Synthesis and in vitro muscarinic activities of a series of 3-(pyrazol- 3-yl)-1-azabicyclo[2.2.2]octanes

Article abstract of DOI:10.1016/S0968-0896(99)00299-0

A series of 3-(pyrazol-3-yl)-1-azabicyclo[2.2.2]octane derivatives C (Fig. 1) was synthesized and tested for muscarinic activity in receptor binding assays using [³H]-oxotremorine-M (OXO-M) and [³H]-pirenzepine (PZ) as ligands. Potential muscarinic agonistic or antagonistic properties of the compounds were determined using binding studies measuring their potencies to inhibit the binding of OXO-M and PZ. Preferential inhibition of OXO-M binding was used as an indicator for potential muscarinic agonistic properties; this potential was confirmed in functional studies on isolated organs. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0968-0896(99)00299-0

Bioorganic & Medicinal Chemistry 8 (2000) 449±454
Synthesis and In Vitro Muscarinic Activities of a Series of
3
-(Pyrazol-3-yl)-1-azabicyclo[2.2.2]octanes
a,
a
a
b
Ralf Plate, * Marc J. M. Plaum, Rob G. A. v. d. Hulst and Thijs de Boer
aDepartment of Medicinal Chemistry, N.V. Organon, PO Box 20, 5340 BH Oss, The Netherlands
bLead Discovery Unit, N.V. Organon, PO Box 20, 5340 BH Oss, The Netherlands
Received 28 July 1999; accepted 15 November 1999
AbstractÐA series of 3-(pyrazol-3-yl)-1-azabicyclo[2.2.2]octane derivatives C (Fig. 1) was synthesized and tested for muscarinic
3
3
activity in receptor binding assays using [ H]-oxotremorine-M (OXO-M) and [ H]-pirenzepine (PZ) as ligands. Potential muscarinic
agonistic or antagonistic properties of the compounds were determined using binding studies measuring their potencies to inhibit the
binding of OXO-M and PZ. Preferential inhibition of OXO-M binding was used as an indicator for potential muscarinic agonistic
properties; this potential was con®rmed in functional studies on isolated organs. # 2000 Elsevier Science Ltd. All rights reserved.
Introduction
e€ects,9,10 thus indicating the general potential of the
cholinergic strategy for treatment of Alzheimer patients.
Five muscarinic cholinergic receptor subtypes are
known and their structure as well as their distribution
have been described.11 These receptors regulate a wide
range of peripheral and central functions in addition to
Cholinergic neurons in the forebrain of patients su€er-
ing from Alzheimer's disease appear to be degenerated
and these changes correlated with the reduction in cog-
1
nitive function. Evidence suggesting a greater loss of
1
,2
12
presynaptic rather than postsynaptic receptors has led
to the current pharmacochemical strategy in this disease
based on the cholinergic de®cit hypothesis2 and is
focused on the stimulation of postsynaptic receptors.
The increase of the availability of acetylcholine via
cholinesterase inhibitors such as tacrine has been mar-
ginally successful as only some aspects of cognitive per-
formance were found improved in association with
their role in cognition. This explains why treatments
involving non-selective agonists, which act at all choli-
nergic receptor subtypes, produce many undesirable
e€ects. Therefore, attention has shifted to subtype
selective muscarinic agonists as a means to improve the
therapeutic ecacy of cholinergic agents and at the
same time to reduce their side e€ects. Despite some
potential problems with this approach,13 attention has
focused on compounds with, in particular, M and M /
5
3
±6
severe side e€ects.
Their poor therapeutic ecacy
1
1
14
might partly be caused by a reduction of acetylcholine
release caused by stimulation of presynaptic inhibitory
autoreceptors following an increase of extracellular
acetylcholine after treatment with these acetylcholine
esterase inhibitors. Similarly, studies involving direct
stimulation of cholinergic receptors with non-selective
cholinergic agonists have been marred by their side
M agonist properties. Recent animal studies indeed
3
have shown positive results in tests of learning and
but de®nite
memory with some of these compounds,1
5,16
conclusions await the outcome of clinical trials.
The aim of the present study was to design and test a
series of compounds with selective e€ects at speci®c
receptor subtypes in vitro, and then to investigate the
central and peripheral e€ects in vivo including e€ects on
memory. The ultimate object of these studies was
to develop compounds with improved ecacy, pref-
erentially a€ecting the central nervous system with a
reduced incidence of peripheral side e€ects.
7
8
e€ect pro®le (e.g. Oxotremorine, RS 86 ).
