Design, synthesis, and structure-activity relationships of novel bicyclic azole-amines as negative allosteric modulators of metabotropic glutamate receptor 5

Article abstract of DOI:10.1021/jm100736h

A novel series of diaryl bicyclic azole-amines that are potent selective negative modulators of metabotropic glutamate receptor 5 (mGluR5) were identified through rational design. An initial hit compound 5a of modest potency (IC₅₀ = 1.2 μM) was synthesized. Evaluation of structure-activity relationships (SAR) on the left-hand side of the molecule revealed a preference for a 2-substituted pyridine group linked directly to the central heterocycle. Variation of the central azolo-amine portion of the molecule revealed a preference for the [4,5-c]-oxazoloazepine scaffold, while right-hand side variants showed a preference for ortho- and meta-substituted benzene rings linked directly to the tertiary amine of the saturated heterocycle. These iterations led to the synthesis of 29b, a potent (IC₅₀ = 16 nM) and selective negative modulator that showed good brain penetrance, high receptor occupancy, and a duration of action greater than 1 h in rat when administered intraperitoneally. Formal PK studies in rat and Rhesus monkey revealed a short half-life that was attributable to high first-pass clearance.

Full text of DOI:10.1021/jm100736h

J. Med. Chem. 2010, 53, 7107–7118 7107
DOI: 10.1021/jm100736h
Design, Synthesis, and Structure-Activity Relationships of Novel Bicyclic Azole-amines
as Negative Allosteric Modulators of Metabotropic Glutamate Receptor 5
Douglas F. Burdi,*,† Rachel Hunt,†,^ Lei Fan, Tao Hu, Jun Wang, Zihong Guo, Zhiqiang Huang, Chengde Wu,§
Larry Hardy,† Michel Detheux,‡ Michael A. Orsini,† Maria S. Quinton,† Robert Lew,† and Kerry Spear†
†Discovery & Early Clinical Research, Sepracor Inc., 84 Waterford Drive, Marlborough, Massachusetts 01752, ‡EuroScreen SA,
Rue Adrienne Bolland 47, B6041 Gosselies, Belgium, §WuXi AppTec Co., Ltd., 288 Fute Zhong Road, Waigaoqiao Free Trade Zone,
Shanghai 200131, Peoples Republic of China, and Shanghai ChemPartner, Building no. 6, 998 Haki Road, Zhangjiang Hi-Tech Park,
Pudong New Area, Shanghai 201203, China. ^ Current address: Galenea Corporation, 300 Technology Square, Second Floor, Cambridge,
Massachusetts 02139
Received June 17, 2010
A novel series of diaryl bicyclic azole-amines that are potent selective negative modulators of metabotro-
pic glutamate receptor 5 (mGluR5) were identified through rational design. An initial hit compound 5a
of modest potency (IC50 = 1.2 μM) was synthesized. Evaluation of structure-activity relationships
(SAR) on the left-hand side of the molecule revealed a preference for a 2-substituted pyridine group
linked directly to the central heterocycle. Variation of the central azolo-amine portion of the molecule
revealed a preference for the [4,5-c]-oxazoloazepine scaffold, while right-hand side variants showed a
preference for ortho- and meta-substituted benzene rings linked directly to the tertiary amine of the
saturated heterocycle. These iterations led to the synthesis of 29b, a potent (IC50 = 16 nM) and selective
negative modulator that showed good brain penetrance, high receptor occupancy, and a duration of
action greater than 1 h in rat when administered intraperitoneally. Formal PK studies in rat and Rhesus
monkey revealed a short half-life that was attributable to high first-pass clearance.
Introduction
subsequently terminated due to liver toxicity.4 With respect to
CNS disorders, negative modulators of mGluR5 have been
extensively validated for the treatment of anxiety. 3-[(2-Methyl-
1,3-thiazol-4-yl)ethynyl]-pyridine (MTEP)5 has shown effi-
cacy in a variety of animal anxiety models including stress-
induced hyperthermia and fear-potentiated startle. Most
recently, a phase 2 study of AFQ-056 showed efficacy in the
alleviation of levodopa-induced dyskinesia in patients with
Parkinson’s disease.6
Following the discovery of the diaryl alkyne 2-methyl-
6-(phenylethynyl)pyridine (MPEP),7 several classes of mole-
cules have been disclosed as negative modulators of mGluR5
(Figure 1).8 Fenobam (1), developed as an anxiolytic of
Glutamate, the major excitatory neurotransmitter in the cen-
tral nervous system, binds to and activates cell surface recep-
tors and ion channels in neurons and astrocytes. The metabo-
tropic glutamate receptors (mGluRsa) are G protein-coupled
receptors that activate intracellular second messengers when
bound to glutamate. The group 1 metabotropic glutamate
receptors, comprising mGluR1 and mGluR5, activate phos-
pholipase C, leading to the mobilization of intracellular
calcium.1
Because of its expression in both the central nervous system
and the periphery,2 modulation of mGluR5 may be useful for
the treatment of both peripheral and CNS disorders. With
respect to peripheral disorders, mGluR5 negative modulators
have recently shown efficacy in the treatment of disorders
of the gastrointestinal tract. A recent clinical trial of ADX-
10059, a negative modulator of mGluR5 in gastresophageal
reflux disease (GERD), met its primary end point3 but was
*To whom correspondence should be addressed. Phone: 1-508-357-
7749. Fax: 1-508-490-5454. E-mail: douglas.burdi@sepracor.com.
a Abbreviations: BINAP, 2,20-bis(diphenylphosphino)-1,10-binaphthyl;
BSA, bovine serum albumin; Dess-Martin reagent, 1,1,1-tris(acetyl-
oxy)-1,1-dihydro-1,2-benziodoxol-3-(1H)-one; EDCI, N-(3-dimethyl-
aminopropyl)-N0-ethylcarbodiimide hydrochloride; EDTA, ethylene-
diaminetetracetic acid; HBSS, Hank’s buffered salt solution; HOBt,
hydroxybenzotriazole; ip, intraperitoneal; mGluR, metabotropic gluta-
mate receptor; MPEP, 2-methyl-6-(phenylethynyl)pyridine; 3H-mPEPy,
[3H]3-methoxy-5-(pyridine-2-ylethynyl)pyridine; MTEP, 3-[(2-methyl-
1,3-thiazol-4-yl)ethynyl]-pyridine; PBS, phosphate buffered saline; SAR,
structure-activity relationship; Xantphos, 4,5-bis(diphenylphosphino)-
9,9-dimethylxanthene.
Figure 1. Examples of small molecule mGluR5 negative modulators.
r
2010 American Chemical Society
Published on Web 09/01/2010
pubs.acs.org/jmc

7108 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19
Burdi et al.
unknown mechanism in 1982,9 was serendipitously discov-
ered to be an mGluR5 negative modulator during a high-
throughput screen of a corporate library.10 Other negative
modulators include MTEP (2),5 the acetylenic octahydroindole
3,11 and the diaryl azole 4.12 Our research program has been
directed toward the development of novel small-molecule
negative modulators of mGluR5. The present paper describes
the rational design, synthesis, biological profile, and structure-
activity relationships (SAR) of a novel class of compounds
containing bicyclic azole-amines as core elements.
A survey of known modulators of mGluR5 suggested a
pharmacophore that was most often described as two aryl
groups tethered by a variety of linkers including acetylenes,
azoles, and amides. We were particularly intrigued by the
saturated bicyclic amine linker exemplified by 3, and the
prospect of merging this attractive, potentially ionizable
feature with the biaryl azole pharmacophore exemplified by 4.
Several candidate molecules were synthesized, and oxazolo-
piperidine 5a (Figure 2) emerged as a novel allosteric modu-
lator (IC50 = 1.2 μM) whose physicochemical properties (e.g.,
molecular weight = 302; AlogP = 2.96) presented an appro-
priate starting point for SAR elucidation.
as shown in Scheme 2. Ethyl picolinimidate17 15 was heated
with benzyl 2-hydrazinyl-2-oxoethylcarbamate18 in refluxing
ethanol, giving rise to aminomethyl-triazole 16. Alkylation
with ethyl bromoacetate afforded 17, which was deprotected
and cyclized via Pd-catalyzed hydrogenation to yield 18.
Borane/THF reduction gave piperazine 19, which was
coupled with 3-bromo-5-fluorobenzonitrile to afford 20.
We finally turned our attention to the fused saturated ring.
To first examine the impact of ring contraction, oxazolopyr-
rolidine 21 was synthesized in a manner similar to the method
used for 5k in Scheme 1 but starting from Cbz-protected
3-amino-4-hydroxypyrrolidine.19
The ring-expanded oxazoloazepines were synthesized as
shown in Scheme 3. Bromination of tosyl-protected 4-piper-
idinone afforded 22, which was expanded via Eistert homo-
logation to azepine 23a. Hydrolysis and decarboxylation
yielded 23b, and bromide displacement with sodium azide
afforded 24. Reduction with lithium aluminum hydride
afforded amino-alcohol 25 as a mixture of isomers, which
were coupled with picolinic acid to afford 26. Oxidation
yielded 27, which was cyclized to 28a using Burgess’ reagent.
Deprotection was achieved using HBr to yield 28b; Buchwald
coupling with the appropriate bromide afforded nitriles 29a
and 29b.
Results and Discussion
Our initial efforts began with a broad survey of the SAR in
order to define the minimum pharmacophore and a path
forward. Table 1 shows a representative SAR of a series of
tetrahydrooxazolo[4,5-c]pyridines (5a-k) and the isomeric
tetrahydrooxazolo[5,4-c]pyridines (11a-k). The IC50 values
were measured in cells inducibly expressing the recombinant
human receptor, using a luminescence-based calcium mobili-
zation assay.20
As mentioned earlier, the initial hit 5a showed modest
potency (IC50 = 1.2 μM) that was comparable with its iso-
meric counterpart 11a (see Table 1). Replacement of the
phenyl ring on the left-hand side with a 2-pyridyl group as
seen in 5b and 11b (IC50s = 0.11 and 0.64 μM, respectively)
improved potency by an order of magnitude. Alkyl group
replacements, as exemplified by 5c and 11c, eliminated acti-
vity. Cycloakyl substituents, as shown in 11d (79% at 10 μM),
retained some potency but were clearly inferior to their
2-pyridylcounterparts. Saturationof the 2-pyridylsubstituent
afforded 11h, prepared as a racemic mixture, which showed
modest, but greatlyreducedpotency relative toitsunsaturated
counterpart. Interestingly, the 3- and 4-pyridyl isomers 5j and
11j were inactive relative to their 2-pyridyl counterparts 5i
and 11i; these stark contrasts in potencies served as testimony
to the steepness of the SAR that we encountered on this side
of the molecule. We concluded from these initial studies that a
Figure 2. Structure of the initial hit 5a.
Chemistry
The syntheses of 5a and initial analogues are described
in Scheme 1. The tetrahydrooxazolo[5,4-c]pyridine isomers
11a-k (see Table 1) were prepared in a manner similar to that
shown in Scheme 1 starting from the isomeric Cbz-protected
4-amino-3-piperidinol.13 Cbz-protected amino alcohol 613
was coupled with the appropriate carboxylic acid to afford
7. Oxidation with Dess-Martin periodinane yielded the
ketone 8, which was cyclized using phosphorus oxychloride
to afford oxazolo-piperidine 9. Deprotection of the Cbz group
to afford 10 was achieved using either catalytic hydrogenation
or refluxing HBr. Alkylation of the amine to form 5 with
activated halides (such as 2-chloropyridine) could be realized
using an organic base such as diisopropylethylamine, while
couplings with less activated halides were achieved using
Buchwald chemistry.
