Discovery of 4-(5-methyloxazolo[4,5-b]pyridin-2-yl)-1,4-diazabicyclo[3.2.2] nonane (CP-810,123), a novel α7 nicotinic acetylcholine receptor agonist for the treatment of cognitive disorders in schizophrenia: Synthesis, SAR development, and in vivo efficacy in cognition models

Article abstract of DOI:10.1021/jm9015075

A novel α7 nAChR agonist, 4-(5-methyloxazolo[4,5-b]pyridin-2-yl)-1,4- diazabicyclo[3.2.2]nonane (24, CP-810,123), has been identified as a potential treatment for cognitive deficits associated with psychiatric or neurological conditions including schizophrenia and Alzheimer's disease. Compound 24 is a potent and selective compound with excellent pharmaceutical properties. In rodent, the compound displays high oral bioavailability and excellent brain penetration affording high levels of receptor occupancy and in vivo efficacy in auditory sensory gating and novel object recognition. The structural diversity of this compound and its preclinical in vitro and in vivo package support the hypothesis that α7 nAChR agonists may have potential as a pharmacotherapy for the treatment of cognitive deficits in schizophrenia. 2009 American Chemical Society.

Full text of DOI:10.1021/jm9015075

1222 J. Med. Chem. 2010, 53, 1222–1237
DOI: 10.1021/jm9015075
Discovery of 4-(5-Methyloxazolo[4,5-b]pyridin-2-yl)-1,4-diazabicyclo[3.2.2]nonane (CP-810,123),
a Novel r7 Nicotinic Acetylcholine Receptor Agonist for the Treatment of Cognitive Disorders in
Schizophrenia: Synthesis, SAR Development, and in Vivo Efficacy in Cognition Models
Christopher J. O’Donnell,* Bruce N. Rogers,* Brian S. Bronk, Dianne K. Bryce, Jotham W. Coe, Karen K. Cook,
ꢀ
Allen J. Duplantier, Edelweiss Evrard, Mihaly Hajos, William E. Hoffmann, Raymond S. Hurst, Noha Maklad,
Robert J. Mather, Stafford McLean, Frank M. Nedza, Brian T. O’Neill, Langu Peng, Weimin Qian, Melinda M. Rottas,
Steven B. Sands, Anne W. Schmidt, Alka V. Shrikhande, Douglas K. Spracklin, Diane F. Wong, Andy Zhang, and Lei Zhang
Pfizer Global Research and Development, Groton Laboratories, Eastern Point Road, Groton, Connecticut 06340
Received October 9, 2009
A novel R7 nAChR agonist, 4-(5-methyloxazolo[4,5-b]pyridin-2-yl)-1,4-diazabicyclo[3.2.2]nonane (24,
CP-810,123), has been identified as a potential treatment for cognitive deficits associated with
psychiatric or neurological conditions including schizophrenia and Alzheimer’s disease. Compound
24 is a potent and selective compound with excellent pharmaceutical properties. In rodent, the
compound displays high oral bioavailability and excellent brain penetration affording high levels of
receptor occupancy and in vivo efficacy in auditory sensory gating and novel object recognition. The
structural diversity of this compound and its preclinical in vitro and in vivo package support the
hypothesis that R7 nAChR agonists may have potential as a pharmacotherapy for the treatment of
cognitive deficits in schizophrenia.
Introduction
predominantly a genetic disorder, with an 80% estimated risk
of heritability.5 Considerable genetic evidence implicates the
R7 nicotinic acetylcholine receptor (nAChRa), including the
subunit gene CHRNA7, and a linkage to impaired auditory
gating (P50).6,7 Additionally, cholinergic deficits in hippo-
campal regions that mediate stimulus processing have been
attributed to attentional disorders and cognitive deficits.8
A hypothesis has been constructed from the observation of
diminished suppression of the P50 gating response following
auditory stimulation and the observed reduction in R7 nA-
ChRs found in schizophrenic patients.9 The gating deficit in
schizophrenic patients and their first degree relatives is tran-
siently improved or normalized by nicotine, also supporting a
connection to the R7 nAChR. In addition, heavy tobacco
smoking in schizophrenic patients is well documented and
interpreted as a potential form of self-medication.10,11 As
such, the R7 nAChR has received considerable attention as
a promising target for schizophrenia therapy, in particular for
the cognitive symptoms,12 and may also offer promise for
other cognitive disorders such as Alzheimer’s disease and
attention deficit hyperactivity disorder (ADHD).
Schizophrenia is a chronic and highly debilitating psychia-
tric disease thatafflicts about 1-2% of the world population.1
This complex disorder is characterized by variable expression
of three major symptomologies: positive, negative, and cog-
nitive. Psychotic, or positive, symptoms include hallucina-
tions and delusions, while negative symptoms include apathy,
anhedonia, and social or emotional withdrawal. Cognitive
symptoms, including disorganized thoughts, decreased atten-
tion, and memory deficits, have more recently become a focus
in schizophrenia research.2 Significant therapeutic advances
have been made in the treatment of the positive and negative
symptoms of schizophrenia,3 but the vast majority of schizo-
phrenic patients (over 85%) also suffer from cognitive deficits
that are poorly addressed with current agents.4 Although
both genetic and environmental components contribute
to schizophrenia, studies of twins show that the disease is
*To whom correspondence should be addressed. For C.J.O.: phone,
860-715-4118; fax, 860-686-5158; e-mail, christopher.j.odonnell@
pfizer.com. For B.N.R.: phone, 860-686-0545; fax, 860-686-5403; e-mail,
bruce.n.rogers@pfizer.com.
Neuronal nAChRs, so named because they are activated by
the alkaloid nicotine, are a well-defined subset of ligand-gated ion
a
Abbreviations: ACh, acetylcholine; ADME, absorption, distribution,
channels permeable to cations such as Naþ, Kþ, and Ca2þ 13,14
.
metabolism, and excretion; ADHD, attention deficit hyperactivity disor-
der; AMP, amphetamine; BINAP, 2,20-bis(diphenylphosphino)-1,10-bi-
naphthyl; BTX, bungarotoxin; DME, 1,2-dimethoxyethane; DMF,
dimethylformamide; EEG, electroencephalography; hERG, human
ether-a-go-go-related gene; FLIPR, fluorescence imaging plate reader;
HLM, human liver microsomes; IVMN, in vitro micronucleus; MED,
minimally effective dose; MDCK, Madin-Darby canine kidney; MW,
molecular weight; nAChR, nicotinic acetylcholine receptor; NCS, N-chlo-
rosuccinimide; NMP, N-methylpyrrolidone; NOR, novel object recogni-
tion; NSVT, nonsustained ventricular tachycardia; P-gp, P-glycoprotein;
RLM, rat liver microsomes; RT, room temperature; SAR, structure-acti
vity relationship; sc, subcutaneous; TEA, triethylamine; TFA, trifluoro-
acetic acid; THF, tetrahydrofuran; TPSA, topological polar surface area.
Formed by five individual subunits, the family of nAChRs
is potentially vast, as 17 distinct nAChR subunits are known
and can assemble in many different combinations to form
a pentameric channel. These subunits assemble into homo-
pentamers (e.g., R7, R9) or more commonly into hetero-
pentamers.15 The homopentameric R7 nAChR consists of five
R7 subunits, and each subunit provides an orthosteric binding
site for its endogenous ligand, acetylcholine.16 Interest in the
development of R7 nAChR agonists has greatly expanded
r
pubs.acs.org/jmc
Published on Web 12/31/2009
2009 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3 1223
Figure 1. Known quinuclidne-based R7 nAChR ligands.
Figure 3. Design of the benzoxazole and azabenzoxazole series
from the carbamate series.
Scheme 1a
Figure 2. Non-quinuclidine amine templates in the nAChR field.
over the past decade, with the identification of many novel
and selective chemical entities. The literature in the area has
been extensively reviewed,17 with the majority of ligands
derived from various quinuclidine amine containing scaffolds,
including such structures as spiro-oxazolidinones (AR-
R17779),18 carbamates,19 ethers,20 and amides (Figure 1).21
Our previous efforts led to the identification of two phase I
clinical candidates from the quinuclidine amide arena, PHA-
543613 and PHA-568487,22 both of which were discontinued
because of cardiovascular findings of nonsustained ventri-
cular tachycardia (NSVT).23
a Reagents and conditions: (a) thiophosgene, THF, RT, or potassium
ethyl xanthate, EtOH, 90 °C; (b) MeI, K2CO3, DMF, RT; (c) i-PrOH,
Et3N, heat; (d) 2-chlorobenzoxazole, i-Pr2NEt, toluene, 35%, RT.
We became interested in retaining this putative hydrogen
bond while improving the potency while maintaining full
agonist activity of the carbamate series, and we envisioned
replacing the carbamate linkage with heterocyclic motifs as
represented by 2. We describe herein the exploration of benzo-
xazole and azabenzoxazole (3) replacements of the carbamate
functional group and the development of the SAR within this
heterocyclic motif.
While several R7 nAChR agonists, such as GTS-21,24 are
reported to have entered human clinical trials, there remains a
need for novel ligands with improved safety profiles. The
structural diversity of R7 nAChR agonists has been limi-
ted and generally focuses around quinuclidine amine based
ligands. Within the nAChR field, however, a wide variety of
amine scaffolds are prevalent among ligands for R4β2
nAChRs and include compounds such as epibatidine,25
ABT-594,26 and the recently approved partial agonist vareni-
cline (Figure 2).27 In the R7 nAChR area, recent disclosures
including SR-180711,28 PHA-709829,29 and A-58294130 de-
monstrate that novel scaffolds can be identified, and we
believed that additional opportunities remained to identify
structural variants with a well tolerated safety profile in the R7
nAChR arena.31
Chemistry
In order to fully explore the SAR associated with the R7
nAChR, we sought to probe both steric and electronic
requirements by efficiently and systematically installing sub-
stituents around the heteroaromatic ring system. The unsub-
stituted benzoxazole analog 7 was prepared by reacting
commercially available 2-chlorobenzoxazole with 1,4-
diazabicyclo[3.2.2]nonane, 6,33 in the presence of Hunig’s
€
base in 35% yield. Compounds bearing substitution about
the benzoxazole ring (8-12) were prepared from the corre-
sponding 2-aminophenols 4a (Scheme 1). Cyclization of 4a
with either thiophosgene or potassium ethyl xanthate fol-
lowed by methyl iodide alkylation of the resulting 2-mercap-
tobenzoxazole afforded methyl sulfide intermediates 5.34
Intermediate 5 was then coupled with diamine 6 in the
presence of triethylamine in warm isopropanol to generate
We recently described the structure-activity relationship
(SAR) of a 1,4-diazabicyclo[3.2.2]nonane aryl carbamate
series of R7 nAChR agonists 1 (Figure 3).32 One key finding
in this work was that the carbamate oxygen atoms were
critical for R7 nAChR potency and functional activity, pos-
sibly forming productive hydrogen bonds with the receptor.