Careful adjustment of the dose may improve certain
cognitive functions without severe compromising side
Keywords: neurotransmitters muscarinic agonist; muscarinic antago-
nist; 3-(pyrazol-3-yl)-1-azabicyclo[2.2.2]octanes.
We chose arecoline (A) as a useful starting point for the
design of the title series based on the observation of
therapeutically bene®cial e€ects of arecoline (A), a
*
5
Corresponding author. Tel.: +31-412-661-957; fax: +31-412-662-
46; e-mail: r.plate@organon.oss.akzonobel.nl
0968-0896/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(99)00299-0

4
50
R. Plate et al. / Bioorg. Med. Chem. 8 (2000) 449±454
Figure 1.
compound of limited use due to its short duration of
action.
i. Treatment of 1 with tosylmethylisocyanide (TOS-
MIC reagent) gave the cyanide 2.20 Subsequently,
treatment of 3-cyano-1-azabicyclo[2.2.2]octane (2) with
methyl lithium gave 3-acetyl-1-azabicyclo[2.2.2]octane
(3) in good yield. Reaction of 3 with dimethylforma-
mide dimethylacetal as reagent gave the enamine
derivative 4, unfortunately in low yield (10%). Finally,
treatment of 4 with hydrazine and methylhydrazine
gave the desired pyrazole derivatives 5 and 6, respec-
tively, in moderate yields.
In our search for a bioisosteric replacement of the ester
function of arecoline (A) we developed the series of 3-
(
pyrazolyl)-1,2,5,6-tetrahydropyridine derivatives B,17
a
series of potent muscarinic agonists (Fig. 1). Com-
pounds in this series produced weak agonists selective
18
for the M -receptor. Here we report the synthesis
3
of the related series of 3-(pyrazol-3-yl)-1-azabicyclo-
2.2.2]octane derivatives C (Table 1) which was tested
[
for muscarinic activity using [ H]-oxotremorine-M
3
ii. The second, more ecient route (Scheme 2),
started with the addition of propargylaldehyde diethyl
acetal to the keto function of 1. Treatment of the adduct
3
(
2
OXO-M) and [ H]-pirenzepine (PZ) as ligands (Table
) and pro®led in our in vitro (Table 3) test battery.19
7
with hydrazine gave the 1-azabicyclo[2.2.2]-octan-3-ol
derivative 8. The alcohol function of 8 was removed
in a two-step procedure: (1) elimination using thionyl-
chloride as reagent and (2) subsequent catalytic hydro-
genation of 9 in a Parr apparatus (35 psi) gave 5.
Chemistry
Synthesis of 3(pyrazol-3-yl)-1-azabicyclo[2.2.2]octane
derivatives
The products 5 (Scheme 3) as well as 9 (Scheme 2) were
converted into the mono-bromo derivatives 10 and 11,
the mono-iodo derivative 12 and the di-bromo deriva-
tive 13 using one or two equivalents of N-halogen
succinimide as reagent, respectively.
For the synthesis of the 3-(pyrazole-3-yl)-1-azabicyclo-
[2.2.2]octane derivatives two approaches have been
devised commencing with 1-azabicyclo[2.2.2]octane-3-
one (1; Schemes 1 and 2).
Table 1. Physical properties 3-(pyrazol-3-yl)-1-azabicyclo[2.2.2]octanes
ꢀ
Compound
R
1
R
2
R
3
R
4
Salt
Mp ( C)
Molecular formula
ꢁ
5
6
8
H
H
H
H
H
Br
H
H
H
Br
I
H
Me
H
H
H
H
H
OH
H
H
H
HCl
mala
HCl
mala
mal
mala
229
153
228
140
145
163
C
C
C
H
H
H
10
11
10
10
10
10
H
H
H
14BrN
14IN
13Br
15
17
15
N
3
ꢁ
2HCl
N
3
C
4
ꢁ
H
4
O
4
N
3
O 2HCl
ꢁ
11
12
13
C
C
C
3
C
4
H
4
O
4
a
ꢁ
3
C
4
H
4
O
4
ꢁ
Br
H
2
N
3
C
4
H
4
O
4
mala
188
C
ꢁ
3
12BrN C
1
0
10
H
4 4 4
H O

R. Plate et al. / Bioorg. Med. Chem. 8 (2000) 449±454
Table 2. Receptor binding anity and anity ratios for assays using
451
agonist, Oxotremorine-M (OXO-M), and antagonist pirenzepine (PZ)
ligands (data are mean values of 2±4 binding inhibition experiments;
for further details see Experimental)
3H-OXO-M
in mM)
3H-PZ
(K in mM)
3
H-PZ/ H-OXO-M ratio
3
Compound
(
K
i
i
Arecoline
0.0079
0.79
0.63
1.58
40
6.3
40
0.15
1.63
0.126
1.02
200
5
6
8
50.6
10.0
12.9
2.38
135
3.1
1
1
1
1
0
1
2
3
0.063
0.012
0.004
0.025
31.5
40.3
Table 3. Muscarinic cholinergic activity in guinea pig ileum (MUGI)
and rat left atrium (M LA)
2
Compound
MUGI
M
2
LA
a
2
pA b
c
ꢀ
pD
a
2
pD
2
a
pA
2
Scheme 2. (a) HC(CCH(OEt)
2
,
NNH .H O, EtOH, 18 h, re¯ux; (c): SOCl
t-BuOK, THF, 3 h, � 5 C; (b):
H
2
2
2
2
, benzene, 18 h, re¯ux;
(d): H , 10% Pd/C, MeOH, 24 h, 30 psi; (e): NBS, DMF, 3 h, � 15 C.
