Synthesis and in vitro pharmacology of novel heterocyclic muscarinic ligands

Article abstract of DOI:10.1016/S0014-827X(03)00113-7

A set of novel heterocyclic ligands (7a-9a, 7b-9b, and 9c) structurally related to oxotremorine 2 was designed, synthesized, and tested at muscarinic receptor subtypes. In the binding experiments at cloned hm1-5, the presence of the 2-methylimidazole/2-me

Full text of DOI:10.1016/S0014-827X(03)00113-7

Il Farmaco 58 (2003) 739ꢀ748
/
www.elsevier.com/locate/farmac
Synthesis and in vitro pharmacology of novel heterocyclic muscarinic
ligands
Marco De Amici a, Paola Conti a, Ezio Fasoli a, Elisabetta Barocelli b,
Vigilio Ballabeni b, Simona Bertoni b, Mariannina Impicciatore b, Bryan L. Roth c,
Paul Ernsberger d, Carlo De Micheli a,*
a Istituto di Chimica Farmaceutica e Tossicologica, Universita` di Milano, Viale Abruzzi 42, Milan 20131, Italy
b Istituto di Farmacologia e Farmacognosia, Universita` di Parma, Parco Area delle Scienze 27/A, Parma 43100, Italy
c Department of Biochemistry and NIMH Psychoactive Drug Screening Program, SOM W438, Case Western Reserve University, 2109 Adelbert Rd.,
Cleveland, OH 44106, USA
d Department of Nutrition and NIMH Psychoactive Drug Screening Program, Case Western Reserve University, 2109 Adelbert Rd., Cleveland, OH
44106, USA
Received 16 November 2002; accepted 15 January 2003
Abstract
A set of novel heterocyclic ligands (7aꢀ
tested at muscarinic receptor subtypes. In the binding experiments at cloned hm1ꢀ
/
9a, 7bꢀ
/
9b, and 9c) structurally related to oxotremorine 2 was designed, synthesized, and
5, the presence of the 2-methylimidazole/2-
/
methyl-3-alkylimidazolium moiety in place of the pyrrolidine ring revealed, in derivatives 8a, 8b, and 9c, a moderate selectivity for
some receptor subtypes. The functional in vitro assays yielded results that correlated closely to binding data. In general, on passing
from agonists bearing the pyrrolidine moiety to their analogues carrying the 2-methylimidazole function, the overall
pharmacological efficacy profile is shifted from agonism toward partial agonism. The insertion of the 2-methyl-3-alkylimidazolium
moiety advances the effect such that the compounds are pure antagonists. Quite similarly, chiral 3-oxo-D2-isoxazoline (9
/)-10
behaved as a weak antagonist unable to discriminate the different muscarinic receptor subtypes.
´
# 2003 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
Keywords: Muscarinic ligands; Oxotremorine analogues; Muscarinic receptor subtypes; Functional assays; Cloned receptors
1. Introduction
smooth muscles, e.g. urinary bladder (M3) and mucosal
glands (M3). The M4 receptors have been identified
peripherally in the lung [4] and centrally in the striatum
using immunocytochemical and in situ hybridization
techniques and are believed to be involved in the control
of motor function [5] as well as in the mediation of
antinociceptive effects [6]. Even though a better in vitro
characterization of the M5 receptors was recently
achieved [7], at present their physiological role is far
from being unequivocally established, although they are
present mainly in the central nervous system [1,8].
The knowledge of the muscarinic receptor heteroge-
neity so far acquired through established conventional
pharmacological approaches has been recently enriched
by the advent of the genomic technology, which
significantly contributes at providing the rationale for
the identification of novel therapeutic targets for sub-
The muscarinic acetylcholine receptors (mAChRs),
which belong to the large superfamily of G protein-
linked receptors, are expressed within the parasympa-
thetic and central nervous systems, where they are
involved in the excitatory or inhibitory modulation of
a number of central and peripheral physiologic func-
tions [1]. Molecular biological techniques have resulted
in the cloning of five molecularly distinct mAChR (M1ꢀ
/
M5) [2,3]. The M1 receptors are found at high density in
parasympathetic ganglia and neuronal tissues, whereas
the M2 and M3 subtypes are mainly localized in
peripheral effector organs such as heart (M2) and
* Corresponding author.
E-mail address: carlo.demicheli@unimi.it (C. De Micheli).
´
0014-827X/03/$ - see front matter # 2003 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
doi:10.1016/S0014-827X(03)00113-7

740
M. De Amici et al. / Il Farmaco 58 (2003) 739ꢀ748
/
type-selective muscarinic agents [1,9]. As a consequence,
renewed efforts have been devoted to the search for new
ligands able to selectively interact with the different
muscarinic receptor subtypes. While a number of
muscarinic antagonists have been developed in the
past decades, there is still a demand for truly subtype-
selective agonists, which could be clinically useful in
treating, for example, Alzheimer’s disease, pain, schizo-
As an extension of our previous studies, we now
report the synthesis and the results of the pharmacolo-
gical investigation of novel heterocyclic muscarinic
ligands endowed with a butynyl side-chain. In particu-
lar, in our search for a bioisosteric replacement of the
pyrrolidine function of derivatives 3ꢀ
set of derivatives where such a moiety was replaced by
the 2-methylimidazole ring (Fig. 1, 7aꢀ9a). We also
prepared the corresponding quaternary ammonium salts
7bꢀ9b and 9c. Previous investigations showed that the
/5, we designed a
/
phrenia, and intestinal hypomotility [1,10ꢀ
In the past, our research group investigated the
activityꢀselectivity profile of a set of chiral muscarinic
ligands structurally related to natural muscarine [13ꢀ
/12].
