6beta-Acetoxynortropane: a potent muscarinic agonist with apparent selectivity toward M2-receptors.

Article abstract of DOI:10.1021/jm9705115

A series of tropane derivatives, related in structure to baogongteng A (1), an alkaloid from a Chinese herb, were synthesized. 6beta-Acetoxynortropane (5) had weak affinity (Ki 22 microM) for central (M1) muscarinic receptors in a [3H]quinuclidinyl benzil

Full text of DOI:10.1021/jm9705115

J . Med. Chem. 1998, 41, 2047-2055
2047
6â-Acetoxyn or tr op a n e: A P oten t Mu sca r in ic Agon ist w ith Ap p a r en t Selectivity
tow a r d M2-Recep tor s
Xue-Feng Pei, Tara H. Gupta, Barbara Badio, W. L. Padgett, and J ohn W. Daly*
Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of
Health, Bethesda, Maryland 20892
Received August 4, 1997
A series of tropane derivatives, related in structure to baogongteng A (1), an alkaloid from a
Chinese herb, were synthesized. 6â-Acetoxynortropane (5) had weak affinity (Ki 22 µM) for
central (M1) muscarinic receptors in a [3H]quinuclidinyl benzilate binding assay but had
extremely high affinity (Ki 2.6 nM) and selectivity for M2-muscarinic receptors expressed in
CHO cells. It had 13-fold lower affinity for M4-receptors, 260-fold lower affinity for M3-receptors,
and 8200-fold lower affinity for M1-receptors expressed in CHO cells. The 6â-carbomethoxy
analogue (14) of baogongteng A had only weak affinity for M2-muscarinic receptors, as did
6â-carbomethoxynortropane (13) and 6â-acetoxytropane (4). In transfected CHO cells, 6â-
acetoxynortropane (5) was an agonist at M2-receptors, based on a GTP-elicited decrease in
affinity, and a full agonist with an IC50 of 11 nM at M4-receptors, based on inhibition of cyclic
AMP accumulation, while being a full agonist at M1-receptors with an EC50 of 23 nM and a
partial agonist at M3-receptors with an EC50 of 3.6 nM, based in both cases on stimulation of
phosphoinositide breakdown. All of the 16 tropane derivatives had weak affinities for central
R4â2-nicotinic receptors with 6â-carbomethoxynortropane (13) having the highest affinity, which
was still 150-fold less than that of nicotine. 6â-Acetoxynortropane (5) represents a potent
muscarinic agonist with apparent selectivity toward M2-receptors.
Agents that enhance central cholinergic function have
been the object of intensive research in recent years
because of deficits in such function in Alzheimer’s
disease. One approach has focused on subtype-selective
muscarinic agonists and/or antagonists as research tools
or potential therapeutics.1,2 At present, five major
subtypes of muscarinic receptors are recognized. All are
G-protein-coupled receptors with the M1-, M3-, and M5-
muscarinic receptors coupling through Gq-proteins to
stimulate phospholipase C, while the M2- and M4-
receptors couple through Gi-proteins to inhibit adenylyl
cyclase.3 Ion channels can also be modulated by mus-
carinic receptors.4 Mixed populations of receptors occur
in different brain regions and tissues complicating any
pharmacological search for selective muscarinic agents.3,5
However, cells transfected with specific muscarinic
receptors do provide homogeneous subtypes.6
has high affinity for M4-muscarinic receptors but has
only weak affinity for M1- and M3-muscarinic receptors
and for R4â2-nicotinic receptors. In functional assays,
5 is a full agonist at M4- and M1-muscarinic receptors
but only a partial agonist at M3-muscarinic receptors.
Alterations in the structure of 5 result in marked
reductions or loss of activity.
Ch em istr y
Compounds 3-5 have been prepared from 6â-acetox-
ytropinone (2) as shown in Scheme 1A with an overall
yield of 27%.9 An alternate synthetic route from com-
mercially available 6â-hydroxytropinone (19) (Aldrich
Chemical Co., Milwaukee, WI) was developed, as shown
in Scheme 1B. Wolff-Kischner decarbonylation of 19
yielded 6â-tropanol (20).10 Acetylation of 20 gave 4,
which was demethylated to give the nortropane 5. The
overall yield of 5 in this alternate route is about 40%.
Compounds 6-9 were synthesized as shown in Scheme
2. The 1,3-dipolar cycloaddition of 1-benzyl-3-oxidopy-
ridinium and phenyl vinyl sulfone gave exclusively 6â-
isomer (21, 78%) as the adduct.11,12 Catalytic hydroge-
nation of 21 afforded the ketone (22, 97%), which was
reduced with NaBH4 to give 2â-ol (23, 59%) and 2R-ol
(24, 41%). The phenylsulfonyl group of 23 and 24 was
reductively cleaved using sodium amalgam to give 25
(62%) and 26 (42%), respectively. Acetylation of 23-
26 gave acetates 27-30, which were debenzylated by
catalytic hydrogenation to give nortropanes 6-9.
Compounds 10-17 were synthesized as shown in
Scheme 3. The 1,3-dipolar cycloaddition of 1-benzyl-3-
oxidopyridinium and methyl acrylate gave a mixture of
the 6â-isomer (31) and the 6R-isomer (2:1 ratio esti-
mated from NMR), which could not be separated by
Many tropane alkaloids, such as atropine and scopo-
lamine, are potent muscarinic receptor antagonists, but
baogongteng A (1) (Chart 1), a nortropane alkaloid
isolated from the Chinese herb Erycibei obtusifola
Benth,7 has been reported to have agonist activity at
muscarinic receptors.8 In addition, a synthetic ana-
logue, 6â-acetoxynortropane (5), was concluded to be a
selective M2-muscarinic receptor agonist, based on bind-
ing affinity and functional assays in brain, heart, ileum
smooth muscle, and iris.8 We now report that, from a
series of 16 tropane derivatives, 6â-acetoxynortropane
(5), based on binding assays, is the most active and most
selective agonist for M2-muscarinic receptors. It also
* To whom correspondence should be addressed at Bldg. 8, Rm.
1A17, NIH, Bethesda, MD 20892. Tel: (301) 496-4024. Fax: (301) 402-
0008.
S0022-2623(97)00511-6 This article not subject to U.S. Copyright. Published 1998 by the American Chemical Society
Published on Web 05/02/1998

2048 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 12
Pei et al.
Ch a r t 1
TLC. However, pure 31 could be crystallized from the
mixture in 47% yield. Catalytic hydrogenation of 31
gave a mixture of 10 (49%) and 32 (46%). Decarbony-
lation of 10 was carried out by converting it to the
dithioketal (11, 90%), followed by catalytic hydrogena-
tion in the presence of Raney nickel to afford a mixture
of 12 (12%) and 13 (14%). Reduction of 32 with NaBH4
gave 2â-ol (33, 36%) and 2R-ol (34, 44%), which were
converted to acetates 35 and 36. Nortropanes 14-17
were obtained from 33-36 by catalytic hydrogenation.
The synthesis of baogongteng A (1) has been de-
scribed.13
be noted that agonists do show relatively low affinities
for M1-muscarinic receptors, when assayed against an
antagonist radioligand, which labels mainly a receptor
state with low affinity for agonists (see ref 14). Such
tropanes and nortropanes did show much higher affini-
ties when assayed against an agonist radioligand (Table
2). The most potent compound (5) of the present series
was about 20-fold more potent than muscarine and
carbamylcholine in inhibiting agonist binding to central
muscarinic receptors. The alkaloids arecoline, ecgonine
methyl ester, and ecgonidine methyl ester were either
weak or inactive at the brain receptors (Table 1).
