(-)-Spiro[1-azabicyclo[2.2.2]octane-3,5'-oxazolidin-2-one], a Conformationally restricted analogue of acetylcholine, is a highly selective full agonist at the α7 nicotinic acetylcholine receptor

Article abstract of DOI:10.1021/jm000249r

Neuronal nicotinic acetylcholine receptors are members of the ligand-gated ion channel receptor superfamily and may play important roles in modulating neurotransmission, cognition, sensory gating, and anxiety. Because of its distribution and abundance in the CNS, the α7 nicotinic receptor is a strong candidate to be involved in some of these functions. In this paper we describe the synthesis and in vitro profile of AR-R17779, (-)-spiro[1-azabicyclo[2.2.2]octane-3,5'oxazolidin-2'-one] (4a), a potent full agonist at the rat α7 nicotinic receptor, which is highly selective for the rat α7 nicotinic receptor over the α4β2 subtype. Preliminary SAR of AR-R17779 presented here indicate that there is little scope for modification of this rigid molecule as even minor changes result in significant loss of the α7 nicotinic receptor affinity.

Full text of DOI:10.1021/jm000249r

J . Med. Chem. 2000, 43, 4045-4050
4045
Expedited Articles
(-)-Sp ir o[1-a za bicyclo[2.2.2]octa n e-3,5′-oxa zolid in -2′-on e], a Con for m a tion a lly
Restr icted An a logu e of Acetylch olin e, Is a High ly Selective F u ll Agon ist a t th e
r7 Nicotin ic Acetylch olin e Recep tor
George Mullen,* J ames Napier,§ Michael Balestra, Thomas DeCory, Gregory Hale, J ohn Macor,| Robert Mack,
J ames Loch III, Ed Wu, Alexander Kover,| Patrick Verhoest,# Anthony Sampognaro, Eifion Phillips, Yanyi Zhu,
Robert Murray, Ronald Griffith,‡ J ames Blosser,† David Gurley,† Anthony Machulskis,† J ohn Zongrone,†
Alan Rosen,† and J ack Gordon†
Departments of Chemistry and Molecular Biology, AstraZeneca R&D Boston, 3 Biotech, One Innovation Drive,
Worcester, Massachusetts 01605
Received J une 9, 2000
Neuronal nicotinic acetylcholine receptors are members of the ligand-gated ion channel receptor
superfamily and may play important roles in modulating neurotransmission, cognition, sensory
gating, and anxiety. Because of its distribution and abundance in the CNS, the R7 nicotinic
receptor is a strong candidate to be involved in some of these functions. In this paper we describe
the synthesis and in vitro profile of AR-R17779, (-)-spiro[1-azabicyclo[2.2.2]octane-3,5′-
oxazolidin-2′-one] (4a ), a potent full agonist at the rat R7 nicotinic receptor, which is highly
selective for the rat R7 nicotinic receptor over the R4â2 subtype. Preliminary SAR of AR-R17779
presented here indicate that there is little scope for modification of this rigid molecule as even
minor changes result in significant loss of the R7 nicotinic receptor affinity.
Sch em e 1a
In tr od u ction
Neuronal nicotinic acetylcholine receptors (nAChRs)
are cation channels composed of five subunits and may
be either homo-oligomeric (R subunits) or hetero-oligo-
meric (R and â subunits) in composition. Recent
studies1-8 have indicated that neuronal nicotinic recep-
tors play important roles in modulating neurotransmis-
sion, cognition, sensory gating, and anxiety; thus, there
has been renewed interest in the use of nicotinic
agonists for treating neurodegenerative diseases,9-12
such as Alzheimer’s disease, in which there is cholin-
ergic dysfunction. The subtypes which mediate nico-
tine’s CNS actions are largely unknown; because of their
distribution and abundance in the CNS,10 the R7 and
R4â2 subtypes are strong candidates to be involved in
at least some of these functions.
While there are a number of nicotinic agonists known
to be selective for the R4â2 subtype,10,12 there are no
reported agonists which bind the R7 nicotinic receptor
selectively over other subtypes. Analogues of the marine
worm toxin anabaseine13,14 have been reported to be
“functionally” selective for the R7 subtype because these
a
Reagents: (a) NH2NH2/toluene; (b) NaNO2, HCl/H2O; (c)
RNH2/MeOH; (d) CDI/THF.
compounds are partial agonists at the rodent R7 nico-
tinic receptor and antagonists at all other nicotinic
receptor subtypes. However the therapeutic potential
of these compounds as R7 nicotinic receptor agonist
probes is compromised by the lack of significant binding
selectivity for R7 over R4â2 (selectivity ratios of 10 or
less); they also noncompetitively antagonize the R7
nicotinic receptor in a slowly reversible manner. In the
current paper, we describe AR-R17779 [spirooxazolidi-
none (-)-4a 22], a conformationally restricted analogue
of acetylcholine. The compound is the first reported full
agonist at the rat R7 subtype, with selective affinity for
the R7 vs the R4â2 nAChR subtype and which also lacks
noncompetitive antagonist activity.15
* To whom correspondence should be addressed: AstraZeneca R&D
Boston, 35 Gatehouse Dr., Waltham, MA 02451. Tel: 781-839-4511.
Fax: 781-839-4640. E-mail: george.mullen@astrazeneca.com.
† Department of Molecular Biology.
§ Present address: Abbott Laboratories, Abbott Park, IL 60064-3500.
| Present address: Bristol-Myers Squibb, P.O. Box 4000, Princeton,
NJ 08543-4000.