We next turned our attention to optimization of the central
azole ring. Imidazole 12b,14,15 thiazole 13,15 and bridgehead
imidazole 1416 (see Table 2) were prepared using known pro-
cedures. The bridgehead triazolopyrazine 20 was synthesized
Scheme 1a
a Reagents: (a) R1COOH/EDCI; (b) Dess-Martin periodinane; (c) POCl3 Δ; (d) H2/Pd(OH)2 or HBr, Δ; (e) R2X/DIEA or R2X/Pd2(dba)3/NaOt-Bu

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19 7109
Table 2. In Vitro Potencies of Tetrahydroazolopyridine Analoguesa
Table 1. In Vitro Potencies of Tetrahydrooxazolopyridine Analogues
as mGluR5 Negative Modulatorsa
compd
X
Y
Z
IC50 (μM)
0.028
0.054
5k
O
N
N
N
C
C
C
C
C
N
N
N
11k
12b
13
O
NH
S
34% at 10 μM
52% at 10 μM
0.153
14
20
N
N
N
0.346
a mGluR5 assay data are presented as the mean of two determina-
tions. Assay reproducibility is monitored by the use of the mGluR5
negative modulators MPEP (IC50 = 15 ( 5 nM) and 1 (IC50 = 106 (
30 nM).
2-pyridyl substituent was preferred on the left-hand side of the
molecule, and this substituent was carried into the next phase
of SAR development.
We next turned our attention to the right-hand side of the
molecule, where preference for substituted aryl rings was
quickly established. Recapitulation of the carbamate func-
tionality in 3, exemplified by the synthesis of 5f, led to a loss of
activity. Saturation of the phenyl substituent in 5g (IC50
=
0.33 μM) led to 5d, which was devoid of activity, as was
the saturated amine 5h. Alkyl substituents, exemplified by the
isopropyl-substituted 5e, were inactive. The linker region
between the central and terminal rings did not tolerate
homologation. Sulfonamides such as11f were inactive; homo-
logation of the linker in 11i gave rise to benzylic amine 11g,
which showed a steep drop in potency. Finally, insertion of a
carbonyl group in the linker region, as exemplified by carbox-
amide 11e (IC50 = 2.6 μM), gave compounds with greatly
reduced potency relative to their lower homologues.
To complete the initial survey of SAR, we addressed the
preference for oxazole isomers in the central ring. A compari-
son of oxazole isomers 5b (IC50 = 0.11 μM) and 11b (IC50
0.64 μM) and isomers 5i (IC50 = 0.06 μM) and 11i (IC50
=
=
0.29 μM) revealed a small but consistent preference for the
[4,5-c] isomer. This trend was further confirmed by the
addition of a fluorine substituent to the 3-position of the
benzonitrile, which afforded 5k (IC50 = 0.028 μM) and 11k
(IC50 = 0.054 μM). From these initial efforts, compound 5k
emerged as a lead compound and was selected for further
optimization.
The SAR of the core-modified tetrahydroazolopyridine
analogues is summarized in Table 2. Compound 5k, which
depicts the favored [4,5-c] orientation of the oxazole, and
[5,4-c]-oxazole 11k, are included for comparison. The pro-
foundstructuralsensitivity of the coreheterocycle isevidenced
by the dramatic loss of potency seen in imidazole 12b and
thiazole 13. Although some activity was retained in the iso-
meric imidazopiperazine 14 and triazolopiperazine 20, these
analogues were inferiorto5kby nearly an order of magnitude.
We concluded that the substitution and orientation evoked by
[4,5-c]-oxazole 5k was optimal for activity.
The in vitro potencies of saturated ring variants (Table 3)
show a trend to higher potency with increased ring size, with
similar potencies seen in the six- and seven-membered rings.
Homologation of pyrrolidine 21 to piperidine 5i led to a 4-fold
increase in potency; substitution of fluorine for hydrogen at
the meta-position, resulting in 5k, gave the expected boost in
a mGluR5 assay data are presented as the mean of two determina-
tions. Assay reproducibility is monitored by the use of the mGluR5
negative modulators MPEP (IC50 = 15 ( 5 nM) and 1 (IC50 = 106 ( 30
nM). b Racemic mixture. c Measured using [glutamate] at 80% of its
saturating concentration.

7110 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19
Burdi et al.
Scheme 2a
a Reagents: (a) benzyl 2-hydrazinyl-2-oxoethylcarbamate/EtOH/Δ; (b) ethyl bromoacetate/KOt-Bu/DMF; (c) H2/10%Pd/C/AcOH; (d) BH3 THF;
(e) 3-bromo-5-fluorobenzonitrile/Pd2(dba)3/Xantphos/Cs2CO3.
3
Scheme 3a
a Reagents: (a) Br2/DCM; (b) ethyl diazoacetate/BF3 Et2O; (c) 3N HCl/Δ; (d) NaN3/DMF/AcOH; (e) LAH/THF; (f) picolinic acid/EDCI/DCM;
3
(g) Dess-Martin reagent/DCM; (h) Burgess’ Reagent, THF, Δ; (i) 48% HBr/Δ; (j) 3-bromo-5-f luorobenzonitrile or 3-bromobenzonitrile/Pd2(dba)3/
NaOt-Bu
Table 3. The Effect of Saturated Ring Size on in Vitro Potenciesa
To identify any off-target activities, 5k and 29b were pro-
filed extensively against a battery of in vitro assays including
mGluR1 at test compound concentrations of 10 μM. Of 55
receptors screened (Cerep ExpresSProfile), only a weak, but
detectable activity at the κ-opioid receptor (77%I @ 10 μM)
was detected for 29b.
Binding studies in vitro showed that both 5k and 29b
interact with the allosteric modulation site of mGluR5 origi-
compd
n
X
IC50 (μM)
nally identified as the binding site of MPEP (Table 4).21 For
thesetwocompoundsandseveralanalogues tested (additional
data not shown), binding affinities for this site with rat brain
andhumanrecombinant mGluR5wereverycloselycorrelated
with the potencies for functional inhibition of recombinant
human mGluR5.
21
0
1
1
2
2
H
H
F
0.219
0.057
0.028
0.065
0.016
5i
5k
29a
29b
H
F
a mGluR5 assay data are presented as the mean of two determina-
tions. Assay reproducibility was monitored by the use of the mGluR5
negative modulators MPEP (IC50 = 15 ( 5 nM) and 1 (IC50 = 106 (
30 nM).
The good correlation observed between binding affinity
and functional potency prompted us to employ a single-dose
receptor occupancy assay in order to rapidly assess brain
penetrance and in vivo affinity of molecules of interest.
Briefly, male Sprague-Dawley rats were dosed at time zero
with the test compound. After 60 min, 3H-mPEPy was
administered via tail-vein injection, and the animals were
sacrificed 2 min later. Binding of the test compound to
mGluR5 receptors in striatum and cerebellum from dose rats
potency. Addition of a second methylene unit led to azepines
29a and 29b, where we were pleased to achieve an in vitro
potency of 16 nM. On the basis of their in vitro potencies, 29b
and its six-membered ring counterpart 5k were selected for
further study and characterization.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19 7111
Table 4. Receptor Occupancies and Exposures for mGluR5 Negative Modulators 5k and 29b
compd
human mGluR5 IC50 (nM)a
rat mGluR5 Ki (nM)b
%ROc
plasma levels (nM)d
brain levels (nM)d
RO50 (mpk)
5k
29b
28
16
17
17
45
82
2486 ( 959
1433 ( 494
1293 ( 431
1096 ( 216
>30e
0.9 f
a mGluR5 assay data are presented as the mean of two determinations. Assay reproducibility is monitored by the use of the mGluR5 negative
modulators MPEP (IC50 = 15 ( 5 nM) and 1 (IC50 = 106 ( 30 nM). b Binding assay for displacement of [3H]-MPEP from rat cortical membranes in
vitro, represented as the mean of two determinations. Assay reproducibility is monitored by the use of the mGluR5 negative modulator MPEP (Ki =
10 ( 1 nM) c Receptor occupancy in vivo following 3 mpk dose administered ip. d Average of four rats/cohort. e Dose-response measurements
conducted at 0.3, 1, 3, 10, and 30 mpk po. f Dose-response measurements conducted at 0.3, 1, 3, 10, and 30 mpk ip.
Table 5. Pharmacokinetic Parameters of 29b in Rata and Monkeyb
species (dose)
AUC0-t (μM h)
T1/2 (h)
Vd (L/kg)
CL (L/h/kg)
Cmax (μM)
F (%)
3
rat (2 mpk iv)
rat (2 mpk po)
2.26 ( 0.69
0.70 ( 0.25
3.36 ( 1.39
3.06 ( 0.48
0.28 ( 0.16
0.62 ( 0.18
0.77 ( 0.08
0.91 ( 0.19
0.67 ( 0.05
0.63 ( 0.19
2.38 ( 0.50
N/A
N/A
2.79 ( 0.88
N/A
N/A
5.25 ( 1.53
0.62 ( 0.20
2.73 ( 1.04
5.08 ( 1.31
0.21 ( 0.06
N/A
31
rat (10 mpk po)
monkey (1 mpk iv)
monkey (1 mpk po)
30
0.95 ( 0.08
N/A
0.98 ( 0.14
N/A
N/A
10
a Male Sprague-Dawley rats (three animals per cohort). b Male Rhesus monkeys (three animals per cohort).
was quantified by measuring decreases in 3H-mPEPy bound,
which was measured by a digital radiographic imager. Table 4
contains a detailed comparison of 5k and 29b. Both com-
pounds showed good brain penetration and similar brain/
plasma ratios. Azepine 29b exhibited an occupancy ED50 of
0.9 mg/kg when administered intraperitoneally.
Encouraged by the results for 29b, we determined the
duration of occupancy following a 3 mpk ip dose; the results
are displayed in Figure 3. The rapid brain penetrance of 29b is
evidenced by the 85% receptor occupancy recorded at 30 min
postdose. This level of occupancy persisted at 1 h, declined to
55% occupancy at 2 h, and ultimately returned to baseline
24 h postdose. The plasma pharmacokinetics of 29b were also
characterized to allow a more detailed comparison with the
duration of action.
and have resulted in oxazolo-azepine 29b. This molecule
displays excellent in vitro potency and selectivity, good brain
penetration, and robust receptor occupancy in rodent. Des-
pite its acceptable oral bioavailability in rodent, its half-life
in both rodent and primate is short. In view of recent reports
highlighting the need for high sustained receptor occupancies
in order to achieve efficacy in animal models of levodopa-
induced dyskinesia,22 the short half-life seen in 29b represents
a significant hurdle to development, and a series of corrective
actions have been undertaken to impede first-pass clearance
via metabolic blockade.
Experimental Section
Unless otherwise noted, all materials were obtained from
commercial suppliers and used without further purification. An-
hydrous solvents were purchased from Aldrich and used directly.
