Synthesis and pharmacological characterization of new chiral derivatives of muscarine and allo-muscarine

Article abstract of DOI:10.1016/S0014-827X(00)00034-3

Novel derivatives of natural muscarine and allo-muscarine, i.e. the benzyl ethers (-)-10 and (-)-12 and the benzoate (-)-13, were synthesized in very high enantiomeric excess. Target compounds were tested in vitro on guinea pig tissues, and their muscarin

Full text of DOI:10.1016/S0014-827X(00)00034-3

www.elsevier.nl/locate/farmac
Il Farmaco 55 (2000) 535–543
Synthesis and pharmacological characterization of new chiral
derivatives of muscarine and allo-muscarine
Marco De Amici a,*1, Clelia Dallanoce a, Piero Angeli b,*2, Gabriella Marucci b,
Franco Cantalamessa c, Carlo De Micheli a
a Istituto di Chimica Farmaceutica e Tossicologica, Uni6ersita` di Milano, 6iale Abruzzi, 42-20131 Milan, Italy
b Dipartimento di Scienze Chimiche, Uni6ersita` di Camerino, 6ia S. Agostino, 1-62032 Camerino, Italy
c Dipartimento di Scienze Farmacologiche e Medicina Sperimentale, Uni6ersita` di Camerino, 6ia Scalzino, 1-62032 Camerino, Italy
Received 30 November 1999; accepted 28 February 2000
Dedicated to Professor Pietro Pratesi
Abstract
Novel derivatives of natural muscarine and allo-muscarine, i.e. the benzyl ethers (−)-10 and (−)-12 and the benzoate (−)-13,
were synthesized in very high enantiomeric excess. Target compounds were tested in vitro on guinea pig tissues, and their
muscarinic potency was evaluated at M2 (heart force and rate) and M3 (ileum and bladder) receptor subtypes. The derivatives
under study were also assayed in vivo on pithed rat. In addition, muscarinic receptor heterogeneity was investigated by
determining the affinity and the relative efficacy of compounds (−)-10, (−)-12 and (−)-13 at M2 (heart force and rate) and M3
(ileum and bladder) receptor subtypes. © 2000 Elsevier Science S.A. All rights reserved.
Keywords: Muscarine derivatives; allo-Muscarine derivatives; Muscarinic receptor subtypes
1. Introduction
In the last decade we studied in depth the structure–
activity relationships of a set of chiral muscarinic ago-
nists related to the alkaloid muscarine (+)-1, whose
absolute configuration is reported in Fig. 1. Well-
defined stereochemical requirements in the molecular
structure of such muscarinic ligands revealed essentials
for a productive interaction with the complementary
receptor subsites. Among the tested chiral muscarinic
agonists, the four chiral isomers of muscarone [5] and
methylenemuscarone [6] showed, in fact, a high value of
the eudismic ratio (ER) and a spatial arrangement
around the chiral centers matching those of (+)-1. The
absolute configuration of the most potent stereoisomer
of muscarone [(−)-2] and methylenemuscarone [(−)-3]
is represented in Fig. 1. The nature of the interaction of
the hydroxy group of muscarine with the complemen-
tary receptor subsite (‘muscarinic subsite’) was further
investigated by synthesizing and testing fluoro-
muscarine (+)-4 [7] and the enantiomers of both difl-
uoromuscarine 5 [8] and desoxymuscarine 6 [9,10],
whose eutomers are depicted in Fig. 1. The pharmaco-
Muscarinic receptors are a family of G protein cou-
pled receptors widely distributed both in the central and
peripheral nervous systems, where they mediate the
actions of endogenous acetylcholine [1,2]. Muscarinic
receptors have been pharmacologically classified into
four different subtypes (M1–M4). In addition, parallel
molecular cloning studies have demonstrated the pres-
ence, in the brain and peripheral tissues, of a fifth
subtype (m5) [1], which is waiting for a pharmacological
identification. Current research in the field of mus-
carinic ligands is addressed to the design of subtype
selective agents as potential targets to disorders of
intestinal motility, cardiac and urinary bladder func-
tions, asthma, analgesia, Parkinson’s and Alzheimer’s
diseases [3,4].
1 *Corresponding author. Tel.: +39-02-2950 2263; fax: +39-02-
2951 4197; e-mail: marco.deamici@unimi.it
2 *Corresponding author.
0014-827X/00/$ - see front matter © 2000 Elsevier Science S.A. All rights reserved.
PII: S0014-827X(00)00034-3

536
M. De Amici et al. / Il Farmaco 55 (2000) 535–543
Fig. 1. Structures of the muscarinic ligands under study.
2. Chemistry
logical profile of these new chiral muscarinic agonists
was studied by means of in vitro and in vivo assays,
and the results compared to those of natural muscarine
(+)-1 [10]. The replacement of the hydroxy group of
muscarine with a fluorine atom, i.e. (+)-4, brought
about significant changes at heart force potency and at
heart rate affinity and efficacy, whereas the introduc-
tion of a second fluorine atom at C-4 greatly affected
the responses at the M2 subtype regulating heart
rate, where (+)-4 displays a 240-fold higher affinity
and a 120-fold lower efficacy than (+)-5. Furthermore,
the presence of two fluorine or hydrogen atoms at C-4,
as in 5 and 6, caused an overall drop of the enantiose-
lectivity, when compared to muscarine 1 and mus-
carone 2.
