Highly chiral muscarinic ligands: The discovery of (2S,2′R,3′S, 5′R)-1-methyl-2-(2-methyl-1,3-oxathiolan-5-yl)pyrrolidine 3-sulfoxide methyl iodide, a potent, functionally selective, M2 partial agonist

Article abstract of DOI:10.1021/jm0510878

By further steric complication of previously studied highly chiral muscarinic agonists, we have obtained a small chiral library of enantiomeric and diasteromeric 1-methyl-2-(2-methyl-1,3-oxathiolan-5-yl)pyrrolidine 3-sulfoxides. Binding studies on cloned

Full text of DOI:10.1021/jm0510878

J. Med. Chem. 2006, 49, 1925-1931
1925
Highly Chiral Muscarinic Ligands: The Discovery of
(2S,2′R,3′S,5′R)-1-Methyl-2-(2-methyl-1,3-oxathiolan-5-yl)pyrrolidine 3-sulfoxide Methyl Iodide,
a Potent, Functionally Selective, M2 Partial Agonist
Serena Scapecchi,*,† Rosanna Matucci,§ Cristina Bellucci,† Michela Buccioni,‡ Silvia Dei,† Luca Guandalini,† Cecilia Martelli,†
Dina Manetti,† Elisabetta Martini,† Gabriella Marucci,‡ Marta Nesi,§ Maria Novella Romanelli,† Elisabetta Teodori,† and
Fulvio Gualtieri†
Dipartimento di Scienze Farmaceutiche, UniVersita` di Firenze, Via Ugo Schiff 6, 50019 Sesto Fiorentino, Italy,
Dipartimento di Farmacologia Preclinica e Clinica, UniVersita` di Firenze, Viale Pieraccini 6, 50139 Firenze, Italy,
and Dipartimento di Chimica, UniVersita` di Camerino, Via S.Agostino 1, 62032 Camerino (MC), Italy
ReceiVed October 26, 2005
By further steric complication of previously studied highly chiral muscarinic agonists, we have obtained a
small chiral library of enantiomeric and diasteromeric 1-methyl-2-(2-methyl-1,3-oxathiolan-5-yl)pyrrolidine
3-sulfoxides. Binding studies on cloned human muscarinic receptors expressed in CHO cells show that the
introduction of a fourth stereogenic center gives undetectable affinity for hm1, hm3, hm4 and hm5 subtypes
while leaving a quite modest affinity only for hm2 subtypes. However, functional studies on model M1-M4
muscarinic tissues have shown that three compounds of the series [(-)-5, (-)-7, (+)-8] are endowed with
functional activity and behave as M2 selective partial agonists. Among them, compound (2S,2′R,3′S,5′R)-
1-methyl-2-(2-methyl-1,3-oxathiolan-5-yl)pyrrolidine 3-sulfoxide methyl iodide [(+)-8] is particularly
interesting, as it is a potent partial agonist on guinea pig atrium (force) (M2; pD2 ) 7.65, R ) 0.41) while
being a poor antagonist on M1, M3, and M4 model tissues (pKb < 5).
Chart 1
Introduction
Cholinergic compounds have been intensively studied over
the past years to find agents to treat cholinergic receptor
dysfunctions and to identify molecules useful to characterize
muscarinic1 and nicotinic2 receptor subtypes. A few compounds
of this class have found use as medicines, while many others
have been used as pharmacological tools for cholinergic receptor
characterization. However, while antagonists have been im-
mensely useful for both purposes, agonists have hardly found
any use in receptor subtypes classification, in particular as
regards muscarinic receptors.3 Thus, new agonists, selective for
one of the several muscarinic receptor subtypes, would be
extremely useful not only to further characterize the receptors
but also for their therapeutic potential in pathological states such
as pain,4 schizophrenia,5 and neurodegenerative pathologies such
as Alzheimer’s disease.6,7 For several years we have been
working on cholinergic agonists characterized by a pentatomic
cycle, such as that of 1,3-oxathiolanes (compounds A and B,
Chart 1),8 and recently we have reported the synthesis and the
pharmacological profile of a new series of 1,3-oxathiolane
derivatives (compounds C, Chart 1)9 whose structure had been
sterically complicated, with respect to the parent compound (+)-
(R,R)-2-methyl-5-[(dimethylamino)methyl]-1,3-oxathiolane meth-
yl iodide, by introducing a third stereogenic center. We reasoned
that exalting the molecular complexity of A, through stereo-
chemical complication in the proximity of the critical cationic
head of the molecule, would result in agonists able to detect
the subtle structural differences between muscarinic receptor
subtypes, whose recognition sites are highly conserved.10,11
Indeed, binding studies on the five cloned human muscarinic
receptors have shown that these compounds (C, Chart 1) possess
some selectivity toward hm2 subtypes, a property confirmed in
functional assays, where they show clear-cut functional selectiv-
ity on guinea pig atrium.12
Building on this hypothesis, we have now designed and
synthesized, by oxidation of the sulfur atom of these 1,3-
oxathiolanes derivatives, the corresponding sulfoxides 1-8
reported in Chart 1. The new compounds present a fourth
stereogenic center, and we anticipated that further increase of
stereochemical complexity of our molecules would improve
further M2 selectivity.
Chemistry. The starting material for the synthesis of sul-
foxides 1-8 were oxathiolanes 18-21 that, in turn, were
obtained from commercially available (R)- and (S)-prolinol, as
previously reported.9
However, in the present work, we have definitely improved
the synthetic procedure by isolating and characterizing all
intermediates and obtaining all possible isomers of 18-21, as
described in Scheme 1 for the (R)-prolinol series. The same
holds true for the series of compounds derived from (S)-prolinol.
The yield of (R)-2-vinylpyrrolidine-1-carboxylic acid benzyl
ester (9) was increased from 47 to 85% by using the base KN-
* Corresponding author. Tel: ++39 55 4573692. Fax: ++39 55
4573671. E-mail serena.scapecchi@unifi.it.
† Dipartimento di Scienze Farmaceutiche, Universita` di Firenze.
‡ Dipartimento di Chimica, Universita` di Camerino.
§ Dipartimento di Farmacologia Preclinica e Clinica, Universita` di
Firenze.
10.1021/jm0510878 CCC: $33.50 © 2006 American Chemical Society
Published on Web 02/18/2006

1926 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 6
Scapecchi et al.
