Molecular modulation of muscarinic antagonists. Synthesis and affinity profile of 2,2-diphenyl-2-ethylthio-acetic acid esters designed to probe the binding site cavity

Article abstract of DOI:10.1016/j.farmac.2004.08.003

The synthesis and preliminary pharmacological profile of a new series of muscarinic antagonists, derived from previously studied 2,2-diphenyl-2- ethylthio-acetic acid esters, are reported. The parent molecules were decorated with linkers of different leng

Full text of DOI:10.1016/j.farmac.2004.08.003

IL FARMACO 59 (2004) 971–980
http://france.elsevier.com/direct/FARMAC/
Molecular modulation of muscarinic antagonists. Synthesis and affinity
profile of 2,2-diphenyl-2-ethylthio-acetic acid esters designed to probe
the binding site cavity
Serena Scapecchi a,*, Elisabetta Martini a, Cristina Bellucci a, Michela Buccioni b, Silvia Dei a,
Luca Guandalini a, Dina Manetti a, Cecilia Martelli a, Gabriella Marucci b, Rosanna Matucci c
a Dipartimento di Scienze Farmaceutiche, Università di Firenze, Via Ugo Schiff 6, 50019 Sesto Fiorentino, Firenze, Italy
b Dipartimento di Scienze Chimiche, Università di Camerino, Via S. Agostino 1, 62032 Camerino, Italy
c Dipartimento di Farmacologia Preclinica e Clinica, Università di Firenze, Viale Pieraccini 6, 50139 Firenze, Italy
Received 30 June 2004; accepted 5 August 2004
Available online 30 September 2004
Abstract
The synthesis and preliminary pharmacological profile of a new series of muscarinic antagonists, derived from previously studied
2,2-diphenyl-2-ethylthio-acetic acid esters, are reported. The parent molecules were decorated with linkers of different length, carrying an
amino group to catch a putative anionic function outside the recognition site of the receptor. It was hoped that the interception of this function
would give molecules with higher potency and selectivity. The attempt has not been successful, but a new series of compounds with a peculiar
pharmacological profile has been identified.
© 2004 Elsevier SAS. All rights reserved.
Keywords: Muscarinic antagonists; Affinity; Functional activity
1. Introduction
genic mice lacking genes encoding each of the muscarinic
receptor subtypes [8] and muscarinic toxins from snake ve-
noms [9]. As a matter of fact, several synthetic ligands
(mainly antagonists) do show some subtype selectivity [2],
but none is so selective as to display at least 100-fold selec-
tivity for one subtype over all the others. This can be due to
the high degree of sequence identity in the binding sites of
the five subtypes [10,11], even if it has to be considered that
a single amino acid difference in GPCR subtypes may yield
significant differences in ligand binding affinity [12]. As a
consequence, the number of muscarinic agonists and antago-
nists that have been introduced in therapy are relatively small
[2].
Muscarinic receptors are found in the central and periphe-
ral nervous system. They play an important role in several
vital functions such as control of movement, cognition,
smooth muscle tone, and glandular secretion [1]. As a conse-
quence of this vast array of actions, muscarinic receptor
agonists and antagonists have potential for therapeutic use in
a variety of pathologies such as smooth muscle hyperactivity
and neurodegenerative diseases [2–4]. However, non selec-
tive compounds, because of the large distribution of the
receptors in the body and the existence of several subtypes,
may produce several, severe, side effects.
Five distinct subtypes of muscarinic receptors have been
cloned (M1–M5) but only four have been characterized phar-
macologically (M1–M4) [5]. Fundamental information on
muscarinic receptor subtypes has been collected from a va-
riety of more or less selective synthetic ligands [6,7], trans-
Given the situation described above, it is not surprising
that research on muscarinic ligands is still very active [6,7] in
particular on antagonists [13–21]. Indeed, selective com-
pounds are badly needed both for further characterization of
subtypes and for therapeutic applications.
In the past decade, we have been engaged in the synthesis
and study of a series of muscarinic antagonists that are amino
esters of 2,2-diphenyl-2-ethylthio-acetic acid such as 1 and 2
* Corresponding author.
E-mail address: serena.scapecchi@unifi.it (S. Scapecchi).
0014-827X/$ - see front matter © 2004 Elsevier SAS. All rights reserved.
doi:10.1016/j.farmac.2004.08.003

972
S. Scapecchi et al. / IL FARMACO 59 (2004) 971–980
ligands designed according to the approach suggested by
Jacobson et al. [27]. In brief, the approach consists in the
decoration of known ligands with suitably tethered basic or
acidic functions, to explore the space vicinal to the receptor
recognition site which should be less conserved in the diffe-
rent subtypes. It is predicted that when the linker allows
interaction with a complementary acid or basic function, a
sudden increase of the affinity shows up.
Therefore, we have designed the series of compounds
(3–20) shown in Chart 1, where 2,2-diphenyl-2-ethylthio-
acetic acid has been esterified by N′-piperazine ethanol and
N-piperidine ethanol moieties carrying an N,N-diethylamino
group linked by methylene or oxygenated chains of varying
lengths. Such compounds would represent probes to check
the presence of anionic binding sites in the proximity of the
recognition site of the receptor, exploiting possible differen-
ces in this region of the muscarinic receptor subtypes.
2. Chemistry
Chart 1
.
The reaction pathways used to synthesize compounds 3–9
are reported in Scheme 1. Compounds 3, 4, 5, 7, 8 were
obtained starting from 2-hydroxyethylpiperazine whose ni-
trogen was quantitatively protected as tert-butoxycarbonyl
(BOC) derivative, and then reacted with 2,2-diphenyl-2-
ethylthio-acetylchloride [25] to afford 21. The protecting
group was quantitatively removed with HCl 6N to give com-
pound 22 that was then reacted with the appropriate chlora-
(Chart 1) [22–26]. Such compounds, while being functio-
nally very potent and selective on M1 and M2 with respect to
the M3 subtypes [25], failed to show selectivity in binding
studies [26].
Aiming to modulate the affinity and potency of the series,
we reasoned that the high degree of sequence identity in the
binding sites of the five subtypes could be circumvented by
Scheme 1
.

