Synthesis and pharmacological characterization of enantiomerically pure muscarinic agonists: Difluoromuscarines

Article abstract of DOI:10.1021/jm960742w

The four homochiral 4-deoxy-4,4-difluoromuscarine stereoisomers (difluoromuscarines) were prepared in very high enantiomeric excess. A convenient sequence based on the use of natural as well as 'unnatural' ethyl lactate allowed the synthesis of target compounds, whose absolute configuration is dictated by that of the starting synthon. Quaternary ammonium salts (+)-5, (-)-5, (-)-6, and (+)-6 were tested in vitro on guinea pig tissues, and their muscarinic potency was evaluated at M₂ (heart) and M₃ (ileum and bladder) muscarinic receptor subtypes. The eutomer (+)-5 and distemer (-)-5 were also tested in vivo on pithed rat, and their muscarinic activity at the M₁ receptor subtype was compared with those of racemic muscarine [(±)-1] and (2S,4R,5S)-4-deoxy-4-fluoromuscarine [(+)-4]. Further pharmacological parameters such as affinity, relative efficacy, and enantioselectivity have been determined for compounds (+)-5 and (-)-5 at M₂ (heart force and rate) and M₃ (ileum and bladder) receptors in order to investigate muscarinic receptor heterogeneity. The four homochiral difluoromuscarines behave as muscarinic agonists in all the tests with a potency trend which is different from that previously observed with the 4- deoxy-4-fluoromuscarines and (±)-1, thus indicating the intervention of the second fluorine atom on the receptor-ligand interaction. Moreover, the second fluorine atom produces significant differences in the affinity and relative efficacy values of compounds (+)-5 and (-)-5 at M₂ and Ms subtypes, which could be attributed to a heterogeneity between the muscarinic receptors mediating heart rate and heart force and those involved in the contraction of ileum and bladder.

Full text of DOI:10.1021/jm960742w

J . Med. Chem. 1997, 40, 1099-1103
1099
Syn th esis a n d P h a r m a cologica l Ch a r a cter iza tion of En a n tiom er ica lly P u r e
Mu sca r in ic Agon ists: Diflu or om u sca r in es
Piero Angeli,*,† Franco Cantalamessa,‡ Roberto Cavagna,‡ Paola Conti,§ Marco De Amici,§ Carlo De Micheli,*,§
Anna Gamba, and Gabriella Marucci†
Dipartimento di Scienze Chimiche, Universita` di Camerino, via S. Agostino, 1-62032 Camerino (MC), Italy, Istituto di
Farmacologia e Farmacognosia, Universita` di Camerino, via Scalzino, 1-62032 Camerino (MC), Italy, Istituto di Chimica
Farmaceutica e Tossicologica, Universita` di Milano, v.le Abruzzi, 42-20131 Milano, Italy, and Dipartimento di Chimica
Organica, Universita` di Pavia, v.le Taramelli, 10-27100 Pavia, Italy
Received October 24, 1996X
The four homochiral 4-deoxy-4,4-difluoromuscarine stereoisomers (difluoromuscarines) were
prepared in very high enantiomeric excess. A convenient sequence based on the use of natural
as well as “unnatural” ethyl lactate allowed the synthesis of target compounds, whose absolute
configuration is dictated by that of the starting synthon. Quaternary ammonium salts (+)-5,
(-)-5, (-)-6, and (+)-6 were tested in vitro on guinea pig tissues, and their muscarinic potency
was evaluated at M2 (heart) and M3 (ileum and bladder) muscarinic receptor subtypes. The
eutomer (+)-5 and distomer (-)-5 were also tested in vivo on pithed rat, and their muscarinic
activity at the M1 receptor subtype was compared with those of racemic muscarine [(()-1] and
(2S,4R,5S)-4-deoxy-4-fluoromuscarine [(+)-4]. Further pharmacological parameters such as
affinity, relative efficacy, and enantioselectivity have been determined for compounds (+)-5
and (-)-5 at M2 (heart force and rate) and M3 (ileum and bladder) receptors in order to
investigate muscarinic receptor heterogeneity. The four homochiral difluoromuscarines behave
as muscarinic agonists in all the tests with a potency trend which is different from that
previously observed with the 4-deoxy-4-fluoromuscarines and (()-1, thus indicating the
intervention of the second fluorine atom on the receptor-ligand interaction. Moreover, the
second fluorine atom produces significant differences in the affinity and relative efficacy values
of compounds (+)-5 and (-)-5 at M2 and M3 subtypes, which could be attributed to a
heterogeneity between the muscarinic receptors mediating heart rate and heart force and those
involved in the contraction of ileum and bladder.
A common feature among the major chiral muscarinic
agonists is the high value of the eudismic ratio (ER) and
a spatial arrangement around the chiral centers match-
ing those of natural muscarine (+)-1 (Figure 1).
