C(8) substituted 1-azabicyclo[3.3.1]non-3-enes and C(8) substituted 1-azabicyclo[3.3.1]nonan-4-ones: Novel muscarinic receptor antagonists

Article abstract of DOI:10.1021/jm020572p

Expedient syntheses of C(8) substituted 1-azabicyclo[3.3.1]non-3-enes and C(8) substituted 1-azabicyclo[3.3.1]nonan-4-ones are reported to begin with 2,5-disubstituted pyridines. Catalytic reduction of the pyridine to the piperidine followed by treatment

Full text of DOI:10.1021/jm020572p

2216
J . Med. Chem. 2003, 46, 2216-2226
C(8) Su bstitu ted 1-Aza bicyclo[3.3.1]n on -3-en es a n d C(8) Su bstitu ted
1-Aza bicyclo[3.3.1]n on a n -4-on es: Novel Mu sca r in ic Recep tor An ta gon ists
Myoung Goo Kim,† Erik T. Bodor,§ Chen Wang,† T. Kendall Harden,*,§ and Harold Kohn*,†
Division of Medicinal Chemistry and Natural Products, School of Pharmacy, University of North Carolina at Chapel Hill,
Chapel Hill, North Carolina 27599-7360, and Department of Pharmacology, University of North Carolina School of Medicine,
Chapel Hill, North Carolina 27599-7365
Received December 24, 2002
Expedient syntheses of C(8) substituted 1-azabicyclo[3.3.1]non-3-enes and C(8) substituted
1-azabicyclo[3.3.1]nonan-4-ones are reported to begin with 2,5-disubstituted pyridines. Catalytic
reduction of the pyridine to the piperidine followed by treatment with ethyl acrylate and
Dieckmann cyclization gave diastereomeric mixtures of C(8) substituted 3-ethoxycarbonyl-4-
hydroxy-1-azabicyclo[3.3.1]non-3-enes, which were separable by chromatography. We found
that the catalytic reduction (PtO2, H2) procedure provided the cis-substituted piperidine but
that pyridine reduction was accompanied by competitive cleavage of the C(2) pyridyl substituent.
Accordingly, an alternative route was devised that afforded a diastereomeric mixture of the
cis- and trans-2,5-disubstituted piperidine. Treatment of the substituted pyridine with m-CPBA
gave the pyridine N-oxide, which was reduced to the piperidine by sequential reduction with
ammonium formate in the presence of Pd-C followed by NaBH3CN. Addition of ethyl acrylate
completed the synthesis of the substituted piperidine. The overall four-step reaction gave higher
yields (57%) than the two-step procedure (13%) with little cleavage of the C(2) pyridyl
substituent. Acid decarboxylation of the bicyclo[3.3.1]non-3-enes provided the C(8) substituted
1-azabicyclo[3.3.1]nonan-4-ones. Structural studies revealed diagnostic 13C NMR signals that
permit assignment of the orientation of the C(8) substituent. Pharmacological investigations
documented that 3-ethoxycarbonyl-4-hydroxy-1-azabicyclo[3.3.1]non-3-enes efficiently bind to
the human M1-M5 muscarinic receptors and function as antagonists. We observed that exo-
8-benzyloxymethyl-3-ethoxycarbonyl-4-hydroxy-1-azabicyclo[3.3.1]non-3-ene (3) displayed the
highest affinity, exhibiting Ki values at all five muscarinic receptors that were approximately
10-50 times lower than carbachol and approximately 30-230 times lower than arecoline.
Receptor selectivity was observed for 3. Compound 3 contained two different pharmacophores
found in many muscarinic receptor ligands, and preliminary findings indicated the importance
of both structural elements for maximal activity. Compound 3 serves as a novel lead compound
for further drug development.
The pathophysiological processes under the influence
of muscarinic ligands have attracted the attention of
medicinal chemists and medical practitioners for many
years.1-4 Recent studies have linked muscarinic recep-
tors to Alzheimer’s and Parkinson’s diseases, schizo-
phrenia, urinary incontinence, irritable bowel syndrome,
chronic obstructive pulmonary disease, and the percep-
tion of pain.1,2 Reports have shown [3.2.1]-, [2.2.2]-, and
[2.2.1]-bicyclic amines bind muscarinic receptors with
high affinity.4 Notable examples include the muscarinic
receptor antagonist LY316108/NNC11-21924b ([3.2.1]-
bicyclic amine) and the agonists talsaclidine4d ([2.2.2]-
bicyclic amine) and CI-10174c ([2.2.1]-bicyclic amine). By
comparison, few [3.3.1]-bicyclic amines have been ex-
amined despite early findings of activity.1b,c,5-8 Efforts
have been hampered by lack of general procedures for
synthesis of this ring system and the accessibility of the
synthetic precursors. Studies in our laboratories re-
quired the preparation of derivatives of 8-substituted
aza-bridged [3.3.1]-bicyclic amine 1. In this investiga-
tion, we combined older, more recent, and new synthetic
methodologies for the preparation of 1. X-ray and 13C
NMR spectroscopic studies are coupled to provide useful
structural tools for the stereochemical assignments of
[3.3.1]-bicyclic amine ring substituents. We report the
pharmacological activity of these compounds against the
human M1-M5 muscarinic receptors and document
their potent activity against these receptors. Evidence
for receptor selectivity was observed.
Resu lts a n d Discu ssion
* To whom correspondence should be addressed. For H.K.: Phone:
(919) 966-2680. Fax: (919) 843-7835. E-mail: harold_kohn@unc.edu.
For T.K.H.: Phone: (919) 966-4816. Fax: (919) 966-5640. E-mail:
tkh@med.unc.edu.
Syn th esis. Our studies required the synthesis of C(8)
substituted methylene-[3.3.1]-bicyclic amines 2-6. In
1952, Sternbach and Kaiser reported that Dieckmann
cyclization of the diethyl ester of 3-carboxypiperidine-
1-propionic acid (13a , Scheme 1) provided 2 and that 2
† Division of Medicinal Chemistry and Natural Products, School of
Pharmacy.
§ Department of Pharmacology, University of North Carolina School
of Medicine.
10.1021/jm020572p CCC: $25.00 © 2003 American Chemical Society
Published on Web 04/26/2003

Novel Muscarinic Receptor Antagonists
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 11 2217
Sch em e 1. Synthesis of Aza-Bridged [3.3.1]-Bicyclic
Amines
commercially available ethyl 3-piperidinecarboxylate
(14) rather than reduce ethyl nicotinate (12a ). For 12c,
nearly 50% of the product mixture consisted of the
byproduct 15 (C(2) reduction) after treatment with ethyl
acrylate.
The catalytic reduction of 12c and 12e proceeded
stereoselectively to give 13c and 13e as a single dia-
stereomer after the Michael addition with ethyl acrylate
while the C(2) methyl piperidine 15 byproduct was
generated as an approximate 1:1 diastereomeric mix-
ture. The 13C NMR provided information concerning
piperidine 13 and 15 structures. For 15, we observed
diagnostic signals for the C(2) (δ 53.5, 55.1) and the C(5)
(δ 41.2, 42.2) carbons in the cis- and trans-products. We
have assigned the upfield set (δ 41.2, 53.3) to the cis-
adduct and the corresponding downfield pair (δ 42.2,
55.1) to the trans-isomer on the basis of established
patterns.17 This finding permitted us to tentatively
assign as the sole adduct for the PtO2-H2 reduced
products 13c (δ 40.6, 57.6) and 13e (δ 40.1, 55.5) as the
cis-isomers. Further evidence for this assignment came
from an independent synthesis of both cis- (δ 40.6, 57.6)
and trans-13c (δ 41.9, 59.4) (see below). The stereose-
lectively of the PtO2-H2 reduction for 12c and 12e
suggested that the C(2) substituent complexed with the
PtO2 surface, promoting pyridine reduction from one
side. We tested this hypothesis. Catalytic reduction
(PtO2-H2, H+) of ethyl 2-methyl-5-pyridine carboxylate
(16), followed by treatment with ethyl acrylate, gave 15
as a 1:0.2 diastereomeric mixture.
underwent acid decarboxylation to give 1-azabicyclo-
[3.3.1]nonan-4-one hydrochloride (7) in 23% overall
yield.5a We are unaware of other general methods for
this ring system. Accordingly, we adopted the Dieck-
mann route9-11 and first coupled it with a more recent
procedure for the synthesis of 2,5-disubstituted pi-
peridines from the corresponding pyridine12-14 (Scheme
1).
Cyclization of 13a , 13c, and 13e proceeded under
stringent conditions (t-BuOK, 110 °C, 1 h)5-8 to give 2,5a
3 and 4, and 5 and 6, respectively. Acid decarboxylation
(concd aqueous HCl, 100 °C, 14 h)5a of 2, 5, and 6 gave
the corresponding 1-azabicyclo[3.3.1]nonan-4-ones 7,5a
10, and 11, respectively. Use of these conditions for the
preparation of 8 and 9 led to complex product mixtures.
Improved yields of 7,5a 10, and 11 were achieved with
the Dieckmann cyclization and acid decarboxylation
steps but without characterizing the intermediate esters
2, 5, and 6, respectively.
The competitive reductive cleavage of the C(2) pyridyl
substituent hampered our synthetic efforts to produce
2-7, 10, and 11. We sought an alternative route to the
Dieckmann precursor 13. In Scheme 2 we outline a four-
step procedure for cis- and trans-13c that proceeded
with little cleavage of the appended benzyl unit, a group
sensitive to reduction. Beginning with 12c, m-CPBA
oxidation gave the pyridine N-oxide 17c. Ammonium
formate reduction in the presence of Pd-C18 gave the
conjugated enamidoester (76% yield) 18c19 and the
corresponding debenzylated compound (10% yield). Treat-
ment of 18c with NaBH3CN completed the reduction
and gave 13c after treatment with ethyl acrylate. The
overall yield of 13c from 12c was 57% compared to the
13% yield obtained in Scheme 1. 13C NMR analysis
indicated that the ratio of cis- to trans-13c was ap-
proximately 0.16:1, respectively. Dieckmann cyclization
Synthesis of 2,5-disubstituted pyridines 12c-e was
straightforward. Treatment of 12b15 with NaH and
benzyl bromide gave ether 12c in 75% yield. Compound
12e was prepared in two steps. First, 12b was converted
to mesylate 12d in situ, and then the mesylate group
was displaced by dimethylamine to give 12e in 82%
yield.