The tropane and nortropane derivatives were also
very weak or inactive versus binding of [3H]nicotine to
the nicotinic (R4â2) receptors of rat cerebral cortical
membranes (Table 1). The most potent compound (13)
had 150-fold lower affinity than nicotine. Replacement
of the 6â-carbomethoxy group of 13 with a 6â-acetoxy
group, to yield 5, resulted in a 20-fold lower affinity.
The N-methyl derivative 4 had somewhat higher affinity
Resu lts a n d Discu ssion
The tropane and nortropane derivatives were rather
weak or inactive versus binding of [3H]quinuclidinyl
benzilate to muscarinic (mainly M1) receptors of rat
cerebral cortical membranes (Table 1). Muscarinic
agonists, such as muscarine and methacholine, were
also very weak (Table 1 and data not shown). It should

Nortropanes as Muscarinic Agonists
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 12 2049
Sch em e 1a
a
(a) (i) ClCO2CH2CCl3, (ii) Zn/AcOH; (b) BF3‚Et2O/(CH2SH)2; (c) H2/Raney-Ni/THF; (d) (i) NH2NH2‚H2O, (ii) KOH; (e) Ac2O/Py.
Sch em e 2a
a
(a) CH2dCHSO2Ph/Et3N; (b) H2/10% Pd-C/EtOH; (c) NaBH4/EtOH; (d) 6% Na-Hg/MeOH-THF/Na2HPO4; (e) Ac2O/Py; (f) H2/Pd(OH)2-
C/THF.
Sch em e 3a
a
(a) CH2dCHCO2CH3/Et3N; (b) H2/10% Pd-C/EtOH; (c) BF3‚Et2O/(CH2SH)2; (d) H2/Raney-Ni/THF; (e) NaBH4/EtOH; (f) Ac2O/Py; (g)
H2/Pd(OH)2-C/THF.
than 5. The 2â-hydroxy group of 14 was well-tolerated,
while the 2R-hydroxy of 15 and other 2-substituents in
analogues of 13 were not tolerated. The 3-keto function
in 2 and 3 was also not tolerated (compare to 4 and 5).
Because of the low affinities of the compounds, there
was no further investigation of nicotinic systems.
Further investigations of muscarinic systems were
conducted with CHO cells transfected with M1-, M2-,

2050 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 12
Pei et al.
Ta ble 1. Affinities of Tropanes and Nortropanes for Nicotinic and Muscarinic Receptors in Rat Cerebral Cortical Membranesa
Ki (µM) or % inhibition of binding
compd
R1
R2â
R2R
R6
OAc
OAc
OAc
OAc
H
H
SO2Ph
SO2Ph
CO2CH3
CO2CH3
CO2CH3
CO2CH3
CO2CH3
CO2CH3
CO2CH3
CO2CH3
R3Râ
nicotinic (R4â2)
muscarinic (M1)
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
CH3
H
CH3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
O
O
5% (100 µM)
13% (100 µM)
1.5 ( 0.3
0% (100 µM)
11% (100 µM)
280 ( 33
H, H
H, H
H, H
H, H
H, H
H, H
H, H
H, H
H, H
H, H
H, H
H, H
H, H
H, H
3.0 ( 0.3
22 ( 3
OAc
H
OAc
H
32 ( 10
18% (100 µM)
6% (100 µM)
0% (100 µM)
6% (100 µM)
0% (100 µM)
3% (100 µM)
7% (100 µM)
9% (100 µM)
6% (100 µM)
3% (100 µM)
1% (100 µM)
1% (100 µM)
OAc
H
OAc
24 ( 4
16% (100 µM)
10% (100 µM)
15% (100 µM)
36% (100 µM)
50% (100 µM)
0.15 ( 0.03
4.0 ( 0.2
O
-SCH2CH2S-
H
H
SC2H5
H
H
OH
H
OAc
H
OH
H
OAc
64 ( 6
13% (100 µM)
19% (100 µM)
0.0010 ( 0.0001
nicotine
muscarine
arecoline
ecgonidine methyl ester
ecgonine methyl ester
3% (100 µM)
43% (100 µM)
25 ( 2
0.27 ( 0.04
8.9 ( 1.6
11% (100 µM)
CH3
CH3
CO2CH3
CO2CH3
2,3-ene
H
H
H
H
H, OH
27% (100 µM)
a
Values are means ( SEM (n ) 4) for inhibition of binding of [3H]nicotine to nicotinic receptors or for inhibition of binding of
[3H]quinuclidinyl benzilate to muscarinic receptors in rat cerebral cortical membranes. Either Ki values or % inhibition at the highest
concentration tested is reported. The Kd for [3H]quinuclidinyl benzilate was 0.26 nM and the Bmax 2500 fmol/mg of protein. The Kd for
[3H]nicotine was 1.0 nM and the Bmax 90 fmol/mg of protein.
Ta ble 2. Affinities of Certain Tropanes and nortropanes
fold more potent at M4-receptors than muscarine. The
N-methyl derivative 4 had manyfold lower affinity than
compound 5 at M1-, M2-, M3-, and M4-receptors (Table
3). The lack of tolerance for the N-methyl substituent
versus Agonist and Antagonist Binding to Muscarinic (M1)
Receptors in Rat Cerebral Cortical Membranes.a
Ki (µM) or % inhibition of binding versus
[3H]quinuclidinyl
benzilate
was particularly striking at the M2-receptor where
compound 4 had 1000-fold lower affinity than 5.
compd
[3H]oxotremorine M
A
similar large reduction in affinity for compound 5
compared to compound 4 was seen when assayed versus
agonist binding to muscarinic (M1) receptors in brain
membranes (Table 2). Replacement of the 6â-acetoxy
group of 5 with a 6â-carbomethoxy group in 13 resulted
in a very marked reduction in affinity at all muscarinic
receptors.
The Ki value of 5 for inhibition of binding of [3H]-
quinuclidinyl benzilate to M2-receptors was markedly
increased in the presence of GTP, indicative of agonist-
like properties. The IC50 values for 5, muscarine,
methacholine, and carbamylcholine in the absence and
presence of GTP are listed in Table 4. GTP increased
the Ki value for compound 5 and muscarine to a similar
extent, while causing greater shifts for methacholine
and carbamylcholine.
Functional assays for inhibition of adenylate cyclase
in membranes of cells transfected with M2-receptors
proved difficult, perhaps due to the low density of M2-
receptors expressed (see legend of Table 3). Inhibition
of adenylate cyclase by compound 5 and muscarinic
agonists was examined in the CHO cells transfected
with M4-receptors. Compound 5 was a full agonist with
an IC50 of 11 ( 2 nM in such cells (Figure 1). It is to be
expected that compound 5 would be much more potent
at M2-receptors, since it had a 15-fold higher binding
affinity for M2-receptors compared to M4-receptors
4
5
13
14
2.6 ( 0.1
280 ( 33
0.0056 ( 0.0005
0.26 ( 0.02
18 ( 1
22 ( 3
9% (100 µM)
6% (100 µM)
3% (100 µM)
5% (100 µM)
muscarine
carbamylcholine
0.027 ( 0.002
0.031 ( 0.01
a
Values are means ( SEM (n ) 4) for inhibition of binding of
the agonist [3H]oxotremorine M and of the antagonist [3H]quinu-
clidinyl benzilate to muscarinic receptors in rat cerebral cortical
membranes. Either Ki values or percent inhibition at the highest
concentration tested is reported. The Kd for [3H]oxotremorine M
was 0.75 nM, and the Kd for [3H]quinuclidinyl benzilate was 0.26
nM.
M3-, or M4-muscarinic receptors.6 Compound 5 had an
affinity for M1-receptors in CHO cell membranes (Table
3) identical to its affinity for muscarinic receptors in
cerebral cortical membranes (Table 1), consonant with
M1-receptors being the major muscarinic receptor in the
brain membranes. Compound 5 had a 30-fold higher
affinity for M3-receptors than for M1-receptors and was
manyfold more potent at M3-receptors than the musca-
rinic agonists muscarine or methacholine (Table 2).