Scheme 1a
Scheme 2a
a (a) H2O2, CH3COOH, chromatographic separation; (b) CH3I. For the
synthesis of the compounds of the 2S-series the same procedure was
followed
Figure 1. Thermal ellipsoid plot (30% ellipsoids) of compound (-)-
5.
a (a) KN(TMS)2, Ph3PCH3Br; (b) m-CPBA, chromatographic separation;
(c) CH3COSH; (d) HCl, MeOH; (e) p-toluensulfonic acid, CH3CH(OMe)2;
(f) LiAlH4, chromatographic separation. For the synthesis of the compounds
of the S-series, starting from (2S)-9, the same procedure was followed.
CH3I. As expected, the same reaction sequence carried out on
the (2S)-derivatives yielded comparable results.
The absolute configuration of 1,3-oxathiolane 3-sulfoxide
compounds was established on the basis of the X-ray crystal-
lography of the methiodide (-)-5 (whose crystallographic
structure is shown in Figure 1) and of the 1D and 2D 1H NMR
spectra, exploiting the fact that all cis-sulfoxides (where cis and
trans is referred to the relationship with the substituent on C5′)
show fairly similar spectra, as do trans-sulfoxides. NMR
experiments were performed on the tertiary amines, but their
results can obviously be extended to the corresponding methio-
dide.
Pharmacology. Muscarinic receptor affinity was evaluated
in CHO cells expressing the five human muscarinic subtypes
(hm1-hm5). Functional activity was evaluated in vitro on
classical preparations, following previously reported methods:
15,16 rabbit stimulated vas deferens (M1), guinea pig stimulated
left atria (M2), guinea pig ileum (M3), and guinea pig lung strips
(M4). In this respect, it is appropriate to mention that, for a
long time, the contraction of rabbit vas deferens was considered
as an effect mediated by M1-receptor subtypes, whereas more
recent studies attribute the same effect to an M4-activation.17,18
Therefore, since the pharmacological characterization of the
receptor subtype involved does not appear to be clearly
established, in this work the rabbit vas deferens will be
considered a putative M1 subtype. Carbachol, arecaidine pro-
pargyl ester (APE), and 4-Cl-McN-A-343 were used as reference
compounds. Results are expressed as pKi values (affinity), as
pD2 (pED50; agonist potency), or as pKb (antagonist affinity)
and are reported in Table 1. The affinity constants (Ka) of (-)-
5, (-)-7, (+)-8, which showed functional activity as agonists,
were evaluated using the method of Waud for partial agonists,19
(TMS)2 in the Wittig reaction.13 Epoxides 10 and 11 were
obtained by reaction with m-CPBA as previously described,9
but now the two diastereoisomers (2R,2′R)-10 and (2R,2′S)-11
were isolated by flash chromatographic separation on silica gel
and characterized; their absolute configuration at C2′ was
attributed on the basis of the C2′ configuration9 of the
compounds 18-21 obtained from each isomer. In this respect,
according to previous reports,14 we found that each epoxide was
a mixture of rotamers (1H and 13C NMR evidence).
The opening of epoxides 10 and 11 with thioacetic acid and
subsequent hydrolysis of the thioesters under acidic conditions,
gave (2R,1′R)-12 and (2R,1′S)-13, which could thus be com-
pletely characterized. Cyclization of each mercapto alcohol (12,
13) afforded a diastereomeric mixture of cis and trans isomerss
14 and 15 from 12, 16 and 17 from 13sthat were not separated
but reduced as such with LiAlH4 to give compounds 18-21.
Column chromatography on Al2O3 allowed separation of the
two mixtures to obtain two cis isomers, 18 and 20, and two
trans isomers, 19 and 21, in a 5:1 ratio (NMR). The inefficient
chromatographic separation that in our hands resisted any
improvement, allowed isolation of only a small amount of trans
isomers. As a consequence, the following sulfoxidation reaction
was performed only on the cis isomers 18 and 20 as shown in
Scheme 2 for the 2R series. Each isomer was oxidized with
H2O2 to give the expected mixture of diastereomeric sulfoxides
1-4 that were separated by column chromatography and then
transformed into the corresponding methyl iodides 5-8 with

Highly Chiral Muscarinic Ligands
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 6 1927
Table 1. Binding Affinity and Functional Selectivity of Compounds 1-8
functional activityb
guinea pig
binding affinitya
rabbit
pKi(SEM)
atrium (force) (M2)
ileum (M3)
lung (M4)
vas deferens
compd
hm1
hm2
<4
<4
<4
<4
hm3
hm4
<4
hm5
(M1)c pD2 [pKb]
R
pD2 [pKb]
R pD2 [pKb] pD2 [pKb]
(-)-(2R,2′R,3′R,5′R)-1 <4
(+)-(2R,2′R,3′S,5′R)-2 <4
(+)-(2R,2′S,3′S,5′S)-3 <4
(+)-(2R,2′S,3′R,5′S)-4 <4
(-)-(2R,2′R,3′R,5′R)-5 <4
(+)-(2R,2′R,3′S,5′R)-6 <4
(+)-(2R,2′S,3′S,5′S)-7 <4
(-)-(2R,2′S,3′R,5′S)-8 <4
(+)-(2S,2′S,3′S,5′S)-1 <4
(-)-(2S,2′S,3′R,5′S)-2 <4
(-)-(2S,2′R,3′R,5′R)-3 <4
(-)-(2S,2′R,3′S,5′R)-4 <4
(+)-(2S,2′S,3′S,5′S)-5 <4
(-)-(2S,2′S,3′R,5′S)-6 <4
<4
<4
-
-
-
-
[<5]
-
[<5]
[<5]
[<5]
-
-
-
-
-
-
-
-
-
-
-
-
-
1
-
-
-
<4
<4
<4
<4
<4
<4
<4
<4
<4
<4
<4
<4
<4
<4
<4
<4
<4
-
<4
-
-
<4
-
-
5.03(0.14) <4
<4 <4
4.09(0.11) <4
<4 <4
4.06(0.13) <4
<4
0.58(0.03) 6.11(0.15)
5.58(0.08)
-
[<5]
-
<4
-
-
-
4.24(0.13)
<4
0
0
0
-
[<5]
[<5]
[<5]
-
0
[<5]
[<5]
[<5]
-
[<5]
[<5]
[<5]
[<5]
[<5]
[<5]
[<5]
-
[<5]
[<5]
[<5]
[<5]
[<5]
[<5]
0
0
-
<4
<4
<4
<4
<4
<4
<4
[<5]
[<5]
[<5]
[<5]
[<5]
[<5]
-
0
0
0
0
[<5]
[<5]
[<5]
[<5]
0
0
0
0
4.05(0.12) <4
4.11(0.16) <4
4.54(0.17) <4
<4
<4
<4
(-)-(2S,2′R,3′R,5′R)-7 4.03(0.08) 4.81(0.11) 4.47(0.06) 4.33(0.06) 4.47(0.13)
0.56(0.06) 6.37(0.10) 0.54(0.05) 4.62(0.15)
(+)-(2S,2′R,3′S,5′R)-8 <4
carbachol
APE
4.29(0.12) <4
<4
<4
0.41(0.03) 7.65(0.15)
0
[<5]
4.42(0.09) 5.91(0.06) 4.36(0.10) 5.20(0.07) 4.16(0.07)
5.91(0.05) 7.06(0.09) 6.07(0.04) 6.01(0.05) 6.03(0.04)
5.71(0.09) 5.49(0.07) 5.62(0.09) 5.04(0.07) 5.42(0.06)
1
1
-
7.33(0.08)
8.67(0.04)
-
1
1
-
6.68(0.01) 5.43d(0.03)
7.64(0.09) 5.56d(0.07)
7.14d(0.18)
6.33d(0.06)
MCN-A-343
-
-
a Binding affinity on cloned human muscarinic receptors expressed in CHO cells. The values represent the mean (SEM) of at least three experiments.