S. Scapecchi et al. / IL FARMACO 59 (2004) 971–980
973
mines. 2-Chloroethylamine is commercially available, while
other chloramines (n = 3 [28], 4 [29], 8 [30], 10 (23)) were
obtained by treating the corresponding amino alcohols with
SOCl2 in CHCl3 stabilized with amylene. In turn, the amino
alcohols (n = 3 [31], 4 [32], 8 [30], 10 [33]) were synthesized
from the corresponding commercially available chloro or
bromoalcohols and diethylamine in anhydrous CH3CN in
presence of K2CO3 according to a previously reported proce-
dure [34]. Compounds 6 and 9 were obtained by an alterna-
tive pathway treating 2-hydroxyethylpiperazine with the pro-
per chloramines (n = 6 [35], 12 (24)) and then coupling the
obtained compounds 28 and 29 with 2,2-diphenyl-2-
ethylthio-acetylchloride.
mercially available 4-chlorobutyryl chloride and 3-chloro-
propionyl chloride, respectively, with 3-diethylpropano-
lamine (25) and 12-diethyldodecanolamine (27)[33] and
from the reaction of 5-bromopentanoyl chloride [36] and
2-diethyletanolamine (26). Their reaction with 2-hydroxy-
ethylpiperazine gave 30–32, which, after reaction with 2,2-
diphenyl-2-ethylthio-acetylchloride, afforded compounds
10–12.
The key intermediates in the synthesis of the piperidine
compounds 13–20 (Scheme 2) were pyridine amines 35–42.
Compounds 35 [37], 36 [38] and 37 [38] have already been
described, and compounds 38–41 were synthesized with the
method reported in literature [38], using the proper chlora-
mines (n = 5 [39], 7 [40], 9 (33), 11 (34)). Compound 42 was
obtained from the reaction of isonicotinoyl chloride [41] with
In the same scheme, the synthesis of compounds 10–12 is
also reported. Halo esters 25–27 were obtained from com-
Scheme 2
.

974
S. Scapecchi et al. / IL FARMACO 59 (2004) 971–980
the appropriate amino alcohol. The pyridine ring of com-
pounds 35–42 was then hydrogenated at 48 psi for 48 h using
PtO2 as a catalyst and a mixture of MeOH and HCl conc. as a
solvent. Reaction with bromoethanol in anhydrous CH3CN
gave the corresponding alcohols 43–50 that were then reac-
ted with 2,2-diphenyl-2-ethylthio-acetyl chloride affording
compounds 13–20.
There is indeed a small increase of affinity for piperazinic
compounds with n = 8 (7), 10 (8), 12 (9) and for piperidinic
compounds with n = 6 (16), 8 (17) but, very likely, this
increase is due to the contribution of the methylene groups
rather than to new ionic interactions. This is somehow confir-
med by the fact that the increase in affinity is similar in all
subtypes and the new molecules completely lack selectivity.
Since, as discussed in the Introduction, the parent com-
pounds 1 and 2 show a fairly good potency and selectivity in
functional assays, we decided to check the functional activity
of selected compounds 7 and 17, which are among those
showing an increase in affinity. The results are reported in
Table 2. Unlike the parent compounds, 7 and 17 are only
modest functional antagonists and do not show any subtype
selectivity, confirming that our attempt was unsuccessful.
However, these two compounds do show some peculiar
features that recall those of a related series of 2,2-diphenyl-
2-ethylthio-acetic acid esters described in a previous work
3. Results and discussion
Binding affinity of the synthesized compounds 3–20 and
of the parent compounds 1, 2 was measured on CHO-
expressed human muscarinic receptor subtypes (hM1–hM5)
and the results are reported in Table 1. Compared to the
parent compounds, none of the newly synthesized molecules
showed the sharp increase in affinity expected in the case of a
successful ionic interaction of the introduced amine function.
Table 1
Binding affinity of compounds 1–20 on CHO expressed human muscarinic receptors a
N
X
Z
pKi
N
–
HM1
HM2
HM3
HM4
HM5
1
–
–
6.47 0.08
6.56 0.04
5.82 0.39
6.03 0.19
6.64 0.06
6.07 0.19
7.17 0.01
7.30 0.01
7.19 0.02
6.23 0.01
5.92 0.20
6.61 0.08
6.10 0.12
6.13 0.11
6.49 0.05
7.07 0.02
7.00 0.03
6.85 0.04
6.43 0.09
7.03 0.02
6.25 0.10
6.48 0.06
5.79 0.30
6.39 0.14
6.68 0.04
5.73 0.57
6.59 0.06
7.00 0.02
7.07 0.02
6.46 0.10
5.65 0.65
6.47 0.07
6.29 0.09
6.67 0.05
6.63 0.07
7.08 0.02
6.60 0.07
6.83 0.03
6.23 0.13
6.58 0.10
6.44 0.08
6.43 0.07
5.93 0.62
5.72 0.42
6.38 0.08
5.95 0.15
6.46 0.04
6.46 0.04
6.79 0.04
5.67 0.23
5.16 1.10
6.63 0.07
5.82 0.20
5.90 0.11
6.39 0.06
6.46 0.07
6.12 0.14
6.38 0.10
6.40 0.10
6.27 0.07
5.95 0.11
6.04 0.16
5.51 0.43
5.32 0.82
6.14 0.07
5.59 0.35
6.84 0.01
7.18 0.01
7.29 0.01
5.34 0.68
5.36 0.66
6.80 0.04
5.70 0.26
5.86 0.17
6.31 0.06
6.94 0.01
6.72 0.03
6.89 0.02
6.27 0.09
6.53 0.04
6.25 0.14
6.28 0.10
5.73 0.39
5.39 0.85
6.31 0.09
5.80 0.30
6.82 0.03
6.77 0.04
6.95 0.03
5.71 0.40
5.44 0.77
6.58 0.07
5.90 0.24
5.98 0.21
6.54 0.05
6.69 0.04
6.31 0.13
6.41 0.01
6.35 0.12
6.63 0.05
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
–
–
–
N
(CH2)n
2
N
(CH2)n
3
N
(CH2)n
4
N
(CH2)n
6
N
(CH2)n
8
N
(CH2)n
10
12
–
N
(CH2)n
N
(CH2)3COO(CH2)3
(CH2)4COO(CH2)2
(CH2)2COO(CH2)12
(CH2)n
N
–
N
–
CH
CH
CH
CH
CH
CH
CH
CH
2
(CH2)n
3
(CH2)n
4
(CH2)n
6
(CH2)n
8
(CH2)n
10
12
–
(CH2)n
COO(CH2)6
a 3–6 Experiments in duplicate.