The desired target compounds (−)-10, (−)-12 and
(−)-13 were prepared following the reaction sequences
reported in Scheme 1. The two stereoisomeric iodoalco-
hols (−)-15 and (+)-16 were obtained in a 1:1 ratio
from (S)-O-benzyl lactic aldehyde (−)-14, according
to a previously described procedure [13,14]. HPLC
analysis of the (R)-(+)-MTPA esters [15] of (−)-15
and (+)-16 showed an enantiomeric excess higher than
In the course of parallel studies on the nature of the
interaction at the ‘muscarinic subsite’, interesting re-
sults were reported by Brasili et al., who synthesized
and tested some ether derivatives of deoxamuscarine
(9)-7 [11]. Among them, benzyldeoxamuscarine (9)-8
(Fig. 1) behaved as a muscarinic agonist with a pro-
nounced selectivity for the ileum M3 receptor sub-
type. On the other hand, Giannella and co-workers
have recently reported [12] that the benzoate of deoxa-
muscarine (9)-9 (Fig. 1) is a muscarinic agonist with
some degree of selectivity towards the cardiac M2 sub-
type.
In view of these outcomes, we designed new chiral
derivatives, such as the benzyl ether of natural mus-
carine (+)-1 and the benzyl ether and the benzoate of
allo-muscarine (−)-11 [13], characterized by the pres-
ence of lipophylic and sterically hindered groups at the
‘muscarinic subsite’. This paper reports the synthesis
and the pharmacological investigation of (−)-10, (−)-
12 and (−)-13 (Fig. 1) at M2 (heart) and M3 (ileum
and bladder) muscarinic receptor subtypes.
Scheme 1. (a) TPP/DEAD/PhCOOH, THF. (b) K2CO3, MeOH/H2O.
(c) C6H5CH2C(NH)CCl3/CF3SO3H, CH2Cl2. (d) (CH3)2NH, MeOH.
(e) CH3I, acetone/ether. (f) N(CH3)3, MeOH.

M. De Amici et al. / Il Farmaco 55 (2000) 535–543
537
Table 1
Potencies and intrinsic activities of compounds (+)-1, (9)-7, (9)-8, (9)-9, (−)-10, (−)-11, (−)-12 and (−)-13 at M2 and M3 muscarinic
receptors
Tissue
Guinea pig heart (M2)
Force
Guinea pig ileum (M3)
Guinea pig bladder (M3)
Rate
a
b
a
b
a
b
a
b
pD2
h
pD2
h
pD2
h
pD2
h
(+)-1
7.6890.10
5.9390.05
5.5990.08
6.5690.15
6.8790.05
5.2790.06
7.5590.12
5.0790.15
1.0
1.0
0.92
0.96
1.0
1.0
0.36
1.0
7.0790.14
1.0
7.0590.10
6.1390.07
6.8190.08
5.4190.11
6.7490.13
5.6690.07
4.9890.09
4.9190.12
1.0
1.0
1.0
0.86
1.0
1.0
1.0
0.91
5.5890.05
4.7990.05
4.8290.02
1.0
0.95
0.23
(9)-7 c
(9)-8 c
(9)-9 d
(−)-10
(−)-11 e
(−)-12
(−)-13
6.8090.07
1.0
4.8790.14
0.86
0.0
7.0790.19
4.5190.11
0.23
1.0
a −log ED50. The results are the mean9SEM, and the number of observations varies between six and ten.
b Intrinsic activity, measured by the ratio between the maximum response of the compound and the maximum response of (+)-muscarine.
c Data taken from Ref. [11].
d Data taken from Ref. [12].
e Data taken from Ref. [13].
98% for both derivatives [13]. A complete inversion of
the configuration of the hydroxy group of (−)-15 and
(+)-16 was achieved by means of the Mitsunobu reac-
tion [16], leading to the two iodoalcohols (−)-17 and
(+)-20, respectively. Stereoisomeric iodobenzyl ethers
(−)-18 and (−)-21 were smoothly prepared by react-
ing (−)-17 and (+)-20 with benzyl 2,2,2-trichloroace-
zyl deoxamuscarine (9)-8, deoxamuscarine benzoate
(9)-9 and allo-muscarine (−)-11.
Inspection of the data of Table 1 shows a decrease of
the potency values on passing from natural muscarine
(+)-1, the most potent compound in the series, to the
corresponding benzyl ether (−)-10, which still behaves
as a relatively potent full agonist at the investigated
tissues. Such a trend is more pronounced at heart force
and bladder (7- and 5-fold, respectively) than at heart
rate and ileum (2-fold at both tissue preparations). In
any case, the substitution pattern does not increase the
selectivity for ileal M3 muscarinic receptors observed on
passing from deoxamuscarine (9)-7 to its benzyl ether
(9)-8 [M3(ileum)/M2(force) selectivity=17].
On the other hand, the replacement of the hydroxy
group of allo-muscarine (−)-11 with the benzyloxy
function, i.e. (−)-12, causes a sharp variation of the
pharmacological profile at cardiac receptors. Indeed,
the benzyl ether (−)-12, at variance with the parent
compound, is a potent partial agonist at cardiac M2
receptors (pD2=7.55, h=0.36 and pD2=7.07, h=
0.23), whereas, like (−)-11, it behaves as a weak full
agonist at ileal M3 receptors (pD2=4.98, h=1). Con-
versely, the profile of the benzoate of (−)-13 is very
similar to that of allo-muscarine (−)-11, both in terms
of potency and intrinsic activity. In this instance, the
introduction of the ester function does not affect sub-
type selectivity, at variance with what observed on
moving from deoxamuscarine (9)-7 to the correspond-
ing benzoate (9)-9 [M2(force)/M3(ileum) selectivity=
14].
timidate
[17,18],
under
the
catalysis
of
trifluoromethanesulfonic acid. The use of a more con-
ventional procedure, that is benzyl bromide in the
presence of sodium hydride, substantially lowered the
reaction yield. Finally, the reaction of (−)-18 and
(−)-21 with dimethylamine followed by treatment with
methyl iodide afforded the desired quaternary ammo-
nium salts (−)-10 and (−)-12, respectively. Methio-
dide (−)-13 was in turn prepared by reaction of the
intermediate iodobenzoate (+)-19 with trimethyl-
amine. Based on the kind of reactions performed and
our previous experience in the field [9,13], we believe
that each stereoisomer has completely retained the
enantiomeric purity induced by the initial iodoetherifi-
cation process.