Ch em istr y
The spirooxazolidinones 4 were prepared as shown
in Scheme 1. Reaction of hydroxy ester 116 with hydra-
zine followed by Curtius rearrangement led to the
desired carbamate 4a . Resolution of 4a was accom-
Present address: ScinoPharm, P.O. Box 20-140, Tainan, Taipei.
Present address: Department of Chemistry, University of Penn-
#
sylvania, Philadelphia, PA.
‡ Present address: Tanabe Research Laboratories, USA, Inc., 4540
Towne Centre Court, San Diego, CA 92121.
10.1021/jm000249r CCC: $19.00 © 2000 American Chemical Society
Published on Web 10/13/2000

4046 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 22
Mullen et al.
Sch em e 2a
Sch em e 5a
a
Reagents: (a) 3 N HClO4; (b) triphosgene; (c) NaH/diethyl
malonate/toluene; (d) aq HCl/DMF.
Sch em e 3a
a
Reagents: (a) BH3-THF; (b) MeNO2/DBU; (c) Zn/HOAc.
a
Reagents: (a) RNH2/MeOH; (b) aq HCl; (c) H2/Ra Ni/NH3/
Sch em e 4a
MeOH; (d) BH3-THF; (e) CDI/iPr2NEt/CHCl3; (f) CDI/THF.
Ta ble 1. Rat Affinity and Efficacy Data on Spirooxazolidinones
a
Reagents: (a) LiAlH4/THF; (b) CDI/THF.
Ki (nM) ( SEMa
R7 IA R7 potency
(%) EC50 (mM)
plished via the dibenzoyl-D(or L)-tartrate salt. Com-
pounds 4b,c were prepared from amino alcohols 3 by
cyclization using carbonyldiimidazole (CDI). The amino
alcohols 3 were prepared by reaction of epoxide 217 with
either methylamine or ethylamine in methanol.
Compounds 5 and 6 were prepared from epoxide 217
as shown in Scheme 2. The synthesis of the cyclic
carbonate 5 was achieved by ring opening of the epoxide
2 using 3 N perchloric acid followed by ring closure with
triphosgene. The lactone 6 was prepared via addition
of the sodium anion of diethyl malonate to epoxide 2
followed by acid-catalyzed cyclization-decarboxylation.
Protection of the quinuclidine nitrogen as its borane
adduct was necessary in order to prepare the spirolac-
tam 9 (Scheme 3). Thus, reaction of the unsaturated
ester 718 with borane-THF, followed by Michael con-
densation with nitromethane, gave the intermediate
nitro ester 8; reduction of 8 using zinc in acetic acid and
concomitant cyclization of the resulting amino ester/
deprotection of the quinuclidine nitrogen provided the
desired lactam 9.
Reduction of the nitrile alcohol 10 (see Scheme 4),
prepared by reaction of the lithium salt of acetonitrile
with 3-quinuclidinone in THF, provided the amino
alcohol 11, which cyclized upon treatment with CDI to
give homologue 12.
Synthesis of the ‘reverse’ spirooxazolidinone 17 and
the spiroimidazolidinones 18 are presented in Scheme
5. Reaction of cyanohydrin 1319-21 with ammonia or
methylamine provided the corresponding cyano amines
14 (R ) H, Me). Hydrolysis of nitrile 14 (R ) H) using
aqueous HCl gave the amino acid 15, which, upon
borane reduction and cyclization with CDI, yielded the
desired spirooxazolidinone 17. Alternatively, Raney
nickel hydrogenation of cyano amines 14 gave the
triamines 16 (R ) H, Me), which led to the spiroimid-
azolidinones 18 upon cyclization with CDI.
compd
R
R4â2 affinity
R7 affinity
(-)-Nic
4a
(+)-4a
(-)-4a
4b
2.3 ( 0.6 (6)
16000 ( 4000 (3)
30000 ( 10000 (4)
16000 ( 4000 (3)
450 ( 80 (3)
480 ( 34 (6)
190 ( 10 (10)
59
43
H
H
H
Me
9400 ( 300 (3) <10c
92 ( 10 (4)
250 ( 10 (3)
>32000 (3)
96
21
(+)-4b Me 24000b (2)
1
6
(-)-4b Me
4c Et
200 ( 80 (3)
170 ( 10 (3)
220 ( 60 (4)
690 ( 20 (3)
82
36
a
b
Number of determinations in parentheses. Range ) 1000.
c @ 100 µM.
tinic receptor agonist (efficacy ) 59%). Furthermore,
AR-R17779 is the most selective R7 receptor agonist
known thus far. This is especially apparent when one
compares the selectivities of AR-R17779 and (-)-
nicotine for the R7 receptor versus the R4â2 nicotinic
receptor. Whereas nicotine is selective for the R4â2
nicotinic receptor (i.e., 0.005× “selective” for the R7
receptor), AR-R17779 is selective for the R7 receptor
(175× selective for the R7 receptor - approximately
35000-fold more selective than (-)-nicotine). The unique
potency and selectivity of AR-R17779 make this com-
pound an important tool for the understanding of the
function of the R7 nicotinic receptor.
