (m, 2H), 0.30 - 0.24 (m, 2H);
LC–MS (M+H)+ 328.2.
[1,2,4]triazolo[4,3-a]pyridine (19 mg, 95 % yield) as a white
6.2 7-(benzyloxy)-3-(cyclopropylmethyl)-8-(trifluoromethyl)-
[1,2,4]triazolo[4,3-a]pyridine (7a)
solid. 1H NMR (400MHz, METHANOL-d4)  8.97 (d, J=7.8

Hz, 1H), 7.75 (d, J=7.8 Hz, 1H), 7.55 - 7.51 (m, 2H), 7.49 - 7.38
(m, 3H), 5.66 (s, 2H), 3.14 (d, J=6.8 Hz, 2H), 1.37 - 1.27 (m,
1H), 0.77 - 0.66 (m, 2H), 0.42 (q, J=4.9 Hz, 2H); LC–MS
(M+H)+ 348.2.
0.453 mmol) in toluene (0.5 mL) was added to a microwave vial
charged with 7-chloro-3-(cyclopropylmethyl)-8-
(trifluoromethyl)-[1,2,4]triazolo[4,3-a]pyridine (18)22 (25 mg,
0.091 mmol, cesium carbonate (59.1 mg, 0.181 mmol), (S)-(R)-
JOSIPHOS (11 mg, 0.018 mmol), and allylpalladium chloride
dimer (3.3 mg, 9.1 µmol). The vessel was flushed with nitrogen
then sealed with a teflon coated cap. The mixture was heated at
135 °C for 2 h. The reaction was cooled to rt. The crude
reaction mixture was purified by silica gel column
chromatography (100% EtOAc) and was further purified using
reverse phase preparatory HPLC to afford the title compound
(9.4 mg, 20% yield) as a TFA salt. 1H NMR (500 MHz,
METHANOL-d4)  8.52 (d, J=7.8 Hz, 1H), 7.44 - 7.28 (m, 2H),
7.14 (d, J=7.9 Hz, 1H), 7.10 - 6.98 (m, 2H), 4.49 - 4.29 (m, 2H),
3.40 - 3.20 (m, 1H), 3.05 (d, J=6.9 Hz, 2H), 1.44 (d, J=7.0 Hz,
3H), 1.27 - 1.14 (m, 1H), 0.67 - 0.53 (m, 2H), 0.40 - 0.26 (m,
2H); LC–MS (M+H)+ 394.1.
6.3 3-(cyclopropylmethyl)-7-(2,2-difluoro-2-phenylethoxy)-8-
(trifluoromethyl)-[1,2,4]triazolo[4,3-a]pyridine (7b)
6.3.1. 2,2-difluoro-2-phenylethanol. To a stirring suspension
of LAH (0.265 g, 6.98 mmol) in THF (3.0 mL) was added
dropwise
a solution of ethyl 2,2-difluoro-2-phenylacetate
(commercial, 345 mg, 1.72 mmol) in THF (2.0 mL). The
resulting mixture was stirred at rt for 18 h. The reaction was
quenched by the addition of aqueous ammonium hydroxide
solution. The reaction mixture was slurried in a bilayer of water
and ethyl acetate and filtered through celite. The layers were
separated and the aqueous layer was extracted with ethyl acetate.
The combined organic layers were washed with brine, dried
(magnesium sulfate), filtered, and concentrated in vacuo. The
residue was purified by silica-gel column chromatography (10-
15% ethyl acetate/hexane) to afford (2,2-difluoro-2-
phenylethanol (180 mg, 66% yield). 1H NMR (400 MHz,
CDCl3)  7.58 - 7.36 (m, 5H), 3.98 (td, J=13.4, 6.0 Hz, 2H), 2.14
(t, J=6.4 Hz, 1H).
6.5 3-(cyclopropylmethyl)-7-(2-(4-fluorophenyl)-2-
methylpropoxy)-8-(trifluoromethyl)-[1,2,4]triazolo[4,3-
a]pyridine (7d)
6.5.1. 2-(4-fluorophenyl)-2-methylpropanenitrile. A solution
of 1.0 M potassium tert-butoxide in tert-butanol (92 mL, 92
mmol) was added to a mixture of 2-(4-fluorophenyl)acetonitrile
(commercially available, 5.0 g, 37.0 mmol), iodomethane (6.36
mL, 102 mmol), and THF (100 mL) maintained at 0 °C. After
complete addition, the cold bath was removed and the mixture
was allowed to stir at rt for 18 h. The reaction was quenched
with a saturated aqueous solution of ammonium chloride, poured
into water, and extracted with ethyl acetate. The combined
organics were washed with brine, dried (magnesium sulfate),
filtered, and concentrated in vacuo. The crude product was
purified using silica gel column chromatography (40:1
6.3.2.
2-chloro-4-(2,2-difluoro-2-phenylethoxy)-3-
(trifluoromethyl)pyridine. This material was prepared from
6.3.1. and 2,4-dichloro-3-(trifluoromethyl)pyridine32 according to
the procedure described in step 6.2.1. to afford 2-chloro-4-(2,2-
difluoro-2-phenylethoxy)-3-(trifluoromethyl)pyridine (221 mg,
58% yield) as an orange oil. 1H NMR (500MHz, CDCl3)  8.37
(d, J=5.8 Hz, 1H), 7.60 - 7.56 (m, 2H), 7.49 (q, J=7.1 Hz, 3H),
6.85 (d, J=5.8 Hz, 1H), 4.49 (t, J=11.1 Hz, 2H).
6.3.3.
4-(2,2-Difluoro-2-phenylethoxy)-2-hydrazinyl-3-
hexane/ethyl
acetate)
to
afford
2-(4-fluorophenyl)-2-
(trifluoromethyl)pyridine. 4-(2,2-Difluoro-2-phenylethoxy)-2-
hydrazinyl-3-(trifluoromethyl)pyridine (186 mg, 86 % yield) was
methylpropanenitrile (5.2 g, 84 % yield) as a clear liquid. 1H
NMR (500 MHz, CDCl3)  7.50 - 7.41 (m, 2H), 7.14 - 7.03 (m,
2H), 1.73 (s, 6H).
prepared
from
2-chloro-4-(2,2-difluoro-2-phenylethoxy)-3-
(trifluoromethyl)pyridine (from step 6.3.2) following a procedure
analogous to step 6.2.2. 1H NMR (500 MHz, CDCl3)  8.23 -
8.12 (m, 1H), 7.62 - 7.43 (m, 5H), 6.54 (br s, 1H), 6.28 (d, J=6.0
Hz, 1H), 4.43 (t, J=11.1 Hz, 2H), 3.99 (t, J=13.4 Hz, 2H).
6.5.2. 2-(4-fluorophenyl)-2-methylpropanoic acid. A mixture
of 2-(4-fluorophenyl)-2-methylpropanenitrile (from step 6.5.1.,
5.2 g, 31.9 mmol) and 15% aqueous sodium hydroxide (80 mL)
was heated at reflux for 16 h. The mixture was cooled to rt and
extracted with diethyl ether. The aqueous layer was made acidic
with 1 N aqueous hydrochloric acid (300 mL) and was then
extracted with diethyl ether. The acidic organic extract was
washed with brine, dried (magnesium sulfate), filtered and
concentrated in vacuo. The residue was dried under high vacuum
for several hours to afford 2-(4-fluorophenyl)-2-methylpropanoic
acid (1.89 g, 33 % yield) as a white solid. 1H NMR (500 MHz,
CDCl3)  7.44 - 7.32 (m, 2H), 7.09 - 6.97 (m, 2H), 1.60 (s, 6H).
6.3.4. 3-(Cyclopropylmethyl)-7-(2,2-difluoro-2-phenylethoxy)-
8-(trifluoromethyl)-[1,2,4]triazolo[4,3-a]pyridine. The titled
compound (59 mg, 17 % yield over 2 steps) was prepared from
4-(2,2-difluoro-2-phenylethoxy)-2-hydrazinyl-3-
(trifluoromethyl)pyridine (from preparation 6.3.3.) following
procedures analogous to steps 6.2.3 and 6.2.4. The final product
was purified by silica gel column chromatography (100 % ethyl
acetate) and reverse phase preparatory HPLC. 1H NMR (500
MHz, METHANOL-d4)  8.56 (d, J=7.8 Hz, 1H), 7.65 (dd,
J=7.6, 1.8 Hz, 2H), 7.56 - 7.47 (m, 3H), 7.19 (d, J=7.8 Hz, 1H),
3.08 (d, J=6.9 Hz, 2H), 1.29 - 1.17 (m, 1H), 0.63 (dd, J=8.1, 1.4
Hz, 2H), 0.42 - 0.31 (m, 2H); LC–MS (M+H)+ 398.2.
6.5.3. 2-(4-Fluorophenyl)-2-methylpropan-1-ol.
2-(4-
fluorophenyl)-2-methylpropan-1-ol (1.77 g, quantitative yield)
was prepared from 2-(4-fluorophenyl)-2-methylpropanoic acid
(from step 6.5.2.) following a procedure analogous to step 6.3.1.
1H NMR (500 MHz, CDCl3)  7.42 - 7.32 (m, 2H), 7.08 - 6.98
(m, 2H), 3.61 (s, 2H), 1.34 (s, 6H).
6.4 3-(cyclopropylmethyl)-7-(2-(4-fluorophenyl)propoxy)-8-
(trifluoromethyl)-[1,2,4]triazolo[4,3-a]pyridine, TFA salt
(7c)
6.5.4.