1224 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3
O’Donnell et al.
Scheme 2a
Scheme 3a
a Reagents and conditions: (a) HNO3, H2SO4, vV, 33%; (b) Pd/C, H2
(45 psi), EtOH/MeOH (1:1); (c) HCl, NaNO2, HPF6, H2O, 6% for two
steps; (d) NCS, CHCl3, microwave, 150 °C, 10 min, 70%; (e) (H2Cd
CH)SnBu3, Pd(PPh3)2Cl2, toluene, vV; (f) Pd/C, H2 (45 psi), 1:1 EtOH/
MeOH, 43% for two steps; (g) R-MgCl/ZnCl2, Pd(dppf)Cl2, THF,
microwave, 37% for both 36 and 37; (h) ArB(OH)2, Pd(PPh3)4, Na2CO3,
toluene, MeOH, H2O, 22% for 38, 32% for 39; (i) R-OH, CuCl,
tetramethylheptane-3,5-dione, Cs2CO3, NMP, microwave, 200 °C,
10 min, 46% for 40, 32% for 41; (j) R1R2NH, Pd(OAc)2, BINAP,
NaOt-Bu, toluene, 100 °C, 32% for 42, 53% for 43.
a Reagents and conditions: (a) Br2, NaOAc, AcOH, H2O, vV, 2 h, 78%;
(b) NCS, CHCl3, microwave, 150 °C, 10 min, 33%; (c) HNO3, H2SO4;
(d) Pd/C, H2 (45 psi), 1:1 EtOH/MeOH; (e) HCl, NaNO2, HPF6, H2O,
32% (three steps); (f) ZnMe2, Pd(dppf)Cl2, 1,4-dioxane, microwave,
150 °C, 10 min, 70%, or sec-butylboronic acid, Pd(PPh3)4, Na2CO3, EtOH,
H2O, 85 °C; (g) (H2CdCH)SnBu3, Pd(PPh3)2Cl2, toluene, vV, 68%;
(h) Pd(OAc)2, Et3N, 2-(N,N-dimethylamino)-20-dicyclohexylphosphi-
nobiphenyl, PhB(OH)2, CsF, DME, 27%; (i) Zn(CN)2, Pd(dppf)Cl2,
DMF, microwave, 220 °C, 10 min, 78%; (j) ArB(OH)2, Pd(PPh3)4, K2-
CO3, 9:1 EtOH/H2O, microwave, 150 °C, 10 min (15-24%); (k) phenol,
CuCl, tetramethylheptane-3,5-dione, Cs2CO3, DMF, microwave, 200 °C,
40 min, 16%.
extremely versatile (Scheme 3): tributyl(vinyl)stannane Stille
coupling followed by hydrogenation gave 35; alkylation with
alkylzinc reagents afforded 36 and 37; Suzuki coupling
afforded 38 and 39; aryl ether formation gave 40 and 41,
and arylamine formation under Buchwald conditions35 pro-
vided access to 42 and 43.
1,4-diazabicyclo[3.2.2]nonane-4-benzoxazole analogues 8-12
in 50-60% yields. This synthetic route was applied to the
preparation of azabenzoxazole analogues to give compounds
3a, 13-16, and 24-27 from the corresponding aminohydroxy-
pyridine 4b.
Results and Discussion
The unsubstituted azabenzoxazole 13 was used as a key
intermediate for preparing the 6-halo analogues 17-19 as
shown in Scheme 2. The 6-bromo (17) and 6-chloro (18)
analogues were prepared by direct halogenation of 13 using
bromine and N-chlorosuccinimide (NCS), respectively. The
6-fluoro analogue 19 was attained via the 6-amino intermedi-
ate, itself derived by nitration of 13 under acidic conditions
followed by catalytic hydrogenation of the nitro group,
through a Sandmeyer reaction using HPF6 as the fluoride
source. The 6-bromo analogue 17 was a versatileintermediate,
giving rapid access to a variety of 6-substituted azabenzox-
azoles: alkylation withdimethylzinc or s-butylboronic acidvia
palladium catalysis afforded compounds 20 and 22, respec-
tively; tributyl(vinyl)stannane Stille coupling followed by
hydrogenation gave 21; Suzuki couplings afforded com-
pounds 23, 29-31; cyanation using zinc cyanide yielded 28,
and aryl ether formation in the presence of copper chloride
provided 32.
Disubstituted analogues 33 and 34 were prepared from
5-methylazabenzoxazole 24 as depicted in Scheme 3. Nitra-
tion at the 6-position of 24 afforded the corresponding
6-nitro-5-methyl intermediate. Nitro reduction followed by
a HPF6 mediated Sandmeyer reaction gave the 6-fluoro-5-
methyl analogue 33. The corresponding 6-chloro-5-methyl
compound 34 was prepared by direct chlorination of 24
with NCS. Again, the 5-bromo intermediate 3a proved to be
The R7 nAChR and 5-HT3 binding activities and R7
nAChR functional data for a number of monosubstituted
benzoxazole and nonsubstituted azabenzoxazole analogues
are shown in Table 1. The unsubstituted benzoxazole ana-
logue 7 was characterized as a full agonist in a Xenopus oocyte
assay with good R7 nAChR affinity and equivalent potency at
the 5-HT3 receptor. As is apparent from the data, replacing
the carbamate functional group with a heterocyclic benzox-
azole proved to be a viable option, as 7 was ∼32-fold more
potent than 1 at the R7 nAChR. Paralleling SAR trends
observed with the carbamate series of compounds recently
described by our group,32 the SAR about the 5- and 6-posi-
tions of the benzoxazole ring improved affinity for the R7
nAChR (8-12). However, these substitutions at the 5- and
6-positions also introduced significant affinity for the 5-HT3
receptor, an activity we had not observed in the carbamate
series.32 Substitution at the distal position of the benzoxazole
ring with a halogen or alkyl group generally increased potency
for the R7 nAChR, but these substitutions did not improve
5-HT3 selectivity.36 Four monoazabenzoxazole regioisomers
(13-16) were also prepared in an attempt to broadly define
the SAR for this core. Both the 4-azabenzoxazole 13 and
7-azabenzoxazole 16 demonstrated potent R7 nAChR affinities,
maintained full agonist activity as shown in the functional assay,
and demonstrated potential for improved selectivity over the

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3 1225
Table 1. Structure-Activity Relationships of Benzoxazoles (7-12) and Azabenzoxazoles (13-16)
a Binding assay for rat R7 nAChR expressed in GH4C1 cells using [125I]BTX as the radioligand. Each assay (n) represents an average of a six-point
concentration-response curve run in triplicate, with geometric mean (95% confidence interval).37 b Rat R7 expressed in Xenopus oocytes. All test
compounds were measured at 32 μM and are reported as % control versus nicotine response at 50 μM.38 c Binding assay for mouse 5-HT3 receptors
expressed in HEK293 cells using [3H]LY278584 as the radioligand. All values represent an average of three six-point concentration-response curves run
in triplicate in a single assay.39
5-HT3 receptor, pointing to a promising direction for additional
SAR studies.
the 5- and 6-positions (R2 and R1, respectively, Table 2). In
general, compounds in the 4-azabenzoxazoles template (A)
displayed greater binding affinity at the R7 nAChR than their
counterparts in the 7-azabenzoxazole template (B) (for exam-
ple, see 18 vs 25, 24 vs 27) with the exception of the 5-phenyl
substitution, which displayed essentially identical R7 nAChR
binding affinities (23 vs 26). Halogens, -CN, and small alkyl
groups (-Me) were preferred at the 6-position of template A
(17-20, 28), with the chloro analogue 18 displaying the
greatest binding affinity for the R7 nAChR. Extension of
the methyl group of 20 to ethyl (21) resulted in reduced
affinity; however, replacing the methyl group with a branched
alkyl group such as sec-butyl (22) greatly reduced R7 nAChR
affinity, suggesting steric limitations at the 6-position. In
addition, substitution of this position with a sp2-hybridized
As a key objective of our program, we sought compounds
that were high affinity ligands at the R7 nAChR with potent
and full functional responses. To rapidly understand the SAR
in this series, each compound was evaluated in the R7 nAChR
binding assay and a high-throughput FLIPR-based func-
tional assay that utilized SH-EP1 cells expressing the R7/5-
HT3 chimera, in parallel. This chimera, combining the ligand
binding domain of the R7 nAChR and the transmembrane
domains of the 5-HT3 receptor, was previously established as
a rapid and viable option to develop SAR21,40 and linked the
data to previous clinical candidates from our group.22,29 Star-
ting from compounds 13 and 16, the SARs of 4-azabenzox-
azoles (A) and 7-azabenzoxazoles (B) were further explored at

1226 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3
O’Donnell et al.
Table 2. Structure-Activity Relationship of the 4-Azabenzoxazole and 7-Azabenzoxazole Templates
R7 binding
R7 efficacy
5-HT3
Ki (nM)c,d
compd
template
R1
R2
Ki (nM)a
EC50 (nM)b,d
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
A
A
A
A
A
A
A
A
B
-Br
-Cl
-F
-H
-H
-H
-H
-H
-H
-H
-Me
-Cl
-Ph
-H
-H
-H
-H
-H
-H
-Me
2.08 (1.63-2.66 n=22)
1.00 (0.533-1.88 n=4)
17.2 (11.7-25.3 n=4)
6.66 (4.25-10.3, n=4)
22.5 (n=1)
240 (64%)
160 (83%)
213 (64%)
1015 (55%)
300 (53%)
NT
926
1370
6200
3390
-Me
-Et
-sec-butyl
-Ph
>5260
NT
2117
>1,000 (n=1)
7.04 (1.34-37.1 n=4)
13.5 (7.31-24.9 n=6)
2.59 (2.11-3.18 n=4)
2.31 (0.466-11.4 n=5)
88.6 (51.0-154 n=2)
6.20 (n=1)
320 (71%)
244 (46%)
227 (62%)
264 (69%)
1200 (59%)
510 (58%)
390 (68%)
430 (70%)
NT
-H
-H
-H
269
NT
B
3673
126
B
-Me
-CN
-pyrazol-3-yl
-pyridin-3-yl
-pyrimid-5-yl
-OPh
-F
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
>5290
>5260
>5350
>7140
1960
23.0 (6.44-82.2 n=3)
119 (38.3-370 n=4)
60 (n=1)
4.16 (n=1)
620 (75%)
2100 (36%)
160 (58%)
100 (34%)
580 (68%)
525 (54%)
320 (71%)
160 (73%)
1000 (56%)
380 (63%)
1900 (30%)
470 (67%)
15.3 (0.158-1490 n=2)
3.19 (0.941-10.8 n=4)
18.7 (4.82-72.6 n=4)
9.05 (2.18-37.5 n=3)
5.30 (3.24-8.67 n=3)
3.22 (0.802-12.9 n=4)
3.45 (2.80-4.26 n=4)
24.9 (n=1)
547
244
-Cl
-H
-H
-H
-H
-H
-H
-H
-Me
-Et
>5260
>5290
>5290
>3580
>3770
141
-i-Pr
-cyclopentyl
-Ph
-2-fluoro-Ph
-OMe
-OPh
-piperidine
-pyrrolidine
17.3 (n=1)
67.1 (15.8-284 n=3)
1.91 (0.009-408 n=2)
6830
1,140
6340
-H
-H
a Binding assay for rat R7 nAChR expressed in GH4C1 cells using [125I]BTX as the radioligand. Each assay (n) represents an average of a six-point
concentration-response curve run in triplicate, with geometric mean (95% confidence interval).37 b R7 nAChR agonist EC50 and efficacy measured in a
functional assay using the chimeric R7-5HT3 receptor. Efficacy is reported as a percent of the response evoked by 100 μM nicotine.21 c Binding assay for
human 5-HT3 receptors expressed in HEK293 cells using [3H]LY278584 as the radioligand. All values represent an average of a six-point
concentration-response curve run in triplicate.39 d NT=not tested.
phenyl (23) or phenoxy (32) provided compounds with R7
nAChR affinity that was comparable to that of the 6-methyl
analogue 20. Replacing the phenyl group of 23 with 3-pyra-
zolyl, 3-pyridinyl, or 4-pyrimidinyl groups (29, 30, or 31,
respectively) decreased R7 nAChR binding affinity, a finding
consistent with the SAR observed in the previously reported
carbamate series.32
Encouraging data from the SAR sparked further explora-
tion of the 5-position of compounds such as 24 and the
evaluation of combining the SAR of the 5- and 6-positions
intoa singlemolecule. Two 5,6-bis-substitutedanalogueswere
profiled (33-34), and the 6-chloro-5-methyl analogue 34
showed improved affinity for the R7 nAChR, thus translating
into an improved selectivity over the 5-HT3 receptor com-
pared to 24, suggesting that the SAR trends observed for the
5- and 6-positions independently translated into a combine
synergistic effect. In contrast to the SAR trends observed with
6-mono substituted azabenzoxazoles, branched alkyl groups
at the 5-position of template A were very potent (35-37). For
example, when the 5-position of azabenzoxazole template A
was substituted with an isopropyl (36) or cyclopentyl group
(37), potent R7 nAChR affinity was observed, along with a
substantial decrease in 5-HT3 affinity. Simple phenyl substitu-
tion (38) led to a roughly 3-fold improvement in R7 nAChR
affinity vs 24 while greatly increasing selectivity over the
5-HT3 receptor (>700-fold). Ortho-F substitution on the
phenyl ring (39) further enhanced 5-HT3 receptor selectivity,
eliminating 5-HT3 binding without impacting R7 potency.
Alkoxy or amino substituents (40-43) also increased R7
nAChR potency and selectivity over the 5-HT3 receptor
(41-43).
Through these SAR efforts, we successfully identified a
cohort of compounds with both high affinity (<20 nM) and
potent functional responses (EC50 < 250 nM) such as com-
pounds 17-19, 24-26, 34-35, and 39. Derivatives that
exhibited a disconnect between the binding and functional
activity (e.g., 20, 23, 28, 29, 32-33, 36-38, and 43) or were
simply less active in either assay were deprioritized. All
analogues in Table 2 were agonists as demonstrated by their
percent maximal response in the functional assay relative to
the response of 100 μM nicotine. As a class, the azabenzox-
azole analogues also afford good to excellent binding selec-
tivity for the R7 nAChR over the 5-HT3 receptor (>100-fold)
with three notable exceptions (24, 27, and 34).