Compound 5 proved to be highly selective for the M2-
and M4-receptors that are inhibitory to adenylyl cyclase
via Gi-proteins. The highest affinity was for M2-recep-
tors, where compound 5 had an affinity (Ki value of 2.6
nM) 30-fold greater than that of muscarine. Compound
5 had a Ki value for M4-receptors of 33 nM and was 35-

Nortropanes as Muscarinic Agonists
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 12 2051
Ta ble 3. Affinities of Tropanes and Nortropanes for Muscarinic Receptors in Membranes from Transfected CHO Cellsa
Ki (µM) or % inhibition of binding to the muscarinic receptor
compd
M1
M2
M3
M4
4
5
13
14
48% (30 µM)
21 ( 1
2.7 ( 0.2
15 ( 1
8.3 ( 1.0
0.0026 ( 0.0005
0.48 ( 0.05
12 ( 1
0.68 ( 0.03
18 ( 3
0.033 ( 0.011
7.9 ( 1.6
24% (300 µM)
2% (300 µM)
15% (300 µM)
13% (100 µM)
26% (100 µM)
13% (100 µM)
8% (300 µM)
5% (300 µM)
0% (100 µM)
28% (100 µM)
5.7 ( 0.3
28% (100 µM)
19% (100 µM)
34% (100 µM)
16% (300 µM)
33% (300 µM)
1.4 ( 0.4
2.4 ( 0.5
muscarine
arecoline
ecgonidine methyl ester
ecgonine methyl ester
methacholine
0.075 ( 0.020
0.040 ( 0.011
0.059 ( 0.009
0.020 ( 0.001
1.6 ( 0.4
2.6 ( 0.7
carbamylcholine
a
Values are means ( SEM (n ) 3) for inhibition of binding of [3H]quinuclidinyl benzilate to muscarinic receptors in membranes of
CHO cells transfected with M1-, M2-, M3-, or M4-muscarinic receptors. Either Ki values or percent inhibition at the highest concentration
tested is reported. The Kd and Bmax values for [3H]quinuclidinyl benzilate binding were as follows: M1-receptor, 0.13 nM and 1800 fmol/
mg of protein; M2-receptor, 0.018 nM and 71 fmol/mg of protein; M3-receptor, 0.071 nM and 2050 fmol/mg protein; M4-receptor, 0.031 nM
and 340 fmol/mg of protein.
Ta ble 4. Inhibition of [3H]Quinuclidinyl Benzilate Binding to
M2-Muscarinic Receptors by Nortropane 5 and Muscarinic
Agonists in the Absence and Presence of GTPa
IC50 (µM)
compd
-GTP
+GTP
-fold shift
5
0.017 ( 0.003
0.49 ( 0.10
0.38 ( 0.06
0.13 ( 0.01
0.12 ( 0.01
3.9 ( 0.7
5.2 ( 1.0
3.1 ( 0.9
6
8
14
24
muscarine
methacholine
carbamylcholine
a
Values are means ( SEM (n ) 3) for inhibition of 0.1 nM
[3H]quinuclidinyl benzilate binding to M2-receptors in membranes
of transfected CHO cells in the absence or presence of 10 µM GTP.
F igu r e 1. Inhibition of [3H]cyclic AMP accumulation in CHO
cells transfected with M4-muscarinic receptors: nortropane 5
([) and muscarine (2). Values are means ( SEM (n ) 3). See
Experimental Section for assay conditions.
F igu r e 2. Stimulation of [3H]inositol monophosphate (IP1)
accumulation in [3H]inositol-labeled CHO cells transfected
with either (A) M1-muscarinic receptors or (B) M3-muscarinic
receptors: nortropane 5 ([), muscarine (2), methacholine (9),
and carbamylcholine (b). Values are means ( SEM (n ) 3).
Error bars are in some cases smaller than the symbol. See
Experimental Section for assay conditions.
(Table 3). Carbamylcholine (IC50 230 ( 60 nM), mus-
carine (IC50 470 ( 120 nM), and methacholine (IC50 700
( 440 nM) were much less active than nortropane 5 at
M4-receptors (Figure 1 and data not shown).
Functional assays for stimulation of phosphoinositide
breakdown by phospholipase C were conducted with
cells transfected with either M1- or M3-receptors. Nortro-
pane 5 was a potent full agonist at M1-receptors with
an EC50 of 23 ( 2 nM (Figure 2A). Muscarine (EC50
219 ( 3 nM), methacholine (EC50 400 ( 40 nM), and
carbamylcholine (EC50 650 ( 50 nM) were much less
potent but also were full agonists. In cells transfected
with M3-receptors, nortropane 5 was a potent agonist
with an EC50 of 3.6 ( 0.3 nM (Figure 2B). Muscarine
(EC50 130 ( 10 nM), methacholine (EC50 120 ( 4 nM),
and carbamylcholine (EC50 480 ( 90 nM) were much
weaker agonists. However, compared to muscarine,
methacholine, and carbamylcholine, compound 5 was
only a partial agonist (Figure 2B). The transfected cells
apparently had a large excess of spare receptors, since
the EC50 values for stimulation of phosphoinositide

2052 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 12
Pei et al.
J ) 9.8, 3.9 Hz, 6R-CH), 3.60 (1H, d, J ) 7.6, 1-CH), 2.82-
2.77 (1H, m, 7â-CH), 2.01 (1H, dd, J ) 14.3, 9.3 Hz, 7R-H).
Anal. (C20H19NO3S) C, H, N.
breakdown were manyfold lower than the Ki values from
binding experiments for all four muscarinic agonists (see
Table 3).
8-Ben zyl-6â-(p h en ylsu lfon yl)-8-a za bicyclo[3.2.1]octa n -
2-on e (22). Compound 21 (707 mg, 2.0 mmol) was dissolved
in EtOH (10 mL), and 10% Pd-C (80 mg) was added. The
mixture was stirred at room temperature under H2 and
monitored by TLC until all the starting material disappeared.
The catalyst was removed by filtration, and the solvent was
evaporated in a vacuum to give 22 (692 mg, 97%) as colorless
crystals. An analytical sample was obtained by recrystalli-
zation from EtOH: mp 125-128 °C; CI-MS m/z 356 (MH+);
1H NMR (CDCl3) δ 7.93-7.17 (10H, m, 2Ph), 3.88 (1H, br s,
5-CH), 3.84 (1H, d, J ) 13.8 Hz, PhCH), 3.71 (1H, d, J ) 13.5
Hz, PhCH), 3.66 (1H, dd, J ) 9.2, 6.7 Hz, 6R-CH), 3.43 (1H, d,
J ) 6.9, 1-CH), 2.66 (1H, m, 7â-CH), 2.45-2.26 (3H, m, 4â-H,
3-CH2), 2.11 (1H, dd, J ) 14.3, 9.3 Hz, 7R-CH), 1.82-1.77 (1H,
m, 4R-CH). Anal. (C20H21NO3S) C, H, N.
