Synthesis, affinity profile and functional activity of potent chiral muscarinic antagonists with a pyrrolidinylfuran structure

Article abstract of DOI:10.1021/jm901048j

Starting from the structure of previously studied muscarinic agonists, characterized by a pyrrolidinylfuran scaffold, a new series of muscarinic antagonists was synthesized by substituting the 5-position of the furane cycle with bulky hydrophobic groups. Both tertiary amines and the corresponding iodomethyl derivatives were obtained and studied. All the new compounds show high affinity toward cloned human muscarinic M₁-M₅ receptors expressed in Chinese hamster ovary (CHO) cells and behave as competitive antagonists on classical models of muscarinic receptors. The diastereoisomeric mixture of the highest affinity compound of the series was resolved into the four optical isomers by chiral HPLC. The relative and absolute configuration of the obtained compounds was established by means of a combined strategy based on X-ray crystallography and chiroptical techniques. Although generally fairly potent, the compounds showed only modest subtype selectivity, with the exception of 2a and 6a, which in functional assays presented clear-cut selectivity for the muscarinic receptors present in rabbit vas deferens.

Full text of DOI:10.1021/jm901048j

J. Med. Chem. 2010, 53, 201–207 201
DOI: 10.1021/jm901048j
Synthesis, Affinity Profile and Functional Activity of Potent Chiral Muscarinic Antagonists
with a Pyrrolidinylfuran Structure†
Serena Scapecchi,*,‡ Marta Nesi,§ Rosanna Matucci,§ Cristina Bellucci,‡ Michela Buccioni,^ Silvia Dei,‡ Luca Guandalini,‡
Dina Manetti,‡ Cecilia Martelli,‡ Elisabetta Martini,‡ Gabriella Marucci,^ Francesca Orlandi,‡ Maria Novella Romanelli,‡
Elisabetta Teodori,‡ and Roberto Cirilli
‡
§
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, Dipartimento di Chimica, Universita di Camerino, Via S.
^
ꢀ
Agostino 1, 62032 Camerino (MC), Italy, and Dipartimento del Farmaco, Istituto Superiore di Sanita, Viale Regina Elena 299, 00161 Roma,
ꢀ
ꢀ
Italy
Received July 15, 2009
Starting from the structure of previously studied muscarinic agonists, characterized by a pyrrolidinyl-
furan scaffold, a new series of muscarinic antagonists was synthesized by substituting the 5-position of
the furane cycle with bulky hydrophobic groups. Both tertiary amines and the corresponding
iodomethyl derivatives were obtained and studied. All the new compounds show high affinity toward
cloned human muscarinic M1-M5 receptors expressed in Chinese hamster ovary (CHO) cells and
behave as competitive antagonists on classical models of muscarinic receptors. The diastereoisomeric
mixture of the highest affinity compound of the series was resolved into the four optical isomers by chiral
HPLC. The relative and absolute configuration of the obtained compounds was established by means of
a combined strategy based on X-ray crystallography and chiroptical techniques. Although generally
fairly potent, the compounds showed only modest subtype selectivity, with the exception of 2a and 6a,
which in functional assays presented clear-cut selectivity for the muscarinic receptors present in rabbit
vas deferens.