When one examines the SAR of 4a presented in
Tables 1 and 2, it is readily apparent that there is little
room for change in this rigid molecule. A clear stereo-
genic preference can be seen for the (-) versus the (+)
enantiomer of 4. In addition to the fact that (+)-4a has
poor affinity for the R7 receptor, it also possesses poor
intrinsic activity for the receptor. These results suggest
that the location of the carbonyl functionality in AR-
R17779 is crucial to mimicking acetylcholine in the R7
receptor. Addition of a simple methyl group to the amide
nitrogen in 4 as found in (-)-4b reduces R7 receptor
affinity and dramatically increases R4â2 receptor af-
finity. This trend is continued with the N-ethyloxa-
zolidinone (4c), suggesting that there may be a lipophilic
pocket in the R4â2 receptor that is lacking in the R7
receptor.
Resu lts a n d Discu ssion
As presented in Table 1, the spirooxazolidinone (-)-
4a (AR-R17779) is a potent full agonist (efficacy ) 96%)
for the R7 nicotinic receptor: it is twice as potent as
(-)-nicotine, although (-)-nicotine is a partial R7 nico-
Several changes to the nature of the carbonyl func-
tionality in 4a cause an immediate loss in R7 receptor
affinity. As seen in Table 2, changing the carbamate in

Spiro[1-azabicyclo[2.2.2]octane-3,5′-oxazolidin-2′-one]
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 22 4047
Ta ble 2. Rat Affinity Data on Oxazolidinone Ring-Modified
Derivatives
HP 5988A quadrupole mass spectrometer for desorption
chemical ionization (CI) or by a Micromass Quattro 1 mass
spectrometer for electrospray (ES). High-resolution mass
spectra were obtained using a Micromass QTOF mass spec-
trometer. Elemental analyses were performed internally and
were within 0.4% of the theoretical value when indicated by
symbols of the elements, unless otherwise noted. Karl Fischer
analyses were performed using a Brinkmann 684KF coulom-
etric titrator.
Ki (nM) ( SEMa
compd
X
Y
R4â2 affinity
R7 affinity
Sp ir o[1-a za b icyclo[2.2.2]oct a n e -3,5′-oxa zolid in -2′-
on e] (4a ). Hydrazine (2.0 mL, 64 mmol) was added to a
solution of 2.90 g (14.5 mmol) of methyl 3-hydroxy-1-azabicyclo-
[2.2.2]octane-3-acetate16 (1) in 10 mL of methanol. The result-
ing solution was refluxed under nitrogen for 1 h, allowed to
cool, then concentrated in vacuo. The resulting solid was
suspended in 50 mL of toluene and heated to reflux for 2 h in
an apparatus equipped with a Dean-Stark trap. The reaction
mixture was allowed to cool, and the precipitate was collected
to give 1.82 g (63%) of the hydrazide as a tan solid. To a
suspension of 910 mg (4.57 mmol) of the hydrazide in 7 mL of
water was added 12 M HCl to give a solution with a pH ) 1.
The mixture was cooled to 0 °C and a 0 °C solution of 0.33 g
of sodium nitrite in 5 mL of water was added. The solution
was stirred at 0 °C for 20 min and then heated to 70 °C for an
additional 20 min. The reaction mixture was cooled to ambient
temperature, basified with 50% aqueous NaOH, saturated
with NaCl and extracted with chloroform (4 × 20 mL). The
combined organic extract was dried over MgSO4 and concen-
trated in vacuo to give 730 mg (87%) of 4a as an off-white
solid: 1H NMR (200 MHz, DMSO-d6) δ 1.36 (m, 1H), 1.48 (m,
2H), 1.77 (m, 1H), 1.91 (m, 1H), 2.64 (m, 4H), 3.33 (s, 2H),
(-)-4a NH
O
O
O
CH2
O
16000 ( 4000 (3)
92 ( 10 (4)
560 ( 20 (3)
3700 (1)
5
O
2200 (1)
6
9
12
17
18a
18b
CH2
NH
CH2NH
O
NH
NH
4000 (1)
23000b (2)
>4300 (1)
NH
NH
11000 (1)
>13000 (1)
>13000 (1)
NCH3 35000 (1)
a
b
Number of determinations in parentheses. Range ) 1000.
4a to a carbonate as in 5, to an ester as in 6, or to an
amide as in 9 all lead to a dramatic loss in R7 receptor
affinity. These changes to the carbonyl made seemingly
very minor changes to the electronic nature of the
carbonyl but caused dramatic effects on binding to the
R7 receptor. Insertion of a methylene into the oxazoli-
dinone ring as seen in 12 or reversing the carbamate
as seen in 17 also severely diminished R7 receptor
affinity. All of these results further strengthen the
hypothesis that the electronic nature and the location
of the carbonyl group in AR-R17779 are linchpins for
affinity and potency at the R7 nicotinic receptor. Al-
though acetylcholine is a small, achiral receptor agonist,
the molecule recognition features employed by the R7
nicotinic receptor likely are very well-defined and
specific. This would correspondingly lead to a very
narrow SAR with unnatural mimics. We are presently
examining additional modification to, and analogues of,
AR-R17779, and these results will be presented in due
course.
3.28 & 3.53 (AB, 2H, J ) 8.55), 7.42 (br s, 1H); IR (KBr, cm-1
1743; CIMS m/z 183.