All reactions involving air- or moisture-sensitive reagents were
performed under a nitrogen atmosphere. Compounds were puri-
fied using preparative thin layer chromatography or medium
pressure liquid chromatography on a CombiFlash Companion
(Teledyne Isco) with RediSep normal-phase silica gel (230-400
mesh) and UV detection at 254 nm. The purity of all final
compounds was determined to be g95% by HPLC unless other-
wise indicated. The purities of intermediates and final compounds
were determined by integration of the UV signal obtained by
analytical LC/MS on an Agilent 1100 series instrument equipped
with a diode array detector and a Waters Micromass spectro-
1
meter. H NMR data were determined with a Varian 400 MHz
Figure 3. Duration of occupancy of 29b in rat following a 3 mpk ip
dose.
spectrometer at ambient temperature and are reported in the form
of delta (δ) values given in parts per million (ppm) relative to
tetramethylsilane (TMS) as an internal standard. Conventional
abbreviations used for signal shape are: s, singlet; d, doublet; t,
triplet; q, quartet; m, multiplet; br, broad.
The pharmacokinetic parameters for 29b in both rat and
monkey, shown in Table 5, were disappointing. Despite its
reasonable oral bioavailability in rat (30%), 29b exhibited a
half-life of less than 1 h. The short half-life persisted in higher
species (monkey) and was likely the major contributor to the
poor bioavailability (10%) seen in this species. A high rate of
hepatic microsomal oxidation was identified as the culprit in
both species (data not shown), and a major campaign was
undertaken to identify and block the site(s) of metabolism.
The results of these efforts will be reported in due course.
2-(2-Phenyl-6,7-dihydrooxazolo[4,5-c]pyridin-5(4H)-yl)nico-
tinonitrile (5a). 2-Phenyl-4,5,6,7-tetrahydro[1,3]oxazolo[4,5-c]pyr-
idine hydrochloride (purchased from Anichem) (200 mg, 0.84
mmol), 2-chloro-3-pyridine-carbonitrile (176 mg, 1.27 mmol),
and DIEA (218 mg, 1.69 mmol) were combined in DMF
(1.5 mL) and heated under microwave at 100 ꢀC for 40 min.
The solvent was concentrated and removed under vacuum. The
residue was then purified by silica gel column (EtOAc (10%
MeOH)/hexanes), followed by HPLC purification to give a
white solid (62 mg, 24%). 1H NMR (400 MHz, CDCl3): δ
8.36 (dd, J1 = 5.2 Hz, J2 = 1.9 Hz, 1H), 8.01 (m, 2H), 7.83 (dd,
J1 = 7.8 Hz, J2 = 1.8 Hz, 1H), 7.45 (m, 3H), 6.78 (dd, J1 = 7.8
Hz, J2 = 4.8 Hz, 1H), 4.73 (s, 2H), 4.10 (t, J = 5.4 Hz, 2H), 3.09
(m, 2H). LC/MS: m/e = 303 (M þ H)þ. HPLC purity: 98%.
Conclusions
A novel set of diaryl bicyclic amines have been identified.
The substituent effects on the terminal aryl rings, the central
azole ring, and the fused saturated ring have been elucidated

7112 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19
Burdi et al.
2-(2-(Pyridin-2-yl)-6,7-dihydrooxazolo[4,5-c]pyridin-5(4H)-yl)-
nicotinonitrile (5b). To a solution of 2-pyridin-2-yl-4,5,6,7-tetra-
hydro-oxazolo[4,5-c]-pyridine (60 mg, 0.3 mmol) and DIEA
(77 mg, 0.6 mmol) in DMF (2 mL) was added 2-chloro-nicoti-
nonitrile (62 mg, 0.45 mmol). The mixture was heated to 100 ꢀC
overnight. The mixture was partitioned between EtOAc and
H2O, and the organic layer dried over MgSO4. The crude
product was purified by preparative HPLC to afford 2-(2-
pyridin-2-yl-6,7-dihydro-4H-oxazolo[4,5-c]pyridin-5-yl)-nicoti-
nonitrile (1.5 mg, 2%). 1H NMR (400 MHz, CDCl3): δ 8.69 (t,
J = 1.6 Hz, 1H), 8.31 (dd, J1 = 4.8 Hz, J2 = 2.0 Hz, 1H), 8.08 (d,
J = 8.0 Hz, 1H), 7.84 (m, 1H), 7.76 (dd, J1 = 7.6 Hz, J2 = 2.0
Hz, 1H), 7.35 (t, J = 7.2 Hz, 1H), 6.76 (dd, J1 = 7.6 Hz, J2 = 4.4
Hz, 1H), 4.69 (s, 2H), 4.06 (t, J = 5.2 Hz, 2H), 3.08 (t, J = 5.2
Hz, 2H). LC/MS: m/e = 304 (M þ H)þ. HPLC purity: 100%.
2-(2-Methyl-6,7-dihydrooxazolo[4,5-c]pyridin-5(4H)-yl)nico-
tinonitrile (5c). 2-Methyl-4,5,6,7-tetrahydro-oxazolo[4,5-c]pyr-
idine hydrochloride (purchased from Anichem) (200 mg, 1.14
mmol), 2-chloro-3-pyridine-carbonitrile (317 mg, 2.29 mmol),
and DIEA (444 mg, 3.43 mmol) were combined in DMF (1.0 mL)
and heated under in a microwave reactor at 160 ꢀC for 1.5 h.
The solvent was removed under vacuum, and the residue was
purified by silica gel column (EtOAc (10%MeOH)/hexanes) to
give a yellow oil (245 mg, 89%). 1H NMR (400 MHz, CDCl3): δ
8.34 (dd, J1 = 4.8 Hz, J2 = 2 Hz, 1H), 7.78 (dd, J1 = 7.6 Hz,
J2 = 2.2 Hz, 1H), 6.76 (dd, J1 = 7.6 Hz, J2 = 4.8 Hz, 1H), 4.59
(m, 2H), 4.02 (t, J = 5.4 Hz, 2H), 2.95 (m, 2H), 2.44 (s, 3H). LC/
MS: m/e = 241 (M þ H)þ. HPLC purity: 95%.
(46 mg, 0.05 mmol), 2,20-bis(diphenylphosphino)-1,10-binaph-
thyl (111 mg, 0.18 mmol), and bromobenzene (246 mg, 1.57 mmol)
were combined in toluene (4 mL). The mixture was flushed
with nitrogen, and sodium t-butoxide (275 mg, 2.86 mmol) was
added. The mixture was flushed again with nitrogen and then
heated at 80 ꢀC for 24 h. The reaction was filtered through
Celite, and the solvent evaporated in vacuo. The crude material
was purified by reverse phase chromatography (NH4HCO3/
acetonitrile) to give a brown solid (70 mg, 35%). 1H NMR
(400 MHz, CDCl3): δ 8.71 (s, 1H), 7.98 (d, J = 8.0 Hz, 1H), 7.81
(t, J = 8.0 Hz, 1H), 7.40 (t, 1H), 7.26-7.32 (m, 2H), 7.00 (d, J =
8.0 Hz, 2H), 6.88 (t, J = 8.0 Hz, 1H), 4.30 (s, 2H), 3.70 (s, 2H),
2.97 (s, 2H). LC/MS: m/e=278 (MþH)þ. HPLC purity: 100%.
5-Piperidin-4-yl-2-pyridin-2-yl-4,5,6,7-tetrahydro- oxazolo-
[4,5-c]pyridine (5h). To a solution of 2-pyridin-2-yl-4,5,6,7-tetra-
hydro-oxazolo[4,5-c]pyridine (200 mg, 1 mmol) in MeOH (15
mL) was added NaBH3CN (188 mg, 3 mmol) at 0 ꢀC. After
stirring for 10 min, a solution of 4-oxo-piperidine-1-carboxylic
acid tert-butyl ester (597 mg, 03 mmol) in MeOH (5 mL) was
added to the above mixture. The reaction was stirred for 10 h at
room temperature. The reaction was concentrated to dryness
and partitioned between EtOAc and H2O. The organic layer was
separated and dried over Na2SO4. Concentration afforded
crude product, which was purified by prep-TLC to afford
4-(2-pyridin-2-yl-6,7-dihydro-4H-oxazolo[4,5-c]pyridin-5-yl)-
1
piperidine-1-carboxylic acid tert-butyl ester (87 mg, 20%). H
NMR (400 MHz, CDCl3): δ 8.63 (d, J = 4.4 Hz, 1H), 7.99 (d,
J = 4.0 Hz, 1H), 7.71 (t, J = 2.0 Hz, 1H), 7.27 (t, J = 4.8 Hz,
1H), 4.15 (m, 2H), 3.66 (s, 2H), 2.93 (m, 2H), 2.83-2.90 (m, 2H),
2.62-2.70 (m, 3H), 1.83 (m, 2H), 1.40 (s, 9H). HPLC purity:
100%.
To a solution of 4-(2-pyridin-2-yl-6,7-dihydro-4H-oxazolo-
[4,5-c]pyridin-5-yl)-piperidine-1- carboxylic acid tert-butyl ester
(50 mg, 0.13 mmol) in DCM (2 mL) was added TFA (3 mL) at
room temperature The mixture was stirred for 4 h at rt. The
solution was concentrated to give the product 5-piperidin-4-yl-
2-pyridin-2-yl-4,5,6,7-tetrahydro-oxazolo[4,5-c]pyridine 5h (40
mg, 71%) as its TFA salt.
5-Cyclohexyl-2-(pyridin-2-yl)-4,5,6,7-tetrahydrooxazolo[4,5-c]-
pyridine (5d). To a solution of 2-(pyridin-2-yl)-4,5,6,7-tetra-
hydrooxazolo[4,5-c]pyridine (100 mg, 0.50 mmol) in DCM
(10 mL) was added cyclohexanone (50 mg, 0.50 mmol) and
acetic acid (90 mg, 1.5 mmol). After stirring for 20 min, NaBH-
(OAc)3 (420 mg, 2.00 mmol) was added and the mixture was
stirred for an additional 2 h. The solvent was removed in vacuo,
and the residue was purified using reverse phase chromatogra-
phy to afford the desired product (90 mg, 60%) as a white solid.
1H NMR (400 MHz, CDCl3): δ 8.69 (m,1H), 8.07 (d, J = 8.0 Hz,
1H), 7.79 (m, 1H), 7.32 (m, 1H), 3.70 (m, 2H), 2.97 (m, 2H), 2.87
(m, 2H), 2.58 (m, 1H), 1.93 (m, 2H), 1.83 (m, 2H), 1.68 (m, 1H),
1.31 (m, 4H), 1.16 (m, 1H). LC/MS: m/e = 284 (MþH)þ. HPLC
purity: 98%.
5-Isopropyl-2-(pyridin-2-yl)-4,5,6,7-tetrahydrooxazolo[4,5-c]-
pyridine (5e). 2-(Pyridin-2-yl)-4,5,6,7-tetrahydrooxazolo[4,5-c]-
pyridine (200 mg, 0.71 mmol), sodium cyanoborohydride (45
mg, 0.71 mmol), acetone (2 mL), and acetic acid (2 drops) were
combined in DCM (2 mL) and stirred at room temperature for
2 days. Water (10 mL) was added, and the mixture was extracted
with EtOAc. The aqueous layer was concentrated in vacuo to a
volume of 1.5 mL and purified by reverse phase chromatogra-
phy to give a yellow oil (11 mg, 7%). 1H NMR (400 MHz,
CDCl3): δ 8.68 (d, 1H), 8.06 (d, J = 8.0 Hz, 1H), 7.78 (t, J = 8.0
Hz, 1H), 7.32 (m, 1H), 3.65 (s, 2H), 3.02 (m,1H), 2.88 (m, 4H),
1.15 (d, 6H). LC/MS: m/e = 244 (M þ H)þ. HPLC purity:
100%.