ꢀ
Arecoline
5
6.5
4.6
Ð
5.7
6.2
Ð
1.0
1.0
Ð
1.0
0.7
Ð
2
Ð
5.7
Ð
5.7
5.2
Ð
Ð
<4
6.3
Ð
Ð
Ð
Ð
Ð
5.9
6.2
Ð
10
11
12
13
0
0.5
Ð
a
2
Agonist values, pD .
Antagonist values, pA .
2
b
c
a, intrinsic activity.
ꢀ
Scheme 3. (a) 1 eq NBS, DMF, 1 h, � 10 C; (b) 1 eq NIS, DMF, 1 h,
ꢀ
�
10 C; (b) 2 eq NBS, DMF, 1 h, rt.
cholinergic agonist OXO-M is compared with their
potency to inhibit the binding of the radiolabeled mus-
carinic cholinergic antagonist pirenzepine. The ratio of
antagonist/agonist binding has been suggested1 to give
an indication of agonist potential: a large ratio indicates
agonistic properties of the compound (viz. Table 2, the
ratio PZ/OXO-M). This was further evaluated in iso-
lated organ preparation allowing the assessment of
functional activity for muscarinic cholinergic of the
subtypes M and M in guinea pig ileum and the iso-
4
2
3
ꢀ
Scheme 1. (a) Tosmic, KOtBu, DME, 0±5 C, 20 h; (b) MeLi, THF,
ꢀ
lated rat left atrium, respectively. The results show that
the unsubstituted compound has a modest anity in the
binding studies. It showed full agonistic properties in
0
C, 2 h; (c): DMF-dimethylacetal, DMF, 20 h, re¯ux; (d):
NNH .H
2
H
2
2
2
O, MeOH, 5 h, rt; e: MeNHNH , MeOH, 20 h, rt.
the M model in agreement with its binding ratio of
3
about 50. Substitution with a methyl group enhanced
the anity in the PZ binding without a€ecting the
OXO-M binding, thus causing a decrease in the ratio
from 50 for the unsubstituted compound 5 to 10 for
compound 6. Introduction of a hydroxyl group at posi-
tion R4 decreased the anity in OXO-M without
a€ecting PZ binding anity, thus reducing again the
OXO-M/PZ binding ratio. Substitution with a bromine
Pharmacology
Results
The physical properties of the derivatives are shown in
Table 1. The e€ects of the compounds on cholinergic
receptors measured as the inhibition of ligand binding
are shown in Table 2. The anity of the compounds as
inhibitor of the binding of the radiolabeled muscarinic
at position R caused an 80-fold increase in anity in
2

4
52
R. Plate et al. / Bioorg. Med. Chem. 8 (2000) 449±454
OXO-M binding and a smaller 25-fold increase in an-
ity in PZ binding. As a result a PZ/OXO-M ratio of
Acetyl-1-azabicyclo[2.2.2]oct-2-ene (3). 3-Cyano-1-aza-
bicyclo[2.2.2]octane 2 was prepared from 1-azabicyclo-
[2.2.2]octan-3-one 1 according to the procedure des-
cribed in literature.20 Methyl lithium (76.8 mmol)