3-(cyclopropylmethyl)-7-(2-(4-fluorophenyl)-2-
6.4.1. (2-fluoro-2-phenylethanol. 2,2-difluoro-2-phenylethanol
(371 mg, 73 % yield) was prepared from methyl 2-fluoro-2-
phenylacetate (commercially available) following a procedure
analogous to step 6.3.1. 1H NMR (500 MHz, CDCl3)  7.45 -
7.24 (m, 1H), 5.65 - 5.51 (m, 1H), 4.01 - 3.78 (m, 3H), 2.26 (dd,
J=8.4, 4.6 Hz, 1H).
methylpropoxy)-8-(trifluoromethyl)-[1,2,4]triazolo[4,3-
a]pyridine. The titled compound (13 mg, 13 % yield) was
prepared from 2-(4-fluorophenyl)-2-methylpropan-1-ol (6.5.3.)
and
7-chloro-3-(cyclopropylmethyl)-8-(trifluoromethyl)-
[1,2,4]triazolo[4,3-a]pyridine (18)22 following the procedure
analogous to example 6.4.2. The final product was purified using
preparative LC/MS (column: XBridge C18, 19 x mm, 5-μm
particles; mobile phase A: 5:95 acetonitrile/water with 10-mM
6.4.2. 3-(cyclopropylmethyl)-7-(2-(4-fluorophenyl)propoxy)-
8-(trifluoromethyl)-[1,2,4]triazolo[4,3-a]pyridine. A solution
of 2-(4-fluorophenyl)propan-1-ol (from step 6.4.1., 69.9 mg,

ammonium acetate; mobile phase B: 95:5 acetonitrile/water with
10-mM ammonium acetate; gradient: 30-70% B over 15 minutes,
then a 7-minute hold at 100% B; flow: 20 mL/min). 1H NMR
(500 MHz, DMSO-d6)  8.66 (d, J=7.6 Hz, 1H), 7.50 (dd, J=8.5,
5.5 Hz, 2H), 7.23 - 7.07 (m, 3H), 4.33 (s, 2H), 3.02 (d, J=7.0 Hz,
2H), 1.40 (s, 6H), 1.15 (br. s., 1H), 0.49 (d, J=6.4 Hz, 2H), 0.25
(d, J=4.0 Hz, 2H); LC–MS (M+H)+ 408.2.
6.7.3.
3-(cyclopropylmethyl)-7-((1-(4-
fluorophenyl)cyclopropyl)methoxy)-8-(trifluoromethyl)-
[1,2,4]triazolo[4,3-a]pyridine. The titled compound (64 mg, 9
%
yield over
2
steps) was prepared from 4-((1-(4-
fluorophenyl)cyclopropyl)methoxy)-2-hydrazinyl-3-
(trifluoromethyl)pyridine (from step 6.7.2.) following the
procedures detailed in steps 6.2.3. and 6.2.4. The final product
was purified by silica-gel column chromatography (0-100% ethyl
6.6 7-(2-(4-chlorophenyl)-2-methylpropoxy)-3-
(cyclopropylmethyl)-8-(trifluoromethyl)-
[1,2,4]triazolo[4,3-a]pyridine (7e)
1
acetate/hexanes). H NMR (500 MHz, CDCl3)  8.38 (d, J=7.6
Hz, 1H), 7.46 - 7.40 (m, 2H), 7.04 - 6.98 (m, 2H), 6.87 (d, J=7.8
Hz, 1H), 4.27 (s, 2H), 3.11 (d, J=6.7 Hz, 2H), 1.16 (br. s., 1H),
1.10 - 1.02 (m, 4H), 0.64 - 0.59 (m, 2H), 0.36 - 0.31 (m, 2H);
LC–MS (M+H)+ 405.8.
6.6.1.
(trifluoromethyl)pyridine.
methylpropoxy)-3-(trifluoromethyl)pyridine (810 mg, 82
yield) was prepared from 2-(4-chlorophenyl)-2-methylpropan-1-
2-chloro-4-(2-(4-chlorophenyl)-2-methylpropoxy)-3-
2-chloro-4-(2-(4-chlorophenyl)-2-
%
6.8 7-((1-(4-chlorophenyl)cyclopropyl)methoxy)-3-
(cyclopropylmethyl)-8-(trifluoromethyl)-
[1,2,4]triazolo[4,3-a]pyridine (7g)
ol
(commercially
available)
and
2,4-dichloro-3-
(trifluoromethyl)pyridine32 following a procedure analogous to
step 6.2.1. LC–MS (M+H)+ 363.9. H NMR (500 MHz, CDCl3)
6.8.1. 2-chloro-4-((1-(4-chlorophenyl)cyclopropyl)methoxy)-
1
 8.32 (d, J=5.8 Hz, 1H), 7.39 - 7.29 (m, 4H), 6.82 (d, J=5.8 Hz,
3-(trifluoromethyl)pyridine.
chlorophenyl)cyclopropyl)methoxy)-3-(trifluoromethyl)pyridine
(438 mg, 74 yield) was prepared from (1-(4-
chlorophenyl)cyclopropyl)methanol (commercially available)
and 2,4-dichloro-3-(trifluoromethyl)pyridine32 following
procedure analogous to step 6.2.1.
1H NMR (500 MHz,
CDCl3)  8.31 (d, J=5.8 Hz, 1H), 7.39 - 7.35 (m, 2H), 7.32 -
7.29 (m, 2H), 6.75 (d, J=6.0 Hz, 1H), 4.11 (s, 2H), 1.09 - 1.02
(m, 4H); LC–MS (M+H)+ 362.0.
2-chloro-4-((1-(4-
1H), 4.00 (s, 2H), 1.49 (s, 6H).
%
6.6.2. 4-(2-(4-chlorophenyl)-2-methylpropoxy)-2-hydrazinyl-
3-(trifluoromethyl)pyridine.
4-(2-(4-chlorophenyl)-2-
a
methylpropoxy)-2-hydrazinyl-3-(trifluoromethyl)pyridine (457
mg, quantitative yield) was prepared from 2-chloro-4-(2-(4-
chlorophenyl)-2-methylpropoxy)-3-(trifluoromethyl)pyridine
(from step 6.6.1.) following a procedure analogous to step 6.2.2.
1H NMR (500 MHz, DMSO-d6)  8.16 (d, J=5.8 Hz, 1H), 7.52 -
7.41 (m, 2H), 7.41 - 7.30 (m, 3H), 6.55 (d, J=6.0 Hz, 1H), 4.26
(s, 2H), 4.11 (s, 2H), 1.37 (s, 6H); LC–MS (M+H)+ 360.1.
6.8.2.
4-((1-(4-Chlorophenyl)cyclopropyl)methoxy)-2-
hydrazinyl-3-(trifluoromethyl)pyridine.
4-((1-(4-
chlorophenyl)cyclopropyl)methoxy)-2-hydrazinyl-3-
(trifluoromethyl)pyridine (439 mg, quantitative yield) was
prepared from 2-chloro-4-((1-(4-
chlorophenyl)cyclopropyl)methoxy)-3-(trifluoromethyl)pyridine
(from step 6.8.1.) following a procedure analogous to step 6.2.2.
LC–MS (M+H)+ 358.0.
6.6.3.
7-(2-(4-chlorophenyl)-2-methylpropoxy)-3-
(cyclopropylmethyl)-8-(trifluoromethyl)-[1,2,4]triazolo[4,3-
a]pyridine. The titled compound (157 mg, 65 % yield over 2
steps)
was
prepared
from
4-(2-(4-chlorophenyl)-2-
methylpropoxy)-2-hydrazinyl-3-(trifluoromethyl)pyridine (from
step 6.6.2., 200 mg, 0.556 mmol) following procedures
analogous to steps 6.2.3. and 6.2.4. The final product was
purified by silica gel column chromatography (100% EtOAc).
1H NMR (500 MHz, CDCl3)  8.06 (d, J=7.6 Hz, 1H), 7.46 -
7.36 (m, 2H), 7.36 - 7.29 (m, 2H), 6.72 (d, J=7.8 Hz, 1H), 4.10
(s, 2H), 3.06 (d, J=6.7 Hz, 2H), 1.53 (s, 6H), 1.21 - 1.08 (m, 1H),
0.70 - 0.55 (m, 2H), 0.32 (q, J=5.0 Hz, 2H); LC–MS (M+H)+
424.2.
6.8.3.
7-((1-(4-chlorophenyl)cyclopropyl)methoxy)-3-
(cyclopropylmethyl)-8-(trifluoromethyl)-[1,2,4]triazolo[4,3-
a]pyridine. The titled compound (131 mg, 20 % yield over 2
steps)
was
prepared
from
4-((1-(4-
chlorophenyl)cyclopropyl)methoxy)-2-hydrazinyl-3-
(trifluoromethyl)pyridine (from 6.8.2.) following procedures
analogous to steps 6.2.3. and 6.2.4. The final product was
purified by silica-gel column chromatography (100% ethyl
6.7 3-(cyclopropylmethyl)-7-((1-(4-
1
acetate). H NMR (500 MHz, CDCl3)  8.23 (d, J=7.6 Hz, 1H),
fluorophenyl)cyclopropyl)methoxy)-8-(trifluoromethyl)-
[1,2,4]triazolo[4,3-a]pyridine (7f)
7.46 - 7.40 (m, 2H), 7.04 - 6.97 (m, 2H), 6.77 (d, J=7.8 Hz, 1H),
4.25 (s, 2H), 3.09 (d, J=6.7 Hz, 2H), 1.19 - 1.12 (m, 1H), 1.10 -
1.02 (m, 4H), 0.65 - 0.59 (m, 2H), 0.35 - 0.30 (m, 2H); LC–MS
(M+H)+ 422.0.
6.7.1. 2-chloro-4-((1-(4-fluorophenyl)cyclopropyl)methoxy)-
3-(trifluoromethyl)pyridine.
fluorophenyl)cyclopropyl)methoxy)-3-(trifluoromethyl)pyridine
(537 mg, 86 yield) was prepared from (1-(4-
fluorophenyl)cyclopropyl)methanol (commercially available) and
2,4-dichloro-3-(trifluoromethyl)pyridine32 following a procedure
analogous to step 6.2.1. 1H NMR (500 MHz, CDCl3)  8.30 (d,
J=6.0 Hz, 1H), 7.43 - 7.38 (m, 2H), 7.04 - 6.98 (m, 2H), 6.75 (d,
J=5.8 Hz, 1H), 4.10 (s, 2H), 1.08 - 1.00 (m, 4H); LC–MS
(M+H)+ 346.0.
