1,4-Dioxane, a suitable scaffold for the development of novel M3 muscarinic receptor antagonists

Article abstract of DOI:10.1021/jm2013216

In this study the modulation of the pharmacological profile from agonist to antagonist was successfully obtained by replacing the methyl group in position 6 of the 1,4-dioxane scaffold of the potent M₂/M₃ muscarinic agonist 1 with bulkier groups. In particular, the 6,6-diphenyl substitution provided the potent M₃ preferring antagonist (±)-17, which in in vivo study proved to be effective in reducing the volume-induced contractions of rat urinary bladder and was devoid of cardiovascular effects.

Full text of DOI:10.1021/jm2013216

Brief Article
pubs.acs.org/jmc
1,4-Dioxane, a Suitable Scaffold for the Development of Novel M3
Muscarinic Receptor Antagonists†
Fabio Del Bello,‡ Elisabetta Barocelli,§ Simona Bertoni,§ Alessandro Bonifazi,‡ Mercedes Camalli,∥
Gaetano Campi,∥ Mario Giannella,‡ Rosanna Matucci,⊥ Marta Nesi,⊥ Maria Pigini,‡ Wilma Quaglia,‡
and Alessandro Piergentili*,‡
‡Scuola di Scienze del Farmaco e dei Prodotti della Salute, Universita
̀
di Camerino, Via S. Agostino 1, 62032 Camerino, Italy
§Dipartimento di Scienze Farmacologiche, Biologiche e Chimiche Applicate, Universita
43124 Parma, Italy
̀
degli Studi di Parma, V.le delle Scienze 27/A,
∥Istituto di Cristallografia, CNR, Area della Ricerca Roma 1, Via Salaria Km 29.3, 00016 Monterotondo Stazione, Roma, Italy
⊥Dipartimento di Farmacologia, Universita
̀
di Firenze, Viale G. Pieraccini 6, 50139, Firenze, Italy
S
* Supporting Information
ABSTRACT: In this study the modulation of the pharmacological profile from agonist to antagonist was successfully obtained
by replacing the methyl group in position 6 of the 1,4-dioxane scaffold of the potent M2/M3 muscarinic agonist 1 with bulkier
groups. In particular, the 6,6-diphenyl substitution provided the potent M3 preferring antagonist ( )-17, which in in vivo study
proved to be effective in reducing the volume-induced contractions of rat urinary bladder and was devoid of cardiovascular
effects.
INTRODUCTION
■
Muscarinic acetylcholine receptors (mAChRs) are subdivided
into five different subtypes, M1−M5, and are involved in the
regulation of several physiological functions.1 Though muscar-
inic agonists have been reported to be useful in cognitive
disorders, muscarinic antagonists are therapeutically more
interesting, being currently used for the treatment of numerous
pathologies associated with the hyperactivity of the muscarinic
system. In particular, M3 antagonists are useful for the
treatment of smooth muscle disorders, such as chronic
obstructive pulmonary disease (COPD),2 overactive bladder
(OAB),3 and irritable bowel syndrome (IBS).4 In the
symptomatic treatment of OAB, mAChR antagonists block
M2 and M3 receptors localized on detrusor smooth muscle cells
and on urothelium and suburothelium within the urinary
bladder wall. In this tissue, the M3 subtype has been
demonstrated to mediate the direct contractile responses
necessary for the normal bladder function.3 At present,
combination therapies of β2-agonists with muscarinic antago-
nists are given for severe COPD and antimuscarinic drugs are
currently marketed for the treatment of OAB and IBS.