Several pyridine-reductive methods have been re-
ported.12-14,16 For C(2) substituted methylene pyridines,
piperidine formation is accompanied by competitive
reduction of the C(2) methylene substituent. We exam-
ined several methods and found, for 12c and 12e, that
PtO2, H2, and acid12-14 provided the desired piperidines.
The crude piperidines prepared from 12c and 12e
were heated (80 °C) with ethyl acrylate to give the
Dieckmann cyclization precursors 13c and 13e, respec-
tively. The combined yields for these two steps ranged
from 13 to 51%. For the preparation of 13a , we used

2218 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 11
Sch em e 2. Improved Synthesis of 13c
Kim et al.
F igu r e 1. ORTEP X-ray crystallographic diagram for 10.
P h a r m a cologica l An a lysis. The structural cor-
respondence of 2-7, 10, and 11 with bicyclic amines
previously4b-d shown to interact with muscarinic recep-
tors prompted us to test the pharmacological activity
of these compounds. An earlier study reported that
1-azabicyclo[3.3.1]nonan-4-ol (19) displayed antispas-
modic activity.5b Muscarinic receptors are distributed
both centrally and peripherally and are recognized to
have important roles in cognitive function, central
control of movement, peripheral control of gastrointes-
tinal functions, and bronchodilation.1 To date, five
muscarinic receptor subtypes have been characterized
and cloned at the molecular level (m1-m5), and four of
these (m1-m4) have been pharmacologically classified
as M1-M4 on the basis of their response to selective
antagonists.1
of the cis- and trans-mixture 13c gave 3 and 4 (69%
yield) in an approximate 1:1 ratio. The relative percent-
age of 3 and 4 was comparable to that observed for cis-
13c, indicating that the C(5) carbon epimerized under
the basic Dieckmann cyclization conditions.
Str u ctu r e An a lysis. The identities of Dieckmann
products 2-6 and the decarboxylated adducts 7, 10, and
11 were determined by IR, 1H NMR, 13C NMR, and
mass spectroscopic measurements. The stereochemical
orientation of the C(8) substituent was assigned on the
basis of the X-ray crystallographic analysis of 10, a
1
comparison of the H and the 13C NMR within the data
set, and the use of diagnostic 13C NMR patterns for
cyclic structures.
Figure 1 shows the ORTEP X-ray crystallographic
diagram for 10, indicating that the C(8) dimethylami-
nomethylene unit is exo to the [3.3.1]-bicyclic ring
system. Of note, this compound crystallized as the
monohydrochloride salt. Structural identification of 10
permitted us to assign the C(8) configuration for the
isomeric acid decarboxylated salt 11 as endo and to
confidently predict the structures of the corresponding
Dieckmann adducts 5 and 6. Further evidence for these
assignments came from an assessment of the NMR data
set (Table 1). Comparison of the 1H and 13C NMR
resonances for 2-7, 10, and 11 revealed distinctive 13C
NMR chemical shift values for the C(2) and C(9)
resonances. We observed that the C(2) chemical shift
peak for the exo-adducts 3, 5, and 10 resonated 9.2-
10.1 ppm downfield from the C(2) signal in endo-isomers
4, 6, and 11. Correspondingly, the C(9) resonance in 3,
5, and 10 appeared 6.9-8.0 ppm upfield from the same
signal in 4, 6, and 11. Similar 13C NMR chemical shift
differences have been reported for the methyl and the
C(3) methylene resonances in substituted methylcyclo-
hexanes20 and for ring substituents within 2-azabicyclo-
[3.3.1]nonanes.21 In agreement with our proposed 1H
NMR assignments, NOESY experiments on the Dieck-
mann products 3-6 showed the expected correlation
(Supporting Information). For 3, we observed a steric
interaction between the C(8) methine hydrogen and the
C(2) methylene protons, whereas for the endo-isomer 4
we noted interactions between the C(8) methine hydro-
gen and the C(9) and C(8)CH2 methylene protons.
Similar NOESY correlations were detected for the
isomeric pair 5 and 6.
We chose to initially study the Gq/phospholipase
C-coupled M1 muscarinic receptor and the Gi/adenylyl
cyclase-coupled M2 muscarinic receptor in our pharma-
cological analyses. [3H]QNB was utilized as a radioli-
gand in competition binding assays with membranes
obtained from Sf9 insect cells expressing either the
human M1 or M2 muscarinic receptors (Figure 2). The
Dieckmann products 2-6 exhibited the highest appar-
ent affinities of the compounds tested. Compounds 7,
10, and 11 were not included in subsequent experiments
because of their weak affinities for the muscarinic
receptors.
Further studies were carried out with compounds
2-6, using membranes prepared from COS-7 cells
independently expressing each of the five human mus-
carinic receptors.22 The calculated Ki values determined
for compounds 2-6 in multiple experiments are pre-
sented in Table 2. Only upper limits of activity could
be estimated for compounds 5 (at M1, M3, M4, and M5)
and 6 (at M1, M2, M4, and M5). Within the Dieckmann
products, the C(8) benzyloxymethylene compounds 3
and 4 displayed increased affinity compared with that
of their C(8) dimethylaminomethylene counterparts 5
and 6. Furthermore, we found that the C(8) exo-isomers
were largely more potent than the C(8) endo-isomers
and that this difference approached 50-fold (p < 0.01)
and varied with molecules and receptors (i.e., 3 vs 4 at
the M2 and M4 receptors). Compound 3 displayed the
highest affinity, exhibiting Ki values at all five musca-

Novel Muscarinic Receptor Antagonists
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 11 2219
Ta ble 1. Key NMR Assignments of 2-7, 10, and 11
1H NMRa
C(8′)HH′
13C NMRb
compd
R
R′
C(2)HH′
C(8)H
C(9)HH′
C(2) C(7) C(8) C(9)
3.24 (d, 16.8)
3.73 (d, 16.8)
3.33 (d, 16.8)
3.89 (d, 16.8)
2.89 (d, 13.0)
2.96 (d, 13.0)
3.52 (dd, 6.5, 9.3) 2.75 (d, 13.4)
3.65 (dd, 7.5, 9.3) 3.07 (d, 13.4)
3.01 (d, 13.2)
2
3
4
5
H
H
H
2.90-2.96 (m)
3.01-3.02 (m)
49.5 19.1 55.2 51.4
52.1 19.4 60.1 46.3
42.5 21.7 61.5 53.2
52.5 20.9 58.8 45.9
CH2OBn
H
CH2OBn
H
3.37-3.51 (m)
3.04-3.13 (m) 3.37-3.51 (m)
3.08 (d, 13.2)
3.31 (d, 16.8)
3.93 (d, 16.8)
3.35 (dd, 1.0, 17.2)
3.55 (d, 17.2)
3.08-3.23 (m)
3.27-3.33 (m)
3.41-3.50 (m)
3.20-3.26 (m)
3.34-3.39 (m)
2.23 (dd, 6.8, 12.5) 2.77 (d, 13.6)
2.70 (dd, 8.4, 12.5) 3.09 (d, 13.6)
2.12 (dd, 7.5, 12.7) 3.03 (br dt, 1.3, 13.0)
2.45 (dd, 6.3, 12.7) 3.12 (dt, 2.3, 13.0)
3.29-3.39 (m)
2.26 (dd, 6.8, 12.4) 2.91 (d, 13.9)
2.72 (dd, 8.4, 12.4) 3.32 (d, 13.9)
2.13-2.34 (m)
3.20-3.26 (m)
2.72-2.77 (m)
CH2N(CH3)2
2.90-2.94 (m)
6
7
H
CH2N(CH3)2
2.91-2.97 (m)
3.08-3.23 (m)
3.08-3.12 (m)
42.4 24.1 60.3 53.9
51.2 21.9 53.3 53.9
53.8 23.2 56.8 48.0
H
H
H
10
CH2N(CH3)2
11
H
CH2N(CH3)2
3.20-3.26 (m)
44.6 26.2 58.1 56.0
a
The number in each entry is the chemical shift value (δ) observed in ppm followed by multiplicity of the signal and the coupling
constant (s) in Hz. The spectra were recorded at 600 MHz (3, 4), 500 MHz (2, 5, 6, 10, 11), and 300 MHz (7). The solvent used was CDCl3.
b
The number in the entry is the chemical shift value (δ) observed in ppm relative to the solvent peak. The spectra were recorded at 150
MHz (2, 3), 125 MHz (4-6, 10, 11), and 75 MHz (7). The solvent used was CDCl3.
rinic receptors that were approximately 10-50 (p <
0.01) times lower than carbachol (20) and approximately
30-230 times lower (p < 0.01) than arecoline (21).
Receptor selectivity also was observed with compound
3, which exhibited a 10-fold greater affinity (p < 0.01)
for the M2 receptor over the M3 receptor. Lower degrees
of selectivity between receptor subtypes were observed
for other compounds (e.g., compounds 2 and 4 at M4
receptor versus M5 receptor).
these three analogues do not exhibit agonist activity at
any of the muscarinic receptor subtypes. In contrast,
analogues 2, 3, and 4 inhibited the capacity of 3 µM of
carbachol to activate phospholipase C, confirming that
these compounds are antagonists of all muscarinic
receptor subtypes. Similar results were obtained with
these compounds in studies of the M2 muscarinic
receptor, examining its capacity to promote [35S]GTPγS-
binding in membranes from Sf9 insect cells expressing
the M2 muscarinic receptor24 (Supporting Information
Figure 2). Taken together, our studies illustrate that
analogues 2-4 are antagonists of the M1 through M5
receptors and do not exhibit any partial agonist activi-
ties at any of these five receptors under the conditions
studied.
Compound 3 exhibited the greatest affinity for the M2
receptor, being 3-fold greater (p < 0.01) for the M2
receptor than for either the M1 or the M4 receptors,
7-fold greater (p < 0.01) than for the M5 receptor, and
10-fold greater (p < 0.01) than for the M3 receptor. In
recent years, M2 and M4 selective antagonists have been
promoted for the treatment for movement disorders,
dementia, cardiac disorder, and pain.2,25 Also, selective
presynaptic M2 muscarinic antagonists enhance acetyl-
choline levels in vitro as well as in vivo, suggesting their
use for the treatment of degenerative disorders associ-
ated with impaired cholinergic functions, such as Alzhe-
imer’s disease.4d,26 Accordingly, 3 serves as a useful lead
compound for further optimization.