was added within 1 h to a suspension of 2 (4.6 g,
26.6 mmol) in dry THF (50 mL). After 2 h stirring at
135 was found and this compound showed full agonistic
properties in the model for M receptors with a pD of
3
2
5.7 and an intrinsic activity (a-value) of 1.0. In contrast,
no agonistic properties were observed in the M model
and the compound appeared to be antagonistic with a
pA value of 5.9. Introduction of an iodine at position
2
ꢀ
5 C the reaction was quenched with water (30 mmol).
The solvent was removed in vacuo and the residue was
subjected to column chromatography (silicagel 60, elu-
ent: MeOH:conc. NH OH=98:2) to give the acetyl
2
R caused a further 3-fold increase in the anity of
2
compound 12 in OXO-M binding and a 13-fold increase
in anity in PZ binding. The compound showed a PZ/
OXO-M binding ratio of 31.5 and behaved as a partial
agonist (a=0.5±0.7) in the models used for measuring
M (atrium rat) and M (guinea pig ileum) activity.
4
1
derivative 3 in 93% yield. H NMR (200 MHz, CDCl )
3
d 3.40 (d, J=6.4 Hz, 1H, CH), 2.90±2.60 (m, 6H,
CH N's), 2.25±2.20 (m, 1H, CH), 2.20 (s, 3H, CH ),
1.75±1.60 (m, 2H), 1.50±1.35 (m, 2H).
2
3
2
3
Substitution of two bromine atoms in the pyrazole ring
caused an approximate 30-fold reduction in anity in
both binding tests, leaving the PZ/OXO-M ratio
roughly unchanged. However, despite a ratio of 40 this
substitution resulted in loss of agonistic properties in the
3-(Dimethylamino)-1-azabicyclo[2.2.2.]oct-3-yl)-prop-2-en-
1-one (4). Dimethylformamide dimethylacetale (2.0 mL,
14.4 mmol) was added to a solution of 3 (7.7 mmol) in
30 mL dry toluene. The reaction mixture was heated to
re¯ux for 48 h. After cooling the mixture, the organic
layer was washed three times with water. Drying of the
organic layer and evaporation of the solvent gave the
M model (MUGI). The halo-substituted compounds 11,
3
12 and 13 were also tested for potential M -agonistic
1
model in the hippocampal slice preparation, but all
three compounds failed to show any M1 muscarinic
agonistic properties (data not shown).
1
title compound 4 as an oil in 10% yield. H NMR
(200 MHz, CDCl ) d 7.65 (d, J=13.1 Hz, 1H, HCˆ), 5.0
3
(
3
2
d, J=13.5 Hz, 1H, ˆCH), 3.70 (d, J=5.9 Hz , 1H, CH),
.20±2.70 (m, 4H, CH N`s), 2.95+2.90 (s, 6H, CH `s),
2 3
.25 (m, 1H, CH), 1.90±1.65 (m, 3H), 1.60±1.40 (m, 1H).
Discussion
The present series of muscarinic cholinergic ligands was
evaluated for potential muscarinic cholinergic agonistic
properties by assessing the PZ/OXO-M binding ratio
and veri®cation of properties in models selectively
revealing muscarinic subtype selective agonistic proper-
ties. In this series the PZ/OXO-M ratio did not show a
correlation with the muscarinic agonistic properties
measured in isolated organs. In fact, compound 5 has
3-(1H-Pyrazol-3-yl)-1-azabicyclo[2.2.2]octane (5). Hydra-
zine hydrate (0.5 mL) was added to a solution of the
enamine 4 (0.35 g, 1.7 mmol) in 2.5 mL dry ethanol. The
reaction mixture was stirred at room temperature for
15 h. After evaporation of the solvent, the pyrazole
derivative was isolated as a hydrochloric acid salt in
ꢀ
1
39% yield; mp=230 C. H NMR (200 MHz, CDCl )
3
d 7.55 (s, 1H), 6.20 (s, 1H), 3.40±2.80 (m, 8H), 2.05
(m, 1H), 1.90±1.60 (m, 3H), 1.40 (m, 1H). Anal.
the highest ratio (135) and accordingly is a full M ago-
3
nist, but it fails to show up as an agonist in the M and
2
M models. Thus, the compounds show up as musca-
3
ꢁ
(C H N 2HCl) C, H, N. Exact mass calcd for
10 15
3
+
C H N [M+H] 178.1344, found: 178.1328.
10 16 3
rinic subtype selective agonists based on their capacity
to stimulate the M and M subtypes and their failure to
stimulate M receptors in hippocampal slices. However,
3-(1-Methyl-1H-pyrazol-5-yl)-1-azabicyclo[2.2.2]octane
(6). A solution of 4 (0.75 g, 3.6 mmol) and methyl-
hydrazine (0.38 mL, 7.2 mmol) in 10 mL dry methanol
was stirred for 20 h at room temperature. After evap-
oration of the solvent the residue was subjected to
column chromatography (silicagel 60, eluent MeOH:
2
3
1
such a pro®le is not favorable for their potential to cor-
rect a memory defect. Such an e€ect supposedly
2
3
requires M agonistic properties, which are absent in
1
the present series.