Table 2
Functional assays of compounds 1, 2, 7, 17 on muscarinic receptor subtypes
N
pKb SE (pA2 SE)
M1 Rabbit vas deferens
(7.79 0.01)
M2 Guinea pig atrium
(7.93 0.03)
(9.12 0.03)
7.17 0.09
M3 Guinea pig ileum
M4 Guinea pig lung
1
(5.88 0.09)
(6.50 0.07)
5.94 0.01
<5.52
–
2
(8.97 0.06)
–
7a
6.47 0.09
5.58 0.01
5.40 0.12
17a
<5.52
6.04 0.08
a Compounds 7 and 17 behave also as putative muscarinic agonists on M1 and M2 receptors. See text for details.

S. Scapecchi et al. / IL FARMACO 59 (2004) 971–980
975
[42]. In fact, compounds 7 and 17 behave also as putative
muscarinic agonists on M1 and M2 receptors since, after 1 h
incubation at 10–5 M, they strongly reduce the twitch induced
by electrical stimulation of rabbit vas deferens and guinea pig
heart, as classical muscarinic agonists do, even if they, like
the compounds described previously, are characterized by
large size structures typical of muscarinic antagonists. The
reasons for such behavior are under investigation.
stirred at r.t. for 12 h; the mixture was then treated with 10%
NaOH and the product extracted in CHCl3. The organic
phase was anhydrified over Na2SO4, the solvent removed
under vacuum and the title compound, obtained quantitati-
vely as an oil, used as such for the next reaction.
1H-NMR (CDCl3) d: 1.10 (t, J = 3.33 Hz, 3H, SCH2CH3);
2.25–2.45 (m, 6H, 4 CH piperazine; SCH2CH3); 2.55 (t,
J = 1.67 Hz, 2H, COOCH2CH2N); 2.73–2.83 (m, 4H, 4 CH
piperazine); 4.30 (t, J =1.67 Hz, 2H, COOCH2); 7.20–7.50
(m, 10H, aromatics).
4. Experimental section
4.1. Chemistry
4.4. 12-(Chlorododecyl)diethylamine (24)
All melting points were taken on a Büchi apparatus and
are uncorrected. Infrared spectra were recorded with a Perki-
n–Elmer 681 or a Perkin–Elmer Spectrum RX I FT-IR spec-
trophotometer in Nujol mull for solids and neat for liquids.
Unless otherwise stated, NMR spectra were recorded on a
Gemini 200 spectrometer, and chromatographic separations
were performed on a silica gel column by gravity chromato-
graphy (Kieselgel 40, 0.063–0.200 mm; Merck) or flash
chromatography (Kieselgel 40, 0.040–0.063 mm; Merck).
Although the IR spectra data are not always included (be-
cause of lack of unusual features), they were obtained for all
reported compounds and are consistent with the assigned
structures. Yields are given after purification, unless othe-
rwise stated. Where analyses are indicated by symbols, the
analytical results are within 0.4% of the theoretical values.
Compounds were named following IUPAC rules as applied
by Beilstein-Institut AutoNom (version 2.1), a software for
systematic names in organic chemistry. When reactions were
performed in anhydrous conditions, the mixtures were main-
tained under nitrogen.
One equivalent of 12-diethylaminododecanol [33] was
dissolved in CHCl3 stabilized with amylene, added with an
excess of SOCl2, and refluxed for 2 h. The excess of SOCl2
was eliminated under vacuum, the crude mixture basified
with Na2CO3 and the chlorododecyldiethylamine (24) ex-
tracted with CHCl3. The organic phase was dried over
Na2SO4 and the solvent removed under vacuum. The title
compound was obtained as oil.Yield 73%. 1H-NMR (CDCl3)
d: 1.02 (t, J = 7.14 Hz, 6H, 2NCH2CH3); 1.22–1.50 (m,
18 H); 1.76 (m, 2H); 2.40 (t, J = 7.69 Hz, 2H, CH2CH2N);
2.68 (q, J = 7.14 Hz, 4H, 2NCH2CH3); 3.53 (t, J = 6.78 Hz,
2H, CH2Cl,). Anal. (C16H34ClN) C,H,N.
Compounds 23, 33 and 34 were obtained in the same way
and their spectroscopic characteristics are consistent with the
proposed structures.
4.5. 4-Chloro-butyric acid 3-diethylamino-propyl ester
(25)
One equivalent of 3-diethylaminopropanol was dissolved
in anhydrous CH2Cl2, cooled at 0 °C and added dropwise
with 1 equivalent of 4-chlorobutirrylchloride; the solution
was stirred at r.t. for 48 h. The mixture was basified with a
10% NaOH solution and extracted with CH2Cl2. The organic
phase was dried over Na2SO4 and the solvent removed under
vacuum afforded compound 25 as oil.Yield 100%. 1H-NMR
(CDCl3) d: 0.99 (t, J = 7.10 Hz, 6H, NCH2CH3); 1.68–1.82
(m, 2H, ClCH2CH2CH2CO); 2.00–2.14 (m, 2H,
OCH2CH2CH2N); 2.43–2.54 (m, 8H, CH2COO, CH2NEt2,
2NCH2CH3); 3.58 (t, J = 6.60 Hz, 2H, ClCH2); 4.11 (t,
J = 6.60 Hz, 2H, COOCH2). Anal. (C11H22ClNO2) C,H,N.
Compounds 26 and 27 were obtained in the same way and
their spectroscopic characteristics are consistent with the
proposed structures.