b Evaluated as reported in the experimental part and expressed as pD2 (-log ED50) for agonism and as pKb (-log Kb) for antagonism; for agonists, R
represents intrinsic activity. c The presence of the M1 subtype in rabbit vas deferens has been questioned in favor of the M4 subtype; this attribution must
then be considered putative. d The intrinsic activity of the compounds is 1.
Table 2. Potency and Affinity of Compounds 5, 7, and 8
guinea pig atrium (force)a (M2)
guinea pig ileum (M3)
Rb
pD2 ( SEM
pKa ( SEM
Ka/ED50
Rb
pD2 [pKb] ( SEM
pKa ( SEM
Ka/ED50
c
c
compd
(-)-5
(-)-7
(+)-8
0.58 ( 0.03
0.56 ( 0.06
0.41 ( 0.03
0.40 ( 0.03e
1
6.11 ( 0.15
6.37 ( 0.10
7.65 ( 0.15
6.66 ( 0.05
7.33 ( 0.08
5.11 ( 0.18
4.64 ( 0.18
6.13 ( 0.20
5.22 ( 0.08
6.02 ( 0.18
12
58
84
28
9
1.00 ( 0.08
5.58 ( 0.08
4.62 ( 0.15
[<5]
6.95 ( 0.05
6.68 ( 0.01
4.87 ( 0.20
4.59 ( 0.16
-
5.38 ( 0.05
4.79 ( 0.03
7
1
-
37
51
0.54 ( 0.05
0
(-)-(2R,3R,5R)-Bd
1.20 ( 0.09e
carbachol
1
a The compounds do not show any effect on frequency. b R represents Ariens’s intrinsic activity of the agonist. c Ka/ED50 represents an estimate of Furchgott’s
efficacy of the agonist. d See ref 36. e Relative efficacy (see ref 37).
and the results are reported in Table 2, together with that of
carbachol, evaluated with the method of Furchgott20 for full
agonists and of the parent compound (-)-(2R,3R,5R)-B.
for functional assays, are used for binding studies. The agonist-
induced receptor trafficking concept proposed by Kenakin24
seems to be one of the most convincing. Here, different agonists
are considered able to stabilize different conformations of the
same receptor, which have the ability to selectively activate one
G protein over another. Moreover, tissue variation in receptor
density and signaling efficiencies may influence the expression
of selective agonism over and above the inherent affinity
differences; as a consequence, receptor agonists may exhibit
“functional” rather than absolute subtype selectivity.25 Another
finding that can be at the origin of such discrepancies has
recently been individuated in the presence in tissues of receptor
homo-oligomers and/or hetero-oligomers that may possess
different affinity for some ligands with respect to the monomers
mainly present in binding experiments.26,27
Results and Discussion
It is immediately apparent from the binding data, in the left
part of Table 1 that sulfoxides 1-8 possess undetectable affinity
for cloned human muscarinic subtypes. Compared to the parent
1,3-oxathiolanes C (Chart 1),12 sulfoxides show a sharp drop
in affinity: their pKis are below 4 and only in the case of the
hm2 subtype do a few of them showed a modest affinity, i.e.,
the case of (-)-5, (-)-6, (-)-7, (+)-8 (pKi ) 5.03, 4.54, 4.81,
4.29, respectively). This was a frustrating result indeed, but we
knew, from the literature and from our previous research,12 that
binding affinities for cloned receptor subtypes do not always
accurately predict the functional behavior of muscarinic ligands.
As a matter of fact, it is well-documented that partial
muscarinic agonists, such as talsaclidine and sabcomeline,21,22
do not show subtype selectivity in binding studies, while they
do show M1 selectivity in functional assays. In particular, it
has been reported that talsaclidine shows only minor differences
in affinity among hm1-hm5 receptor subtypes, while in
functional assays it is more potent and efficacious in cells
expressing M1 receptors, slightly less for M3, and inactive for
M2.22 There are several factors that might explain the discrep-
ancies between binding and functional studies,23 in particular
when cloned human receptors expressed in CHO cells, which
represent an artificial system loosely related to the tissues used
Therefore, we tested some selected compounds on functional
models of muscarinic receptors, namely M1-M4 subtypes; the
results are reported in the right side of Table 1. It can be seen
that, in general, the poor affinity of sulfoxides for muscarinic
receptors is confirmed and the compounds are inactive as
agonists while being, in several cases, poor antagonists (pKb <
5). However, three out of four compounds that showed some
affinity for the hm2 subtype were endowed with functional
activity [(-)-5, (-)-7, (+)-8]; the fourth compound [(-)-6] did
not confirm its modest hm2 selectivity and lacks any functional
activity on M1-M4 subtypes. Compound (-)-5, which in the
binding test appeared to be the most promising in terms of
affinity and selectivity, is, in functional assays, a partial agonist

1928 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 6
Scapecchi et al.
with modest selectivity for M2 versus M3 receptors (pD2 ) 6.11
and 5.58, respectively) where it behaves as a full agonist. On
the contrary, compound (-)-7, which showed some affinity for
all five cloned receptors resulted to be fairly selective, as it is
a poor antagonist on M1 and M4 subtypes and is an agonist 2
orders of magnitude more potent on M2 with respect to M3
receptors (pD2 ) 6.37 and 4.62, respectively). Compound (+)-8
emerged as the most interesting and intriguing one; indeed, even
if in binding assays it showed a modest affinity for the hm2
subtype, much to our surprise, in functional assays compound
(+)-8 resulted to be a fairly potent partial agonist on the M2
subtype (pD2 ) 7.65) while being a poor antagonist on the other
subtypes.