NH OH=98:2). The title compound 6 was crystallised
4
ꢀ
1
as a maleic acid salt in 50% yield; mp 153 C. H NMR
(
200 MHz, MeOD) d 7.45 (s, 1H, NˆCH), 6.50 (s, 1H,
Experimental
Chemistry
C±CHˆ), 6.25 (s, 2H, mal), 3.90 (s, 3H, CH ), 3.90±3.40
3
ꢁ
(m, 7H), 2.30±1.70 (m, 5H). Anal. (C H N C H O )
4
11
17
3
4
4
+
C, H, N. Exact mass calcd for C H N [M+H]
18
11
3
Melting points were taken on a Bu
È
chi capillary melting
192.1501, found: 192.1490.
point apparatus and are uncorrected. The elemental
analyses were within 0.45 of the theoretical values. Pro-
ton magnetic resonance spectra were measured on a
Bruker WP200 or AC200 instrument. Chemical shifts
are reported as d-values (parts per million) relative to
3-(3,3-Diethoxyprop-1-ynyl)-1-azabicyclo[2.2.2]octan-3-ol
(7). A suspension of propargylaldehyde diethylacetal
(2.5 g, 19.2 mmol) and tBuOK (2.4 g, 22.5 mmol) in
ꢀ
40 mL anhydrous THF was stirred for 1 h at � 10 C.
Me Si as an internal standard. Thin layer chroma-
Then a solution of 1-azabicyclo[2.2.2]octan-3-one 1
(2.0 g, 16 mmol) in 25 mL anhydrous THF was added
4
tography (TLC) was carried out by using Merck pre-
coated silicagel F-254 plates (thickness 0.25 mm). Spots
were visualized with a UV hand lamp and Cl /tetra-
ꢀ
dropwise. After stirring for 2 h at 0 C an aqueous acetic
acid solution was added and the solvents were removed
under reduced pressure.
2
methylbenzidine.

R. Plate et al. / Bioorg. Med. Chem. 8 (2000) 449±454
453
ꢀ
succinimide (0.66 g, 3.7 mmol) in 10 mL DMF (10 mL)
was added under N . The mixture was stirred at this
2
The residue was made alkaline (2 N NaOH), and the
product was extracted with ethyl acetate (3Â). The com-
DMF (10 mL) and at � 10 C a solution of N-bromo-
bined organic layers were dried (MgSO ) and evaporated
4
1
to dryness to give 7 (84%). H NMR (200MHz, CDCl )
3
temperature for 3 h and then poured into water. The
aqueous layer was extracted with ethyl acetate (3Â).
d 3.95 (s, 2H), 3.80±3.50 (m, 4H), 3.30±3.00 (m, 2H),
2.85 (t, 3H), 2.10±1.40 (m, 5H), 1.25 (t, 6H).
The combined organic layers were dried (MgSO ) and
4
evaporated. The residue was subjected to column chro-
matography (silicagel 60, eluent MeOH) to give 11 as an
oil in 42% yield. The maleic acid salt was prepared;
3
-(1H-Pyrazol-3-yl)-1-azabicyclo[2.2.2]octan-3-ol (8). A
solution of 7 (4.05 g, 16 mmol) and hydrazine dihydro-
chloride (1.85 g, 17.6 mmol) in ethanol (50 mL) was
heated to re¯ux for 18 h. The solvent was removed
under reduced pressure. The residue was made alkaline
using a 2 N NaOH solution, and the product was
extracted with ethyl acetate (3Â). The combined organic
ꢀ
1
mp 140 C. H NMR (200 MHz, D O) d 7.70 (s, 1H),
2
6.30 (s, 2H, mal), 4.00 (m, 1H), 3.70±3.40 (m, 4H), 2.40
(m, 1H), 2.20±1.70 (m, 4H) 1.40 (m, 1H). Anal.
ꢁ
(C H BrN C H O ) C, H, N. Exact mass calcd for
4
10
14
3
4
4
+
C H BrN [M+H] 256.0649, found: 256.0645.
10
15
3
layers were dried (MgSO ) and evaporated to give an
4
ꢀ
1
oil. The hydrochloric acid salt 8 (60%); mp 228 C. H
NMR (200 MHz, D O) d 7.95 (s, 1H), 6.70 (s, 1H),
4
3
1
3-(4-Iodo-1H-pyrazol-1-yl)-1-azabicyclo[2.2.2]octane (12).