However, because of the lack of M3 subtype selectivity, these
drugs present numerous side effects, especially those cognitive
and cardiovascular owing to their interaction with M1 and M2
subtypes, respectively.2−5 Therefore, potent and selective M3
antagonists would maximize therapeutic efficacy and minimize
unpleasant side effects. We have recently demonstrated that the
1,4-dioxane ring is a suitable scaffold for building ligands
targeting mAChRs. In particular, 1 (Figure 1) has emerged as a
preferential M2/M3 subtype agonist.6 Since the replacement of
the methyl group in muscarinic agonists, such as ACh or
muscarine and its 1,3-dioxolane analogue (2), with bulkier
groups modulates the pharmacological profile from agonist to
Figure 1. Structures of 1−6, 17, 22, and 23 and X-ray structure of (R)-
(+)-5.
antagonist,7 in this study, the methyl group in position 6 of the
1,4-dioxane nucleus of 1 has been replaced by ethyl, phenyl,
two phenyl, or diphenylmethane moieties (3−6, respectively)
to obtain novel muscarinic antagonists, possibly endowed with
subtype selectivity. Moreover, since the interaction of a ligand
with mAChRs is usually extremely specific, the role of
stereochemistry has been investigated through the cis and
trans diastereomers of the 6-monosubstituted derivatives and
the enantiomers of the 6,6-diphenyl-substituted 5. Finally,
considering that ligands with a tertiary basic function can act as
antagonists at mAChRs,7 probably by binding in the cationic
form, the racemic tertiary amine 17 and its enantiomers have
also been pharmacologically evaluated.
Received: July 28, 2011
Published: January 13, 2012
© 2012 American Chemical Society
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Journal of Medicinal Chemistry
Brief Article
a
Scheme 1
a
Reagents: (a) CH2CHCH2OH, Na; (b) (CH3COO)2Hg, H2O; (c) KI, I2; (d) m-CPBA, CH2Cl2; (1S)-(+)-10-CSA, CH2Cl2; (e) p-TsCl,
pyridine; (f) Me2NH, benzene; (g) CH3I, diethyl ether.
a
Scheme 2
a
Reagents: (a) (COCl)2, CH2Cl2, cat. DMF; (b) nBuLi, (R)-(+)-4-benzyl-2-oxazolidinone, THF, −78 °C; (c) LiBH4, THF, H2O; (d) p-TsCl,
pyridine; (e) Me2NH, benzene; (f) CH3I, diethyl ether.
18b show a deshielding effect with respect to the same protons
in the diastereomers 15a and 18a. Therefore, the stereo-
chemical relationship between the substituents in positions 2
and 6 is cis in 15a and 18a and trans in 15b and 18b. For the
preparation of (R)-(+)- and (S)-(−)-5 the 6,6-diphenyl-1,4-
dioxane-2-carboxylic acid9 was converted into the correspond-
ing acyl chloride which, treated with the lithium salt of the (R)-
(+)-4-benzyl-2-oxazolidinone, yielded the diastereomers 19a
and 19b, easily separable by column chromatography (Scheme
2). These derivatives were reduced with lithium borohydride to
yield the enantiomeric alcohols (R)-(+)- and (S)-(−)-20,
respectively, which were treated with tosyl chloride and
subsequently with dimethylamine to afford the free amines
(R)-(+)- and (S)-(−)-17. These were transformed into the
corresponding methiodides (R)-(+)- and (S)-(−)-5. Enantio-
meric purity of amines (R)-(+)- and (S)-(−)-17, determined by
1H NMR spectroscopy on addition of the chiral shift reagent
(R)-(+)-α-methoxy-α-(trifluoromethyl)phenylacetic acid
[(+)-MTPA] and in comparison with the spectrum of racemic
compound ( )-17, was found to be >98% (detection limit) for
both of them. In fact, the spectrum of racemic ( )-17 in the
presence of (+)-MTPA showed two double doublets at δ 3.35