Experimental Section
Chemistry. All melting points were taken on a Bu¨chi apparatus
and are uncorrected. NMR spectra were recorded on a Gemini 200
spectrometer (200 MHz for H NMR, 50.3 MHz for 13C), and on
1
1
Brucker Avance 400 spectrometer (400 MHz for H NMR, 100
MHz for 13C). Chromatographic separations were performed on a
silica gel column by gravity chromatography (Kieselgel 40, 0.063-
0.200 mm; Merck) or flash chromatography (Kieselgel 40, 0.040-
0.063 mm; Merck). When necessary, chromatographic separations
were performed on an Al2O3 column by gravity chromatography
(aluminum oxide 90 standardized, Merck). Yields are given after
purification, unless differently stated. Where analyses are indicated
by symbols, the analytical results are within (0.4% of the
theoretical values. Optical rotation was measured at a concentration
of 1 g/100 mL (c ) 1), unless otherwise stated, with a Perkin-
Elmer polarimeter (accuracy (0.002°). When reactions were
performed under anhydrous conditions, the mixtures were main-
tained under nitrogen. Compounds were named following IUPAC
rules as applied by Beilstein-Institute AutoNom (version 2.1)
software for systematic names in organic chemistry.
Trying to find an explanation for these results, we have
evaluated the affinity constants of these three compounds using
the method of Waud for partial agonists;19 the results are
reported in Table 2.
It can be seen that, in the case of compounds (-)-5 and (-)-
7, affinities evaluated in functional assays are in agreement with
those obtained in binding studies. This suggests that differences
between binding and functional data can be explained on the
basis of efficacy and, from molecular point of view, by one of
the mechanisms discussed above.
(R)-2-Vinylpyrrolidine-1-carboxylic Acid Benzyl Ester (R)-
9. Potassium bis(trimethylsilyl)amide (1.01 g, 5.06 mmol) was
added to a solution of methyltriphenylphosphonium bromide (1.81
g, 5.06 mmol) in 30 mL of anhydrous THF at room temperature
and the mixture was stirred at room temperature for 1 h and at
-78 °C for 10 min. A solution of (R)-phenylmethyl 2-formylpyr-
rolidine-1-carboxylate (0.59 g, 2.53 mmol)9 in THF (5 mL) was
then added and the whole was stirred at -78 °C for 10 min and
for 2 h at room temperature. Then methanol (9 mL) and aqueous,
saturated Rochelle salt were added, and stirring was continued at
room temperature for 30 min. The organic compounds were
extracted with ether and the organic phase was washed with brine,
dried over Na2SO4, filtered, and evaporated under reduced pressure.
Then Ph3PO was eliminated with hexane and the desired alkene
obtained in 85% yield.9
This is not the case for compound (+)-8 that, being inactive
on the other subtypes, shows an affinity on guinea pig atrium
(force) that is some 2 orders of magnitude larger than that found
in binding studies (hm2, pKi ) 4.29; M2, pKa ) 6.13). At the
moment, we do not have an explanation for this fact, and more
studies, which are under way, will be necessary to understand
the situation. However, one can speculate that this difference
could be related to the presence in the guinea pig atrium tissue
of oligomeric receptors showing for compound (+)-8 an affinity
different from that of the monomer usually present in binding
experiments. Whatever the case, compound (+)-8 is a new,
potent, partial agonist showing remarkable functional selectivity
for M2 muscarinic receptor subtypes that may represent a useful
pharmacological tool.
The same procedure was applied to the synthesis of the (S)-
enantiomer (S)-9 that was obtained in 85% yield.
(2R,2′R)-Phenylmethyl 2-Oxiran-2-ylpyrrolidine-1-carboxy-
late (2R,2′R)-10 and (2R,2′S)-Phenylmethyl 2-Oxiran-2-ylpyr-
rolidine-1-carboxylate (2R,2′S)-11. To a solution of 0.5 g (2.16
mmol) of olefin (R)-9 in dry CH2Cl2 at 0 °C was added a solution
of 0.75 g (4.32 mmol) of m-chloroperbenzoic acid in dry CH2Cl2.
The mixture was stirred for 20 h at room temperature, ethyl acetate
was added, and the solution washed consecutively with saturated
NaHCO3, 10% NaHSO3, saturated NaHCO3, water, and brine. The
organic phase was then dried over Na2SO4, filtered, and evaporated
to dryness, yielding 0.46 g of product as a mixture of two
diastereoisomers. Flash chromatography (hexane:ether 5:2) afforded
0.2 g (0.81 mmol, 38% yield) of (2R,2′R)-10 and 0.11 g (0.45
mmol, 21% yield) of (2R,2′S)-11.
Any attempt to relate stereochemistry to binding and func-
tional activity is frustrated by the lack of activity of most of
the compounds studied. More information will hopefully be
collected by an eudismic analysis, which is under way, on the
set of compounds described in this and in previous papers on
the subject9,12,28-30 (shown in Chart 1). Only a few qualitative
considerations can be made. Two of the most affinitive com-
pounds on hm2 receptors [(-)-5 and (-)-7], as far as the 1,3-
oxathiolane moiety is concerned, show the same stereochemistry
(2′R,3′R,5′R) of the most potent compounds of the 1,3-
oxathiolane (2R,5R)30 and 1,3-oxathiolane 3-oxide (2R,3R,5R)29
series (A and B, Chart 1). These two compounds are also among
the few showing functional activity on the M2 receptor model.
The situation appears to be more complicated when the
comparison is made with the 1,3-oxathiolanylpyrrolidines that
we have recently described (C, Chart 1).12 In this case, even if
the most affinitive compounds for the hm2 subtype belong to
the 2′R,5′R series, compounds with fairly good affinity were
found also in the 2′S,5′S series. The position of compound (+)-8
is, once again, peculiar, since the absolute stereochemistry of
its 1,3-oxathiolane ring is 2′R,3′S,5′R. It is hoped that eudismic
analysis28 will cast some light on the whole matter. At the
moment, it can be said that the introduction of more sterogenic
centers into 1,3-oxathiolanes disturbs the interaction with
muscarinic receptor subtypes, resulting in hm2 subtype selectiv-
ity, but the centers complicate the interpretation of the results
in terms of stereochemistry.