Introduction
selective ligands are still needed. There is intense research to
identify selective M3 subtype antagonists for use in smooth
muscle disorders such as urinary incontinence (UI), chronic
obstructive pulmonary disease (COPD), and irritable bowel
syndrome (IBS).12-15
For several years, we have been working on cholinergic
ligands, both agonists and antagonists.16-20 Recently, we
reported the muscarinic agonist activity of a series of pyrro-
lidinylfuran derivatives. Of these, S-(-)-2-(5-methyl-2-furyl)-
1-methylpyrrolidine metiodide, whose structure is shown in
Chart 1, was the most interesting, because it was a potent and
selective M2 receptor partial agonist.21
In the present paper, we report the synthesis of a new series
of pyrrolidinylfuran derivatives (1-8) that were transformed
into potent antagonists by the insertion of bulky hydro-
phobic groups in position 5 of the furan ring (Chart 1) as
successfully done in previous studies on pentatomic cyclic
compounds.17,20,22 We reasoned that by extending the length of
the molecules it would be possible to explore the properties of
less conserved proximal regions of the orthosteric binding site of
the receptor, as also suggested by the chemical structure of some
recently reported subtype-selective muscarinic antagonists.23-26
Five muscarinic acetylcholine receptor subtypes (M1-M5)
are found in the central and peripheral nervous system where
they mediate a variety of vital functions.1 Four of these
subtypes (M1-M4) have been characterized from a pharma-
cological point of view.2 Due to the physiological role of such
receptors, muscarinic drugs have the potential for therapeutic
use in several pathological states.3,4 The high sequence con-
servation within the orthosteric domain across all five mus-
carinic subtypes5 has made it difficult to identifyeither natural
or synthetic selective ligands.6 As a consequence, despite the
ubiquitous presence of muscarinic receptors in the human
body, the number of muscarinic drugs that have been intro-
duced into therapy is limited, particularly when one considers
the importance of this class of receptors.3 The recent identi-
fication of allosteric sites,7-9 which could be different in the
five subtypes, and the discovery of allosteric agonists such as
AC-4210,11 that can interact with them may open a new
promising approach toward selective modulation of muscari-
nic subtypes. In the meantime, classical research into orthos-
teric-site-directed ligands both for agonists and antagonists is
still active. It aims to identify muscarinic ligands that can be
used as therapeutic drugs or as pharmacological tools for
studying muscarinic receptors.4 Muscarinic subtype charac-
terization is far from being satisfactory, and more potent and
Chemistry
Initially, to save time and to avoid labor-intensive experi-
mental work, we decided to synthesize the designed com-
pounds, disregarding the problem of stereoisomerism. Once
the pharmacological activity of the obtained mixtures was
evaluated, we would then resolve the complex mixture of the
†Dedicated to Prof. Fulvio Gualtieri on the occasion of his retire-
ment.
*To whom correspondence should be addressed. Tel þ39-055-
4573692; fax þ39-055-4573780; e-mail serena.scapecchi@unifi.it.
r
2009 American Chemical Society
Published on Web 11/20/2009
pubs.acs.org/jmc

202 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1
Scapecchi et al.
Chart 1. Structure of Compounds S-(-)-2-(5-Methyl-2-furyl)-1-methylpyrrolidine Methiodide and 1-8
Scheme 1. Synthesis of Compounds 1-8a
a (a) BuLi, PhCOCy or PhCOPh; (b) NaBH4, CF3COOH; (c) CH3I.
Table 1. Binding Affinitya of Compounds 1-8
compound
hM1
hM2
hM3
hM4
hM5
1
7.45 ( 0.06
8.91 ( 0.08
8.91 ( 0.09
7.64 ( 0.07
6.72 ( 0.07
5.91 ( 0.08
7.37 ( 0.10
7.87 ( 0.07
7.63 ( 0.07
8.68 ( 0.06