4.2. 4-[2-(2-Ethylsulfanyl-2,2-diphenylacetoxy)ethyl]
piperazine-1-carboxylic acid tert-butyl ester (21)
A total of 1.92 g (9 mmol) of (tert-BOC)hydroxy-
ethylpiperazine were reacted with 2.62 g (9 mmol) of 2,2-
diphenyl-2-ethylthioacetylchloride [25] in anhydrous
CH2Cl2 at r.t. for 2 h. The solution was then basified with
10% NaOH and extracted with CHCl3. The crude product
was purified by flash chromatography using abs.
EtOH:CH2Cl2:Et2O:petroleum ether = 180:360:360:900 as
eluting system. The oily compound, 39% yield, was used as
such in the next reaction.
1H-NMR (CDCl3) d: 1.10 (t, J = 6.67 Hz, 3H, SCH2CH3);
1.45 (s, 9H, C(CH3)3 ); 2.16–2.26 (m, 4H, 4 CH piperazine);
2.34 (q, J = 6.67 Hz, 2H, SCH2CH3); 2.53 (t, J =3.33 Hz, 2H,
COOCH2CH2N); 3.21–3.35 (m, 4H, 4 CH piperazine); 4.28
(t, J =3.33 Hz, 2H, COOCH2); 7.15–7.45 (m, 10H, aroma-
tics).
4.6. 2-[4-(6-Diethylaminohexyl)piperazin-1-yl]ethanol (28)
One equivalent of 6-chlorohexyldiethylamine dissolved in
abs. EtOH was added with N-2-hydroxyethylpiperazine
(1 equivalent) and with anhydrous NEt3 (2 equivalents) and
the mixture refluxed for 24 h. The solvent was removed under
reduced pressure and the resulting thick oil was heated at
80 °C for 20 h; 20 ml of a saturated solution of Na2CO3 were
4.3. Ethylsulfanyldiphenylacetic acid 2-piperazin-1-ylethyl
ester (22)
A total of 0.28 g (0,7 mmol) of 21 was dissolved in 10 ml
of AcOEt added with 6 ml of 6N HCl. The reaction was

976
S. Scapecchi et al. / IL FARMACO 59 (2004) 971–980
added and the resulting suspension extracted with CH2Cl2.
After anhydrification over Na2SO4, the solvent was removed
under vacuum and the mixture purified by flash chromatogra-
4.9. Ethylsulfanyldiphenylacetic acid
2-[4-(8-diethylaminooctyl)piperazin-1-yl]ethyl ester (7)
A total of 0.6 g (1.46 mmol) of 22 was dissolved in 15 ml
of abs. EtOH, added with (8-chlorooctyl)d iethyl amine [30]
0.37 g (1.46 mmol) and anhydrous NEt3 (2 equivalents,
0.4 ml) and refluxed for 3 days. The solvent was removed and
the residue treated with 10% NaOH solution and extracted
with CHCl3; the organic phase was dried over Na2SO4 and
CHCl3 evaporated under vacuum. The title compound was
obtained as oil after two column chromatographic separa-
tions using NH4OH:abs. EtOH:CH2Cl2: Et2O:petroleum
phy
using
NH4OH:abs.
EtOH:CH2Cl2:petroleum
ether = 45:225:600:90 as eluting system. Oil; yield 16%.
1H-NMR (CDCl3) d: 1.02 (t, J = 6.96 Hz, 6H, 2NCH2CH3);
1.28–1.38 (m, 4H); 1.42–1.55 (m, 4H); 2.28–2.58 (m, 18H,
HOCH2CH2N, CH2NEt2, Npip-CH2, NCH2CH3, 8H pipera-
zine); 2.95 (bs, 1H, OH); 3.6 (t, J = 5.49 Hz, 2H, CH2OH).
Anal. (C16H35N3O) C,H,N.
Compound 29 was obtained in the same way and its
spectroscopic characteristics are consistent with the pro-
posed structure.
ether
=
9.9:180:360:360:900
and
NH4OH:abs.
EtOH:CH2Cl2: petroleum ether = 8:65:340:60, respectively,
as eluting system.Yield 22%. 1H-NMR (CDCl3) d: 0.94–1.56
(m, 9H, SCH2CH3, 2NCH2CH3); 1.19–1.56 (m, 14H); 1.65–
1.87 (m, 2H); 2.18–2.66 (m, 14H, 8CH piperazine,
SCH2CH3, 2NCH2CH3); 3.52 (t, J = 1.67 Hz, 2H,
COOCH2CH2N); 4.32 (t, J = 1.67 Hz 2H, COOCH2); 7.20–
7.37 (m, 6H, aromatics); 7.37–7.54 (m, 4H, aromatics). Anal.
(C35H53N3O2S) C,H,N.
Compounds 3–5 and 8 were obtained in the same way
starting from compound 22 and the appropriate chloroalkyl-
diethylamine (n = 2, 3, 4, 10) and their spectroscopic charac-
teristics are consistent with the proposed structures.
4.7. 4-[4-(2-Hydroxyethyl)piperazin-1-yl]butyric acid
3-diethylaminopropyl ester (30)
Compound 25 (1 equivalent) was dissolved in toluene and
2-hydroxyethylpiperazine (1 equivalent) and an excess of
K2CO3 were added. The mixture was heated at 100 °C under
stirring for 2 days, then K2CO3 was filtered off, the solvent
evaporated under reduced pressure and the oily product puri-
fied by flash chromatography using NH4OH:abs. EtOH:
CH2Cl2:petroleum ether = 8:65:340:60 as eluting system.
Compound 30 was obtained as oil in 100% yield. 1H-NMR
(CDCl3) d: 1.01 (t, J = 7.10 Hz, 6H, 2NCH2CH3); 1.70–1.85
4.10. Diethyl(6-pyridin-4-ylhexyl)amine (38)
(m,
5H,
NCH2CH2CH2CO+OCH2CH2CH2N+OH);
2.30–2.56 (m, 20H); 3.60 (t, J =5.30 Hz, 2H, CH2OH); 4.11
(t, J = 6.20 Hz, 2H, COOCH2,). Anal. (C17H35N3O3) C,H,N.
Compounds 31 and 32 were obtained in the same way and
their spectroscopic characteristics are consistent with the
proposed structures.