3. Results and discussion
Natural muscarine (+)-1 and compounds (−)-10,
(−)-12 and (−)-13 were tested in vitro on guinea pig
tissues, and their muscarinic potency (expressed as pD2)
was evaluated at M2 (heart force and rate) and M3
(ileum and bladder) receptor subtypes. The results ob-
tained, gathered in Table 1, were compared to those,
known from literature, of deoxamuscarine (9)-7, ben-
Agonists (+)-1, (−)-10, (−)-12 and (−)-13 were
also assayed in vivo on pithed rat (Table 2), in order to

538
M. De Amici et al. / Il Farmaco 55 (2000) 535–543
evaluate their muscarinic activity (expressed as ED50) at
ganglionic M1 and cardiac (heart rate) M2 receptors.
The in vivo results, obtained according to a previously
described protocol [8], confirm that natural muscarine
(+)-1 is the most potent ligand at both receptor sub-
types. The results reported in Table 2 suggest that all
the tested compounds are equally active at the gan-
glionic M1 and the heart rate M2 receptors, with the
exception of (−)-10, which is about two times more
active at the M1 subtype. A conceivable explanation for
the different activity order at heart rate between the in
vitro and the in vivo assays could be once again at-
tributed to differences in metabolism and pharmacoki-
netics among the agonists in the two preparations
[7,8,10].
Since the comparison of the sole potency of agonists
does not allow any speculation on differences among
receptor subtypes [19], muscarinic receptor heterogene-
ity was investigated by determining the affinity (KD, KB,
or KP) and the relative efficacy (er) in vitro of the
compounds under study at M2 (heart force and rate)
and M3 (ileum and bladder) receptors (Table 3).
A vertical analysis of the data reported in Table 3
shows that, among the compounds examined, natural
muscarine (+)-1 displays the highest values of affinity
at heart force and ileum, whereas derivative (−)-12
shows its peak values at heart rate and bladder. Com-
pound (−)-10, the benzyl ether of natural muscarine,
has an affinity profile which is not significantly different
from that of the parent compound at all the investi-
gated tissues. Conversely, (−)-10 is about six times
more efficacious than (+)-1 at the M2 heart rate
receptors. Furthermore, inspection of the data of (9)-7
and (9)-8 reveals that the ileum/heart (force) selectiv-
ity observed for benzyl deoxamuscarine (9)-8 is due to
a sharp difference in the relative efficacy values at the
ileal and cardiac receptors. Indeed, (9)-8 is 130 times
more efficacious at the ileum M3 receptors than at the
heart force M2 ones. On the contrary, the benzyl ether
(−)-10 displays comparable efficacies at the same re-
ceptor subtypes (0.63 and 0.98, respectively).
Interestingly, the inversion (from S to R) of the
absolute configuration at C-2, as in the two stereoiso-
meric benzyl ethers (−)-10 and (−)-12, brings about a
significant change of the affinity profile at both M2 and
M3 receptor subtypes. In fact, on passing from (−)-10
to (−)-12, a 190-fold higher affinity at the M2 heart
rate receptor subtype is observed, whereas the affinity
at the M2 heart force subtype is almost unaffected. On
the contrary, a 140-fold lower affinity is measured at
M3 ileal receptors, while the affinity at bladder is about
4-fold higher.
A horizontal analysis of the data of Table 3 suggests
that derivatives (+)-1, (−)-10 and (−)-12 are able to
discriminate within M2 and M3 receptor populations
since, according to Furchgott’s receptor theory [20],
differences in −log KD of at least 0.5 may be taken as
an evidence of a receptor heterogeneity. In particular,
(+)-1 and (−)-10 show remarkable differences be-
tween the affinity values at heart force and rate (43-
and 68-fold, respectively), whereas (−)-12 displays a
100-fold higher affinity at bladder than at ileum M3
receptors. In addition, (−)-12 behaves as a weak full
agonist at ileum and as an antagonist at bladder.
On the whole, the replacement of the hydroxy group
of natural muscarine with the benzyloxy function gives
a muscarinic agonist which retains the overall pharma-
cological profile of the parent compound, while show-
ing no selectivity between M2 heart force and M3 ileal
receptors and an improved capability to discriminate
the populations of the M2 receptor subtype. This result
is different from that emerged from the study of the
related pair deoxamuscarine/deoxamuscarine benzyl
ether, in which the same structural variation brought
about a discrimination between M2 heart force and M3
ileal receptors. The presence of the benzyloxy function
at the muscarinic subsite (C-4) coupled with the pres-
ence of a substituent at C-2 with the (R) configuration
caused major variations of the pharmacological profile,
predominantly at cardiac M2 receptors. Once again
[7,8,21], our studies on the structure–activity relation-
ships of a set of chiral muscarinic ligands structurally
related to muscarine put in evidence distinct structural
requirements of the receptor subtypes regulating heart
force and rate on one side, and those mediating the
contraction of ileum and bladder on the other.