9.16 ( 0.08
8.04 ( 0.07
7.32 ( 0.07
6.48 ( 0.07
7.94 ( 0.11
8.18 ( 0.07
9.49 ( 0.06
7.53 ( 0.12
8.17 ( 0.09
8.34 ( 0.10
7.22 ( 0.11
6.44 ( 0.11
5.49 ( 0.11
7.19 ( 0.18
6.85 ( 0.09
7.50 ( 0.10
7.99 ( 0.09
8.63 ( 0.10
7.70 ( 0.09
7.06 ( 0.09
6.12 ( 0.10
7.87 ( 0.2
7.76 ( 0.09
9.75 ( 0.10
7.02 ( 0.08
8.11 ( 0.11
8.78 ( 0.11
7.63 ( 0.12
6.49 ( 0.11
5.78 ( 0.11
7.07 ( 0.08
7.47 ( 0.12
7.44 ( 0.09
8.14 ( 0.12
8.82 ( 0.12
8.03 ( 0.12
6.81 ( 0.12
6.29 ( 0.12
7.63 ( 0.08
8.05 ( 0.11
9.87 ( 0.10
6.98 ( 0.06
7.85 ( 0.06
8.40 ( 0.09
7.26 ( 0.08
6.26 ( 0.07
5.51 ( 0.10
6.80 ( 0.05
7.11 ( 0.07
7.19 ( 0.07
7.81 ( 0.08
8.37 ( 0.10
7.48 ( 0.08
6.43 ( 0.08
5.63 ( 0.10
7.42 ( 0.08
7.71 ( 0.08
9.85 ( 0.07
7.01 ( 0.06
8.15 ( 0.07
8.48 ( 0.08
7.56 ( 0.04
6.33 ( 0.08
5.81 ( 0.07
6.98 ( 0.05
7.26 ( 0.08
7.43 ( 0.07
7.94 ( 0.08
8.81 ( 0.07
7.92 ( 0.07
6.64 ( 0.06
6.06 ( 0.06
7.50 ( 0.07
7.81 ( 0.09
9.68 ( 0.05
2
(1R,2R)-2a
(1R,2S)-2b
(1S,2R)-2c
(1S,2S)-2d
3
4
5
6
(1R,2R)-6a
(1R,2S)-6b
(1S,2R)-6c
(1S,2S)-6d
7
8
NMSb
a Binding affinity at cloned human muscarinic receptor subtypes expressed in CHO cells. The affinity estimates (as pKi) were derived from both
[3H]-NMS homologous and heterologous competition curves and represent the mean ( SEM of at least three experiments. The affinity is estimated
as pKi (-logKi), where the unit of measure for Ki is M. b N-Methylscopolamine.
most interesting ones. Scheme 1 illustrates the synthetic path-
way that was followed.
preparation of each of the four stereoisomers of methiodide 6
separately.
Metalation with BuLi at 0 ꢀC of racemic 2-furan-2-yl-1-
methylpyrrolidine21 and subsequent treatment with the ap-
propriate electrophile (diphenyl ketone for 1 and phenylcy-
clohexyl ketone for 2) gave compounds 1 and 2; their
reduction with NaBH4 in CF3COOH27 afforded compounds
3 and 4. Diastereomeric mixtures 1-4 were transformed into
the corresponding methiodides (5-8) with an excess of CH3I
in the dark at room temperature. The binding affinity of these
mixtures of four (2, 4, 6, 8) or two (1, 3, 5, 7) stereoisomers
was evaluated. Based on the obtained results (Table 1), we
decided to resolve mixture 2, which would in turn allow the
Therefore, our first attempt was to separate the two dia-
stereoisomers of compound 2 by flash chromatography, but
we obtained only enriched mixtures of each diastereoisomer.
The diastereo- and enantioselective HPLC performed on the
Chiracel OD chiral stationary phase (CSP) using the n-
hexane/2-propanol/DEA 100:1:0.1 (v/v/v) mixture as mobile
phase (Figure 1) was more useful. These conditions, scaled-up
to semipreparative level, permitted isolation of milligram
amounts of each stereoisomer for a single chromatographic
run within 12 min (Figure 2a). The CDa spectra (Figure 3) and
the specific rotations (Figure 2b) of the four isomers indicated

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1 203
Figure 3. CD spectra of the 2a-2d stereoisomers in n-hexane.
of Scheme 1 was performed on the (2R)-(þ)-(2-furyl)-1-
methylpyrrolidine isomer.21 In this case, only two diastereoi-
somers of compound 2 with known stereochemistry on the
pyrrolidine center were obtained. Diastereoselective HPLC
showed that the isomers obtained were 2a and 2c (Figure 4).
The structure of (þ)-2a (first eluted on the OD CSP) was
readily secured by X-ray crystallography. As illustrated in
Figure 5, the absolute configuration of (þ)-2a was determined
as (1R,2R); thus the (1S,2S)-configuration was assigned
[(1S,2S)-(-)-2d] tothe (-)-enantiomerof2a (2d, fourth eluted
on the OD CSP). On the basis of combined chromatographic,
chiroptical, and NMR studies, the absolute configuration
assignment was unambiguously completed, and configura-
tions were assigned to the second and third eluting enantio-
mers: (1R,2S)-(-)-2b and (1S,2R)-(þ)-2c.