1H NMR (400 MHz, CDCl3 þ MeOD). δ 8.58 (s, 1H), 7.97 (d,
J = 8.0 Hz, 1H), 7.80 (t, J = 7.2 Hz, 1H), 7.30-7.37 (m, 1H),
4.29 (s, 2H), 3.72 (m, 1H), 3.60 (s, 2H), 3.45 (d, J = 12.8 Hz, 2H),
3.23 (d, J = 1.6 Hz, 1H), 3.16 (s, 2H), 3.01 (t, J = 12.8 Hz, 2H),
2.26 (d, J = 11.6, 2H), 2.10-2.20 (m, 2H).. LC/MS: m/e = 285
(M þ H)þ. HPLC purity: 99%.
3-(2-(Pyridin-2-yl)-6,7-dihydrooxazolo[4,5-c]pyridin-5(4H)-yl)-
benzonitrile (5i). To a solution of 2-(pyridin-2-yl)-4,5,6,7-tetra-
hydrooxazolo[4,5-c]pyridine (20 mg, 0.1 mmol) in toluene
(2 mL) was added 3-bromobenzonitrile (27 mg, 0.15 mmol),
Cs2CO3 (65 mg, 0.2 mmol), Pd(OAc)2 (1 mg, cat.), and Xant-
phos (2 mg, cat.). The reaction was heated to 100 ꢀC and stirred
overnight. The mixture was dissolved in MeOH and filtered.
The filtrate was concentrated, and the residue was purified by
preparative HPLC to afford 3-(2-(pyridin-2-yl)-6,7-dihydro-
oxazolo[4,5-c]pyridin-5(4H)-yl)benzonitrile (5 mg, 17%) as a
yellow solid. 1H NMR (MeOH-d4): δ 8.65 (d, J = 4.4 Hz, 1H),
8.15 (d, J = 8.0 Hz, 1H), 8.05 (t, J = 6.0 Hz, 1H), 7.50-7.60 (m,
1H), 7.30-7.41 (m, 3H), 7.11 (d, J = 5.2 Hz, 1H), 4.31 (s, 2H),
3.80 (t, J = 5.6 Hz, 2H), 2.99 (t, J = 5.6 Hz, 2H). LC/MS: m/e =
303 (M þ H)þ. HPLC purity: 96%
Methyl 2-(Pyridin-2-yl)-6,7-dihydrooxazolo[4,5-c]pyridine-
5(4H)-carboxylate (5f). To a solution of 2-(pyridin-2-yl)-4,5,6,7-
tetrahydrooxazolo[4,5-c]pyridine (100 mg, 0.50 mmol) in THF
(10 mL) was added methyl carbonochloridate (50 mg, 0.50 mmol)
and triethylamine (100 mg, 1.00 mmol). The mixture was stirred
at room temperature for 2 h. The solvent was evaporated in
vacuo, and the residue was purified using reverse phase chro-
matography to give the desired product (75 mg, 60%) as a white
solid. ESI (m/e) (M þ H)þ: 260.0. 1H NMR (400 MHz, CDCl3):
δ 8.69 (m, 1H), 8.09 (d, J = 8.0 Hz, 1H), 7.84 (m, 1H), 7.27 (m,
1H), 4.55 (m, 2H), 3.90 (m, 2H), 3.76 (s, 3H), 2.86 (m, 2H). LC/
MS: m/e = 260 (M þ H)þ. HPLC purity: 97%.
3-(2-(Pyridin-4-yl)-6,7-dihydrooxazolo[4,5-c]pyridin-5(4H)-yl)-
benzonitrile (5j). The title compound was prepared via the pro-
cedure used for 5i, substituting pyridine-4-carboxylic acid for
picolinic acid. Preparative HPLC afforded 3-(2-(pyridin-4-yl)-
6,7-dihydrooxazolo[4,5-c]pyridin-5(4H)-yl)benzonitrile (10 mg,
13%). 1H NMR (400 MHz, CDCl3): δ 8.78 (d, J = 6.4 Hz, 2H),
8.12 (d, J = 6.4 Hz, 2H), 7.28-7.33 (m, 1H), 7.07-7.13 (m, 3H),
4.29 (s, 2H), 3.72 (t, J = 5.6 Hz, 2H), 2.93-3.00 (t, J = 5.2 Hz,
2H). LC/MS: m/e = 303 (M þ H)þ. HPLC purity: 100%.
3-Fluoro-5-(2-(pyridin-2-yl)-6,7-dihydrooxazolo[4,5-c]pyridin-
5(4H)-yl)benzonitrile (5k). The title compound was prepared via
5-Phenyl-2-(pyridin-2-yl)-4,5,6,7-tetrahydrooxazolo[4,5-c]pyr-
idine (5g). 2-(Pyridin-2-yl)-4,5,6,7-tetrahydrooxazolo[4,5-c]pyr-
idine (202 mg, 0.72 mmol), tris (dibenzylideneacetone)dipalladium(0)

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19 7113
the procedure used for 5i, substituting 3-bromo-5-fluorobenzo-
nitrile for 3-bromobenzonitrile. Preparative HPLC afforded
3-fluoro-5-(2-(pyridin-2-yl)-6,7-dihydrooxazolo[4,5-c]pyridin-
5(4H)-yl)benzonitrile (30 mg, 18%) as a yellow solid. 1H NMR
(400 MHz, CDCl3): δ 8.74 (d, 1H), 8.12 (m, 1H), 7.91-7.96 (m,
1H), 7.45-7.48 (m, 1H), 6.91 (s, 1H), 6.72-6.79 (m, 2H), 4.29 (s,
2H), 3.71 (m, 2H), 2.94 (m, 2H). LC/MS: m/e = 321 (M þ H)þ.
HPLC purity: 98%.
4-Hydroxy-3-[(pyridine-2-carbonyl)-amino]-piperidine-1-car-
boxylic acid benzyl ester (7). To a solution of pyridine-2-
carboxylic acid (492 mg, 4 mmol) and TEA (1.21 g, 12 mmol)
in DCM (25 mL) was added EDCI (1.53 g, 8 mmol) and HOBt
(1.08 g, 8 mmol). (()-trans-3-Amino-4-hydroxy-piperidine-1-
carboxylic acid benzyl ester 611 (1.00 g, 4 mmol) in DCM (5 mL)
was added to the above mixture, and the reaction was stirred
overnight at room temperature. The mixture was partitioned
between DCM and water, and the organic layer was washed
with aq NaHCO3, 1N HCl solution, and brine. The organic
phase was dried over anhydrous Na2SO4 and concentrated to
afford 4-hydroxy-3-[(pyridine-2-carbonyl)-amino]-piperidine-
1-carboxylic acid benzyl ester (1.2 g, 84%) as a white solid. 1H
NMR (400 MHz, CDCl3): δ 8.46 (m, 1H), 8.12 (m, 2H), 7.80 (m,
1H), 7.38 (m, 1H), 7.29 (m, 4H), 5.06 (m, 2H), 4.16 (m, 1H), 3.88
(m, 2H), 3.76 (m, 1H), 3.12 (m, 2H), 1.98 (m, 2H), 1.58 (m, 2H).
LC/MS: m/e = 356 (M þ H)þ. HPLC purity: 94%.
mL) and heated under microwaves at 160 ꢀC for 1.5 h. The
solvent was concentrated and removed under vacuum. The
residue was then purified by silica gel column (EtOAc (10%
MeOH)/hexanes), followed by recrystallization in ethanol to
give a white solid (54 mg, 21%). 1H NMR (400 MHz, CDCl3): δ
8.37 (dd, J1 = 5.0 Hz, J2 = 2.0 Hz, 1H), 8.02 (m, 2H), 7.80 (dd,
J1 = 8.0 Hz, J2 = 2.0 Hz, 1H), 7.38-7.46 (m, 3H), 6.78 (dd,
J1 = 7.4 Hz, J2 = 5.2 Hz, 1H), 4.73 (s, 2H), 4.10 (t, J = 5.6 Hz,
2H), 3.09 (m, 2H). LC/MS: m/e = 303 (M þ H)þ. HPLC purity:
100%.
2-(2-(Pyridin-2-yl)-6,7-dihydrooxazolo[5,4-c]pyridin-5(4H)-yl)-
nicotinonitrile (11b). To a solution of 2-(pyridin-2-yl)-4,5,6,7-
tetrahydrooxazolo[5,4-c]pyridine (100 mg, 0.5 mmol) in DMF
(5 mL) was added 2-chloronicotinonitrile (140 mg, 1 mmol) and
DIEA (140 mg, 1 mmol). The mixture was heated to 100 ꢀC and
stirred for 6 h. The mixture was cooled to room temperature,
poured into water, and extracted with EtOAc. The organic
phase was concentrated, and the residue was purified by pre-
parative TLC to afford 2-(2-(pyridin-2-yl)-6,7-dihydrooxazolo-
[5,4-c]pyridin-5(4H)-yl)nicotinonitrile (20 mg, 13%) as a yellow
solid. 1H NMR (400 MHz, CDCl3): δ 8.65 (s, 1H); 8.30 (dd, J1 =
5.2 Hz, J2 = 2.0 Hz, 1H), 8.07 (d, J = 7.6 Hz, 1H), 7.78 (m, 2H),
7.30 (t, J = 6.0 Hz, 1H), 6.75 (q, J = 4.8 Hz, 1H), 4.81 (s, 2H),
4.03 (t, J = 5.6 Hz, 2H), 2.92 (t, J = 5.2 Hz, 2H). LC/MS: m/e =
304 (M þ H)þ. HPLC purity: 94%.
4-Oxo-3-[(pyridine-2-carbonyl)-amino]-piperidine-1-carboxy-
lic Acid Benzyl Ester (8). To a solution of 4-hydroxy-3-[(pyr-
idine-2-carbonyl)-amino]-piperidine-1-carboxylic acid benzyl ester
(500 mg, 1.4 mmol) in DCM (10 mL) was added Dess-Martin
reagent (656 mg, 2.8 mmol). The mixture was stirred overnight
at room temperature. The reaction was partitioned between
0.5N NaOH and DCM. The organic phase was dried over
anhydrous Na2SO4 and concentrated to afford crude product.
Purification by preparative TLC (PT/EtOAc = 1:1) afforded
4-oxo-3-[(pyridine-2-carbonyl)-amino]-piperidine-1-carboxylic
acid benzyl ester (400 mg, 81%). 1H NMR (400 MHz, CDCl3): δ
8.54 (m, 1H), 8.14 (m, 1H), 7.80 (m, 1H), 7.22-7.40 (m, 6H),
5.10-5.30 (m, 2H), 4.96 (m, 1H), 4.69 (m, 1H), 4.48 (m, 1H),
4.48 (m, 1H), 2.85 (m, 1H), 2.62 (m, 2H). LC/MS: m/e = 354
(M þ H)þ. HPLC purity: 95%.
3-(2-Isopropyl-6,7-dihydrooxazolo[5,4-c]pyridin-5(4H)-yl)ben-
zonitrile (11c). The title compound was prepared in a manner
similar to 11i starting from isobutyric acid and (()-trans-4-
amino-3-hydroxy-piperidine-1-carboxylic acid benzyl ester.