These peculiarities were essentially confirmed by our
studies on the eight chiral muscarine stereoisomers,1
as well as the four chiral isomers of muscarone2 and
methylenemuscarone.3 The absolute configuration of
the most potent stereoisomer of muscarone [(-)-2] and
methylenemuscarone [(-)-3] is depicted in Figure 1. The
nature of the interaction involving the binding of the
hydroxy group of muscarine with the complementary
receptor subsite has been the subject of detailed inves-
tigations by us since many years.4 In 1992 Bravo et
F igu r e 1.
al.5 reported the synthesis and the pharmacological
[(+)-5, (-)-5, (-)-6, and (+)-6] (Figure 1) in enantiomeric
excess higher than 98%. The compounds were tested
in vitro on guinea pig tissues and their muscarinic
potency evaluated at M2 (heart) and M3 (ileum and
bladder) muscarinic receptor subtypes.
evaluation of four 4-deoxy-4-fluoromuscarine stereoiso-
mers. The overall results suggested that the pharma-
cological profile of this set of muscarinic agonists was
quite similar to that of the reference muscarines. In
particular, the replacement of the hydroxy group of
muscarine with a fluorine atom, i.e. (+)-4 (Figure 1)
produced a change only in the receptor-ligand interac-
tion at the cardiac M2 muscarinic receptors controlling
rate.
Ch em istr y
The synthesis of compounds (+)-5, (-)-5, (-)-6, and
(+)-6 (difluoromuscarines) was achieved following the
strategy previously reported for the preparation of
homochiral muscarines1 and muscarones.2 By employ-
ing the commercially available natural [S-(-)-] and
“unnatural” [R-(+)-] ethyl lactate we prepared key
diastereomeric iodo ketones 7 and 8 which could be
separated by column chromatography (Scheme 1). In-
termediates 7 and 8 were then separately reacted with
In order to further investigate the role played by a
fluorine atom in this critical position we prepared the
four chiral 4-deoxy-4,4-difluoromuscarine stereoisomers
† Universita` di Camerino, via S. Agostino.
‡ Universita` di Camerino, via Scalzino.
§ Universita` di Milano.
Universita` di Pavia.
X Abstract published in Advance ACS Abstracts, February 15, 1997.
S0022-2623(96)00742-X CCC: $14.00 © 1997 American Chemical Society

1100 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 7
Angeli et al.
Ta ble 1. Potencies and Intrinsic Activities of
Difluoromuscarines (+)-5/(-)-5, (-)-6/(+)-6, Fluoromuscarine
(+)-4, and Muscarine (()-1 at M2 and M3 Muscarinic Receptors
Sch em e 1
tissue
guinea pig
heart (force) M2
guinea pig
ileum M3
guinea pig
bladder M3
Rb
pD2
Rb
pD2
Rb
a
a
a
compd
pD2
(+)-5 6.56 ( 0.14 1.00 5.80 ( 0.07 0.99 5.23 ( 0.17 0.83
(-)-5 5.76 ( 0.13 0.97 5.53 ( 0.04 0.98 3.82 ( 0.06 0.95
(-)-6 6.28 ( 0.20 1.00 5.33 ( 0.20 0.97 4.16 ( 0.13 1.00
(+)-6 5.77 ( 0.04 0.98 4.85 ( 0.12 0.99 3.95 ( 0.08 0.67
(+)-4 6.73 ( 0.08 0.98 7.36 ( 0.03 0.95 5.66 ( 0.03 0.88
(()-1 6.69 ( 0.05 1.00 7.10 ( 0.11 1.00 5.69 ( 0.06 1.00
a
-log ED50. The results are the mean ( SEM, and the number
b
of observations varies between 6 and 10. Intrinsic activity,
measured by the ratio between the maximum response of the
compound and the maximum response of muscarine.
Ta ble 2. Potencies of (+)-5, (-)-5, (+)-4, and (()-1 at Cardiac
M2 Receptors Mediating Bradycardia and at Ganglionic M1
Receptors Mediating Tachycardia in the Pithed Rat
ED50 (µg/kg),a pithed rat
increase in
decrease in
heart rate (M1)
heart rate (M2)
(+)-5
ERb
50 ( 5.5
2
140 ( 15.2
2
(-)-5
(+)-4
(()-1
100 ( 8.5
4.5 ( 0.70
2.5 ( 0.23
273.5 ( 33.3
23.5 ( 3.1
3.7 ( 0.46
Sch em e 2
a
The results are the mean ( SEM, and the number of
b
observations varies between five and nine. Eudismic ratio: ratio
between the potency of the more potent and the less potent
enantiomer.
fluoromuscarine (+)-4 (fluoromuscarine) and of racemic
muscarine (()-1 to study the effects of the different
substituents at the 4-position on the pharmacological
profile of the ligand. Compound (+)-5, the most potent
isomer in the series, was also tested in vivo on pithed
rat and its muscarinic activity at M1 receptor subtype
was compared with those of fluoromuscarine (+)-4 and
(()-1 (Table 2). In addition, affinity and relative efficacy
were calculated for the pair of enantiomers (+)-5 and
(-)-5 at M2 (heart force and rate) and M3 (ileum and
bladder) receptors in order to investigate muscarinic
receptors heterogeneity (Table 3).