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3 1227
Table 3. Structure-Activity Relationship of the 4-Azabenzoxazole and 7-Azabenzoxazole Templates
R7 binding
Ki (nM)a
5-HT3 binding
Ki (nM)b,e
5-HT3 function
IC50 (nM)c,e
hERG
IC50 (nM)d,e
compd
IVMNe
17
18
19
24
25
34
35
39
2.08 (1.63-2.66 n=22)
1.00 (0.533-1.88 n=4)
17.2 (11.7-25.3 n=4)
13.5 (7.31-24.9 n=6)
2.59 (2.11-3.18 n=4)
3.19 (0.941-10.8 n=4)
18.7 (4.82-72.6 n=4)
3.45 (2.80-4.26 n=4)
926
1370
6200
269
779
484
2300
3300
pos
NT
pos
neg
NT
neg
neg
neg
2480
5
8700
40000
2200
NT
244
NT
70
16200
27700
2800
5260
>3770
3310
>7310
a Binding assay for rat R7 nAChR expressed in GH4C1 cells using [125I]BTX as the radioligand. Each assay (n) represents an average of a six-point
dose-response curve run in triplicate.37 b Binding assay for human 5-HT3 receptors expressed in HEK293 cells using [3H]LY278584 as the radioligand.
All values represent an average of a six-point dose-response curve run in triplicate.39 c 5-HT3 functional antagonist assay in human skin epithelial cells.
d Blockade of the hERG potassium channel in HEK293 cells.44 e NT=not tested.
As summarized in Table 3, a group of high interest com-
pounds (17-19, 24, 25, 34, 35, and 39) were further profiled in
key selectivity assays. Given the high degree of homology
between orthosteric sites in R7 nAChRs and 5-HT3 recep-
tors,41 some degree of cross-reactivity was anticipated and we
were therefore interested in identifying compounds that af-
forded reasonable binding selectivity and were functional
antagonists at the 5-HT3 receptor. We postulated that the
risk associated with 5-HT3 receptor antagonism was minimal
and, in fact, could be beneficial, since recent clinical studies
suggest that the potent and selective 5-HT3 receptor antago-
nist ondansetron is well tolerated in patients with schizophre-
nia for over 12 weeks of treatment and may provide benefits to
tardive dyskinesia and psychotic symptoms.42 In constrast,
compounds that displayed functional 5-HT3 agonism were
not of interest because of the potential link between this
pharmacology and cardiovascular events.43 All of the com-
pounds tested in this series displayed antagonist activity as
measured in a functional selectivity assay for the 5-HT3
receptor.
Prolongation of the QT interval is believed to increase the
risk of cardiac arrhythmia in humans, potentially leading to
ventricular fibrillation,45 and blockade of the hERG potas-
sium channel has become an important preclinical parameter
to assess a compound’s proarrhythmic potential.46 As such,
the set of compounds in Table 3 were evaluated in a patch
clamp assay to assess potential hERG liability (hERG IC50).
Monosubstituted compounds with bromide, chloride, fluor-
ide, or aryl substituents on the azabenzoxazole ring demon-
strated more potent hERG inhibition (17-19, 25, and 39),
whereas substitution at the 5-position (24, 35) or 5,6-disub-
stitution (34) displayed reduced hERG inhibition. In parti-
cular, compounds 24, 34, and 35 stood out as promising
compounds with hERG IC50 values of >15 μM.
Table 4. Selected Physicochemical Properties, in Vitro Characteristics,
ADME, and Pharmacokinetic Data for 24
parameter
value
MW
cLogP
258.33
1.57
0.36
log D at pH 7.4
TPSA
45.4
16.4 nM
195%
R7 Ki (human)a
% agonist vs nicotine (oocyte)b
R4β2 Kic
1350 nM
5370 nM
>2.4 mg/mL
>120 min
<5.3 (mL/min)/kg
0.8
R3β4 Kid
aq solubility
e
T1/2
HLM Clhf
MDR1 BA/ABg
MDCK ABh
18.0 ꢀ 10-6 cm/s
plasma protein binding ( fu) (rat)
brain/plasmai
F (rat)
0.78
1.5
73%
a Binding assay for human R7 nAChR expressed in IMR-32 cells
using [125I]BTX as the radioligand. Each assay (n) represents an average
of a six-point concentration-response curve run in triplicate. b Rat R7
nAChR expressed in Xenopus oocytes. The test compound was mea-
sured at 32 μM and is reported as % control versus nicotine response at
50 μM.38 c Rat brain homogenate binding assay using [3H]nicotine as the
radioligand. This value represents an average of a six-point dose-
response curve run in triplicate.51 d IMR32 cells binding assay using
[3H]epibatidine as the radioligand. This value represents an average of a
six-point concentration-response curve run in triplicate.52 e Derived
from human liver microsomes. f Predicted hepatic clearance (hCLh)
from human liver microsomal stability assay. g Ratio of (basal f apical)
to (apical f basal) transfer rate of a test compound at 2 μM across
contiguous monolayers of MDR1-transfected MDCK (Madin-Darby
canine kidney) cells. h MS-based quantification of apical f basolateral
transfer rate of a test compound at 2 μM across contiguous monolayers
of MDCK cells. i Determined in rat at 40 min postdose (1 mg/kg, sc).
The potential for genetic toxicity was assessed in an in vitro
micronucleus (IVMN) assay (Table 3).47 6-Halogen substi-
tuted azabenzoxazoles 17 and 19 were positive in the IVMN
assay upon metabolic activation. Compounds substituted at
the 5-position of the azabenzoxazole ring such as 24, 34, 35,
and 39 proved to be negative in the IVMN assay, indicating
the vital role of the 5-substituent on the azabenzoxazole ring
to avoid this potential risk. The overall in vitro potency and
selectivity profiles at R7 nAChR, 5-HT3, hERG, and IVMN
prompted further evaluation of compound 24 in nicotinic
selectivity assays, in vitro and in vivo ADME studies, and our
in vivo efficacy models.
clearly in alignment with our design goals of low molecular
weight (MW), low cLogP and cLogD, and topological polar
surface area (TPSA) consistent withCNS drugs.48 Compound
24 had been identified as a potent R7 nAChR ligand in a
binding assay using the rat receptor (13.5 nM, Table 3), and
this potency was confirmed to be 16.4 nM using a human
receptor. The functional agonism of 24 was also confirmed
with native R7 nAChRs utilizing rat R7 nAChR expressed in
Xenopus oocytes (195% vs 50 μM nicotine). In evaluating
The physicochemical properties, in vitro characteristics,
ADME, and pharmacokinetic data for compound 24 are
captured in Table 4. The physicochemical properties are

1228 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3
O’Donnell et al.
Figure 4. Ex vivo binding of 24 in rat hippocampal homogenate.
nicotinic selectivity, 24 was found to be selective against the
predominant ganglionic nAChR (R3β4) and the high affinity
neuronal nAChR (R4β2) as shown in Table 4. In addition, 24
was screened in a broad selectivity panel at 10 μM and found
to have no significant additional activities.49
Compound 24 possessed excellent solubility and ADME
attributes (Table 4). In vitro ADME data showed that 24 was
metabolically stable in the presence of human liver micro-
somes (hCLh<5.3 (mL/min)/kg), resulting ina long projected
half-life. The compound was also well absorbed (MDCK A f
B, g18 ꢀ 10-6 cm/s) with no evidence of P-gp efflux liability
(MDR1 B f A/A f B, ∼1).50 Subsequent in vivo studies
validated these in vitro results, as compound 24 demonstrated
high oral bioavailability and favorable brain/plasma ratios in
rats (Table 4), with no evidence of impaired brain penetration
using the P-gp knockout mouse model (data not shown).
Furthermore, in these P-gp knockout mice, the ratio
[CSF]/[total plasma] vs fraction unbound was 0.8, sug-
gesting that there is no disequilibrium and the compound
readily distributes to the target site of the brain.
The overall properties of compound 24 prompted further
characterization in models to fully understand its potential in
vivo. Ex vivo binding studies evaluated exposure of 24 at the
target site following systemic administration. Receptor bind-
ing under these conditions is dependent on the access of the
compound to the receptor and is therefore influenced by brain
uptake, protein binding, and other partitioning that may
occur in the brain. Receptor occupancy was determined
in the hippocampal brain regions of rats 30 min after sub-
cutaneous (sc) injections of 24 or vehicle (3 animals/dose
group) using [125I]R-bungarotoxin. The dose and ex vivo
derived receptor occupancy relationship yielded an ED50 of
0.34 mg/kg for 24 (Figure 4). These results are consistent with
in vitro potencies reported in Table 2 and reveal that the
compound enters the brain and binds to native hippocampal
R7 nAChRs at pharmacologically relevant doses.
It has been hypothesized that impaired auditory gating
(P50), observed in patients with schizophrenia, may relate to
inefficient sensory processing and disturbed perception com-
mon to the disease.53 P50 was measured in a rat model of
auditory sensory gating in which normal auditory gating was
disrupted by systemic administration of amphetamine.54 Pre-
vious work has shown that amphetamine-induced gating
deficits can be restored by partial and full R7 nAChR ago-
nists.53,55,56 Compound 24 significantly improved auditory
gating at 0.3 and 1.0 mg/kg sc (p < 0.05; Figure 5A). Lower
doses (0.03 and 0.1 mg/kg) trended toward reversing
amphetamine-induced deficits, but the effect was not statisti-
cally significant. These data were used to establish a minimally
effective dose (MED) of 24 in the gating assay of 0.3 mg/kg.
Figure 5. (A) Efficacy of 24 (0.03-1 mg/kg, sc) on amphetamine
(AMP)-induced auditory gating deficit in rats, expressed as reversal
of amphetamine-induced deficit. Significant ( p < 0.05) differences
in gating after amphetamine and R7 nAChR agonists administra-
tion are indicated by an asterisk (/). Vehicle treatment (PBS) did not
significantly impact amphetamine-induced gating deficit. (B) Power
of hippocampal θ oscillation induced by amphetamine (AMP)
significantly augmented by administration of 24 (1 mg/kg, sc, (#)
p<0.05 vs AMP) but not vehicle (PBS).
The free plasma exposure achieved in rats given 0.3 mg/kg, sc,
of 24 was 326 nM, approximately 40 min after dosing. On the
basis of the measured brain to plasma ratio (Table 4) and a
lack of P-gp efflux, there is low likelihood of disequilibrium
between drug concentrations in plasma and brain; therefore,
free brain concentrations should be comparable.
Consistent with previous findings,57 amphetamine admin-
istration resulted in θ band synchronization of hippocampal
EEG. Quantitative EEG analysis showed that subsequent
administration of 24 but not vehicle (PBS, 1 mL/kg iv)
significantly enhanced θ band power (Figure 5B), in line with
our previous observations.58 These results demonstrate that
R7 nAChRs agonists can synchronize ongoing hippocampal
oscillations, leading to a significantly augmenting θ power,
suggesting thatitcould beoneof the contributing mechanisms
to procognitive actions of R7 nAChRs agonists. Importantly,
doses at which efficacy was observed in auditory gating for 24
were consistent with ex vivo binding.
The rat novel object recognition task (NOR) is thought to
be comparable to delayed-matching-to-sample tasks that are
commonly utilized in both nonhuman primates and humans
to study working memory performance.59 Results in rat indi-
cate that 24 significantly improved the scopolamine-impaired

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3 1229
as described above. The final pellet was resuspended in 20 mL of
the same buffer. Radioligand binding was carried out with
[
125I]R-bungarotoxin from New England Nuclear, specific ac-
tivity of about 16 μCi/μg, used at 0.4 nM final concentration in a
96-well microtiter plate. The plates were incubated at 37 °C for
2 h with 25 μL of drugs or vehicle for total binding, 100 μL of
[
125I]bungarotoxin, and 125 μL of tissue preparation. Nonspe-
cific binding was determined in the presence of methyllycaconi-
tine at 1 μM final concentration. The reaction was terminated by
filtration using 0.5% polyethylene imine treated Whatman GF/
B glass fiber filters (Brandel Biomedical Research & Develop-
ment Laboratories, Inc., Gaithersburg, MD) on a Skatron cell
harvester (Molecular Devices Corp., Sunnyvale, CA) with ice-
cold buffer; filters were dried overnight and counted on a Beta
plate counter using Betaplate Scint (Wallac Inc., Gaithersburg,
MD). Data are expressed as IC50 values (concentration that
inhibits 50% of the specific binding) or as an apparent Ki,
IC50/(1 þ [L]/KD), where [L] is the ligand concentration and
KD is the affinity constant for [125I] ligand determined in a
separate experiment.
r7 Nicotinic Receptor Functional Assay in Oocyte. Xenopus
oocytes were harvested surgically and treated with collagenase
(1.3 mg/mL) for 3 h to remove the follicular layer. The oocytes
were injected with 10-50 ng of rat R7 neuronal nicotinic
receptor cRNA and stored in Barth’s saline for up to 2 weeks.
Electrophysiological recordings were performed 4-10 days
later using a two-electrode voltage clamp. The oocytes were
placed in the recording chamber and superfused with Ringer’s
saline (in mM: 115 NaCl, 2.5 KCl, 0.4 BaCl2, 0.1 CaCl2, 10
HEPES, pH 7.5) containing agonists or antagonists. Electro-
des are filled with 3 M KCl. Holding potential (Vh) was -60 or
-90 mV. Currents induced by the application of drugs were
digitized by a Data Translation analog/digital board and ana-
lyzed with Axodata software. All test compounds were tested at
32 μM and are compared to a test dose of nicotine at 50 μM.
Data are reported as % nicotine response with the response of
nicotine at 50 μM set at 100%.
Figure 6. Efficacy of 24 in rat novel object recognition assay. Rats
were habituated to the test arena for 3 min on day 1. The following
day, animals (10-15 per group) were first dosed (sc) with either
vehicle or test compound followed 30 min later by injection of either
vehicle or (-)-scopolamine (0.2 mg/kg, sc). After an additional
30 min, two identical objects were presented for 3 min. Two and a
half hours later, rats were exposed to one novel and one familiar
object for 3 min. The time spent exploring each object (mean time (s) (
SEM) is plotted.
performance at both 0.32 and 1.0 mg/kg (sc) (Figure 6). This is
consistent with prior reports demonstrating efficacy of R7
nAChR agonists in object recognition in mouse and further
supports the notion that selective activation of this receptor
may improve some aspects of cognitive function.22a,60 Addi-
tionally, the effective doses of 24 in NOR are in complete
alignment with the effective doses in both the ex vivo binding
and (AMP)-induced auditory gating assays.
Conclusions
Construction and Expression of the Chimeric r7-5HT3 Recep-
tor. The cDNA encoding the N-terminal 201 amino acids from
the human R7 nAChR that contain the ligand binding domain
of the ion channel was fused to the cDNA encoding the pore
forming region of the mouse 5HT3 receptor as described by
Eisele.40 The chimeric R7-5HT3 ion channel was inserted into
pGS175 and pGS179 which contain the resistance genes for
G-418 and hygromycin B, respectively. Both plasmids were
simultaneously transfected into SH-EP1 cells, and cell lines were
selected that were resistant to both G-418 and hyrgromycin B.
Cell lines expressing the chimeric ion channel were identified by
their ability to bind fluorescent R-bungarotoxin on their cell
surface. The cells with the highest amount of fluorescent
R-bungarotoxin binding were isolated using a fluorescent acti-
vated cell sorter (FACS). Cell lines that stably expressed the
chimeric R7-5HT3 were identified by measuring fluorescent
R-bungarotoxin binding after growing the cells in minimal
essential medium containing nonessential amino acids supple-
mented with 10% fetal bovine serum, L-glutamine, 100 U/mL
penicillin/streptomycin, 250 ng/mg fungizone, 400 μg/mL
hygromycin B, and 400 μg/mL G-418 at 37 °C with 6% CO2
in a standard mammalian cell incubator for at least 4 weeks in
continuous culture.
The synthesis and in vitro and in vivo evaluation of a series
of 1,4-diazabicyclo[3.2.2]nonane azabenzoxazole R7 nAChR
agonists have been described. Of the azabenzoxazoles evalu-
ated, compound 24 (CP-810,123) exhibited an excellent bal-
ance of potency, selectivityand high affinity agonist activityat
the R7 nAChR. The compound’s in vitro profile is comple-
mented by excellent brain penetration, high oral bioavailabil-
ity in rats, and an acceptable preclinical cardiovascular safety
profile with a favorable hERG and genetic toxicology profile.
It demonstrates high receptor occupancy at low doses, in vivo
efficacy in an amphetamine induced P50 gating deficit model,
and improved performance in novel object recognition test.
A good correlation between the in vitro data, ex vivo binding,
and exposure required for in vivo efficacy demonstrates the
solid in vitro to in vivo correlation for 24. The structural
diversity of this class of compounds, when compared to our
previous clinical entries, combined with its preclinical evalua-
tion package supports thehypothesis thatR7 nAChR agonists
may have potential as a pharmacotherapy for the treatment of
cognitive deficits in schizophrenia.
Functional Assay for the Chimeric r7-5HT3 Receptor Agonist
Activity. To assay the activity of the R7-5HT3 ion channel, cells
expressing the channel were plated into each well of either a 96-
or 384-well dish (Corning no. 3614) and grown to confluence
prior to assay. On the day of the assay, the cells were loaded with
a 1:1 mixture of 2 mM Calcium Green 1, AM (Molecular
Probes) dissolved in anhydrous DMSO, and 20% pluronic
F-127 (Molecular Probes). This solution was added directly to
the growth media of each well to achieve a final concentration of
2 μM. The cells were incubated with the dye for 60 min at 37 °C
Experimental Section
Biology. r7 Nicotinic Receptor Binding. Membrane prepara-
tions were made for nicotinic receptors expressed in GH4Cl cell
line. Briefly, 1 g of cells by wet weight was homogenized with a
Polytron in 25 mL of buffer containing 20 mM HEPES, 118 mM
NaCl, 4.5 mM KCl, 2.5 mM CaCl2, 1.2 mM MgSO4, pH 7.5.
The homogenate was centrifuged at 40000g for 10 min at 4 °C,
and the resulting pellet was homogenized and centrifuged again