CHEMISTRY
■
Compounds 7 and 8 were obtained as previously reported in
the literature,8,9 whereas the novel compound 9 was prepared
by treating 2-benzhydryloxirane10 with metallic sodium and
allyl alcohol (Scheme 1). The allyloxyalkanols 7 and 9 were
treated with mercury(II) acetate followed by an aqueous
solution of iodine and potassium iodide, affording a mixture of
the diastereomers 10a/10b and 11a/11b, respectively, which
were separated by column chromatography. Iodo derivative 12
was obtained as described in the literature.9 Alcohols 13a and
13b, prepared starting from 8,9 were treated with tosyl chloride
in pyridine to yield 14a and 14b, respectively. The amination of
iodo derivatives 10−12 and tosyl derivatives 14 with dimethyl-
amine afforded amines 15−18, which were transformed into
the methiodides 3−6. The stereochemical relationship between
the substituents in positions 2 and 6 of 1,4-dioxane nucleus of
1
3a/3b and 6a/6b was assigned by comparing the H NMR
spectra of the corresponding free amines 15a/15b and 18a/
18b with those of the 6-methyl and 6-phenyl analogues, whose
structures had previously been determined by X-ray crystallog-
raphy or the NOE effect, respectively.6,9 In particular, in the 1H
NMR spectra the protons of the 2-methylene group of 15b and
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Journal of Medicinal Chemistry
Brief Article
a
Table 1. Affinity Constants (pKi) of 1, 3−6, 17, Enantiomers of 5 and 17, and NMS for Human Cloned Muscarinic Receptors,
b
c
Expressed in CHO Cells; Potency (pD2 = −log EC50) and Intrinsic Activity (α) for 1 and 3, and Dissociation Constants
d
(pKB) for 4, ( )-5, (S)-(−)-5, (R)-(+)-5, ( )-17, (S)-(−)-17, (R)-(+)-17, 22, 23, Oxybutynin, Methoctramine, and 4-DAMP in
the Isolated Guinea Pig Left Atrium (M2), Longitudinal Ileum (M3), and Lung (M4) Muscarinic Receptors
e
functional data
binding data, pKi
M2
pD2 (pKB)
M3
M4
compd
1 (cis)
hM1
hM2
hM3
hM4
hM5
α
pD2 (pKB)
α
pD2 (pKB)
4.52
4.81
<4
5.80
5.20
4.74
5.19
4.40
8.24
8.55
7.48
4.55
4.10
7.78
8.01
7.02
9.75
5.12
4.75
4.09
5.01
4.50
8.44
8.83
7.21
5.05
4.67
8.66
8.82
7.14
9.87
4.94
4.60
<4
5.11
4.58
<4
7.57 0.11
1
7.34 0.13
4.82 0.05
5.19 0.08
(5.20 0.08)
(<5)
1
1
1
0
0
0
0
0
3a (cis)
3b (trans)
4a (cis)
4b (trans)
( )-5
5.62 0.20
1
f
f
5.45 0.11
0.7
0
5.17
4.58
9.10
9.30
7.79
5.42
4.80
8.34
8.90
7.45
9.49
4.91
4.53
8.58
8.83
6.82
4.95
4.77
8.31
8.63
7.15
9.85
4.99
4.48
8.36
8.77
6.97
5.12
4.50
8.27
8.74
7.09
9.68
(5.56 0.11)
(5.14 0.17)
(8.21 0.16)
(7.31 0.08)
(6.67 0.01)
(5.24 0.20)
(<5)
0
0
(8.34 0.03)
(8.52 0.10)
(7.22 0.03)
(7.77 0.22)
(6.70 0.19)
(6.62 0.04)
(S)-(−)-5
(R)-(+)-5
6a (cis)
6b (trans)
( )-17
0
0
(6.95 0.19)
(7.63 0.01)
(7.00 0.25)
0
0
0
(8.24 0.05)
(8.34 0.03)
(7.45 0.12)
0
0
0
(7.02 0.22)
(6.94 0.14)
(7.24 0.01)
(S)-(−)-17
(R)-(+)-17
NMS
g
g
22
(8.29)
(7.91)
g
g
23
(7.11)
(6.38)
oxybutynin
methoctramine
4-DAMP
8.62
7.93
8.82
8.44
7.85
(7.45 0.28)
0
(8.47 0.08)
0
(7.19 0.26)
h
h
h
(7.8−8.3)
(6.3−6.9)
(9.02 0.06)
(7.6)
h
h
(8.0−8.4)
(9.4)
a
b
Data are the mean SEM of three experiments performed in duplicate. Ki values were from two to three experiments which agreed 10%. pD2
c
values are the −log of the agonist concentration that caused 50% of the maximum response attainable in that tissue. Intrinsic activity was measured
by the ratio between the maximum response of the agonist and the maximum response of bethanechol at guinea pig atrium and ileum receptors.