Since the study of enantioselectivity gives information
on the behavior of ligands and on the characterization
of receptor subgroups, we also evaluated this parameter
in our in vivo and in vitro studies by inspection of the
results obtained with distomer (-)-5 (Tables 2 and 3).
Table 1 shows that the four chiral difluoromuscarines
behave as muscarinic agonists with different degrees
of potency at the investigated tissues. Their potency
trend (heart > ileum > bladder) is different from that
observed with fluoromuscarines and (()-1,5 suggesting
that a further introduction of a fluorine atom in the
nucleus has different consequences on the receptor-
ligand interaction. As expected, the (2S,5S)-enantiomer
[(+)-5)], which has the same absolute configuration of
natural muscarine, is the most potent agonist in the new
set of derivatives; it displays its higher potency at heart
(M2 subtype) at variance with (+)-4 and (()-1 which are
more active at ileum (M3 subtype). Difluoromuscarine
(+)-5 is in fact 6 and 21 times more potent at M2 than
at M3 receptor subtypes (ileum and bladder, respec-
tively).
a 4-fold excess of (diethylamino)sulfur trifluoride
(DAST).6,7 The reaction was completed in a few hours
at room temperature and gave the desired difluoro
derivatives 9 and 10 in 60-70% yield (Scheme 2).
Through the conventional transformations of the side
chain reported in Scheme 2 we synthesized the two pairs
of enantiomeric salts (+)-5/(-)-5 and (-)-6/(+)-6. Their
absolute configuration derives from the absolute con-
figuration of the known iodo ketones 7 and 8.2 The
enantiomeric excess (ee) of the final derivatives (>98%)
is inferred from the ee value of iodo ketones 7 and 8
which was previously determined.2
Resu lts a n d Discu ssion
The pairs of enantiomers 5 and 6 were tested in vitro
on guinea pig tissues, and their muscarinic potency was
evaluated at M2 (heart) and M3 (ileum and bladder)
muscarinic receptor subtypes (Table 1). These results
were compared with those of (2S,4R,5S)-4-deoxy-4-
The overall results suggest that the hydrogen bonding

Enantiomerically Pure Muscarinic Agonists
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 7 1101
Ta ble 3. Pharmacological Parameters of (+)-5, (-)-5, (+)-4, and (()-1 in the Guinea Pig Heart Force and Rate (M2) and Ileum and
Bladder (M3)
tissue
heart
heart
heart
force
rate
ileum
bladder
-log ED50
force
-log KD
rate
-log KD
ileum
-log KD
bladder
-log KD
force rate ileum bladder
a
a
a
a
a
a
a
a
b
b
b
b
-log ED50 -log ED50 -log ED50
er
er
er
0.9
2.3 1.0
er
(+)-5 6.56 ( 0.14 6.58 ( 0.13 5.80 ( 0.07 5.23 ( 0.17 4.37 ( 0.21 3.39 ( 0.15 4.70 ( 0.08 4.40 ( 0.16 1.6 23
ERc 6.3
1.9 1.9 26 5.4 0.8 1.7 4.9 1.2
(-)-5 5.76 ( 0.13 6.31 ( 0.15 5.53 ( 0.04 3.82 ( 0.06 3.64 ( 0.14 3.48 ( 0.16 4.47 ( 0.14 3.71 ( 0.06 1.3 10
(+)-4 6.73 ( 0.08 6.87 ( 0.04 7.36 ( 0.03 5.66 ( 0.03 4.67 ( 0.12 5.77 ( 0.17 5.72 ( 0.11 4.39 ( 0.09 1.2 0.2
(()-1 6.69 ( 0.05 6.42 ( 0.04 7.10 ( 0.11 5.69 ( 0.06 4.70 ( 0.10 4.61 ( 0.19 5.95 ( 0.10 4.62 ( 0.09
0.6
3.0
0.2
1.5
1
0.8
3
1
1
1
a
b
The results are the mean ( SEM, and the number of observations varies between 6 and 10. Relative efficacy [(()-muscarine ) 1].
c Eudismic ratio: ratio between the potency, the affinity and the relative efficacy of eutomer and distomer.
or the dipole-dipole interaction between the fluorine
atom of (+)-4 and the complementary receptor subsite
is less effective due to the presence of a second fluorine
atom in the geminal position, i.e. (+)-5. The extent of
this effect is particularly evident in the ileum. A similar
decline in potency was previously observed in the
muscarinic agonists bearing the oxathiolane sulfoxide
and oxathiolane sulfone nucleus.8
respectively), which parallels a decrease in affinity (10
and 18 times, respectively) as well as relative efficacy.