8-Ben zyl-6â-(p h en ylsu lfon yl)-8-a za bicyclo[3.2.1]octa n -
2â- a n d -2r-ols (23 a n d 24). Compound 22 (1.06 g, 3.0 mmol)
was dissolved in EtOH (50 mL), and NaBH4 (120 mg, 3.0
mmol) was added. The mixture was stirred at room temper-
ature for 1 h. The solvent was evaporated in a vacuum. The
residue was added to H2O (50 mL) and extracted with Et2O (2
× 50 mL). After the removal of Et2O in a vacuum, the residue
was chromatographed (CH2Cl2/MeOH ) 40/1) to give first 23
(636 mg, 59%) as colorless crystals and then 24 (444 mg, 41%)
as colorless crystals. Analytical samples were obtained by
recrystallization from EtOAc. 23: mp 126-128 °C; CI-MS m/z
358 (MH+); 1H NMR (CDCl3) δ 7.95-7.29 (10H, m, 2Ph), 4.07
(1H, d, J ) 13.4 Hz, PhCH), 3.73 (1H, d, J ) 13.4 Hz, PhCH),
3.85 (1H, brs, 2R-CH), 3.60-3.41 (3H, m, 1,5,6R-CH), 2.61 (1H,
p, 7â-CH), 2.05-1.95 (1H, m, 2â-OH), 1.84 (1H, dd, J ) 14.1,
The present results confirm and extend a prior report
on muscarinic activity of (-)-6â-acetoxynortropane (6â-
AN),8 the levorotatory enantiomer of the presently
described compound 5. Yu and Sun8 report Ki values
versus [3H]quinuclidinyl benzoate for (-)-6â-AN of 0.43
µM for rat cortical membranes, 0.026 µM for rat heart
membranes, 2.3 µM for guinea pig ileum muscle mem-
branes, and 3.2 µM for rabbit pupil. The value for heart
(M2-receptors) is 10-fold higher than our value for
transfected M2-receptors, while the value for rat brain
is 50-fold lower than our value for rat brain. 6â-AN had
potent negative inotropic and chronotropic effects on
guinea pig heart and had potent activity in contracting
guinea pig longitudinal smooth muscle and constricting
rabbit pupils.8 All these functional effects were blocked
by atropine. The (+)-enantiomer of 6â-AN was sever-
alfold less potent than (-)-6â-AN, as was baogongteng
A.8 In such functional assays8 6â-AN appeared to be a
selective M2-receptor agonist. The present results
confirm that compound 5 ((()-6â-AN) is an extremely
potent muscarinic agonist with high affinity for M2-
muscarinic receptors. Compound 5 should readily pass
into the central nervous system, and indeed Yu and
Sun8 did report cognitive enhancement in a three-arm
maze for mice. The facile synthetic route to compound
5 makes this potent muscarinic agonist readily acces-
sible for research on cholinergic function.
9.1 Hz, 7R-CH), 1.67-1.31 (4H, m, 3,4-CH2). Anal. (C20H23
-
NO3S) C, H, N. 24: mp 111-113 °C; CI-MS m/z 358 (MH+);
1H NMR (CDCl3) δ 7.92-7.20 (10H, m, 2Ph), 4.11-3.49 (6H,
m, PhCH2, 1,2â-, 5,6R-CH), 2.40 (1H, m, 7â-CH), 2.14-2.02
(1H, m, 2R-OH), 2.10 (1H, dd, J ) 14.2, 9.2 Hz, 7R-CH), 1.78-
1.23 (4H, m, 3,4-CH2). Anal. (C20H23NO3S) C, H, N.
Exp er im en ta l Section
Gen er a l. Melting points (uncorrected) were measured with
a Thomas-Hoover capillary melting point apparatus. 1H NMR
were recorded on a Varian XL-300 MHz spectrometer. Chemi-
cal shifts are reported as δ values (ppm) relative to Me4Si as
an internal standard. Chemical ionization (CI) mass spectral
data were obtained on a Finnigan-1015D mass spectrometer.
Elemental analyses were performed by Atlantic Microlab, Inc.
(Norcross, GA). Unless otherwise indicated, all separations
were carried out by column chromatography (Merck silica gel
60, 230-400 mesh), using the described solvents. All reactions
involving nonaqueous solutions were performed under an inert
atmosphere and with anhydrous solvents, unless otherwise
noted. All the synthesized compounds were racemates.
8-Ben zyl-8-a za bicyclo[3.2.1]octa n -2â-ol (25). Compound
23 (357 mg, 1.0 mmol) was dissolved in MeOH (5 mL) and
THF (10 mL), and Na2HPO4 (681 mg, 5 mmol) and 6% Na-
Hg (2.30 g) were added. The mixture was refluxed with
stirring for 24 h, cooled to room temperature, poured into H2O
(40 mL), and extracted with EtOAc (2 × 30 mL). After removal
of AcOEt in a vacuum, the residue was chromatographed (CH2-
Cl2/MeOH ) 20/1) to give 25 (154 mg, 62%) as a colorless oil:
1
CI-MS m/z 218 (MH+); H NMR (CDCl3) δ 7.40-7.21 (5H, m,
Ph), 4.01 (1H, d, J ) 13.7 Hz, PhCH), 3.70 (1H, d, J ) 13.7
Hz, PhCH), 3.81 (1H, brs, 2R-CH), 3.60-3.41 (2H, m, 1,5-CH),
2.01(1H, brs, 2â-OH), 1.99-1.32 (8H, m, 3,4,6,7-CH2).
8-Ben zyl-8-a za bicyclo[3.2.1]octa n -2r-ol (26). In a simi-
lar way to the preparation of 25, compound 26 was obtained
from 24 as a colorless oil, yield 43%: CI-MS m/z 218 (MH+);
1H NMR (CDCl3) δ 7.38-7.23 (5H, m, Ph), 3.91 (1H, brs, 2â-
CH), 3.60 (2H, s, PhCH2), 3.20-3.10 (2H, m, 1,5-CH), 2.12 (1H,
brs, 2R-OH), 1.94-1.40 (8H, m, 3,4,6,7-CH2).
8-Meth yl-6â-a cetoxy-8-a za bicyclo[3.2.1]octa n e (4) (6â-
Acetoxytr op a n e). 6â-Tropanol (20; 523 mg, 3.71 mmol) was
dissolved in CHCl3 (5 mL), and pyridine (1 mL) and Ac2O (1
mL) were added. The mixture was stirred at room tempera-
ture overnight and poured into a saturated NaHCO3 solution
(10 mL). The CHCl3 layer was separated, and the aqueous
layer was extracted with CHCl3 (2 × 10 mL). After the
removal of CHCl3 under reduced pressure, the remaining oil
was distilled in a vacuum (54-56 °C/1 mmHg) to give 4 (537
mg, 84%) as a colorless oil. The spectrum was identical with
the published spectrum.9
8-Ben zyl-2â-a cetoxy-8-a za bicyclo[3.2.1]octa n e (27). In
a similar way to the preparation of 4, compound 27 was
obtained from 25 as a colorless oil, yield 49%: CI-MS m/z 260
1
(MH+); H NMR (CDCl3) δ 7.42-7.21 (5H, m, Ph), 4.60 (1H,
brs, 2R-CH), 3.59 (1H, d, J ) 13.7 Hz, PhCH), 3.42 (1H, d, J
) 14.0 Hz, PhCH), 3.25 (2H, brs, 1,5-CH), 2.08 (3H, s, CH3-
CO), 1.99-1.32 (8H, m, 3,4,6,7-CH2).
8-Ben zyl-6â-(p h en ylsu lfon yl)-8-a za bicyclo[3.2.1]oct-3-
en -2-on e (21). A mixture of 1-benzyl-3-oxidopyridinium
chloride (6.45 g, 29 mmol), phenyl vinyl sulfone (4.89 g, 29
mmol), Et3N (6 mL), and hydroquinone (60 mg) in THF (60
mL) was refluxed with stirring overnight, cooled to room
temperature, and filtered. The solvent was evaporated in a
vacuum, and the residue was recrystallized from EtOAc to give
21 (7.94 g, 78%) as yellow crystals: mp 146-148 °C; CI-MS
8-Ben zyl-2r-a cetoxy-8-a za bicyclo[3.2.1]octa n e (28). In
a similar way to the preparation of 4, compound 28 was
obtained from 26 as a colorless oil, yield 51%: CI-MS m/z 260
1
(MH+); H NMR (CDCl3) δ 7.38-7.23 (5H, m, Ph), 4.91 (1H,
brs, 2â-CH), 3.55 (2H, s, PhCH2), 3.20 (1H, brs, 1-CH), 3.13
(1H, brs, 5-CH), 2.07 (3H, s, CH3CO), 1.94-1.44 (8H, m,
3,4,6,7-CH2).