Butyllithium in hexane (3.2 ml of a 1.6 M solution;
5 mmol) was added to 0.5 g (5 mmol) of diisopropylamine
dissolved in THF (15 ml), cooled at 0 °C and the mixture
stirred for 30 min. 4-Methylpyridine (0.45 g, 5 mmol) was
then added at 0 °C and the mixture stirred for 30 min; then the
electrofile 5-chloropentyldiethyl amine [39] was added
(1.52 g, 5.5 mmol) and the mixture warmed up to r.t. and
treated with a saturated solution of NH4Cl. The solution was
extracted with diethyl ether and the organic phase dried. The
oily product was purified by flash chromatography using
4.8. Ethylsulfanyldiphenylacetic acid
2-[4-(6-diethylaminohexyl)piperidin-1-yl]ethyl ester (6)
Compound 28 (0.04 g) was reacted with 2-ethylthio-2,2-
diphenylacetyl chloride [25] (0.041 g 0.14 mmol) in 20 ml of
anhydrous CH2Cl2 under N2 at r.t. for 6 days. The reaction
mixture was then treated with 10% NaOH solution and ex-
tracted with CH2Cl2; the organic phase was dried over
Na2SO4 and CH2Cl2 evaporated under vacuum. The title
compound was obtained as oil after flash chromatographic
separation using NH4OH:abs. EtOH:CH2Cl2:petroleum
ether = 8:65:340:60 as eluting system. Yield 19%. 1H-NMR
(CDCl3) d: 0.99–1.18 (m, 9H, SCH2CH3, 2NCH2CH3);
1.25–1.35 (m, 4H); 1.46–1.56 (m, 4H); 2.25–2.65 (m, 20H,
8CH piperazine, SCH2CH3; 2NCH2CH3, CH2NEt2,
OCH2CH2N, Npip-CH2); 4.32 (t, J = 5.49 Hz 2H,
COOCH2); 7.27–7.38 (m, 6H, aromatics); 7.38–7.50 (m, 4H,
aromatics). Anal. (C32H49N3O2S) C,H,N.
NH4OH:abs.
EtOH:CHCl2:Et2O:petroleum
ether = 25:45:180:180:450 as eluting system. Yield 18%.
1H-NMR (CDCl3) d: 0.84 (t, J = 7.30 Hz, 6H, 2NCH2CH3);
1.15–1.22 (m, 4H); 1.38–1.50 (m, 2H); 1.60–1.80 (m, 2H);
2.23 (t, J = 7.30 Hz, 2H, CH2NEt2); 2.29–2.45 (m, 6H,
2NCH2CH3, pyridine-CH2,); 6.92 (d, J = 5.86 Hz, 2H pyri-
dine); 8.28 (d, J = 5.86 Hz, 2H pyridine). Anal. (C15H26N2)
C,H,N.
Compounds 37 [38], 39, 40 and 41 were obtained in the
same way, treating 4-methylpyridine with the appropriate
electrofile. Their spectroscopic characteristics are consistent
with the proposed structures.
4.11. Isonicotinic acid 6-diethylaminohexyl ester (42)
Compounds 9–12 were obtained in the same way and their
spectroscopic characteristics are consistent with the pro-
posed structures.
A total of 0.8 g (4.6 mmol) of 6-diethylaminohexanol was
dissolved in CHCl3, and anhydrous NEt3 (2 equivalent,
1.3 ml) was added; the reaction was cooled at 0 °C and

S. Scapecchi et al. / IL FARMACO 59 (2004) 971–980
977
isonicotinoylchloride in CHCl3 (0.76 g, 4.6 mmol) was ad-
ded. The mixture was stirred for 36 h at r.t., treated with 10%
NaOH solution, extracted with CHCl3 and the organic phase
dried. The oily product was then purified by flash chromato-
graphy using NH4OH:abs. EtOH:CH2Cl2:Et2O:petroleum
ether = 9.9:180:360:360:900 as eluting system. Yield 26%.
1H-NMR (CDCl3) d: 1.00 (t, 6H, 2NCH2CH3, J = 7.10 Hz);
1.30–1.55 (m, 6H); 1.74–1.81 (m, 2H); 2.41 (t, J = 7.33 Hz,
2H, CH2NEt2,); 2.51 (q, J = 7.10 Hz, 4H, 2NCH2CH3); 4.34
(t, J = 6.59 Hz, 2H, COOCH2); 7.83 (d, J = 4.76 Hz, 2H
pyridine); 8.76 (d, J = 4.76 Hz, 2H pyridine). Anal.
(C16H26N2O2) C,H,N.
compound was obtained as oil after chromatographic separa-
tion using NH4OH:abs. EtOH:CH2Cl2:petroleun
ether = 8:65:340:60 as eluting system. Yield 68%. 1H-NMR
(CDCl3) d: 1.03–1.18 (m, 9H, SCH2CH3, 2NCH2CH3);
1.20–1.28 (m, 10H); 1.42–1.65 (m, 4H); 1.81–1.96 (m, 2H);
2.38 (q, J = 7.33 Hz, 2H, SCH2CH3); 2.55–2.85 (m, 11H);
4.32 (t, J = 5.50 Hz, 2H, COOCH2); 7.27–7.35 (m, 6H,
aromatics); 7.35–7.45 (m, 4H, aromatics). Anal.
(C33H50N2O2S) C,H,N.
Compounds 13–15 and 17–20 were obtained in the same
way and their spectroscopic characteristics are consistent
with the proposed structures.
4.12. 2-[4-(6-Diethylaminohexyl)piperidin-1-yl]ethanol
5. Pharmacology
(46)
A total of 1.84 g (8 mmol) of 38 were dissolved into
MeOH (50 ml) and conc. HCl (2 ml), added with 0.05 g of
PtO2 and hydrogenated in a Parr apparatus for 24 h at 48 psi.
At the end of the reaction, the catalyst was filtered off and the
solvent removed under vacuum. The residue was treated with
10% NaOH solution, extracted with Et2O and the organic
phase dried and evaporated under reduced pressure. The oily
compound was used as such for the next reaction.Yield 42%.