Furthermore, the introduction of two fluorine atoms
in the 4-position of muscarine causes a marked drop of
the enantioselectivity as reflected by the negligible
eudismic ratio values (0.8-26, Table 3) shown by the
pair of enantiomers (+)-5 and (-)-5. This outcome does
not allow any valuable speculation on the differences
among the two muscarinic receptor subtypes but the
observation that these values are always slightly higher
on heart force (potency and affinity) and bladder (po-
tency, affinity and efficacy) than on heart rate and
ileum. The poor enantioselectivity observed for difluo-
romuscarines is rather unusual when the parallel
results obtained on muscarine and other closely related
chiral muscarinic agonists are taken into account.1-4
With the purpose to evaluate the muscarinic activity
of enantiomers (+)-5 and (-)-5 at ganglionic M1 and
cardiac M2 receptors, these agonists were tested in vivo
on a pithed rat preparation, according to the method
reported by Angeli et al.,9 and results were compared
with those obtained on compounds (+)-4 and (()-1
(Table 2).4 In particular, (+)-5 is 11 and 20 times less
active than reference compounds (+)-4 and (()-1 at M1
receptor subtype, while at M2 subtype lowerings amount
to 6 and 38, respectively. As a consequence, eutomer
(+)-5 is slightly (3-fold) selective for M1 muscarinic
receptors. The same selectivity was observed for dis-
tomer (-)-5, which displays a reduced potency both at
M1 and M2 receptors.
The horizontal analysis of the results gathered in
Table 3 indicates that compounds (+)-4 and (()-1 have
identical profiles at the two receptor subtypes as far as
potency is concerned. The two agonists show also
similar affinity values except at heart rate, where a 15-
fold higher value has been reported5 for compound (+)-
4. Remarkably, compounds (+)-4 and (()-1 show the
highest potency value on ileum, and compound (+)-4 has
the highest relative efficacy in the same tissue prepara-
tion.
A conceivable explanation for the decrease of the ED50
values displayed by the pair of enantiomers (+)-5 and
(-)-5 when compared to those of reference compounds
(+)-4 and (()-1 on heart rate in vivo (the four agonists
show similar potencies on heart rate in vitro) could be
attributed to differences in metabolism and pharmaco-
kinetics of the agonists in the two preparations, as
already postulated for compounds (+)-4 and (()-1.5
According to Furchgott’s theory,10 differences in -log
KD of at least 0.5 are indicative of receptor heterogene-
ity; at the same time we know that the comparison of
agonist potencies on different tissues is not sufficient
to claim for receptor heterogeneity.8,11 Consequently,
further pharmacological parameters such as affinity and
relative efficacy have been determined for the pair of
enantiomers (+)-5 and (-)-5 (Table 3).
A vertical analysis of Table 3 shows that, as far as
the M2 subtype regulating heart force and the M3
subtype of bladder are concerned, potency, affinity, and
relative efficacy of compounds (+)-5, (+)-4, and (()-1
do not significantly differ from each other. On the
contrary, at the M2 subtype regulating heart rate, the
same compounds display similar potency values, whereas
affinity and relative efficacy show discrete differences.
As a matter of fact fluoromuscarine (+)-4 has a 240-
fold higher affinity and a 115-fold lower efficacy than
difluoromuscarine (+)-5. Moreover, (+)-5 shows in the
ileum (M3 subtype) a decrease in potency in comparison
with (+)-4 and racemic muscarine (36 and 20 times,
Conversely, compound (+)-5 is more active on heart
(both force and rate) where it shows the highest relative
efficacy value (er ) 23) on heart rate. It is worth
pointing out that distomer (-)-5 possesses a pharma-
cological profile similar to that of (+)-5, thus confirming
the peculiar interaction of the two fluorine atoms with
the complementary muscarinic receptor subsite.
Once again our results with agonists confirm the
different structural requirements of the muscarinic
receptor subtypes and the role played by the subsite
which interacts with the hydroxy function of muscarine
(the “muscarinic subsite”) in affecting organ selectivity.4
In conclusion, our data show that, while the replace-
ment of the hydroxy group for fluorine in the 4 position
of muscarine does not greatly influence the behavior of
the molecule, the introduction of two fluorine atoms at
the same position does affect its pharmacological profile
at M2 (heart rate) and M3 (ileum) muscarinic receptor
subtypes. Difluoromuscarines represent, therefore, a
valuable tool to study the heterogeneity among M2 and
M3 subtypes and to further put in evidence the differ-
ences between the muscarinic receptors mediating heart
rate and heart force and those of ileum and bladder.4

1102 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 7
Angeli et al.