Dissociation constants were calculated according to Furchgott.20 The results are the mean SEM of four to six independent experiments. This
d
e
f
g
h
compound showed no agonist activity and, when tested as antagonist, proved inactive up to 10 μM. Data from ref 7. Data from ref 21.
and 3.55 ppm for the 2-methylene protons, whereas only one
double doublet was observed for (+)-17 and (−)-17 at δ 3.35
and 3.55 ppm, respectively. The absolute configuration R was
assigned to the dextrorotatory enantiomer (+)-5 through X-ray
diffraction analysis (Figure 1).
functional models.17,18 Binding data show that the replacement
of the methyl group in position 6 of 1 with an ethyl or only one
phenyl group (3 or 4, respectively) does not significantly alter
the muscarinic affinities of the lead 1 (Table 1). Interestingly,
the introduction of two phenyl groups in the same position (5)
markedly increases the affinity for all muscarinic subtypes. In
contrast, the increase of the distance between the diphenyl
hydrophobic portion and the basic function of 5, affording the
diastereomers 6a and 6b, lowers the binding affinities at all the
muscarinic receptor subtypes to values similar to those of 1.
This effect is independent of the stereochemical relationship
between the substituents in positions 2 and 6. The high
muscarinic affinity displayed by 5 is enhanced by its eutomer
(S)-(−)-5, while the enantiomer (R)-(+)-5 shows significantly
lower values at the five mAChR subtypes (20-, 6-, 17-, 58-, and
25-fold, respectively), suggesting that in this series of
compounds the stereocenter in position 2 plays a critical role
in the interaction of the antagonist with mAChRs. Interestingly,
the tertiary amine ( )-17 and its (S)-eutomer are preferring M3
over M2 subtype. However, they are also endowed with similar
high affinity for all the other muscarinic subtypes. In particular,
considering that they might cross the brain−blood barrier, the
blockade of central M1 receptors would produce cognitive side
effects.5 The functional data reported in Table 1 show that the
6-ethyl diastereomers 3a and 3b, inactive at the M4 subtype,
behave as agonists at M2 and M3 receptor subtypes but are
endowed with about 100-fold lower potency with respect to the
lead 1, confirming that the increase of the distance between the
terminal methyl group and the basic nitrogen atom (Ing’s rule)
is a critical requirement for the agonist potency. The 6-
RESULTS AND DISCUSSION
■
The muscarinic binding profile of the novel compounds was
evaluated using [3H]N-methylscopolamine ([3H]NMS) as
radioligand to label cloned human muscarinic hM1−hM5
receptors, expressed in Chinese hamster ovary (CHO) cells
(Table 1).11 The functional activities of the tested compounds
were determined on guinea pig left atrium (M2),12 ileum
(M3),13 and lung strips (putative M4)14 and are reported as pD2
or pKB values for agonist or antagonist compounds, respectively
(Table 1). The antagonist potencies of ( )-17 and (S)-(−)-5
on rabbit vas deferens (putative M1)15 were also determined.