1
m/z 354 (MH+); H NMR (CDCl3) δ 7.89-7.06 (10H, m, 2Ph),
6.92 (1H, dd, J ) 9.8, 5.0 Hz, 4-CH), 6.14 (H, d, J ) 9.8 Hz,
3-CH), 4.18 (1H, d, J ) 5.0 Hz, 5-CH), 3.79 (1H, d, J ) 13.0
Hz, PhCH), 3.68 (1H, d, J ) 13.4 Hz, PhCH), 3.60 (1H, dd,
8-Ben zyl-2â-a cetoxy-6â-(p h en ylsu lfon yl)-8-a za bicyclo-
[3.2.1]octa n e (29). In a similar way to the preparation of 4,
compound 29 was obtained from 23 as colorless crystals, yield

Nortropanes as Muscarinic Agonists
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 12 2053
100%: mp 125-126 °C; CI-MS m/z 400 (MH+); 1H NMR
(CDCl3) δ 7.94-7.24 (10H, m, 2Ph), 4.57 (1H, brs, 2R-CH), 4.00
(1H, d, J ) 13.4 Hz, PhCH), 3.86 (1H, d, J ) 14.1 Hz, PhCH),
3.90 (1H, brs, 1-CH), 3.58 (1H, t, J ) 8.7, 6.6 Hz, 6R-CH), 3.45
(1H, brs, 5-CH), 2.52 (1H, m, 7â-CH), 2.23-2.12 (1H, m, 3â-
H), 2.03 (3H, s, CH3CO), 1.83 (1H, dd, J ) 14.0, 9.5 Hz, 7R-
CH), 1.72-1.36 (3H, m, 3R-CH, 4-CH2). Anal. (C22H25NO4S)
C, H, N.
8-Ben zyl-2r-a cetoxy-6â-(p h en ylsu lfon yl)-8-a za bicyclo-
[3.2.1]octa n e (30). In a similar way to the preparation of 4,
compound 30 was obtained from 24 as colorless crystals, yield
100%: mp 147-148 °C; CI-MS m/z 400 (MH+); 1H NMR
(CDCl3) δ 7.95-7.24 (10H, m, 2Ph), 4.99 (1H, m, 2â-CH), 4.04
(1H, d, J ) 13.9 Hz, PhCH), 3.82 (1H, d, J ) 13.9 Hz, PhCH),
3.79 (1H, brs, 1-CH), 3.51 (1H, t, J ) 8.4, 7.0 Hz, 6R-CH), 3.39
(1H, brs, 5-CH), 2.40 (1H, m, 7â-CH), 2.15 (1H, dd, J ) 14.0,
9.4 Hz, 7R-CH), 2.10-2.04 (1H, m, 3â-H), 1.98 (3H, s, CH3-
CO), 1.95-1.25 (3H, m, 3R-CH, 4-CH2). Anal. (C22H25NO4S)
C, H, N.
3.72 (1H, d, J ) 13.5 Hz, PhCH), 3.74 (3H, s, OCH3), 3.66 (1H,
d, J ) 7.6 Hz, 1-CH), 2.96-2.87 (2H, m, 6R,7â-CH), 1.93 (1H,
dd, J ) 13.7, 9.4 Hz, 7R-H). Anal. (C16H17NO3) C, H, N.
6â-Ca r bom eth oxy-8-a za bicyclo[3.2.1]octa n -2-on e (10)
a n d 8-Ben zyl-6â-ca r bom eth oxy-8-a za bicyclo[3.2.1]octa n -
2-on e (32). Compound 31 (5.54 g, 20.4 mmol) was dissolved
in EtOH (15 mL), and 10% Pd-C (500 mg) was added. The
mixture was stirred at room temperature under H2 and
monitored by TLC until all the starting material disappeared.
The catalyst was removed by filtration, and the solvent was
evaporated in a vacuum. The residue was chromatographed
(CH2Cl2/MeOH ) 40/1) to give 32 (2.53 g, 46.3%) as colorless
crystals and 10 (1.82 g, 48.8%) as a colorless oil. Compound
10 was converted to an oxalate salt as white crystals. 10‚-
oxalate: mp 170-175 °C. Free base: CI-MS m/z 184 (MH+);
1H NMR (CDCl3) δ 3.85-3.75 (2H, m, 1,5-CH), 3.75 (3H, s,
OCH3), 3.00 (1H, dd, J ) 9.2, 5.1 Hz, 6R-CH), 2.39-1.89 (7H,
m, NH, 3,4,7-CH2). 10‚oxalate: Anal. (C9H13NO3‚C2H2O4‚H2O)
1
C, H, N. 32: mp 70-72 °C; CI-MS m/z 274 (MH+); H NMR
(CDCl3) δ 7.30-7.23 (5H, m, Ph), 3.79-3.66 (4H, m, 1,5-CH,
PhCH2), 3.73 (3H, s, OCH3), 3.48 (1H, d, J ) 7.4 Hz, 1-CH),
2.98 (1H, dd, J ) 9.5, 5.7 Hz, 6R-CH), 2.71 (1H, m, 7â-CH),
2.06 (1H, dd, J ) 14.0, 9.5 Hz, 7R-CH), 2.39-1.55 (4H, m, 3,4-
CH2). Anal. (C16H19NO3) C, H, N.
2â-Acetoxy-8-a za bicyclo[3.2.1]octa n e (6). Compound 27
(82 mg, 0.32 mmol) was dissolved in THF (5 mL), and
Pd(OH)2-C (20 mg) was added. The mixture was stirred
under H2 and monitored by TLC until all the starting material
had disappeared. The catalyst was removed by filtration, and
the solvent was evaporated in a vacuum. The residue was
chromatographed (CH2Cl2/MeOH ) 20/1) to give 6 (54 mg,
100%) as a colorless oil. It was converted to a fumarate salt
as white crystals. 6‚fumarate: mp 122-132 °C. Free base:
6â-C a r b o m e t h o x y -2,2-(1′,2′-e t h y le n e d i t h i a n o )-8-
a za bicyclo[3.2.1]octa n -2-on e (11). Compound 10 (960 mg,
5.25 mmol) was dissolved in 1,2-ethanedithiol (5 mL), and
BF3‚Et2O (2.5 mL) was slowly added dropwise with stirring.
The mixture was stirred at room temperature under N2
overnight, then poured into H2O (20 mL), and extracted with
Et2O (2 × 20 mL). The aqueous layer was neutralized with
saturated NaHCO3 solution and extracted with CH2Cl2 (3 ×
20 mL). After the removal of CH2Cl2 in a vacuum, the residue
was chromatographed (CH2Cl2/MeOH ) 40/1) to give 11 (743
mg, 54.5%) as a colorless oil. It was converted to a fumarate
salt. 11‚fumarate: mp °C. Free base: CI-MS m/z 260 (MH+);
1H NMR (CDCl3) δ 3.69 (3H, s, OCH3), 3.62 (1H, brs, 1-CH),
3.30-3.25 (3H, m, 5-CH, S-CH2), 2.90-2.71 (4H, m, NH, 6R-
CH, S-CH2), 2.22-1.67 (6H, m, 3,4,7-CH2). 11‚fumarate: Anal.