)
A solution of 7.5 g (21 mmol) of dibenzoyl-L-tartaric acid in
absolute ethanol was added to 3.8 g (21 mmol) of 4a in absolute
ethanol, followed by a small portion of ethyl acetate. The solid
which formed upon standing was filtered (filtrate A). After
recrystallization from ethanol, this solid was dissolved in
aqueous NaOH, extracted with chloroform (3 × 50 mL), dried
over MgSO4 and concentrated in vacuo. The residue was
dissolved in methanol and HCl gas was bubbled through the
mixture until the pH < 2. Et2O was added to the solution and
the resulting white solid was filtered to give 630 mg of (+)-
o
4a : mp >250 C; chiral HPLC (Diacel OD, 4.6 × 250 mm, 4:1
Su m m a r y
hexane/ethanol, 1.00 mL/min) 8.69 min; [R]D ) +57.078 (c )
0.6570, MeOH); 13C NMR (200 MHz, DMSO-d6) δ 17.77, 18.10,
29.01, 44.39, 45.01, 48.74, 57.19, 78.20, 157.10; 1H NMR (200
MHz, DMSO-d6) δ 1.92 (m, 3H), 2.03 (m, 1H), 2.23 (m, 1H),
3.17 (m, 3H), 3.20 (m, 1H), 3.55 (m, 4H), 7.75 (s, 1H), 11.09
(br s, 1H). Anal. (C9H15ClN2O2) C, H, N.
The spirooxazolidinone (-)-4a is a unique and novel
tool that will be useful for elucidating the use and
function of the R7 nicotinic receptor. Its combination of
receptor potency and nicotinic receptor selectivity makes
(-)-4a the only known selective R7 nicotinic receptor
agonist. (-)-4a is 5-fold more potent and 35000-fold
more selective than (-)-nicotine for the R7 nicotinic
receptor. Preliminary SAR of this compound has shown
little opportunity for improvement as relatively minor
changes result in significant loss of R7 nicotinic receptor
affinity. We continue to examine the SAR around this
unique discovery and are using the compound to probe
the physiological function of the R7 nicotinic receptor.
Filtrate A was converted to the free base by concentrating
the solution and then dissolving the residue in 20% aqueous
NaOH; extraction with chloroform, followed by drying over
MgSO4 and concentration in vacuo, gave 1.21 g of residue,
which was dissolved in absolute ethanol and combined with a
solution of 2.38 g of dibenzoyl-D-tartaric acid in absolute
ethanol. This solution was treated as above to give 210 mg of
(-)-4a : mp >250 °C; chiral HPLC (Diacel OD, 4.6 × 250 mm,
4:1 hexane/ethanol, 1.00 mL/min) 9.78 min; [R]D ) -61.473 (c
) 0.7727, MeOH); 13C NMR (200 MHz, DMSO-d6) δ 17.76,
18.10, 28.95, 44.47, 45.07, 48.73, 57.24, 78.18, 157.10; 1H NMR
(200 MHz, DMSO-d6) δ 1.92 (m, 3H), 2.03 (m, 1H), 2.23 (m,
1H), 3.17 (m, 3H), 3.51 (s, 2H), 3.45 & 3.60 (AB, 2H, J ) 9.60),
7.74 (s, 1H), 10.77 (br s, 1H). Anal. (C9H15ClN2O2) C, H, N.
3′-Meth ylsp ir o[1-a za bicyclo[2.2.2]octa n e-3,5′-oxa zoli-
d in -2′-on e] (4b). Methylamine (25 mL, 0.726 mol) was added
to a solution of 10.2 g (0.0733 mol) of epoxide 217 in 75 mL of
methanol. The resulting solution was stirred at ambient
temperature overnight, then concentrated in vacuo to give
12.42 g of amino alcohol 3 (R ) Me). A mixture of 8.0 g of
amino alcohol 3 (R ) Me) and 9.15 g (0.056 mole, 1.2 equiv) of
carbonyldiimidazole in 100 mL of tetrahydrofuran was re-
fluxed for 3 h. Upon cooling to ambient temperature, the
reaction was concentrated in vacuo and the residue was
Exp er im en ta l Section
Melting points were determined in capillary tubes on a
Thomas-Hoover apparatus and are uncorrected. Optical rota-
tions were measured using a J asco DIP-370 digital polarim-
eter. NMR spectra were determined in the indicated solvent
on a Brucker AC 200 or Brucker AMX 500 NMR spectrometer
at ambient temperature with tetramethylsilane (TMS) as the
internal standard. Chemical shifts are given in ppm and
coupling constants are in hertz. Splitting patterns are desig-
nated as follows: s, singlet; br s, broad singlet; d, doublet; t,
triplet; q, quartet; m, multiplet. Infrared spectra were recorded
on a Nicolet 510P FTIR or Bruker Vector 22 FTIR spectro-
photometer. Mass spectra were recorded by a Hewlett-Packard

4048 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 22
Mullen et al.
dissolved in 200 mL of methylene chloride, washed succes-
sively with 100 mL of water, 50 mL of brine and dried over
MgSO4. Crystallization of the hydrochloride salt from 1:1
methanol/2-propanol gave 5.55 g (51%) of 4b as its hydrochlo-
ride salt: mp 305-307 °C dec; 1H NMR (200 MHz, DMSO-d6)
δ 1.82 (m, 3H), 1.98 (m, 1H), 2.22 (m, 1H), 2.75 (s, 3H), 3.15
(m, 4H), 3.48 (m, 2H), 3.53 & 3.66 (AB, 2H, J ) 8.77), 10.64
(br s, 1H); IR (KBr, cm-1) 1751; CIMS m/z 197.
combined organic extract was dried over MgSO4 and concen-
trated in vacuo. The residue was flash chromatographed
through neutral silica gel using a 97:3 mixture of chloroform:
ammoniated methanol as the eluant to provide 34 mg (20%)
of lactone 6: 13C NMR (200 MHz, CDCl3) δ 21.269, 22.741,
28.741, 30.925, 32.087, 46.221, 46.560, 61.455; IR (KBr, cm-1
)
2361, 1772; HR-ESMS m/z calcd for C10H16NO2, 182.1181;
found, 182.1168.