1230 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3
O’Donnell et al.
and were washed with a modified version of Earle’s balanced
salt solution (MMEBSS) as described in WO 00/73431.40b The
ion conditions of the MMEBSS was adjusted to maximize the
flux of calcium ion through the chimeric R7-5HT3 ion channel as
described in WO 00/73431.40b The activity of compounds on the
chimeric R7-5HT3 ion channel was analyzed on FLIPR. The
instrument was set up with an excitation wavelength of 488 nm
using 500 mW of power. Fluorescent emission was measured
above 525 nm with an appropriate F-stop to maintain a maximal
signal-to-noise ratio. Agonist activity of each compound was
measured by directly adding the compound to cells expressing
the chimeric R7-5HT3 ion channel and measuring the resulting
increase in intracellular calcium that is caused by the agonist-
induced activation of the chimeric ion channel. The assay is
quantitative such that concentration-dependent increase in
intracelluar calcium is measured as concentration-dependent
change in Calcium Green fluorescence. The effective concentration
needed for a compound to cause a 50% maximal increase relative
to 100 μM nicotine in intracellular calcium is termed the EC50.
5-HT3 Receptor Binding. Radioligand binding studies were
performed according to Wong with some modifications.39
Briefly, frozen cell paste (HEK293 cells expressing mouse or
human 5-HT3A receptors) was homogenized using a Brinkman
Polytron model PT3000 (setting 15 000 rpm, 15 s) in 50 mM Tris-
HCl buffer, pH 7.4, containing 2 mM MgCl2. The homogenate
was centrifuged for 10 min at 40000g, washed, and recentri-
fuged. The final pellet was resuspended in 20 mM Tris-HCl
buffer, pH 7.4, at 37 °C containing 154 mM NaCl (3.5 mg/mL).
Incubations were initiated by the addition of tissue homogenate
to wells of 96-well plates containing 3H-LY-278584 (1 nM, final
concentration) and varying concentrations of test compound,
buffer, or 10 μM ICS205-930 in a final volume of 250 μL.
Nonspecific binding was defined as the radioactivity remaining
in the presence of a saturating concentration of ICS205-390.
After a 60 min incubation at 37 °C, assay samples were filtered
onto GF/B filtermats presoaked in 0.5% polyethylenimine,
using a Skatron cell harvester (Molecular Devices), and washed
with ice-cold 50 mM Tris buffer, pH 7.4, at 4 °C. Radioactivity
was quantified by liquid scintillation counting (Betaplate,
Wallac Instruments). The IC50 value (concentration at which
50% inhibition of specific binding occurs) was calculated by
linear regression of the concentration-response data. Ki values
were calculated according to Cheng and Prusoff, where Ki =
IC50/(1 þ (L/Kd)), where L is the concentration of the radio-
ligand used in the experiment and the Kd value is the dissociation
constant for the radioligand (determined previously by satura-
tion analysis). The data reported in the tables represent the
average of three six-point dose-response curves that were run in
a single assay. Tropisetron was used as an internal standard in
each assay and had Ki = 2.26 ( 0.29 nM (n = 11).
the assay plate for 2 min from a 4ꢀ stock solution. Antagonist
activity was determined as the inhibition of the fluorescence
signal in response to a subsquent challenge with 5-HT for an
additional 2 min (250 nM final from a 4ꢀ stock).
r7 Nicotinic ex Vivo Binding. Each hippocampus was homo-
genized with a Polytron in 4.5 mL of buffer containing 20 mM
HEPES, 118 mM NaCl, 4.5 mM KCl, 2.5 mM CaCl2, 1.2 mM
MgSO4, pH 7.5. Radioligand binding was carried out with
[
125I]R-bungarotoxin from New England Nuclear, specific ac-
tivity of about 16 μCi/μg, used at 0.4 nM final concentration in a
96-well microtiter plate. The plates were incubated at 37 °C for
2 h with 0.4 nM [125I]bungarotoxin and 750 μL of tissue
homogenates in a 1 mL assay. Nonspecific binding was deter-
mined in the presence of methyllycaconitine at 1 μM final
concentration. The reaction was terminated by filtration using
0.5% polyethylene imine treated Whatman GF/B glass fiber
filters (Brandel Biomedical Research & Development Labora-
tories, Inc., Gaithersburg, MD) on a Skatron cell harvester
(Molecular Devices Corp., Sunnyvale, CA) with ice-cold buffer;
filters were dried overnight and counted on a Beta plate counter
using Betaplate Scint (Wallac Inc., Gaithersburg, MD). Data
are expressed as ED50 values (concentration that inhibits 50% of
the specific binding).
Auditory Gating Assay. Experiments were performed on male
Sprague-Dawley rats (weighing 250-300 g) under chloral
hydrate anesthesia (400 mg/kg, ip). The femoral vein was
cannulated for drug administration or additional doses of
anesthetic. Unilateral hippocampal field potential (EEG) was
recorded by a metal monopolar macroelectrode placed into the
CA3 region (coordinates: 3.0-3.5 mm posterior from the breg-
ma, 2.6-3.0 mm lateral, and 3.8-4.0 mm ventral). Field poten-
tials were amplified, filtered (0.1-100 Hz), displayed, and
recorded for on-line and off-line analysis; quantitative EEG
analysis was performed by means of fast Fourier transformation
(Spike3). The auditory stimulus consisted of a pair of 10 ms,
5 kHz tone bursts with a 0.5 s delay between the first “con-
ditioning” stimulus and second “test” stimulus. Auditory-
evoked responses were computed by averaging of responses to
50 pairs of stimuli presented with an interstimulus interval of
10 s. Amphetamine (d-amphetamine sulfate, 1 mg/kg, iv) was
administered in order to disrupt sensory gating. By use of fast
Fourier transform, the relative θ power was determined by
calculating the percentage of total power that occurred in the
θ (3.5-5.5 Hz) frequency band compared with the 0-15 Hz
frequency band. Statistical significance was determined by
means of two-tailed paired Student’s t-test.
Chemistry. General Methods. Solvents and reagents were of
reagent grade and were used as supplied by the manufacturer.
All reactions were run under a N2 atmosphere. Organic extracts
were routinely dried over anhydrous Na2SO4. Concentration
refers to rotory evaporaration under reduced pressure. Chro-
matography refers to flash chromatography using disposable
RediSepRf 4-120 g silica columns or Biotage disposable col-
umns on a CombiFlash Companion or Biotage Horizon auto-
matic purification system. Microwave reactions were carried out
in a microwave reactor manufactured by Smithcreator of Per-
sonal Chemistry. Purification by mass-triggered HPLC was
carried out using Waters XTerra PrepMS C18 columns, 5 μm,
30 mm ꢀ 100 mm steel. Compounds were presalted as TFA salts
and diluted with 1 mL of dimethyl sulfoxide. Samples were
purified by mass triggered collection using a mobile phase of
0.1% TFA in water and acetonitrile with a starting gradient of
100% aqueous to 100% acetonitrile over 10 min at 20 mL/min
flow rate. Elemental analyses were performed by QTI, White-
house, NJ. All target compounds were analyzed using ultrahigh
performance liquid chromatography/ultraviolet/evaporative
light scattering detection coupled to time-of-flight mass spectro-
metry (UHPLC/UV/ELSD/TOFMS). Unless otherwise noted,
all tested compounds were found to be g95% pure by this
method.
Functional Assay for 5-HT3 Receptor Antagonist Activity. The
human 5-HT3A receptor was stably expressed in human skin
epithelial cells. Functional activity was evaluated as calcium flux
using the fluorescence imaging plate reader (FLIPR, Molecular
Devices). Cells were grown in DMEM supplemented with 10%
FBS, L-glutamine, sodium pyruvate, 500 μg/mL Geneticin, and
100 units/mL penicillin/streptomycin. The cells were trypsinized
and plated in 384-well plates with dark side walls and clear
bottoms at a density of 15 000 cells/well 2 days before analysis.
Cells were loaded in a 1:1 mixture of 8 mM Fluo-4 stock and
20% Pluronic F-127 (Molecular Probes). This reagent was
added directly to the growth medium of each well to achieve a
final concentration of 8 μM Fluo-4 in plating medium (same as
above but without Geneticin). Cells were then incubated in the
dye for 1 h at 37 °C and then washed 4 times in assay buffer
comprising Hank’s balanced salt solution supplemented with
20 mM Tris-HEPES and 1 μM atropine. CaCl2 was added to
achieve 4 mM Ca2þ, and the pH was asjusted to 7.4. FLIPR was
used to measure changes in intracellular Ca2þ. After 30 s of
baseline recording, test compounds were added to each well of