2-chloro-4-((1-(4-
6.9 3-(cyclopropylmethyl)-7-((1-phenylcyclohexyl)methoxy)-
8-(trifluoromethyl)-[1,2,4]triazolo[4,3-a]pyridine (7h)
%
6.9.1.
(trifluoromethyl)pyridine.
phenylcyclohexyl)methoxy)-3-(trifluoromethyl)pyridine
mg, 90 yield) was prepared from
2-chloro-4-((1-phenylcyclohexyl)methoxy)-3-
2-chloro-4-((1-
(875
(1-
%
phenylcyclohexyl)methanol (commercially available) and 2,4-
dichloro-3-(trifluoromethyl)pyridine32 following
a procedure
6.7.2.
4-((1-(4-fluorophenyl)cyclopropyl)methoxy)-2-
1
analogous to step 6.2.1. H NMR (500 MHz, CDCl3)  ppm 8.25
(d, J=6.0 Hz, 1H), 7.53 - 7.42 (m, 2H), 7.42 - 7.33 (m, 2H), 7.27 -
7.22 (m, 1H), 6.73 (d, J=6.0 Hz, 1H), 3.91 (s, 2H), 2.30 (d,
J=13.3 Hz, 2H), 1.90 - 1.77 (m, 2H), 1.68 - 1.57 (m, 3H), 1.48 -
1.35 (m, 3H); LC–MS (M+H)+ 370.0.
hydrazinyl-3-(trifluoromethyl)pyridine.
fluorophenyl)cyclopropyl)methoxy)-2-hydrazinyl-3-
(trifluoromethyl)pyridine (500 mg, 94 % yield) was prepared
from 2-chloro-4-((1-(4-fluorophenyl)cyclopropyl)methoxy)-3-
4-((1-(4-
(trifluoromethyl)pyridine (from step 6.7.1.) following
a
procedure analogous to step 6.2.2 (b). LC–MS (M+H)+ 342.0.
6.9.2.
2-hydrazinyl-4-((1-phenylcyclohexyl)methoxy)-3-
2-hydrazinyl-4-((1-
(trifluoromethyl)pyridine.

phenylcyclohexyl)methoxy)-3-(trifluoromethyl)pyridine
mg, 92 yield) was prepared from 2-chloro-4-((1-
(from
(437
6.11.1. 3-phenylcyclobutanol. Sodium borohydride (108 mg,
2.86 mmol) was added to stirred solution of 3-
%
a
phenylcyclohexyl)methoxy)-3-(trifluoromethyl)pyridine
phenylcyclobutanone (380 mg, 2.60 mmol) in ethanol (15 mL)
maintained in a 0 °C ice bath. After 2 h, the reaction was diluted
with water and the pH was adjusted to 2 by the addition of 1 N
aqueous hydrochloric acid. The mixture was extracted with ethyl
acetate. The combined organics were washed with brine, dried
(magnesium sulfate), filtered and concentrated in vacuo to afford
3-phenylcyclobutanol (423 mg, quantitative yield). The product
was used without purification. 1H NMR was consistent with the
desired product, but suggested a 2:1 (trans/cis) mixture of
diastereomers. Data for major diasteromer: 1H NMR (500 MHz,
CDCl3)  7.33 - 7.31 (m, 2H), 7.25 (d, J=7.8 Hz, 3H), 4.35 -
4.28 (m, 1H), 3.03 - 2.95 (m, 1H), 2.84 - 2.77 (m, 2H), 2.07 -
2.01 (m, 2H).
step 6.9.1.) following a procedure analogous to step 6.2.2. 1H
NMR (500 MHz, DMSO-d6)  8.10 (d, J=5.8 Hz, 1H), 7.44 (d,
J=7.5 Hz, 2H), 7.33 (t, J=7.7 Hz, 3H), 7.24 - 7.12 (m, 1H), 6.46
(d, J=6.0 Hz, 1H), 4.25 (s, 2H), 3.96 (s, 2H), 2.19 (d, J=13.7 Hz,
2H), 1.84 - 1.69 (m, 2H), 1.62 - 1.41 (m, 3H), 1.39 - 1.20 (m,
3H); LC–MS (M+H)+ 366.1.
6.9.3.
3-(cyclopropylmethyl)-7-((1-
phenylcyclohexyl)methoxy)-8-(trifluoromethyl)-
[1,2,4]triazolo[4,3-a]pyridine. The titled compound (107 mg,
45 % yield over 2 steps) was prepared from 2-hydrazinyl-4-((1-
phenylcyclohexyl)methoxy)-3-(trifluoromethyl)pyridine
(from
6.9.2.) following procedures analogous to steps 6.2.3. and 6.2.4.
The crude product was dissolved in a minmum amount of boiling
ethyl acetate. The solution was allowed to cool to rt. After 3 h,
the crystalline solid was collected by vacuum filtration to afford a
white solid. 1H NMR (500 MHz, DMSO-d6)  8.62 (d, J=7.8 Hz,
1H), 7.48 (d, J=7.5 Hz, 2H), 7.35 (t, J=7.8 Hz, 2H), 7.26 - 7.16
(m, 1H), 7.09 (d, J=7.8 Hz, 1H), 4.22 (s, 2H), 3.01 (d, J=6.9 Hz,
2H), 2.23 (d, J=13.9 Hz, 2H), 1.89 - 1.74 (m, 2H), 1.66 - 1.44 (m,
3H), 1.43 - 1.24 (m, 3H), 1.22 - 1.04 (m, 1H), 0.56 - 0.42 (m,
2H), 0.31 - 0.17 (m, 2H); LC–MS (M+H)+ 430.2.
6.11.2.
2-chloro-4-(3-phenylcyclobutoxy)-3-
(trifluoromethyl)pyridine. 2-chloro-4-(3-phenylcyclobutoxy)-
3-(trifluoromethyl)pyridine (100 mg, 11 % yield) was prepared
from 3-phenylcyclobutanol (from step 6.11.1.) and 2,4-dichloro-
3-(trifluoromethyl)pyridine30 following a procedure analogous to
step 6.2.1. 1H NMR (500 MHz, CDCl3)  8.36 (d, J=5.8 Hz,
1H), 7.39 - 7.33 (m, 2H), 7.28 - 7.24 (m, 3H), 6.81 (d, J=5.8 Hz,
1H), 4.82 (quin, J=7.2 Hz, 1H), 3.31 - 3.22 (m, 1H), 3.06 - 2.97
(m, 2H), 2.41 (tdq, J=9.9, 7.5, 2.5 Hz, 2H); LCMS (M+H)+
328.2.
6.10 3-(cyclopropylmethyl)-7-(((1r,4r)-4-
phenylcyclohexyl)oxy)-8-(trifluoromethyl)-
[1,2,4]triazolo[4,3-a]pyridine (7i)
6.11.3.
2-hydrazinyl-4-(3-phenylcyclobutoxy)-3-
(trifluoromethyl)pyridine.
2-hydrazinyl-4-(3-
phenylcyclobutoxy)-3-(trifluoromethyl)pyridine
quantitative yield) was prepared from 2-chloro-4-(3-
phenylcyclobutoxy)-3-(trifluoromethyl)pyridine (from step
(103 mg,
6.10.1.
(trifluoromethyl)pyridine.
phenylcyclohexyl)oxy)-3-(trifluoromethyl)pyridine (395 mg,
1.11 mmol, 65 yield) was prepared from (1r,4r)-4-
phenylcyclohexanol (commercially available) and 2,4-dichloro-3-
(trifluoromethyl)pyridine30 following a procedure analogous to
step 6.2.1. 1H NMR (500 MHz, CDCl3)  8.33 (d, J=5.8 Hz,
1H), 7.37 - 7.30 (m, 2H), 7.26 - 7.19 (m, 3H), 6.93 (d, J=6.0 Hz,
1H), 4.55 - 4.42 (m, 1H), 2.64 (tt, J=11.9, 3.5 Hz, 1H), 2.35 -
2.23 (m, 2H), 2.13 - 2.02 (m, 2H), 1.83 - 1.70 (m, 2H), 1.70 -
1.59 (m, 2H); LC–MS (M+H)+ 356.1.
2-chloro-4-((1-phenylcyclohexyl)methoxy)-3-
2-Chloro-4-(((1r,4r)-4-
6.11.2.) following a procedure analogous to step 6.2.2. LC–MS
%
(M+H)+ 324.2.
6.11.4.
3-(cyclopropylmethyl)-7-(3-phenylcyclobutoxy)-8-
The titled
(trifluoromethyl)-[1,2,4]triazolo[4,3-a]pyridine.
compound (40 mg, 34 % yield over 2 steps) was prepared from
2-hydrazinyl-4-(3-phenylcyclobutoxy)-3-
(trifluoromethyl)pyridine (from step 6.11.3.) following
procedures analogous to steps 6.2.3. and 6.2.4.
The final
product was purified by preparative LC-MS. 1H NMR (500 MHz,
DMSO-d6)  8.68 (d, J=7.6 Hz, 1H), 7.38 - 7.14 (m, 5H), 7.08
(d, J=7.6 Hz, 1H), 5.13 (t, J=7.0 Hz, 1H), 3.25 - 3.13 (m, 1H),
3.12 - 2.88 (m, 4H), 2.17 (d, J=8.9 Hz, 2H), 1.28 - 1.05 (m, 1H),
0.61 - 0.41 (m, 2H), 0.37 - 0.18 (m, 2H); LC–MS (M+H)+ 388.4.
6.10.2.