To a solution of 5 (0.5 g, 2.8 mmol) in anhydrous DMF
2
ꢀ
(0.64 g, 2.8 mmol) in DMF (10 mL) was added. The
.10 (d, J=15 Hz, 1H), 3.60 (d, J=15 Hz, 1H), 3.50±
.20 (m, 4H), 2.60±2.40 (m, 2H), 2.10±1.90 (m, 2H),
(20 mL) at � 10 C a solution of N-iodosuccinimide
ꢁ
.80±1.60 (m, 1H). Anal. (C H N O 2HCl) C, H, N.
3
mixture was stirred under N at room temperature for
2
10
15
+
Exact mass calcd for C H N O [M+H] 194.1293,
1
found: 194.1281.
20 h and then poured into water. The aqueous layer was
extracted with ethyl acetate (3Â). The combined organic
0
16
3
layers were dried (MgSO ) and evaporated. The maleic
4
ꢀ
1
3
-(1H-Pyrazol-1-yl)-1-azabicyclo[2.2.2]oct-2-ene (9). To
acid salt was prepared in 62% yield; mp 146 C. H
a suspension of 8 (4.5 g, 18.1 mmol) in anhydrous
benzene (25 mL) thionyl chloride (2 mL, 27 mmol) was
added. The solution was heated to re¯ux. After 18 h the
solvent was removed under reduced pressure. The resi-
due was made alkaline using a 2 N NaOH-solution, and
the product was extracted with ethyl acetate. The com-
NMR (200 MHz, D O) d 7.70 (s, 1H), 6.30 (s, 2H, mal),
2
4.00 (m, 1H), 3.70±3.40 (m, 4H), 2.40 (m, 1H), 2.20±1.70
ꢁ
+
(m, 4H) 1.40 (m, 1H). Anal. (C H IN C H O ) C, H,
4
10
14
3
4
4
N. Exact mass calcd for C H IN [M+H] 304.0311,
3
10
15
found: 304.0301.
bined organic layers were dried (MgSO ) and evapor-
4
ated to give 9 as an oil (63%).
3-(3,4-Dibromo-1H-pyrazol-1-yl)-1-azabicyclo[2.2.2]octane
(13). N-Bromosuccinimide (0.7 g, 9 mmol) was added all
at once to a solution of 5 (0.49 g, 2.8 mmol) in 10 mL
dry DMF. The reaction was strongly exothermic
(60 C). After 1 h stirring, the solvent was evaporated.
The residue was subjected to column chromatography
3
-(1H-Pyrazol-3-yl)-1-azabicyclo[2.2.2]octane (5). Hydro-
genation of 9 (1.6 g, 10 mmol) in methanol (50 mL) over
0% Pd/C (250 mg) at 30 psi at room temperature in a
Parr apparatus for 24 h gave the title compound 5, after
ltration and evaporation of the solvent, in 93% yield.
ꢀ
1
(silicagel 60, eluent MeOH:NH OH=98:2) to give 13
4
®
as an oil in 32% yield. The maleic acid salt was
prepared; mp 163 C. H NMR (D O, 200 MHz) d
ꢀ
1
ꢀ
6.25 (s, 2H, mal), 3.70±3.20 (m, 7H), 2.40 (m, 1H,
1
The hydrochloride salt was prepared; mp 229 C. H
2
NMR (200 MHz, CDCl ) d 7.55 (s, 1H), 6.20 (s, 1H),
3
3
.40±2.80 (m, 8H), 2.05 (m, 1H), 1.90±1.60 (m, 3H), 1.40
CH), 2.25±2.00 (m, 2H), 1.90±1.75 (m, 2H). Anal.
ꢁ
(
m, 1H).
(C H Br N C H O ) C, H, N. Exact mass calcd for
13
10
2
3
4
4
4
+
C H Br N [M+H] 333.9954, found: 333.9937.