/
exchange of the pyrrolidine nucleus of oxotremorine
with the 2-methylimidazole ring, to generate 6 (Fig. 1),
shifted the pharmacological profile from a full to a
partial agonist [20]. Even more interesting, the insertion
of the 2-methylimadazole nucleus in the skeleton of
diphenylbutyramide derivatives, provided with an anti-
muscarinic activity, enhanced the selectivity towards the
M3 receptor subtype [21]. Therefore, we designed and
assayed novel ligands incorporating both the 2-methy-
/
/
15]. Later on, we inserted the 3-oxo-isoxazolidinyl
moiety of the highly potent muscarinic agonist azamus-
carone 1 [16] (Fig. 1) into the molecular skeleton of
oxotremorine 2 (Fig. 1), an efficacious muscarinic
agonist moderately selective for the central nervous
system [17]. In a series of related analogues, the
pharmacological profile of representative derivatives,
limidazole and the 2-methylimidazolium nuclei, i.e. 7aꢀ
/
i.e. 3ꢀ5, was studied in depth by taking into account
/
9a, 7bꢀ9b, and 9c, with the objective to uncover
/
both receptor occupancy [18] and in vivo tests [19]. It
turned out that the oxotremorine-like derivative 3
exhibited a quite interesting analgesic activity.
compounds with selective effects at specific receptor
subtypes.
Moreover, in analogy to a modification previously
performed on oxotremorine and its analogues [20,22],
we synthesized the racemate of pyrrolidinyl derivative 10
(Fig. 1) to evaluate if the presence of the stereogenic
center was able to alter the profile of compound 4, the
related non-chiral parent derivative. The new com-
pounds were assayed at cloned human muscarinic
receptor subtypes (hm1ꢀ5), expressed in Chinese Ham-
/
ster Ovary (CHO) cells, and in functional tests at M1ꢀ3
tissue preparations.
2. Chemistry
The synthesis of target compounds was accomplished
along the reaction sequences depicted in Schemes 1 and
2. Isoxazolidin-3-one (13) was conveniently prepared by
transformation of known 3-nitro-D2-isoxazoline (11)
[23] into the corresponding 3-benzyloxy derivative 12,
which was then submitted to a catalytic hydrogenation
(Scheme 1). Treatment of 13 with 1,4-dichloro-2-butyne,
carried out in refluxing acetone in the presence of
potassium carbonate, produced a 9:1 mixture of N-
and O-alkynyl derivatives 14 and 15, respectively. The
two regioisomers were easily separated by column
chromatography, and their structure was assigned by
taking into account the difference in the chemical shifts
of the a-CH2 protons of the side-chain (d 4.34 for 14
and d 4.82 for 15). The desired final salts 7a and 7b were
obtained by reacting 14 with 2-methylimidazole fol-
lowed by treatment of the intermediate free base 7 either
with excess fumaric acid or methyl iodide (Scheme 1). In
agreement with previously reported results [20], we
Fig. 1. Structure of model and target compounds.

M. De Amici et al. / Il Farmaco 58 (2003) 739ꢀ
/748
741
Scheme 1. (a) C6H5CH2OLi, DMSO; (b)H2-Pd/C, 5%; (c) 1,4-dichloro-2-butyne/K2CO3; (d) 2-butyne-1,4-diol/NaH/THF; (e) CH3SO2Cl/NEt3; (f) 2-
methylimidazole/CH3CN; (g) C4H4O4/2-propanolꢀMeOH; (h) CH3I/2ꢀpropanolꢀether.
/
/
/
Scheme 2. 1,4-Dichloro-2-butyne/K2CO3; (b) 2-methylimidazole/CH3CN; (c) C4H4O4/EtOHꢀ
/
ether; (d) CH3I/2-propanolꢀether; (e) p-
/
BrC6H4CH2Br/2-propanolꢀether; (f) NaH-THF/11; (g) pyrrolidine/(HCHO)n /CuCl/HOAc/dioxane.
/

742
M. De Amici et al. / Il Farmaco 58 (2003) 739ꢀ748
/
assume that the alkylation step takes place at N-3. As a
consequence, the reaction of 7 with methyl iodide gives
rise to the 2,3-dimethylimidazolium iodide, i.e. 7b.
Since the alkylation of intermediate 13 produced 15 in
a very low yield, the same strategy could not be applied
to the synthesis of the O-alkylated derivatives. For such
a reason, we devised a new reaction sequence, which
yielded exclusively the desired compounds. The reaction
of 2-butyne-1,4-diol with 3-nitro-D2-isoxazoline (11), in
the presence of sodium hydride, produced alcohol 16 in
51% yield as a single product. Such an intermediate was
then converted into the corresponding mesylate 17, thus
allowing the preparation of the O-alkynyl derivatives 8a
and 8b (Scheme 1).
as the reference agonists at M2ꢀ3 and M1 subtypes,
respectively.
Inspection of the data reported in Table 1 puts in
evidence that among the 2-methylimidazole derivatives
7aꢀ9a, solely 8a displays a certain degree of selectivity.
/
Its affinity for hm2, hm3, and hm5 (pKi values: 8.44,
8.60, and 8.21, respectively) is at least 10-fold higher
than that associated to hm1 and hm4 (pKi values: 7.04
and 7.40, respectively). The transformation of 8a into
the corresponding methylimidazolium methiodide 8b
gives rise to the appearance of a 10-fold selectivity for
the hm3 receptor. As a matter of fact, derivative 8b
possesses a hm3/hm2 selectivity ratio of 14.6 which is
higher than that reported for 4-DAMP (M3/M2 equal to
3.5 or 4.2) [27,28]. A similar profile holds true for 9c
since the derivative is a slightly selective hm3 ligand.