(C11H17NO2S2‚C4H4O4) C, H, N.
1
CI-MS m/z 170 (MH+); H NMR (CDCl3) δ 4.72 (1H, brs, 2R-
CH), 4.12 (1H, brs, 1-CH), 3.76 (1H, brs, 5-CH), 2.13 (3H, s,
CH3CO), 2.17-1.26 (9H, m, NH, 3,4,6,7-CH2). 6‚fumarate:
Anal. (C9H15NO2‚C4H4O4) C, H, N.
2r-Acetoxy-8-a za bicyclo[3.2.1]octa n e (7). In a similar
way to the preparation of 6, compound 7 was obtained from
28 as a colorless oil, yield 73%. It was converted to a fumarate
salt as white crystals. 7‚fumarate: mp 190-192 °C. Free
base: CI-MS m/z 170 (MH+); 1H NMR (CDCl3) δ 5.02-4.97
(1H, m, 2â-CH), 4.10 (1H, brs, 1-CH), 3.79 (1H, brs, 5-CH),
1.98 (3H, s, CH3CO), 2.17-1.26 (9H, m, NH, 3,4,6,7-CH2).
7‚fumarate: Anal. (C9H15NO2‚C4H4O4) C, H, N.
2â-Ace t oxy-6â-(p h e n ylsu lfon yl)-8-a za b icyclo[3.2.1]-
6â-Ca r bom eth oxy-2r-(eth ylth ia n o)-8-a za bicyclo[3.2.1]-
octa n e (12) a n d 6â-Ca r bom eth oxy-8-a za bicyclo[3.2.1]-
octa n e (13). Compound 11 (752 mg, 2.90 mmol) was dissolved
in THF (30 mL), and Raney nickel (5 g) was added. The
mixture was refluxed with stirring under H2 for 1 h. After
cooling to room temperature, the catalyst was removed by
filtration, and the solvent was evaporated in a vacuum. The
residue was chromatographed (CH2Cl2/MeOH ) 40/1) to give
first 12 (80 mg, 12%) as a colorless oil and then 13 (71 mg,
14%) as a colorless oil. Both were converted to fumarate salts.
12‚fumarate: mp 150-151 °C. Free base: CI-MS m/z 230
octa n e (8). In
a similar way to the preparation of 6,
compound 8 was obtained from 29 as colorless crystals, yield
64%. It was also converted to an oxalate salt as white crystals.
8: mp 110-112 °C; CI-MS m/z 310 (MH+); 1H NMR (CDCl3) δ
7.94-7.59 (5H, m, Ph), 4.67 (1H, brs, 2R-CH), 3.99 (1H, brs,
1-CH), 3.75 (1H, d, J ) 5.21 Hz, 5-CH), 3.53 (1H, dd, J ) 8.8,
5.1 Hz, 6R-CH), 2.32 (1H, m, 7â-CH), 2.12 (3H, s, CH3CO),
2.10 (1H, s, NH), 2.00-1.92 (1H, m, 3â-H), 1.96 (1H, dd, J )
14.3, 9.0 Hz, 7R-CH), 1.90-1.58 (2H, m, 3R,4â-CH), 1.42-1.35
(1H, m, 4R-H). Anal. (C15H19NO4S) C, H, N. 8‚oxalate: mp
138-140 °C. Anal. (C15H19NO4S‚C2H2O4‚0.2H2O) C, H, N.
1
(MH+); H NMR (CDCl3) δ 3.70 (3H, s, OCH3), 3.59 (2H, brs,
1,5-CH), 2.77 (1H, dd, J ) 8.8, 4.7 Hz, 6R-CH), 2.56 (2H, dd,
J ) 14.2, 6.2 Hz, S-CH2), 2.35-2.27 (2H, m, NH, 7â-CH),
1.98-1.55 (5H, m, 7â-CH, 3,4-CH2), 1.26 (3H, t, J ) 7.2 Hz,
CH3). 12‚fumarate: Anal. (C11H19NO2S‚C4H4O4) C, H, N.
13‚fumarate: mp 158-160 °C. Free base: CI-MS m/z 170
2â-Ace t oxy-6r-(p h e n ylsu lfon yl)-8-a za b icyclo[3.2.1]-
octa n e (9). In
a similar way to the preparation of 6,
compound 9 was obtained from 30 as a colorless oil, yield 86%.
It was converted to an oxalate salt as a white powder. 9‚-
oxalate: mp 156-159 °C. Free base: CI-MS m/z 310 (MH+);
1H NMR (CDCl3) δ 7.94-7.59 (5H, m, Ph), 4.85 (1H, m, 2â-
CH), 3.92 (1H, brs, 1-CH), 3.70 (1H, brs, 5-CH), 3.45 (1H, dd,
J ) 8.3, 5.5 Hz, 6R-CH), 2.33-2.10 (3H, m, NH, 3â,7â-CH),
2.01 (3H, s, CH3CO), 2.01-1.27 (4H, m, 3R,7R-CH, 4-CH2).
9‚oxalate: Anal. (C15H19NO4S‚C2H2O4‚0.3H2O) C, H, N.
1
(MH+); H NMR (CDCl3) δ 3.71 (4H, s, NH, OCH3), 3.42 (2H,
brs, 1,5-CH), 2.91 (1H, dd, J ) 8.8, 5.1 Hz, 6R-CH), 2.56 (2H,
dd, J ) 14.2, 6.2 Hz, S-CH2), 2.14 (1H, m, 7â-CH), 2.02 (1H,
dd, J ) 13.1, 9.2 Hz, 7R-CH), 1.83-1.46 (6H, m, 3,4,5-CH2).
13‚fumarate: Anal. (C9H15NO2S‚C4H4O4) C, H, N.
8-Ben zyl-6â-ca r bom eth oxy-8-a za bicyclo[3.2.1]oct-3-en -
2-on e (31). A mixture of 1-benzyl-3-oxidopyridinium chloride
(11.1 g, 50 mmol), methyl acrylate (25 mL), Et3N (10 mL), and
hydroquinone (100 mg) in THF (65 mL) was refluxed with
stirring overnight, cooled to room temperature, and filtered.
The solvent was evaporated in a vacuum, and the residue was
chromatographed (CH2Cl2/MeOH ) 40/1). Crude 31 was
obtained and recrystallized twice from EtOH to give 31 (6.31
g, 47%) as yellow crystals: mp 90-91 °C; CI-MS m/z 272
8-Ben zyl-6â-ca r b om et h oxy-8-a za b icyclo[3.2.1]oct a n -
2â- a n d -2r-ols (33 a n d 34). In a similar way to the
preparation of 22 and 23, compounds 33 (36%) and 34 (44%)
were obtained from 32 as a colorless oil and colorless crystals,
1
respectively. 33: CI-MS m/z 276 (MH+); H NMR (CDCl3) δ
7.30-7.26 (5H, m, Ph), 3.74 (3H, s, OCH3), 3.58-3.44 (4H, m,
1,2R-CH, PhCH2), 3.33 (1H, brs, 5-CH), 2.86 (1H, dd, J ) 9.5,
5.6 Hz, 6R-CH), 2.65 (1H, m, 7â-CH), 1.90-1.80 (2H, m, OH,
7R-CH), 1.59-1.28 (4H, m, 3,4-CH2). 34: mp 110-112 °C; CI-
MS m/z 276 (MH+); 7.33-7.21 (5H, m, Ph), 3.90-3.84 (1H, m,
2â-CH), 3.73 (3H, s, OCH3), 3.66 (1H, d, J ) 13.6 Hz, PhCH),
3.58 (1H, d, J ) 13.7 Hz, PhCH), 3.53 (1H, brs, 1-CH), 3.23
1
(MH+); H NMR (CDCl3) δ 7.33-7.23 (5H, m, Ph), 6.96 (1H,
dd, J ) 9.8, 5.0 Hz, 4-CH), 6.10 (H, d, J ) 9.8 Hz, 3-CH), 4.06
(1H, d, J ) 5.0 Hz, 5-CH), 3.83 (1H, d, J ) 13.5 Hz, PhCH),

2054 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 12
Pei et al.