(+)-11 (2S,5S): 1H NMR (CDCl3) δ 1.26 (dd, 3, H-6; J )
6.5 and 2.3), 2.06 (m, 1, H-3; J ) 13.5, 9.5, 20.0, and 13.5),
2.29 (s, 6, NMe2), 2.39 (dd, 1, H-7; J ) 13.0 and 4.5), 2.43 (m,
1, H-3′; J ) 13.5, 6.5, 16.0, and 9.5), 2.54 (dd, 1, H-7′; J ) 13.0
and 7.2), 3.89 (m, 1, H-5; J ) 6.5, 12.5, and 12.5), 4.15 (m, 1,
H-2; J ) 4.5, 6.5, 7.2, and 9.5); 13C NMR (CDCl3) δ 13.23 (C-6;
J 6,F′ ) 1.4, J 6,F ) 7.0), 40.14 (C-3; J 3,F′ ) 24.0, J 3,F ) 24.9),
45.99 (NMe2), 63.27 (C-7), 74.09 (C-2; J 2,F′ ) J 2,F ) 4.85), 78.33
(C-5; J 5,F′ ) 26.6, J 5,F ) 32.65), 128.61 (C-4 J 4,F′ ) 248.6, J 4,F
) 255.8); Rf 0.53 (dichloromethane/methanol, 9:1); [R]20D +8.15
(c 1.110, CH2Cl2). Anal. (C8H15F2NO) C, H, N.
Exp er im en ta l Section
Ma ter ia l a n d Meth od s. (R)-(+)- and (S)-(-)-ethyl lactate
and DAST were obtained from commercial suppliers and were
used without further purification. Iodo ketones (-)-7, (+)-7,
(+)-8, and (-)-8 were prepared according to the previously
reported procedure;2 their specific rotations agreed with the
value known in the literature2 for the same compounds. 1H
NMR and 13C NMR spectra were recorded with a Bruker AC-E
300 (300 Mhz) spectrometer in CDCl3 or D2O solution; chemical
shifts (δ) 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 N-3B
thermostat. TLC were performed on commercial silica gel
GF254 plates; spots were further evidenced by spraying with a
dilute alkaline potassium permanganate solution. Liquid
compounds were characterized by the oven temperature for
Kugelrohr distillations. Melting points were determined on
a Bu¨chi apparatus and are uncorrected. Microanalyses of new
compounds agreed with the theoretical value to within (0.3%.
Syn th esis of 2-(Iod om eth yl)-4,4-d iflu or o-5-m eth yltet-
r a h yd r ofu r a n Isom er s (-)-9, (+)-9, (-)-10, a n d (+)-10. To
a dichloromethane solution (15 mL) of iodoketone (-)-72 (1.00
g, 4.17 mmol) was added dropwise DAST (2.20 mL, 16.67
mmol). The progress of the reaction was monitored by TLC
(eluent: 10% ethyl acetate/cyclohexane). After being stirred
overnight at room temperature, the mixture was carefully
poured in a slurry of ice and sodium bicarbonate and extracted
with ethyl ether (4 × 10 mL). The organic extracts were dried
over anhydrous sodium sulfate. Compound (-)-9 distilled at
75-80 °C/20 mmHg, yield 0.743 g (68%).
(-)-11 (2R,5R): [R]20 -8.56 (c 1.028, CH2Cl2). Anal.
D
(C8H15F2NO) C, H, N.
(+)-12 (2S,5R): 1H NMR (CDCl3) δ 1.24 (dd, 3, H-6; J )
6.5 and 2.2), 2.15 (m, 1, H-3; J ) 13.5, 7.5, and 19.0), 2.29 (s,
6, NMe2), 2.34 (dd, 1, H-7; J ) 12.5 and 4.5), 2.46 (m, 1, H-3′;
J ) 13.5, 7.5, and 16.5), 2.55 (dd, 1, H-7′; J ) 12.5 and 7.5),
4.12 (m, 1, H-5; J ) 6.5, 13.0, and 8.7), 4.35 (dddd, 1, H-2; J
) 4.5, 7.5, 7.5, and 7.5); 13C NMR (CDCl3) δ 13.80 (C-6; J 6,F′
)
1.6, J 6,F ) 7.6), 38.75 (C-3; J 3,F′ ) 21.1, J 3,F ) 25.2), 45.77
(NMe2), 63.21 (C-7), 73.43 (C-2; J 2,F′ ) 3.0, J 2,F ) 6.4), 76.11
(C-5; J 5,F′ ) 26.2, J 5,F ) 31.5), 128.30 (C-4; J 4,F′ ) 250.9, J 4,F
)
253.4); Rf 0.51 (dichloromethane/methanol, 9:1); [R]20 -9.31
D
(c 1.128, CH2Cl2). Anal. (C8H15F2NO) C, H, N.