The results collected in the binding experiments were
largely confirmed in the functional in vitro assays (Table
2). Comparison of the binding and function data by
linear regression showed strong correlations for M1 and
M2 receptors and a moderate correlation for M3 subtype
(Fig. 2). For M1 and M2 receptors, the six compounds
tested in both assays fell very close to the line of identity,
where binding affinity and functional activity show
molar equivalency. Part of the deviation from perfect
correlations may reflect species differences in ligand
selectivity, as binding assays were carried out with the
human form of the receptors and functional assays were
performed in rabbits and guinea pigs. Compounds
which were agonists (8a and 9a for M1 and compounds
7a, 8a, and 9a for M2) fell above the line of identity,
indicating slightly greater functional activity relative to
binding affinity. In contrast, antagonist compounds fell
near or below the line of identity. The same trend was
observed at M3 receptors, but all compounds showed
greater activity in binding than in functional assays,
resulting in a downward shift of all points.
Following the procedure previously applied to 13, the
reaction of 3-hydroxyisoxazole (18) [24] with 1,4-di-
chloro-2-butyne gave isoxazolyl ether (19), precursor of
the functionalized derivatives 9aꢀ
Finally, alkynyl-D2-isoxazoline (9
(9)-3-butyne-2-ol 20 and 11, was submitted to a CuCl-
/
9c (Scheme 2).
/
)-21, prepared from
/
catalyzed Mannich reaction in the presence of parafor-
maldehyde and pyrrolidine [25] (Scheme 2). The tertiary
base (9)-10, so far obtained, was then transformed into
/
the corresponding fumarate.
3. Results and discussion
The new compounds were tested for binding affinity
at human muscarinic receptor subtypes (hm1ꢀ5) in
/
transfected CHO cells labeled with [3H]quinuclidinyl
benzylate and the results are compared with those of
well-recognized selective ligands (Table 1).
The potency and efficacy of the compounds under
study were also evaluated towards some muscarinic
receptor subtypes by performing the following four
functional in vitro models: rabbit electrically stimulated
vas deferens (M1) [26], guinea pig electrically driven left
atrium (M2), guinea pig ileum (M3), and urinary bladder
(M3; Table 2). Bethanechol and McN-A-343 were used
By taking into account the data collected in the
functional tests (Table 2), the insertion of the 2-
methylimidazole moiety shifts the pharmacological
profile of the derivatives from a full (aꢁ1) to a partial
/
Table 1
Binding affinity (pKi, molar) of compounds 7aꢀ
/
7b, 8aꢀ
/
8b, 9a, and 9c at cloned human muscarinic receptor subtypes (hm1ꢀ
/
5) expressed in CHO cells
hm5
Comp.
hm1
7.679
hm2
hm3 hm4
Pirenzepine
Methoctramine
/
0.037
7.4590.042
/
7.119
/0.036
4-DAMP
Tropicamide
9.179
/
0.042
8.469
7.069
6.599
7.409
7.039
6.339
6.689
/
0.021
0.034
0.035
0.046
0.037
0.022
0.042
7a
7b
8a
8b
9a
9c
6.929
6.299
7.049
6.649
5.969
6.069
/
0.036
0.044
0.007
0.036
0.018
0.039
7.149
6.569
8.449
7.039
6.559
6.959
/
0.030
6.999
7.129
8.609
8.209
6.969
7.779
/
0.101
/
9.299
6.889
8.219
7.299
6.689
7.229
/
0.037
0.055
0.086
0.04
/
/0.05
/
0.02
/
/
/
/
0.080
0.042
0.050
0.090
/
0.022
0.090
0.022
0.103
/
/
/
/
/
/
/
/
/
/
/
/0.056
/
/
/
/
/0.02

M. De Amici et al. / Il Farmaco 58 (2003) 739ꢀ
/748
743
Table 2
In vitro functional activity of compounds 7aꢀ
atrium (M2), guinea pig ileum (M3) and urinary bladder (M3)
/
7b, 8aꢀ
/
8b, 9aꢀ
/
9c, and 10 at muscarinic receptor subtypes in rabbit vas deferens (M1), guinea pig left
Comp.
M1
M2
M3
Rabbit vas deferens a
Guinea pig left atrium
Guinea pig ileum
Guinea pig urinary bladder
b
d
b
d
b
d
b
d
pEC50
ia c pKB
pEC50
5.839
ia c pKB
pEC50
6.519
ia c pKB
pEC50
4.969
ia c pKB
Bethanechol
McN-A-343
/
0.04
0.06
0.15
0.11
1
/
0.07
1
ꢀ/
/
0.03
1
4.389
/
0.33
6.429
/
0.04
1
0
0
1
0
ꢀ
/
ꢀ/
ꢀ/
ꢀ/
7a
7b
8a
8b
9a
9b
9c
10
7.049
6.159
/
0.10 7.479
/
1
0
1
0
1
0
0
0
ꢀ
/
7.539
7.949
6.909
/
0.20 0.9
ꢀ/
6.619
6.519
5.449
/
0.08 0.3
/0.07
6.219
/
0.07
0
6.089
/
0.23
0
6.049
/0.10
7.439
/
0.04
ꢀ/
8.099
/
ꢀ/
/
0.17 0.7
ꢀ/
/
0.24 0.3
6.729
/0.12
6.799
/0.23
0
6.919
/0.02
0
7.09
/
0.16
6.519
/
0.19 0.8
ꢀ/
7.309
/
ꢀ/
/
0.15 0.7
ꢀ/
/
0.15 0.3
0
0
0
5.739
6.369
6.939
/
0.32
0.17
0.06
6.329
7.079
6.549
/
0.11
0.24
0.14
0
0
0
6.479
6.449
6.359
/
0.06
0.12
0.29
0
0
0
6.389
5.939
6.559
/
0.09
0.09
0.05
/
/
/
/
/
/
/
/
a
See Ref. [26].
b
c
pEC50
9SE values are the negative logarithm of the agonist concentration that caused 50% of the maximum response attainable in that tissue.
/
Intrinsic activity (a) measured by the ratio between the maximum response of the compound and the maximum response of the reference agonist.
d
Apparent pKB values9SE were calculated according to Furchgott [35].