Figure 1. Chromatograms of 2 obtained by simultaneous UV and
chiroptical (polarimetric or CD) detection. Column, Chiralcel OD
(250 mm ꢀ 4.6 mm I.D); eluent, n-hexane/2-propanol/DEA
100:1:0.1 (v/v/v); flow-rate, 1 mL min-1; temperature, 25 ꢀC.
Reaction of 2a-d with methyl iodide, according to
Scheme 1, afforded the corresponding methiodides 6a-d.
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 pre-
parations: rabbit stimulated vas deferens (putative M1), gui-
nea pig stimulated left atria (M2), guinea pig ileum (M3), and
guinea pig lung strips (putative M4), following the previously
reported methods.28 It should be remembered that the con-
traction of rabbit vas deferens has long been considered an
effect mediatedbyM1-receptor subtypes, whereasmorerecent
studies attribute the same effect to an M4 activation.2,29
Analogously, the validity of the guinea pig lung strips as an
M4 model30 has been questioned.31 For this reason, these two
preparations are indicated as putative M1 and M4 receptor
models in the present work. Carbachol, arecaidine propargyl
ester (APE), and 4-Cl-McN-A-343 were used as agonists.
Results are expressed as pKi values (affinity) and as pKb
values calculated from the equation pKb = log(DR-1) - log
[B], where DR is the ratio of ED50 values of agonist after and
before treatment with one or two antagonist concentra-
tions, [B].32 Results are shown in Tables 1 and 2, where the
N-methylscopolamine data are reported for comparison.
Figure 2. (a) Simultaneous diastereo- and enantioseparation of 10
mg of 2; (b) chromatographic and polarimetric analysis of the
isolated stereoisomers. Column, (a) Chiralcel OD (250 mm ꢀ 10
mm I.D.), (b) Chiralcel OD (250 mm ꢀ 4.6 mm I.D.); eluent, n-
hexane/2-propanol/DEA 100:1:0.1 (v/v/v); flow-rate, (a) 5.0 mL
min-1, (b) 1 mL min-1; detector, UV at 254 nm; temperature, 25 ꢀC.
Results and Discussion
that the enantiomeric couples are 2a/2d and 2b/2c where a-d
indicates the stereomeric elution order. To establish the
absolute configuration of the four isomers, the first reaction
The results reported in Table 1 show that the compounds
studied are endowed with high affinity for the five human

204 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1
Scapecchi et al.
Table 2. Functional Activity of Compounds 1-8a
rabbit vas guinea pig atriumguinea pig ileum
guinea
pig lung
compound
deferens
(M2)
(M3)
1
2
6.91 ( 0.19
c
6.92 ( 0.11
6.98 ( 0.21
7.91 ( 0.20
6.65 ( 0.03
6.23 ( 0.20
5.95 ( 0.02
6.52 ( 0.19
5.36 ( 0.15
7.08 ( 0.20
7.88 ( 0.17
8.49 ( 0.15
7.32 ( 0.21
6.85 ( 0.03
5.92 ( 0.02
7.09 ( 0.07
6.21 ( 0.25
9.33 ( 0.03d
6.65 ( 0.09
7.80 ( 0.02 <5
8.76 ( 0.02
7.66 ( 0.18
6.77 ( 0.18 <5
6.41 ( 0.11 <5
6.81 ( 0.08
7.05 ( 0.20
7.60 ( 0.15
8.35 ( 0.04
8.95 ( 0.09
7.95 ( 0.18
6.95 ( 0.09
6.32 ( 0.02
<5
(1R,2R)-2a 9.58 ( 0.20
5.92 ( 0.13
5.84 ( 0.09
(1R,2S)-2b
(1S,2R)-2c
(1S,2S)-2d
3
4
5
6
c
c
c
6.64 ( 0.15
c
5.19 ( 0.23
<5
6.20 ( 0.04
5.90 ( 0.04
7.75 ( 0.03
6.16 ( 0.12
6.33 ( 0.10
8.46 ( 0.23
c
(1R,2R)-6a 10.15 ( 0.16
(1R,2S)-6b
(1S,2R)-6c
(1S,2S)-6d
7
c
c
c
<5
7.91 ( 0.05
c
7.59 ( 0.10 <5
8
7.88 ( 0.15
5.69 ( 0.20
NMSb
c
9.21 ( 0.07d
8.19 ( 0.06d
a pKb values from functional tests ( SEM; n = 3. b N-Methylscopo-
lamine. c Not tested d pA2 values.