Table 2
Potencies of compounds (+)-1, (−)-10, (−)-12 and (−)-13 at gan-
glionic M1 receptors mediating tachycardia and at cardiac M2 recep-
tors mediating bradycardia in the pithed rat
ED50 (mg/kg) a, pithed rat
4. Experimental
Increase in heart rate
(M1)
Decrease in heart rate
(M2)
4.1. Chemistry
(+)-1
3.090.50
4.091.6
66.8921.8
21.793.1
4.590.75
9.194.7
64.2920.8
30.1911.8
(−)-10
(−)-12
(−)-13
Melting points were determined on a model B 540
Bu¨chi apparatus and are uncorrected. Liquid com-
pounds were characterized by the oven temperature for
1
a The results are the mean9SEM, and the number of observations
Kugelrohr distillations. H NMR spectra were recorded
varies between five and nine.
with a Bruker AC-E 200 (200 MHz) spectrometer in

Table 3
Pharmacological parameters of (+)-1, (9)-7, (9)-8, (9)-9, (−)-10, (−)-11, (−)-12 and (−)-13 in the guinea pig heart force and rate (M2) and ileum and bladder (M3)
Tissues
Heart
Ileum
Bladder
Heart
Ileum
Bladder
Heart
Force
Ileum
Bladder
Force
Rate
Force
Rate
Rate
a
a
a
a
a
a
a
a
b
b
b
b
−log ED50
−log ED50
7.0790.14
−log ED50
−log ED50
−log KD
−log KD
−log KD
−log KD
er
er
er
er
(+)-1
7.6890.10
5.9390.05
5.5990.08
6.5690.15
6.8790.05
5.2790.06
7.5590.12
5.0790.15
7.0590.10
6.1390.07
6.8190.08
5.4190.11
6.7490.13
5.6690.07
4.9890.09
4.9190.12
5.5890.05
4.7990.05
4.8290.02
5.3090.12
4.4790.16
4.5890.08
3.6790.32
5.7490.23
4.3890.12
4.6790.11
4.8190.09
4.3090.12
4.8590.09 d
1.0
0.13
0.05
1.0
5.7
1.0
2.8
6.5
1.0
0.59
/
(9)-7 c
(9)-8 c
(9)-9 e
(−)-10
(−)-11 g
(−)-12
(−)-13
6.8090.07
4.8790.14
4.5090.08
2.6790.07
5.6590.16
3.5090.25
4.8890.18 f
5.5090.25 d
0.98
0.63
1.5
0.28
7.0790.19
4.5190.11
4.7190.15 d
4.9590.08 d
(
a The results are the mean9SEM, and the number of observations varies between six and ten.
5
b Relative efficacy [(+)-1=1].
c Data are taken from Ref. [11].
d −log KB.
543
e Data are taken from Ref. [12].
f −log KP.
g Data are taken from Ref. [13].

540
M. De Amici et al. / Il Farmaco 55 (2000) 535–543
CDCl3 (unless otherwise specified) solution; chemical
shifts (l) are expressed in ppm and coupling constants
(J) in Hertz. Rotary power determinations were carried
out with a Perkin–Elmer 241 polarimeter coupled with
a Haake N3-B thermostat. TLC analyses were per-
formed on commercial silica gel 60 F254 aluminum
sheets: spots were further evidenced by spraying with a
dilute alkaline potassium permanganate solution. Mi-
croanalyses (C, H, N) of new compounds agreed with
the theoretical value within 90.4%.
4.1.2. 2-(Iodomethyl)-4-benzyloxy-5-methyltetrahydro-
furan isomers (−)-18 and (−)-21
To a magnetically stirred solution of (−)-17 (0.850
g, 3.51 mmol) and benzyl 2,2,2-trichloroacetimidate
(980 ml, 5.27 mmol) in dichloromethane (30 ml) tri-
fluoromethanesulfonic acid (50 ml) was added at 0°C
under N2. The cooling bath was removed and the
temperature of the reaction mixture raised at 30°C with
the concomitant formation of a colorless precipitate.
After about 2 h, a further 490 ml of the benzylating
agent and 25 ml of the acid catalyst were added at 0°C.
The reaction was stirred for additional 2 h, then filtered
and the filtrate was washed with an aqueous saturated
solution of NaHCO3. After the usual work up, the
residue of the organic phase was submitted to a silica
gel column chromatography (eluant: 2% ethyl acetate–
petroleum ether) to afford 0.966 g (83% yield) of the
desired benzyl ether.
4.1.1. 2-(Iodomethyl)-4-hydroxy-5-methyltetrahydro-
furan isomers (−)-17 and (+)-20 and 2-(iodomethyl)-
4-benzoyloxy-5-methyltetrahydrofuran (+)-19
(A) To a magnetically stirred ice-cooled solution of
(−)-15 [13] (1.2 g, 4.96 mmol), triphenylphosphine (5.2
g, 19.84 mmol) and benzoic acid (1.21 g, 9.92 mmol) in
dry THF (80 ml) a solution of diethylazodicarboxylate
(3.14 ml, 19.92 mmol) in dry THF (20 ml) was added
dropwise. At the disappearance of the starting material,
the solvent was evaporated off and the residue was
column chromatographed (eluant: 10% ethyl acetate–
cyclohexane) to yield the benzoate of (−)-17 (1.47 g,
86%), colorless prisms from petroleum ether, m.p. 67–
68°C. Rf 0.43 (eluant: 10% ethyl acetate–cyclohexane);
[h]2D0 −11.75 (c 0.950, CHCl3).