(1H, brs, 5-CH), 2.86 (1H, dd, J ) 9.7, 5.9 Hz, 6R-CH), 2.46
(1H, m, 7â-CH), 2.12 (1H, dd, J ) 13.9, 9.9 Hz, 7R-CH), 1.94-
1.20 (5H, m, OH, 3,4-CH2). Anal. (C16H21NO3) C, H, N.
8-Ben zyl-2â-a cet oxy-6â-ca r b om et h oxy-8-a za b icyclo-
[3.2.1]octa n e (35). In a similar way to the preparation of 4,
compound 35 was obtained from 33 as a colorless oil, yield
82%: CI-MS m/z 318 (MH+); 1H NMR (CDCl3) δ 7.42-7.21
(5H, m, Ph), 4.65 (1H, brs, 2R-CH), 3.76 (3H, s, OCH3), 3.68
(1H, J ) 13.9 Hz, PhCH), 3.58 (1H, J ) 14.0 Hz, PhCH), 3.66
(1H, brs, 1-CH), 3.43 (1H, d, J ) 4.9 Hz, 5-CH), 2.90 (1H, dd,
J ) 9.3, 5.9 Hz, 6R-CH), 2.61 (1H, m, 7â-CH), 2.08 (3H, s, CH3),
1.83 (1H, dd, J ) 14.0, 9.6 Hz, 7R-CH), 1.76-1.27 (4H, m, 3,4-
CH2).
8-Ben zyl-2r-a cet oxy-6â-ca r b om et h oxy-8-a za b icyclo-
[3.2.1]octa n e (36). In a similar way to the preparation of 4,
compound 36 was obtained from 34 as a colorless oil, yield
93%: CI-MS m/z 318 (MH+); 1H NMR (CDCl3) δ 7.35-7.21
(5H, m, Ph), 4.92 (1H, p, 2â-CH), 3.73 (3H, s, OCH3), 3.65 (2H,
d, J ) 3.7 Hz, PhCH2), 3.52 (1H, brs, 1-CH), 3.36 (1H, brs,
5-CH), 2.78 (1H, dd, J ) 9.5, 5.7 Hz, 6R-CH), 2.50 (1H, m,
7â-CH), 2.11 (1H, dd, J ) 13.9, 9.7 Hz, 7R-CH), 2.02 (3H, s,
CH3), 1.98-1.27 (4H, m, 3,4-CH2).
of Health, Bethesda, MD) and grown in Dulbecco’s modified
Eagle medium with 10% fetal bovine serum, 100 units/mL
penicillin, and 100 g/mL streptomycin. Cells were grown at
37 °C in an atmosphere enriched in CO2. For description of
transfection protocols and characterization of cells, see ref 6.
Mem br a n es. The cerebral cortex of rat brains (Pel Freez
Biologicals, Rogers, AR) were homogenized in ice-cold 50 mM
Tris-HCl buffer (pH 7.4) using a Brinkman polytron (setting
6, 10 s). The homogenate was centrifuged for 15 min at 35000g
at 4 °C, and the pellet was washed once by recentrifugation
in Tris buffer. The final pellet was resuspended in Tris buffer
and stored at -70 °C. Prior to assay, membranes were diluted
to a concentration of 1-3 mg/mL in a binding buffer consisting
of 20 mM HEPES buffer (pH 7.4) containing 1 mM MgCl2, 120
mM NaCl, 5 mM KCl, and 2 mM CaCl2.
The CHO cells were harvested at 80-100% confluence.
Cells were washed with phosphate-buffered (pH 7.4) saline
solution and then scraped into 10 mL of ice-cold binding buffer
(see above). The tissue was homogenized using a Brinkman
polytron (setting 6, 10 s) and centrifuged for 15 min at 16000g
at 4 °C. The pellet was resuspended in the binding buffer and
homogenized again at the same setting. Aliquots (2 mL) were
stored at -70 °C. Protein concentrations were determined
using the bicinchoninic acid protein assay (Pierce Chemical
Co., Rockford, IL), using bovine albumin as a standard.
Bin d in g Assa ys. Inhibition of [3H]nicotine binding was
assayed essentially as described.15 Briefly, assays contained
0.2 nM [3H]nicotine, 300 µL of HEPES binding buffer (see
above), test agents, 100 µL of membrane suspension, and 200
µM diisopropyl fluorophosphate in a final volume of 0.5 mL.
Nonspecific binding was determined with 1 µM nicotine.
Assays were initiated by addition of membrane and were for
120 min at 0-4 °C in triplicate. Assays were terminated by
filtration through Whatman GF/B filters presoaked in 0.3%
poly(ethylenimine) for 30 min using a Brandel M24R cell
harvester (Brandel, Gaithersburg, MD). Filters were washed
twice with ice-cold 50 mM Tris-HCl buffer (pH 7.4), then placed
in vials with 4 mL of Hydrofluor scintillation fluid, and counted
for tritium.
Inhibition of binding of [3H]quinuclidinyl benzilate was
assayed with rat cerebral cortical membranes essentially as
described.15 Briefly, assays contained 0.2 nM [3H]quinuclidinyl
benzilate, 100 µL of membrane suspension, and test agents
in 20 mM HEPES buffer (pH 7.4) containing 10 mM MgCl2
and 100 mM NaCl in a final volume of 0.5 mL. Nonspecific
binding was determined with 1 µM atropine. Assays were
initiated by addition of membranes and were for 30 min at 37
°C in triplicate. Filtration, washing, and scintillation counting
were as described for [3H]nicotine binding.
Inhibition of binding of [3H]oxotremorine M was assayed
with rat cerebral cortical membranes essentially as described.16
Briefly, assays contained 2 nM [3H]oxotremorine macetate, 100
µL of membrane suspension, and test agents in 25 mM sodium
phosphate buffer (7.4) containing 5 mM MgCl2 in a final
volume of 0.5 mL. Nonspecific binding was determined with
5 µM atropine. Assays were initiated by addition of mem-
branes and were for 2 h at 25 °C in triplicate. Filtration,
washing, and scintillation counting were as described above
for [3H]nicotine binding. Saturation assays were conducted
in the same manner with a range of concentrations of [3H]-
oxotremorine M.
Binding of [3H]quinuclidinyl benzilate with membranes of
transfected CHO cells was assayed as described for [3H]-N-
ethylscopolamine binding.6 Briefly, assays were in 25 mM
sodium phosphate buffer (pH 7.4) containing 5 mM MgCl2, [3H]-
quinuclidinyl benzilate (0.2 nM for M1- and M3-receptors, 0.1
nM for M2- and M4-receptors), test agents, and 100 µL of
membrane suspension in a final volume of 0.5 mL. Nonspecific
binding was determined with atropine (1 µM for M2-, M3-, and
M4-receptors, 10 µM for M1-receptors). Assays were initiated
by addition of membranes and were for 30 min at 37 °C in
triplicate. GTP at 10 µM was present in certain experiments.
Filtration, washing, and scintillation counting were as de-
scribed above for [3H]nicotine binding. Saturation assays were
6â-Ca r bom eth oxy-8-a za bicyclo[3.2.1]octa n -2â-ol (14).