chosen for the resolution procedure. Compound 2a had the
highest affinity for the muscarinic receptors, while its enan-
tiomer 2d had the lowest. As a consequence, the enantioselec-
tivity, expressed by the eudismic ratio (ER) between the
affinity of two enantiomers, is very high for all receptor
subtypes varying from 468 for hM5 to 708 for hM2, 776 for
hM4, and 1000 for hM1 and hM3. In contrast, the other
enantiomeric couple (2b, 2c) shows only modest enantioselec-
tivity, about 2 orders of magnitude lower (ER = 6 for hM2; 8
for hM1; 10 for hM4; 14 for hM3, 17 for hM5). Apparently, the
configuration of 2a is optimal to interact with muscarinic
receptors. Unfortunately, once again, this is true for all
subtypes. As a matter of fact, subtype selectivity also remains
very low for the pure stereoisomers and this behavior is
maintained in the corresponding methiodides (6a-6d). As
expected, 6a-6d show a slightly higher affinity, but they
maintain similar eudismic ratios and subtype selectivity com-
pared with the corresponding tertiary amines. This confirms
that the presence of a quaternary nitrogen is not critical for
muscarinic antagonists.33
Figure 4. Identification of the diastereomeric pairs of 2. Column,
Chiralcel OD (250 mm ꢀ 4.6 mm I.D.); eluent, n-hexane/2-propa-
nol/DEA 100:1:0.1 (v/v/v); flow-rate, 1 mL min-1; detector, UV at
240 nm; temperature, 25 ꢀC.
The resultsof functionalactivityon the classicmodels of M2
(guinea pig atrium) and M3 (guinea pig ileum) muscarinic
receptors are reported in Table 2, together with the results
obtained in two other tissues (rabbit vas deferens and guinea
pig lung) for which the nature of the muscarinic subtypes
involved, as mentioned above, is currently controversial; to
save animals and money, only selected compounds were
assayed on rabbit vas deferens. As far as M2 and M3 receptors
are concerned, the results of functional tests are in fairly
good agreement with binding assays. This was expected,
since antagonists usually do not present the discrepancies
between binding and functional assays often found with
agonists.18,19,21 There are only small variations in potency
and the enantioselectivity of the couples 2a/2d, 2b/2c, 6a/6d,
and 6b/6c is of the same order of magnitude as that found in
binding. The same occurs for subtype selectivity, which un-
fortunately remains low. A different situation is found in the
other two tissues: the functional data on guinea pig lung do
not correlate with the binding of any of the human muscarinic
subtypes expressed in CHO cells, confirming the doubts31 on
the validity of this tissue as a model for M4, as proposed
previously,30 or for any other subtype. It is interesting that the
functionaldata on rabbit vas deferens, performed on isomer2,
Figure 5. Ortep drawing of compound of (þ)-(1R,2R)-2a.
muscarinic receptor subtypesexpressedinCHO cellsbut show
only low or modest subtype selectivity. The most potent was
the mixture of the four stereoisomers 2, which was therefore

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1 205
shows that 2a and 6a, which were the highest affinity isomers
on hM1 receptors (pKi = 8.91 and 9.16, respectively), are also
the most potent on rabbit vas deferens (pKb = 9.58 and 10.15
respectively).