1
(2R,2′R)-10. H NMR (CDCl3) 400 MHz δ (ppm): 1.80-1.97
(m, 4H, CH2CH2), 2.51 and 2.81 (2 bs, 1H, CHHO), 2.67 and 2.86
(2 bs, 1H, CHHO), 2.91 and 3.05 (2 bs, 1H, CHO), 3.41-3.57 (m,
2H, CH2N), 3.61 and 3.74 (2 bs, 1H, CHN), 5.08-5.21 (m, 2H,
13
CH2Ph), 7.30-7.41 (m, 5H, aromatics). C NMR (CDCl3) 100
MHz δ (ppm): 23.20 and 24.09 (CH2), 27.32 and 28.65 (CH2),
46.78 and 47.21 (CH2-N), 47.45 and 47.63 (CH2O), 52.54 and
52.95 (CHO), 58.52 and 59.12 (CHN), 66.78 and 67.17 (CH2Ph),
127.82, 127.97, 128.18, 128.25, and 128.51 (CH aromatics), 136.53
and 136.81 (C aromatics), 155.22 and 155.38 (CdO). [R]20
)
D
+15.8 (c 1.0, CHCl3).
1
(2R,2′S)-11. H NMR (CDCl3) 400 MHz δ (ppm): 1.80-2.08
(m, 4H, CH2CH2), 2.42-2.53 (m, 1H, CHHO), 2.59-2.72 (m, 1H,
CHHO), 2.97-3.10 (m, 1H, CHO), 3.36-3.54 (m, 2H, CH2N),
4.12 and 4.24 (2 bs, 1H, CHN), 5.08-5.23 (m, 2H, CH2Ph), 7.26-
13
7.42 (m, 5H, aromatics). C NMR (CDCl3) 100 MHz δ (ppm):
23.52 and 24.31 (CH2), 28.40 and 28.89 (CH2), 44.36 (CH2N), 46.96
and 47.21 (CH2O), 53.87 (CHO), 56.37 (CHN), 66.84 (CH2Ph),
127.82, 127.95 and 128.46 (CH aromatics), 136.82 (C aromatic),
154.65 and 155.0 (CdO). [R]20 ) +68.9 (c 1.0, CHCl3).
D

Highly Chiral Muscarinic Ligands
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 6 1929
(2S,2′S)-Phenylmethyl 2-Oxiran-2-ylpyrrolidine-1-carboxylate
(2S,2′S)-10 and (2S,2′R)-Phenylmethyl 2-Oxiran-2-ylpyrrolidine-
1-carboxylate (2S,2′R)-11. Using the same procedure described
above, starting from 4.71 g (20.4 mmol) of olefin (S)-9 and 7.03 g
(40.7 mmol) of m-chloroperbenzoic acid, 4.95 g of a mixture of
two diastereoisomers (10 and 11) was obtained. Flash chromatog-
raphy (hexane:ether 5:2) afforded 1.35 g (27% yield, 5.46 mmol)
of (2S,2′S)-10, 0.44 g (8.7% yield,1.78 mmol) of (2S,2′R)-11.
(2S,2′S)-10. 1H NMR (CDCl3) 400 MHz and 13 C NMR (CDCl3)
100 MHz spectra are identical to the spectra of the enantiomer.
(CDCl3) 200 MHz δ (ppm): 1.56 (d, 3H, CHCH3, JH-Me ) 5.6
Hz), 1.79-2.18 (m, 4H, CH2CH2), 2.75-3.14 (m, 2H, CH2S),
3.39-3.54 (m, 2H), 4.00-4.12 (m, 1.5H), 4.20-4.34 (m, 0.5H),
5.05-5.23 (m, 3H, CH2Ph + CHCH3), 7.36 (s, 5H, aromatics).
A solution of 0.79 g (2.59 mmol) of the diastereomeric mixture
(14 and 15), dissolved in the minimum amount of anhydrous THF,
was added dropwise to a suspension of 0.62 g (16.3 mmol) of
LiAlH4 in anhydrous THF at -18 °C under nitrogen. The mixture
was allowed to reach room temperature and after 4 h was treated
with brine and extracted with ethyl acetate. The organic layer was
dried over Na2SO4 and concentrated in vacuo to afford an oily
mixture of diastereoisomers (2R,2′R,5′R)-18 and (2R,2′S,5′R)-19.
The chromatographic separation on Al2O3 (eluent CHCl3/petroleum
ether 3:7) afforded the cis isomer (2R,2′R,5′R)-18 and the trans
isomer (2R,2′S,5′R)-19 in a 30:1 ratio.
(2R,2′S,5′S)-1-Methyl-2-(2-methyl-1,3-oxathiolan-5-yl)pyrro-
lidine (2R,2′S,5′S)-20 and (2R,2′R,5′S)-1-Methyl-2-(2-methyl-1,3-
oxathiolan-5-yl)pyrrolidine (2R,2′R,5′S)-21. Using the same
procedure described for (2R,2′R,5′R)-18 and (2R,2′S,5′R)-19 starting
from 1.31 g (4.66 mmol) of (2R,1′S)-13, 1.24 g (yield 87%) of a
mixture of two diastereoisomers (16 and 17) was obtained. 1H NMR
(CDCl3) 200 MHz δ (ppm): 1.55 (d, 3H, CHCH3, JH-Me ) 5.8
Hz), 1.78-2.10 (m, 4H, CH2CH2), 2.80-3.00 (m, 2H, CH2S),
3.28-3.47 (m,1H), 3.48-3.74 (m, 1H), 4.05-4.40 (m, 2H), 5.03-
5.26 (m, 3H, CH2Ph + CHCH3), 7.37 (s, 5H, aromatics).
The subsequent reduction with LiAlH4 and chromatographic
separation afforded the cis isomer (2R,2′S,5′S)-20 and the trans
isomer (2R,2′R,5′S)-21 in a 30:1 ratio. 1H NMR and 13C NMR data
of the trans isomers are reported in Table 3 (Supporting Informa-
tion).
[R]20 ) -15.8 (c 1.0, CHCl3).
D
(2S,2′R)-11. 1H NMR (CDCl3) 400 MHz and 13 C NMR (CDCl3)
100 MHz spectra are identical to the spectra of the enantiomer.
[R]20 ) -68.9 (c 1.0, CHCl3).