Article
UPLC/MS Analysis. The UPLC was performed on a Waters
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3 1231
prepared by diluting in ethyl acetate and adding a solution of
2.5 N HCl in ethyl acetate.
ACQUITY UPLC system (Waters, Milford, MA), which was
equipped with a binary solvent delivery manager, column
manager, and sample manager coupled to ELSD and UV
detectors (Waters, Milford, MA). Detection was performed on
a Waters LCT premier XE mass spectrometer (Waters, Milford,
MA). The instrument was fitted with an Acquity BEH (bridged
ethane hybrid) C18 column (30 mm ꢀ 2.1 mm, 1.7 μm, Waters,
Milford, MA) operating at 60 °C.
4-(5-Fluorobenzoxazol-2-yl)-1,4-diazabicyclo[3.2.2]nonane (8).
The free base of the title compound was prepared from 2-amino-
4-fluorophenol by the methods described for the preparation of
10 in 54% overall yield: 1H NMR (CDCl3, 400 MHz) δ 7.12 (dd,
1H, J = 8.7, 4.6 Hz), 7.01 (dd, 1H, J = 9.1, 2.5 Hz), 6.70-6.65
(m, 1H), 4.54-4.51 (m, 1H), 3.94 (t, 2H, J=5.8 Hz), 3.24-3.17
(m, 4H), 3.11-3.03 (m, 2H), 2.21-2.14 (m, 2H), 1.90-1.82 (m,
2H); MS (CI) m/z 262.1 (M þ 1). The hydrochloride salt was
prepared by diluting in ethyl acetate and adding a 2.5 N HCl
solution in ethyl acetate.
4-Benzoxazo-2-yl-1,4-diazabicyclo[3.2.2]nonane (7). 2-Chloro-
benzoxazole (99 μL, 0.87 mmol) was added to a solution of
1,4-diazabicyclo[3.2.2]nonane 6 (100 mg, 0.79 mmol) in metha-
nol (2.65 mL) at 0 °C. The reaction mixture was allowed to
slowly warm to RT. After a period of 16 h i-Pr2NEt (138 μL, 0.79
mmol) was added and the mixture was stirred at RT for 4.5 h, at
which time it was diluted with CHCl3 and NaHCO3. The layers
were partitioned, and the aqueous layer was extracted with
CHCl3 (ꢀ3). The combined organic layers were washed with
H2O and brine, dried (Na2SO4), filtered, and concentrated. The
crude residue was purified by chromatography (Biotage, 12 L),
eluting with 4% MeOH in CHCl3 containing 20 drops of
NH4OH per liter of eluent to afford 67 mg (35%) of the free
4-(5-Chlorobenzoxazol-2-yl)-1,4-diazabicyclo[3.2.2]nonane (9).
The free base of the title compound was prepared from 2-amino-
4-chlorophenol by the methods described for the preparation of
10 in 48% overall yield: 1H NMR (CDCl3, 400 MHz) δ 7.25 (d,
1H, J=2.1 Hz), 7.09 (d, 1H, J=8.3 Hz)), 6.91 (dd, 1H, J=8.3,
2.1 Hz), 4.49-4.47 (m, 1H), 3.90 (t, 2H, J=5.8 Hz), 3.20-3.12
(m, 4H), 3.07-2.99 (m, 2H), 2.17-2.09 (m, 2H), 1.85-1.77 (m,
2H); 13C NMR (CDCl3, 100 MHz) δ 162.4, 147.5, 145.0, 129.4,
120.2, 116.2, 109.3, 57.0, 50.4, 46.3, 44.0, 26.7; MS (APCI) m/z
278.1 (M þ 1). The hydrochloride salt was prepared by diluting
in ethyl acetate and adding a 2.5 N HCl solution in ethyl acetate.
4-(5-Methylbenzoxazol-2-yl)-1,4-diazabicyclo[3.2.2]nonane (11).
The free base of the title compound was prepared from 2-amino-
4-methylphenol by the methods described for the preparation of
10: 1H NMR (CDCl3, 400 MHz) δ 7.14 (s, 1H), 7.10 (d, 1H, J=
7.9 Hz), 6.80 (dd, 1H, J=7.9, 0.8 Hz), 4.55-4.53 (m, 1H), 3.95
(t, 2H, J=5.8 Hz), 3.24-3.17 (m, 4H), 3.10-3.03 (m, 2H), 2.38
(s, 3H), 2.22-2.15 (m, 2H), 1.89-1.81 (m, 2H); MS (APCI) m/z
258.2 (M þ 1). The hydrochloride salt was prepared by diluting
in ethyl acetate and adding a 2.5 N HCl solution in ethyl acetate.
4-(6-Methylbenzoxazol-2-yl)-1,4-diazabicyclo[3.2.2]nonane (12).
The free base of the title compound was prepared from 2-amino-
5-methylphenol by the methods described for the preparation of
10 in 55% overall yield: 1H NMR (CDCl3, 400 MHz) δ 7.21 (d,
1H, J=7.9 Hz), 7.06 (s, 1H), 6.96 (d, 1H, J=8.3 Hz), 4.55-4.52
(m, 1H), 3.94 (t, 2H, J=5.8 Hz), 3.23-3.15 (m, 4H), 3.09-3.01
(m, 2H), 2.39 (s, 3H), 2.22-2.14 (m, 2H), 1.89-1.80 (m, 2H);
MS (APCI) m/z 258.2 (M þ 1). The hydrochloride salt was
prepared by diluting in ethyl acetate and adding a 2.5 N HCl
solution in ethyl acetate.
4-Oxazolo[4,5-b]pyridin-2-yl-1,4-diazabicyclo[3.2.2]nonane (13).
The free base of the title compound was prepared from
2-(methylthio)oxazolo[4,5-b]pyridine34 by the methods des-
cribed for the preparation of 10 in 98% yield: 1H NMR
(CDCl3, 400 MHz) δ 8.14 (dd, 1H, J = 5.0, 1.2 Hz), 7.34 (dd,
1H, J=7.5, 1.2 Hz), 6.81 (dd, 1H, J=7.8, 5.0 Hz), 4.50 (s, 1H),
3.90 (t, 2H, J=5.8 Hz), 3.13-3.05 (m, 4H), 2.98-2.91 (m, 2H),
2.13-2.05 (m, 2H), 1.79-1.71 (m, 2H); 13C NMR (CDCl3, 100
MHz) δ 163.1, 158.7, 144.7, 141.4, 115.4, 114.8, 57.1, 50.6, 46.4,
46.3, 44.4, 30.3, 26.9; MS (APCI) m/z 245.2 (M þ 1). The
hydrochloride salt was prepared by diluting in ethyl acetate
and adding a 2.5 N HCl solution in ethyl acetate.
4-Oxazolo[4,5-c]pyridin-2-yl-1,4-diazabicyclo[3.2.2]nonane (14).
The free base of the title compound was prepared from
2-(methylthio)oxazolo[4.5-c]pyridine34 by the methods de-
scribed for the preparation of 10 in 32% yield: 1H NMR
(CDCl3, 400 MHz) δ 8.57 (s, 1H), 8.21 (d, 1H, J = 5.4 Hz),
7.16 (d, 1H, J = 5.4 Hz), 4.46-4.45 (m, 1H), 3.88 (t, 2H, J =
5.8 Hz), 3.14-3.03 (m, 4H), 3.00-2.93 (m, 2H), 2.13-2.06 (m,
2H), 1.82-1.74 (m, 2H); 13C NMR (CDCl3, 100 MHz) δ 161.6,
154.3, 142.0, 141.4, 138.0, 104.9, 62.3, 57.1, 50.8, 46.3, 44.6, 30.3,
26.9; MS (APCI) m/z 245.2 (M þ 1). The hydrochloride salt was
prepared by diluting in ethyl acetate and adding a 2.5 N HCl
solution in ethyl acetate.
1
base of the title compound as a yellow oil: H NMR (CDCl3,
400 MHz) δ 7.30 (d, 1H, J = 7.5 Hz), 7.19 (d, 1H, J =7.9 Hz),
7.10 (t, 1H, J=7.5 Hz), 6.94 (t, 1H, J=7.9 Hz), 4.46 (s, 1H), 3.87
(t, 2H, J = 5.8 Hz), 3.12-2.92 (m, 6H), 2.15-2.05 (m, 2H),
1.79-1.70 (m, 2H); 13C NMR (CDCl3, 100 MHz) δ 161.8, 148.9,
143.7, 124.1, 120.3, 116.1, 108.7, 57.3, 50.3, 46.5, 44.4, 27.1; MS
(APCI) m/z 244.3 (M þ 1).
4-(5-Bromobenzoxazol-2-yl)-1,4-diazabicyclo[3.2.2]nonane
(10). Step 1. Potassium ethyl xanthate (416 mg, 2.60 mmol) was
added to a solution of 2-amino-4-bromophenol (4a, 244 mg,
1.30 mmol) in EtOH (3.24 mL). The reaction mixture was heated
at reflux for 4 h. Upon cooling to RT, the mixture was
concentrated and the resulting residue was dissolved in water.
Acetic acid was added until pH 5 was obtained and a white solid
precipitated from the solution. The solid was filtered, washed
with water, and dried to afford 270 mg (90%) of 5-bromo-3H-
benzooxazole-2-thione as a tan powder which was used without
1
further purification: H NMR (DMSO-d6, 400 MHz) δ 14.02
(s, 1H), 7.47-7.38 (m, 3H); 13C (DMSO-d6, 400 MHz) δ 181.4,
148.1, 133.7, 127.1, 117.8, 118.8, 112.2; MS (CI) m/z 229.8
(M - 1).
Step 2. 5-Bromo-3H-benzooxazole-2-thione (530 mg, 2.30
mmol) was dissolved in DMF (5.75 mL). Potassium carbonate
(318 mg, 2.30 mmol) and iodomethane (172 μL, 2.76 mmol) were
added, and the reaction mixture was allowed to stir at RT for
3.5 h. The mixture was diluted with water (10 mL) and extracted
with ethyl acetate (4 ꢀ 10 mL). The combined organic extracts
were washed with water (3 ꢀ 10 mL), brine (10 mL), dried
(Na2SO4), filtered, and concentrated to afford 538 mg (96%) of
5-bromo-2-methylsulfanylbenzooxazole (5) as a dark-brown
solid: 1H NMR (CDCl3, 400 MHz) δ 7.72 (d, 1H, J = 2.1 Hz),
7.36-7.26 (m, 2H), 2.75 (s, 3H); 13C NMR (CDCl3, 100 MHz) δ
167.6, 151.2, 143.8, 126.9, 121.6, 117.3, 111.2, 14.8; MS (CI) m/z
244.0 (M þ 1).
Step 3. 1,4-Diazabicyclo[3.2.2]nonane (57%, 731 mg, 3.31
mmol) was added to a solution of 5-bromo-2-methylsulfanyl-
benzooxazole (538 mg, 2.20 mmol) in i-PrOH (4.4 mL). The
mixture was placed in an oil bath at 90 °C, and the solvent was
evaporated. The mixture was allowed to stir neat at 90 °C for
18 h. Upon cooling to RT, the mixture was purified by chromato-
graphy (Biotage, 25 M), eluting with 4% MeOH in CHCl3
with 20 drops of NH4OH per liter of eluent to afford 392 mg
(55%) of compound 10 as an oil: 1H NMR (CDCl3, 400 MHz) δ
7.40 (t, 1H, J=1.2 Hz), 7.05 (d, 2H, J=1.2 Hz), 4.46-4.43 (m,
1H), 3.87 (t, 2H, J=5.8 Hz), 3.14-2.93 (m, 6H), 2.13-2.06 (m,
2H), 1.81-1.73 (m, 2H); 13C NMR (CDCl3, 100 MHz) δ 162.3,
148.0, 145.6, 122.9, 119.0, 116.8, 109.8, 57.2, 50.5, 46.5, 44.4,
27.0; MS (CI) m/z 322.0 (M þ 1). The hydrochloride salt was
4-Oxazolo[5,4-c]pyridin-2-yl-1,4-diazabicyclo[3.2.2]nonane (15).
The free base of the title compound was prepared from
2-(methylthio)oxazolo[5,4-c]pyridine34 by the methods des-
cribed for the preparation of 10 in 67% yield: 1H NMR