In conclusion, we have synthesized and studied a new series
ofmuscarinic antagonists wherethe length of the moleculehas
been extended compared with similar classes of compounds
studied previously.17,20,22 Some members of the series are
fairly potent and show higher enantioselectivity but still fail
to present subtype selectivity. More work, such as a further
extension of the molecule length, will be necessary to explore
the viability of this approach.
added, and the mixture stirred for 2 h at 0 ꢀC. Diphenylmetha-
none (7.28 mmol) was added, and the mixture maintained at
0 ꢀC for 10 min and then warmed to 25 ꢀC. The solution was
acidified with 2 N HCl aqueous solution (10 mL) and extracted
with diethyl ether (3 ꢀ 20 mL). Then the aqueous phase was
made alkaline with NaOH 10% solution and extracted with
diethyl ether (3 ꢀ 20 mL), and the organic phase was dried
(Na2SO4). The mixture obtained was purified by column chro-
matography, using CH2Cl2/abs. EtOH/Et2O/petroleum ether/
NH4OH = 180:45:450:180:25 as eluting system. Compound 1
was obtained as an oil in 80% yield. Anal. (C22H23NO2) C, H, N.
1H NMR and 13C NMR spectra are reported in Table 3
(Supporting Information).
Cyclohexyl-[5-(1-methylpyrrolidin-2-yl)furan-2-yl]phenylme-
thanol (2). By the same procedure as that for 1 with cyclohex-
ylphenylmethanone as the electrophile, 2 was obtained in 98%
yield as an oil that was the expected mixture of four stereoi-
somers (two racemates).
When the reaction was performed on the (þ)-2R-furan-2-yl-
1-methylpyrrolidine isomer,21 only two diastereoisomers were
obtained: (1R,2R)- and (1S,2R)-[5-(1-methylpyrrolidin-2-yl)-
furan-2-yl]diphenylmethanol. Anal. (C22H29NO2) C, H, N.
1H NMR and 13C NMR spectra of the isolated enantiomers
(1R,2R)-2a and (1S,2R)-2c are reported in Table 3 (Supporting
Information).
2-(5-Benzhydrylfuran-2-yl)-1-methylpyrrolidine (3). Sodium
borohydride (0.1 g, 2.28 mmol) was added to stirring trifluoro-
acetic acid (3 mL) at 15 ꢀC under nitrogen according to the
procedure reported by Gribble.27 Compound 1 (0.14 g; 4.20
mmol), dissolved in 1.5 mL of anhydrous CH2Cl2, was added in
portions to this mixture at 15-20 ꢀC over 15 min. The mixture
was stirred under nitrogen for 19 h. Dilution in water, basifica-
tion with NaOH 10% aqueous solution, and extraction with
diethyl ether gave, after drying (Na2SO4) and concentration in
vacuum, 0.13 g of racemic 3 in 97% yield as an oil. Anal.
(C22H23NO) C, H, N. 1H NMR and 13C NMR spectra are
reported in Table 3 (Supporting Information).
Experimental Section
€
Chemistry. All melting points were taken on a Buchi appara-
tus and are uncorrected. Unless otherwise stated, NMR spectra
were recorded on a Bruker Avance 400 spectrometer (400 MHz
for 1H NMR, 100 MHz for 13C). Chromatographic separations
were performed on a silica gel column by gravity chromato-
graphy (Kieselgel 40, 0.063-0.200 mm; Merck) or flash chro-
matography (Kieselgel 40, 0.040-0.063 mm; Merck). Yields are
given after purification, unless otherwise stated. Where analyses
are indicated by symbols, the analytical results are within
(0.4% of the theoretical values. The purity of the tested
compounds not subjected to combustion analysis (2a-d) was
determined by HPLC (see details below). All reported com-
pounds had a purity of at least 95%. When reactions were
performed in anhydrous conditions, the mixtures were main-
tained under nitrogen. Compounds were named following
IUPAC rules as applied by Beilstein-Institute AutoNom
(version 4.01.305), a software for systematic names in organic
chemistry. Diethylamine (DEA) was obtained from Fluka
Chemie (Buchs, Switzerland).
HPLC enantioseparations were performed using stainless-
steel Chiralcel OD (250 mm ꢀ 4.6 mm I.D. and 250 mm ꢀ 10 mm
I.D.) columns (Chiral Technologies Europe, Illkirch, France).