Documentation of relatively high affinity interaction
of compounds 2-4 at the M1-M5 receptor subtypes in
the radioligand binding assays led us to examine their
activities in functional assays of muscarinic receptor
action. Because the M1, M3, and M5 receptors couple
through Gq to activate phospholipase C-â (PLC-â), these
receptors were independently expressed in COS-7 cells,
and PLC-â activity was measured. The same assay was
performed with the Gi-coupled M2 and M4 receptors.
However, the chimeric G protein GRq/i was cotrans-
fected along with these receptors to allow signal trans-
duction from these Gi-linked receptors to Gq-activated
PLC-â.
As illustrated in Figure 3, 3 µM carbachol promoted
a 4-6-fold increase in [3H]inositol phosphate accumula-
tion in COS-7 cells transfected with various muscarinic
receptor subtypes. In contrast, no effect of compounds
2, 3, and 4 on [3H]inositol phosphate accumulation was
observed at 100 µM concentration. A comparable result
was observed in Sf9 cells expressing the M1 muscarinic
receptor23 (Supporting Information Figure 1). Thus,
The biological activities observed with these bicyclic
amines showed that muscarinic receptor activity de-
pended on ring size and nitrogen position.1-4 Analysis
of the composite data has revealed structural elements
important for receptor binding. Two of these pharma-
cophores are embedded in the chemical neurotransmit-
ter, acetylcholine chloride (22), and the muscarinic

2220 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 11
Kim et al.
tuting the C(8) benzyloxymethylene unit in 3 with a
dimethylaminomethylene group to give 5 led to lower
activities than 2 at the M1-M5 receptor subtypes.
Finally, we observed that the geometrical orientation
of the C(8) unit was key to efficient binding. In most
cases, the exo-isomer (3, 5) displayed increased binding
affinity compared with the endo-product (4, 6). Signifi-
cantly, our incorporation of two structurally unique
pharmacophores within a single muscarinic receptor
ligand differs from two recent reports27,28 describing
bivalent29 muscarinic agonists where two identical 1,2,5-
thiadiazole derivatives were linked to provide a novel
series of potent agents.27
Con clu sion s
Our studies have documented that C(8) substituted
1-azabicyclo[3.3.1]non-3-ene analogues efficiently bind
to muscarinic M1-M5 receptors, and functional assays
showed that these compounds serve as antagonists at
these receptors. Selectivity for the M2 receptor was
observed for 3. Investigations are in progress to deter-
mine if these findings can be generalized to other aza-
bridged [3.3.1]-bicyclic amines.
Exp er im en ta l Section
Gen er a l. FT-IR spectra were run on a Mattson Galaxy
Series FT-IR 5000 spectrometer. 1H and 13C NMR were
recorded on Varian VXR 300, Bruker DRX 500, Varian INOVA
500, Varian INOVA 600, and Bruker AMX 600 SR-1 NMR
instruments. Low- and high-resolution (CI) mass spectral
investigations were conducted at the University of Texas at
Austin by Dr. M. Moini. The low-resolution mass spectra were
run on a Finnegan MAT-TSQ-70 instrument, and the high-
resolution mass spectra were recorded on a Micromass ZAB-E
spectrometer. Microanalyses were provided by Atlantic Mi-
crolab, Inc. (Norcross, GA).
Syn th esis. Eth yl 2-Ben zyloxym eth yl-5-p yr id in eca r -
boxyla te (12c). NaH (144 mg, 60% in mineral oil, 3.6 mmol)
was slowly added to an anhydrous DMF solution (6 mL) of
12b15 (543 mg, 3.0 mmol) and BnBr (429 µL, 3.6 mmol) at room
temperature, the slurry was stirred (30 min), and H2O (10 mL)
was added. The reaction mixture was extracted with EtOAc
(3 × 20 mL), and the combined extracts were dried (Na2SO4)
and concentrated in vacuo. Purification of the concentrated
residue by column chromotography (EtOAc/hexanes ) 1/5)
gave 12c (610 mg, 75%) as a yellow oil: Rf ) 0.65 (EtOAc/
hexanes ) 1/1); IR (neat) 3031, 2981, 2860, 1721, 1598, 1379,
1281, 1112 cm-1; 1H NMR (CDCl3) δ 1.40 (t, J ) 7.2 Hz, 3 H),
4.40 (q, J ) 7.2 Hz, 2 H), 4.67 (s, 2 H), 4.74 (s, 2 H), 7.24-7.42
(m, 5 H), 7.59 (d, J ) 8.1 Hz, 1 H), 8.30 (dd, J ) 1.5, 8.1 Hz,
1 H), 9.15 (br s, 1 H); 13C NMR (CDCl3) 14.4, 61.5, 72.9, 73.3,
120.7, 125.1, 127.9, 128.0 (2 C), 128.6 (2 C), 137.8, 137.9, 150.4,
163.2, 165.4 ppm; MS (+CI) 272 [M + 1]+; Mr (+CI) 272.128 97
[M + 1]+ (calcd for C16H18NO3, 272.128 67).
Eth yl 2-Dim eth yla m in om eth yl-5-p yr id in eca r boxyla te
(12e). Methanesulfonyl chloride (186 µL, 2.4 mmol) was added
dropwise to a cooled (0 °C) CH2Cl2 solution (8 mL) containing
12b15 (362 mg, 2.0 mmol) and triethylamine (334 µL, 2.4 mmol)
under Ar, and then the solution was stirred at 0-5 °C (1 h).
CH2Cl2 (10 mL) was added to the reaction, and the organic
phase was successively washed with a saturated aqueous
NaHCO3 solution (10 mL) and brine (2 × 10 mL), dried (Na2-
SO4), filtered, and evaporated to give mesylate 12d (508 mg,
98%). Compound 12d was combined with a 2 M dimethylamine
THF solution (10 mL, 20 mmol) and triethylamine (279 µL,
2.0 mmol) in EtOH (15 mL) and heated at 80 °C (15 h). The
reaction solution was concentrated in vacuo and the residue
taken up in CH2Cl2 (10 mL). The organic solution was washed
with H2O (2 × 10 mL) and brine (2 × 10 mL), dried (Na2SO4),
filtered, and evaporated. The crude product was purified by
F igu r e 2. Competition binding assay at the human M1 and
M2 receptors: Increasing amounts of the indicated analogue
were incubated with [3H]QNB and Sf9 insect cell membranes
expressing either the M1 (A) or M2 (B) muscarinic receptor.
Data are shown for compounds 2 (2), 3 (0), 4 (1), 5 (]), 6 ([),
7 (O), 10 (b), and 11 (9). The data shown are typical curves of
three independent experiments carried out in duplicate for
both M1 and M2 receptors. Bars represent the range of
duplicate values.
agonist, arecoline (21),1 and these structural units are
highlighted in Figure 4.
Our findings indicate the apparent importance of both
pharmacophores shown in Figure 4 for maximal bioac-
tivity for C(8) substituted 1-azabicyclo[3.3.1]non-3-enes.
We observed that the parent Dieckmann product 2
showed appreciable binding to all five muscarinic recep-
tor subtypes. Significantly, 2 contains only the struc-
tural element found in arecoline (21). Furthermore,
altering this pharmacophore to give 7 led to a loss of
binding. Our data set also showed that the structural
unit associated with acetylcholine (22) may have influ-
enced muscarinic receptor binding for Dieckmann prod-
ucts 2-6. In particular, we observed that incorporating
an exo-C(8) benzyloxymethylene unit to give 3 led to an
increase in muscarinic receptor binding compared with
that of 2, most significantly at the M2 receptor where
an increase of 20-fold (p < 0.01) was observed. Substi-

Novel Muscarinic Receptor Antagonists
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 11 2221
Ta ble 2. Binding Affinities of 2-6 at Human Muscarinic Receptors
hM1 receptor
hM2 receptor
hM3 receptor
hM4 receptor
hM5 receptor
compd
Kia,b
Kia,b
Kia,b
Kia,b
Kia,b
2
3
4
5
6
1.2 ( 0.1
0.25 ( 0.01c
6.6 ( 0.5
>100d
1.6 ( 0.2
0.08 ( 0.01
4.1 ( 0.1
86 ( 30
1.5 ( 0.1
0.84 ( 0.07
7.5 ( 0.6
>100d
2.6 ( 0.3
0.27 ( 0.02
11 ( 1
0.98 ( 0.06
0.56 ( 0.03
3.0 ( 0.5
>100c,d
>100d
>100d
>100d
37 ( 9
>100d
7.6 ( 0.4
60 ( 30
>100c,d
20
21
12 ( 5
29 ( 1
1.3 ( 0.3
2.4 ( 0.4
20 ( 4
43 ( 5
4.3 ( 0.7
56 ( 30c
a
Ki values were calculated according to the formula
IC50
[[3H]QNB]
Ki )
1 +
KD
where IC50 is the concentration of the competing analogue that inhibited [3H]QNB binding by 50%, [[3H]QNB] is the concentration of
[3H]QNB in the binding assay, and KD is the KD of [3H]QNB. The KD determined for [3H]QNB for hM1 was 83.9 pM, hM2bwas 56.7 pM,
hM3 was 249 pM, hM4 was 93.7 pM, and hM5 was 176 pM. Values are means ( SEM (n ) 3) unless otherwise indicated. In µM. c The
d
value is the average of two determinations. Ki value was not calculated for this compound.
F igu r e 3. Quantitation of functional activity at the human M1-M5 receptors. The accumulation of [3H]inositol phosphates in
COS-7 cells expressing the M1, M3, or M5 receptor alone or the M2 or M4 receptor coexpressed with chimeric GRq/i was measured
in response to 3 µM carbachol and/or 100 µM compound 2, 3, or 4. The data shown are the result of an experiment performed in
triplicate.