1232 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3
O’Donnell et al.
(CDCl3, 400 MHz) δ 8.44 (s, 1H), 8.27 (d, 1H, J=5.0 Hz), 7.19
(d, 1H, J = 5.3 Hz), 4.48 (s, 1H), 3.90 (t, 2H, J = 5.8 Hz),
3.14-3.07 (m, 4H), 3.00-2.93 (m, 2H), 2.12-2.07 (m, 2H),
1.82-1.74 (m, 2H); 13C NMR (CDCl3, 100 MHz) δ 163.4, 150.9,
147.5, 145.5, 129.8, 111.6, 62.3, 50.9, 46.4, 44.7, 30.3, 26.9; MS
(APCI) m/z 245.2 (M þ 1). The hydrochloride salt was prepared
by diluting in ethyl acetate and adding a 2.5 N HCl solution in
ethyl acetate.
4-Oxazolo[5,4-b]pyridin-2-yl-1,4-diazabicyclo[3.2.2]nonane (16).
The free base of the title compound was prepared from
2-(methylthio)oxazolo[5.4-b]pyridine34 by the methods des-
cribed for the preparation of 10 in 72% yield: 1H NMR
(CDCl3, 400 MHz) δ 7.83 (dd, 1H, J = 5.0, 1.2 Hz), 7.47 (dd,
1H, J = 7.5, 1.2 Hz), 7.04 (dd, J = 7.5, 5.0 Hz), 4.49-4.47 (m,
1H), 3.89 (t, 2H, J=5.8 Hz), 3.13-3.05 (m, 4H), 3.00-2.92 (m,
2H), 2.13-2.06 (m, 2H), 1.81-1.72 (m, 2H); 13C NMR (CDCl3,
100 MHz) δ 160.7, 158.4, 138.6, 136.3, 122.7, 120.7, 57.1, 50.4,
46.4, 44.2, 26.9; MS (APCI) m/z 245.2 (M þ 1). The hydro-
chloride salt was prepared by diluting in ethyl acetate and
adding a 2.5 N HCl solution in ethyl acetate.
4-(6-Bromooxazolo[4,5-b]pyridin-2-yl)-1,4-diazabicyclo[3.2.2]-
nonane (17). Bromine (0.12 mL, 2.29 mmol) was added to a
solution of 4-oxazolo[4,5-b]pyridin-2-yl-1,4-diazabicyclo[3.2.2]-
nonane 13 (560 mg, 2.29 mmol) and sodium acetate (2.26 g, 27.5
mmol) in water (12 mL) and acetic acid (12 mL). The resulting
mixture was heated to reflux for 2 h. The mixture was cooled and
extracted with ethyl acetate (3ꢀ). The combined organic layers
were washed with water (2ꢀ) and brine (1ꢀ) and dried over
sodium sulfate, filtered, and concentrated. The crude residue
was purified by chromatography (Biotage, 25 M) using a
gradient elution from 4% MeOH/CHCl3 containing 0.1%
NH4OH to 8% MeOH/CHCl3 containing 0.1% NH4OH, giving
578 mg (78%) of the free base of the title compound as an oil: 1H
NMR (CDCl3, 400 MHz) δ 8.23 (d, 1H, J=1.7 Hz), 7.50 (d, 1H,
J=1.7 Hz), 4.51 (br s, 1H), 3.92 (br s, 2H), 3.16-3.04 (m, 4H),
3.02-2.94 (m, 2H), 2.17-2.01 (m, 2H), 1.83-1.74 (m, 2H); MS
(APCI) m/z 325.0/323.0 (M þ 1).
2.10-2.20 (m, 2H), 1.76-1.87 (m, 2H); LCMS m/z 259.2
(M þ 1).
4-(6-Ethyloxazolo[4,5-b]pyridin-2-yl)-1,4-diazabicyclo[3.2.2]-
nonane (21). Step 1. Tributyl(vinyl)stannane (117 mg, 0.37 mmol)
and Pd(PPh3)2Cl2 (11 mg, 0.015 mmol) were added to a stirred
solution of 4-(6-bromooxazolo[4,5-b]pyridin-2-yl)-1,4-diazabi-
cyclo[3.2.2]nonane 17 (100 mg, 0.31 mmol) in toluene. The
reaction mixture was heated to reflux. After cooling to RT, the
mixture was concentrated, hydrolyzed with water, and extracted
withCH2Cl2 (10ꢀ, 10mL). Organicextractswerecombined, dried
(MgSO4), concentrated, and purified by chromatography, eluting
with 5% MeOH in CH2Cl2 containing 5% NH4OH to give 57 mg
(68%) of 2-(6-vinyloxazolo[4,5-b]pyridin-2-yl)-2,5-diazabicyclo-
[3.2.2]nonane: 1H NMR (CD3OD, 400 MHz) δ 8.09 (s, 1H),
7.80 (s, 1H), 6.77 (dd, 1H, J=17.6, 11.0 Hz), 5.77 (d, 1H, J=17.6
Hz), 5.26 (d, 1H, J=11.2 Hz), 4.63 (m, 1H), 4.10 (m, 2H), 3.35 (m,
2H), 3.28 (m, 4H), 2.30 (m, 2H), 2.07 (m, 2H); LCMS m/z 271.0
(M þ 1).
Step 2. 10% Pd/C (10 mg) was added to a solution of 2-(6-
vinyloxazolo[4,5-b]pyridin-2-yl)-2,5-diazabicyclo[3.2.2]nonane
(55 mg) in 1:1 EtOH/MeOH (10 mL). The mixture was shaken
under H2 (45 psi) for 2 h in a PARR apparatus and filtered
through a pad of Celite and the filtrate was concentrated to give
55 mg (99% yield) of the free base of the title compound as a
viscous oil: 1H NMR (CD3OD, 400 MHz) δ 7.97 (s, 1H), 7.57 (s,
1H), 4.61 (m, 1H), 4.10 (m, 2H), 3.36 (m, 2H), 3.30 (m, 4H), 2.70
(q, 2H, J=7.5 Hz), 2.29 (m, 2H), 2.05 (m, 2H), 1.26 (t, 3H, J=
7.5 Hz); LCMS m/z 273.0 (M þ 1).
4-(6-sec-Butyloxazolo[4,5-b]pyridin-2-yl)-1,4-diazabicyclo[3.2.2]-
nonane (22). The compound was prepared in a library for-
mat using the following protocol: a stock solution of 4-(6-
bromooxazolo[4,5-b]pyridin-2-yl)-1,4-diazabicyclo[3.2.2]nonane
17 (20 mg in 0.75 mL) in 1,2-dichloroethane/methanol (1/1) was
added to an 8 mL reaction vial with the corresponding boronic
acid (0.12 mmol). The resulting mixture was then evaporated in
vacuo, and the residue was dissolved in ethanol (0.65 mL),
followed by addition of sodium carbonate aqueous solution
(19 mg, 0.18 mmol in 0.085 mL) and a solution of tetrakis-
(triphenylphosphine)palladium(0) in toluene (3.4 mg, 0.003
mmol in 0.1 mL). The reaction vial was then heated to 85 °C
for 18 h. When the mixture was cooled to RT, 1 N NaOH
(1.5 mL) was added. The reaction mixture was extracted with
ethyl acetate (3 ꢀ 2.5 mL). The combined organic layer was
loaded onto a solid phase extraction (SPE) cartridge and eluted
with ethyl acetate (5 mL) followed by methanol (5 mL) into a
waste container. The column was then eluted with 1 N triethyl-
amine in methanol (5 mL) to a collecting container. The filtrate
was concentrated in vacuo to afford crude product, which was
converted to the trifluoroacetic acid salt by adding a 1 mL of a
15/485 trifluoroacetic acid/dichloromethane solution. The
crude was purified by mass triggered HPLC and analyzed using
the general HPLC conditions. LCMS retention time 1.71 min,
m/z 323.03 (M þ Na).
4-(6-Chlorooxazolo[4,5-b]pyridin-2-yl)-1,4-diazabicyclo[3.2.2]-
nonane (18). 4-Oxazolo[4,5-b]pyridin-2-yl-1,4-diazabicyclo[3.2.2]-
nonane 13 (300 mg, 1.2 mmol) and N-chlorosuccinimide (239 mg,
1.8 mmol) were dissolved in chloroform, sealed in a microwave
reactor tube, and heated at 150 °C for 10 min in a microwave
reactor. The crude material was cooled and purified by chromato-
graphy, eluting with a solution of NH4OH/MeOH/CH2Cl2 (0.5/
9.5/90) to give 111 mg (33%) of the free base of the title compound
as a sticky solid: 1H NMR (CDCl3, 400 MHz) δ 8.10 (d, 1H, J=
2.1 Hz), 7.34 (d, 1H, J=2.1 Hz), 4.42-4.53 (m, 1H), 3.82-3.96
(m, 2H), 3.02-3.16 (m, 4H), 2.88-3.02 (m, 2H), 1.98-2.16 (m,
2H), 1.68-1.83 (m, 2H); LCMS m/z 279.2 (M þ 1).
4-(6-Fluorooxazolo[4,5-b]pyridin-2-yl)-1,4-diazabicyclo[3.2.2]-
nonane (19). The free base of the title compound was prepared
from 4-oxazolo[4,5-b]pyridin-2-yl-1,4-diazabicyclo[3.2.2]nonane
13 by the methods described for the preparation of 33 in 32%
yield: 1H NMR (CDCl3, 400 MHz) δ 8.12 (t, 1H, J = 2.3 Hz),
7.25 (dd, 1H, J=7.5, 2.5 Hz), 4.55 (m, 1H), 3.92-3.98 (m, 2H),
3.11-3.22 (m, 4H), 2.98-3.07 (m, 2H), 2.10-2.21 (m, 2H),
1.77-1.89 (m, 2H); MS (APCI) m/z 263.2 (M þ 1).
4-(6-Phenyloxazolo[4,5-b]pyridin-2-yl)-1,4-diazabicyclo[3.2.2]-
nonane (23). Et3N (5 μL) was added to a solution of palladium(II)
acetate (0.7 mg, 3.1 μmol) and 2-(N,N-dimethylamino)-20-dicy-
clohexylphosphinobiphenyl (1.8 mg, 4.65 μmol) in 1,2-dimethoxy-
ethane (0.5 mL) under a nitrogen atmosphere at RT. 4-(6-Bro-
mooxazolo[4,5-b]pyridin-2-yl)-1,4-diazabicyclo[3.2.2]nonane 17
(50 mg, 0.155 mmol), phenylboronic acid (32 mg, 0.233 mmol),
and CsF (70 mg, 0.465 mmol) were added to the solution, and the
mixture was heated in an oil bath (80 °C) for a period of 16 h. The
reaction mixture was cooled to RT, filtered through a pad of
Celite, and concentrated in vacuo. The crude residue was purified
by chromatography, eluting with 4% MeOH in CHCl3 with 1 mL
of NH4OH per liter to give 14 mg (27%) of the free base of the title
compound as a film: 1H NMR (CDCl3, 400 MHz) δ 8.48 (d, 1H,
J = 2.1 Hz), 7.62 (d, 1H, J = 2.1 Hz), 7.57-7.55 (m, 2H),
7.47-7.43 (m, 2H), 7.40-7.34 (m, 1H), 4.62 (br s, 1H), 4.00 (t,
2H, J = 5.8 Hz), 3.20-3.15 (m, 4H), 3.08-3.01 (m, 2H),
4-(6-Methyloxazolo[4,5-b]pyridin-2-yl)-1,4-diazabicyclo[3.2.2]-
nonane (20). 4-(6-Bromo-oxazolo[4,5-b]pyridin-2-yl)-1,4-diaza-
bicyclo[3.2.2]nonane 17 (50 mg, 0.15 mmol), dimethylzinc (2 M
solution in toluene, 0.15 mL, 0.31 mmol), and Pd(dppf)Cl2
(4 mg, 0.03 mmol) were dissolved in 1,4-dioxane (1 mL), sealed
in a microwave reactor tube, and heated at 150 °C for 10 min in a
microwave reactor. The reaction mixture was diluted with
MeOH and filtered through a pad of Celite. The filtrate was
purified by chromatography, eluting with a solution of
NH4OH/MeOH/CH2Cl2 (0.5/9.5/90) to give 27 mg (70%) of
the free base of the title compound as an oil: 1H NMR (CDCl3,
400 MHz) δ 8.04 (s, 1H), 7.24 (s, 1H), 4.53-4.58 (m, 1H), 3.95
(s, 2H), 3.11-3.21 (m, 4H), 2.97-3.07 (m, 2H), 2.36 (s, 3H),