Oxazolidinone 4b as its HCl salt (500 mg) was converted to
its free base via neutralization with Na2CO3, then dissolved
in 5 mL of ethanol and treated with a solution of 900 mg of
dibenzoyl-D-tartaric acid in 10 mL of ethanol to give, following
recrystallization from ethanol, 177 mg of the dibenzoyl-D-
tartrate salt of 4b. Conversion to the free base with K2CO3
followed by salt formation using HCl in ether gave 93 mg of
(-)-4b as its hydrochloride salt: [R]D ) -56.14 (c ) 0.3438,
MeOH); chiral HPLC (OD-H #139, 2.1 × 150 mm, 14.0% EtOH
in hexane at 0.25 mL/min) 98.4% peak 1 at 5.68 min; 1H NMR
(200 MHz, DMSO-d6) δ 1.79 (m, 2H), 1.88 (m, 1H), 2.03 (m,
1H), 2.25 (m, 1H), 2.77 (s, 3H), 3.14 (m, 2H), 3.31 (m, 2H),
3.48 & 3.70 (AB, 2H, J ) 12.0), 3.53 (m, 2H), 10.76 (br s, 1H);
IR (KBr, cm-1) 1759; ESMS m/z 197. Anal. (C10H17ClN2O2) C,
H, N, Cl.
S p ir o [1-a za b ic y c lo [2.2.2]o c t a n e -3,4′-p y r r o lid in -2-
on e] (9). Under nitrogen, 4.8 mL of 1.0 M borane-THF was
added to an ice-cold solution of 935 mg of ethyl 1-azabicyclo-
[2.2.2]octane-3-acrylate18 in 50 mL of THF. After 30 min, the
solvent was removed in vacuo and the residue was dissolved
in 1.3 mL of nitromethane, to which was added 0.74 mL of
DBU. The reaction was stirred at ambient temperature for 4
days, then partitioned between 50 mL of ether and 50 mL of
0.1 N HCl. The acid layer was extracted twice with 25 mL of
ether and the combined organic layer was dried over MgSO4
and concentrated in vacuo. Flash chromatography, eluting
with 3:1 hexane/ethyl acetate, followed by 2:1 hexane/ethyl
acetate, gave 681 mg (52%) of ethyl 3-nitromethyl-1-azabicyclo-
[2.2.2]octane-3-acetate (8) as a light yellow syrup: IR (KBr)
2365 (m), 2318 (m), 2270 (m), 1723 (s), 1547 (s); CIMS m/z
269 (M - H, 100%).
Resolution of 5.60 g of 4b using 10.20 g (1 equiv) of
dibenzoyl-L-tartaric acid in ethanol gave, following 3 recrys-
tallizations from ethanol, 529 mg of (+)-4b: chiral HPLC
(OD-H #139, 2.1 × 150 mm, 14.0% EtOH in hexane at 0.25
mL/min) 98.7% peak 2 at 7.35 min.
Zinc dust (1.3 g) was added to a solution of 666 mg (2.45
mmol) of ester 8 in 80 mL of acetic acid; the resulting
suspension was heated to reflux and stirred for 20 h. An
additional 1.2 g of zinc dust was then added and reflux was
continued for an additional 48 h. Upon cooling to ambient
temperature, the suspension was filtered through Celite. The
filtrate was concentrated in vacuo and the residue dissolved
in 50 mL of saturated aqueous Na2CO3. Extraction with a 3:1
mixture of chloroform/methanol gave, following flash chroma-
tography using a 20:5:0.5 mixture of chloroform/methanol/
concentrated ammonium hydroxide, 149 mg (34%) of lactam
9: mp 162-165 °C; 1H NMR (200 MHz, CDCl3) δ 1.58 (m, 4H),
1.73 (m, 1H), 2.22 & 2.43 (AB, 2H, J ) 16.4), 2.85 (m, 6H),
3′-Eth ylsp ir o[1-a za bicyclo[2.2.2]octa n e-3,5′-oxa zolid in -
2′-on e] (4c). Oxazolidinone 4c was prepared from epoxide 217
as above, using ethylamine, and isolated as its hydrochloride
1
salt: mp 283-286 °C; H NMR (200 MHz, DMSO-d6) δ 1.07
(t, 3H, J ) 7.15), 1.82 (m, 3H), 2.02 (m, 1H), 3.15 (m, 4H),
3.29 (m, 2H), 3.42 & 3.55 (AB, 2H, J ) 14.2), 3.58 & 3.68 (AB,
2H, J ) 9.46), 11.18 (br s, 1H); IR (KBr, cm-1) 1760; ESMS
m/z 211. Anal. (C11H19ClN2O2) C, H, N.
Spir o[1-azabicyclo[2.2.2]octan e-3,5′-dioxolan -2′-on e] (5).