PTLC (EtOAc) to give 12e (342 mg, 82%) as a yellow oil: Rf )
0.17 (EtOAc/hexanes ) 3/1); IR (neat) 2976, 2821, 2776, 1723,
1599, 1459, 1376, 1282, 1117 cm-1; 1H NMR (CDCl3) δ 1.41 (t,
J ) 7.2 Hz, 3 H), 2.31 (s, 6 H), 3.65 (s, 2 H), 4.41 (q, J ) 7.2
Hz, 2 H), 7.51 (d, J ) 7.8 Hz, 1 H), 8.27 (dd, J ) 2.1, 7.8 Hz,
1 H), 9.16 (d, J ) 2.1 Hz, 1 H); 13C NMR (CDCl3) 14.2, 45.5 (2
C), 61.2, 65.4, 122.4, 124.7, 137.3, 150.3, 163.5, 165.2 ppm;
MS (+CI) 209 [M + 1]+; Mr (+CI) 209.129 93 [M + 1]+ (calcd
for C11H17N2O2, 209.129 00).
E t h yl 1-[2-(E t h oxyca r b on yl)et h yl]-3-p ip er id in eca r -
boxyla te (13a ).7 Ethyl 3-piperidinecarboxylate (14) (3.14 g,
20 mmol) was dissolved in ethyl acrylate (2.50 g, 25 mmol)
and stirred at 80 °C (12 h), and then the reaction was
concentrated in vacuo and purified by PTLC (EtOAc/hexanes
) 3/1) to give 13a (4.64 g, 90%) as a pale yellow oil: Rf ) 0.51
(EtOAc/hexanes ) 3/1); IR (neat) 2944, 2806, 1735, 1455, 1376,
1309 cm-1; 1H NMR (CDCl3) δ 1.17-1.22 (m, 6 H), 1.35-1.51
(m, 2 H), 1.62-1.68 (m, 1 H), 1.83-1.89 (m, 1 H), 1.95-2.02
(m, 1 H), 2.11-2.18 (m, 1 H), 2.40-2.50 (m, 3 H), 2.62-2.70
(m, 3 H), 2.91 (dd, J ) 1.8, 8.1 Hz, 1 H), 4.03-4.12 (m, 4 H);
13C NMR (CDCl3) 14.3 (2 C), 24.7, 26.9, 32.5, 41.9, 53.5, 53.9,
55.3, 60.3, 60.4, 172.6, 174.1 ppm; MS (+CI) 258 [M + 1]+; Mr
(+CI) 258.170 30 [M + 1]+ (calcd for C13H24NO4, 258.170 53).
Eth yl 2-Ben zyloxym eth yl-1-[2-(eth oxyca r bon yl)eth yl]-
5-p ip er id in eca r boxyla te (13c). To an EtOAc solution (0.9
mL) of 12c (271 mg, 1.0 mmol) was added 2 M ethereal HCl
(3.6 mL), and then the solvent was removed in vacuo, the
residual 12c‚HCl salt was dissolved in MeOH (5.4 mL), and
PtO2 (14 mg) was added. The mixture was hydrogenated under
1 atm of H2 (1 h). The catalyst was filtered, and the solvent
was removed in vacuo. The residue was dissolved in aqueous
1 N NaOH (3.5 mL) and extracted with EtOAc (3 × 15 mL).
The organic extracts were combined, dried (Na2SO4), concen-
trated, dissolved in ethyl acrylate (1.1 mL, 10 mmol), and
stirred at 80 °C (20 h). The reaction was concentrated to
dryness, and the residue was separated by PTLC (EtOAc/
hexanes ) 2/3) to give 13c (48 mg, 13%) and 15 (32 mg, 12%),
respectively.
Com p ou n d 13c: clear oil; Rf ) 0.57 (EtOAc/hexanes ) 1/1);
1
IR (neat) 2978, 2935, 2862, 1731, 1454, 1372, 1181 cm-1; H
NMR (CDCl3) δ 1.24 (t, J ) 7.2 Hz, 3 H), 1.25 (t, J ) 7.2 Hz,
3 H), 1.64-1.77 (m, 4 H), 2.43-2.54 (m, 3 H), 2.66 (dd, J )
3.9, 12.2 Hz, 1 H), 2.82-2.97 (m, 4 H), 3.45 (dd, J ) 5.9, 9.6
Hz, 1 H), 3.66 (dd, J ) 5.4, 9.6 Hz, 1 H), 4.12 (q, J ) 7.2 Hz,
4 H), 4.47 (1/2 AB, J ) 12.2 Hz, 1 H), 4.52 (1/2 AB, J ) 12.2
Hz, 1 H), 7.27-7.34 (m, 5 H); 13C NMR (CDCl3) 14.1, 14.2,
23.3, 25.9, 33.1, 40.6, 50.1, 50.3, 57.6, 60.1, 60.2, 69.3, 73.2,
127.4, 127.5 (2 C), 128.3 (2 C), 138.3, 172.7, 174.1 ppm; MS
(+CI) 378 [M + 1]+; Mr (+CI) 378.228 63 [M + 1]+ (calcd for
C
21H32NO5, 378.228 05).
Com p ou n d 158 (cis- a n d tr a n s-m ixtu r e): yellow oil; Rf
) 0.44 (EtOAc/hexanes ) 1/1); IR (neat) 2974, 2924, 2814,

2222 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 11
Kim et al.
2987, 2859, 1726, 1465, 1386, 1301, 1230, 1118 cm-1; 1H NMR
(CD3OD, 300 MHz) δ 1.39 (t, J ) 7.2 Hz, 3 H), 4.40 (q, J ) 7.2
Hz, 2 H), 4.73 (s, 2 H), 4.77 (s, 2 H), 7.27-7.42 (m, 5 H), 7.78
(d, J ) 8.1 Hz, 1 H), 8.04 (d, J ) 8.1 Hz, 1 H), 8.71 (s, 1 H);
13C NMR (CD3OD, 75 MHz) 14.4, 63.4, 67.1, 74.7, 125.1, 129.1
(2 C), 129.2 (2 C), 129.6, 129.9, 130.0, 138.9, 141.0, 155.1, 164.1
ppm; MS (+CI) 288 [M + 1]+; Mr (+CI) 288.123 95 [M + 1]+
(calcd for C16H18NO4, 288.123 58). Anal. (C16H17NO4): C, H,
N.
Eth yl (2R,S)-2-Ben zyloxym eth yl-1,2,3,4-tetr a h yd r o-5-
p yr id in eca r boxyla te (18c). Dry ammonium formate (9.24
g, 146.5 mmol) was added to a solution of 17c (4.21 g, 14.7
mmol) containing 10% Pd-C (1.45 g) in anhydrous MeOH (130
mL) under an atm of Ar. The reaction mixture was stirred (17
h) at room temperature, and the mixture was filtered. The
filtrate was evaporated in vacuo, and the residue was tritu-
rated with EtOAc (100 mL). The insoluble solid was filtered,
and the filtrate was concentrated in vacuo. The residue was
purified by column chromatography (EtOAc/hexanes ) 1/2) to
give benzyl compound 18c (3.08 g, 76%) and the corresponding
debenzylated compound (271 mg, 10%), respectively.
Com p ou n d 18c: white solid; mp 64-66 °C; Rf ) 0.60
(EtOAc/hexanes ) 2/1); IR (KBr) 3384, 2943, 2857, 1661, 1616,
1490, 1353, 1303, 1207, 1099 cm-1; 1H NMR (CDCl3, 300 MHz)
δ 1.25 (t, J ) 7.2 Hz, 3 H), 1.43-1.55 (m, 1 H), 1.76-1.85 (m,
1 H), 2.23-2.45 (m, 2 H), 3.30-3.57 (m, 3 H), 4.13 (q, J ) 7.2
Hz, 2 H), 4.53 (s, 2 H), 4.81-4.82 (m, 1 H), 7.26-7.40 (m, 5
H), 7.46 (d, J ) 6.0 Hz, 1 H); 13C NMR (CDCl3, 75 MHz) 14.6,
19.6, 23.6, 49.9, 58.9, 73.2, 73.3, 95.9, 127.7, 127.9 (2 C), 128.5
(2 C), 137.7, 141.9, 168.6 ppm; MS (+CI) 276 [M + 1]+; Mr
(+CI) 276.160 31 [M + 1]+ (calcd for C16H22NO3, 276.159 97).
Anal. (C16H21NO3): C, H, N.
F igu r e 4. Pharmacophore elements present in 3, 4, 21, and
22.
1735, 1455, 1377 cm-1 1H NMR (CDCl3) δ 1.00 (d, J ) 6.6
;
Hz, 1.4 H), 1.10 (d, J ) 6.6 Hz, 1.6 H), 1.23-1.28 (m, 6 H),
1.40-2.01 (m, 4 H), 2.18-2.31 (m, 1 H), 2.42-2.59 (m, 4 H),
2.67-2.88 (m, 2 H), 3.03-3.10 (m, 1 H), 4.08-4.17 (m, 4 H);
13C NMR (CDCl3) 13.7 (2 C), 14.2 (2 C), 19.9 (2 C), 23.1 (2 C),
27.4 (2 C), 32.7, 33.8, 41.2, 42.2, 48.8, 49.6, 49.8, 53.3, 54.1,
55.1, 60.2 (2 C), 60.3 (2 C), 172.8 (2 C), 174.1 (2 C) ppm; MS
(+CI) 272 [M + 1]+; Mr (+CI) 272.186 63 [M + 1]+ (calcd for
Deben zyla ted Com p ou n d : colorless oil; Rf ) 0.16 (EtOAc/
hexanes ) 2/1); IR (neat) 3384, 2940, 2871, 1731, 1660, 1615,
1454, 1369, 1204, 1110 cm-1 1H NMR (CDCl3, 300 MHz) δ
;
C
14H26NO4, 272.186 18).
1.25 (t, J ) 7.2 Hz, 3 H), 1.44-1.56 (m, 1 H), 1.75-1.84 (m, 1
H), 2.05-2.42 (m, 2 H), 3.34-3.72 (m, 4 H), 4.11 (q, J ) 7.2
Hz, 2 H), 5.32-5.34 (m, 1 H), 7.50 (d, J ) 6.0 Hz, 1 H); 13C
NMR (CDCl3, 75 MHz) 14.6, 19.5, 23.3, 51.9, 59.0, 65.8, 96.2,
142.1, 168.7 ppm; MS (+CI) 186 [M + 1]+; Mr (+CI) 186.113 01
[M + 1]+ (calcd for C9H16NO3, 186.113 02). Anal. (C9H15NO3‚
0.4 H2O): C, H, N.
Eth yl 2-Dim eth yla m in om eth yl-1-[2-(eth oxyca r bon yl)-
eth yl]-5-p ip er id in eca r boxyla te (13e). With the same pro-
cedure employed for the preparation of 13c, 12e (335 mg, 1.6
mmol), 2 M ethereal HCl (6 mL), PtO2 (24 mg), and ethyl
acrylate (1.7 mL, 16 mmol) gave 13e (259 mg, 51%) and 15
(52 mg, 12%) following PTLC purification (10% MeOH-
CHCl3).