(B) To a solution of the above reported crude ben-
zoate (1.3 g, 3.76 mmol) in methanol (75 ml) a 20%
aqueous solution of potassium carbonate (40 ml) was
added. The reaction mixture was stirred until disap-
pearance of the starting material. Methanol was evapo-
rated off and the residual aqueous phase was extracted
with ethyl acetate (4×25 ml). The pooled organic
extracts were dried over anhydrous sodium sulfate. The
solvent was removed at reduced pressure and the crude
iodoalcohol (−)-17 was distilled at 110–115°C/0.5
mmHg (0.855 g, 94%). Rf 0.14 (eluant: 20% ethyl
acetate–n-hexane); [h]D20 −34.08 (c 0.9, CHCl3), lit. [13]
[h]2D0 −33.72 (c 0.874, CHCl3).
(2S,4R,5S) 2-(Iodomethyl)-4-benzyloxy-5-methylte-
trahydrofuran (−)-18: pale yellow oil, b.p. 175–180°C/
0.1
mmHg;
Rf
0.28
(eluant:
5%
ethyl
1
acetate–cyclohexane); H NMR 1.28 (d, 3H, CH3, J=
6.5); 1.82 (ddd, 1H, H-3%, J=6.5, 9.1 and 13.3); 2.18
(ddd, 1H, H-3, J=2.6, 5.9 and 13.3); 3.28 (dd, 1H,
CH2I, J=6.1 and 10.1); 3.31 (dd, 1H, CH2I, J=4.9
and 10.1); 3.77 (m, 1H, H-4); 4.03–4.19 (m, 2H, H-2
and H-5); 4.51 and 4.53 (d, 2H, CH2Ph, J=11.9); 7.35
(m, 5H, arom.); [h]2D0 −40.08 (c 1.038, CHCl3).
The above reported procedure carried out on a com-
parable amount of (+)-20 gave benzyl ether (−)-21 in
74% yield.
(2R,4R,5S) 2-(Iodomethyl)-4-benzyloxy-5-methylte-
trahydrofuran (−)-21: pale yellow oil, b.p. 150–155°C/
0.1
mmHg;
Rf
0.30
(eluant:
5%
ethyl
1
acetate–cyclohexane); H NMR 1.20 (d, 3H, CH3, J=
6.5); 2.02 (ddd, 1H, H-3%, J=4.6, 4.6 and 13.5); 2.37
(ddd, 1H, H-3, J=6.9, 6.7 and 13.5); 3.33 (m, 2H,
CH2I); 3.78 (m, 1H, H-4); 4.18–4.36 (m, 2H, H-2 and
H-5); 4.50 and 4.55 (d, 2H, CH2Ph, J=11.7); 7.35 (m,
5H, arom.); [h]D20 −20.79 (c 1.068, CHCl3).
The same procedure carried out on a comparable
amount of (+)-16 [13] afforded benzoate (+)-19 and
iodoalcohol (+)-20 in 82 and 92% yields, respectively.
(2R,4R,5S) 2-(Iodomethyl)-4-benzoyloxy-5-methylte-
trahydrofuran (+)-19: colorless oil, b.p. 180–185°C/
4.1.3. 2-[(Dimethylamino)methyl]-4-benzyloxy-
5-methyltetrahydrofuran methiodides (−)-10 and
(−)-12 and 2-[(dimethylamino)methyl]-4-benzoyloxy-5-
methyltetrahydrofuran methiodide (−)-13
0.5
mmHg;
Rf
0.38
(eluant:
10%
ethyl
1
acetate–cyclohexane); H NMR 1.30 (d, 3H, CH3, J=
6.7); 2.15 (ddd, 1H, H-3%, J=3.4, 3.4 and 14.3); 2.65
(ddd, 1H, H-3, J=6.8, 7.2 and 14.3); 3.36 (m, 2H,
CH2I); 4.31–4.47 (m, 2H, H-2 and H-5); 5.18 (m, 1H,
H-4); 7.46 (t, 2H, arom., J=7.9); 7.59 (t, 1H, arom.,
J=7.9), 8.02 (d, 2H, arom., J=7.9); [h]2D0+32.85 (c
1.064, CHCl3).
A sealed metal container, filled with a solution of
(−)-18 (0.810 g, 2.44 mmol) in methanol (10 ml) and a
10-fold excess anhydrous dimethylamine was heated
overnight at 120°C. The container was cooled at 0°C,
the solution was acidified by addition of 3 N HCl, and
the volatiles were evaporated under vacuum. The resid-
ual aqueous phase was treated with ether (4×10 ml),
made alkaline by a portionwise addition of solid
K2CO3, then extracted with dichloromethane (4×15
ml). After the usual work up, the residue was column
chromatographed (eluant: 5% methanol–dichloro-
(+)-20: colorless oil, b.p. 110–115°C/0.5 mmHg; Rf
0.21 (eluant: 20% ethyl acetate–n-hexane); [h]D20
+
13.22 (c 0.9, CHCl3), lit. [13] [h]2D0 +13.51 (c 1.061,
CHCl3).

M. De Amici et al. / Il Farmaco 55 (2000) 535–543
541
methane), affording 0.328 g (54%) of the corresponding
tertiary base as a pale yellow oil.
yellow leaflets after treatment with ether/methanol
(0.508 g, 55% yield).
(2S,4R,5S) 2-[(Dimethylamino)methyl]-4-benzyloxy-
5-methyltetrahydrofuran: b.p. 175–180°C/0.2 mmHg;
Rf 0.38 (eluant: 20% methanol–chloroform); H NMR
(2R,4R,5S) 2-[(Dimethylamino)methyl]-4-benzoyloxy-
5-methyltetrahydrofuran methiodide (−)-13: m.p.