(trifluoromethyl)pyridine.
phenylcyclohexyl)oxy)-3-(trifluoromethyl)pyridine (385 mg, 100
yield) was prepared from 2-chloro-4-(((1r,4r)-4-
2-hydrazinyl-4-((1r,4r)-4-phenylcyclohexyloxy)-3-
2-hydrazinyl-4-(((1r,4r)-4-
%
phenylcyclohexyl)oxy)-3-(trifluoromethyl)pyridine (from step
6.10.1.) following a procedure analogous to step 6.2.2. LC–MS
(M+H)+ 352.2.
6.12 3-(cyclopropylmethyl)-7-(3-(4-
fluorophenyl)cyclobutoxy)-8-(trifluoromethyl)-
[1,2,4]triazolo[4,3-a]pyridine (7k)
6.10.3.
3-(Cyclopropylmethyl)-7-((1r,4r)-4-
phenylcyclohexyloxy)-8-(trifluoromethyl)-[1,2,4]triazolo[4,3-
a]pyridine. The titled compound (147 mg, 64 % yield over 2
6.12.1. 3-(4-fluorophenyl)cyclobutanol. Sodium borohydride
(0.253 g, 6.70 mmol) was added to a stirred solution of 3-(4-
fluorophenyl)cyclobutanone (1.0 g, 6.09 mmol) in ethanol (40
mL) maintained in a 0 °C ice bath. After 2 h, the reaction was
diluted with water and the pH was adjusted to 2 by the addition
of 1 N aqueous hydrochloric acid. The mixture was extracted
with ethyl acetate. The combined organics were washed with
brine, dried (magnesium sulfate), filtered and concentrated in
vacuo to afford 3-(4-fluorophenyl)cyclobutanol (1.12 g,
quantitative yield). The product was used without purification.
1H NMR integration suggested a 6:1 mixture of diastereomers
steps)
was
prepared
from
2-hydrazinyl-4-(((1r,4r)-4-
phenylcyclohexyl)oxy)-3-(trifluoromethyl)pyridine (from step
6.10.2.) following procedures analogous to steps 6.2.3. and 6.2.4.
The product was purified using silica gel column
chromatography (100% EtOAc). 1H NMR (500 MHz, CDCl3) 
8.07 (d, J=7.6 Hz, 1H), 7.39 - 7.29 (m, 2H), 7.26 - 7.17 (m, 3H),
6.84 (d, J=7.8 Hz, 1H), 4.54 - 4.42 (m, 1H), 3.07 (d, J=6.7 Hz,
2H), 2.64 (tt, J=12.0, 3.5 Hz, 1H), 2.32 - 2.22 (m, 2H), 2.14 -
2.03 (m, 2H), 1.86 - 1.74 (m, 2H), 1.71 - 1.56 (m, 2H), 1.22 -
1.12 (m, 1H), 0.70 - 0.56 (m, 2H), 0.40 - 0.27 (m, 2H); LC–MS
(M+H)+ 416.2.
1
(trans/cis). Data for trans isomer: H NMR (500 MHz, CDCl3) 
7.21 - 7.17 (m, 2H), 7.02 - 6.98 (m, 2H), 4.34 - 4.27 (m, 1H),
3.00 - 2.91 (m, 1H), 2.82 - 2.75 (m, 2H), 2.07 (d, J=2.0 Hz, 1H),
2.02 - 1.97 (m, 2H).
6.11 3-(cyclopropylmethyl)-7-(3-phenylcyclobutoxy)-8-
(trifluoromethyl)-[1,2,4]triazolo[4,3-a]pyridine (7j)
6.12.2.
(trifluoromethyl)pyridine.
2-chloro-4-(3-(4-fluorophenyl)cyclobutoxy)-3-
2-chloro-4-(3-(4-

fluorophenyl)cyclobutoxy)-3-(trifluoromethyl)pyridine (1.22 g,
59 % yield) was prepared from 3-(4-fluorophenyl)cyclobutanol
dichloro-3-(trifluoromethyl)pyridine32 following
a procedure
analogous to step 6.2.1. (a). 1H NMR (500 MHz, CDCl3)  8.34
(d, J=5.8 Hz, 1H), 7.12 - 7.07 (m, 2H), 7.01 - 6.94 (m, 2H), 6.88
(d, J=5.8 Hz, 1H), 4.26 (dd, J=9.9, 6.0 Hz, 1H), 4.07 (dd, J=9.8,
6.9 Hz, 1H), 1.57 - 1.49 (m, 1H), 1.16 - 1.06 (m, 2H), 0.96 - 0.83
(m, 1H).
(from
step
6.12.1.)
and
2,4-dichloro-3-
(trifluoromethyl)pyridine30 following a procedure analogous to
1
step 6.2.1. H NMR (500 MHz, CDCl3)  8.35 (d, J=5.8 Hz,
1H), 7.24 - 7.19 (m, 2H), 7.07 - 7.01 (m, 2H), 6.79 (d, J=5.8 Hz,
1H), 4.80 (t, J=7.1 Hz, 1H), 3.28 - 3.19 (m, 1H), 3.04 - 3.00 (m,
2H), 2.39 - 2.32 (m, 2H); LC-MS (M+H)+ = 346.1.
6.13.4.
(±)-4-(((1S,2S)-2-(4-
fluorophenyl)cyclopropyl)methoxy)-2-hydrazinyl-3-
6.12.3.
4-(3-(4-fluorophenyl)cyclobutoxy)-2-hydrazinyl-3-
(trifluoromethyl)pyridine.
(±)-4-(((1S,2S)-2-(4-
(trifluoromethyl)pyridine. 4-(3-(4-fluorophenyl)cyclobutoxy)-
2-hydrazinyl-3-(trifluoromethyl)pyridine (486 mg, quantitative
fluorophenyl)cyclopropyl)methoxy)-2-hydrazinyl-3-
(trifluoromethyl)pyridine (181 mg, quantitative yield) was
yield)
was
prepared
from
2-chloro-4-(3-(4-
(from
prepared
from
(±)-2-chloro-4-(((1S,2S)-2-(4-
fluorophenyl)cyclobutoxy)-3-(trifluoromethyl)pyridine
fluorophenyl)cyclopropyl)methoxy)-3-(trifluoromethyl)pyridine
(from step 6.13.3.) following a procedure analogous to step 6.2.2.
1H NMR (500 MHz, METHANOL-d4)  8.19 - 8.16 (m, 1H),
7.17 - 7.13 (m, 2H), 6.98 (s, 2H), 6.59 - 6.53 (m, 1H), 4.29 - 4.23
(m, 1H), 4.10 - 4.03 (m, 1H), 1.55 - 1.46 (m, 1H), 1.11 - 1.04 (m,
2H), 19F NMR m -56.19, -119.87; LC–MS (M+H)+ 342.1.
step 6.12.2.) following a procedure analogous to step 6.2.2. LC–
MS (M+H)+ 345.1.
6.12.4.
3-(cyclopropylmethyl)-7-(3-(4-
fluorophenyl)cyclobutoxy)-8-(trifluoromethyl)-
[1,2,4]triazolo[4,3-a]pyridine. The titled compound (103 mg, 38
%
yield over
2
steps) was prepared from 4-(3-(4-
6.13.5.
(±)-3-(Cyclopropylmethyl)-7-(((1S,2S)-2-(4-
fluorophenyl)cyclobutoxy)-2-hydrazinyl-3-
fluorophenyl)cyclopropyl)methoxy)-8-(trifluoromethyl)-
[1,2,4]triazolo[4,3-a]pyridine. The titled compound (80 mg, 20
% yield over 2 steps) was prepared from (±)-4-(((1S,2S)-2-(4-
fluorophenyl)cyclopropyl)methoxy)-2-hydrazinyl-3-
(trifluoromethyl)pyridine (from step 6.12.3.) following
procedures analogous to steps 6.2.3. and 6.2.4. The final product
was purified using silica gel column chromatography (100%
EtOAc). 1H NMR (500 MHz, CDCl3)  8.09 (d, J=7.6 Hz, 1H),
7.26 - 7.20 (m, 2H), 7.08 - 7.00 (m, 2H), 6.72 (d, J=7.8 Hz, 1H),
4.87 (quin, J=7.2 Hz, 1H), 3.27 - 3.17 (m, 1H), 3.09 (d, J=6.7 Hz,
2H), 3.04 - 2.94 (m, 2H), 2.48 - 2.37 (m, 2H), 1.23 - 1.12 (m,
1H), 0.68 - 0.61 (m, 2H), 0.37 - 0.32 (m, 2H); LC–MS (M+H)+
406.3.
(trifluoromethyl)pyridine (from step 6.13.4.) following
procedures analogous to steps 6.2.3. and 6.2.4. The final product
was purified by silica-gel column chromatography (0-100% ethyl
acetate) and reverse phase preparatory HPLC. 1H NMR (500
MHz, METHANOL-d4)  8.56 (d, J=7.8 Hz, 1H), 7.21 (d, J=7.5
Hz, 1H), 7.17 - 7.11 (m, 2H), 7.01 - 6.93 (m, 2H), 4.47 (dd,
J=10.3, 6.3 Hz, 1H), 4.29 (dd, J=10.2, 7.3 Hz, 1H), 3.07 (d,
J=6.9 Hz, 2H), 2.10 - 2.04 (m, 1H), 1.62 - 1.50 (m, 1H), 1.30 -
1.18 (m, 1H), 1.16 - 1.07 (m, 2H), 0.66 - 0.59 (m, 2H), 0.39 -
0.32 (m, 2H); LC–MS (M+H)+ 406.1.