/
agonist (a B
/
1), as previously observed in the oxotre-
logical profile towards pure antagonists (aꢁ0). Once
/
morine series [20]. In this frame, the profile of 7a is
worth noting since it shows a degree of functional
selectivity. It behaves as a full agonist at guinea pig
again, none of the ammonium derivatives showed
selectivity for any of the muscarinic receptor subtypes.
The 2-methylimidazolium methiodides, 7bꢀ
/
9b, possess
cardiac M2 (pEC50ꢁ
two M3 assays (pEC50ꢁ
aꢁ0.3, respectively) and as a pure antagonist at M1
rabbit vas deferens receptors (pKBꢁ7.04, aꢁ0). As
expected, based on the urinary bladder low coupling
efficiency [29], compounds 7aꢀ9a display a 10ꢀ30-fold
lower potency than that detected on ileum. The agonist
effect of the studied compounds was competitively
blocked by the classical antimuscarinic drug atropine
and was unaffected by the presence of hexamethonium.
The transformation of the tertiary bases into qua-
ternary ammonium salts further shifted the pharmaco-
/
7.47), as a partial agonist in the
pKB values in the range 5.73ꢀ7.00 at M1ꢀM3 receptors.
/
/
/
7.53, aꢁ0.9 and pEC50ꢁ6.61,
/
/
Quaternization of 9 with 4-bromobenzyl bromide to
yield 9c did not substantially alter the profile of the
corresponding methiodide (9b). Such a modification,
previously applied to a series of 2,2-diphenylbutyra-
mides provided with potent antimuscarinic properties
[30], significantly improved the M3 versus M2 selectivity.
Finally, we evaluated the influence of a substituent in
the side-chain on both activity and selectivity. For such
a purpose, the full muscarinic agonist 4 [17,18] (Fig. 1)
was methylated at position 1 of the butynyl side-chain to
/
/
/
/
/
produce derivative (9)-10, which became a weak non-
/
Fig. 2. Correlation of binding affinity (pKi) to functional activity (pEC50 for agonists and pKB for antagonists) for M1 (A), M2 (B), and M3 (C)
mAChRs. Dashed line represents the line of identity connecting identical values for the two axes. Solid line represents linear regression. Pearson
correlation coefficients were as stated (Nꢁ/6). Error bars indicate standard error. For the binding data plotted on the horizontal axis, error bars are
not visible since the errors were smaller than the size of the plotted symbol.

744
M. De Amici et al. / Il Farmaco 58 (2003) 739ꢀ748
/
selective muscarinic antagonist (Table 2). This result
parallels that previously observed on an analogue of
oxotremorine [20,22].
In summary, the data collected on the novel oxotre-
morine-like compounds indicate that the presence of the
4.34 (t, 2H, CH2N, Jꢁ
Jꢁ8.1 Hz). Anal. C7H8ClNO2 (C, H, N).
3-(4-Chloro-2-butynyl)oxy-D2-isoxazoline (15):
colorless liquid, b.p., 115ꢀ120 8C/0.5 mmHg; Rf,
0.61 (eluant: 30% ethyl acetate/cyclohexane); 1H
NMR: 3.0 (t, 2H, H-4, Jꢁ9.6 Hz), 4.18 (t, 2H,
CH2Cl, Jꢁ1.8 Hz), 4.43 (t, 2H, H-5, Jꢁ9.6 Hz),
4.82 (t, 2H, OÃCH2CÅ Jꢁ1.8 Hz). Anal.
C7H8ClNO2 (C, H, N).
/
1.8 Hz), 4.38 (t, 2H, H-5,
/
/
2-methylimidazole/2-methylimadazolium
moieties
/
strongly affects the intrinsic activity (ia) of the com-
pounds. A selectivity between subtypes of 10-fold or
more was noticed in some derivatives in the binding
data, but selectivity was threefold or less in the in vitro
functional assays.
/
/
/
/,
/
B) To a solution of 14 (1.30 g, 7.49 mmol) in 10 ml
acetonitrile was added 2-methylimidazole (1.23 g,
14.98 mmol). After stirring at reflux for 3 h, the
solvent was removed at reduced pressure and the
crude reaction mixture, acidified by addition of 10-
4. Experimental
ml 2-N HCl, was treated with ether (3ꢂ10 ml). The
/
residual aqueous phase was made alkaline (pH 10)
by portion-wise addition of solid K2CO3 and
4.1. Chemistry
3-Substituted-D2-isoxazolines, 11 [23] and 16 [17],
isoxazolidin-3-one (13) [17], and 3-hydroxyisoxazole
(18) [24] were prepared according to procedures de-
scribed in the literature. Melting points were determined
extracted with ethyl acetate (3ꢂ10 ml). The pooled
/
organic extracts were dried over anhydrous sodium
sulfate, the solvent was evaporated under vacuum,
and the oily residue was purified by silica gel
column chromatography (eluant: 2% methanol/
dichloromethane) to afford 0.690 g (42% yield) of
the desired tertiary amine.
on a model B 540 Buchi apparatus and are uncorrected.
¨
Liquid compounds were characterized by the oven
temperature for Kugelrohr distillations. 1H NMR
spectra were recorded with a Bruker AC-E 200 (200
MHz) spectrometer in CDCl3 (unless otherwise speci-
fied) solutions. Chemical shifts (d) are expressed in ppm
and coupling constants (J) in hertz. TLC analyses were
performed on commercial silica gel 60 F254 aluminum
sheets: spots were further evidenced by spraying with a
dilute alkaline potassium permanganate solution. Mi-
croanalyses (C, H, N) of new compounds agreed with
2-[4-(2-Methyl-1H-imidazol-1-yl)-2-butynyl]isox-
azolidin-3-one (7): pale yellow viscous oil, Rf, 0.30
1
(eluant: 5% methanol/dichloromethane); H NMR:
2.40 (s, 3H, CH3), 2.75 (t, 2H, H-4, Jꢁ
(t, 2H, NÃCH2ÃCÅ, Jꢁ1.8 Hz), 4.34 (t, 2H, H-5,
Jꢁ8.1 Hz), 4.61 (t, 2H, CH2ꢀImid., Jꢁ1.8 Hz),
6.88 (s, 1H, H-4?), 6.91 (s, 1H, H-5?).