Eth yl 2-Ben zyloxym eth yl-1-[2-(eth oxyca r bon yl)eth yl]-
5-p ip er id in eca r boxyla te (13c). To a HOAc solution (23 mL)
of 18c (2.62 g, 9.52 mmol) was slowly added a MeOH solution
(23 mL) of NaBH3CN (718 mg, 11.42 mmol). The solution was
stirred at room temperature (3 h) and concentrated in vacuo.
The dried residue (1 h) was treated with ethyl acrylate (10.3
mL, 95.2 mmol) and triethylamine (1.59 mL, 11.4 mmol) at
80-85 °C (17 h). The reaction mixture was concentrated in
vacuo and the residue taken up in EtOAc (70 mL). The mixture
was washed with H2O (2 × 50 mL), dried (Na2SO4), filtered,
and evaporated. The crude residue was purified by column
chromatography (EtOAc/hexanes ) 1/2) to give 13c (3.20 g,
89%, the ratio of cis/trans ) 0.16:1) as yellow oil. The
piperidine adduct consisted of a mixture of cis- and trans-
diastereomers, respectively, that was separable by PTLC
(EtOAc/hexanes ) 1/1).
cis-Dia ster eom er 13c (0.45 g, 13%): yellow oil; Rf ) 0.56
(EtOAc/hexanes ) 1/1); IR (neat) 2978, 2935, 2862, 1731, 1454,
1372, 1181 cm-1; 1H NMR (CDCl3, 300 MHz) δ 1.24 (t, J ) 7.2
Hz, 3 H), 1.25 (t, J ) 7.2 Hz, 3 H), 1.64-1.77 (m, 4 H), 2.43-
2.54 (m, 3 H), 2.66 (dd, J ) 3.9, 12.2 Hz, 1 H), 2.82-2.97 (m,
4 H), 3.45 (dd, J ) 5.9, 9.6 Hz, 1 H), 3.66 (dd, J ) 5.4, 9.6 Hz,
1 H), 4.12 (q, J ) 7.2 Hz, 4 H), 4.47 (1/2 ABq, J ) 12.2 Hz, 1
H), 4.52 (1/2 ABq, J ) 12.2 Hz, 1 H), 7.27-7.34 (m, 5 H); 13C
NMR (CDCl3, 75 MHz) 14.1, 14.2, 23.3, 25.9, 33.1, 40.6, 50.1,
50.3, 57.6, 60.1, 60.2, 69.3, 73.2, 127.4, 127.5 (2 C), 128.3 (2
C), 138.3, 172.7, 174.1 ppm; MS (+CI) 378 [M + 1]+; Mr (+CI)
378.228 63 [M + 1]+ (calcd for C21H32NO5, 378.228 05). Anal.
(C21H31NO5): C, H, N.
Com p ou n d 13e: reddish-yellow oil; Rf ) 0.37 (10% MeOH-
CHCl3); IR (neat) 2945, 2818, 1731, 1457, 1376, 1182 cm-1
;
1H NMR (CDCl3) δ 1.18 (t, J ) 7.2 Hz, 3 H, CH2CH3), 1.19 (t,
J ) 7.2 Hz, 3 H, CH2CH3), 1.56-1.70 (m, 4 H, C(3)H2, C(4)-
H2), 2.15 (s, 6 H, N(CH3)2), 2.26-2.48 (m, 5 H, C(5)H,
NCH2CH2, (CH3)2NCH2), 2.60-2.91 (m, 5 H, C(2)H, C(6)H2,
NCH2CH2), 4.05 (q, J ) 7.2 Hz, 2 H, CH2CH3), 4.07 (q, J ) 7.2
1
Hz, 2 H, CH2CH3), the H NMR assignments were consistent
with the COSY spectrum; 13C NMR (CDCl3) 14.0 (CH2CH3),
14.1 (CH2CH3), 22.8 (C(3)), 25.5 (C(4)), 33.5 (NCH2CH2), 40.1
(C(5)), 46.0 (N(CH3)2), 49.3 (C(6)), 49.7 (NCH2CH2), 55.4 (C(2)),
57.7 ((CH3)2NCH2), 60.1 (2 C, CH2CH3), 172.6 (C(O)), 174.2
(C(O)) ppm, the assignments were consistent with the DEPT
and HMQC spectra; MS (+CI) 315 [M + 1]+; Mr (+CI)
315.229 49 [M + 1]+ (calcd for C16H31N2O4, 315.228 38).
Com p ou n d 15 (cis- a n d tr a n s-m ixtu r e): Rf ) 0.75 (10%
MeOH-CHCl3); 1H NMR (CDCl3) δ 1.00 (d, J ) 6.6 Hz, 2 H),
1.10 (d, J ) 6.6 Hz, 1 H), 1.21-1.28 (m, 6 H), 1.44-2.00 (m, 4
H), 2.21-2.30 (m, 1 H), 2.42-2.59 (m, 4 H), 2.67-2.88 (m, 3
H), 4.08-4.17 (m, 4 H).
Eth yl 2-Ben zyloxym eth yl-5-p yr id in eca r boxyla te N-
Oxid e (17c). A CHCl3 solution (22 mL) of m-chloroperbenzoic
acid (3.72 g, 16.6 mmol) was gradually added to an ice-cooled
(0-5 °C), stirred CHCl3 solution (17 mL) of 12c (4.50 g, 16.6
mmol). The reaction mixture was stirred (4 h), during which
the mixture was allowed to come to room temperature. The
reaction mixture was successively washed with aqueous 0.5
N NaOH (20 mL) and H2O (20 mL), and the CHCl3 layer was
dried (Na2SO4) and concentrated in vacuo. Purification of the
concentrated residue by column chromatography (EtOAc/
hexanes ) 3/1) gave 17c (4.00 g, 84%) as yellowish white
solid: mp 50-52 °C; Rf ) 0.19 (EtOAc/hexanes ) 2/1); IR 3047,
tr a n s-Dia ster eom er 13c (2.75 g, 76%): yellow oil; Rf ) 0.51
(EtOAc/hexanes ) 1/1); IR (neat) 2936, 2863, 1730, 1455, 1370,
1
1183 cm-1; H NMR (CDCl3, 300 MHz) δ 1.24 (t, J ) 7.2 Hz,

Novel Muscarinic Receptor Antagonists
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 11 2223
3 H), 1.25 (t, J ) 7.2 Hz, 3 H), 1.35-1.50 (m, 2 H), 1.69-1.77
(m, 1 H), 2.01-2.04 (m, 1 H), 2.28-2.58 (m, 5 H), 2.83-2.92
(m, 1 H), 3.07-3.19 (m, 2 H), 3.43 (dd, J ) 4.2, 9.9 Hz, 1 H),
3.52 (dd, J ) 4.5, 9.9 Hz, 1 H), 4.11 (q, J ) 7.2 Hz, 4 H), 4.50
(1/2 ABq, J ) 12.4 Hz, 1 H), 4.54 (1/2 ABq, J ) 12.4 Hz, 1 H),
7.24-7.37 (m, 5 H); 13C NMR (CDCl3, 75 MHz) 14.2 (2 C), 26.9,
28.7, 30.6, 41.6, 48.9, 54.1, 59.4, 60.2 (2 C), 72.7, 73.3, 127.6,
127.7 (2 C), 128.3 (2 C), 138.1, 172.7, 174.0 ppm; MS (+CI)
mg, 16%) and 4 (4.0 mg, 14%) following PTLC purification (5%
MeOH-CHCl3).
Com p ou n d 3: yellow oil; Rf ) 0.52 (5% MeOH-CHCl3); IR
(neat) 2933, 2863, 1734, 1656, 1370, 1293, 1209 cm-1; 1H NMR
(CDCl3, 600 MHz) δ 1.31 (t, J ) 7.1 Hz, 3 H, CH2CH3), 1.45-
1.47 (m, 1 H, C(7)HH′), 1.72-1.81 (m, 3 H, C(7)HH′, C(6)H2),
2.17 (br s, 1 H, C(5)H), 2.75 (d, J ) 13.4 Hz, 1 H, C(9)HH′),
3.01-3.02 (m, 1 H, C(8)H), 3.07 (d, J ) 13.4 Hz, 1 H, C(9)-
HH′), 3.33 (d, J ) 16.8 Hz, 1 H, C(2)HH′), 3.52 (dd, J ) 6.5,
9.3 Hz, 1 H, BnOCHH′), 3.65 (dd, J ) 7.5, 9.3 Hz, 1 H,
BnOCHH′), 3.89 (d, J ) 16.8 Hz, 1 H, C(2)HH′), 4.23 (q, J )
7.1 Hz, 2 H, CH2CH3), 4.54 (d, J ) 12.2 Hz, 1 H, PhCHH′O),
4.60 (d, J ) 12.2 Hz, 1 H, PhCHH′O), 7.28-7.35 (m, 5 H, Ph),
11.86 (br s, 1 H, C(4)OH), the 1H NMR assignments were
consistent with the COSY and NOESY spectra; 13C NMR
(CDCl3, 150 MHz) 14.2 (CH2CH3), 19.4 (C(7)), 22.7 (C(6)), 31.6
(C(5)), 46.3 (C(9)), 52.1 (C(2)), 60.1 (C(8)), 60.2 (CH2CH3), 70.7
(BnOCH2), 73.1 (PhCH2O), 99.9 (C(3)), 127.0, 127.5 (2 C), 127.6
(2 C), 138.3 (Ph), 170.9 (C(O)), 171.9 (C(4)) ppm, the assign-
ments were consistent with the DEPT, HMQC, and HMBC
spectra; MS (+CI) 331 [M]+; Mr (+CI) 331.179 07 [M]+ (calcd
for C19H25NO4, 331.178 36). Anal. (C19H25NO4): C, H, N.