D
(2R,1′R)-Phenylmethyl 2-(1-Hydroxy-2-mercaptoethyl)pyr-
rolidine-1-carboxylate (2R,1′R)-12. (2R,2′R)-10 (0.75 g, 3.03
mmol) was reacted with 2 equiv of thioacetic acid for 20 h at 60
°C under nitrogen. The excess of thioacetic acid was removed under
vacuum, and the residue was dissolved in CHCl3 and washed twice
with saturated Na2CO3 and twice with water. The organic phase
was dried over Na2SO4, filtered, and evaporated to afford (2R,1′R)-
phenylmethyl 2-(2-acetyl-sulfanil-1-hydroxyethyl)pyrrolidine-
1
1-carboxylate, which was used as such for the next reaction. H
NMR (CDCl3) 200 MHz δ (ppm): 1.83-2.10 (m, 5H, CH2CH2 +
1H), 2.33 (s, 3H, COCH3), 2.62-2.83 (m, 1H), 3.10-3.22 (m, 1H),
3.29-3.43 (m, 1H), 3.55-3.71 (m, 1H), 3.77-4.11 (m, 1H), 4.14-
4.25 (m, 0.5H), 4.33-4.43 (m, 0.5H), 5.12-5.27 (m, 2H, CH2Ph),
7.29-7.45 (m, 5H, aromatics).
The product obtained was dissolved in 5.6 mL of CH3OH, and
0.22 mL of concentrated HCl was added. The mixture was kept at
60 °C for 5 h and then the solvent was evaporated under vacuum
to afford 0.84 g (yield 98%) of (2R,1′R)-12. 1H NMR (CDCl3) 200
MHz δ (ppm): 1.70-2.02 (m, 5H, CH2CH2 + 1H), 2.52-2.63
(m, 2H, CH2S), 2.81 (bs, 1H), 3.29-3.43 (m, 1H), 3.52-3.68 (m,
1H), 3.75-3.84 (m, 1H), 3.94-4.07 (m, 1H), 5.15 (s, 2H, CH2-
(2S,2′S,5′S)-1-Methyl-2-(2-methyl-1,3-oxathiolan-5-yl)pyrro-
lidine (2S,2′S,5′S)-(18), (2S,2′R,5′S)-1-Methyl-2-(2-methyl-1,3-
oxathiolan-5-yl)pyrrolidine (2S,2′R,5′S)-(19), (2S,2′R,5′R)-1-
Methyl-2-(2-methyl-1,3-oxathiolan-5-yl)pyrrolidine (2S,2′R,5′R)-
(20), and (2S,2′S,5′R)-1-Methyl-2-(2-methyl-1,3-oxathiolan-5-
yl)pyrrolidine (2S,2′S,5′R)-(21). Using the same procedure described
above, starting from (2S,1′S)-12 the diastereomeric mixture of 14
and 15 (yield 93%) was obtained, and starting from (2S,1′R)-13,
the diastereomeric mixture of 16 and 17 (yield 88%) was obtained.
Their 1H NMR spectra are identical to those of the diastereomeric
mixtures 14 and 15, and 16 and 17.
Ph), 7.31 (s, 5H, aromatics). [R]20 ) +48.5 (c 1.0, CHCl3).
D
(2R,1′S)-Phenylmethyl 2-(1-hydroxy-2-mercaptoethyl)pyrro-
lidine-1-carboxylate (2R,1′S)-13. Using the same procedure
described for (2R,1′R)-12, starting from 0.75 g (3.03 mmol) of
(2R,2′S)-11 and 2 equiv of thioacetic acid, (2R,1′S)-phenylmethyl
2-(2-acetyl-sulfanil-1-hydroxyethyl)pyrrolidine-1-carboxylate was
1
obtained, which was used as such for the next reaction. H NMR
The subsequent reduction with LiAlH4 and chromatographic
separation afforded the cis isomers (2S,2′S,5′S)-18 and (2S,2′R,5′R)-
20 and the trans isomers (2S,2′R,5′S)-19 and (2S,2′S,5′R)-21 in a
30:1 ratio.
(CDCl3) 200 MHz δ (ppm): 1.74-2.10 (m, 4H, CH2CH2), 2.36
(s, 3H, COCH3), 2.86-3.00 (m, 1H), 3.16-3.47 (m, 2H), 3.51-
3.78 (m, 2H), 3.89-4.02 (m, 1H), 4.85-5.04 (m, 1H), 5.15 (s,
2H, CH2Ph), 7.29-7.43 (m, 5H, aromatics).
The cis isomers have already been described.9 1H NMR and 13
C
After cleavage of the acetylsulfanil function as described above,
NMR data of the trans isomers are reported in Table 3 (Supporting
Information).
1
0.8 g (92% yield) of (2R,1′S)-13 was obtained. H NMR (CDCl3)
200 MHz δ (ppm): 1.62-2.12 (m, 5H, CH2CH2 + 1H), 2.41-
2.59 (m, 1H), 2.70-2.88 (m, 1H), 3.30-3.48 (m,1H), 3.55-3.73
(m, 2H), 3.88 (bs, 1H), 4.10-4.19 (m, 1H), 5.16 (s, 2H, CH2Ph),
(-)-(2R,2′R,3′R,5′R)-1-Methyl-2-(2-methyl-1,3-oxathiolan-5-
yl)pyrrolidine 3-Oxide (-)-(2R,2′R,3′R,5′R)-(1) and (+)-
(2R,2′R,3′S,5′R)-1-Methyl-2-(2-methyl-1,3-oxathiolan-5-yl)pyr-
rolidine 3-Oxide (+)-(2R,2′R,3′S,5′R)-(2). A solution of 1 equiv
of (2R,2′R,5′R)-18 in CH3COOH at 0 °C was added to 3.6 equiv
of H2O2 (30%). After 50 min at 0 °C the mixture was made alkaline
with 10% NaOH, extracted with CHCl3, and dried over Na2SO4.
Filtration and evaporation afforded a mixture of two diastereomeric
sulfoxides (-)-1 and (+)-2 in 2:1 ratio (calculated from 1H NMR).
The oily mixture obtained was separated by flash chromatography
(eluent NH4OH/absolute ETOH/CH2Cl2/petroleum ether/Et2O )
11.2:118:460:310:99). Anal. (C9H17NO2S) C, H, N.
7.31 (s, 5H, aromatics). [R]20 ) +83.3 (c 1.0, CHCl3).