C) To a solution of fumaric acid (0.620 g, 5.34 mmol)
in 15 ml of methanolꢀ2-propanol (3:7) was added 7
/
8.1 Hz), 4.28
/
/
/
/
/
/
/
/
the theoretical value within 90.4%.
/
(0.390 g, 1.78 mmol). The corresponding salt pre-
cipitated slowly on standing and was purified by
crystallization.
4.1.1. 2-[4-(2-Methyl-1H-imidazol-1-yl)-2-
butynyl]isoxazolidin-3-ones (7a and 7b)
2-[4-(2-Methyl-1H-imidazol-1-yl)-2-butynyl]isox-
A) To a solution of 13 [17] (1.80 g, 20.67 mmol) in
acetone (30 ml) was added potassium carbonate
(5.72 g, 41.38 mmol). The suspension was heated at
reflux for 1 h and then cooled at room temperature.
1,4-Dichloro-2-butyne (12.13 ml, 0.124 mol) was
then added and, after heating at reflux for 24 h, the
crude reaction mixture was poured into water (100
azolidin-3-one sesquifumarate (7a): m.p., 112.5ꢀ
/
115 8C dec. (colorless prisms from absolute etha-
nol); 1H NMR (CD3OD): 2.63 (s, 3H, CH3), 2.81 (t,
2H, H-4, Jꢁ
4.42 (t, 2H, NÃ
CH2ꢀImid., Jꢁ
(d, 1H, H-4?, Jꢁ
Hz). Anal. C17H19N3O8 (C, H, N).
/
8.3 Hz), 4.40 (t, 2H, H-5, Jꢁ
CH2ÃCÅ, Jꢁ1.7 Hz), 5.12 (t, 2H,
1.7 Hz), 6.77 (s, 2H, HCÄCH), 7.37
2.1 Hz), 7.52 (d, 1H, H-5?, Jꢁ2.1
/
8.3 Hz),
/
/
/
/
/
/
/
/
/
ml) and extracted with dichloromethane (3ꢂ50 ml).
/
The pooled organic extracts were dried over anhy-
drous sodium sulfate and concentrated at reduced
pressure. The dark brown residue was chromato-
graphed on silica gel (eluant: 40% ethyl acetate/
petroleum ether) to give regioisomers 14 (1.42 g,
39.5% yield) and 15 (0.16 g, 4.5% yield).
D) A solution of 7 (0.250 g, 1.14 mmol) in 2-propanol
(5 ml) was treated with a fivefold excess of methyl
iodide at room temperature. The corresponding salt
precipitated upon addition of anhydrous ether.
2-[4-(2,3-Dimethyl-1H-imidazolium-1-yl)-2-buty-
nyl]isoxazolidin-3-one iodide (7b): m.p., 140.5ꢀ
/
2-(4-Chloro-2-butynyl)isoxazolidin-3-one
(14):
142 8C (colorless prisms from 2-propanol); 1H
NMR (D2O): 2.64 (s, 3H, CH3), 2.92 (t, 2H, H-4,
m.p., 73.5ꢀ74.5 8C (pale yellow prisms from 10%
/
ethyl acetate/petroleum ether), Rf, 0.19 (eluant: 30%
1
ethyl acetate/cyclohexane); H NMR: 2.78 (t, 2H,
Jꢁ
CH2ꢃ
Hz), 5.04 (t, 2H, CH2ꢀ
/
8.5 Hz), 3.79 (s, 3H, NÃ
CÅ, Jꢁ1.9 Hz), 4.47 (t, 2H, H-5, Jꢁ
Imid., Jꢁ1.9 Hz), 7.35 (d,
/
Me), 4.43 (t, 2H, NÃ
/
/
/
/
/
8.5
H-4, Jꢁ
/
8.1 Hz), 4.14 (t, 2H, CH2Cl, Jꢁ
/1.8 Hz),
/
/

M. De Amici et al. / Il Farmaco 58 (2003) 739ꢀ
/748
745
1H, H-4?, Jꢁ
Hz). Anal. C12H16IN3O2 (C, H, N).
/
2.3 Hz), 7.48 (d, 1H, H-5?, Jꢁ
/
2.3
D) A solution of 8 (0.410 g, 1.87 mmol) in 2-propanol
(10 ml) was treated with a fivefold excess of methyl
iodide at room temperature. The corresponding salt
precipitated upon addition of anhydrous ether.
3-[4-(2,3-Dimethyl-1H-imidazolium-1-yl)-2-buty-
4.1.2. 3-[4-(2-Methyl-1H-imidazol-1-yl)-2-
butynyl]oxy-D2-isoxazolines (8a and 8b)
nyl]oxy-D2-isoxazoline iodide (8b): m.p., 132.5ꢀ
/
A) To a solution of 16 [17] (2.0 g, 12.89 mmol) and
triethylamine (3.6 ml, 25.78 mmol) in dichloro-
methane (50 ml), a solution of methanesulfonyl
chloride (2.50 ml, 25.78 mmol) in dichloromethane
(15 ml) was added dropwise at 0 8C. After stirring
for 1 h at room temperature, 1 N HCl (50 ml) was
added, the phases were separated, and the aqueous
133.5 8C dec. (colorless prisms from 2-propanol);
1H NMR (D2O): 2.65 (s, 3H, CH3), 3.12 (t, 2H, H-4,
Jꢁ
Jꢁ
2H, CH2ꢀ
7.49 (d, 1H, H-5?, Jꢁ
(C, H, N).