378 [M + 1]+; Mr (+CI) 378.229 05 [M + 1]+ (calcd for C21H32
-
NO5, 378.228 05). Anal. (C21H31NO5): C, H, N.
exo-8-B e n z y lo x y m e t h y l-3-e t h o x y c a r b o n y l-4-h y -
dr oxy-1-azabicyclo[3.3.1]n on -3-en e (3) an d en do-8-Ben zyl-
oxymethyl-3-ethoxycarbonyl-4-hydroxy-1-azabicyclo[3.3.1]-
n on -3-en e (4) fr om cis- a n d tr a n s-13c. A suspension of
t-BuOK (842 mg, 7.5 mmol) in anhydrous toluene (5 mL) was
heated to reflux (1 h), and then an anhydrous toluene solution
(1.5 mL) of the cis- and trans-13c (944 mg, 2.5 mmol) was
added (20 min). The reaction mixture was heated to reflux (3
h). The reaction mixture was concentrated in vacuo and the
residue taken up in H2O (10 mL) and neutralized (pH 7-8)
with concentrated aqueous HCl. The mixture was extracted
with CHCl3 (3 × 20 mL), dried (Na2SO4), filtered, and
evaporated. The crude mixture was purified by column chro-
matography (2.5% MeOH/CHCl3) to give exo-compound 3 (297
mg, 36%) and endo-compound 4 (277 mg, 33%) as yellow oils,
respectively.
Com p ou n d 3: yellow oil; Rf ) 0.52 (5% MeOH-CHCl3); 1H
NMR (CDCl3, 300 MHz) δ 1.29 (t, J ) 7.2 Hz, 3 H), 1.43-1.45
(m, 1 H), 1.71-1.81 (m, 3 H), 2.16 (br s, 1 H), 2.73 (d, J ) 13.5
Hz, 1 H), 2.99-3.02 (m, 1 H), 3.05 (d, J ) 13.5 Hz, 1 H), 3.33
(d, J ) 16.7 Hz, 1 H), 3.51 (dd, J ) 7.5, 9.3 Hz, 1 H), 3.64 (dd,
J ) 6.6, 9.3 Hz, 1 H), 3.89 (d, J ) 16.7 Hz, 1 H), 4.21 (q, J )
7.2 Hz, 2 H), 4.52 (d, J ) 12.3 Hz, 1 H), 4.59 (d, J ) 12.3 Hz,
1 H), 7.23-7.34 (m, 5 H), 11.89 (br s, 1 H); 13C NMR (CDCl3,
75 MHz) 14.1, 19.2, 22.6, 31.5, 46.1, 52.0, 60.0, 60.1, 70.6, 72.9,
98.8, 127.3, 127.4 (2 C), 128.1 (2 C), 138.2, 170.7, 171.8 ppm.
Com p ou n d 4: yellow oil; Rf ) 0.30 (5% MeOH-CHCl3); IR
(neat) 2928, 2858, 1657, 1371, 1301, 1209 cm-1 1H NMR
;
(CDCl3, 600 MHz) δ 1.22 (t, J ) 7.1 Hz, 3 H, CH2CH3), 1.30-
1.40 (m, 2 H, C(7)H2), 1.71-1.78 (m, 1 H, C(6)HH′), 1.88-1.91
(m, 1 H, C(6)HH′), 2.22 (br s, 1 H, C(5)H), 3.01 (d, J ) 13.2
Hz, 1 H, C(9)HH′), 3.04-3.13 (m, 1 H, C(8)H), 3.08 (d, J )
13.2 Hz, 1 H, C(9)HH′), 3.37-3.51 (m, 4 H, C(2)H2, BnOCH2),
4.06-4.19 (m, 2 H, CH2CH3), 4.46 (d, J ) 12.3 Hz, 1 H,
PhCHH′O), 4.52 (d, J ) 12.3 Hz, 1 H, PhCHH′O), 7.21-7.28
(m, 5 H, Ph), 11.81 (br s, 1 H, C(4)OH), the 1H NMR
assignments were consistent with the COSY and NOESY
spectra; 13C NMR (CDCl3, 125 MHz) 14.3 (CH2CH3), 21.7
(C(7)), 27.6 (C(6)), 32.0 (C(5)), 42.5 (C(2)), 53.2 (C(9)), 60.2 (CH2-
CH3), 61.5 (C(8)), 71.6 (BnOCH2), 73.1 (PhCH2O), 99.3 (C(3)),
127.6, 127.7 (2 C), 128.3 (2 C), 138.3 (Ph), 170.7 (C(O)), 172.1
(C(4)) ppm, the assignments were consistent with the DEPT,
HMQC, and HMBC spectra; MS (+CI) 331 [M]+; Mr (+CI)
331.178 13 [M]+ (calcd for C19H25NO4, 331.178 36). Anal.
(C19H25NO4): C, H, N.
exo-8-Dim et h yla m in om et h yl-3-et h oxyca r b on yl-4-h y-
d r oxy-1-a za b icyclo[3.3.1]n on -3-en e (5) a n d en d o-8-Di-
m e t h yla m in om e t h yl-3-e t h oxyca r b on yl-4-h yd r oxy-1-
a za bicyclo[3.3.1]n on -3-en e (6). With the same procedure
employed for the preparation of 2, t-BuOK (41 mg, 0.4 mmol)
and 13e (38 mg, 0.1 mmol) in anhydrous toluene (1.5 mL) gave
5 (4 mg, 12%) and 6 (4 mg, 12%) following PTLC purification
(10% MeOH-CHCl3).
Com p ou n d 4: yellow oil; Rf ) 0.30 (5% MeOH-CHCl3); 1H
NMR (CDCl3, 300 MHz) δ 1.30 (t, J ) 7.2 Hz, 3 H), 1.31-1.41
(m, 2 H), 1.74-1.86 (m, 1 H), 1.91-1.98 (m, 1 H), 2.23 (br s, 1
H), 3.02-3.17 (m, 3 H), 3.31-3.57 (m, 4 H), 4.12-4.26 (m, 2
H), 4.53 (d, J ) 12.6 Hz, 1 H), 4.59 (d, J ) 12.6 Hz, 1 H), 7.27-
7.36 (m, 5 H), 11.84 (br s, 1 H); 13C NMR (CDCl3, 75 MHz)
14.2, 21.8, 27.6, 32.0, 42.5, 53.3, 60.2, 61.5, 71.9, 73.1, 99.3,
127.5, 127.7 (2 C), 128.3 (2 C), 138.3, 170.7, 172.2 ppm.
3-Eth oxyca r bon yl-4-h yd r oxy-1-a za bicyclo[3.3.1]n on -3-
en e (2). A suspension of t-BuOK (505 mg, 4.5 mmol) in
anhydrous toluene (4.5 mL) was refluxed (1 h), and then an
anhydrous toluene solution (1.5 mL) of 13a (386 mg, 1.5 mmol)
was added (20 min). The reaction mixture was refluxed for an
additional 3 h and then cooled. EtOH (2 mL) was added, and
the reaction was filtered (Celite pad) and concentrated in
vacuo. The residue was purified by PTLC (5% MeOH-CHCl3)
to give 2 (127 mg, 40%) as a yellow oil: Rf ) 0.21 (5% MeOH-
CHCl3); IR (neat) 2932, 2857, 1656, 1292, 1207 cm-1; 1H NMR
(CDCl3, 500 MHz) δ 1.25 (t, J ) 7.1 Hz, 3 H, CH2CH3), 1.55-
1.64 (m, 2 H, C(7)H2), 1.65-1.73 (m, 1 H, C(6)HH′), 1.84-1.86
(m, 1 H, C(6)HH′), 2.19 (br s, 1 H, C(5)H), 2.89 (d, J ) 13.0
Hz, 1 H, C(9)HH′), 2.90-2.96 (m, 2 H, C(8)H2), 2.96 (d, J )
13.0 Hz, 1 H, C(9)HH′), 3.24 (d, J ) 16.8 Hz, 1 H, C(2)HH′),
3.73 (d, J ) 16.8 Hz, 1 H, C(2)HH′), 4.17 (q, J ) 7.1 Hz, 2 H,
Com p ou n d 5: yellow oil; Rf ) 0.20 (10% MeOH-CHCl3);
IR (neat) 2935, 2862, 2770, 1655, 1456, 1363, 1294, 1208 cm-1
;
1H NMR (CDCl3, 500 MHz) δ 1.32 (t, J ) 7.2 Hz, 3 H, CH2CH3),
1.73-1.83 (m, 4 H, C(6)H2, C(7)H2), 2.19 (br s, 1 H, C(5)H),
2.23 (dd, J ) 6.8, 12.5 Hz, 1 H, (CH3)2NCHH′), 2.29 (s, 6 H,
N(CH3)2), 2.70 (dd, J ) 8.4, 12.5 Hz, 1 H, (CH3)2NCHH′), 2.77
(d, J ) 13.6 Hz, 1 H, C(9)HH′), 2.90-2.94 (m, 1 H, C(8)H),
3.09 (d, J ) 13.6 Hz, 1 H, C(9)HH′), 3.31 (d, J ) 16.8 Hz, 1 H,
C(2)HH′), 3.93 (d, J ) 16.8 Hz, 1 H, C(2)HH′), 4.23 (q, J ) 7.1
Hz, 2 H, CH2CH3), 11.87 (br s, 1 H, C(4)OH), the 1H NMR
assignments were consistent with the COSY and NOESY
spectra; 13C NMR (CDCl3, 125 MHz) 14.7 (CH2CH3), 20.9
(C(7)), 22.9 (C(6)), 32.2 (C(5)), 45.9 (C(9)), 46.1 (2 C, N(CH3)2),
52.5 (C(2)), 58.8 (C(8)), 60.7 (CH2CH3), 61.2 ((CH3)2NCH2), 99.2
(C(3)), 171.0 (C(O)), 171.8 (C(4)) ppm, the assignments were
consistent with the DEPT, HMQC, and HMBC spectra; MS
(+CI) 269 [M + 1]+; Mr (+CI) 269.185 87 [M + 1]+ (calcd for
1
CH2CH3), 11.83 (br s, 1 H, C(4)OH), the H NMR assignments
were consistent with the COSY spectrum; 13C NMR (CDCl3,
150 MHz) 14.2 (CH2CH3), 19.1 (C(7)), 26.6 (C(6)), 32.2 (C(5)),
49.5 (C(2)), 51.4 (C(9)), 55.2 (C(8)), 60.2 (CH2CH3), 99.3 (C(3)),
170.6 (C(O)), 172.1 (C(4)) ppm, the assignments were consis-
tent with the DEPT, HMQC, and HMBC spectra; MS (+CI)
212 [M + 1]+; Mr (+CI) 212.128 08 [M + 1]+ (calcd for C11H18
NO3, 212.128 67). Anal. (C11H17NO3): C, H, N.