1H-NMR (CDCl3) d: 0.98 (t, J = 7.10 Hz, 6H, 2NCH2CH3 );
1.15–1.43 (m, 11H); 1.43–1.61 (m, 2H piperidine); 2.02–
2.18 (m, 2H); 2.24–2.62 (m, 8H); 2.95–3.08 (m, 2H).
5.1. Binding
5.1.1. Cell culture and membrane preparation
Chinese hamster ovary cells stably expressing cDNA en-
coding human muscarinic M1–M5 receptors were generously
provided by Prof. R. Maggio (Department of Neuroscience,
University of Pisa, Italy). Growth medium consisted in Dul-
becco’ modified Eagle’s medium supplemented with 10%
fetal calf serum (Gibco, Grand Iland, N.Y.), 100 units/ml
each of penicillin G and streptomicyn, 4 mM glutamine
(SigmaAldrich, Milano, Italy) and non essential amino acids
(Sigma Aldrich, Milano, Italy) and 50 µg/ml of geneticin
(Gibco, Grand Iland, N.Y.) 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 30 s using an Ultra-Turrax
(setting 5). The pellet was sedimented 17000 × g for 15 min
at 4 °C and the membranes were resuspended in the same
buffer, rehomogenized with Ultra-Turrax and stored at
–80 °C [43]. An aliquot was taken for the assessment of
protein content according to the method of Bradford [44]
using the Bio-Rad protein assay reagent (Bio-Rad Laborato-
ries, Munchen, West Germany) and bovine serum albumin
was used as the standard.
To 0.07
g (0,3 mmol) of diethyl(6-piperidin-4-
yl)hexylamine obtained as described above, dissolved in abs.
EtOH, anhydrous NEt3 (0.084 ml, 2 equivalent) and
2-bromoethanol (0.021 ml, 1 equivalent) were added and the
resulting mixture was refluxed for 24 h. The solvent was
removed and the residue treated with water and extracted
with CHCl3. The organic phase was dried, the solvent remo-
ved and the crude product purified by column chromatogra-
phy
using
NH4OH:abs.
EtOH:CH2Cl2:petroleum
ether = 45:225:600:90 as eluting system. Oil, yield 35%.
1H-NMR (CDCl3) d: 1.02 (t, J = 7.10 Hz, 6H, 2NCH2CH3);
1.20–1.41 (m, 12H); 1.43–1.62 (m, 2H); 1.64–1.68 (m, 2H);
2.01 (t, J = 7.69 Hz, 2H, CH2NEt2); 2.46–2.58 (m, 8H,
2(NCH2CH3), NCH2CH2OH, piperidine-CH2,); 2.82–2.91
(m, 2H piperidine); 3.59 (t, J = 5.50 Hz, 2H, CH2OH). Anal.
(C17H36N2O) C,H,N.
5.1.2. Binding assay
Compounds 43–45 and 47–50 were obtained in the same
way and their spectroscopic characteristics are consistent
with the proposed structures.
The radioligand binding assay was run in polypropylene
96-well plates (Sarstedt, Verona, Italy) and performed for
120 min at r.t. In a final volume of 0.25 ml in 25 mM sodium
phosphate buffer containing 5 µM MgCl2 at pH 7.4. Final
membrane protein concentrations were 30 µg/ml (M1),
70 µg/ml (M2), 25 µg/ml (M3), 50 µg/ml (M4) and 25 µg/ml
(M5).
4.13. Ethylsulfanyldiphenylacetic acid
2-[4-(6-diethylaminohexyl)piperidin-1-yl]ethyl ester (16)
Compound 46 (0.03 g, 0.11 mmol) was refluxed for 48 h
under N2 with 2-ethylthio-2,2-diphenylacetylchloride [25]
(0.031 g, 0.11 mmol) in 20 ml of anhydrous CH2Cl2. The
reaction mixture was then treated with 10% NaOH solution
and extracted with CH2Cl2; the organic phase was dried over
Na2SO4 and CH2Cl2 evaporated under vacuum. The title
In homologous competition curves, [3H]-NMS was pre-
sent at 0.2 nM in tubes containing an increasing concentra-
tion of unlabeled NMS (0.03–1000 nM) and at 0.075–0.2 nM
in tubes without unlabeled ligand. In heterologous competi-
tion curves, fixed concentrations of the tracer (0.2 nM) were
displaced by increasing concentrations of several unlabeled

978
S. Scapecchi et al. / IL FARMACO 59 (2004) 971–980
ligands (0.01–1000 µM); all measurements 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 r.t. under air flow, added of 25 µl of scintillation
liquid (Microscint-20, Perkin Elmer Life Science, Boston,
MA) and counted by TopCount NXT Microplate Scintilla-
tion Counter (Perkin Elmer Life Science, Boston, MA). The
binding data were analyzed by the weighted least-squares
iterative curve fitting LIGAND [44] to obtain the affinity
constant (Ki) and the binding capacity (Bmax).
being discarded and the second one being taken as the
control.
5.2.3. 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 plati-
num 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 cumu-
lative dose–response curve to APE was constructed.
5.2.4. 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,
then two cumulative dose–response curves toAPE (0.01, 0.1,
1, 10, 100 µM) were obtained at 45 min intervals, the first one
being discarded and the second one being taken as the
control.
5.2. Functional tests
5.2.1. General considerations
Male guinea pigs (200–300 g) and male New Zealand
white rabbits (3.0–3.5 kg) were killed by cervical disloca-
tion. 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 refe-
rence agonist. The concentration of agonist in the organ bath
was increased approximately three-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 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 experi-
ments in which tissues did not receive any antagonist were
run in order to check any variation in sensitivity.
5.2.5. Rabbit stimulated vas deferens
This preparation was set up according to Eltze [45]. 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 mounted in PSS with
the following composition (mM): NaCl (118.4), KCl (4.7),
CaCl2 (2.52), MgCl2 (0.6), KH2PO4 (1.18), NaHCO3 (25),
glucose (11.1); 10–6 M yohimbine and 10–8 M tripitramine
were included to block alpha2-adrenoceptors and M2 musca-
rinic receptors, respectively. The solution was maintained at
30 °C and tissues were stimulated through platinum electro-
des by square-wave pulses (0.1 ms, 2 Hz, 10–15V). Contrac-
tions were measured isometrically after tissues were equili-
brated for 1 h, then a cumulative dose–response curve to
pCl-McN-A-343 was constructed.