Purification using reverse phase chromatography afforded the
1
desired compound (58 mg, 58%) as a yellowish oil. H NMR
(400 MHz, CDCl3): δ 7.35 (m, 1H), 7.13 (m, 3H), 4.29 (m, 2H),
3.64 (m, 2H), 3.08 (m, 1H), 2.73 (m, 2H), 1.35 (d, J = 7.2 Hz,
6H). LC/MS: m/e = 286 (M þ H)þ. HPLC purity: 100%.
3-(2-cyclohexyl-6,7-dihydrooxazolo[5,4-c]pyridin-5(4H)-yl)-
benzonitrile (11d). The title compound was prepared in a manner
similar to 11i starting from cyclohexanecarboxylic acid and (()-
trans-4-amino-3-hydroxy-piperidine-1-carboxylic acid benzyl
ester. Purification using reverse phase chromatography afforded
the desired compound (80 mg, 53%) as colorless oil. 1H NMR
(400 MHz, CDCl3): δ 7.34 (m, 1H), 7.10 (m, 3H), 4.29 (m, 2H),
3.64 (m, 2H), 2.73 (m, 3H), 2.07 (m, 2H), 1.83 (m, 2H), 1.55-1.75
(m, 5H), 1.33 (m, 3H). LC/MS: m/e = 308 (M þ H)þ. HPLC
purity: 100%.
3-(2-(Pyridin-2-yl)-4,5,6,7-tetrahydrooxazolo[5,4-c]pyridine-
5-carbonyl)benzonitrile (11e). To a solution of 2-(pyridin-2-yl)-
4,5,6,7-tetrahydrooxazolo[5,4-c]pyridine (50 mg, 0.248 mmol)
in DCM (5 mL) was added 3-cyanobenzoyl chloride (45 mg,
0.273 mmol) and TEA (38 mg, 0.372 mmol) in DCM. The
mixture was stirred at room temperature for 1 h. The reaction
was concentrated and purified by preparative TLC to afford
3-(2-(pyridin-2-yl)-4,5,6,7-tetrahydrooxazolo[5,4-c]pyridine-5-
carbonyl)benzonitrile (13 mg, 14%). 1H NMR (400 MHz,
CDCl3): δ 8.65 (s, 1H), 8.04 (d, J = 8.0 Hz, 1H), 7.78-7.75
(m, 1H), 7.72-7.70 (m, 2H), 7.65 (d, J = 8.0 Hz, 1H), 7.54 (t,
J = 8.0 Hz, 1H), 7.30 (dd, J1 = 6.8 Hz, J2 = 4.4 Hz, 1H), 4.85 (s,
2H), 3.64 (s, 2H), 2.74 (s, 2H). LC/MS: m/e = 331 (M þ H)þ.
HPLC purity: 92%.
3-(2-(Pyridin-2-yl)-6,7-dihydrooxazolo[5,4-c]pyridin-5(4H)-
ylsulfonyl)benzonitrile (11f). To a solution of 2-(pyridin-2-yl)-
4,5,6,7-tetrahydrooxazolo[5,4-c]pyridine (60 mg, 0.3 mmol) in
dry DCM (10 mL) was added TEA (42 μL, 0.3 mmol) and
3-cyanobenzene-1-sulfonyl chloride (75 mg, 0.37 mmol). The
reaction was stirred overnight at room temperature. The solvent
was removed in vacuo and the residue purified by prep-TLC to
afford 3-(2-(pyridin-2-yl)- 6,7-dihydrooxazolo[5,4-c]pyridin-
5(4H)-ylsulfonyl)benzonitrile (10 mg, 11%). 1H NMR (400
MHz, CDCl3): δ 8.55 (d, J = 5.2 Hz, 1H), 8.05 (s, 1H), 7.98
(d, J = 8.0 Hz, 2H), 7.70-7.80 (m, 2H), 7.55-7.65 (m, 1H),
7.28-7.32 (m, 1H), 4.43 (s, 2H), 3.55 (t, J = 6.0 Hz, 2H), 2.68
2-Pyridin-2-yl-6,7-dihydro-4H-oxazolo[4,5-c]pyridine-5-carboxy-
lic acid benzyl ester (9). To a solution of POCl3 (767 mg, 2.5
mmol) in dioxane (10 mL), 4-oxo-3-[(pyridine-2-carbonyl)-
amino]-piperidine-1-carboxylicacidbenzyl ester (0.9 g, 2.5 mmol)
in dioxane (10 mL) was added. The reaction mixture was heated
to reflux and stirred for 3 h. The mixture was quenched into
water and extracted with EtOAc. The organic layer was dried
over anhydrous Na2SO4. The solvent was concentrated, and the
residue was purified by silica gel chromatography (PE:EtOAc =
1:1) to afford 2-pyridin-2-yl-6,7-dihydro-4H-oxazolo[4,5-c]pyr-
1
idine-5-carboxylic acid benzyl ester (430 mg, 51%). H NMR
(400 MHz, CDCl3): δ 8.69 (m, 1H), 8.08 (m, 1H), 7.79 (t, J = 6.0
Hz, 1H), 7.28 (m, 6H), 5.20 (s, 2H), 4.60 (s, 2H), 3.86 (m, 2H),
2.88 (m, 2H). LC/MS: m/e = 336 (M þ H)þ. HPLC purity: 98%.
2-Pyridin-2-yl-4,5,6,7-tetrahydro-oxazolo[4,5-c]pyridine (10).
To a solution of 2-pyridin-2-yl-6,7-dihydro-4H-oxazolo[4,5-c]-
pyridine-5-carboxylic acid benzyl ester (430 mg, 1.28 mmol) in
MeOH (3 mL) was added Pd(OH)2 (20 mg). The mixture was
stirred at room temperature under H2 for 0.5 h. The mixture was
filtered and the filtrate was concentrated to afford 2-pyridin-
2-yl-4,5,6,7-tetrahydro-oxazolo[4,5-c]-pyridine (230 mg, 89%).
1H NMR: (400 MHz, CDCl3): δ 8.72 (d, J = 4.4 Hz, 1H), 8.08
(d, J = 8.0 Hz, 1H), 7.80 (t, J = 6.0 Hz, 1H), 7.34 (m, 1H), 3.84
(m, 1H), 3.68 (s, 1H), 3.49 (m, 1H), 3.14 (m, 1H), 3.06 (m, 1H),
2.86 (m, 2H). LC/MS: m/e = 202 (M þ H)þ. HPLC purity: 95%.
2-(2-Phenyl-6,7-dihydrooxazolo[5,4-c]pyridin-5(4H)-yl)nico-
tinonitrile (11a). 2-Phenyl-4,5,6,7-tetrahydro-oxazolo[5,4-c]pyr-
idine hydrochloride (purchased from Anichem) (200 mg, 0.84
mmol), 2-chloro-3-pyridine-carbonitrile (234 mg, 1.69 mmol),
and DIEA (327 mg, 2.53 mmol) were combined in DMF (1.0

7114 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19
Burdi et al.
(t, J = 5.6 Hz, 2H). LC/MS: m/e = 367 (M þ H)þ. HPLC
purity: 96%.
Benzyl (3-(pyridin-2-yl)-1H-1,2,4-triazol-5-yl)methylcarba-
mate (16). Benzyl 2-hydrazinyl-2-oxoethylcarbamate (16.4 g,
73.6 mmol) and ethyl picolinimidate 1516 (13.3 g, 88.3 mmol)
were combined in EtOH (150 mL). The mixture was heated
at reflux overnight. Acetic acid (60 mL) was added, and the
mixture was stirred at reflux for 2 h. The mixture was concen-
trated to dryness. Trituration using ether afforded the product
as a brown solid (21 g, 92%). 1H NMR (400 MHz, MeOH-d4): δ
8.64 (s, 1H), 8.08 (m, 1H), 7.92 (m, 1H), 7.45 (s, 1H), 7.16-7.42
(m, 5H), 5.12 (s, 2H), 4.48 (s, 2H). LC/MS: m/e = 310 (M þ H)þ.
HPLC purity: 100%.
3-((2-(Pyridin-2-yl)-6,7-dihydrooxazolo[5,4-c]pyridin-5(4H)-yl)-
methyl)benzonitrile (11g). To a solution of 2-(pyridin-2-yl)-
4,5,6,7-tetrahydrooxazolo[5,4-c]pyridine (50 mg, 0.248 mmol)
in dry DMF (5 mL) was added K2CO3 (69 mg, 0.496 mmol) and
3-(bromomethyl)benzonitrile (49 mg, 0.248 mmol) . The mix-
ture was stirred at room temperature overnight and then con-
centrated. The residue was purified by preparative HPLC to
afford 3-((2-(pyridin-2-yl)-6,7-dihydrooxazolo[5,4-c]pyridin-
5(4H)-yl)methyl) benzonitrile (10 mg, 13%). 1H NMR (400
MHz, CDCl3): δ 8.65 (d, J = 4.0 Hz, 1H), 8.40 (d, J = 8.0
Hz, 1H), 7.83-7.79 (m, 1H), 7.74-7.67 (m, 3H), 7.54-7.51 (m,
1H), 7.39-7.37 (m, 1H), 4.25 (s, 2H), 4.20 (s, 2H), 3.42 (t, J =
6.0 Hz, 2H), 2.98 (t, J = 6.0 Hz, 2H). LC/MS: m/e = 339 (M þ
H)þ. HPLC purity: 97%.
Ethyl 2-(5-((benzyloxycarbonylamino)methyl)-3-(pyridin-2-yl)-
1H-1,2,4-triazol-1-yl)acetate (17). Benzyl (3-(pyridin-2-yl)-1H-
1,2,4-triazol-5-yl)methylcarbamate (10.0 g, 32.3 mmol) was
dissolved in THF (100 mL) and cooled to 0 ꢀC. Potassium
t-butoxide (3.63 g, 32.3 mmol) was added. Ethyl bromoacetate
(5.4 g, 32.3 mmol) was dissolved in DMF and added dropwise,
and the reaction was stirred at room temperature for 4 h. The
DMF was removed in vacuo and the residue dissolved in EtOAc.
The organic layer was washed with saturated NaHCO3 solution
and brine, then dried over Na2SO4. Purification by reverse phase
chromatography (Gilson GX-281 (NH4HCO3/acetonitrile))
(()-3-(2-(Piperidin-2-yl)-6,7-dihydrooxazolo[5,4-c]pyridin-
5(4H)-yl)benzonitrile (11h). The title compound was prepared in
a manner similar to 11i starting from 1-(benzyloxycarbo-
nyl)piperidine-2-carboxylic acid and (()-trans-4-amino-3-hy-
droxy-piperidine-1-carboxylic acid benzyl ester. The title com-
pound was isolated as a white solid (15 mg, 21%). 1H NMR (400
MHz, CDCl3): δ 7.29-7.34 (m, 1H), 7.06-7.12 (m, 3H), 4.27 (s,
2H), 3.86-3.90 (m, 1H), 3.62 (t, J = 5.6 Hz, 2H), 3.10-3.14 (m,
1H), 2.70-2.76 (m, 3H), 1.99-2.04 (m, 2H), 1.85-1.87 (m, 1H),
1.61-1.71 (m, 2H), 1.47-1.53 (m, 2H). LC/MS: m/e = 311 (M
þ H)þ. HPLC purity: 95%.