6.13 (±)-3-(Cyclopropylmethyl)-7-(((1S,2S)-2-(4-
fluorophenyl)cyclopropyl)methoxy)-8-(trifluoromethyl)-
[1,2,4]triazolo[4,3-a]pyridine (13l)
6.13.1.
methyl
trans-2-(4-
6.14(±)-3-(Cyclopropylmethyl)-7-(((1S,2S)-2-(4-
chlorophenyl)cyclopropyl)methoxy)-8-(trifluoromethyl)-
[1,2,4]triazolo[4,3-a]pyridine (7m)
fluorophenyl)cyclopropanecarboxylate. A mixture of trans-2-
(4-fluorophenyl)cyclopropanecarboxylic acid (commercially
available, 159 mg, 0.88 mmol), methanol (5.0 ml), and sulfuric
acid (0.15 ml, 2.81 mmol) was heated in a sealed vial at 72 °C for
18 h. The reaction was carefully neutralized by the addition of
aqueous 10 % sodium carbonate solution. The resulting mixture
was evaporated in vacuo. The residue was partitioned between
ethyl acetate and water. The aqueous phase was extracted with
ethyl acetate. The combined organic layers were washed with
brine, dried (sodium sulfate), filtered, and concentrated in vacuo
6.14.1.
methyl
trans-2-(4-
chlorophenyl)cyclopropanecarboxylate. A mixture of trans-2-
(4-chlorophenyl)cyclopropanecarboxylic acid (commercially
available, 1.0 g, 5.09 mmol), methanol (29.1 ml), and sulfuric
acid (0.813 ml, 15.3 mmol) was heated in a sealed vial at 72 °C
for 18 h. The reaction was carefully neutralized by the addition
of aqueous 10 % sodium carbonate solution. The resulting
mixture was evaporated in vacuo. The residue was partitioned
between ethyl acetate and water. The aqueous phase was
extracted with ethyl acetate. The combined organic layers were
washed with brine, dried (sodium sulfate), filtered, and
concentrated in vacuo to afford methyl trans-2-(4-
chlorophenyl)cyclopropanecarboxylate (1.07 g, quantitative
yield) as a light brown oil. 1H NMR (500 MHz, CDCl3)  7.29 -
7.26 (m, 2H), 7.07 - 7.03 (m, 2H), 3.74 (s, 3H), 2.52 (ddd, J=9.3,
6.4, 4.2 Hz, 1H), 1.89 (ddd, J=8.5, 5.3, 4.3 Hz, 1H), 1.65 - 1.60
(m, 1H), 1.33 - 1.29 (m, 1H).
to
afford
methyl
trans-methyl
2-(4-
fluorophenyl)cyclopropanecarboxylate (167 mg, 97 % yield) as a
light brown oil. 1H NMR (500 MHz, CDCl3)  7.11 - 7.06 (m,
2H), 7.01 - 6.95 (m, 2H), 3.74 (s, 3H), 2.53 (ddd, J=9.3, 6.4, 4.1
Hz, 1H), 1.87 (ddd, J=8.4, 5.2, 4.2 Hz, 1H), 1.64 - 1.58 (m, 1H),
1.32 - 1.25 (m, 2H).
6.13.2.
(trans-2-(4-fluorophenyl)cyclopropyl)methanol (119 mg, 83 %
yield) was prepared from trans-methyl 2-(4-
fluorophenyl)cyclopropanecarboxylate (from step 6.13.1.)
following a procedure analogous to step 6.3.1. 1H NMR (500
MHz, CDCl3)  7.10 - 7.01 (m, 2H), 6.98 - 6.93 (m, 2H), 3.63
(d, J=6.7 Hz, 2H), 1.86 - 1.80 (m, 1H), 1.68 (br. s., 1H), 1.45 -
1.37 (m, 1H), 0.97 - 0.85 (m, 3H).
(trans-2-(4-fluorophenyl)cyclopropyl)methanol.
6.14.2.
(trans-2-(4-chlorophenyl)cyclopropyl)methanol.
(trans-2-(4-chlorophenyl)cyclopropyl)methanol (582 mg, 3.19
mmol, 62.8 % yield) was prepared from trans-methyl 2-(4-
fluorophenyl)cyclopropanecarboxylate (from step 6.14.1.)
following a procedure analogous to step 6.3.1. LC–MS (M-
H2O+H)+ 165.0.
6.13.3.
(±)-2-chloro-4-(((1S,2S)-2-(4-
fluorophenyl)cyclopropyl)methoxy)-3-
(trifluoromethyl)pyridine.
(±)-2-chloro-4-(((1S,2S)-2-(4-
6.14.3.
(±)-2-Chloro-4-(((1S,2S)-2-(4-
fluorophenyl)cyclopropyl)methoxy)-3-(trifluoromethyl)pyridine
chlorophenyl)cyclopropyl)methoxy)-3-
(183 mg, 76
%
yield) was prepared from (trans-2-(4-
(trifluoromethyl)pyridine.
(±)-2-Chloro-4-(((1S,2S)-2-(4-
fluorophenyl)cyclopropyl)methanol (from step 6.13.2.) and 2,4-
chlorophenyl)cyclopropyl)methoxy)-3-(trifluoromethyl)pyridine

(450 mg, 76
%
yield) was prepared from (trans-2-(4-
1.27 - 1.13 (m, 1H), 1.09 (t, J=7.0 Hz, 2H), 0.58 - 0.44 (m, 2H),
0.27 (q, J=4.7 Hz, 2H); LC–MS (M+H)+ 421.8.
chlorophenyl)cyclopropyl)methanol (from step 6.14.2.) and 2,4-
dichloro-3-(trifluoromethyl)pyridine32 following
a procedure
6.16(+)-3-(cyclopropylmethyl)-7-(((1S,2S)-2-(4-
chlorophenyl)cyclopropyl)methoxy)-8-(trifluoromethyl)-
[1,2,4]triazolo[4,3-a]pyridine (+)-(7m)
analogous to step 6.2.1. 1H NMR (500 MHz, CDCl3)  8.38 (d,
J=4.7 Hz, 1H), 7.28 - 7.25 (m, 2H), 7.09 - 7.05 (m, 2H), 6.89 (d,
J=4.6 Hz, 1H), 4.27 (dd, J=9.8, 6.1 Hz, 1H), 4.16 - 4.07 (m, 1H),
2.07 - 2.01 (m, 1H), 1.56 (dd, J=12.6, 6.3 Hz, 1H), 1.18 - 1.10
(m, 2H).
6.16.1. (E)-3-(4-chlorophenyl)acryloyl chloride. Thionyl
chloride (8.79 mL, 120 mmol) was added to a 100 mL round-
bottomed flask charged with
a suspension of (E)-3-(4-
6.14.4.
(±)-4-((1-(4-chlorophenyl)cyclopropyl)methoxy)-2-
chlorophenyl)acrylic acid (14) (10 g, 54.8 mmol) in anhydrous
dichloromethane (200 mL) under nitrogen. The reaction mixture
was stirred at rt for 12h. The reaction mixture was concentrated
in vacuo to afford the crude product (E)-3-(4-
chlorophenyl)acryloyl chloride (11.05 g, quantitative yield) as a
white solid. 1H NMR (500MHz, CDCl3)  7.81 (d, J=15.6 Hz,
1H), 7.57 - 7.51 (m, 2H), 7.47 - 7.41 (m, 2H), 6.65 (d, J=15.6 Hz,
1H).
hydrazinyl-3-(trifluoromethyl)pyridine.
chlorophenyl)cyclopropyl)methoxy)-2-hydrazinyl-3-
(±)-4-((1-(4-
(trifluoromethyl)pyridine (451 mg, quantitative yield) was
prepared from (±)-2-chloro-4-(((1S,2S)-2-(4-
chlorophenyl)cyclopropyl)methoxy)-3-(trifluoromethyl)pyridine
(from step 6.14.3.) following a procedure analogous to step 6.2.2.
LC–MS (M+H)+ 358.0.
6.14.5.
chlorophenyl)cyclopropyl)methoxy)-3-(cyclopropylmethyl)-8-
(trifluoromethyl)-[1,2,4]triazolo[4,3-a]pyridine. The titled
(±)-7-(((1S,2S)-2-(4-
6.16.2. (E)-3-(4-chlorophenyl)-1-((3aR,6R,7aS)-8,8-dimethyl-
2,2-dioxidohexahydro-1H-3a,6-methanobenzo[c]isothiazol-1-
yl)prop-2-en-1-one (15). To a cold (0°C) solution of the (E)-3-
(4-chlorophenyl)acryloyl chloride (from step 5.16.1., 11 g, 54.7
mmol) in dichloromethane (25 mL), was added a solution of
(3aR,6R,7aS)-8,8-dimethylhexahydro-1H-3a,6-
compound (145 mg, 26 % yield over 2 steps) was prepared from
(±)-4-((1-(4-chlorophenyl)cyclopropyl)methoxy)-2-hydrazinyl-3-
(trifluoromethyl)pyridine (from step 6.14.4.) following
procedures analogous to steps 6.2.3. and 6.2.4. The final product
was purified by silica-gel column chromatography (0-100% ethyl
methanobenzo[c]isothiazole 2,2-dioxide (12.96 g, 60.2 mmol)
and Et3N (22.88 mL, 164 mmol) in dichloromethane (100 mL)
dropwise. After the addition was complete, the ice bath was
removed, and stirring was continuted for 2 h. The reaction was
diluted with dichloromethane, then washed well with water,
saturated sodium bicarbonate, brine, dried over sodium sulfate,
and concentrated to give a beige solid. The solid was
recrystallized from hot EtOH to give (E)-3-(4-chlorophenyl)-1-
((3aR,6R,7aS)-8,8-dimethyl-2,2-dioxidohexahydro-1H-3a,6-
methanobenzo[c]isothiazol-1-yl)prop-2-en-1-one (17.03 g, 82 %
yield) as a white crystalline solid. 1H NMR (500MHz, CDCl3) 
7.75 (d, J=15.4 Hz, 1H), 7.56 - 7.50 (m, 2H), 7.40 - 7.34 (m, 2H),
7.16 (d, J=15.4 Hz, 1H), 4.01 (dd, J=7.6, 5.0 Hz, 1H), 3.60 - 3.46
(m, 2H), 2.26 - 2.13 (m, 2H), 2.02 - 1.90 (m, 3H), 1.52 - 1.38 (m,
2H), 1.23 (s, 3H), 1.02 (s, 3H); LC–MS (M+H)+ 380.2.