169–170°C (colorless prisms from 2-propanol); 1H
NMR (D2O) 1.30 (d, 3H, CH3, J=6.4); 2.03 (m, 1H,
H-3%); 2.84 (m, 1H, H-3); 3.20 (s, 9H, NMe3); 3.54 (dd,
1H, CH2NMe3, J=1.5 and 13.8); 3.61 (dd, 1H,
CH2NMe3, J=8.0 and 13.8); 4.52 (dq, 1H, H-5, J=1.9
and 6.4); 4.89 (m, 1H, H-2); 5.21 (m, 1H, H-4); 7.53 (t,
2H, arom., J=7.9); 7.67 (t, 1H, arom., J=7.9); 8.02
(d, 2H, arom., J=7.9); [h]2D0 −31.54 (c 0.964, MeOH).
1
1.25 (d, 3H, CH3, J=6.5); 1.70 (ddd, 1H, H-3%, J=6.8,
9.8 and 13.3); 2.07 (ddd, 1H, H-3, J=1.9, 5.8 and
13.3); 2.30 (s, 6H, NMe2); 2.39 (dd, 1H, CH2NMe2,
J=4.6 and 12.7); 2.49 (dd, 1H, CH2NMe2, J=7.5 and
12.7); 3.68 (m, 1H, H-4); 3.98 (m, 1H, H-2); 4.22 (m,
1H, H-5); 4.48 and 4.53 (d, 2H, CH2Ph, J=11.7); 7.33
(m, 5H, arom.); [h]2D0 −32.93 (c 1.154, CHCl3).
A solution of the tertiary amine in acetone/ether (1:1)
was treated with a 5-fold excess methyl iodide. The
quaternary salt precipitated quantitatively as a gummy
oil.
4.2. Pharmacology
4.2.1. In 6itro tests: general considerations
(2S,4R,5S) 2-[(Dimethylamino)methyl]-4-benzyloxy-
5-methyltetrahydrofuran methiodide (−)-10: m.p.
107–108°C (colorless prisms from acetone/ethyl ace-
Male guinea pigs (200–300 g) were killed by cervical
dislocation, and the organs required were set up rapidly
under 1 g of tension in 20 ml organ baths containing
physiological salt solution (PSS) kept at an appropriate
temperature (see below) and aerated with 5% CO2–95%
O2. Two dose–response curves were constructed by
cumulative addition of the reference agonist [(+)-mus-
carine]. 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, a new
dose–response curve to the agonist under study was
obtained. Responses were expressed as a percentage of
the maximal response obtained in the control curve.
The results are expressed in terms of pD2, which is the
−log ED50, the concentration of agonist required to
produce 50% of the maximum contraction. Contrac-
tions were recorded by means of a force transducer
connected to a two-channel Gemini polygraph (U.
Basile). In all cases, parallel experiments in which tis-
sues received only the reference agonist were run in
order to check any variation in sensitivity.
1
tate); H NMR 1.21 (d, 3H, CH3, J=6.6); 1.78 (ddd,
1H, H-3%, J=6.2, 10.6 and 13.3); 2.32 (ddd, 1H, H-3,
J=1.5, 5.1 and 13.3); 3.44 (m, 2H, CH2NMe3); 3.48 (s,
9H, NMe3); 3.75 (m, 1H, H-4); 4.13 (dq, 1H, H-5,
J=2.1 and 6.6); 4.44 and 4.52 (d, 2H, CH2Ph, J=
11.6); 4.53 (m, 1H, H-2); 7.33 (m, 5H, arom.); [h]D20
−25.52 (c 0.960, CHCl3).
The above reported procedure performed on the iodo
derivative (−)-21 afforded the corresponding tertiary
base in 47% yield.
(2R,4R,5S) 2-[(Dimethylamino)methyl]-4-benzyloxy-
5-methyltetrahydrofuran: b.p. 185–190°C/0.2 mmHg as
a pale yellow oil; Rf 0.32 (eluant: chloroform/20%
1
methanol); H NMR 1.18 (d, 3H, CH3, J=6.4); 1.69
(ddd, 1H, H-3%, J=1.3, 6.5 and 13.0); 2.20–2.37 (m,
2H, H-3 and CHNMe2); 2.25 (s, 6H, NMe2); 2.57 (dd,
1H, CHNMe2, J=8.0 and 12.6); 3.68 (m, 1H, H-4);
4.07 (dq, 1H, H-5, J=2.0 and 6.4); 4.18 (m, 1H, H-2);
4.44 and 4.50 (d, 2H, CH2Ph, J=11.8); 7.29 (m, 5H,
arom.); [h]2D0 −50.59 (c 1.018, CHCl3).
(2R,4R,5S) 2-[(Dimethylamino)methyl]-4-benzyloxy-
5-methyltetrahydrofuran methiodide (−)-12: m.p.
88.5–90°C (colorless prisms from acetone/ethyl ace-
4.2.1.1. Determination of dissociation constants. Dissoci-
ation constants (KD) and relative efficacies (er) were
determined as previously described according to the
method of Furchgott and Bursztyn for full agonists
(h=1) [22]. For partial agonists (hB1), the affinity
constants (KB) were calculated from the equation
log(DR−1)=log(partial agonist)−log KB, where DR
(dose ratio) is the ratio of the ED50 values of (+)-mus-
carine after and before incubation (1×10−4 M) with
the partial agonist [23]. The partial agonist KP value for
compound (−)-10 at guinea pig bladder (h=0.86) was
determined according to Waud [24].
1
tate); H NMR 1.14 (d, 3H, CH3, J=6.6); 1.86 (ddd,
1H, H-3%, J=3.7, 3.7 and 13.7); 2.52 (ddd, 1H, H-3,
J=1.8, 7.0 and 13.7); 3.32–3.51 (m, 2H, CH2NMe3);
3.45 (s, 9H, NMe3); 3.78 (m, 1H, H-4); 4.23 (m, 1H,
H-5); 4.46 and 4.52 (d, 2H, CH2Ph, J=11.8); 4.71 (m,
1H, H-2); 7.31 (m, 5H, arom.); [h]2D0 −42.42 (c 1.002,
CHCl3).