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).
Acknowledgements
This research was supported by funds from the Italian
Ministry of University and Scientific Research (MURST).
5.2.2. Guinea pig ileum
Two-centimeter-long portions of terminal ileum were ta-
ken at about 5 cm from the ileum–cecum junction and moun-
ted 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 chan-
ges 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
References
[1] M.P. Caulfield, Muscarinic receptors-characterization, coupling and
function, Pharmac. Ther. 58 (1993) 319–379.
[2] R.M. Eglen, A. Choppin, N. Watson, Therapeutic opportunities from
muscarinic receptor research, TiPS 22 (2001) 409–414.

S. Scapecchi et al. / IL FARMACO 59 (2004) 971–980
979
[3] R.M. Eglen, A. Choppin, M.P. Dillon, S. Hedge, Muscarinic ligands
and their therapeutic potential, Curr. Opin. Chem. Biol. 3 (1999)
426–432.
[4] M.F. Siddiqui, A.I. Levey, Cholinergic therapies in Alzheimer’s dis-
eases, Drug Future 24 (1999) 417–424.
[5] M.P. Caufield, N.J. Birdsall, International union of Pharmacology
XVII. Classification of muscarinic acetylcholine receptors, Pharma-
col. Rev. 50 (1998) 279–290.
[6] C.C. Felder, F.P. Bymaster, J. Ward, N. DeLapp, Therapeutic oppor-
tunities for muscarinic receptors in the central nervous system, J.
Med. Chem. 43 (2000) 4333–4353.
[7] D.P. Zlotos, W. Bender, U. Holzgabe, Muscarinic receptor agonists
and antagonists, Exp. Opin. Ther. Patents 9 (1999) 1029–1053.
[8] J. Wess, Novel insights into muscarinic acetylcholine receptor func-
tion using gene targeting technology, TiPS 24 (2003) 414–420.
[9] K.N. Bradley, Muscarinic toxins from the green mamba, Pharmacol.
Ther. 85 (2000) 87–109.
[10] E.C. Hulme, Z.-L. Lu, M. Bee, C.A.M. Curtis, J. Saldanha, The
conformational switch in muscarinic acetylcholine receptors, Life
Sci. 68 (2001) 2495–2500.
[11] L. Shi, J.A. Javitch, The binding site of aminergic G protein-coupled
receptors: the transmembrane segments and second extracellular loop,
Ann. Rev. Pharmacol. Toxicol. 42 (2002) 437–467.
[12] T. Kenakin, The classification of seven transmembrane receptors in
recombinant expression systems, Pharmacol. Rev. 48 (1996) 413–
463.
[13] J. Clader, W. Billard, H. Binch, L.Y. Chen, G. Crosby, R.A. Duffy,
J. Ford, J. Kozlowski, J. Lachowicz, S. Li, C. Liu, S. McCombie,
S. Vice, G. Zhou, W. Greenlee, Muscarinic M2 antagonists: anthranil-
amide derivatives with exceptional selectivity and in vivo activity,
Bioorg. Med. Chem. 12 (2004) 319–326.
[14] Y. Ogino, N. Ohtake, K. Kobayashi, T. Kimura, T. Fujikawa, T. Hase-
gawa, K. Noguchi, T. Mase, Muscarinic M3 receptor antagonists with
(2R)-2-((1R)-3,3-difluorocyclopentyl)-2-hydroxyphenylacetamide
structure. Part 2, Bioorg. Med. Chem. Lett. 13 (2003) 2167–2172.
[15] Y. Sagara, T. Kimura, T. Fujikawa, K. Noguchi, N. Ohtake, Identifi-
cation of novel muscarinic M-3 selective antagonists with a confor-
mationally restricted hyp-pro spacer, Bioorg. Med. Chem. Lett. 13
(2003) 57–60.
[16] T.M. Bohme, C. Kelm, K. Kreutzmann, M. Linder, T. Dingermann,
G. Dannhardt, E. Mutscler, G. Lambrecht, Structure–activity relation-
ships on dimethindene derivatives as new M2-selective muscarinic
receptor antagonists, J. Med. Chem. 46 (2003) 856–867.
[17] C.D. Boyle, J.E. Lachowicz, Orally active and selective benzylidene
ketal M2 muscarinic receptor antagonists for the treatment of Alzhe-
imer’s disease, Drug Dev. Res. 56 (2002) 310–320.
[23] J. Hu, S.Z. Wang, S. Scapecchi, F. Gualtieri, E.E. El-Fakahany, Car-
dioselective derivatives of 2,2-diphenyl-2-ethylthioacetate do not dis-
criminate between m2 and m3 muscarinic receptors expressed in
CHO cells, Pharmacology 50 (1995) 273–285.
[24] S. Scapecchi, P. Angeli, S. Dei, F. Gualtieri, G. Marucci, R. Moriconi,
F. Paparelli, M.N. Romanelli, E. Teodori, Dialkylaminoalkyl esters of
2,2-diphenyl-2-alkyltioacetic acids: a new class of potent and func-
tionally selective muscarinic antagonists, Bioorg. Med. Chem. 2
(1994) 1061–1073.
[25] S. Scapecchi, P. Angeli, A. Dei, F. Gualtieri, G. Marucci,
M.N. Romanelli, E. Teodori, N,N-diethylaminoethanol and N-methyl-
4-piperidinol esters of 2,2-diphenyl-2-ethylthioacetic acid as potent
and selective muscarinic antagonists, Pharm. Pharmacol. Lett. 2
(1993) 220–223.
[26] P. Angeli, F. Gualtieri, R. Maggio, F. Paparelli, S. Scapecchi, Affinity
profile at five cloned human muscarinic receptors (m1–m5) of a new
series of antimuscarinic drugs, Pharmaceut. Pharmacol. Lett. 3 (1993)
84–87.
[27] K.A. Jacobson, B. Fischer, A.M. van Rhee, Molecular probes for
muscarinic receptors: functionalized congeners of selective muscar-
inic antagonists, Life Sci 56 (1995) 823–830.