D
(2S,1′S)-Phenylmethyl 2-(1-Hydroxy-2-mercaptoethyl)pyrro-
lidine-1-carboxylate (2S,1′S)-12 and (2S,1′R)-Phenylmethyl 2-(1-
Hydroxy-2-mercaptoethyl)pyrrolidine-1-carboxylate (2S,1′R)-
13. Using the same procedure already described, the enantiomers
(2S,1′S)-12 [yield 98%, [R]20 ) -48.5 (c 1.0, CHCl3)].and
(2S,1′R)-13 [yield 98%, [R]20 D ) -83.3 (c 1.0, CHCl3)], were
D
obtained. Their 1H NMR spectra are identical to the spectra of the
enantiomers.
(2R,2′R,5′R)-1-Methyl-2-(2-methyl-1,3-oxathiolan-5-yl)pyrro-
lidine (2R,2′R,5′R)-18 and (2R,2′S,5′R)-1-Methyl-2-(2-methyl-
1,3-oxathiolan-5-yl)pyrrolidine (2R,2′S,5′R)-19. To a solution of
0.79 g (2.81 mmol) of (2R,1′R)-12 in 15.8 mL of 2-propanol was
added 0.083 g (0.44 mmol) of p-toluensulfonic acid monohydrate.
To this mixture, under nitrogen atmosphere, 2.4 mL (22.5 mmol)
of acetaldehyde dimethylacetal was added. After 5 h at 55 °C the
solvent was evaporated and the residue was dissolved in diethyl
ether. The organic layer was washed with water, dried over Na2-
SO4, filtered, and evaporated to afford 0.79 g (2.59 mmol, 92%
yield) of a mixture of two diastereoisomers (14 and 15). 1H NMR
Their spectroscopic, chemical, and physical characteristics are
reported in Tables 4-6 (Supporting Information).
(+)-(2R,2′S,3′S,5′S)-1-Methyl-2-(2-methyl-1,3-oxathiolan-5-
yl)pyrrolidine 3-Oxide (+)-(2R,2′S,3′S,5′S)-(3) and (+)-
(2R,2′S,3′R,5′S)-1-Methyl-2-(2-methyl-1,3-oxathiolan-5-yl)pyr-
rolidine 3-Oxide (+)-(2R,2′S,3′R,5′S)-(4). Using the same procedure
described above, starting from (2R,2′S,5′S)-20, compounds (+)-3
and (+)-4 were obtained. Anal. (C9H17NO2S) C, H, N. Their
spectroscopic, chemical, and physical characteristics are reported
in Tables 4-6 (Supporting Information).

1930 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 6
Scapecchi et al.
(+)-(2S,2′S,3′S,5′S)-1-Methyl-2-(2-methyl-1,3-oxathiolan-5-
yl)pyrrolidine 3-oxide (+)-(2S,2′S,3′S,5′S)-(1), (-)-(2S,2′S,3′R,5′S)-
1-Methyl-2-(2-methyl-1,3-oxathiolan-5-yl)pyrrolidine 3-Oxide
(-)-(2S,2′S,3′R,5′S)-(2), (-)-(2S,2′R,3′R,5′R)-1-Methyl-2-(2-
methyl-1,3-oxathiolan-5-yl)pyrrolidine 3-Oxide (-)-(2S,2′R,
3′R,5′R)-(3), and (-)-(2S,2′R,3′S,5′R)-1-Methyl-2-(2-methyl-1,3-
oxathiolan-5-yl)pyrrolidine 3-Oxide (-)-(2S,2′R,3′S,5′R)-(4). The
same oxidative procedure applied to the S-series afforded the
enantiomers of compounds 1-4. Anal. (C9H17NO2S) C, H, N. Their
spectroscopic, chemical, and physical characteristics are reported
in Tables 4-6 (Supporting Information).
General Procedure for the Synthesis of Methiodides 5-8. An
anhydrous ether solution of the suitable sulfoxide 1-4 was treated
with an excess of methyl iodide and kept for 1 night at room
temperature in the dark. The solid obtained was filtered, dried under
vacuum, and recrystallized from absolute ethanol. Anal. (C10H20-
NO2SI) C, H, N. Their chemical and physical characteristics are
reported in Tables 7-9 (Supporting Information).
X-ray Structural Analysis of Compound (-)-5. C11H21NO2-
SCl3I, M ) 464.60, orthorhombic, space group Pna21, a ) 6.839-
(1) Å, b ) 12.968(1) Å, c ) 21.557(2) Å, V ) 1911.9(4) Å3, Z )
4 Dc ) 1.614, µ ) 18.029 mm-1, F(000) ) 920. Analysis on a
prismatic transparent single crystal was carried out with a Bruker
CCD X-ray diffractometer at 150 K. Cu KR radiation monochro-
mated by crossed Gobel mirrors was used for cell parameter
determination and data collection. The intensities of some equiva-
lents were monitored during data collection to check the stability
of the crystal: no loss of intensity was recognized. The integrated
intensities, measured using the ω scan mode, were corrected for
Lorentz and polarization effects. The number of reflections collected
was 4173 with a 3.98 < θ < 50.97° range; 1808 were independent,
and the final R index was 0.1483 for reflections having I > 2σI,
and 0.1797 for all data. The data acquisition, integration, data
reduction, and absorption correction were performed using SMART,
SAINT, and SADABS programs.31 Structure was refined using the
full-matrix least squares on F2 provided by SHELXL97.32 In Figure
1 a thermal ellipsoid representation of (-)-5 is shown
were obtained in duplicate. At the end of the binding reaction, free
radioligand was separated from bound ligand by rapid filtration
through UniFilter GF/C plates (Perkin-Elmer Life Science, Boston,
MA) using a FilterMate cell harvester (Perkin-Elmer Life Science,
Boston, MA). After filtration, the filters were washed several times
with ice-cold buffer and allowed to dry overnight at room
temperature under air flow, 25 µL of scintillation liquid (Microscint-
20, Perkin-Elmer Life Science, Boston, MA) was added, and the
radioactivity was counted by a TopCount NXT microplate scintil-
lation counter (Perkin-Elmer Life Science, Boston, MA). The
binding data were analyzed by the weighted least-squares iterative
curve fitting program LIGAND35 to obtain the affinity constant (Ki)
and the binding capacity (Bmax).12
Functional Studies. General Considerations. Male guinea pigs
(200-300 g) and male New Zealand white rabbits (3.0-3.5 kg)
were killed by cervical dislocation. The organs required were set
up rapidly under 1 g of tension in 20-mL organ baths containing
physiological salt solution (PSS) maintained at an appropriate
temperature (see below) and aerated with 5% CO2-95% O2. Dose-
response curves were constructed by cumulative addition of the
reference agonist. The concentration of agonist in the organ bath
was increased approximately 3-fold at each step, with each addition
being made only after the response to the previous addition had
attained a maximal level and remained steady. Following 30 min
of washing, tissues were incubated with the antagonist for 60 min,
and a new dose-response curve to the agonist was obtained. In
the case of the agonist, following 30 min of washing, a cumulative
dose-response curve to the agonist under study was constructed.