(-)-12 (2R,5S): [R]20 -9.07 (c 1.270, CH2Cl2). Anal.
D
(C8H15F2NO) C, N.
Syn th esis of 2-[(Dim eth yla m in o)m eth yl]-4,4-d iflu or o-
5-m eth yltetr a h yd r ofu r a n m eth iod id es (+)-5, (-)-5, (-)-
6, a n d (+)-6. An ethereal solution of the tertiary amine was
treated with an excess of methyl iodide. The salts precipitated
quantitatively and were crystallized from acetone/ethyl ether.
(+)-5 (2S,5S): 1H NMR (D2O) δ 1.34 (ddd, 3, H-6; J ) 6.5,
2.4, and 1.0), 2.31 (m, 1, H-3; J ) 14.0, 9.0, 18.0, and 13.0),
2.80 (m, 1, H-3′; J ) 14.0, 7.0, 14.0, and 11.0), 3.30 (s, 9, NMe3),
3.67 (d, 2, H-7 and H-7′; J ) 6.0), 4.20 (m, 1, H-5; J ) 6.5,
12.5, and 12.5), 4.79 (m, 1, H-2); 13C NMR (D2O) δ 13.51 (C-6;
J 6,F′ ) 1.5, J 6,F ) 6.8), 39.52 (C-3; J 3,F′ ) J 3,F ) 25.6), 54.98
(NMe3; J Me,F ) J Me,F′ ) 3.7), 69.78 (C-7), 70.92 (C-2; J 2,F′ ) J 2,F
The same procedure was applied to iodo ketones (+)-7, (+)-8
and (-)-8 to produce the corresponding difluoro derivatives
(+)-9, (-)-10, and (+)-10 in 60-70% yield.
1
(-)-9 (2S,5S): H NMR (CDCl3) δ 1.29 (dd, 3, H-6; J ) 6.4
and 2.3), 2.16 (m, 1, H-3; J ) 12.2, 8.7, 18.6 and 12.8), 2.58
(m, 1, H-3′; J ) 12.2, 6.7, 14.4, 10.6), 3.24 (m, 1, H-7; J ) 10.2,
6.9, 0.8 and 0.8), 3.29 (m, 1, H-7′; J ) 10.2, 5.1, 0.8 and 0.9),
4.02 (qdd, 1, H-5; J ) 6.4, 12.4 and 12.4), 4.10 (m, 1, H-2; J )
5.1, 6.7, 6.9, 8.7 and 0.8); 13C NMR (CDCl3) δ 6.83 (C-7), 13.59
(C-6; J 6,F′ ) 1.7, J 6,F ) 6.8), 41.62 (C-3; J 3,F′ ) 24.1, J 3,F ) 25.2),
) 5.3), 79.63 (C-5; J 5,F′ ) 26.6, J 5,F ) 32.7), 128.50 (C-4; J 4,F′
)
248.1, J 4,F ) 253.2); mp 161-162 °C; [R]20 +25.87 (c 0.978,
D
CH3OH). Anal. (C9H18F2INO) C, H, N.
(-)-5 (2R,5R): mp 162-163 °C; [R]20 -26.40 (c 1.00, CH3-
75.36 (C-2; J 2,F′ ) J 2,F ) 5.3), 78.90 (C-5; J 5,F′ ) 26.4, J 5,F
)
D
32.5), 128.20 (C-4; J 4,F′ ) 249.0, J 4,F ) 256.0); Rf 0.56 (cyclo-
OH). Anal. (C9H18F2INO) C, H, N.
hexane/ethyl acetate, 95:5); [R]20 -9.13 (c 0.974, CH2Cl2).
1
D
(-)-6 (2R,5S): H NMR (D2O) δ 1.28 (ddd, 3, H-6; J ) 6.4,
Anal. (C6H9F2IO) C, H.
2.2, and 0.6), 2.35 (m, 1, H-3; J ) 14.4, 7.2, 18.7, and 13.4),
2.82 (m, 1, H-3′; J ) 14.4, 7.8, 7.8, and 16.4), 3.21 (s, 9, NMe3),
3.49 (dd, 1, H-7; J ) 2.0 and 14.0), 3.75 (dd, 1, H-7′; J ) 10.0
and 14.0), 4.35 (m, 1, H-5; J ) 8.8, 13.6 and 6.4), 4.93 (m, 1,
H-2); 13C NMR (D2O) δ 13.10 (C-6; J 6,F′ ) 1.3, J 6,F ) 7.1), 38.57
(C-3; J 3,F′ ) J 3,F ) 25.2), 55.02 (NMe3; J Me,F ) J Me,F′ ) 3.8),
69.04 (C-7), 70.96 (C-2; J 2,F′ ) 3.3, J 2,F ) 6.9), 77.46 (C-5; J 5,F′
) 26.4, J 5,F ) 31.75), 128.30 (C-4 J 4,F′ ) 249.8, J 4,F ) 252.1);
mp 197.5-198.5 °C dec; [R]20D -22.15 (c 1.022, CH3OH). Anal.