[28] H. Gilman, D.A. Shirley, Some derivatives of phenotiazine, J. Am.
Chem. Soc. 66 (1944) 888–892.
[29] F.P. Doyle, E.R. Stove,Alkylthioamine and quaternary salts, Beecham
Researche Laboratories Ltd., 1958, pp. GB 800963.
[30] W.P. Wetter, C. De Witt Blanton, 8-(o-Aminoalkyl)quinolines as
potential prophylactic antimalarials, J. Med. Chem. 17 (1974) 620–
624.
[31] J. Cossy, M. Guha, Intramolecular quenching of iminium ions gener-
ated by photooxidation of aminoalcohols with ketones. A new synthe-
sis of oxazines and oxazoles, Tetrahedron Lett. 35 (1994) 1715–1718.
[32] I.G. Farbenind, Method for manufacturing aminoalcohol1940, pp. DE
730237.
[33] A.J. Bitonti, Method of using triaryl-ethylene derivatives in the treat-
ment and prevention of osteoporosis, 1995 US 005691384.
[34] R.L. Hudkins, R.B. Mailman, D.L. DeHaven-Hudkins, Novel
(4-phenylpiperidinyl)-and(4-phenylpiperazinyl)alkyl-spaced esters
of 1-phenylcyclopentanecarboxylic acids as potent sigma-selective
compounds, J. Med. Chem. 37 (1994) 1964–1970.
[35] M.P. La Montagne, D. Dagli, M. Sami Khan, P. Blumbergs, Analogs
of 8-[[6-(diethylamino)hexyl]amino]-6-methoxy-4-methylquinoline
as candidate antileishmanial agents, J. Med. Chem. 23 (1980) 981–
985.
[18] Y. Sagara, T. Sagara, T. Mase, T. Kimura, T. Numazawa, T. Fujikawa,
K. Noguchi, N. Ohtake, Cyclohexylmethylpiperidinyltriphenyl-
propionamide: a selective muscarinic M3 antagonist discriminating
against the other receptor subtypes, J. Med. Chem. 45 (2002) 984–
987.
[19] T.M. Bohme, C.E. Augelli-Szafran, H. Hallak, T. Pugsley, K. Serpa,
R.D. Schwarz, Synthesis and pharmacology of benzoxazines as
highly selective antagonist at M4 muscarinic receptors, J. Med. Chem.
45 (2002) 3094–3102.
[20] O. Diouf, S. Gadeau, F. Chellé, M. Gelbcke, P. Talaga, B. Christophe,
M. Gillard, R. Massingham, M. Guyaux, A new series of M3 musca-
rinic antagonists based on the 4-amino-piperidine scaffold, Bioorg.
Med. Chem. Lett. 12 (2002) 2535–2539.
[36] R. Merchant, J.N. Wickert, C.S. Marvel, Some bromine derivatives of
pentanoic and hexanoic acids, J. Amer. Chem. Soc. 49 (1927) 1828–
1831.
[37] A.P. Phillips, The addition of amines to 4-vinylpyridine, J.Am. Chem.
Soc. 78 (1956) 4441–4443.
[38] V.I. Cohen, B.J. Gin, R.C. Reba, Facile and general synthesis of 2-,3-,
or 4-[(dialkylamino)alkyl]pyridines and piperidines, Liebigs Ann.
Chem. 7 (1993) 809–810.
[39] G. Courtois, P. Miginiac, Reaction of functional organometallic com-
pounds with gem-amino ethers and immonium salts. III. Synthesis of
o-functional tertiary amines, Bull. Soc. Chim. France 5 (1983) 148–
152.
[40] E.Y. Borisova, T.T. Vasil’eva, L.A. Lukashova, S.V. Minaeva,
E.M. Cherkasova, Synthesis of aminoamides and amino alcohols
from 1,1,1,7-tetrachloroheptane, Zhur. Organic. Khim. 11 (1975)
943–947.
[21] T.M. Bohme, C.E. Augelli-Szafran, H. Hallak, R.D. Schwarz, Ana-
logs of M4 selective synthetic muscarinic receptors antagonists: syn-
thesis, binding and pharmacokinetic properties, Med. Chem. Res. 11
(2002) 423–433.
[22] S. Scapecchi, P. Angeli, S. Dei, C. Ghelardini, F. Gualtieri,
G. Marucci, F. Paparelli, M.N. Romanelli, E. Teodori, Sar Studies on
the potent and selective muscarinic antagonist 2-ethylthio-2,2-
diphenylacetic acid N,N-diethylaminoethyl ester, Arch. Pharm. 330
(1997) 122–128.
[41] N. Leventis, M. Rawaswdeh Abdel-Monem, G. Zhang, G. Elder,
C. Sotiriou-Leventis, Tuning the redox chemistry of 4-benzoyl-n-
methylpyridinium cations through para substitution. Hammett linear
free energy relationships and the relative aptitude of the two-electron
reduced forms for h-bonding, J. Org. Chem. 67 (2002) 7501–7510.

980
S. Scapecchi et al. / IL FARMACO 59 (2004) 971–980
[42] S. Scapecchi, G. Marucci, P. Angeli, C. Bellucci, S. Dei, F. Gualtieri,
D. Manetti, M.N. Romanelli, E. Teodori, Structure–activity relation-
ships in 2,2-diphenyl-2-ethylthioacetic acid esters: unexpected ago-
nistic activity in a series of muscarinic antagonists, Bioorg. Med.
Chem. 9 (2001) 1165–1174.
[44] M.M. Bradford, A rapid and sensitive method for quantitation of
microgram quantities of protein, utilizing the principle of protein-dye
binding, Anal. Biochem. 72 (1976) 248–254.
[43] F. Dorje, J. Wess, G. Lambrecht, R. Tacke, E. Mutschler, M.R. Brann,
Antagonist binding profiles of five cloned human muscarinic receptor
subtypes, Pharmacol. Exper. Ther. 256 (1991) 727–733.
[45] M. Eltze, Muscarinic M1 and M2 receptors mediating opposite effects
on neuromuscular transmission in rabbit vas deferens, Eur. J. Pharma-
col. 151 (1988) 205–221.