1
acetate/hexanes). H NMR (500 MHz, DMSO-d6)  8.70 (d,
J=7.8 Hz, 1H), 7.33 - 7.27 (m, 2H), 7.21 (d, J=7.8 Hz, 1H), 7.16
- 7.11 (m, 2H), 4.46 - 4.40 (m, 1H), 4.33 (dd, J=10.6, 7.2 Hz,
1H), 3.04 (d, J=6.9 Hz, 2H), 2.08 - 2.01 (m, 1H), 1.60 - 1.51 (m,
1H), 1.22 - 1.14 (m, 1H), 1.13 - 1.07 (m, 2H), 0.54 - 0.48 (m,
2H), 0.30 - 0.25 (m, 2H); LC–MS (M+H)+ 421.8.
6.15(+)-3-(cyclopropylmethyl)-7-(((1S,2S)-2-(4-
chlorophenyl)cyclopropyl)methoxy)-8-(trifluoromethyl)-
[1,2,4]triazolo[4,3-a]pyridine
((+)-7m)
and
(-)-3-
(cyclopropylmethyl)-7-(((1R,2R)-2-(4-
chlorophenyl)cyclopropyl)methoxy)-8-(trifluoromethyl)-
[1,2,4]triazolo[4,3-a]pyridine ((-)-7m)
6.15.1.
(+)-3-(cyclopropylmethyl)-7-(((1S,2S)-2-(4-
chlorophenyl)cyclopropyl)methoxy)-8-(trifluoromethyl)-
6.16.3. ((1S,2S)-2-(4-chlorophenyl)cyclopropyl)((6R,7aR)-8,8-
dimethyl-2,2-dioxidohexahydro-1H-3a,6-
[1,2,4]triazolo[4,3-a]pyridine
(cyclopropylmethyl)-7-(((1R,2R)-2-(4-
chlorophenyl)cyclopropyl)methoxy)-8-(trifluoromethyl)-
((+)-7m)
and
(-)-3-
methanobenzo[c]isothiazol-1-yl)methanone (16). N-methyl-N'-
nitro-N-nitrosoguanidine (23.24 g, 158 mmol) was carefully
added portionwise over 20 min to a swirled bilayer of diethyl
ether (500 mL) and 5 N NaOH (500 ml) in a plastic coated 2000
mL Erlenmyer flask that was placed in an ice-water bath behind a
blast shield. After 15 min, when all bubbling had ceased the
ether layer was decanted into a separate 2000 mL Erlenmyer
flask charged with ~80 g of KOH pellets in an ice-water bath.
The yellow solution was swirled occasionally for 10 min, then
the ethereal diazomethane solution was transferred to a diazo-
addition funnel suspended above a new 2000 mL Erlenmyer fask
charged with a gently stirred solution of (E)-3-(4-chlorophenyl)-
1-((3aR,6R,7aS)-8,8-dimethyl-2,2-dioxidohexahydro-1H-3a,6-
methanobenzo[c]isothiazol-1-yl)prop-2-en-1-one (from step
6.16.2., 10 g, 26.3 mmol) and palladium(II) acetate (0.177 g,
0.790 mmol) in DCM (66 mL) maintained at -10 °C. After
complete addition, the mixture was allowed to warm to rt and for
16h. The reaction was chilled to 0 °C and quenched with acetic
acid. The reaction was filtered over celite, poured into a solution
of saturated sodium bicarbonate, and extracted with ethyl acetate
(3 x 250 mL). The combined organic layers were washed with
brine, dried over magnesium sulfate, and concentrated in vacuo.
The crude product was purified by silica gel column
[1,2,4]triazolo[4,3-a]pyridine ((-)-7m). A sample of (±)-7-
(((1S,2S)-2-(4-chlorophenyl)cyclopropyl)methoxy)-3-
(cyclopropylmethyl)-8-(trifluoromethyl)-[1,2,4]triazolo[4,3-
a]pyridine (from step 6.14.5., 233 mg) was purified using chiral
preparatory HPLC to afford 61 mg of (-)-13m (first eanantiomer
to elute) and 60 mg of (+)-13m (second enantiomer to elute).
HPLC Method: ChiralCel OD (21 x 250 mm, 10 uM), 10 %
ethanol / 90 % heptane (with 0.1 % diethylamine), 15 mL/min,
absorbance 254 nm. The absolute stereochemistry of (+)-13m
was confirmed as (1S,2S) through an independent asymmetric
synthesis (step 6.16). The stereochemistry of (-)-13m was
1
assigned as (1R,2R) by default. (+)-7m: H NMR (500 MHz,
DMSO-d6)  8.69 (d, J=7.6 Hz, 1H), 7.35 - 7.25 (m, J=8.5 Hz,
2H), 7.20 (d, J=7.6 Hz, 1H), 7.17 - 7.07 (m, J=8.5 Hz, 2H), 4.42
(dd, J=10.7, 6.7 Hz, 1H), 4.32 (dd, J=10.7, 7.3 Hz, 1H), 3.03 (d,
J=7.0 Hz, 2H), 2.11 - 1.97 (m, 1H), 1.60 - 1.47 (m, 1H), 1.27 -
1.13 (m, 1H), 1.09 (t, J=7.0 Hz, 2H), 0.58 - 0.44 (m, 2H), 0.27 (q,
1
J=4.7 Hz, 2H); LC–MS (M+H)+ 421.8. (-)-7m: H NMR (500
MHz, DMSO-d6)  8.69 (d, J=7.6 Hz, 1H), 7.35 - 7.25 (m, J=8.5
Hz, 2H), 7.20 (d, J=7.6 Hz, 1H), 7.17 - 7.07 (m, J=8.5 Hz, 2H),
4.42 (dd, J=10.7, 6.7 Hz, 1H), 4.32 (dd, J=10.7, 7.3 Hz, 1H),
3.03 (d, J=7.0 Hz, 2H), 2.11 - 1.97 (m, 1H), 1.60 - 1.47 (m, 1H),
chromatography
(0-15%
ethyl
acetate/hexanes)
and
recrystallization from ethyl acetate/hexanes to afford ((1S,2S)-2-

(4-chlorophenyl)cyclopropyl)((6R,7aR)-8,8-dimethyl-2,2-
dioxidohexahydro-1H-3a,6-methanobenzo[c]isothiazol-1-
HEK293 cells stably-expressing the human mGlu2 receptor
and rat glutamate transporter (GLAST) were used as the cell
membrane source. Cell pellets were homogenized at 4 °C in
hypotonic lysis buffer (50 mM TRIS pH 7.4, 10 mM MgCl2, 2
mM EGTA and 0.1% (v/v) Sigma Protease Inhibitor Cocktail)
followed by centrifugation at 32,000 x g for 30 min. The
supernatant was discarded and the pellet was washed once in
lysis buffer and centrifuged at 32,000 x g for 30 min. This pellet
was then resuspended in lysis buffer and protein concentrations
were determined using bovine serum albumin as a standard. The
membranes were aliquoted and frozen in dry ice/ethanol and kept
at -80°C until the day of the assay.
1
yl)methanone (7.6 g, 73% yield) as a white crystalline solid. H
NMR (500MHz, CDCl3)  7.28 - 7.25 (m, 2H), 7.18 - 7.15 (m,
2H), 3.93 (dd, J=7.8, 4.9 Hz, 1H), 3.53 (d, J=13.7 Hz, 1H), 3.46
(d, J=13.9 Hz, 1H), 2.61 - 2.51 (m, 2H), 2.18 - 2.12 (m, 1H), 2.11
- 2.05 (m, 1H), 1.97 - 1.88 (m, 3H), 1.83 - 1.77 (m, 1H), 1.47 -
1.32 (m, 3H), 1.22 (s, 3H), 1.00 (s, 3H); LC–MS (M+H)+ 394.2.
6.16.4.
((1S,2S)-2-(4-chlorophenyl)cyclopropyl)methanol
(17). To a stirring suspension of LAH (0.915 g, 24.12 mmol) in
THF (100 mL) was added dropwise a solution of ((1S,2S)-2-(4-
chlorophenyl)cyclopropyl)((6R,7aR)-8,8-dimethyl-2,2-
dioxidohexahydro-1H-3a,6-methanobenzo[c]isothiazol-1-
yl)methanone (from step 5.16.3., 7.6 g, 19.29 mmol) in THF (20
mL) and the resulting mixture was stirred at -40 °C under
nitrogen. After complete addition, the reaction mixture was
stirred for 1h. The reaction was quenched by the addition of
aqueous ammonium hydroxide solution. The reaction mixture
was slurried in a mixture of water and ethyl acetate and filtered.
The layers were then separated, the aqueous layer was washed
again with ethyl acetate, and the combined organic layers were
washed with brine and dried over magnesium sulfate. The drying
agent was filtered off and the solvent was concentrated in vacuo
to afford ((1S,2S)-2-(4-chlorophenyl)cyclopropyl)methanol (3.5
g, 99 % yield). LC–MS (M-H20+H)+ 165.2.