-
C14H25N2O3, 269.186 52). Anal. (C14H24N2O3‚0.5 C6H5CH3‚1.6
H2O): C, H, N.
exo-8-Ben zyloxym eth yl-3-eth oxyca r bon yl-4-h yd r oxy-
1-a za bicyclo[3.3.1]n on -3-en e (3) a n d en d o-8-Ben zyloxy-
methyl-3-ethoxycarbonyl-4-hydroxy-1-azabicyclo[3.3.1]non-3-
en e (4) fr om cis-13c. With the same procedure employed for
the preparation of 2, t-BuOK (29 mg, 0.25 mmol) and cis-13c
(32 mg, 0.09 mmol) in anhydrous toluene (1 mL) gave 3 (4.5
Com p ou n d 6: yellow oil; Rf ) 0.26 (10% MeOH-CHCl3);
IR (neat) 2934, 2864, 2773, 1656, 1457, 1366, 1292, 1208 cm-1
;
1H NMR (CDCl3, 500 MHz) δ 1.32 (t, J ) 7.2 Hz, 3 H, CH2CH3),
1.28-1.32 (m, 1 H, C(7)HH′), 1.40-1.45 (m, 1 H, C(7)HH′),
1.77-1.84 (m, 1 H, C(6)HH′), 1.92-1.97 (m, 1 H, C(6)HH′),
2.12 (dd, J ) 7.5, 12.7 Hz, 1 H, (CH3)2NCHH′), 2.23 (br s, 1 H,

2224 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 11
Kim et al.
C(5)H), 2.27 (s, 6 H, N(CH3)2), 2.45 (dd, J ) 6.3, 12.7 Hz, 1 H,
(CH3)2NCHH′), 2.91-2.97 (m, 1 H, C(8)H), 3.03 (br dt, J )
1.3, 13.0 Hz, 1 H, C(9)HH′), 3.12 (dt, J ) 2.3, 13.0 Hz, 1 H,
C(9)HH′), 3.35 (dd, J ) 1.0, 17.2 Hz, 1 H, C(2)HH′), 3.55 (d, J
) 17.2 Hz, 1 H, C(2)HH′), 4.17-4.30 (m, 2 H, CH2CH3), 11.84
C(7)HH′), 1.57-1.60 (m, 1 H, C(7)HH′), 1.89-1.98 (m, 2 H,
C(6)H2), 2.13-2.34 (m, 3 H, C(3)H2, (CH3)2NCHH′), 2.39 (br
s, 1 H, C(5)H), 2.45 (s, 6 H, N(CH3)2), 2.72-2.77 (m, 1 H, (CH3)2-
NCHH′), 3.20-3.26 (m, 4 H, C(2)HH′, C(8)H, C(9)H2), 3.34-
3.39 (m, 1 H, C(2)HH′), the 1H NMR assignments were
consistent with the COSY spectrum; 13C NMR (CDCl3, 125
MHz) 26.2 (C(7)), 27.6 (C(6)), 42.0 (C(3)), 44.6 (C(2)), 45.2
(C(5)), 45.8 (N(CH3)2), 56.0 (C(9)), 58.1 (C(8)), 62.2 ((CH3)2NCH2),
212.5 (C(4)) ppm, the assignments were consistent with the
DEPT, HMQC, and HMBC spectra; MS (+CI) 197 [(M-HCl)
+ 1]+; Mr (+CI) 197.165 74 [(M-HCl) + 1]+ (calcd for
1
(br s, 1 H, C(4)OH), the H NMR assignments were consistent
with the COSY and NOESY spectra; 13C NMR (CDCl3, 125
MHz) 14.7 (CH2CH3), 24.1 (C(7)), 28.4 (C(6)), 32.5 (C(5)), 42.4
(C(2)), 46.6 (2 C, N(CH3)2), 53.9 (C(9)), 60.3 (C(8)), 60.7 (CH2-
CH3), 63.8 ((CH3)2NCH2), 99.3 (C(3)), 170.7 (C(O)), 172.3 (C(4))
ppm, the assignments were consistent with the DEPT, HMQC,
and HMBC spectra; MS (+CI) 269 [M + 1]+; Mr (+CI)
269.185 39 [M + 1]+ (calcd for C14H25N2O3, 269.186 52). Anal.
(C14H24N2O3‚0.4 C6H5CH3‚1 H2O): C, H, N.
C
11H21N2O, 197.165 39).
exo-8-Dim eth yla m in om eth yl-1-a za bicyclo[3.3.1]n on a n -
4-on e‚H Cl (10) a n d en d o-8-Dim et h yla m in om et h yl-1-
a za bicyclo[3.3.1]n on a n -4-on e‚HCl (11) fr om 13e. With the
procedure for the synthesis of 7 from 13a , 13e (314 mg, 1.0
mmol), t-BuOK (337 mg, 3.0 mmol), and anhydrous toluene
(6 mL) gave a crude mixture that was heated (85-90 °C, 16
h) with concentrated aqueous HCl (4 mL) and then separated
by PTLC (25% MeOH-CHCl3) to give 10 (36 mg, 16%) and 11
(31 mg, 13%), respectively.
Com p ou n d 10: yellow semisolid; Rf ) 0.08 (10% MeOH-
CHCl3); 1H NMR (CDCl3, 500 MHz) δ 1.53-1.80 (m, 2 H),
1.80-2.01 (m, 2 H), 2.26 (dd, J ) 6.8, 12.4 Hz, 1 H), 2.30 (s, 6
H), 2.37 (br s, 1 H), 2.49 (dd, J ) 6.2, 17.5 Hz, 1 H), 2.62 (ddd,
J ) 8.9, 10.6, 17.5 Hz, 1 H), 2.70 (dd, J ) 8.4, 12.4 Hz, 1 H),
2.90 (d, J ) 13.9 Hz, 1 H), 3.05-3.10 (m, 1 H), 3.27-3.33 (m,
1 H), 3.32 (d, J ) 13.9 Hz, 1 H), 3.40-3.49 (m, 1 H).
Com p ou n d 11: yellow semisolid; Rf ) 0.13 (10% MeOH-
CHCl3); 1H NMR (CDCl3, 500 MHz) δ 1.30-1.61 (m, 2 H),
1.89-1.98 (m, 2 H), 2.10-2.33 (m, 3 H), 2.40 (br s, 1 H), 2.46
(s, 6 H), 2.70-2.75 (m, 1 H), 3.21-3.25 (m, 4 H), 3.35-3.41
(m, 1 H).
1-Aza bicyclo[3.3.1]n on a n -4-on e (7)7,8 fr om 2. A solution
of 2 (21 mg, 0.1 mmol) and concentrated aqueous HCl (1 mL)
was refluxed (14 h), and then the solution was basified (30%
aqueous KOH) and extracted with CH2Cl2 (5 × 10 mL). The
combined CH2Cl2 extracts were dried (Na2SO4) and concen-
trated in vacuo to give 7 (5 mg, 38%) as a semisolid: Rf )
0.14 (5% MeOH-CHCl3); IR (neat) 2929, 2857, 1697, 1352,
1
1201 cm-1; H NMR (CDCl3, 300 MHz) δ 1.64-1.81 (m, 2 H,
C(7)H2), 1.80-1.86 (m, 2 H, C(6)H2), 2.41 (br s, 1 H, C(5)H),
2.49-2.54 (m, 2 H, C(3)H2), 3.08-3.23 (m, 4 H, C(2)H2, C(8)-
H2), 3.29-3.39 (m, 2 H, C(9)H2), the 1H NMR assignments
were consistent with the COSY spectrum; 13C NMR (CDCl3,
75 MHz) 21.9 (C(7)), 27.5 (C(6)), 41.8 (C(3)), 45.4 (C(5)), 51.2
(C(2)). 53.3 (C(8)), 53.9 (C(9)), 212.6 (C(4)) ppm, the assign-
ments were consistent with the DEPT and HMQC spectrum;
MS (+EI) 139 [M]+; Mr (+EI) 139.100 15 [M]+ (calcd for C8H13
NO, 139.099 71).
-
1-Aza bicyclo[3.3.1]n on a n -4-on e (7) fr om 13a . A suspen-
sion of t-BuOK (1.01 g, 9.0 mmol) in anhydrous toluene (12
mL) was refluxed (1 h), and then an anhydrous toluene
solution (2.5 mL) of 13a (772 mg, 3.0 mmol) was added (20
min). The reaction mixture was refluxed for an additional 3
h, cooled, and extracted with concentrated aqueous HCl (2 ×
5 mL). The combined aqueous HCl extracts were refluxed (16
h), and then the solution was basified (30% aqueous KOH) and
extracted with CH2Cl2 (3 × 10 mL). The combined CH2Cl2
extracts were dried (Na2SO4) and concentrated in vacuo to give
7 (137 mg, 33%) as a semisolid: Rf ) 0.14 (5% MeOH-CHCl3);
1H NMR (CDCl3, 300 MHz) δ 1.52-1.82 (m, 2 H), 1.90-1.97
(m, 2 H), 2.42 (br s, 1 H), 2.50-2.55 (m, 2 H), 3.10-3.23 (m, 4
H), 3.27-3.43 (m, 2 H).