HPLC-grade solvents were supplied by Carlo Erba (Milan,
Italy). The HPLC apparatus consisted of a Perkin-Elmer
(Norwalk, CT) 200 lc pump equipped with a Rheodyne
(Cotati, CA) injector, a 100-μL sample loop, and a HPLC
Dionex TCC-100 oven (Sunnyvale, CA). A Jasco (Jasco, Tokyo,
Japan) model CD 2095 Plus UV/CD and a Perkin-Elmer
polarimeter model 241 equipped with Hg/Na lamps and a
40 μL flow cell were used as detectors for HPLC. The signal
was acquired and processed by Clarity software (DataApex,
Prague, Czech Republic).
The mobile phases were filtered and degassed by sonication
just before use. In semipreparative enantioseparations, a stan-
dard solution was prepared by dissolving 1 g of 2 into 10 mL of
mobile phase. The injection volume was 100 μL. After semipre-
parative separation, the collected fractions were analyzed by
chiral analytical columns to determine their enantiomeric excess
(ee) and diastereomeric excess (de).
Specific rotations of stereoisomers 2a-2d, dissolved in n-
hexane, were measured at 589 nm with a Perkin-Elmer polari-
meter model 241 equipped with a Na lamp. The volume of the
cell was 1 mL, and the optical path was 10 cm. The system was
set at a temperature of 20 ꢀC using a Neslab RTE 740 cryostat.
The circular dichroism (CD) spectra of stereoisomers 2a-2d,
dissolved in n-hexane (concentration about 0.15 mg/mL) in a
quartz cell (0.1 cm-path length) at 20 ꢀC were measured using a
Jasco model J-700 spectropolarimeter. The spectra were aver-
aged over three instrumental scans, and the intensities are
presented in terms of ellipticity values (mdeg).
2-[5-(Cyclohexylphenylmethyl)furan-2-yl]-1-methylpyrroli-
dine (4). By starting from the mixture of compound 2 (0.14 g;
4.13 mmol) and following the same procedure described for
compound 3, we obtained 4 (0.13 g, mixture of diastereoisomers)
1
as an oil in 92% yield. Anal. (C22H29NO) C, H, N. H NMR
spectrum is reported in Table 3 (Supporting Information).
Resolution of the Stereoisomeric Mixture 2. HPLC separation,
performed on Chiracel OD CSP (Figure 1), afforded the four
isomers of compound 2: (1R,2R)-2a (1H NMR and 13C NMR
spectra are reported in Table 3, Supporting Information),
(1S,2S)-2d (1H NMR and 13C NMR spectra are identical to
those of 2a), (1R,2S)-2b (1H NMR and 13C NMR are reported in
Table 3, Supporting Information), and (1S,2R)-2c (1H NMR
and 13C NMR spectra are identical to those of 2b).
When separation was performed on (1R*,2R)-[5-(1-methyl-
pyrrolidin-2-yl)furan-2-yl]diphenylmethanol only the two dia-
stereoisomers 2a and 2c were obtained.
General Procedure for the Synthesis of Dimethyl Pyrrolidinium
Iodides 5-8. An anhydrous diethyl ether solution of the suitable
compound (1, 2a, 2b, 2c, 2d, 3, and 4) was treated with an excess
of methyl iodide and kept overnight at room temperature in the
dark. The obtained solid was filtered, dried under vacuum, and
recrystallized from absolute ethanol/diethyl ether.
The chemical and physical characteristics, the 1H NMR and
13C NMR spectra, and the optical rotations of the obtained
compounds are reported in Table 4 (Supporting Information).
Crystallographic Data for Compound (1R,2R)-2a. C22H29-
NO2; M = 339.46; orthorhombic; space group P212121; a =
3
˚
˚
8.091(1), b = 13.768(1), c = 17.671(1) A; V = 1968.5(3)A ; Z =
4; Dc = 1.145 g cm-3; μ = 0.564 mm-1; F(000)= 736.
Colorless, needle-shaped crystals suitable for collection were
prepared, and RX-analysis was carried out with a Goniometer
Oxford Diffraction KM4 Xcalibur2 at room temperature.
[5-(1-Methylpyrrolidin-2-yl)furan-2-yl]diphenylmethanol (1).