Moreover, the affinity values, functionally determined by us or
collected from the literature, of reference muscarinic antago-
nists methoctramine and 4-DAMP, purportedly selective
toward M2 and M3 receptor subtypes, respectively, dioxolane
derivatives 22 and 237 (Figure 1), lower homologues of 5 and
17, respectively, and oxybutynin are also reported for the sake
of comparison. Some discrepancies observed between pKi and
pKB values can be explained by strain/species differences16 and
by the different organization of the native and cloned receptor
populations.17 Those between functional activity on rabbit vas
deferens and guinea pig lung preparations and the binding data
on hM1 and hM4, respectively, are expected and would
reinforce the doubts regarding the prediction of these
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monophenyl analogues 4a and 4b behave as weak antagonists,
and their activity appears to be scarcely affected by the
stereochemical relationship between the substituents in
positions 2 and 6 of the 1,4-dioxane nucleus. The introduction
of two phenyl groups in position 6 of the 1,4-dioxane nucleus
produces the potent nonselective antagonist 5, which shows
affinity values similar to those of its dioxolane analogue 22.
Therefore, as expected, the modulation of the biological
muscarinic profile from a full potent M2/M3 agonist (1) to a
potent antagonist (5) has successfully been obtained. The
important role played by the configuration of the stereocenter
in position 2 on the affinity for M2 and M3 receptor subtypes,
emerged from the binding assays, is confirmed by the functional
studies, with the eutomer being the (S)-enantiomer. Interest-
ingly, unlike the methiodide 5, the tertiary amine 17 and to a
lesser extent its (S)-eutomer preferentially block the M3
receptor subtype with respect to the M2, with an M3/M2
selectivity ratio slightly higher than those of oxybutynin and the
conventional M3 selective antagonist 4-DAMP. The observa-
tion that a reversal of the selectivity ratio is observed with
respect to its dioxolane lower homologue 23, which shows a
pKB value at M2 higher than that at M3, is also noteworthy.
Surprisingly, preferring M3 antagonist activity, missing in the
racemic methiodide ( )-5, is also displayed by its (S)-eutomer.
On the basis of their favorable preferring M3 antagonism
unmasked by in vitro assays, the oxalate ( )-17 and the
methiodide (S)-(−)-5 were also evaluated on rabbit vas
deferens preparation (putative M1). On this preparation, the
tertiary amine ( )-17 shows a pKB of 7.86 0.03, which is
lower than that at M3 (pKB = 8.24), whereas the pKB found for
antagonism of the tertiary amine ( )-17 might open a
promising strategy for designing novel muscarinic subtype-
selective antagonists.
EXPERIMENTAL SECTION
■
The purity of the novel tested compounds was determined by
combustion analysis and was ≥95%.
(6,6-Diphenyl-1,4-dioxan-2-yl)-N,N-dimethylmethanamine
Oxalate (17). A solution of 129 (1.1 g, 2.9 mmol) and dimethylamine
(5 mL) in dry benzene (15 mL) was heated in a sealed tube at 120 °C
for 60 h. The residue was dissolved in CHCl3, which was washed with
2 N NaOH and dried over Na2SO4. The removal of the solvent gave a
residue, which was purified by column chromatography. Eluting with
CHCl3/CH3OH (97:3) afforded 17 as the free base: 0.7 g (80% yield).
1H NMR (CDCl3): δ 2.22 (s, 6, N(CH3)2), 2.42 (m, 2, CH2N), 3.38−
3.62 (m, 2, cycle), 3.76−3.84 (m, 2, cycle), 4.64 (d, 1, cycle), 7.19−
7.57 (m, 10, ArH). The free amine was transformed into the oxalate
salt, which was recrystallized from 2-PrOH (mp 152−153 °C). Anal.
(C19H23NO2·C2H2O4) C, H, N.