A sealed metal container, filled with a solution of
(+)-19 (0.790 g, 2.28 mmol) in dichloromethane/
methanol 1:1 (30 ml) and a 15-fold excess anhydrous
trimethylamine was heated overnight at 70°C. The con-
tainer was cooled at room temperature and the reaction
mixture was evaporated under vacuum at 20°C. The
crude quaternary ammonium salt was obtained as pale
4.2.1.2. Guinea pig ileum. Portions of terminal ileum (2
cm) were taken at about 5 cm from the ileum–cecum
junction and mounted in PSS at 37°C. The composition

542
M. De Amici et al. / Il Farmaco 55 (2000) 535–543
of PSS was the following (mM): NaCl (118), NaHCO3
(23.8), KCl (4.7), MgSO4·7H2O (1.18), KH2PO4 (1.18),
CaCl2 (2.52), glucose (11.7). Tension changes were
recorded isotonically. Tissues were equilibrated for 30
min, and dose–response curves to (+)-muscarine were
obtained at 30 min intervals, the first one being dis-
carded and the second one being taken as the control.
The preparation was allowed to equilibrate for at least
30 min before drug administration, until mean heart
rate had stabilized. The basal heart rate amounted to
30098 beats/min (n=50). Changes in heart rate were
measured for individual doses of the agonist given i.v.
(0.1 ml/100 g). Full recovery from the pressor and
cardiac effects with return to preinjection values was
allowed between successive doses. After drug injection,
the venous cannula was flushed with 50 ml of isotonic
saline solution.
4.2.1.3. Guinea pig bladder. A 2 mm wide longitudinal
strip of bladder from urethra to the apex of the bladder
was cut, excluding the portion under the urethra orifice,
and mounted in PSS (the same used for ileum) at 37°C.
Contractions were recorded isometrically. Tissues were
equilibrated for 30 min (see protocol for ileum).
4.2.2.1. Experimental protocol. All drugs were dissolved
in saline (0.9% w/v) and injected i.v. in a volume of 0.1
ml/100 g. Because of desensitization phenomena, when
compounds (+)-1, (−)-10, (−)-12 and (−)-13 were
employed, only one single dose–response curve was
assessed in each preparation. ED50 values were deter-
mined graphically from the resultant dose–response
curves and represent the dose causing 50% of the
maximum response of the compound under study. Pre-
treatment (i.v.) with antagonists was carried out 20 min
before the administration of the agonist. This interval
was selected because preliminary experiments showed
that after this time the antagonistic effects of piren-
zepine (50 mg/kg i.v.) and tripitramine (30 mg/kg i.v.),
respectively, were constant during the whole experi-
ment.
4.2.1.4. 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 activ-
ity was recorded isometrically. Tissues were equili-
brated for 2 h and a cumulative dose–response curve to
(+)-muscarine was constructed.
4.2.1.5. Right atria. Right atria were equilibrated for 1
h at the above conditions (see guinea pig stimulated left
atria for PSS and temperature). Contractions were
recorded isometrically.
4.2.3. Statistical analysis
Data are presented as means9SEM of n experi-
ments. Student’s t-test was used to assess the statistical
significance of the difference between two means.
4.2.2. In 6i6o tests: pithed rat
Male normotensive rats (270–330 g) were housed five
per cage and maintained on a 12 h light/dark cycle.
Food and water were available ad libitum. The animals
were anaesthetized with equithesin (9.6 g nembutal
sodium, 42.6 g chloral hydrate, 21.2 g MgSO4, 400 ml
propylene glycol, 50 ml ethyl alcohol and water to 1000
ml) 3 ml/kg of body weight i.p. The right jugular vein
was cannulated (PE 10 polyethylene tubing) for drug
administration. Blood pressure was measured from the
left common carotid artery through a PE 50 catheter
connected to a pressure transducer (P23 ID, Statham,
Hato Rey, Puerto Rico). The heart rate was measured
continuously by means of a rate meter (Basile) which
was triggered by the blood pressure pulse in the carotid
artery.
After catheterization of the trachea, heparin (150
IU/kg) was given i.v. to prevent blood coagulation.
Temperature was maintained at approximately 37°C
throughout the experiment by means of an overhead-
heating lamp. The rats were then pithed by insertion of
a steel rod (1.5 mm in diameter) through the skull and
foramen magnum down into the spinal canal [25]. The
animals were respired artificially by means of a Har-
vard Apparatus model 681 rodent respirator at a fre-
quency of 60 cycles/min with a volume of 1 ml/100 g.
Acknowledgements
The authors wish to thank Consiglio Nazionale delle
Ricerche (CNR) and Ministero della Ricerca Scientifica
e Tecnologica (MURST, Rome) for financial support.
References
[1] M.P. Caulfield, Muscarinic receptors: characterization, coupling
and function, Pharmacol. Ther. 53 (1993) 319–379.
[2] R.M. Eglen, N. Watson, Selective muscarinic agonists and an-
tagonists, Pharmacol. Toxicol. 78 (1996) 59–68.
[3] U. Grimm, U. Moser, E. Mutschler, G. Lambrecht, Muscarinic
receptors: focus on presynaptic mechanisms and recently devel-
oped agonists and antagonists, Pharmazie 49 (1994) 711–726.
[4] M.P. Caulfield, N.J.M. Birdsall, International Union of Pharma-
cology, XVII, Classification of muscarinic acetylcholine recep-
tors, Pharmacol. Rev. 50 (1998) 279–290.
[5] M. De Amici, C. Dallanoce, C. De Micheli, E. Grana, A.