2-Furan-2-yl-1-methylpyrrolidine21 (0.50 g, 3.3 mmol) was dis-
solved in anhydrous THF (36 mL) and cooled to 0 ꢀC. Butyl-
lithium, 1.6 M solution in hexane (4.55 mL, 7.3 mmol), was then

206 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1
Scapecchi et al.
Cu KR radiation (40 mA/-40 kV), monochromated by an
Oxford Diffraction Enhance ULTRA assembly, and an Oxford
Diffraction Excalibur PX Ultra CCD were used for cell para-
meter determination and data collection. The integrated inten-
sities, measured using the ω scan mode, were corrected for
Lorentz and polarization effects.34
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Direct methods of SIR200435 were used in solving the struc-
ture, and it was refined using the full-matrix least-squares on F2
provided by SHELXL97.36
Multiscan symmetry-related measurement was used as ex-
perimental absorption correction type.
A total of 7074 reflections were collected with a 4.07 < θ <
64.71 range with a completeness to θ 97.1%; 2983 were inde-
pendent; the parameters were 264, and the final R index was
0.0538 for reflections having I > 2σI, and 0.0981 for all data.
The non-hydrogen atoms were refined anisotropically,
whereas hydrogen atoms were refined as isotropic, and all of
them were assigned to calculated positions.
The cyclohexyl group is disordered, so it was treated as if its
atoms were in double position, with occupancy factors 0.44 and
0.56; hydrogen atoms on this group were not assigned.
Pharmacology. Binding Studies. All equilibrium radioligand
binding experiments were conducted using a protocol based on
previously described procedures.18 Details of the procedures are
reported in the Supporting Information.
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Marucci, G.; Manetti, D.; Matucci, R.; Romanelli, M. N.;
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tional activity profile of 1-methyl-2-(2-methyl-1,3-dioxolan-4-yl)-
Functional Studies. All in vitro experiments were conducted
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Details of the procedures are reported in the Supporting In-
formation.
pyrrolidine
and
1-methyl-2-(2-methyl-1,3-oxathiolan-5-yl)-
pyrrolidine derivatives. Biochem. Pharmacol. 2005, 69, 1637–1645.
(19) Scapecchi, S.; Matucci, R.; Bellucci, C.; Buccioni, M.; Dei, S.;
Guandalini, L.; Martelli, C.; Manetti, D.; Martini, E.; Marucci, G.;
Nesi, M.; Romanelli, M. N.; Teodori, E. Highly chiral muscarinic
ligands: The discovery of (2S,20R,30S,50R)-1-methyl-2-(2-methyl-
1,3-oxathiolan-5-yl)pyrrolidine 3-sulfoxide methyl iodide, a po-
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(20) Dei, S.; Bellucci, C.; Buccioni, M.; Ferraroni, M.; Guandalini, L.;
Manetti, D.; Martini, E.; Marucci, G.; Matucci, R.; Nesi, M.;
Romanelli, M. N.; Scapecchi, S.; Teodori, E. Synthesis, affinity
profile and functional activity of muscarinic antagonists with a
1-methyl-2-(2,2-alkylaryl-1,3-oxathiolan-5-yl)pyrrolidine structure.
J. Med. Chem. 2007, 50, 1409–1413.
(21) Scapecchi, S.; Nesi, M.; Matucci, R.; Bellucci, C.; Buccioni, M.;
Dei, S.; Guandalini, L.; Manetti, D.; Martini, E.; Marucci, G.;
Romanelli, M. N.; Teodori, E.; Cirilli, R. Synthesis and pharma-
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The discovery of new functionally selective muscarinic agonists.
J. Med. Chem. 2008, 51, 3905–3912.
Acknowledgment. Financial support from the M.U.R.
ꢀ
(Ministero dell’Universita e della Ricerca) is gratefully
acknowledged. The authors wish to thank Dr. Cristina Faggi
for X-ray structure analysis of compound 2a.
Supporting Information Available: Chemical and physical
characteristics, spectral data, and elemental analysis for all the
new compounds; experimental details for the determination of
biological activities; and experimental details for crystal struc-
ture determination of (þ)-(1R,2R)-2a and its crystallographic
data. This material is available free of charge via the Internet at
http://pubs.acs.org.
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