(C9H18F2INO) C, H, N.
(+)-9 (2R,5R): [R]20 +8.81 (c 1.134, CH2Cl2). Anal.
D
(C6H9F2IO) C, H.
(-)-10 (2S,5R): 1H NMR (CDCl3) δ 1.27 (dd, 3, H-6; J )
6.4 and 2.2), 2.31 (m, 1, H-3; J ) 14.1, 7.5, 0.8, 21.0, and 12.2),
2.59 (m, 1, H-3′; J ) 14.1, 7.2, 15.9, and 6.3), 3.30 (m, 1, H-7;
J ) 10.2, 6.4, 0.8, and 0.8),12 3.32 (m, 1, H-7′; J ) 10.2, 5.5,
0.8, and 0.8),12 4.20 (m, 1, H-5; J ) 6.4, 8.7, 14.3, and 0.8),13
4.28 (m, 1, H-2; J ) 7.2, 7.5, 5.6, and 5.6);13 13C NMR (CDCl3)
δ 8.83 (C-7), 13.17 (C-6; J 6,F′ ) 1.1, J 6,F ) 7.4), 40.50 (C-3; J 3,F′
) 23.3, J 3,F ) 25.3), 75.10 (C-2; J 2,F′ ) 2.9, J 2,F ) 7.1), 77.10
(+)-6 (2S,5R): mp 197.5-198 °C dec; [R]20D +22.63 (c 0.972,
CH3OH). Anal. (C9H18F2INO) C, H, N.
(C-5; J 5,F′ ) 26.2, J 5,F ) 31.5), 129.80 (C-4; J 4,F′ ) 251.4, J 4,F
)
254.7), Rf 0.59 (cyclohexane/ethyl acetate, 95:5); [R]20D -19.75
(c 1.127, CH2Cl2). Anal. (C6H9F2IO) C, H.
P h a r m a cology. In Vitr o Tests. Gen er a l Con sid er -
a tion s. 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 refer-
ence agonist [(()-muscarine]. 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. Contractions were recorded by means
of a force transducer connected to a two-channel Gemini
polygraph (U. Basile).
(+)-10 (2R,5S): [R]20 +19.37 (c 1.146, CH2Cl2). Anal.
D
(C6H9F2IO) C, H.
Syn th esis of 2-[(Dim eth yla m in o)m eth yl]-4,4-d iflu or o-
5-m eth yltetr a h yd r ofu r a n Isom er s (+)-11, (-)-11, (+)-12,
a n d (-)-12. A sealed metal container, filled with a solution
of (-)-9 (0.600 g, 2.29 mmol) in methanol (10 mL) and a 10-
fold excess anhydrous dimethylamine was heated at 100 °C
for 4 h. The container was cooled at 0 °C, the solution was
acidified with dilute HCl, and the volatiles were evaporated
under vacuum.The residual aqueous phase was treated with
ether (3 × 10 mL), made alkaline by a portionwise addition of
solid K2CO3, and extracted with dichloromethane (4 × 15 mL).
The extracts were dried (Na2SO4), the solvent was evaporated,
and the residue was distilled at 70-75 °C/18 mmHg to yield
0.230 g (56%) of (+)-11.
The same procedure was applied to iodo derivatives (+)-9,
(+)-10 and (-)-10 to produce the corresponding tertiary amines
(-)-11, (-)-12, and (+)-12 in 55-60% yield.

Enantiomerically Pure Muscarinic Agonists
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 7 1103
In all cases, parallel experiments in which tissues received
only (()-muscarine were run in order to check any variation
in sensitivity.
Deter m in a tion of Dissocia tion Con sta n ts. Dissociation
constants and relative efficacies for compounds (+)-5 and (-)-5
were determined as previously described8,10 according to the
method of Furchgott and Bursztyn.14
and (()-muscarine were employed, only one single dose-
response curve was assessed in each preparation. ED50 values
were determined graphically from the resultant dose-response
curves and represent the dose causing 50% of the maximum
response of the compound under study. Pretreatment, iv, with
antagonists was carried out 20 min before the administration
of agonist. This interval was selected because preliminary
experiments showed that after this time the antagonistic
effects of pirenzepine (50 µg/kg iv) and tripitramine (30 µg/kg
iv), respectively, were constant during the whole experiment.
Gu in ea P ig Ileu m . Two-centimeter-long portions of ter-
minal ileum were taken at about 5 cm from the ileum-cecum
junction and mounted in PSS at 37 °C. The composition of
PSS was as follows (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 discarded and the second one being taken as the control.
Gu in ea P ig Bla d d er . A 2 mm wide longitudinal strip of
bladder from urethra to the apex of the bladder was cut,
excluding the portion under the urethra orefice, 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).