1
gave a yellow oil (5.21 g, 41%). H NMR (400 MHz, CDCl3):
δ 8.70 (d, J = 5.0 Hz, 1H), 8.08 (d, J = 8.0 Hz, 1H), 7.77 (t, J =
8.0 Hz, 1H), 7.28-7.38 (m, 5H), 5.59 (s, 1H), 5.2 (s, 2H), 5.1 (s,
2H), 4.55 (d, J = 6.0 Hz, 2H), 4.21 (q, J = 12.0 Hz, 2H),
1.22-1.32 (t, J = 12.0 Hz, 3H). LC/MS: m/e = 396 (M þ H)þ.
HPLC purity: 93%.
3-(2-(Pyridin-2-yl)-6,7-dihydrooxazolo[5,4-c]pyridin-5(4H)-yl)-
benzonitrile (11i). To a solution of 2-(pyridin-2-yl)-4,5,6,7-
tetrahydrooxazolo[5,4-c]pyridine (100 mg, 0.5 mmol) in t-BuOH
(2 mL) was added 3-chlorobenzonitrile (102 mg, 0.6 mmol),
t-BuONa (100 mg, 1 mmol), Pd2dba3 (5 mg, cat.), and BINAP
(5 mg, cat.). The mixture was heated to 110 ꢀC via microwave
and stirred for 1 h. The reaction mixture was cooled, dissolved in
MeOH, and filtered. The filtrate was concentrated, and the
residue was purified by preparative HPLC to afford 3-(2-(pyri-
din-2-yl)-6,7-dihydrooxazolo[4,5-c]pyridine-5(4H)-
2-(Pyridin-2-yl)-7,8-dihydro-[1,2,4]triazolo[1,5-a]pyrazin-6(5H)-
one (18). Ethyl 2-(5-((benzyloxycarbonylamino)methyl)-3-
(pyridin-2-yl)-1H-1,2,4-triazol-1-yl)acetate (4.94 g, 12.4 mmol)
was dissolved in MeOH (50 mL). Palladium on carbon catalyst
(10%, 300 mg, 6% w/w) was added, and the reaction was stirred
under hydrogen atmosphere (1 atm) for 3 h. The catalyst was
removed by filtration through Celite, washed several times with
hot methanol, and the combined filtrates concentrated to give a
1
white solid (2.03 g, 76%). H NMR (400 MHz, MeOH-d4): δ
1
yl)benzonitrile (10 mg, 7%) as a yellow solid. H NMR (400
8.63 (m, 1H), 8.12 (d, J = 10.0 Hz, 1H), 7.93 (m, 1H), 7.46 (m,
1H), 4.93 (s, 2H), 4.7 (s, 2H). LC/MS: m/e = 216 (M þ H)þ.
HPLC purity: 100%.
MHz, CDCl3): δ 8.71 (s, 1H), 8.10 (d, J = 8.0 Hz, 1H), 7.85 (t,
J = 6.8 Hz, 1H), 7.40 (t, J = 6.8 Hz, 1H), 7.30 (t, J = 8.0 Hz,
1H), 7.07 (m, 3H), 4.40 (s, 2H), 3.68 (t, J = 5.6 Hz, 2H), 2.82 (t,
J = 5.6 Hz, 2H). LC/MS: m/e = 303 (M þ H)þ. HPLC purity:
99%.
3-(2-(Pyridin-3-yl)-6,7-dihydrooxazolo[5,4-c]pyridin-5(4H)-yl)-
benzonitrile (11j). The title compound was prepared via the
procedure used for 11k, substituting nicotinic acid for picolinic
acid and substituting 3-bromobenzonitrile for 3-bromo-5-fluor-
obenzonitrile. Preparative HPLC afforded 3-(2-pyridin-3-yl-
2-(Pyridin-2-yl)-5,6,7,8-tetrahydro-[1,2,4]triazolo[1,5-a]pyra-
zine (19). To a solution of 2-(pyridin-2-yl)-7,8-dihydro-[1,2,4]-
triazolo[1,5-a]pyrazin-6(5H)-one (1.63 g, 7.57 mmol) in THF
(40 mL) was added dropwise a solution of BH3/THF (30 mL,
1M, 30.0 mmol). The reaction was stirred at room temperature
under N2 for 15 min, then heated at reflux under N2 for 4 h. The
reaction was cooled in an ice bath. 4N HCl in dioxane (8 mL)
and MeOH (8 mL) were added dropwise, and the reaction was
heated to reflux for 2 h. The mixture was concentrated to
dryness on the rotavap. MeOH was added to the crude and
evaporated. This process of dilution and evaporation was
repeated a total of 5 times. Brine (1 mL) was added, and the
solution was extracted with DCM (2 ꢀ 5 mL). The pH of the
aqueous layer was adjusted to 14 using 6N NaOH and extracted
with DCM (4 ꢀ 5 mL). The combined organic layers were dried
over MgSO4, filtered, and concentrated to give a yellow solid
(400 mg, 26%). 1H NMR (400 MHz, CDCl3): δ 8.71 (d, J = 5.0
Hz, 1H), 8.08 (d, J = 8.0 Hz, 1H), 7.77 (t, J = 8.0 Hz 1H), 7.30
(m, 1H), 4.25 (m, 4H), 3.38 (t, J = 5.0 Hz, 2H). LC/MS: m/e =
202 (M þ H)þ. HPLC purity: 98%.
6,7-dihydro-4H-oxazolo[5,4-c]pyridin-5-yl)-benzonitrile as
a
light-yellow solid (11 mg, 26%). 1H NMR (400 MHz, CDCl3):
δ 9.33 (d, J = 1.2 Hz, 1H), 8.75 (dd, J1 = 4.0 Hz, J2 = 1.2 Hz,
1H), 8.59 (dd, J1 = 9.6 Hz, J2 = 1.6 Hz, 1H), 7.69 (dd, J1 = 8.4
Hz, J2 = 5.2 Hz, 1H), 7.38-7.34 (m, 1H), 7.17-7.12 (m, 3H),
4.43 (s, 2H), 3.71 (t, J = 4.8 Hz, 2H), 2.86 (t, J = 4.8 Hz, 2H).
MS (ESI): m/e = 303 [M þ 1].
3-Fluoro-5-(2-(pyridin-2-yl)-6,7-dihydrooxazolo[5,4-c]pyridin-
5(4H)-yl)benzonitrile (11k). To a solution of 2-(4-chlorophenyl)-
4,5,6,7-tetrahydrooxazolo[5,4-c]pyridine (20 mg, 0.1 mmol) in
toluene was added 3-bromo-5-fluorobenzonitrile (30 mg, 0.15
mmol), Cs2CO3 (65 mg, 0.2 mmol), Pd(OAc)2 (1 mg, cat.), and
Xantphos (2 mg, cat.). The mixture was heated to 100 ꢀC and
stirred overnight. The mixture was cooled, dissolved in MeOH,
and filtered. The filtrate was concentrated, and the residue was
purified by preparative TLC to afford 3-fluoro-5-(2-(pyridin-2-
yl)-6,7-dihydrooxazolo[5,4-c]pyridin-5(4H)-yl)benzonitrile (10
3-Fluoro-5-(2-(pyridin-2-yl)-5,6-dihydro-[1,2,4]triazolo[1,5-a]-
pyrazin-7(8H)-yl)benzonitrile (20). 2-(Pyridin-2-yl)-5,6,7,8-tetra-
hydro-[1,2,4]triazolo[1,5-a]pyrazine (112 mg, 0.56 mmol), tris-
(dibenzylideneacetone)dipalladium(0) (10 mg, 0.011 mmol),
Xantphos (19 mg, 0.033 mmol), Cs2CO3 (362 mg, 1.11 mmol),
and 3-bromo-5-fluorobenzonitrile (245 mg, 1.22 mmol) were
combined in xylene (4 mL). The mixture was stirred under
microwaves at 150 ꢀC for 30 min. The solvent was evaporated,
and the crude material was purified by silica gel chromatogra-
phy (9/1 DCM/MeOH). The fractions containing the desired
1
mg, 31%) as a yellow solid. H NMR (400 MHz, CDCl3): δ
8.68 (d, J = 8.0 Hz, 1H), 8.05 (d, J = 7.6 Hz, 1H), 7.77 (t, J =
7.6 Hz, 1H), 7.32 (t, J = 6.0 Hz, 1H), 6.90 (s, 1H), 6.75 (m, 2H),
4.40 (s, 2H), 3.68 (t, J = 5.6 Hz, 2H), 2.80 (t, J = 7.2 Hz, 2H).
LC/MS: m/e = 321 (M þ H)þ. HPLC purity: 96%.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19 7115
product were collected and combined and then purified by
reverse phase purification (Gilson GX-281 (NH4HCO3/aceto-
nitrile)) to give the desired product as a colorless oil (1.1 mg,
0.6%). 1H NMR (400 MHz, CDCl3): δ 8.72 (s, 1H), 8.09 (d, J =
8.0 Hz, 1H), 7.80 (t, J = 8.0 Hz, 1H), 7.33 (m, 1H), 7.10 (s, 1H),
6.90 (m, 2H), 4.65 (s, 2H), 4.45 (s, 2H), 3.90 (s, 2H). LC/MS:
m/e = 321 (M þ H)þ. HPLC purity: 100%.
NMR (400 MHz, CDCl3), δ 7.68 (d, J = 6.8 Hz, 2H), 7.34 (d,
J = 7.6 Hz, 2H), 4.20 (dd, J1 = 5.2 Hz, J2 = 8.8 Hz, 1H), 3.77
(dd, J1 = 14.8 Hz, J2 = 5.2 Hz, 1H), 3.68 (m, 1H), 3.01-3.07 (m,
2H), 2.59-2.65 (m, 2H), 2.44 (s, 3H), 1.83-2.0 (m, 2H). LC/MS:
m/e = 309 (M þ H)þ. HPLC purity: 78%.
3-Amino-1-tosylazepan-4-ol (25). To a suspension of lithium
aluminum hydride (760 mg, 20 mmol) in THF (15 mL) was
added 3-azido-1-tosylazepan-4-one (3.08 g 10 mmol) in THF
(35 mL) at 0 ꢀC. The reaction mixture was stirred at 0 ꢀC for
30 min and was quenched with saturated Na2SO4 aqueous
solution. The mixture was extracted with EtOAc and saturated
sodium bicarbonate. The organic layer was dried over sodium
sulfate, filtered, and evaporated to obtain the crude product.
The residue was purified by column chromatography (eluting
solvent: DCM/methanol = 20/1) to give 3-amino-1-tosylaze-
pan-4-ol 25 (1.45 g, 52%). 1H NMR (400 MHz, CDCl3): δ 7.66
(dd, J1 = 2.0 Hz, J2 = 8.4 Hz, 2H), 7.31 (dd, J1 = 8.0 Hz, J2 =
2.0 Hz, 2H), 3.62-3.64 (m, 0.5H), 3.48 (dd, J1 = 3.6 Hz, J2 =
14.4 Hz, 0.5H), 3.03-3.33 (m, 4H), 2.77-2.90 (m, 1H), 2.43 (s,
3H), 1.91-1.98 (m, 2H), 1.66-1.78 (m, 2H). LC/MS: m/e = 285
(M þ H)þ. HPLC purity: 85%.