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3 1233
2.19-2.08 (m, 2H), 1.90-1.81 (m, 2H); MS (APCI) m/z 321.2
(M þ 1).
cooled and filtered through a pad of Celite. The filtrate was
purified by chromatography, eluting with a solution of NH4-
OH/MeOH/CH2Cl2 (0.5/9.5/90) to give 316 mg (78%) of 28 as a
solid: 1H NMR (CDCl3, 400 MHz) δ 8.51 (s, 1H), 7.56 (s, 1H),
4.59 (s, 1H), 3.84-4.17 (m, 2H), 3.12-3.22 (m, 4H), 2.96-3.06
(m, 2H), 2.04-2.25 (m, 2H), 1.79-1.91 (m, 2H); MS (APCI) m/z
270.2 (M þ 1).
4-(5-Methyloxazolo[4,5-b]pyridin-2-yl)-1,4-diazabicyclo[3.2.2]-
nonane (24). The free base of the title compound was prepared
from 5-methyl-2-methylsulfanyloxazolo[4,5-b]pyridine (prepared
from 6-methyl-2-nitropyridin-3-ol) by the methods described
for the preparation of 10 in 67% yield: 1H NMR (CDCl3, 400
MHz) δ 7.22 (d, 1H, J=7.9 Hz), 6.65 (d, 1H, J=7.9 Hz), 4.49 (s,
1H), 3.89 (t, 2H, J=5.8 Hz), 3.13-3.05 (m, 4H), 2.99-2.90 (m,
2H), 2.47 (s, 3H), 2.13-2.06 (m, 2H), 1.78-1.70 (m, 2H); 13C
NMR (CDCl3, 100 MHz) δ 163.3, 158.2, 153.5, 139.8, 114.9,
114.3, 57.1, 50.5, 46.4, 44.3, 26.9, 24.1; MS (APCI) m/z 259.2
(M þ 1). The hydrochloride salt was prepared by diluting in
ethyl acetate and adding a 2.5 N HCl solution in ethyl acetate.
2-(6-Chlorooxazolo[5,4-b]pyridin-2-yl)-2,5-diazabicyclo[3.2.2]-
nonane (25). Step 1. 5-Chloro-3-nitropyridin-2-ol (2 g, 11.46
mmol), Na2S2O4 (20 g, 114.58 mmol), and KOH (11.9 g, 212.01
mmol) were dissolved in water (40 mL) and stirred at RT
overnight. The reaction mixture was extracted with EtOAc
(5ꢀ). The organic extracts were combined, dried over Na2SO4,
and concentrated to give 0.8 g (48%) of 3-amino-5-chloropyr-
2-(6-(1H-Pyrazol-3-yl)oxazolo[4,5-b]pyridin-2-yl)-2,5-diazabi-
cyclo[3.2.2]nonane (29). 4-(6-Bromooxazolo[4,5-b]pyridin-2-yl)-
1,4-diazabicyclo[3.2.2]nonane 17 (291 mg, 0.9 mmol), 1H-pyra-
zol-3-ylboronic acid (100 mg, 0.9 mmol), K2CO3 (497 mg, 3.6
mmol), and Pd(PPh3)4 (21 mg, 0.018 mmol) were dissolved in a
solution of 10% water/ethanol (2.2 mL), sealed in a microwave
reactor tube, and heated at 150 °C for 110 min in a microwave
reactor. The crude material was cooled and filtered through a
pad of Celite. The solvent was removed in vacuo and the residue
was purified by preparative HPLC to give 41 mg (15%) of 29 as
1
an oil: H NMR (CD3OD, 400 MHz) 8.58 (s, 1H), 8.38 (br s,
1H), 8.01 (d, 1H, J=1.7 Hz), 7.72 (d, 1H, J=2.5 Hz), 6.71 (d,
1H, J=2.1 Hz), 4.75 (s, 1H), 4.20-4.26 (m, 2H), 3.61-3.66 (m,
2H), 3.48-3.59 (m, 4H), 2.38-2.50 (m, 2H), 2.17-2.29 (m, 2H);
LCMS m/z 311.2 (M þ 1).
1
idin-2-ol: H NMR (DMSO-d6, 400 MHz) δ 11.40-11.73 (m,
2-(6-(Pyridin-3-yl)oxazolo[4,5-b]pyridin-2-yl)-2,5-diazabicyclo-
[3.2.2]nonane (30). The title compound was prepared from 4-(6-
bromooxazolo[4,5-b]pyridin-2-yl)-1,4-diazabicyclo[3.2.2]nonane
17 and pyridin-3-ylboronic acid by the methods described in
compound 29 in 24% yield and converted to HCl salt by the
method described for compound 8: 1H NMR (CD3OD, 400
MHz) δ 9.34 (s, 1H), 9.01 (d, 1H, J=7.9 Hz), 8.94 (d, 1H, J=5.4
Hz), 8.74 (s, 1H), 8.58 (s, 1H), 8.25 (dd, 1H, J = 7.7, 6.0 Hz),
4.36-4.42 (m, 2H), 3.73-3.79 (m, 2H), 3.60-3.67 (m, 4H), 3.35
(s, 1H), 2.46-2.59 (m, 2H), 2.28-2.41 (m, 2H); LCMS m/z 322.2
(M þ 1).
1 H), 6.71 (d, 1 H, J=2.5 Hz), 6.37 (d, 1 H, J=2.9 Hz), 5.39-
5.46 (m, 2 H); LCMS m/z 144.9 (M þ 1).
Step 2. Thiophosgene (815 μL, 10.7 mmol) was added to a
solution of 3-amino-5-chloropyridin-2-ol in THF (15 mL) under
N2 and stirred at RT for 1.5 h. A solution of NaOH (1.0 N
aqueous solution) was added to adjust the pH to ∼3. The
mixture was concentrated, and another solution of NaOH
(1.0 N aqueous solution) was added to adjust the pH to ∼5.
The mixture was filtered, concentrated, dissolved in EtOAc,
filtered again, concentrated, and purified by chromatography to
give 306 mg (30%) of 6-chlorooxazolo[5,4-b]pyridine-2-thiol:
1H NMR (CDCl3, 400 MHz) δ 8.17 (d, 1 H, J = 2.5 Hz), 7.55
(d, 1 H, J = 2.1 Hz); LCMS m/z 187.0 (M þ 1).
2-(6-(Pyrimidine-5-yl)oxazolo[4,5-b]pyridin-2-yl)-2,5-diazabi-
cyclo[3.2.2]nonane (31). The title compound was prepared in a
library format using the same protocol described for the pre-
paration of 22 from pyrimidin-5-ylboronic acid: LCMS reten-
tion time 1.37 min, m/z 323.16 (M þ 1).
Step 3. 6-Chloro-2-(methylthio)oxazolo[5,4-b]pyridine was
prepared from 6-chlorooxazolo[5,4-b]pyridine-2-thiol by the
methods described for the preparation of 10 (step 2) in 85%
yield: 1H NMR (CDCl3, 400 MHz) δ 8.17 (d, 1 H, J=2.1 Hz),
7.85-7.86 (m, 1 H), 2.77 (s, 3 H); LCMS m/z 201.0 (M þ 1).
Step 4. The free base of the title compound was prepared from
6-chloro-2-(methylthio)oxazolo[5,4-b]pyridine by the methods
2-(6-Phenoxyoxazolo[4,5-b]pyridin-2-yl)-2,5-diazabicyclo[3.2.2]-
nonane (32). 4-(6-Bromo-oxazolo[4,5-b]pyridin-2-yl)-1,4-diaza-
bicyclo[3.2.2]nonane 17 (100 mg, 0.31 mmol), phenol (44 mg,
0.46 mmol), CuCl (15 mg, 0.15 mmol), tetramethylheptane-3,5-
dione (14 mg, 0.08 mmol), and Cs2CO3 (150 mg, 0.46 mmol)
were dissolved in DMF (1 mL)), sealed in a microwave reactor
tube, and heated at 200 °C for 40 min in a microwave reactor.
The crude material was cooled and filtered through a pad of
Celite. The filtrate was purified by chromatography, eluting
with a solution of NH4OH/MeOH/CH2Cl2 (0.5/9.5/90) to give
17 mg (16%) of 32: 1H NMR (CDCl3, 400 MHz) δ 8.08 (s, 1H),
7.28-7.35 (m, 2H), 7.20 (d, 1H, J=2.5 Hz), 7.08 (t, 1H, J=7.3
Hz), 6.96 (d, 2H, J = 7.9 Hz), 4.52-4.58 (m, 1H), 3.95 (s, 2H),
3.11-3.21 (m, 4H), 2.97-3.07 (m, 2H), 2.10-2.21 (m, 2H),
1.76-1.87 (m, 2H); MS (APCI) m/z 337.3 (M þ 1).
1
described for the preparation of 10 (step 3) in 20% yield: H
NMR (CD3OD, 400 MHz) δ 7.87-7.96 (m, 1 H), 7.63-7.70 (m,
1 H), 4.71-4.80 (m, 1 H), 4.24 (t, 2 H, J=5.6 Hz), 3.68 (t, 2 H,
J = 5.8 Hz), 3.59 (t, 4 H, J = 7.9 Hz), 2.37-2.54 (m, 2 H),
2.16-2.35 (m, 2 H); LCMS m/z 279.1 (M þ 1).
4-(6-Phenyloxazolo[5,4-b]pyridin-2-yl)-1,4-diazabicyclo[3.2.2]-
nonane (26). The free base of the title compound was prepared
6-phenyl-2-methylsulfanyloxazolo[5,4-b]pyridine by the meth-
ods described for the preparation of 10 in 50% yield as a
1
colorless oil: H NMR (CDCl3, 400 MHz) δ 8.10 (d, 1H, J =
2.1 Hz), 7.72 (d, 1H, J=2.1 Hz), 7.57-7.55 (m, 2H), 7.47-7.44
(m, 2H), 7.39-7.36 (m, 1H), 4.58 (br s, 1H), 3.98 (t, 2H, J=5.8
Hz), 3.22-3.14 (m, 4H), 3.11-3.01 (m, 2H), 2.22-2.15 (m, 2H),
1.89-1.82 (m, 2H); MS (APCI) m/z 321.2 (M þ 1).
4-(6-Fluoro-5-methyloxazolo[4,5-b]pyridin-2-yl)-1,4-diazabi-
cyclo[3.2.2]nonane (33). Step 1. 4-(5-Methyloxazolo[4,5-b]pyri-
din-2-yl)-1,4-diazabicyclo[3.2.2]nonane 24 (0.6 g) was dissolved
in sulfuric acid (95 - 98%, 4.0 mL) and cooled to 0 °C in an ice
bath. A chilled mixture of sulfuric acid (95-98%, 2.0 mL) and
nitric acid (>90%, 2 mL) was added slowly, and the resulting
mixture was allowed to stir at ambient temperature for 16 h and
then slowly poured over NaHCO3 (15.0 g). A solution of NaOH
(1.0 N aqueous solution) was added to adjust the pH to ∼14. The
mixture was then extracted with CH2Cl2 (3 ꢀ 50 mL). The
combined organic layers were dried over Na2SO4. The solvent
was removed in vacuo, and the residue was purified using flash
chromatography (silica gel, 0-9.5% MeOH in CH2Cl2 with
0.5% NH4OH) to give 4-(5-methyl-6-nitrooxazolo[4,5-
2-(5-Methyloxazolo[5,4-b]pyridin-2-yl)-2,5-diazabicyclo[3.2.2]-
nonane (27). The free base of the title compound was prepared
from 3-amino-6-methylpyridin-2-ol by the methods described
1
for the preparation of 25 (steps 2-4) in 9% yield: H NMR
(CDCl3, 400 MHz) δ 7.48 (d, 1H, J=7.9 Hz), 7.08 (d, 1H, J=
7.9 Hz), 4.43-4.49 (m, 1H), 3.97 (s, 2H), 2.99-3.18 (m, 6H),
2.49 (s, 3H), 2.12-2.28 (m, 2H), 1.85-1.96 (m, 2H); LCMS m/z
259.2 (M þ 1).
4-(6-Cyanooxazolo[4,5-b]pyridin-2-yl)-1,4-diazabicyclo[3.2.2]-
nonane (28). 4-(6-Bromo-oxazolo[4,5-b]pyridin-2-yl)-1,4-diaza-
bicyclo[3.2.2]nonane 17 (500 mg, 1.5 mmol), dicyanozinc (217 mg,
1.9 mmol), and Pd(dppf)Cl2 (36 mg, 0.045 mmol) were dissolved
in DMF (5 mL), sealed in a microwave reactor tube and heated at
220 °C for 10 min in a microwave reactor. The crude material was
1
b]pyridin-2-yl)-1,4-diazabicyclo[3.2.2]nonane in 33% yield: H
NMR (CDCl3, 400 MHz) δ 8.12 (s, 1H), 4.39-4.76 (m, 1H),
3.83-4.16 (m, 2H), 3.09-3.25 (m, 4H), 2.94-3.09 (m, 2H), 2.89