A solution of 0.50 g (3.6 mmol) of epoxide 217 and 4.8 mL (14.4
mmol) of 3 N perchloric acid in 10 mL of water was stirred at
ambient temperature for 18 h, then made basic with sodium
carbonate. The resulting solution was concentrated in vacuo
and the solid residue was extracted with tetrahydrofuran; flash
chromatography through neutral silica gel using a 95:5:0.1
mixture of methanol:ethyl acetate:concentrated ammonium
hydroxide gave 0.27 g (48%) of the diol as a syrup which
solidified on standing: ESMS m/z 158 (MH+). Triphosgene (94
mg, 0.32 mmol) in 1.3 mL of methylene chloride was added to
a suspension of 100 mg (0.64 mmol) of the above diol and 300
mg (3.8 mmol) of pyridine in 2 mL of methylene chloride, while
stirring under nitrogen at -70 °C. The solution was allowed
to warm to ambient temperature and made basic using sodium
carbonate. Extraction with chloroform followed by removal of
the solvent gave 48 mg of dioxolane 5 as a solid: 1H NMR
(200 MHz, CDCl3) δ 1.53 (m, 4H), 1.72 (m, 1H), 2.77 (m, 2H),
2.91 (m, 3H), 3.28 (d, 1H, J ) 16.6), 4.15 & 4.51 (AB, 2H, J )
8.8); IR (KBr, cm-1) 1771 (s); ESMS m/z 184 (Karl Fischer:
found, 1.00% H2O). Anal. (C9H13NO3) C, H, N.
Sp ir o[1-a za b icyclo[2.2.2]oct a n e-3,5′-2H -t et r a h yd r o-
fu r a n -2-on e] (6). Ethyl tert-butyl malonate (4.26 mL, 22.5
mmol) was added slowly to a suspension of 909 mg (22.5 mmol)
of 60% sodium hydride in 50 mL of toluene, under nitrogen.
The resulting suspension was heated to 90 °C and a solution
of 1.04 g (7.5 mmol) of epoxide 217 in 50 mL of toluene was
added dropwise over 30 min. The reaction was stirred at 90
°C for 24 h, allowed to cool to ambient temperature, then
poured into a mixture of 60 mL of 2.5 N hydrochoric acid and
100 g of ice. The layers were separated and the aqueous layer
was basicified with NaHCO3; extraction with chloroform gave
1.42 g (67%) of the R-tert-butoxycarbonyl lactone as a semisolid.
A solution of 256 mg (0.91 mmol) of the R-tert-butoxycarbonyl
lactone in 2 mL of concentrated HCl and 5 mL of DMF was
heated to 80 °C and stirred for 17 h. Upon cooling to room
temperature, the reaction was made basic with saturated
NaHCO3, then extracted with chloroform (4 × 20 mL). The
3.26 & 3.42 (AB, 2H, J ) 9.4), 6.09 (br s, 1H); IR (KBr, cm-1
)
1697; ESMS m/z 181 (Karl Fischer, found 1.0% H2O). Anal.
Calcd for C10H16N2O (adjusted for 1.0% H2O): C, 65.97; H, 8.97;
N, 15.39. Found: C, 65.02; H, 8.71; N, 14.95.
3-Hydr oxy-1-azabicyclo[2.2.2]octan e-3-aceton itr ile (10).
A solution of acetonitrile (4.26 mL, 81.8 mmol) in 80 mL of
THF was added dropwise over 15 min to a solution of 35.5
n
mL (81.8 mmol) of 2.3 M BuLi in hexane and 80 mL of THF,
while stirring at -75 °C under nitrogen. After 1 h, a solution
of 10.22 g (81.8 mmol) of 3-quinuclidinone in 80 mL of THF
was added slowly. After an additional 2 h, the reaction was
quenched with 50 mL of water, allowed to warm to 10 °C, and
concentrated in vacuo. The residue was taken up in water,
made basic with Na2CO3 and saturated with NaCl. Extraction
with chloroform gave 12.21 g of a solid which crystallized from
ethyl acetate/ethanol, providing 7.33 g (54%) of hydroxy nitrile
10, mp 179-180 °C, which was used as such without further
purification.
Sp ir o[1-a za bicyclo[2.2.2]octa n e-2,6′-(3′,4′,5′,6′-tetr a h y-
d r o-1′,3′-oxa zin -2′-on e)] (12). Hydroxy nitrile 10 (3.32 g, 20.0
mmol) was added to a suspension of 1.00 g (26.4 mmol) of
lithium aluminum hydride in 50 mL of THF, while stirring
under nitrogen in an ice bath. The reaction was then heated
to reflux for 16 h, allowed to cool to ambient temperature, then
quenched by cautious addition of sodium sulfate decahydrate,
followed by 50 mL of THF and 1 mL of water. The mixture
was stirred for 1 h, filtered and concentrated in vacuo to afford
3.49 g (100%) of 3-hydroxy-1-azabicyclo[2.2.2]octane-3-ethyl-
amine (11) as a yellow oil, which was used as such without
further purification.
A catalytic amount of (dimethylamino)pyridine was added
to a solution of 1.58 g (9.28 mmol) of 11 and 3.00 g (18.5 mmol)
of carbonyldiimidazole in 25 mL of THF, which was refluxed
under nitrogen for 24 h. Upon cooling to ambient temperature,
the reaction was flash chromatographed through 200 g of
neutral silica gel using a 9:1:0.1 mixture of methylene chloride:
methanol:ammonium hydroxide as the eluant, providing 344

Spiro[1-azabicyclo[2.2.2]octane-3,5′-oxazolidin-2′-one]
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 22 4049
mg of 12 as a yellow oil. Crystallization from ethyl acetate
gave compound 12 as a white solid: 145 mg (8%); mp 171-
175 °C; 13C NMR (200 MHz, CDCl3) δ 21.087, 23.859, 28.620,
29.914, 36.676, 46.740, 47.227, 61.831; IR (KBr, cm-1) 3239,
1701; CIMS m/z 197 (M + H). Anal. (C10H16N2O2) C, H, N.