/
9.7 Hz), 3.79 (s, 3H, NÃ
9.7 Hz), 4.86 (bs, 2H, OÃ
Imid.), 7.37 (d, 1H, H-4?, Jꢁ
2.0 Hz). Anal. C12H16IN3O2
/
Me), 4.44 (t, 2H, H-5,
CH2ÃCÅ), 5.08 (bs,
2.0 Hz),
/
/
/
/
/
/
/
layer was extracted with dichloromethane (3ꢂ30
/
ml). After the usual work up, the residue was
column chromatographed (eluant: 50% petroleum
ether/ethyl acetate) to give 2.62 g (87%) of the
desired ester.
4.1.3. 3-[4-(2-Methyl-1H-imidazol-1-yl)-2-
butynyl]oxyisoxazoles (9a, 9b, and 9c)
A) The 3-substituted isoxazole (19) was prepared in
36% yield from 3-hydroxyisoxazole (18) [24] accord-
ing to the procedure above described for the
preparation of 14 and 15, by increasing the overall
refluxing time to 48 h.
3-(4-Hydroxy-2-butynyl)oxy-D2-isoxazoline me-
sylate (17): m.p., 49ꢀ50 8C (colorless prisms from
/
30% ethyl acetate/petroleum ether); Rf, 0.43 (eluant:
1
40% cyclohexane/ethyl acetate); H NMR: 3.01 (t,
2H, H-4, Jꢁ/9.4 Hz), 3.17 (s, 3H, CH3), 4.43 (t, 2H,
3-(4-Chloro-2-butynyl)oxyisoxazole (19): color-
H-5, Jꢁ9.4 Hz), 4.83 (bs, 2H, OÃ
/
/
CH2CÅ), 4.94 (bs,
/
less liquid, b.p., 125ꢀ
(eluant: 10% ethyl acetate/cyclohexane); H NMR:
4.20 (s, 2H, CH2Cl), 4.94 (s, 2H, OÃCH2CÅ), 6.01
(d, 1H, H-4, Jꢁ1.8 Hz), 8.16 (d, 1H, H-5, Jꢁ1.8
Hz). Anal. C7H6ClNO2 (C, H, N).
/
130 8C/0.5 mmHg; Rf, 0.32
1
2H, CH2OSO2). Anal. C8H11NO5S (C, H, N).
B) To a solution of 17 (1.80 g, 7.72 mmol) in 10 ml
acetonitrile was added 2-methylimidazole (1.27 g,
15.44 mmol). After stirring at reflux for 1 h, the
solvent was removed at reduced pressure and the
residue was submitted to the same procedure above
described for analogue 7. Purification of the crude
basic extract by silica gel column chromatography
(eluant: 2% methanol/dichloromethane) afforded
1.23 g (73% yield) of the desired imidazole deriva-
tive.
/
/
/
/
B) Intermediate 19 (2.20 g, 12.82 mmol) was reacted
with 2-methylimidazole (2.11 g, 25.7 mmol) follow-
ing the protocol previously applied to 14. The
corresponding tertiary base (1.475 g, 53% yield)
was obtained in a pure form after silica gel column
chromatography (eluant: 2% methanol/dichloro-
methane).
3-[4-(2-Methyl-1H-imidazol-1-yl)-2-butynyl]oxy-
D2-isoxazoline (8): yellow viscous oil, Rf, 0.33
(eluant: 5% methanol/dichloromethane); H NMR:
3-[4-(2-Methyl-1H-imidazol-1-yl)-2-butynyl]oxyi-
soxazole (9): light yellow viscous oil, Rf, 0.38
1
(eluant: 5% methanol/dichloromethane); H NMR:
1
2.39 (s, 3H, CH3), 2.99 (t, 2H, H-4, Jꢁ
(t, 2H, H-5, Jꢁ9.6 Hz), 4.65 (t, 2H, CH2ꢀ
Jꢁ1.9 Hz), 4.78 (t, 2H, OÃCH2ÃCÅ, Jꢁ1.9 Hz),
6.91 (s, 1H, H-4?), 6.94 (s, 1H, H-5?).
C) To a solution of fumaric acid (0.763 g, 6.57 mmol)
in 25 ml of methanolꢀ2-propanol (3:7) was added 8
/
9.6 Hz), 4.40
2.39 (s, 3H, CH3), 4.67 (bs, 2H, CH2ꢀ
(bs, 2H, OÃCH2ÃCÅ), 6.0 (d, 1H, H.4, Jꢁ
6.93 (s, 1H, H-4?), 6.95 (s, 1H, H-5?), 8.13 (d, 1H, H-
5, Jꢁ2.0 Hz).
/
Imid.), 4.91
/
/
Imid.,
/
/
/
/
2.0 Hz),
/
/
/
/
/
/
C) Derivative 9 (0.450 g, 2.07 mmol) was reacted with
excess fumaric acid or excess methyl iodide accord-
ing to the procedures applied to 7 and 8.
/
(0.480 g, 2.19 mmol). The corresponding salt pre-
cipitated slowly on standing and was purified by
crystallization.
3-[4-(2-Methyl-1H-imidazol-1-yl)-2-butynyl]oxyi-
soxazole sesquifumarate (9a): m.p., 120.5ꢀ
/
122 8C
(colorless prisms from absolute ethanol); H NMR
(CD3OD): 2.58 (s, 3H, CH3), 4.98 (bs, 2H, OÃCH2Ã
CÅ), 5.08 (bs, 2H, CH2ꢀImid.), 6.18 (d, 1H, H-4,
Jꢁ2.0 Hz), 6.75 (s, 2H, HCÄCH), 7.24 (d, 1H, H-
4?, Jꢁ2.0 Hz), 7.44 (d, 1H, H-5?, Jꢁ2.0 Hz), 8.45
(d, 1H, H-5, Jꢁ2.0 Hz). Anal. C17H17N3O8 (C, H,
N).
3-[4-(2,3-Dimethyl-1H-imidazolium-1-yl)-2-buty-
nyl]oxyisoxazole iodide (9b): m.p., 137ꢀ138 8C dec.