Frozen aliquots of membrane homogenate were thawed,
homogenized and resuspended at 0.2 mg/ml protein in assay
buffer (20 mM TRIS-Cl pH 7.4, 5 mM KCl, 2 mM CaCl2, 2 mM
MgCl2, and 0.005% Triton X-100) at 4 °C. Competition binding
experiments were performed using 7.5 nM [3H]-JNJ-40068782
(Specific activity 28.1 Ci/mmol) in the presence 10
concentrations (in duplicate) of test compound. Addition of 1 uM
mGlu2/3 orthosteric agonist LY379268 was used to increase assay
window without altering test compound IC50s and was included
in the buffer. Test compound, membranes (20 ug/well) and
radioligand were sequentially added to the wells of a 96 well
plate and incubated for 30 minutes at room temperature. Non-
specific binding was defined with 10 μM MPPTS. The assay
was terminated by the addition of 5 mL of ice-cold wash buffer
(50 mM TRIS-Cl pH 7.4 and 0.1% BSA) and rapid filtration
through a Brandel Cell Harvester using Whatman GF/B filters
presoaked in 0.3% PEI. After 3 rapid washes, the filter was
punched onto a 96 well microbeta sample plate, 200 μl/well of
Packard Ultima GoldXR scintillation fluid added and the filters
soaked overnight. The filter pads were then counted in a LKB
Trilux liquid scintillation counter.
6.16.5.
2-chloro-4-(((1S,2S)-2-(4-
chlorophenyl)cyclopropyl)methoxy)-3-
(trifluoromethyl)pyridine.
2-chloro-4-(((1S,2S)-2-(4-
chlorophenyl)cyclopropyl)methoxy)-3-(trifluoromethyl)pyridine
(3.25 g, 66 % yield) was prepared from (((1S,2S)-2-(4-
chlorophenyl)cyclopropyl)methanol (from step 6.16.4.) and 2,4-
dichloro-3-(trifluoromethyl)pyridine32 following
a procedure
analogous to step 6.2.1. 1H NMR (500MHz, CDCl3)  8.37 (d,
J=5.8 Hz, 1H), 7.28 - 7.25 (m, 2H), 7.09 - 7.06 (m, 2H), 6.88 (d,
J=5.8 Hz, 1H), 4.26 (dd, J=9.9, 6.1 Hz, 1H), 4.09 (dd, J=9.8, 6.8
Hz, 1H), 2.07 - 2.01 (m, 1H), 1.59 - 1.54 (m, 1H), 1.16 - 1.11 (m,
2H).
IC50 values were determined using non-linear regression in
Curvemaster. Ki (apparent) values were calculated using the
method of Cheng and Prusoff.
The Kd value of the radiolabel +/- LY379268 was 7.78 nM ±
2.09 and 5.56 nM ± 1.36, respectively.
6.16.6. 4-(((1S,2S)-2-(4-chlorophenyl)cyclopropyl)methoxy)-
2-hydrazinyl-3-(trifluoromethyl)pyridine.
chlorophenyl)cyclopropyl)methoxy)-2-hydrazinyl-3-
(trifluoromethyl)pyridine (2.91 g, 91 % yield) was prepared from
2-chloro-4-(((1S,2S)-2-(4-chlorophenyl)cyclopropyl)methoxy)-3-
4-(((1S,2S)-2-(4-
The constitutive activity for LY341495 in this assay was IC50
= 1.48 nM.
(trifluoromethyl)pyridine (from step 6.16.5.) following
a
procedure analogous to step 6.2.2. LC–MS (M+H)+ 358.2.
7.2 mGlu2 and mGlu3 cAMP functional assays
Functional assays with human mGlu2 were carried out using
the same HEK293 cell line that was used for mGlu2 PAM
binding. The cell line provided a robust and sensitive assay
platform, but also possessed significant mGlu2 receptor
constituitive activity in the cAMP assay. Modifications to the
assay to eliminate constituitive activity (see below) preserved the
ability to evaluate functional allosteric activity, but did not allow
unambiguous determination of the nature of the activity (PAM vs
allosteric agonist). Therefore these studies were carried out in
the FLIPR assay (described later in the methods). The day before
a study, cells were grown in MEM without glutamine (GIBCO
MEM12360) with 10% FBS (dialyzed) and 1%
pennicillin/streptomycin. On the day of the experiment the media
was removed, the cells were washed in PBS and they were
trypsinized to remove them from the plate. After adding media to
stop the trypsin the cells were centrifuged at 800 x g for 10
minutes and the cell pellet was resuspended in stimulation buffer
(Hanks Balanced Salt Solution pH 7.0, 20 mM HEPEs, 2.0 mM
CaCl2, 5 mM MgCl2, 1 mM IMBX, 1 μM forskolin and 1μM
LY341495) at 1.25 x106 cells/per ml. Addition of orthosteric
6.16.7.
(+)-7-(((1S,2S)-2-(4-
chlorophenyl)cyclopropyl)methoxy)-3-(cyclopropylmethyl)-8-
(trifluoromethyl)-[1,2,4]triazolo[4,3-a]pyridine ((+)-7m). The
titled compound (1.77 g, 50 % yield over 2 steps) was prepared
from
4-(((1S,2S)-2-(4-chlorophenyl)cyclopropyl)methoxy)-2-
hydrazinyl-3-(trifluoromethyl)pyridine (from step 6.16.6.)
following procedures analogous to steps 6.2.3. and 6.2.4. The
final product was purified by silica-gel column chromatography
(0-100% ethyl acetate/hexanes) and recrystallized with hot EtOH.
1H NMR (500MHz, CDCl3)  8.10 (d, J=7.6 Hz, 1H), 7.27 - 7.24
(m, 2H), 7.09 - 7.03 (m, 2H), 6.81 (d, J=7.6 Hz, 1H), 4.33 (dd,
J=9.9, 6.1 Hz, 1H), 4.20 (dd, J=9.9, 6.7 Hz, 1H), 3.08 (d, J=6.7
Hz, 2H), 2.08 - 2.00 (m, 1H), 1.60 - 1.51 (m, 1H), 1.20 - 1.12 (m,
3H), 0.67 - 0.62 (m, 2H), 0.36 - 0.32 (m, 2H); LC–MS (M+H)+
421.8.
7.0 In Vitro Biology Materials and Methods
7.1 mGlu2 PAM competition radioligand binding assay

antagonist LY341495 to the stimulation buffer reduced mGlu2
cell line constituitive activity and improved assay window and
consistency, while not altering test compound potency.
Compounds were added to white non-binding surface 384 well
plates (Corning 3574) as 10 concentration dose responses (in
duplicate). Cells in stimulation buffer were then dispensed into
the plate and allowed to incubate at RT for 30 minutes. Cells
were lysed with addition of Lysis buffer (50mM Phosphate
Buffer pH 7.0, 800mM Potassium Fluoride, 0.2% BSA, and 1.0%
Triton) containing D2 and cryptate antibodies diluted 1:40 from
the CYSBIO dynamic cAMP kit. Cells lysates further incubated
for 1 h at RT and were then evaluated using a Perkin Elmer
Viewlux. The mGluR3 functional assays were carried out in the
same manner except using an HEK293 cell pool expressing
human mGluR3 and the glutamate transporter GLAST. Imaging
data from both assays was converted to cAMP levels using a
cAMP standard curve and IC50 values were determined using
non-linear regression in Curvemaster.
8.0 In Vivo Biology Materials and Methods
8.1 Subjects
Unless otherwise specified, animal studies were conducted in
group housed (n=3-4), C57Bl/6J male mice (20-30g; Jackson
Laboratories, Bar Harbor, ME) held in colony rooms maintained
at constant temperature (21 ± 2 °C), and humidity (50 ± 10%)
and illuminated for 12 h per day (lights on at 0600 hours).
Animals had ad libitum access to food and water throughout the
studies. Behavioral studies were conducted between 0700 and
1500 hours. Animals were maintained in accordance with the
guidelines of the Animal Care and Use Committee of the Bristol-
Myers Squibb Company, and the “Guide for the Care and Use of
Laboratory Animals” published by the National Institutes of
Health. Research protocols were approved by the Bristol-Myers
Squibb Company Animal Care and Use Committee.
8.2 Spontaneous and PCP-Stimulated Locomotion
Mice were habituated to Tru Scan locomotor arenas
(Coulbourn Instruments, Allentown, PA) for 90 minutes under
low light conditions (30 lux). Following habituation, all mice
were treated with vehicle, BMT-133218 (1 to 10 mg/kg, p.o.), or
the assay positive control risperidone (0.056 mg/kg, s.c.) and
returned to the activity monitors for 30 minutes. Mice were then
dosed with saline or PCP (3.2 mg/kg, s.c., in saline, Sigma-
Aldrich, St. Louis, MO) and returned to the activity monitors for
2 h. Brain and plasma samples were collected from satellite
animals at a time point corresponding to the peak PCP response
(1 hr post-PCP), stored at -80 C, and analyzed for compound
brain and plasma concentrations. BMT-133218 was prepared in
10% DMSO/18% TPGS/72% water, and risperidone was
prepared in 0.3% tartaric acid. Distance travelled (cm) data was
collected in five minute bins and summed across the 2-hr PCP
phase. Data were analyzed using one-way Analysis of Variance
(ANOVA) followed by post-hoc Dunnett’s tests.
7.3 mGlu2 FLIPR glutamate shift functional assays
Glutamate shift functional assays were carried out using
HEK293 cells stably expressing a humanized form of monkey
mGlu2 and a chimeric Gqi5 G protein which converts Gi
signalling to the Gq pathway. The humanized monkey mGlu2
was identical to human mGlu2 with the exception of a
conservative amino acid change at the C terminus (Leu846Val).