Responses were expressed as a percentage of the maximal response
obtained in the control curve. Contractions were recorded by means
of a force displacement transducer connected to the MacLab system
PowerLab/800. In addition, parallel experiments in which tissues
did not receive any antagonist were run in order to check any
variation in sensitivity. In all cases, parallel experiments in which
tissues received only the reference agonist were run in order to
check any variation in sensitivity. All animal testing was carried
out according to European Communities Council Directive of 24
November 1986 (86/609/EEC)
Pharmacology. Binding Studies. Cell Culture and Membrane
Preparation. Chinese hamster ovary cells stably expressing cDNA
encoding human muscarinic hm1-hm5 receptors were generously
provided by Prof. R. Maggio (Department of Neuroscience,
University of Pisa, Italy). Growth medium consisted in Dulbecco’s
modified Eagle’s medium supplemented with 10% fetal calf serum
(Gibco, Grand Iland, NY), 100 units/mL each of penicillin G and
0.1 ng/mL streptomycin, 4 mM L-glutamine (Sigma Aldrich,
Milano, Italy) and nonessential amino acids (Sigma Aldrich, Milano,
Italy), and 50 µg/mL of geneticin (Gibco, Grand Iland, NY) in a
humidified atmosphere consisting of 5% CO2 and 95% air.
Confluent CHO cell lines were scraped, washed with buffer (25
mM sodium phosphate containing 5 mM MgCl2 at pH 7.4), and
homogenized for 30s using an Ultra-Turrax (setting 35). The pellet
was sedimented 17 000g for 15 min at 4 °C, and the membranes
were resuspended in the same buffer, rehomogenized with Ultra-
Turrax, and stored at -80 °C.33 An aliquot was taken for the
assessment of protein content according to the method of Bradford34
using the Bio-Rad protein assay reagent (Bio-Rad Laboratories,
Munchen, West Germany) and bovine serum albumin was used as
the standard.
Guinea Pig Ileum. Two-centimeter-long portions of terminal
ileum were taken at about 5 cm from the ileum-cecum junction
and mounted in PSS, at 37 °C, of the following composition
(mM): NaCl (118), NaHCO3 (23.8), KCl (4.7), MgSO4‚7H2O
(1.18), KH2PO4 (1.18), CaCl2 (2.52), and glucose (11.7). Tension
changes were recorded isotonically. Tissues were equilibrated for
30 min, and dose-response curves to arecaidine propargyl ester
(APE) were obtained at 30 min intervals, the first one being
discarded and the second one being taken as the control.
Guinea Pig Stimulated Left Atria. The heart was rapidly
removed, and the right and left atria were separately excised. Left
atria were mounted in PSS (the same used for ileum) at 30 °C and
stimulated through platinum electrodes by square-wave pulses (1
ms, 1 Hz, 5-10 V) (Tetra Stimulus, N. Zagnoni). Inotropic activity
was recorded isometrically. Tissues were equilibrated for 2 h, and
a cumulative dose-response curve to APE was constructed.
Guinea Pig Lung Strips. The lungs were rapidly removed and
strips of peripheral lung tissue were cut either from the body of a
lower lobe with the longitudinal axis of the strip parallel to the
bronchus or from the peripheral margin of the lobe. The preparations
were mounted, with a preload of 0.3 g, in PSS with the following
composition (mM): NaCl (118.78), KCl (4.32), CaCl2‚2H2O (2.52),
MgSO4‚7H2O (1.18), KH2PO4 (1.28), NaHCO3 (25), glucose (5.55).
Contractions were recorded isotonically at 37 °C after tissues were
equilibrated for 1 h and then two cumulative dose-response curves
to APE (0.01, 0.1, 1, 10, 100 mM) were obtained at 45-min
intervals, the first one being discarded and the second one being
taken as the control.
Binding Assay. The radioligand binding assay was run in a
polypropylene 96-well plates (Sarstedt, Verona, Italy) and per-
formed for 120 min at room temperature in a final volume of 0.25
mL in 25 mM sodium phosphate buffer containing 5 mM MgCl2
at pH 7.4. Final membrane protein concentrations were 30 µg/mL
(hm1), 70 µg/mL (hm2), 25 µg/mL (hm3), 50 µg/mL (hm4), and
25 µg/mL (hm5).
In homologous competition curves, [3H]NMS was present at 0.2
nM in wells containing increasing concentration of unlabeled NMS
(0.03-1000 nM) and at 0.075-0.2 nM in wells without unlabeled
ligand. In heterologous competition curves, fixed concentrations
of the tracer (0.2 nM) were displaced by increasing concentrations
of several unlabeled ligands (0.01-1000 mM); all measurements
Rabbit Stimulated Vas Deferens. This preparation was set up
according to the method of Eltze.16 Vasa deferentia were carefully
dissected free of surrounding tissue and were divided into four
segments, two prostatic portions of 1 cm and two epididymal
portions of approximately 1.5 cm length. The four segments were

Highly Chiral Muscarinic Ligands
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M
tripitramine were included to block R2-adrenoceptors and M2
muscarinic receptors, respectively. The solution was maintained at
30 °C, and tissues were stimulated through platinum electrodes by
square-wave pulses (0.1 ms, 2 Hz, 10-15 V). Contractions were
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Determination of Affinity Constants. Affinity constants (Ka,
Table 1) were determined according to the method of Furchgott
and Bursztyn20 for full agonists (R ) 1) by means of a software
computation process supplied by Dr. Roberto Gagliardi. Partial
agonist values were determined according to Waud19 by comparison
of its concentration-response curve with that of APE. Several
equieffective concentrations of partial agonist and APE were
determined graphically.
Acknowledgment. The authors are thank Dott. Cristina
Faggi for the X-ray structural analysis of compound 5.
Supporting Information Available: Tables 3-9, X-ray crys-
tallographic data of compound (-)-5, and elemental analysis results
of compounds 1-8. This material is available free of charge via
the Internet at http://pubs.acs.org.
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