(6,6-Diphenyl-1,4-dioxan-2-yl)-N,N,N-trimethylmethanami-
nium Iodide (5). A solution of 17 (0.4 g, 1.4 mmol) in Et2O (10 mL)
was treated with an excess of methyl iodide. After 24 h at room
temperature the solid was filtered and recrystallized from 2-PrOH (mp
232−234 °C). 1H NMR (DMSO): δ 3.05 (s, 9, N(CH3)3), 3.25−4.01
(m, 6, CH2N, cycle), 4.82 (d, 1, cycle), 7.21−7.37 (m, 10, ArH). Anal.
(C20H26INO2) C, H, N.
(R)-4-Benzyl-3-((S)-6,6-diphenyl-1,4-dioxane-2-carbonyl)-
oxazolidin-2-one (19a) and (R)-4-Benzyl-3-((R)-6,6-diphenyl-
1,4-dioxane-2-carbonyl)oxazolidin-2-one (19b). Oxalyl chloride
(0.7 mL) and DMF (0.2 mL) were added to a solution of 6,6-
diphenyl-1,4-dioxane-2-carboxylic acid9 (1.6 g, 5.6 mmol) in CH2Cl2
(15 mL). After 1 h at 25 °C the solvent was removed and the residue
was dissolved in dry toluene (15 mL) and added at −78 °C to the
lithium anion of (R)-(+)-4-benzyl-2-oxazolidinone prepared by adding
n-BuLi (2.25 mL) to a solution of (R)-(+)-4-benzyl-2-oxazolidinone
(1.0 g, 5.6 mmol) in THF (30 mL) at −78 °C and stirring for 1 h.
After 45 min at −78 °C Et2O (50 mL) was added. The organic phase
was washed with NH4Cl (20 mL) and dried over Na2SO4. The
removal of the solvent afforded a mixture of diastereomers, which were
separated by column chromatography, eluting with cyclohexane/
(S)-(−)-5 (pKB = 8.86
0.01) is higher than that at M3
subtype (pKB = 8.52). Moreover, these two compounds were
evaluated in vivo in the anesthetized rat for their ability to affect
the volume-induced contractions of urinary bladder and the
cardiovascular parameters (Table 2). Compared to the
reference compound oxybutynin, an antagonist used for the
1
a
EtOAc (9:1). 19a eluted first: 1.3 g; 52% yield; mp 85−87 °C. H
Table 2. Potency, Expressed as ID50 (μg/kg iv) and
b
NMR (CDCl3): δ 2.82 (dd, 1, CHAr), 3.25 (dd, 1, CHAr), 3.64 (m, 2,
cycle), 4.23 (m, 3, CH2OCO, CHN, cycle), 4.61 (m, 2, CH2OCO,
cycle), 5.27 (dd, 1, cycle), 7.18−7.56 (m, 15, ArH). The second
Efficacy, Expressed as Imax (%) in the Micturition Reflex
and Percentage of Changes in Mean Arterial Pressure
(MAP) and Heart Rate (HR) of (S)-(−)-5, ( )-17, and
Oxybutynin in the Anaesthetized Rat
1
fraction was 19b: 0.77 g; 31% yield; 93−94 °C. H NMR (CDCl3) δ
2.83 (dd, 1, CHAr), 3.39 (dd, 1, CHAr), 3.69 (m, 2, cycle), 4.19 (m, 3,
CH2OCO, CHN, cycle), 4.62 (m, 2, CH2OCO, cycle), 5.38 (dd, 1,
cycle), 7.09−7.61 (m, 15, ArH).