Gu in ea P ig Stim u la ted Left Atr ia . 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, 2 Hz, 10-15 V) (Tetra Stimulus,
N. Zagnoni). Inotropic activity was recorded isometrically.
Tissues were equilibrated for 2 h and a cumulative dose-
response curve to (()-muscarine was constructed.
Sta tistica l An a lysis. Data are presented as means ( SEM
of n experiments. Differences between mean values were
tested for significance by the Student’s t test.
Ack n ow led gm en t. This work was supported by
grants from the University of Camerino and MURST
(Ministero dell’Universita´ e della Ricerca Scientifica e
Tecnologica), Rome.
Refer en ces
(1) De Amici, M.; Dallanoce, C.; De Micheli, C.; Grana, E.; Dondi,
G.; Ladinsky, H.; Schiavi, G. B.; Zonta, F. Synthesis and
pharmacological investigation of stereoisomeric muscarines.
Chirality 1992, 4, 230-239.
(2) De Amici, M.; Dallanoce, C.; De Micheli, C.; Grana, E.; Barbieri,
A.; Ladinsky, H.; Schiavi, G. B.; Zonta, F. Synthesis and
pharmacological investigation of the enantiomers of muscarone
and allomuscarone. J . Med. Chem. 1992, 35, 1915-1920.
(3) De Amici, M.; De Micheli, C.; Gianferrara, T.; Grana, E.; Zonta,
F.; Ladinsky, H.; Schiavi, G. B. Synthesis and muscarinic activity
of the chiral forms of methylenemuscarones. Il Farmaco 1993,
48, 1349-1357.
Wh ole Atr ia . The right and left atria were removed and
equilibrated for 1 h at the above conditions (see Guinea Pig
Stimulated Left Atria for PSS and temperature). Contractions
were recorded isometrically.
(4) Angeli, P. Pentatomic cyclic agonists and muscarinic receptors:
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In Vivo Tests. P ith ed Ra t. 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 of
nembutal sodium, 42.6 g of chloral hydrate, 21.2 g of MgSO4,
400 mL of propylene glycol, 50 mL of ethyl alcohol, and water
to 1000 mL) 3 mL/kg of body weight ip. The right jugular vein
was cannulated (PE 10 polyethylene tubing) for drug admin-
istration. 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.
(5) Bravo, P.; Resnati, G.; Angeli, P.; Frigerio, M.; Viani, F.; Arnone,
A.; Marucci, G.; Cantalamessa, F. Synthesis and pharmacological
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(7) Wilkinson, J . A. Recent Advances in the Selective Formation of
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T.; Teodori, E. Chiral muscarinic agonists possessing a 1,3-
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(9) Angeli, P.; Brasili, L.; Cantalamessa, F.; Marucci, G.; Wess, J .
Determination of muscarinic agonist potencies at M1 and M2
After catheterization of the trachea, heparin (150 IU/kg) was
given iv 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.15
The animals were respired artificially by means of a Harvard
Apparatus Model 681 rodent respirator at a frequency of 60
cycles/min with a volume of 1 mL/100 g. The preparation was
allowed to equilibrate for at least 30 min before drug admin-
istration, until mean heart rate had stabilized. The basal
heart rate amounted to 300 ( 8 beats/min (n ) 50). Changes
in heart rate were measured for individual doses of the agonist
given iv (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 µL of isotonic saline solution.
Exp er im en ta l P r otocol. All drugs were dissolved in
saline (0.9% w/v) and injected iv in a volume of 0.1 mL/100 g.
Because of desensitization phenomena, when compounds (+)-5
muscarinic receptors in
a modified pithed rat preparation.
Naunyn-Schmiedeberg’s Arch. Pharmacol. 1990, 342, 625-629.
(10) Furchgott, R. F. The classifications of adrenoceptors. An evalu-
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experimental pharmacology; Blaschko H., Muscholl E., Eds.;
Springer: Berlin, Heidelberg New York, 1972; pp 283-335.
(11) Angeli, P.; Brasili, L.; Giannella, M.; Gualtieri, F.; Pigini, M.
Affinity and efficacy correlate with chemical structure more than
potency does in a series of pentatomic cyclic muscarinic agonists.
Br. J . Pharmacol. 1985, 85, 783-786.
(12) H-7 and H-7′ coupling constants were determined in acetone-d6
solution.
(13) H-5 and H-2 coupling constants were determined in C6D6
solution.
(14) Furchgott, R. F.; Bursztyn, P. Comparison of dissociation
constants and of relative efficacies of selected agonists acting
on parasympathetic receptors. Ann. N.Y. Acad. Sci. 1967, 144,
882-899.
(15) Shipley, R. E.; Tilden, J . H. A pithed rat preparation suitable
for assaying pressor substances. Proc. Soc. Exp. Biol. Med. 1947,
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J M960742W