10
14
2
3
3-(4-Bromo-1H-pyrazol-1-yl)-1-azabicyclo[2.2.2]oct-2-ene
(
10). A solution of NBS (1.1 g, 6 mmol) in 15 mL dry
ꢀ
6.1 mmol) in 15 mL dry DMF. After 3 h stirring at
DMF was added at � 20 C, to a solution of 9
In Vitro Studies
(
�
ꢀ
H O and three times extracted with ethyl acetate. The
15 C the reaction mixture was diluted with 100 mL
Agonist and antagonist binding studies
2
3
3
combined organic layers were dried (MgSO ) and evap-
4
Binding of [methyl- H]-oxotremorine-M acetate ( H-
OXO-M) in homogenates of frontal cortex. The rapid
®ltration method of Freedman et al.21 was used to
measure the agonist character of muscarinic cholinergic
drugs in rat cerebral cortex homogenates. For routine
orated. The residue was subjected to column chroma-
tography (silicagel 60, eluent MeOH) to give 10 as an oil
in 45% yield. The maleic acid salt was prepared: mp
ꢀ
1
1
88 C. H NMR (200 MHz, MeOD) d 7.90 (s, 1H, N±
CHˆ), 7.45 (s, 1H, BrCˆCH), 6.25 (s, 2H, Mal), 3.95 (s
broad), 1H, ˆC±CH), 3.70±3.55 (m, 2H, CH ), 3.30±
3
measurements the concentration of [ H]-OXO-M was
0.5 nM, tissue concentration was about 1 mg/mL original
(
2
ꢀ
speci®c binding was de®ned as the amount of binding of
3
.10 (m, 2H, CH ), 2.30±2.10 (m, 2H, CH ), 1.95±1.70
tissue and incubation was for 40 min at 30 C. Non-
2
2
ꢁ
(m, 2H, CH ). Anal. (C H BrN C H O ) C, H, N.
Exact mass calcd for C H BrN [M+H] 241.0415,
2
10 12
3
4
4
4
+
3
[ H]-OXO-M in the presence of 2 mM atropine sulfate
and represented about 10% of total binding.
9
12
3
found: 241.0405.
3
3
3
(
-(4-Bromo-1H-pyrazol-1-yl)-1-azabicyclo[2.2.2]octane
11). To a solution of 5 (0.61 g, 3.7 mmol) in anhydrous
Binding of [N-methyl- H]-pirenzepine [ H-PZ] in homo-
genates of rat forebrain. The rapid ®ltration method of

4
54
R. Plate et al. / Bioorg. Med. Chem. 8 (2000) 449±454
Freedman et al.21 was used to characterize M -muscari-
acetylcholine as an agonist (pD -values between 6 and
2
1
nic cholinergic properties of drugs in rat forebrain
membranes. For routine measurements the concentra-
7) were used to evaluate the potency of cholinergic
antagonist. Antagonistic activity was measured as
described above.
3
tion of [ H]-PZ was 1 nM, tissue concentration was
about 10 mg/mL original tissue and incubation was for
ꢀ
6
0 min at 25 C. Non-speci®c binding was de®ned as the
3
amount of binding of [ H]-PZ in the presence of 1 mM
atropine sulfate and represented about 20% of total
binding.
References and Notes
1
1
2
. Mash, D. C.; Flynn, D. D.; Potter, L. T. Science 1985, 228,
115.
. Araujo, D. M.; Lapchak, P. A.; Robitaille, Y.; Gauthier, S.;
Evaluation of the data. Displacement curves were
obtained for the various compounds by measuring the
speci®c binding in the presence of at least four di€erent
concentrations and IC50 values were obtained using a
four-parameter ®tting procedure. Ki values were
obtained from the IC50 values by using the Chang±
Pruso€ equation K =IC =ꢂ1 ‡ C=K † in which C
Quirion, R. Naunyn-Schmiedeberg's Arch. Pharmacol. 1988,
42, 625.
3
3. Eagger, S. A.; Levy, R.; Sahakian, B. J. Lancet 1991, 337,
989.
4. Lamy, P. P. C N S Drugs 1994, 1, 146.
5. Maltby, N.; Broe, G. A.; Creasey, H.; Jorm, A. F.; Chris-
tensen, H.; Brooks, W. S. Br. Med. J. 1994, 308, 879.
i
50
d
equals the radiolabelled ligand concentration and Kd
6
. Asthana, S.; Ra€aele, K. C.; Berardi, A.; Greig, N. H.;
equals the dissociation constant for the radiolabelled
ligand. K values used for these calculations were as
Haxby, J. V.; Schapiro, M. B.; Soncrant, T. T. Alzheimer Dis.
Assoc. Disord. 1995, 9, 223.
d
3
3
follows: [ H]-OXO-M binding: K =0.7 nM; [ H]-PZ
d
7
. Davis, K. L.; Hollander, E.; Davidson, M.; Davis, B. M.;
Mohs, R. C.; Hovath, T. B. Am. J. Psychiatry 1987, 144, 468.