cystometric
cardiovascular
compd
ID50
Imax (%)
MAP (%)
HR (%)
(R)-(6,6-Diphenyl-1,4-dioxan-2-yl)methanol [(R)-(+)-20]. H2O
(0.035 mL) and then LiBH4 (0.036 g) in Et2O (10 mL) were added to
a solution of 19a (0.75 g, 1.7 mmol) in Et2O (50 mL) at 0 °C. After 2
h at 0 °C, 1 N NaOH (3 mL) was added. The mixture was extracted
with EtOAc, which was dried over Na2SO4. The removal of the solvent
afforded a residue which was purified by column chromatography,
eluting with cyclohexane/EtOAc (8:2) to give (R)-(+)-20 as a solid:
0.28 g; 60% yield; mp 114−115 °C; [α]20D +222.32 (c 1, CHCl3). The
1H NMR spectrum was identical to that of the racemic compound.9
(S)-(6,6-Diphenyl-1,4-dioxan-2-yl)methanol [(S)-(−)-20]. This
was obtained as a solid following the procedure described for (R)-
(S)-(−)-5
( )-17
201.4
134.0
347.4
96
72
75
−12
−18
+20
+5
+4
+7
oxybutynin
a
ID50 is the dose required to produce 50% inhibition of AUC peaks
b
from saline baseline. Imax is the % maximal inhibition of VIBC
amplitude compared to saline baseline.
treatment of OAB,5 the newly synthesized 1,4-dioxane
derivatives exhibit a higher potency. Moreover, at 1 μg/kg iv,
they display a comparable or enhanced efficacy in reducing the
area of the voiding contractions. Similar to oxybutynin and
unlike methoctramine,19 a selective M2 antagonist, they are
devoid of significant effects on mean arterial pressure (MAP)
and on heart rate (HR) (Table 2), confirming that they are
preferring M3 over M2 subtype as shown in functional in vitro
assays. In conclusion, this study has demonstrated that a 1,4-
dioxane nucleus might be a suitable scaffold for building
muscarinic antagonists. The potent and preferential M3
(+)-20: 60% yield; mp 114−115 °C; [α]20 −225.58 (c 1, CHCl3).
D
The 1H NMR spectrum was identical to that of the racemic
compound.9
(S)-(6,6-Diphenyl-1,4-dioxan-2-yl)methyl 4-Methylbenzene-
sulfonate [(S)-(+)-21]. Tosyl chloride (0.4 g, 2.1 mmol) was added to
a stirred solution of (R)-(+)-20 (0.4 g, 1.5 mmol) in pyridine (5 mL)
at 0 °C over 30 min. After 3 h at 0 °C, the mixture was left for 20 h at
4 °C in the freezer. Then it was poured into ice and concentrated HCl
(5 mL) and extracted with CHCl3. The organic layers were washed
with 2 N HCl (15 mL), NaHCO3 saturated solution (15 mL), and
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Journal of Medicinal Chemistry
Brief Article
H2O (15 mL) and then dried over Na2SO4. The evaporation of the
(3) Abrams, P.; Andersson, K.-E. Muscarinic receptor antagonists for
overactive bladder. BJU Int. 2007, 100, 987−1006.
(4) Lesbros-Pantoflickova, D.; Michetti, P.; Fried, M.; Beglinger, C.;
Blum, A. L. Meta-analysis: the treatment of irritable bowel syndrome.
Aliment. Pharmacol. Ther. 2004, 20, 1253−1269.
solvent afforded (S)-(+)-21 as a solid (mp 130−131 °C): 0.44 g; 69%
1
yield; [α]20 +123.48 (c 1, CHCl3). H NMR (CDCl3) δ 2.41 (s, 3,
D
CH3), 3.41 (m, 2, CH2O), 3.70 (m, 2, cycle), 4.01 (m, 2, cycle), 4.48
(d, 1, cycle), 7.08−7.78 (m, 14, ArH).
(R)-(6,6-Diphenyl-1,4-dioxan-2-yl)methyl 4-Methylbenzene-
sulfonate [(R)-(−)-21]. This was obtained as a solid following the
procedure described for (S)-(+)-21 (mp 130−131 °C): 70% yield;
[α]20D −121.56 (c 1, CHCl3). The 1H NMR spectrum was identical to
that of (S)-(+)-21.