3-Bromo-1-tosylpiperidin-4-one (22). To a solution of 1-(40-
methylphenylsulfonyl)piperidine-4-one (34.8 g, 137.5 mmol)
in DCM (860 mL) at -5 ꢀC was added a solution of bromine
(21.73 g, 137.5 mmol) in DCM (135 mL) dropwise over 2 h. The
temperature of the reaction was maintained between -4 and
-2 ꢀC. The resulting solution was allowed to warm to room
temperature until the color of bromine disappeared. Saturated
NaHCO3 was added and stirring continued for 30 min. The
organic layer was separated, and the aqueous layer was ex-
tracted with DCM (500 mL). The combined organic layer was
dried over sodium sulfate, filtered, and evaporated to dryness to
1
afford the title compound as a white solid (44.6 g, 98%). H
NMR (400 MHz, CDCl3): δ 7.69 (d, J = 8.0 Hz, 2H), 7.36 (d,
J = 8.4 Hz, 2H), 4.55 (dd, J1 = 5.2 Hz, J2 = 8.4 Hz, 1H), 3.98
(ddd, J1 = 1.6 Hz, J2 = 5.2 Hz, J3 = 13.2 Hz, 1H), 3.62-3.66
(m, 1H), 3.22-3.38 (m, 2H), 2.96 (dt, J1 = 5.2 Hz, J2 = 14.4 Hz,
1H), 2.62-2.70 (m, 1H), 2.45 (s, 3H). LC/MS: m/e = 332 (M þ
H)þ. HPLC purity: 97%.
N-(4-Hydroxy-1-tosylazepan-3-yl)picolinamide (26). To a so-
lution of picolinic acid (615 mg, 5 mmol) in DCM (15 mL) was
added EDCI (1.43 g, 7.5 mmol), HOBt (1.01 g, 7.5 mmol), and
triethylamine (758 mg, 7.5 mmol). The solution was stirred at
room temperature for 10 min. 3-Amino-1-tosylazepan-4-ol
(1.42 g, 5 mmol) in DCM (5 mL) was added to the reaction,
and the resulting mixture was stirred at room temperature
overnight. The reaction was diluted with DCM and washed
with saturated sodium bicarbonate solution. The organic layer
was dried over sodium sulfate, filtered, and evaporated. The
crude product was purified by column chromatography to
obtain N-(4-hydroxy-1-tosylazepan-3-yl)picolinamide 26 (1.55
g, 80%) (eluting solvent:dichloromethane/methanol = 50/1).
LC/MS: m/e = 390 (M þ H)þ. HPLC purity: 92%.
Ethyl 6-Bromo-5-oxo-1-tosylazepane-4-carboxylate (23a). To
a solution of 3-bromo-1-tosylpiperidin-4-one (39.3 g, 118.7
mmol) in anhydrous DCM (1.3 L) at -5 ꢀC under argon was
added a solution of BF3 Et2O (22 g, 1.25 equiv, 148.4 mmol) in
3
DCM (180 mL) over 60 min. The reaction was stirred at -5 ꢀC
for 45 min, and a solution of ethyl diazoacetate (16.9 g, 148.4
mmol) in DCM (180 mL) was added over 90 min. The mixture
was stirred at 0 ꢀC for 1 h, and water was added slowly. The
mixture was stirred at room temperature for 30 min. The organic
layer was separated, dried over sodium sulfate, filtered, and
concentrated in vacuo to give a pale-yellow solid which was
recrystallized from ethyl acetate to give ethyl 6-bromo-5-oxo-1-
tosylazepane-4-carboxylate 23a as a white solid (35.69 g, 72%).
1H NMR (400 MHz, CDCl3): δ 7.66 (d, J = 8.4 Hz, 2H), 7.33 (d,
J = 8.0 Hz, 2H), 4.45 (dd, J1 = 6.8 Hz, J2 = 11.2 Hz, 1H),
4.19-4.3 (m, 3H), 3.94 (dd, J1 = 2.8 Hz, J2 = 9.2 Hz, 1H), 4.03
(dt, J1 = 2.8 Hz, J2 = 12.0 Hz, 1H), 3.07 (dd, J1 = 15.2 Hz, J2 =
10.8 Hz, 1H), 2.80 (ddd, J1 = 14 Hz, J2 = 12 Hz, J3 = 2.8 Hz,
1H), 2.44 (s, 3H), 2.2 (m, 1H), 2.0-2.1 (m, 1H), 1.26 (t, J = 6.8
Hz, 3H). LC/MS: m/e = 418 (M þ H)þ. HPLC purity: 93%.
3-Bromo-1-tosylazepan-4-one (23b). A suspension of ethyl
6-bromo-5-oxo-1-tosylazepane-4-carboxylate (35.7 g, 85.6 mmol)
in dioxane (540 mL) was heated to 80 ꢀC. 3N HCl (290 mL)
was added to the mixture over 30 min. The resulting solution
was heated to 100 ꢀC for 6 h. The dioxane was removed in
vacuo, and the aqueous layer was extracted with DCM. The
combined organic layer was dried, filtered, and evaporated to
give the crude product, which was recrystallized from diisopro-
pyl ether to give 3-bromo-1-tosylazepan-4-one 23b as a white
solid (26.5 g, 90%). 1H NMR (400 MHz, CDCl3): δ 7.67 (d, J =
8.8 Hz, 2H), 7.34 (d, J = 8.0 Hz, 2H), 4.37 (dd, J1 = 10.8 Hz,
J2 = 6.4 Hz, 1H), 4.24 (dd, J1 = 6.4 Hz, J2 = 14.4 Hz, 1H), 3.90
(dt, J1 = 4.4 Hz, J2 = 13.6 Hz, 1H), 3.05 (m, 1H), 2.75-2.92 (m,
2H), 2.55-2.59 (m, 1H), 2.44 (s, 3H), 1.8-1.97 (m, 2H). LC/MS:
m/e = 346 (M þ H)þ. HPLC purity: 90%.
N-(4-Oxo-1-tosylazepan-3-yl)picolinamide (27). To a solution
of N-(4-hydroxy-1-tosylazepan-3-yl)picolinamide (1.16 g, 2.98
mmol) in DCM (20 mL) was added Dess-Martin periodinane
(3.82 g, 8.9 mmol) in one portion. The reaction was stirred at
room temperature for 3 h and then diluted with DCM. The
mixture was cooled to 0 ꢀC, and ice-cold 0.5N sodium hydroxide
solution was added. The biphasic mixture was stirred at 0 ꢀC for
1 h. The organic layer was separated, dried over sodium sulfate,
and concentrated in vacuo to obtain N-(4-oxo-1-tosylazepan-3-
yl)picolinamide 27 (1.06 g, 92%). LC/MS: m/e = 388 (M þ H)þ.
HPLC purity: 93%.
2-(Pyridin-2-yl)-5-tosyl-5,6,7,8-tetrahydro-4H-oxazolo[4,5-c]-
azepine (28a). A mixture of N-(4-oxo-1-tosylazepan-3-yl)pico-
linamide (10.0 g, 25.8 mmol) and Burgess’ reagent (21.49 g, 90.3
mmol) in dry THF (40 mL) was heated to 150 ꢀC by microwave
and stirred for 45 min. The reaction mixture was diluted with
water and extracted with DCM. The organic layer was dried
over sodium sulfate and evaporated to dryness. The crude
product was recrystallized from methanol to give 2-(pyridin-2-
yl)-5-tosyl-5,6,7,8-tetrahydro-4H-oxazolo[4,5-c]azepine 28a (7.4
1
g, 78%). H NMR (400 MHz, CDCl3), δ 8.70 (d, J = 4.4 Hz,
1H), 8.04 (d, J = 8.0 Hz, 1H), 7.81 (dt, J1 = 2.0 Hz, J2 = 8.0 Hz,
1H), 7.69 (d, J = 8.0 Hz, 2H), 7.33-7.37 (m, 1H), 7.28 (d, J =
8.0 Hz, 2H), 4.43 (s, 2H), 3.51-3.54 (m, 2H), 2.92 (t, J = 6.4 Hz,
2H), 2.39 (s, 3H), 2.03-2.49 (m, 2H). LC/MS: m/e = 370 (M þ
H)þ. HPLC purity: 93%.
2-(Pyridin-2-yl)-5,6,7,8-tetrahydro-4H-oxazolo[4,5-c]azepine
(28b). A solution of 2-(pyridin-2-yl)-5-tosyl-5,6,7,8-tetrahydro-
4H-oxazolo[4,5-c]azepine (7.4 g, 20 mmol) in 48% aqueous
hydrobromide solution (100 mL) was heated to 100 ꢀC and
stirred for 3 h. The mixture was cooled to room temperature
and extracted with tert-butyl methyl ether. The aqueous layer
was adjusted to pH 13 with sodium hydroxide solution and
extracted with dichloromethane. The combined organic layer
was dried over sodium sulfate, filtered, and evaporated to obtain
3-Azido-1-tosylazepan-4-one (24). To a solution of 3-bromo-
1-tosylazepan-4-one (26.5 g, 76.8 mmol) in dry DMF (580 mL)
was added acetic acid (9.22 g, 153.6 mmol) and sodium azide
(9.98 g, 153.6 mmol) under nitrogen. The resulting solution was
stirred at room temperature for 16 h. The mixture was poured
onto ice-water, and the aqueous solution was extracted with
ethyl acetate. The combined organic layer was washed with
brine, dried, filtered, and evaporated. The crude product was
recrystallized from tert-butyl methyl ether to give 3-azido-1-
tosylazepan-4-one 24 as a pale-yellow solid (19 g, 80%). 1H

7116 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19
Burdi et al.
2-(pyridin-2-yl)-5,6,7,8-tetrahydro-4H-oxazolo[4,5-c]azepine
28b (3.4 g, 74%). H NMR (400 MHz, CD3OD), δ 8.62-8.64
(m, 1H), 8.07 (td, J1 = 1.2 Hz, J2 = 8.4 Hz, 1H), 7.96 (dt, J1 =
2.0 Hz, J2 = 7.6 Hz, 1H), 7.47-7.51 (m, 1H), 3.90 (s, 2H),
3.08-3.10 (m, 2H), 3.00 (t, J = 6.0 Hz, 2H), 1.93-1.98 (m, 2H).
LC/MS: m/e = 216 (M þ H)þ. HPLC purity: 96%.
tube was added buffer (15 mM tris(hydroxymethyl)amino-
methane hydrochoride, 120 mM sodium chloride, 100 mM
potassium chloride, 25 mM magnesium chloride, 25 mM cal-
cium chloride, pH 7.4), rat cortical membrane extracts (between
50 and 200 μg protein/well), [3H]-MPEP (American Radiola-
beled Chemicals, Inc.) (0.6 nM), and test compound at increas-
ing concentrations. Nonspecific binding was determined by
coincubation with a 200-fold excess of 1 (Tocris). The samples
were incubated in a final volume of 0.1 mL for 120 min at 4 ꢀC
and then filtered over Whatman GF-C filters that had been
presoaked in washing buffer containing 0.1% polyethylenei-
mine for 2 h at room temperature. The filters were washed three
times with 3 mL of cold washing buffer (50 mM tris(hydroxy-
methyl)aminomethane hydrochoride, pH 7.4). Microscint 20
fluid (2 mL) (Packard) was added to each tube, and the tubes
were counted with a Tricarb counter for 1 min/tube.