1234 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3
O’Donnell et al.
(s, 3H), 1.95-2.29 (m, 2H), 1.75-1.94 (m, 2H); MS (ESIþ) for
C14H17N5O3 m/z 304.3 (M þ 1).
3.97-4.05 (m, 2H), 3.00-3.19 (m, 6H), 2.14-2.27 (m, 2H),
1.85-1.98 (m, 2H); LCMS m/z 323.1, 325.1 (M þ 1).
Step 2. 10% Pd/C (50 mg) was added to a solution of 4-(5-
methyl-6-nitrooxazolo[4,5-b]pyridin-2-yl)-1,4-diazabicyclo[3.2.2]-
nonane (0.208 g) in 1:1 EtOH/MeOH (100 mL). The mixture
was shaken under H2 (45 psi) for 2 h in a PARR apparatus and
filtered through a pad of Celite and the filtrate was concentrated
to give 2-(1,4-diazabicyclo[3.2.2]non-4-yl)-5-methyloxazolo-
2-(5-Ethyloxazolo[4,5-b]pyridin-2-yl)-2,5-diazabicyclo[3.2.2]-
nonane (35). The free base of the title compound was synthesized
from 2-(5-bromooxazolo[4,5-b]pyridin-2-yl)-2,5-diazabicyclo-
[3.2.2]nonane 3a following the method described for the pre-
1
paration of 21 in 43% overall yield: H NMR (CD3OD, 400
MHz) δ 7.53 (d, 1H, J=8.3 Hz), 6.88 (d, 1H, J=7.9 Hz), 4.51-
4.57 (m, 1H), 3.99-4.05 (m, 2H), 3.00-3.18 (m, 6H), 2.77 (q,
2H, J = 7.5 Hz), 2.15-2.27 (m, 2H), 1.85-1.97 (m, 2H),
1.25-1.31 (t, 3H, J=7.5 Hz); LCMS m/z 273.3 (M þ 1).
2-(5-Isopropyloxazolo[4,5-b]pyridin-2-yl)-2,5-diazabicyclo[3.2.2]-
nonane (36). 2-(5-Bromooxazolo[4,5-b]pyridin-2-yl)-2,5-diaza-
bicyclo[3.2.2]nonane 3a (800 mg, 0.62 mmol), isopropylmagne-
sium chloride (2 M solution in THF, 928 μL, 1.85 mmol),
Pd(dppf)Cl2 (50 mg, 0.06 mmol), and zinc chloride (solution
in THF, 3.7 mL, 1.85 mmol) were dissolved in THF, sealed in a
microwave reactor tube, and heated at 170 °C for 10 min in a
microwave reactor. The mixture was diluted with 1 N NaOH
(20 mL) and CH2Cl2 (20 mL). The layers were partitioned, and
the aqueous layer was extracted with CH2Cl2 (2ꢀ). The com-
bined organic layers were dried (MgSO4), concentrated, and
purified by chromatography using a gradient elution from 100%
CH2Cl2 to 7% MeOH in CH2Cl2 to give a brown oil. The brown
oil was dissolved in a minimum amount of methanol, followed
by addition of 1 N HCl in Et2O. The mixture was triturated with
ether and filtered to give 83 mg (37%) of the HCl salt of the title
compound as a beige solid: 1H NMR (CD3OD, 400 MHz) δ 8.17
(d, J=7.9 Hz, 1H), 7.31 (d, J=7.9 Hz, 1H), 4.30-4.45 (m, 2H),
3.71-3.77 (m, 2H), 3.57-3.67 (m, 4H), 3.20-3.30 (m, 2H),
2.41-2.58 (m, 2H), 2.26-2.40 (m, 2H), 1.42 (d, J=7.1 Hz, 6H);
MS (APCI) m/z 287.3 (M þ 1).
1
[4,5-b]pyridin-6-ylamine: H NMR (CDCl3, 400 MHz) δ 6.88
(s, 1H), 4.45-4.57 (m, 1H), 3.88-3.95 (m, 2H), 3.08-3.20
(m, 4H), 2.96-3.07 (m, 2H), 2.42 (s, 3H), 2.10-2.21 (m,
2H), 1.73-1.84 (m, 2H); MS (ESIþ) for C14H19N5O m/z 274.3
(M þ 1).
Step 3. 2-(1,4-Diazabicyclo[3.2.2]non-4-yl)-5-methyloxazolo-
[4,5-b]pyridin-6-ylamine (0.180 g) was dissolved in a solution of
HCl (36%, 0.4 mL) and water (2 mL). The resulting mixture was
heated to 100 °C for 20 min and then cooled to -10 °C in an
ice-NaCl bath. A solution of NaNO2 (0.055 g) in water (2 mL)
was added, followed by the addition of HPF6 (60%, 0.17 mL).
The resulting suspension was stirred at -10 °C for additional 30
min. The mixture was then filtered to give a solid, which was
transferred to a vial and heated to 165 °C in an oil bath for
20 min. The residue was purified using reverse phase HPLC to
give the title compound in 6% yield: 1H NMR (CDCl3, 400
MHz) δ 7.20 (d, 1 H, J=8.3 Hz), 4.50-4.60 (m, 1H), 3.95 (t, 2H,
J=5.8 Hz), 3.12-3.24 (m, 4H), 2.97-3.10 (m, 2H), 2.50 (d, 3H,
J=3.7 Hz), 2.11-2.23 (m, 2H), 1.76-1.90 (m, 2H); MS (ESIþ)
for C14H17FN4O m/z 277.3 (M þ 1).
4-(6-Chloro-5-methyloxazolo[4,5-b]pyridin-2-yl)-1,4-diazabicyclo-
[3.2.2]nonane (34). 4-(5-Methyloxazolo[4,5-b]pyridin-2-yl)-1,4-
diazabicyclo[3.2.2]nonane 24 (0.1 g) and N-chlorosuccinimide
(0.051 g) were dissolved in CHCl3, sealed in a microwave reactor
tube (Smith process vial), and heated to 150 °C for 10 min in a
microwave reactor. The mixture was filtered, and the solvent
was removed in vacuo. The residue was dissolved in MeOH, and
HCl in 1,4-dioxane (4 M, 0.4 mL) was added. The solvent was
removed in vacuo and the residue was dissolved in MeOH and
triturated with CH2Cl2 to give 34 in 70% yield: 1H NMR
(CD3OD, 400 MHz) δ 8.27 (s, 1H), 4.84-4.89 (m, 1H), 4.34
(t, 2H, J=5.8 Hz), 3.73 (t, 2H, J=5.8 Hz), 3.61 (t, 4H, J=7.9
Hz), 2.69 (s, 3H), 2.42-2.54 (m, 2H), 2.26-2.37 (m, 2H); MS
(ESIþ) for C14H17ClN4O m/z 293.0 (M þ 1).
2-(5-Cyclopentyloxazolo[4,5-b]pyridin-2-yl)-2,5-diazabicyclo-
[3.2.2]nonane (37). The HCl salt of the title compound was
prepared from cyclopentylmagnesium chloride by the methods
described for the preparation of 36 in 37% yield: 1H NMR
(CD3OD, 400 MHz) δ 8.15 (d, 1H, J=8.3 Hz), 7.33 (d, 1H, J=
7.9 Hz), 4.27-4.46 (m, 2H), 3.70-3.77 (m, 2H), 3.58-3.66 (m,
4H), 3.20-3.30 (m, 2H), 2.41-2.56 (m, 2H), 2.28-2.40 (m, 2H),
2.19-2.28 (m, 2H), 1.87-1.98 (m, 2H), 1.73-1.86 (m, 4H); MS
(APCI) m/z 313.3 (M þ 1).
2-(5-Phenyloxazolo[4,5-b]pyridin-2-yl)-2,5-diazabicyclo[3.2.2]-
nonane (38). Pd(PPh3)4 (43 mg, 0.04 mmol), Na2CO3 (197.4 mg,
1.86 mmol), and phenylboronic acid (1.36 mg, 1.1 mmol) were
added to a solution of 2-(5-bromooxazolo[4,5-b]pyridin-2-yl)-
2,5-diazabicyclo[3.2.2]nonane 3a (300 mg, 0.93 mmol) in
toluene. The reaction mixture was heated at reflux overnight.
Upon cooling to RT, the mixture was diluted with CH2Cl2 and
extracted with saturated Na2CO3 solution, dried (Na2SO4), and
concentrated to give a brown oil. The oil was purified by flash
chromatography using a gradient elution from 100% CH2Cl2 to
7% MeOH in CH2Cl2 and converted to HCl salt to give 80 mg
(22% yield) of the title compound: 1H NMR (CD3OD, 400
MHz) δ 8.28 (d, 1H, J=8.3 Hz), 7.80-7.86 (m, 2H), 7.61-7.69
(m, 4H), 4.36-4.44 (m, 2H), 3.73-3.79 (m, 2H), 3.60-3.67 (m,
5H), 2.44-2.59 (m, 2H), 2.29-2.42 (m, 2H); LCMS m/z 321.2
(M þ 1).
2-(5-Bromooxazolo[4,5-b]pyridin-2-yl)-2,5-diazabicyclo[3.2.2]-
nonane (3a). Step 1. Sodium methoxide (25% in methanol,
44.6 mL, 178.5 mmol) was added to a solution of 2-nitropyri-
din-3-ol (25 g, 178.5 mmol) in methanol under N2. The mixture
was stirred at RT for 30 min before cooling to 0 °C. Bromine was
added dropwise, and the mixture was stirred for 1 h. Acetic acid
(3.1 mL) was added, and the mixture was concentrated to give a
yellow solid. The yellow solid was triturated with CH2Cl2 to give
a white solid which was dissolved in boiling methanol (5 mL)
followed by addition of water (12 mL). The mixture was cooled
to RT. The resulting precipitate was filtered and washed with
1
water to give 6-bromo-2-nitropyridin-3-ol as a white solid: H
NMR (CD3OD, 400 MHz) δ 7.78 (d, 1H, J = 8.7 Hz), 7.56
(d, 1H, J = 8.7 Hz).
Step 2. Iron (11.7 g, 20.9 mmol) and calcium chloride (4.6 g,
41.9 mmol) were added to a solution of 6-bromo-2-nitropyridin-
3-ol (10.2 g, 46.6 mmol) in EtOH containing 20% water. The
reaction mixture was refluxed for 1 h and cooled to RT. The
mixture was filtered through a pad of Celite and washed with
methanol. The filtrate was concentrated and purified by flash
chromatography (Biotage, 25 L) using a gradient elution from
100% CH2Cl2 to 15% MeOH/CH2Cl2 to give 7.45 g of 2-amino-
2-(5-(2-Fluorophenyl)oxazolo[4,5-b]pyridin-2-yl)-2,5-diazabi-
cyclo[3.2.2]nonane (39). The HCl salt of the title compound was
prepared from 2-fluorophenylboronic acid by the methods
described for the preparation of 38 in 32% yield: 1H NMR
(CD3OD, 400 MHz) δ 8.24 (d, 1H, J = 7.9 Hz), 7.70-7.76 (m,
1H), 7.61-7.69 (m, 1H), 7.58 (d, 1H, J=8.3 Hz), 7.34-7.46 (m,
2H), 4.91-4.97 (m, 1H), 4.36-4.43 (m, 2H), 3.75 (t, 2H, J=5.6
Hz), 3.63 (t, 4H, J=7.9 Hz), 2.44-2.57 (m, 2H), 2.29-2.41 (m,
2H); MS (APCI) m/z 339.3 (M þ 1).
1
6-bromopyridin-3-ol as an orange solid: H NMR (CD3OD,
400 MHz) δ 6.77 (d, 1H, J= 7.9 Hz), 6.60 (d, 1H, J =7.9 Hz).
Step 3. The free base of the title compound was prepared from
2-amino-6-bromopyridin-3-ol by the methods described for the
preparation of 10: 1H NMR (CD3OD, 400 MHz) δ 7.53 (d, 1H,
J = 7.9 Hz), 7.16 (d, 1H, J = 7.9 Hz), 4.48-4.56 (m, 1H),
2-(5-Methoxyoxazolo[4,5-b]pyridin-2-yl)-2,5-diazabicyclo[3.2.2]-
nonane (40). 2-(5-Bromooxazolo[4,5-b]pyridin-2-yl)-2,5-diaza-
bicyclo[3.2.2]nonane 3a (100 mg, 0.31 mmol), CuI (5.9 mg,
0.031 mmol), 1,10-phenanthrene (11.1 mg, 0.06 mmol), and

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3 1235
Cs2CO3 (141 mg, 0.43 mmol) were dissolved in MeOH (1 mL),
sealed in a tube, and heated at 120 °C for 3 h. The mixture was
diluted with CH2Cl2/i-PrOH (3:1) and extracted with saturated
NH4Cl solution. The organic extract was dried (Na2SO4),
concentrated, and purified by HPLC to afford 39.4 mg (46%)
of the title compound as a solid. The hydrochloride salt was
(3) Miyamoto, S.; Duncan, G. E.; Marx, C. E.; Lieberman, J. A.
Treatments for schizophrenia: a critical review of pharmacology
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(4) Green, M. F.; Braff, D. L. Translating the basic and clinical
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1
prepared by addition of 2 equiv of 4.0 N HCl in dioxane: H
(5) (a) Sullivan, P. F.; Kendler, K. S.; Neale, M. C. Schizophrenia as a
complex trait: evidence from a meta-analysis of twin studies. Arch.
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(6) Freedman, R.; Coon, H.; Myles-Worsley, M.; Orr-Urtreger, A.;
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Hoff, M.; Rosenthal, J.; Waldo, M. C.; Reimherr, F.; Wender, P.;
Yaw, J.; Young, D. A.; Breese, C. R.; Adams, C.; Patterson, D.;
Adler, L. E; Kruglyak, L.; Leonard, S.; Byerley, W. Linkage of a
neurophysiological deficit in schizophrenia to a chromosome 15
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(7) Stitzel, J. A. Naturally occurring genetic variability in the nicotinic
acetylcholine receptor R4 and R7 subunit genes and phenotypic
diversity in humans and mice. Front. Biosci. 2008, 13, 477–491.
(8) Kozak, R.; Martinez, V.; Young, D.; Brown, H.; Bruno, J. P.;
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symptoms of schizophrenia: disruption of cortical cholinergic
neurotransmission following repeated amphetamine exposure in
attentional task-performing, but not non-performing, rats. Neuro-
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(9) Adler, L. E.; Olincy, A.; Waldo, M.; Harris, J.; Griffith, J. G.;
Stevens, K.; Flach, K.; Nagamoto, H.; Bickford, P.; Leonard, S.;
Freedman, R. Schizophrenia, sensory gating, and nicotinic recep-
tors. Schizophr. Bull. 1998, 24, 189–202.
NMR (CD3OD, 400 MHz) δ 7.52 (d, 1H, J = 8.3 Hz), 6.35 (d,
1H, J=8.7 Hz), 4.40-4.49 (m, 1H), 3.95 (t, 2H, J=5.8 Hz), 3.86
(s, 3H), 2.96-3.14 (m, 6H), 2.10-2.23 (m, 2H), 1.80-1.93 (m,
2H); LCMS m/z 275.2 (M þ 1).
2-(5-Phenoxyoxazolo[4,5-b]pyridin-2-yl)-2,5-diazabicyclo[3.2.2]-
nonane (41). 2-(5-Bromooxazolo[4,5-b]pyridin-2-yl)-2,5-diaza-
bicyclo[3.2.2]nonane 3a (100 mg, 0.31 mmol), CuCl (15.3 mg,
0.155 mmol), phenol (58 mg, 0.62 mmol), tetramethylheptane
3,5-dione (34 mg), and Cs2CO3 (201 mg, 0.62 mmol) were
dissolved in NMP (2 mL), sealed in a tube, and heated at
120 °C for 4 h. The mixture was cooled to RT and filtered
through a pad of Celite. The filtrate was concentrated to give an
oil. The oil was diluted with CH2Cl2/i-PrOH (3:1) and washed
with water/brine. The organic layers were combined, dried over
Na2SO4, and concentrated in vacuo. The residue was purified by
preparative HPLC to give 34 mg (32%) of the title compound.
The hydrochloride salt was prepared by addition of 2 equiv of
1
4.0 N HCl in dioxane: H NMR (CD3OD, 400 MHz) δ 7.77
(d, 1H, J=8.7 Hz), 7.37-7.44 (m, 2H), 7.17-7.23 (m, 1H), 7.10
(d, 2H, J=7.5 Hz), 6.59 (d, 1H, J=8.7 Hz), 4.72-4.78 (m, 1H),
4.19-4.27 (m, 2H), 3.64-3.70 (m, 2H), 3.54-3.62 (m, 3H),
3.24-3.28 (m, 1H), 2.38-2.50 (m, 2H), 2.19-2.31 (m, 2H);
LCMS m/z 337.2 (M þ 1)).
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demonstrates an association between schizophrenia and tobacco
smoking behaviors. Schizophr. Res. 2005, 76, 135–157.
2-(5-(Piperidin-1-yl)oxazolo[4,5-b]pyridin-2-yl)-2,5-diazabicyclo-
[3.2.2]nonane (42). 2-(5-Bromooxazolo[4,5-b]pyridin-2-yl)-2,5-
diazabicyclo[3.2.2]nonane 3a (250 mg, 0.77 mmol), piperidine
(91.4 mL, 0.92 mmol), Pd(OAc)2 (6.9 mg, 0.03 mmol), BINAP
(20.3 mg, 0.03 mmol), NaOt-Bu (103.6 mg, 1.1 mmol) were
dissolved in toluene (4 mL) and heated at 100 °C overnight. The
mixture was cooled to RT, filtered through a pad of Celite,
concentrated, and purified by flash chromatography (Biotage,
12 L) using a gradient elution from 100% CH2Cl2 to 7% MeOH
in CH2Cl2 to give 82 mg (32%) of the title compound as a white
solid. The hydrochloride salt was prepared by addition of
2 equiv of 4.0 N HCl in dioxane: 1H NMR (CD3OD, 400 MHz)
δ 7.93-7.99 (m, 1H), 6.92-7.02 (m, 1H), 4.78-4.84 (m, 1H),
4.25-4.33 (m, 2H), 3.68-3.74 (m, 2H), 3.56-3.65 (m, 8H),
2.39-2.53 (m, 2H), 2.23-2.35 (m, 2H), 1.82-1.95 (m, 4H),
1.74-1.83 (m, 2H); MS (APCI) m/z 328.3 (M þ 1).
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prepared from pyrrolidine by the methods described for the
preparation of 42 in 53% yield: 1H NMR (CD3OD, 400 MHz) δ
7.94 (d, 1H, J=8.1 Hz), 6.40 (d, 1H, J=8.3 Hz), 4.78 (m, 1H),
4.23 (m. 2H), 3.64 (m, 2H), 3.56 (m, 8H), 2.40 (m, 2H), 2.25 (m,
2H), 2.10 (m, 4H); MS (APCI) m/z 314.2 (M þ 1).
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Acknowledgment. The authors thank the Pfizer ADME
Technology Group for permeability and microsomal clear-
ance data, the Pfizer Safety Science Group for hERG and in
vitro micronucleus data, and Mark Bundesman for library
compound preparation. The authors also thank Dr. Martin
Pettersson, Dr. David Gray, Dr. Christopher Helal, and
Dr. Christopher Shaffer for their helpful suggestions and
comments.
(18) Mullen, G.; Napier, J.; Balestra, M.; DeCory, T.; Hale, G.; Macor,
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