3-Am in o-1-a za bicyclo[2.2.2]octa n e-3-ca r bon itr ile (14,
R ) H). 3-Hydroxy-1-azabicyclo[2.2.2]octane-3-carbonitrile19-21
(13) (10.21 g, 67.1 mmol) was dissolved in methanolic ammonia
(7 M, 100 mL) and the solution was stirred at 50 °C for 48 h,
then allowed to cool and evaporated in vacuo. Crystallization
from ethyl acetate/hexane gave amino nitrile 14 (R ) H) as a
colorless solid (7.67 g, 76%): 1H NMR (500 MHz, DMSO-d6) δ
1.25 (1H, m), 1.67 (2H, m), 1.91 (2H, m), 2.66 (7H, m), 3.05
(1H, d); ESMS m/z 152 (100%, MH+). Anal. (C8H13N3) C, H,
N.
3-N-Met h yla m in o-1-a za b icyclo[2.2.2]oct a n e-3-ca r b o-
n itr ile (14, R ) Me) was prepared from cyanohydrin 1319-21
as above using 2 M methylamine in methanol in place of
ammonia. The residue was recrystallized from ethyl acetate/
hexane to give 14 (R ) Me) as a colorless solid (4.32 g, 74%):
1H NMR (500 MHz, DMSO) δ 1.28 (1H, m), 1.63 (2H, m), 1.75
(1H, m), 2.00 (1H, m), 2.28 (3H, d, J ) 4 Hz), 2.66 (6H, m),
3.02 (1H, d, J ) 14 Hz); ESMS m/z 166. Anal. (C9H15N3) C, H,
N.
h. The solution was then filtered and evaporated under
reduced pressure to afford triamine 16 (R ) H) (132 mg, 80%)
as an oil, which was used without further purification.
1,1′-Carbonyldiimidazole (446 mg, 2.74 mmol) was added
to a solution of triamine 16 (R ) H) (356 mg, 2.29 mmol) in
dry THF (10 mL). The resulting solution was heated under
reflux for 2 h. Following evaporation in vacuo, the residue was
subjected to reverse-phase HPLC on a Waters Bondapak C18
column using a gradient of 0-10% acetonitrile and 0.1%
aqueous trifluoroacetic acid. The product was dissolved in
methanol and excess 1 M HCl in ether was added. The
resulting salt was recrystallized from MeOH by vapor diffusion
with ether to afford 29 mg (6%) of 18 (R ) H) as a colorless
solid: 1H NMR (500 MHz, DMSO-d6) δ 1.76-1.83 (3H, m), 1.96
(1H, s), 2.06 (1H,m), 3.11-3.33 (6H,m) 3.40 (1H, d, J ) 9),
3.47 (1H, d, J ) 13.5), 6.38 (1H, s), 7.11 (1H, s), 9.90 (1H, s);
HR-ESMS calcd for C9H15N3O, 182.1293; found, 182.1316.
3′-Met h ylsp ir o[1-a za b icyclo[2.2.2]oct a n e-3,4′-2H -t et -
r a h yd r oim id a zolid in -2-on e] (18b). Amino nitrile 14 (R )
Me) (422 mg, 2.55 mmol) was dissolved in methanolic ammonia
(7 M, 20 mL), and Raney nickel (approximately 100 mg wet)
was added. The resulting mixture was stirred under an
atmosphere of hydrogen at 50 psi for 15 h. The solution was
then filtered and concentrated in vacuo to afford 16 (R ) Me)
(392 mg, 91%) as an oil which was used without purification.
3-Am in o-1-a za b icyclo[2.2.2]oct a n e-3-ca r b oxylic Acid
(15). Amino nitrile 14 (R ) H) (20.05 g, 133 mmol) was
dissolved in a 1:1 mixture of concentrated hydrochloric acid
and water (200 mL). The solution was stirred at room
temperature for 24 h, heated to reflux for 24 h, then allowed
to cool and evaporated in vacuo. The residue was then
dissolved in water and passed through a column of hydroxide
form A26 ion-exchange resin (prepared by suspension of the
resin in water, washing with aqueous potassium hydroxide
solution and then with deionized water) using dilute aqueous
acetic acid. Evaporation gave 9.04 g of amino acid 15, acetate
salt. A sample (403 mg) of the product was suspended in 4 M
HCl in dioxane, and the solution was stirred overnight.
Following evaporation, the residue was dissolved in the
minimum volume of dilute aqueous hydrochloric acid and
crystallized by the addition of THF to give, after drying, amino
acid 15, dihydrochloride hydrate as a colorless solid (178 mg):
1H NMR (500 MHz, DMSO-d6) δ 1.73 (m, 1H), 1.92 (m, 2H),
2.22 (m, 1H), 2.44 (s, 1H), 3.06 (m, 1H), 3.25 (m, 2H), 3.39 (d,
1H), 3.50 (m, 1H), 3.83 (m, 1H), 9.29 (br m, 3H), 11.20 (br s,
1H); ESMS m/z 171 (100%, MH+) (Karl Fisher analysis: found,
9.499% H2O). Anal. (C8H16Cl2N2O2) C, H, N.