1
3-(2-(Pyridin-2-yl)-7,8-dihydro-4H-oxazolo[4,5-c]azepin-5(6H)-
yl)benzonitrile (29a). To a solution of 2-(pyridin-2-yl)-5,6,7,8-
tetrahydro-4H-oxazolo[4,5-c]azepine (20 mg, 0.09 mmol) in
toluene (2 mL) was added 3-bromobenzonitrile (25 mg, 0.14
mmol), Cs2CO3 (61 mg, 0.18 mmol), Pd(OAc)2 (1 mg, cat.), and
Xantphos (2 mg, cat.). The mixture was heated to 100 ꢀC and
stirred overnight. The reaction was cooled, diluted with MeOH,
and filtered. The filtrate was concentrated and the residue was
purified by prep-TLC to afford the title compound (5 mg, 17%)
as a yellow solid. 1H NMR (400 MHz, CDCl3): δ 8.65 (d, J = 4.4
Hz, 1H), 8.00 (d, J = 8.0 Hz, 1H), 7.75 (t, J = 6.0 Hz, 1H), 7.27
(t, J = 4.8 Hz, 1H), 7.15-7.20 (m, 1H), 6.95-7.00 (m, 2H), 6.80
(d, J = 7.2 Hz, 1H), 4.49 (s, 2H), 3.78 (t, J = 4.8 Hz, 2H), 2.94 (t,
J = 6.0 Hz, 2H), 1.87-1.96 (m, 2H). LC/MS: m/e = 317 (M þ
H)þ. HPLC purity: 94%
mGluR5 Rat Receptor Occupancy Assay. All animal experi-
mental procedures were carried out in accordance with the
governmental guidelines and approved by the Institutional
Animal Care and Use Committee (IACUC). In vivo occupancy
studies for the negative allosteric modulator site of the mGluR5
receptor were performed using 3H-mPEPy (Moravek Bio-
chemicals; specific activity ∼58-61 Ci/mmol). Male Sprague-
Dawley rats (Harlan, 200-250 g; n = 4 per group) were injected
intraperitoneally or orally (po) with either vehicle (45% hydro-
xypropyl-β-cyclodextrin, HPβCD) or test compounds. Sixty
min after drug administration (or otherwise stated in the case
3-Fluoro-5-(2-(pyridin-2-yl)-7,8-dihydro-4H-oxazolo[4,5-c]aze-
pin-5(6H)-yl)benzonitrile (29b). To a microwave tube flushed
with argon was added potassium t-butoxide (1.35 g, 12 mmol),
tris(dibenzylideneacetone)dipalladium(0) [Pd2(dba)3] (275 mg,
0.3 mmol), Xantphos (347 mg, 0.6 m mol), 2-(pyridin-2-yl)-
5,6,7,8-tetrahydro-4H-oxazolo[4,5-c]azepine (1.3 g, 6 mmol),
3-bromo-5-fluorobenzonitrile (1.8 g, 9 mmol), and dry toluene
(30 mL). The reaction mixture was heated to 100 ꢀC via micro-
wave and stirred for 70 min. The reaction mixture was diluted
with ethyl acetate and washed with brine. The combined organic
layer was dried over sodium sulfate, filtered, and evaporated to
dryness. The crude product was purified by column chromato-
graphy on silica gel (PE/EA = 5/1-1/1) to give the title com-
3
of the time-course experiment), H-mPEPy (120uCi/kg; 1 mL/
kg in saline) was administered as a bolus intravenous injection
via the tail vein. Two minutes after 3H-mPEPy administration,
rats were euthanized by rapid decapitation and trunk blood and
brains harvested. Brains were dissected and frozen immediately
in isopentane cooled in dry ice and stored at -80 ꢀC until
required for tissue sectioning. Blood samples were centrifuged
(20 min, 3000g, 6 ꢀC), and the resulting plasma samples were
stored at -80 ꢀC until required for drug level analysis. For tissue
sectioning and image processing, brains were removed from
storage (-80 ꢀC) and allowed to thaw to -20 ꢀC for tissue
sectioning on a Leica CM1900 cryostat. Coronal sections
(12 mm) of striatum (the region of interest) and cerebellum
(reference region) were cut and thaw-mounted onto glass micro-
scope slides. Images of brain sections from vehicle and drug-
treated animals were generated by placing the slide-mounted
sections in a Biospace β-imager (Biospace Inc., France) for 12 h.
Striatal and cerebellar images were quantitated and expressed as
units radioactivity/mm2 using the b-vision plus software sup-
plied by Biospace. Signal:noise (striatum:cerebellum) ratio
(S:N) was calculated and the receptor occupancy (%RO) was
determined for each subject using the following formula:
1
pound as a yellow solid (440 mg, 22%). H NMR (400 MHz,
CDCl3): δ 8.70 (d, J = 4.8 Hz, 1H), 8.06 (td, J1 = 1.2 Hz, J2 =
8.0 Hz, 1H), 7.82 (dt, J1 = 1.6 Hz, J2 = 8.0 Hz, 1H), 7.33-7.37
(m, 1H), 6.83-6.84 (m, 1H), 6.72 (td, J1 = 2.4 Hz, J2 = 12 Hz,
1H), 6.64-6.67 (m, 1H), 4.53 (s, 2H), 3.80-3.83 (m, 2H), 3.02 (t,
2H, J = 6.0 Hz), 1.99-2.02 (m, 2H). LC/MS: m/e = 335 (M þ
H)þ. HPLC purity: 98%.
mGluR5 Functional Assay. The functional assay utilized an
aequorin cell line expressing human recombinant mGluR5
receptor, an inducible cell line that expresses the human receptor
under the control of a promoter induced by doxycycline.
mGluR5 cells in midlog phase, grown 18 h prior to the test in
antibiotic-free media supplemented with doxycycline (600 ng/
mL), were detached by gentle flushing with PBS-EDTA (5 mM
EDTA). The cells were recovered by centrifugation and resus-
pended in assay buffer (HBSS, 2.1 mM CaCl2, 3 μg/mL gluta-
mate-pyruvate transaminase, 4 mM MEM sodium pyruvate,
0.1% BSA protease-free). Cells were incubated at room tem-
perature for at least 4 h with coelenterazine-h (Molecular
Probes). The cell suspension was incubated with the test com-
pound in a volume of 60 μL for at least 3 min, followed by the
addition of a 30 μL of glutamate solution to give a final
concentration sufficient for a response 80% of the maximal
response given by saturating glutamate (EC80). The resulting
emission of light was recorded using a Hamamatsu Functional
Drug Screening System 6000 (FDSS 6000). To standardize the
emission of recorded light (determination of the “100% signal”)
across plates and across different experiments, some wells
contained 100 μM digitonin or a saturating concentration
(20 μM) of adenosine triphosphate. Percentages of inhibition
were calculated on the basis of the activation induced by the
agonist glutamate at its EC80. Dose-response data were ana-
lyzed with XLFit (IDBS) software using nonlinear regression
applied to a sigmoidal dose-response model.
%RO ¼ 100ꢀ½ðaverageS : Nvehicle
Þ
- S : Nsubjectꢁ=½ðaverageS : NvehicleÞ - 1ꢁ
Data are expressed as mean %RO ( standard error of the
mean. Dose-occupancy curves for 29b at the mGluR5 NAM
site were generated using Prism ver5.0.
To determine plasma and brain concentrations, sample pre-
paration began with the addition of rat brain (200 mg) to a
15 mL plastic test tube and dilution to 40 mg/mL with 95:5
water:acetonitrile containing 0.1% formic acid. The mixture
was then homogenized using an ultrasonic homogenizer for 1
min. A 60 μL aliquot of plasma or homogenized rat brain was
placed into a 96 deep well plate, and 200 μL of acetonitrile was
added to precipitate the proteins. Samples were vortexed for 1
min and centrifuged at 4500 rpm for 15 min at 4 ꢀC. Then 200 μL
aliquots of supernatant was transferred to a deep-well 96 plate,
and evaporated to dryness. The samples were then reconstituted
in 150 μL 90:10 5 water:acetonitrile with 0.1% formic acid and
centrifuged at 4500 rpm for 15 min at 4 ꢀC. A 45 μL aliquot from
the supernatant was injected into the LC/MS/MS instrument.
mGluR5 Rat Binding Assay. The radioligand binding assay
was a competition assay performed in duplicate. To a Minisorb

Article
Rat Pharmacokinetics. The pharmacokinetics of 29b was
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19 7117
(2) Chizh, B. A. Novel Approaches to Targeting Glutamate Receptors
for the Treatment of Chronic Pain: Review Article. Amino Acids
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determined in male Sprague-Dawley rats following an intra-
venous dose of 2 mg/kg and oral doses of 2 mg/kg and 10 mg/kg
(N = 3 animals/cohort). The test article was dissolved in 40%
hydroxypropyl-βCD:50 mM potassium phosphate pH 6.0 50:50
to yield a nominal concentration of 0.5 mg/mL (pH = 6-7). The
formulation was filtered through a 0.22 μm filter to achieve a
clear solution prior to iv dosing (4 mL/kg) and oral gavage
(4 mL/kg and 20 mL/kg for the 2 mg/kg and 10 mg/kg doses,
respectively). Blood samples (approximately 300 μL) were col-
lected via jugular cannula predose and at 5 min, 0.25, 0.50, 1, 2,
4, 6, 8, and 24 h postdose. Following blood collection at each
time point, approximately 300 μL of sterile 0.9% sodium
chloride was injected through the cannula to replace the volume
of blood collection. Blood samples were placed into tubes
containing sodium K2-EDTA and centrifuged under refriger-
ated conditions at 8000 rpm for 6 min at 4 ꢀC to separate plasma
from sample. Following centrifugation, the supernatant was
transferred to clean tubes and stored frozen at -80 ꢀC pending
bioanalysis. For bioanalysis, plasma samples (0.05 mL) were
transferred to tubes, and 250 μL of a methanol solution contain-
ing internal standard (200 ng/mL) was added to each sample.
After vortexing for 1 min and centrifuging for 5 min at 15000
rpm, 100 μL aliquots of supernatant were transferred to glass
autosampler vials for LC/MS/MS analysis. The lower limit
of quantitation (LLOQ) was 2.5 ng/mL. All pharmacokinetic
parameters were calculated using a noncompartmental model in
the WinNonlin program.
Monkey Pharmacokinetics. The pharmacokinetics of 29b was
determined in male Rhesus monkeys following an intravenous
dose of 1 mg/kg and an oral dose of 1 mg/kg (N = 3 animals/
cohort). The test article was dissolved in 40% hydroxypropyl-
βCD:50 mM potassium phosphate pH 6.0 50:50 to yield a
nominal concentration of 0.5 mg/mL (pH = 6). The formula-
tion was filtered through a 0.22 μm filter to achieve a clear
solution prior to iv dosing and oral gavage (2 mL/kg). Blood
samples (approximately 1 mL) were collected via femoral vein
predose and at 5 min, 0.25, 0.50, 1, 2, 4, 6, 8, and 24 h postdose.
Blood samples were placed into tubes containing K3EDTA and
centrifuged at 3500 rpm for 10 min at 4 ꢀC to separate plasma
from sample. Following centrifugation, the supernatant was
transferred to clean tubes and stored frozen at -80 ꢀC pending
bioanalysis. For bioanalysis, plasma samples (50 μL) were
transferred to tubes and 500 μL of internal standard working
solution (10 ng/mL) was added to each sample. After vortexing
for 1 min and centrifuging for 5 min at 15000 rpm, 100 μL
aliquots of supernatant were transferred to glass autosampler
vials for LC/MS/MS analysis. The lower limit of quantitation
(LLOQ) was 2.5 ng/mL. All pharmacokinetic parameters were
calculated using a noncompartmental model in the WinNonlin
program.
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Supporting Information Available: Detailed synthetic schemes
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