exo-8-Dim eth yla m in om eth yl-1-a za bicyclo[3.3.1]n on a n -
4-on e‚HCl (10) fr om 5. With the procedure for the synthesis
of 7 from 2, 5 (10 mg, 0.04 mmol) and concentrated aqueous
HCl (0.5 mL) gave 10 (3.9 mg, 38%) as a yellow semisolid
following PTLC purification (10% MeOH-CHCl3); Rf ) 0.08
(10% MeOH-CHCl3); IR (neat) 2932, 2866, 2774, 1700, 1460,
1357, 1277 cm-1; 1H NMR (CDCl3, 500 MHz) δ 1.52-1.56 (m,
1 H, C(7)HH′), 1.72-1.78 (m, 1 H, C(6)HH′), 1.80-1.86 (m, 1
H, C(7)HH′), 1.97-2.05 (m, 1 H, C(6)HH′), 2.26 (dd, J ) 6.8,
12.4 Hz, 1 H, (CH3)2NCHH′), 2.31 (s, 6 H, N(CH3)2), 2.39 (br
s, 1 H, C(5)H), 2.49 (dd, J ) 6.2, 17.5 Hz, 1 H, C(3)HH′), 2.62
(ddd, J ) 8.9, 10.6, 17.5 Hz, 1 H, C(3)HH′), 2.72 (dd, J ) 8.4,
12.4 Hz, 1 H, (CH3)2NCHH′), 2.91 (d, J ) 13.9 Hz, 1 H, C(9)-
HH′), 3.08-3.12 (m, 1 H, C(8)H), 3.27-3.33 (m, 1 H, C(2)HH′),
3.32 (d, J ) 13.9 Hz, 1 H, C(9)HH′), 3.41-3.50 (m, 1 H, C(2)-
HH′), the 1H NMR assignments were consistent with the
COSY spectrum; 13C NMR (CDCl3, 125 MHz) 23.2 (C(7)), 24.1
(C(6)), 41.1 (C(3)), 45.2 (C(5)), 46.2 (N(CH3)2), 48.0 (C(9)), 53.8
(C(2)), 56.8 (C(8)), 62.4 ((CH3)2NCH2), 213.1 (C(4)) ppm, the
assignments were consistent with the HMQC and HMBC
spectra; MS (+CI) 197 [(M-HCl) + 1]+; Mr (+CI) 197.165 81
[(M-HCl) + 1]+ (calcd for C11H21N2O, 197.165 39).
X-r a y Cr ysta llogr a p h ic Stu d y of 10. Compound 10 was
recrystallized from chloroform-d. Crystals of 10 belong to the
space group P21/n (monoclinic) with a ) 10.953 (3) Å; b ) 7.864
(2) Å; c ) 25.010 (8) Å; V ) 2140 Å3, Dcalcd ) 1.46 Mg‚m-3, and
Z ) 4. Data were collected at -100 °C, and the structure was
refined to Rf ) 0.053, Rw ) 0.063 for 5057 reflections with I >
3σ(I).
P h a r m a cologica l An a lyses
h M1-h M2 Exp r ession in Sf9 In sect Cells a n d Mem -
br a n e P r ep a r a tion . Human M1 and M2 receptors were
expressed in Sf9 insect cells by using the baculovirus expres-
sion system. Cells were lysed by nitrogen cavitation, and
membranes were collected through differential centrifugation
and resuspended in a buffer containing 20 mM HEPES (pH)
8), 250 mM sucrose, 0.1 mM EDTA, and a protease inhibitor
cocktail for a final protein concentration of 5-10 mg/mL.
h M1-h M5 Exp r ession in COS-7 Cells a n d Mem br a n e
P r ep a r a tion . COS-7 cells were subcultured in 150 mm dishes
to a density of 20 000 cells/cm2. The cells were transfected with
Fugene 6 transfection reagent (according to Roche Molecular
Biochemical’s specifications) and pcDNA3.1 DNA containing
sequence coding for the hM1, hM2, hM3, hM4, or hM5 receptor
(approximately 20 µg DNA/150 mm dish). Cell lysates were
harvested after 24 h by scraping the cells and sonicating in
buffer (5 mM Tris (pH 7.5), 1 mM MgCl2, and protease
inhibitors). Membranes were isolated by differential centrifu-
gation and resuspended in freezing buffer (20 mM HEPES (pH
8.0), 250 mM sucrose, 0.1 mM EDTA, and protease inhibitors).
Binding assays were carried out as described below.
h M1-h M5 Bin d in g Assa y. Competition binding assays
were performed essentially as described30 in a volume of 2 mL
by using 10 µL membrane (50-100 µg protein), 200 pM [3H]-
QNB (approximately 20 000 cpm/assay), and various concen-
trations of competing agents (carbachol, arecoline, and com-
pounds 2-7, 10, and 11) in binding buffer (20 mM HEPES
(pH 7.5), 150 mM NaCl, 3 mM MgCl2, and 1 mM EDTA).
Incubations were for 90 min at 30 °C. The assays were
terminated by addition of 4 mL of wash buffer (20 mM Tris
(pH 7.5), 150 mM NaCl, and 2 mM MgCl2), quick filtration
over Whatman GF/A filters, and two washes with 4 mL of wash
en do-8-Dim eth ylam in om eth yl-1-azabicyclo[3.3.1]n on an -
4-on e‚HCl (11) fr om 6. With the procedure for the synthesis
of 7 from 2, 6 (12 mg, 0.04 mmol) and concentrated aqueous
HCl (0.5 mL) gave 11 (4.3 mg, 42%) as a yellow semisolid
following PTLC purification (10% MeOH-CHCl3); Rf ) 0.13
(10% MeOH-CHCl3); IR (neat) 2936, 2873, 1694, 1462, 1361,
1
1219 cm-1; H NMR (CDCl3, 500 MHz) δ 1.29-1.36 (m, 1 H,

Novel Muscarinic Receptor Antagonists
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 11 2225
Analgesics with Potent and Selective Effects on the Gastrointes-
tinal Tract: Potential Application for the Treatment of Irritable
Bowel Syndrome. J . Med. Chem. 1997, 40, 538-546. (c) Tecle,
H.; Barrett, S. D.; Lauffer, D. J .; Augelli-Szafran, C.; Brann, M.
R.; Callahan, M. J .; Caprathe, B. W.; Davis, R. E.; Doyle, P. D.;
Eubanks, D.; Lipinski, W.; Mirzadegan, T.; Moos, W. H.; More-
land, D. W.; Nelson, C. B.; Pavia, M. R.; Raby, C.; Schwarz, R.
D.; Spencer, C. J .; Thomas, A. J .; J aen, J . C. Design and
Synthesis of m1-Selective Muscarinic Agonists: (R)-(-)-(Z)-1-
Azabicyclo[2.2.1]heptan-3-one, O-(3-3′-Methoxyphenyl)-2-propy-
nyl)-oxime Maleate (CI-1017), a Functionally m1-Selective Mus-
carinic Agonist. J . Med. Chem. 1998, 41, 2524-2536. (d)
Greenlee, W.; Clader, J .; Asberom, T.; McCombie, S.; Ford, J .;
Guzik, H.; Kozlowski, J .; Li, S.; Liu, C.; Lowe, D.; Vice, S.; Zhao,
H.; Zhou, G.; Billard, W.; Binch, H.; Crosby, R.; Duffy, R.;
Lachowicz, J .; Coffin, V.; Watkins, R.; Ruperto, V.; Strader, C.;
Taylor, L.; Cox, K. Muscarinic Agonists and Antagonists in the
Treatment of Alzheimer’s Disease. Il Farmaco 2001, 56, 247-
250.
buffer. The filters were placed into scintillation vials with 5
mL of scintillation fluid and radioactivity quantitated.
In ositol P h osp h a te Accu m u la tion Assa y. COS-7 cells
were subcultured in 12-well culture dishes to a density of
20 000 cells/cm2. The cells were transfected with Fugene 6
transfection reagent (according to Roche Molecular Biochemi-
cal’s specifications) and pcDNA3.1 DNA containing sequence
coding for hM1, hM2, hM3, hM4, or hM5 receptor (approximately
20 µg DNA/150-mm dish) and either empty pcDNA3.1 (for hM1,
hM3, and hM5) or pcDNA3.1 containing sequence coding for
the chimeric G protein GRq/i (for hM2 and hM4). Approximately
24 h after the addition of DNA and transfection agent, the
inositol lipid pool of cells was radiolabeled by incubating the
cells overnight in inositol-free DMEM medium containing 1
µCi of myo-[3H]inositol per well. Approximately 12 h post-
labeling, cells were preincubated with either drug or vehicle
for 5 min. [3H]Inositol accumulation was initiated by addition
of LiCl (final concentration of 10 mM) and either vehicle or
the indicated drug concentration. The assay was terminated
by aspirating the medium and adding 750 µL ice-cold 50 mM
formic acid. After 30 min at 4 °C, the samples were neutralized
with 250 µL 150 mM NH4OH. [3H]Inositol phosphates were
isolated by ion-exchange chromatography on Dowex AG 1-X8
columns and quantitated by liquid scintillation counting.23
Sta tistica l An a lysis. Nonlinear regression and paired
t-test were performed using GraphPad Prism version 3.00
for Windows, GraphPad Software, San Diego, California;
www.graphpad.com.
(5) (a) Sternbach, L. H.; Kaiser, S. Antispasmodics. I. Bicyclic Basic
Alcohols. J . Am. Chem. Soc. 1952, 74, 2215-2218. (b) Sternbach,
L. H.; Kaiser, S. Antispasmodics. II. Esters of Basic Bicyclic
Alcohols. J . Am. Chem. Soc. 1952, 74, 2219-2221.
(6) Martell, M. J .; Soine, T. O. Esters of Bicyclic Amino Alcohols as
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Pharm. Sci. 1963, 52, 331-336.
(7) Yanina, A. D.; Rubtsov, M. V. Hofmann Cleavage of 1-Azabicyclo-
(3,2,1)-octanes. II. Cleavage of 7-Methyl-1-azabicyclo-(3,2,1)-
octane. J . Gen. Chem. USSR (English Transl.) 1960, 30, 548-
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(8) King, F. D.; Hadley, M. S.; J oiner, K. T.; Martin, R. T.; Sanger,
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Ack n ow led gm en t. We thank Dr. Lucia Carrano
and Biosearch Italia S.p.A. (Gerenzano, Italy) for sup-
port of this study. We thank Dr. P. White (University
of North Carolina, Department of Chemistry) for con-
ducting the X-ray crystallographic analysis of 10, and
Dr. Thomas O’Connell (GlaxoSmithKline, Research
Triangle Park, NC) for NMR studies of 10 and 11. We
are indebted to Christine Chung for outstanding techni-
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