Sp ir o[1-a za b icyclo[2.2.2]oct a n e -3,4′-oxa zolid in -2′-
on e] (17). Borane (1 M in THF, 30 mL, 30 mmol) was added
to a suspension of amino acid 15, acetate salt (0.481 g, 2.82
mmol) in THF (20 mL), while stirring under nitrogen in an
ice bath. The solution was heated briefly to reflux to dissolve
the solid, and then stirred overnight at ambient temperature.
Following workup with methanol, dilute hydrochloric acid was
added to the residue; the resulting solution was stirred at
room-temperature overnight and concentrated in vacuo. To 520
mg of the resulting residue, chloroform (10 mL) and diisopro-
pylethylamine (2 mL, 1.48 g, 11.5 mmol) were added, followed
by 1,1′-carbonyldiimidazole (1.84 g, 11.3 mmol). The solution
was stirred overnight at room temperature, brine was added,
and the solution was then extracted with chloroform. The
combined organic extract was dried with magnesium sulfate
and evaporated. The residue was purified by HPLC on a
Waters Porasil column using a gradient of 0-50% 1:1 v/v 3.5
M NH3 in MeOH/CHCl3 in CHCl3 to give compound 17 as a
colorless solid: 1H NMR (200 MHz, CDCl3) δ 1.62 (m, 3H),
1.82 (m, 1H), 1.95 (m, 1H), 2.79 (m, 4H), 3.02 (m, 2H), 4.12 (d,
1H, J ) 9), 4.48 (d, 1H, J ) 9), 6.84 (br s, 1H); HR-ESMS calcd
for C9H14N2O2, 183.1134; found, 183.1126.
1,1′-Carbonyldiimidazole (501 mg, 3.09 mmol) was added
to a solution of triamine 16 (R ) Me) (392 mg, 2.32 mmol) in
dry THF (10 mL). The resulting solution was heated under
reflux for 1.5 h. The solvent was then evaporated in vacuo and
the residue was subjected to reverse-phase HPLC on a Waters
Bondapak C18 column using a gradient of 0-10% acetonitrile
and 0.1% aqueous trifluoroacetic acid. The product was dis-
solved in methanol and excess 1 M HCl in ether was added.
The resulting salt was recrystallized from MeOH by vapor
diffusion with ether to afford 125 mg (23.2%) of 18b as a
colorless solid: 1H NMR (500 MHz, DMSO-d6) δ 1.74 (m, 1H),
1.84 (m, 1H), 1.94 (m, 1H), 2.12 (s, 1H), 2.21 (m, 1H), 2.83 (s,
3H), 3.17-3.41 (m, 8H), 6.54 (s, 1H), 9.91 (s, 1H); HR-ESMS
calcd for C10H18N3O, 196.1450; found, 196.1441. Anal. (C10H18N3-
ClO) C, H, N.
r4â2 Nicotin ic Recep tor Bin d in g Assa y. Rat forebrains
were homogenized in cold buffer (as above) and centrifuged
at 12000g for 20 min. The washed membranes were incubated
in buffer with 3 nM (-)-[3H]nicotine for 1 h at 4 oC. Membranes
were collected on GF/B filters treated with 0.01% PEI. Bound
radioactivity was determined using a Beckman scintillation
counter. Nonspecific binding was determined by 100 µM
carbachol. IC50 values were determined from five concentra-
tions of test compound (triplicate) using a nonlinear curve
fitting program. Ki values were calculated using the Cheng-
Prusoff equation: Ki ) IC50/(((2 + [lig]/Kd)2)1/n) - 1, where n
) 1 and Kd ) 1.7 nM.
r7 Nicotin ic Recep tor Bin d in g Assa y. Rat hippocampii
were homogenized with a Polytron in 20 vol of cold buffer
(mM: Tris 50; MgCl2 1; NaCl 120; KCl 5; CaCl2 2; pH 7.4).
The homogenate was centrifuged (1000g, 5 min), reextracted
and then the pooled supernatants were centrifuged at 12000g
for 20 min. The washed membranes were incubated in buffer
with 5 nM [125I]R-bungarotoxin, 1 mg/mL BSA for 2 h at 21
°C. Membranes were collected on GF/C filters pretreated with
1% BSA/0.01% PEI. Bound radioactivity was determined using
a Packard TopCount. Nonspecific binding was defined by 100
µM nicotine. IC50 values were determined from six concentra-
tions (triplicate) of test compound using a nonlinear curve
fitting program. Ki values were calculated using the Cheng-
Prusoff equation: Ki ) IC50/(((2 + [lig]/Kd)2)1/n) - 1, where n
) 2 and Kd ) 1.67 nM.
r7 Nicotin ic Recep tor F u n ction a l Assa y. Potency and
intrinisic activity values were determined by measuring cur-
rent activation in Xenopus oocytes expressing rat R7 nAChRs.
Oocytes were injected with cRNA coding for nAChR R7.
Oocytes were used 3-10 days postinjection. 100% intrinsic
activity was defined for each egg by current elicited by 3 mM
acetylcholine. Currents were measured from baseline to peak.
Sp ir o[1-a za b icyclo[2.2.2]oct a n e-3,4′-2H -t et r a h yd r o-
im id a zolid in -2-on e] (18a ). 3-Amino-1-azabicyclo[2.2.2]octyl-
3-carbonitrile (14, R ) H) (160 mg, 1.06 mmol) was dissolved
in 20 mL of 7 N methanolic ammonia, and Raney nickel
(approximately 100 mg wet) was added. The resulting mixture
was stirred under an atmosphere of hydrogen at 50 psi for 15

4050 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 22
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