1
3-[4-(2-Methyl-1H-imidazol-1-yl)-2-butynyl]oxy-
D2-isoxazoline sesquifumarate (8a): m.p., 121.5ꢀ
/
/
/
123 8C dec. (colorless prisms from absolute etha-
/
/
nol); 1H NMR (CD3OD): 2.63 (s, 3H, CH3), 3.05 (t,
/
/
2H, H-4, Jꢁ
4.89 (bs, 2H, OÃ
Imid.), 6.75 (s, 2H, HCÄ
Jꢁ2.0 Hz), 7.51 (d, 1H, H-5?, Jꢁ
C17H19N3O8 (C, H, N).
/
9.7 Hz), 4.40 (t, 2H, H-5, Jꢁ
CH2ÃCÅ), 5.11 (bs, 2H, CH2ꢀ
CH), 7.32 (d, 1H, H-4?,
2.0 Hz). Anal.
/
9.7 Hz),
/
/
/
/
/
/
/
/
/
/
/

746
M. De Amici et al. / Il Farmaco 58 (2003) 739ꢀ748
/
(colorless prisms from 2-propanol); 1H NMR
(CD3OD): 2.70 (s, 3H, CH3), 3.89 (s, 3H, NÃ
CH3), 5.03 (bs, 2H, OÃCH2ÃCÅ), 5.20 (bs, 2H,
CH2ꢀImid.), 6.19 (d, 1H, H-4, Jꢁ2.0 Hz), 7.56 (d,
1H, H-4?, Jꢁ2.0 Hz), 7.65 (d, 1H, H-5?, Jꢁ2.0
Hz), 8.45 (d, 1H, H-5, Jꢁ2.0 Hz). Anal.
C12H14IN3O2 (C, H, N).
pressure. After addition of 10-ml 2-N HCl and
extraction with ether (3ꢂ10 ml), the residual
aqueous phase was made alkaline (pH 10) by
/
/
/
/
/
/
/
portion-wise addition of solid K2CO3, and extracted
/
/
with ethyl acetate (3ꢂ10 ml). After the usual work
/
/
up, the oily residue was purified by silica gel column
chromatography (eluant: 5% methanol/ethyl ace-
tate) to give the desired pyrrolidine derivative (0.329
g, 43% yield).
D) A solution of 9 (0.300 g, 1.38 mmol) in 10 ml of
ether/2-propanol (4:1) was treated with 4-bromo-
benzyl bromide (0.552 g, 2.21 mmol) at room
temperature. The corresponding ammonium salt
precipitated on standing.
(9)-3-[4-(1-Pyrrolidinyl)-1-methyl-2-butynyl]-
/
oxy-D2-isoxazoline: yellow oil, Rf, 0.40 (eluant: 5%
methanol/dichloromethane); 1H NMR: 1.56 (d, 3H,
3-[4-(2-Methyl-3-(4-bromobenzyl)-1H-imidazo-
lium-1-yl)-2-butynyl]oxyisoxazole bromide (9c):
CHCH3, Jꢁ
2.59 (m, 4H, a-pyrrolidine), 2.94 (t, 2H, H-4, Jꢁ
Hz), 3.43 (s, 2H, CH2N), 4.37 (t, 2H, H-5, Jꢁ
Hz), 5.24 (q, 1H, CHCH3, Jꢁ7.2 Hz).
/
7.2 Hz), 1.77 (m, 4H, b pyrrolidine),
/9.8
m.p., 139ꢀ
/
141 8C dec. (colorless prisms from acet-
/9.8
1
oneꢀ
5.05 (t, 2H, OÃ
CH2-Imid., Jꢁ
(d, 1H, H-4, Jꢁ
Hz), 7.63 (d, 1H, H-4?, Jꢁ
Jꢁ8.6 Hz), 7.73 (d, 1H, H-5?, Jꢁ
1H, H-5, Jꢁ
N).
/
ether); H NMR (CD3OD): 2.71 (s, 3H, CH3),
CH2ÃCÅ, Jꢁ1.5 Hz), 5.22 (t, 2H,
1.5 Hz), 5.45 (s, 2H, CH2Ar), 6.19
1.9 Hz), 7.31 (d, 2H, Ar, Jꢁ8.6
2.1 Hz), 7.67 (d, 2H, Ar,
2.1 Hz), 8.44 (d,
/
/
/
/
/
C) The corresponding fumarate was prepared by
reacting the tertiary base (0.300 g, 1.35 mmol)
with a threefold excess of fumaric acid.
/
/
/
/
(9
oxy-D2-isoxazoline fumarate (10): m.p., 154ꢀ
dec. (colorless prisms from absolute ethanol); 1H
NMR (CD3OD): 1.60 (d, 3H, CHCH3, Jꢁ7.1 Hz),
2.11 (m, 4H, b-pyrrolidine), 3.03 (t, 2H, H-4, Jꢁ9.7
Hz), 3.39 (m, 4H, a-pyrrolidine), 4.13 (s, 2H,
CH2N), 4.39 (t, 2H, H-5, Jꢁ9.7 Hz), 5.24 (q, 1H,
CHCH3, Jꢁ7.1 Hz), 6.73 (s, 2H, HCÄCH). Anal.