In a similar way to the preparation of 6, compound 14 was
obtained from 33 as a colorless oil, yield 87%. It was converted
to a fumarate salt. 14‚fumarate: mp 170-173 °C. Free
1
base: CI-MS m/z 186 (MH+); H NMR (CDCl3) δ 3.74 (3H, s,
OCH3), 3.70-3.33 (3H, m, 1, 2R,5-CH), 2.86 (1H, dd, J ) 9.5,
5.6 Hz, 6R-CH), 2.65 (1H, m, 7â-CH), 1.90-1.28 (7H, m, NH,
OH, 7R-CH, 3,4-CH2). 14‚fumarate: Anal. (C9H15NO3‚C4H4O4)
C, H, N.
6â-Ca r bom eth oxy-8-a za bicyclo[3.2.1]octa n -2r-ol (15).
In a similar way to the preparation of 6, compound 15 was
obtained from 34 as a colorless oil, yield 100%. It was
converted to a fumarate salt. 15‚fumarate: mp 159-161 °C.
1
Free base: CI-MS m/z 186 (MH+); H NMR (CDCl3) δ 3.90-
3.84 (1H, m, 2â-CH), 3.73 (3H, s, OCH3), 3.53-3.23 (2H, m,
1,5-CH), 2.75 (1H, dd, J ) 9.7, 5.9 Hz, 6R-CH), 2.45 (1H, p,
7â-CH), 2.12 (1H, dd, J ) 13.9, 9.9 Hz, 7R-CH), 1.94-1.20 (6H,
m, NH, OH, 3,4-CH2). 15‚fumarate: Anal. (C9H15NO3‚C4H4O4)
C, H, N.
2â-Ac e t o x y -6â-c a r b o m e t h o x y -8-a za b ic y c lo [3.2.1]-
octa n e (16). In a similar way to the preparation of 6,
compound 16 was obtained from 35 as a colorless oil, yield
98%. It was converted to a fumarate salt. 16‚fumarate: mp
130-135 °C. Free base: CI-MS m/z 228 (MH+); 1H NMR
(CDCl3) δ 4.68 (1H, brs, 2R-CH), 3.72 (3H, s, OCH3), 3.70-
3.63 (2H, m, 1,5-CH), 2.93 (1H, dd, J ) 9.5, 4.8 Hz, 6R-CH),
2.86 (1H, p, 7â-CH), 2.24 (2H, m, NH, 3â-CH), 2.13 (3H, s,
CH3CO), 1.93 (1H, dd, J ) 13.7, 9.2 Hz, 7R-CH), 1.87-1.26
(3H, m, NH, OH, 3R-CH, 4-CH2). 16‚fumarate: Anal. (C11H17
NO3‚C4H4O4) C, H, N.
-
2r-Ac e t o x y -6â-c a r b o m e t h o x y -8-a za b ic y c lo [3.2.1]-
octa n e (17). In a similar way to the preparation of 6,
compound 17 was obtained from 36 as a colorless oil, yield
100%. It was converted to a fumarate salt. 17‚fumarate: mp
138-139 °C. Free base: CI-MS m/z 228 (MH+); 1H NMR
(CDCl3) δ 4.84 (1H, m, 2â-CH), 3.71 (3H, s, OCH3), 3.61-3.58
(2H, m, 1,5-CH), 2.79 (1H, dd, J ) 8.9, 4.8 Hz, 6R-CH), 2.21
(1H, dd, J ) 13.5, 9.1 Hz, 7R-CH), 2.05 (3H, s, CH3CO), 2.00-
1.92 (3H, m, NH, 3â,7â-CH), 1.78-1.26 (3H, m, 3R-CH, 4-CH2).
17‚fumarate: Anal. (C11H17NO3‚C4H4O4) C, H, N.
Oth er Agen ts. Muscarine, methacholine, arecoline, ecgoni-
dine methyl ester, and ecgonine methyl ester were from
Research Biochemicals International (Natick, MA). Carbam-
ylcholine, atropine, and (-)-nicotine were from Sigma Chemi-
cal Co. (St. Louis, MO). The [3H]quinuclidinyl benzilate (sp.
act. 46 Ci/mmol), [3H]nicotine (sp. act. 75 Ci/mmol), [3H]-
adenine (sp. act. 29 Ci/mmol), and [3H]inositol (sp. act. 21 Ci/
mmol) were from New England Nuclear (Boston, MA). Other
compounds were from standard commercial sources.
Cu ltu r ed Cells. Four lines of CHO cells, each expressing
a different homogeneous human muscarinic receptor popula-
tion, were provided by Dr. J urgen Wess (National Institutes

Nortropanes as Muscarinic Agonists
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conducted in the same manner with a range of concentrations
of [3H]quinuclidinyl benzilate. The final volume was 1 mL.
In h ibition of Ad en ylyl Cycla se. The assay of inhibition
of [3H]cyclic AMP formation in [3H]adenine-labeled CHO cells
was essentially as described.17 Briefly, the CHO cells were
labeled in 12-well plates with 2 µCi/mL [3H]adenine for 24 h.
Wells contained ca. 3 × 105 cells in a volume of 1 mL. Cells
were then washed once with Dulbecco’s modified Eagle me-
dium containing 20 mM HEPES buffer (pH 7.4) and were then
incubated in the buffered Dulbecco’s media containing 1 mM
isobutylmethylxanthine at room temperature for 20 min.
Forskolin (10 µM) and muscarinic agents were then added to
each well. After incubation for 30 min at 37 °C, the medium
was aspirated and replaced with 1 mL of cold 5% aqueous
trichloroacetic acid with 1 mM cyclic AMP and 1 mM ATP.
After 40 min at 4 °C, the cyclic AMP in the trichloroacetic acid
solution was isolated by Dowex and alumina chromatography18
and counted for tritium in Hydrofluor scintillation fluid.
Stim u la tion of P h osp h oin ositid e Br ea k d ow n . The
assay of accumulation of [3H]inositol monophosphate is es-
sentially as described.17 The CHO cells were labeled in 12-
well plates with 1 µCi/mL myo-[3H]inositol for 24 h. Wells
contained ca. 3 × 105 cells in a volume of 1 mL. Cells were
then washed once with Hank’s balanced salt media containing
20 mM HEPES buffer (pH 7.4) and were then incubated in
the buffered Hank’s media containing 10 mM LiCl at room
temperature for 15 min. Muscarinic agents were then added
to each well. After incubation for 1 h at 37 °C, the medium
was aspirated and replaced with 750 µL of cold 20 mM aqueous
formic acid. After 35 min at 4 °C, the formic acid was removed
and replaced with 250 µL of 60 mM NH4OH. The inositol
monophosphate in the NH4OH solution was isolated by anion-
exchange chromatography19 and counted for tritium in Hy-
drofluor scintillation fluid.
Da ta An a lysis. Kd and Bmax values were derived from
linear regression analyses of the saturation binding data using
GraphPad-InPlot (GraphPad Software Inc.). Kd values were
the negative slope of the Rosenthal plot, and Bmax values were
the x-intercept. IC50 values from competitive binding data
were determined by computer analysis, using GraphPad-
InPlot, whereby a nonlinear curve was fitted to a graph of
binding (% total binding) values plotted against the log values
of the corresponding drug concentrations. Ki values were
calculated from IC50 values using the Cheng and Prusoff
equation.
Ack n ow led gm en t. The authors gratefully acknowl-
edge Dr. J urgen Wess for providing the transfected CHO
lines and for advice and O. D. Oshunleti for valuable
technical assistance.
Refer en ces
(1) Eglen, R.; Watson, N. Selective muscarinic receptor agonists and
antagonists. Pharmacol. Toxicol. 1996, 78, 59-68.
(2) Moltzen, E. K.; Bjernholm, B. Drugs Future 1995, 20, 37-57.
(3) Caulfield, M. P. Muscarinic receptors
- Characterization,
coupling and function. Pharmacol. Ther. 1993, 58, 319-379.
J M9705115