Barbieri, H. Ladinsky, G.B. Schiavi, F. Zonta, Synthesis and
pharmacological investigation of the enantiomers of muscarone
and allo-muscarone, J. Med. Chem. 35 (1992) 1915–1920.

M. De Amici et al. / Il Farmaco 55 (2000) 535–543
543
[6] M. De Amici, C. De Micheli, T. Gianferrara, E. Grana, G.
Dondi, H. Ladinsky, G.B. Schiavi, Synthesis and muscarinic
activity of the chiral forms of methylenemuscarones, Farmaco 48
(1993) 1349–1357.
[7] P. Bravo, G. Resnati, P. Angeli, M. Frigerio, F. Viani, A.
Arnone, G. Marucci, F. Cantalamessa, Synthesis and pharmaco-
logical evaluation of enantiomerically pure 4-deoxy-4-fluoro-
muscarines, J. Med. Chem. 35 (1992) 3102–3110.
[8] P. Angeli, F. Cantalamessa, R. Cavagna, P. Conti, M. De Amici,
C. De Micheli, A. Gamba, G. Marucci, Synthesis and pharmaco-
logical characterization of enantiomerically pure muscarinic ago-
nists: difluoromuscarines, J. Med. Chem. 40 (1997) 1099–1103.
[9] P. Conti, C. Dallanoce, M. De Amici, C. De Micheli, G. Carrea,
F. Zambianchi, Chemoenzymatic synthesis of the enantiomers of
desoxymuscarine, Tetrahedron: Asymmetry 9 (1998) 657–665.
[10] M. De Amici, C. Dallanoce, C. De Micheli, P. Angeli, G.
Marucci, F. Cantalamessa, L. Sparapassi, Pharmacological
profile of enantiomerically pure muscarinic agonists: desoxymus-
carines, Life Sci. Pharmacol. Lett. (in press).
[11] L. Brasili, M. Giannella, A. Piergentili, S.K. Tayebati, Effect of
deoxamuscarine etherification on M2 and M3 muscarinic affinity
and efficacy, Med. Chem. Res. 2 (1992) 298–305.
[12] A. Piergentili, M. Pigini, W. Quaglia, S.K. Tayebati, F. Amenta,
M. Sabbatini, M. Giannella, Muscarinic thioligands with cy-
clopentane nucleus, Bioorg. Med. Chem. 4 (1996) 2193–2199
and Refs. cited therein.
[13] M. De Amici, C. Dallanoce, C. De Micheli, E. Grana, G. Dondi,
H. Ladinsky, G.B. Schiavi, F. Zonta, Synthesis and pharmaco-
logical investigation of the stereoisomeric muscarines, Chirality 4
(1992) 230–239.
[14] M. De Amici, C. De Micheli, G. Molteni, D. Pitre´, G. Carrea, S.
Riva, S. Spezia, L. Zetta, Chemoenzymatic synthesis of the eight
stereoisomeric muscarines, J. Org. Chem. 56 (1991) 67–72.
[15] J.A. Dale, D.L. Dull, H.S. Mosher, a-Methoxy-a-trifl-
uoromethylphenylacetic acid, a versatile reagent for the determi-
nation of the enantiomeric composition of alcohols and amines,
J. Org. Chem. 34 (1969) 2543–2549.
[16] O. Mitsunobu, The use of diethyl azodicarboxylate and
triphenylphospine in synthesis and transformation of natural
products, Synthesis (1981) 1–28.
[17] H.P. Wessel, T. Iversen, D.R. Bundle, Acid-catalysed benzyla-
tion and allylation by alkyl trichloroacetimidates, J. Chem. Soc.
Perkin Trans. I (1985) 2247–2250.
[18] P. Eckenberg, U. Groth, T. Huhn, N. Richter, C. Schmeck, A
useful application of benzyl trichloroacetimidate for the benzyla-
tion of alcohols, Tetrahedron 49 (1993) 1619–1624.
[19] P. Angeli, L. Brasili, M. Giannella, F. Gualtieri, M.T. Picchio,
E. Teodori, Chiral muscarinic agonists possessing a 1,3-oxathi-
olane nucleus: enantio- and tissue-selectivity on isolated prepara-
tions of guinea-pig ileum and atria and of rat urinary bladder,
Naunyn-Schmiedeberg’s Arch. Pharmacol. 337 (1988) 241–245.
[20] R.F. Furchgott, The classification of adrenoceptors (adrenergic
receptors). An evaluation from the standpoint of receptor the-
ory, in: H. Blaschko, E. Muscholl (Eds.), Handbook of Experi-
mental Pharmacology, Catecholamines, vol. 33, Springer, New
York, 1972, pp. 283–335.
[21] P. Angeli, Pentatomic cyclic agonists and muscarinic receptors: a
20 years review, Farmaco 9 (1995) 565–577 and Refs. cited
therein.
[22] R.F. Furchgott, P. Bursztyn, Comparison of dissociation con-
stants and of relative efficacies of selected agonists acting on
parasympathetic receptors, Ann. NY Acad. Sci. 139 (1967) 882–
899.
[23] J.M. van Rossum, Cumulative dose–response curves. II. Tech-
nique for the making of dose–response curves in isolated organs
and the evaluation of drug parameters, Arch. Int. Pharmacodyn.
Ther. 143 (1963) 299–330.
[24] D.R. Waud, On the measurement of the affinity of partial
agonists for receptors, J. Pharmacol. Exp. Ther. 170 (1969)
117–122.
[25] R.E. Shipley, J.H. Tilden, A pithed rat preparation suitable for
assaying pressor substances, Proc. Soc. Exp. Biol. Med. 64
(1947) 453–455.
.
.