. Hollander, E.; Davidson, M.; Mohs, R. C.; Horvath, T. B.;
Davis, B. M.; Zemishlany, Z.; Davis, K. L. Biol. Psychiatry
binding: K =8.3 nM.
d
8
Interactions with muscarinic subtype mediated responses
in isolated organs
1
987, 22, 1067.
. Soncrant, T. T.; Ra€aele, K. C.; Asthana, S.; Beradi, A.;
9
Morris, P. P.; Haxby, J. V. Psychopharmacology 1993, 112,
M -mediated activity in the hippocampal slice. Musca-
1
4
1
21.
0. Asthana, S.; Ra€aele, K. C.; Berardi, A.; Greig, N. H.;
rinic cholinergic agonists e€ectively suppress the elec-
trically evoked ®eld excitatory postsynaptic potential
2
2
Morris, P. P.; Schapiro, M. B.; Rapoport, S. I.; Blackman, M.
R.; Soncrant, T. T. Psychoneuroendocrinology 1995, 20, 623.
(
fEPSP) in the rat hippocampal slice. This e€ect is due
to a decrease in release of excitatory amino acids from
the Scha€er collateral/commissural ®bers, probably
1
1. Anon. Trends in Pharmacological Sciences. Receptor & Ion
Channel Nomenclature Supplement, 1997. p 4.
12. Levey, A. I. Life Sci. 1993, 52, 441.
13. Flynn, D. D.; Ferrari-DiLeo, G.; Levey, A. I.; Mash, D. C.
Life Sci. 1995, 56, 869.
14. Freedman, S. B.; Dawson, G. R.; Iversen, L. L.; Baker, R.;
Hargreaves, R. J. Life Sci. 1993, 52, 489.
1
Psychopharmacology 1994, 113, 361.
1
1
1
22
mediated by a presynaptic M muscarinic receptor.
1
Carbachol (3E-5 mol/L) causes
intrinsic activity=1.0) in the amplitude of the elec-
trically evoked fEPSP, an e€ect that can be fully antag-
a 100% decrease
(
23
onized by the M selective antagonist pirenzepine.
1
5. Fawson, G. R.; Bayley, P.; Channell, S.; Iversen, S. D.
M -mediated e€ects. Interactions with M -muscarinic
2
2
6. Sedman, A. J.; Bockbrader, H.; Schwarz, R. D. Life Sci.
995, 56, 877.
7. Plate, R.; Plaum, J. M.; Boer, Th. de; Andrews, J. S; Rae,
cholinergic receptors were studied in the isolated left
atrium of the rat. An automated assay was used similar
2
4
to the b -adrenoreceptor described previously but with
1
D. R.; Gibson, S. Bioorg. Med. Chem. 1996, 4, 227.
8. Plate, R.; Plaum, J. M.; Boer, Th. de; Andrews, J. S.
Bioorg. Med. Chem. 1996, 4, 239.
the following adaptation. The inhibition by cholinergic
agonists of the electrical stimulation evoked switch of
the atrium was measured using carbachol as a reference.
Cholinergic agonistic activity was compared to carba-
chol. Potential M -activity was veri®ed via antagonism
2
in the presence of the M -selective antagonist AF-DX
2
1
19. Plate, R. Eur. Patent Application No. 92.202227.2, 1992.
20. Olek, B. S.; Blaney, F. E.; Brown, F.; Clark, M. S. G.;
Hadley, M. S.; Hatcher, J.; Riley, G. J.; Rosenberg, H. E.;
Wadswporth, H. J.; Wyman, P. J. Med. Chem. 1994, 34, 2726.
2
macol. 1988, 156, 133.
1. Freedman, S. B.; Beer, M. S.; Harley, E. A. Eur. J. Phar-
116. Antagonistic activity was measured as a shift to the
right of the dose±response curve to carbachol in the
presence of the compound as described above.
2
2
2
2. Sheridan, R. D.; Sutor, B. Neuroscience Letters 1990, 108,
73.
3. For more details see: Plate, R.; Plaum, M. J. M.; Pintar P.;
M -mediated e€ects. Interactions with M -muscarinic
3
3
Jans, C.G.; Boer, Th. de; Dijcks, F. A.; Ruigt, G.; Andrews, S.
Bioorg. Med. Chem. 1998, 6, 1403.
24. De Graaf, J. S.; De Vos, C. J.; Steenbergen, H. J. J. Phar-
macol. Meth. 1983, 10, 113.
cholinergic receptors were measured in the isolated
guinea pig ileum. A fully automated method was used
2
3
as described previously. Contractions induced with