/)-3-[4-(1-Pyrrolidinyl)-1-vmethyl-2-butynyl]-
/
/
/
156 8C
/1.9 Hz). Anal. C18H17Br2N3O2 (C, H,
/
/
4.1.4. (9
/
)-3-[4-(1-Pyrrolidinyl)-1-methyl-2-
butynyl]oxy-D2-isoxazoline (10)
/
/
/
A) To a solution of (9
/
)-3-butyn-2-ol (20) (1.80 g, 25.68
C16H22N2O6 (C, H, N).
mmol) in 25 ml anhydrous THF was added sodium
hydride (0.72 g, 30 mmol) under an inert atmo-
sphere. After stirring at room temperature for 30
min, a solution of 11 (1.0 g, 8.61 mmol), in
anhydrous THF (10 ml) was added dropwise. The
reaction mixture was heated at reflux for 18 h, then
cooled at 0 8C. After cautious addition of water (20
ml) and concentration at reduced pressure, the
residual aqueous phase was extracted with ethyl
4.2. Pharmacology
Stable cell lines expressing the hm1ꢀhm5 muscarinic
receptors were obtained via the resources of NIMH
PDSP and were tested for receptor expression by
/
radioligand binding to
zylate ([3H]QNB). Saturation experiments were per-
formed using 5ꢀ
2500 pM [3H]QNB in 10 mM
potassium phosphate buffer, pH 7.4, to determine the
Kd of [3H]QNB for each receptor subtype. Competition
binding experiments were performed using 180 pM
[3H]QNB and 10ꢀ
50 mg of membrane protein. All
binding experiments were performed in the presence of
1% DMSO. Non-specific binding was defined by 0.5 mM
atropine. After equilibrium was reached (120 min
incubation at 25 8C), bound and free radioactivity
were separated by filtration using Whatman GF-C
filters. Ki values were derived from IC50 values using
L
-quinuclidinyl[phenyl-4-3H]ben-
acetate (4ꢂ30 ml). After the usual work up, the
/
/
residue was purified on a silica gel column (eluant:
30% ethyl acetate/petroleum ether) to give 0.540 g
(45%) of the intermediate alkyne.
(9
(21): colorless oil, b.p., 85ꢀ
0.23 (eluant: 20% ethyl acetate/cyclohexane); 1H
NMR: 1.61 (d, 3H, CHCH3, Jꢁ7.2 Hz), 2.56 (d,
1H, HÃCÅC, Jꢁ1.9 Hz), 2.98 (t, 2H, H-4, Jꢁ9.8
Hz), 4.40 (t, 2H, H-5, Jꢁ9.8 Hz), 5.25 (dq, 1H,
CHCH3, Jꢁ7.2 and 1.9 Hz). Anal. C7H9NO2 (C,
H, N).
B) A solution of alkyne (9
/
)-3-(1-Methyl-2-propynyl)oxy-D2-isoxazoline
/
/
90 8C/0.3 mmHg; Rf,
/
/
/
/
/
/
/
Chengꢀ
Male guinea pigs (250ꢀ
rabbits (3.0ꢀ3.5 kg; Morini, S. Polo, Italy) were used.
/Prusoff equation [31].
/)-21 (0.478 g, 3.44 mmol) in
/
350 g) and New Zealand white
4 ml of dioxane was added to a stirred suspension of
pyrrolidine (334 ml, 4.0 mmol), cuprous chloride (26
mg, 0.26 mmol), glacial acetic acid (0.1 ml), and
paraformaldehyde (0.106 g, 3.53 mmol) in dioxane
(5 ml). The reaction mixture was stirred at 50 8C for
18 h, then the solvent was removed at reduced
/
The tissues for in vitro experiments were removed from
animals fasted 24 h before the experiments and killed by
CO2 inhalation. Isolated preparations were set up
following the techniques previously described [17].

M. De Amici et al. / Il Farmaco 58 (2003) 739ꢀ
/748
747
4.2.1. Electrically stimulated rabbit vas deferens
curve to the full agonists, Bethanechol or McN-A-343
was repeated after 30 min incubation with the test
According to Eltze [32], the prostatic portion of each
vas deferens was mounted in a 10-ml organ bath,
containing a modified Krebs solution (mM composi-
tion: NaCl, 134; KCl, 3.4; CaCl2, 2.8; KH2PO4, 1.3;
NaHCO3, 16; MgSO4, 0.6; glucose, 7.7) kept at 31 8C
compounds (10 nMꢀ10 mM). The potency of the
/
compounds acting as antagonists was expressed as
pKB value, the calculated molar concentration of the
test compounds that causes a twofold increase in the
EC50 values of the muscarinic agonists used in the
functional tests, calculated according to Furchgott’s
and bubbled with 95% O2ꢀ5% CO2. Yohimbine
/
(1.0 mM) was present throughout the experiment to
prevent prejunctional a2-adrenoceptors stimulation. For
isometric recordings, the tissues were left to equilibrate
for 4 min under a passive load of 0.75 g before electric
field stimulation through platinum electrodes was ap-
plied by square-wave pulses (0.5 ms, 0.05 Hz, supra-
maximal intensity 450 mA; LACE Elettronica Mod.
ES3, Ospedaletto PI, Italy).
method [35]. All data are expressed as means of 6ꢀ8
/
experiments.
Acknowledgements
This research has been supported by a grant (COFIN
1999) from Ministero dell’Istruzione, dell’Universita` e
della Ricerca (MIUR, Rome). The authors wish to
thank Dr. Giuseppe Domenichini (University of Parma)
for his skilful technical assistance. Additional support
from the NIMH PDSP and KO2MH01366 to BLR is
acknowledged.
4.2.2. Electrically stimulated guinea pig left atrium
The left atria were mounted in 20 ml organ baths
under 0.5 g tension at 33 8C, immersed in a modified
Krebs-Henseleit solution (mM composition: NaCl,
118.9; KCl, 4.6; CaCl2, 2.5; KH2PO4, 1.2; NaHCO3,
25; MgSO4×
/
7H2O, 1.2; glucose, 11.1), gassed with a 95%
O2ꢀ5% CO2 mixture. After a period of stabilization of
/
45 min, tissues were electrically stimulated through
platinum electrodes by square-wave submaximal pulses
(2 Hz, 5 ms, 5 V), and inotropic activity was recorded
isometrically.
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/
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/
/
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/
agonists were reconstructed in the presence of atropine
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/response
psychotic-like activity: structureꢀactivity relationships of 1,2,5-
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/
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