Humanized monkey mGlu2 cells were plated the day before the
assay at 40,000 cells per well in 384 well clear bottom plates in
glutamine free Gibco MEM media (12360) with 10% dialyzed
FBS and 1% pennicillin/streptomycin. The day of the assay the
media was removed and replaced with assay buffer (Hanks
Balanced Salt Solution, 20 mM HEPES, 2 mM CaCl2, 1 mM
MgCl2, 2.5 mM Probenecid, and 5 μM Fluo-4) and incubated at
37 °C for 60 min. The standard assay format was 4 glutamate
dose response curves for each test compound (made up of a
vehicle dose response and dose responses with three test
compound concentrations). Test compound concentrations were
adjusted as needed to capture maximal PAM activity. Test
compounds were prepared in DMSO and added directly to the
assay plates using an ECHO acoustic dispenser and allowed to
incubate for 10 minutes at RT. A FLIPR plate reader system was
used to dispense the glutamate concentration curves (prepared in
assay buffer without Probenecid and Fluo-4) and fluorescence
was measured for 3 minutes. Peak fluorescence was normalized
against a 100 μM glutamate positive control and concentration
response curves were generated using non-linear regression in
Loadmaster. Fold shift values were determined by dividing the
EC50 of glutamate with vehicle treatment by the EC50 of the most
left shifted glutamate curve in the presence of compound. Curves
8.3 Two Trial Y maze
For these experiments, male C57Bl6/Tac mice (20-27g) were
used. Experimental methods were adapted from those reported in
the literature.33 Testing was conducted in a grey polyethylene
plastic Y maze that had 3 arms, 2 of 40 cm length (test arms) and
1 of 20 cm length (start arm). Each arm had distinct visual cues
covering the walls and a removable partition to occlude the arm
as needed. The test consisted of two trials: a forced choice trial
followed by a free-choice trial. For the forced choice trial only
the start arm and one test arm was open with access to the 2nd
test arm blocked by the partition. Individual subjects were placed
in the start arm and allowed to explore the open test arm for 8
min after which they were removed from the maze, placed in a
holding cage and the maze was cleaned. Animals were then
immediately placed back on the Y maze for the free choice trial
and allowed to explore all open arms for 4 min. The protocol was
thus designed for minimal delay between the forced choice and
free choice trials. Animal behavior was video recorded during
both trials and the time spent in the previously accessible arm (ie
familiar arm) and the previously blocked arm (ie novel arm)
determined during the free choice trial using Cleversys software.
with
a low Y-max (less than 70%, suggesting assay
desensitization) were not used in the calculation.
7.4 mGlu2 FLIPR agonist assay
Assays were carried out in a similar manner to the Glutamate
shift assays (see above). In the agonist assay compound serial
dilutions were added to the plate using the FLIPR and
immediately scanned for 3 minutes. The peak fluorescence at
each dilution was normalized to 100 uM glutamate (maximal
response). In the PAM assay the compound serial dilutions were
added to the cells in the plate using the ECHO acoustic dispenser.
After a 10 minute preincubation an EC10-20 of glutamate was
added to the plate using the FLIPR and the plate was scanned as
described previously. IC50 values in both assays were determined
using non-linear regression in Curvemaster.
Results for the free choice trial are presented as the mean ±
SEM percentage of time spent exploring the novel arm. The
percent time was calculated for each individual subject using the
formula: % novel time = novel time/(novel + familiar time) x 100
and was analyzed by one-way ANOVA followed by Dunnett’s
post-hoc test. The mean time spent exploring the novel or
familiar arm was analyzed for each treatment group using a
within subjects t-test. Data analysis was conducted using

Graphpad Prism (v5.0, San Diego, CA) with significance set at
p<0.05.
Set shifting was tested following maze habituation and
consisted of 2 test sessions conducted over 2 consecutive days.
On the first day (Set 1) rats were required to learn the association
of either the color or texture of the maze arms with the location
of a food reward. Thus, prior to testing each animal was
randomly assigned a correct exemplar within a given dimension
as being predictive of reward with 50% of the rats assigned color
(i.e. black or white) and 50% assigned texture (i.e. rough or
smooth). Rats were required to ignore the opposite dimension in
order to establish which exemplar within the assigned dimension
correctly predicted food at the end of the arm. Each rat was given
a maximum of 120 trials on the maze which was configured in
the ‘T’ format and rotated 90 degrees between successive trials as
described above. Animals were allowed to visit 1 of the 2 open
arms and the trial recorded as correct if the arm with the pre-
assigned exemplar was visited and the food reward retrieved. The
inter-trial interval was 15 s as previously described. Rats were
required to achieve a performance criterion of 8 consecutive
entries into the rewarded arm at which point testing stopped and
the animal was presumed to have learnt the association between
the correct exemplar in the assigned dimension and food reward.
BMT-133218 was dissolved in 10% DMSO/ 18% TPGS/ 72%
water on the day of testing. Vehicle or mGlu2 PAM treatment
was administered orally 30 minutes prior to MK-801 (0.05
mg/kg, s.c.) and 60 minutes prior to the forced choice trial.
Doses of BMT-133218 tested were 1, 3, and 10 mg/kg (p.o.). For
the positive control, mice were pre-treated with the 7 nACh
receptor full agonist, PNU-282987 (10 mg/kg, s.c.) 5 minutes
prior to the MK-801 injection. A satellite group of animals was
sacrificed and plasma samples collected and stored at -80 ºC
prior to measurement of drug concentrations.
8.4 Reversal of MK-801-Induced Deficits in Set Shift
Performance in Rat
Male Sprague-Dawley rats (150 - 175g at delivery; Harlan,
Dublin, VA) were singly housed in colony rooms maintained at a
constant temperature (21 ± 2°C) and humidity (50 ± 10%) and
illuminated for 12 h per day (lights on at 0600 h). Behavioral
studies were conducted between 0600 and 1300 h. Animals were
maintained in accordance with the guidelines of the Animal Care
and Use Committee of the Bristol-Myers Squibb Company, the
“Guide for Care and Use of Laboratory Animals” and the
guidelines published in the National Institutes of Health Guide
for the Care and Use of Laboratory Animals. Research protocols
were approved by the Bristol-Myers Squibb Company Animal
Care and Use Committee.
On day 2 (Set 2) animals were again tested in the maze
configured as described for Set 1 with the exception that the
dimension predicting food reward was now switched (i.e. animals
trained on color for Set 1 are now switched to texture and vice
versa). Again, within each assigned dimension half the animals
were assigned one exemplar as correct (e.g. rough) and half the
opposite dimension (e.g. smooth). This new ‘correct’ assignment
requires that the animal suppress previously learned associations
that predicted food reward in Set 1 and learn new associations
based on correctly establishing the relationship between an
exemplar in a different dimension with the presence of food
rewards i.e. set shifting. Correct performance was again defined
as 8 consecutive entries into the rewarded arm with a maximal
number of 80 trials allowed.
Rats were food-restricted (15g/rat/day) for 7 days prior to
initiation of training and for the duration of the study. Training
consisted of 3 days of handling (~5 min per day) followed by 6
days of habituation to the maze. The maze consists of 4 arms (40
cm long x 14 cm wide x 20 cm high) arranged in a ‘+’
configuration and the walls and floor of each arm are distinct and
colored either black or white with either a rough or smooth
texture (i.e. 1 arm with each of the following combinations:
white/smooth, white/rough, black/smooth, black/rough). On the
first 4 days of maze habituation, rats were given a single
exposure to the maze during which they were placed in the center
of the ‘+’ maze and allowed to explore all 4 arms which were
each baited with 2 food pellets (45 mg precision pellet, Bio-Serv,
Frenchtown, New Jersey). The rats were removed from the maze
after consuming all 8 food pellets, placed in a holding chamber
for 2 min and then returned to their home cage. The last 2 days of
maze habituation were designed to ensure that animals explored
the maze with no preference for any particular arm, to prevent
positional bias (e.g. always turning right) and to minimize the
impact of any extra-maze cues on guiding behavior. For these
sessions the maze was set up in a ‘T’ configuration with 1 arm
completely blocked. The arm opposite the blocked arm was
designated as the ‘start arm’ and only 1 of the 2 remaining arms
was baited with food pellets. Each animal was given 8 successive
trials with a 15 s inter-trial interval during which they were
placed in the holding chamber. At the beginning of each trial the
animal was placed in the ‘start arm’ and allowed to visit 1 of the
2 remaining arms and consume the food pellet if the arm was
baited. If the animal visited the baited arm (e.g. right arm) then
the opposite (i.e. left arm) was baited on the next trial; if not then
the arm in the same position (e.g. right) continued to be baited
until the pellets had been retrieved. Between successive trials the
maze was rotated by 90 degrees so that the adjacent arms now
became either the ‘start arm’ or the arm that was blocked. Thus
each arm served as the ‘start arm’ or ‘blocked arm’ on 2 out of
the 8 trials. Any rat showing >80% side bias (i.e. right or left)
was excluded from further testing.
The mGlu2 test compound was prepared in 5% DMSO 5%
Cremaphor El, 90% saline; add DMSO, vortex; add cremaphor,
glass beads and shake overnight; in the morning add saline, let
shake 20-30 min. Animals were dosed prior to Set 2 with either
mGluR2 test compound (1.0 – 10 mg/kg, po) or vehicle (1 ml/kg,
sc) followed 25 min later by either MK-801 (0.04 mg/kg, ip) or
vehicle (1 ml/kg, ip) and behavioral testing initiated 35 min later
(i.e. 60 min post-treatment with mGluR5 test compound). For
comparison, animals dosed with the α7 nACh receptor partial
agonist SSR-180711 (10 mg/kg, po; 60 min pretreatment time
prior to MK-801) were also included.
Behavioral data were analyzed by analysis of variance
followed by a planned contrasts post-hoc test. Statistics were
performed using SAS (Statistical Analysis Software, V9.1) with
significance set at p<0.05.
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Triazolopyridine Ethers as Potent, Orally
Active mGluR2 Positive Allosteric
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Modulators for Treating Schizophrenia
Mendi A. Higgins*, Lawrence R. Marcin, F. Christopher Zusi, Robert Gentles, Min Ding, Bradley C.
Pearce, Amy Easton, Walter A. Kostich, Matthew A. Seager, Clotilde Bourin, Linda J. Bristow, Kim A.
Johnson, Regina Miller, John Hogan, Valerie Whiterock, Michael Gulianello, Meredith Ferrante, Yanling
Huang, Adam Hendricson, Andrew Alt, John E. Macor, Joanne J. Bronson
Bristol-Myers Squibb, Research and Development, 5 Research Parkway, Wallingford, CT 06492-7660, USA