(5) Kessler, T. M.; Bachmann, L. M.; Minder, C.; Lohrer, D.;
Umbehr, M.; Schunemann, H. J.; Kessels, A. G. H. Adverse event
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(R)- and (S)-(6,6-Diphenyl-1,4-dioxan-2-yl)-N,N-dimethylme-
thanamine Oxalate [(R)-(+)-17 and (S)-(−)-17]. These were
prepared as described for 17: 81% yield. (R)-(+)-17: [α]20 +221.34
D
1
(c 1, CHCl3). (S)-(−)-17: [α]20 −219.97 (c 1, CHCl3). Their H
D
(7) Angeli, P. Pentatomic cyclic antagonists and muscarinic
receptors: a 30-year review. Farmaco 1998, 53, 1−21.
NMR spectra were identical to that of the racemic compound 17. The
free amines were transformed into the oxalate salts, which were
recrystallized from 2-PrOH (mp 152−153 °C). Anal.
(C19H23NO2·C2H2O4) C, H, N.
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hexachlorocyclopentadiene with allyloxyalkanols. J. Org. Chem. 1960,
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Santoni, G.; Poggesi, E.; Leonardi, A. Structure−activity relationships
in 1,4-benzodioxan-related compounds. 9. From 1,4-benzodioxane to
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(R)- and (S)-(6,6-Diphenyl-1,4-dioxan-2-yl)-N,N,N-trimethyl-
methanaminium Iodide [(R)-(+)-5 and (S)-(−)-5]. These were
prepared as described for 5 and were recrystallized from 2-PrOH: 86%
yield; mp 232−234 °C. (R)-(+)-5: [α]20D +208.14 (c 1, MeOH). (S)-
1
(−)-5: [α]20 −207.29 (c 1, MeOH). The H NMR spectrum was
D
identical to that of the racemic compound 5. Anal. (C20H26INO2) C,
H, N.
(10) Zhang, S.; Zhen, J.; Reith, M. E. A.; Dutta, A. K. Discovery of
novel trisubstituted asymmetric derivatives of (2S,4R,5R)-2-benzhydr-
yl-5-benzylaminotetrahydropyran-4-ol, exhibiting high affinity for
serotonin and norepinephrine transporters in a stereospecific manner.
J. Med. Chem. 2005, 48, 4962−4971.
ASSOCIATED CONTENT
■
S
* Supporting Information
General chemistry; details for the syntheses of 3, 4, and 6;
elemental analysis results for 3−6, 17, (S)-(−)-5, (R)-(+)-5,
(S)-(−)-17, and (R)-(+)-17; X-ray crystallographic data for
(R)-(+)-5; experimental details of binding, functional, and in
vivo assays. This material is available free of charge via the
Internet at http://pubs.acs.org.
(11) Dei, S.; Angeli, P.; Bellucci, C.; Buccioni, M.; Gualtieri, F.;
Marucci, G.; Manetti, D.; Matucci, R.; Romanelli, M. N.; Scapecchi, S.;
Teodori, E. Muscarinic subtype affinity and functional activity profile
of 1-methyl-2-(2-methyl-1,3-dioxolan-4-yl)pyrrolidine and 1-methyl-2-
(2-methyl-1,3-oxathiolan-5-yl)pyrrolidine derivatives. Biochem. Phar-
macol. 2005, 69, 1637−1645.
Accession Codes
(12) Eglen, R. M.; Watson, N. Selective muscarinic receptor agonists
and antagonists. Pharmacol. Toxicol. 1996, 78, 59−68.
†The X-ray coordinates of compound (R)-(+)-5 have been
deposited with Cambridge Crystallographic Data Centre with
accession number CCDC 820353.
(13) Barocelli, E.; Ballabeni, V.; Bertoni, S.; Dallanoce, C.; De Amici,
M.; De Micheli, M.; Impicciatore, M. New analogues of oxotremorine
and oxotremorine-M: estimation of their in vitro affinity and efficacy at
muscarinic receptor subtypes. Life Sci. 2000, 67, 717−723.
(14) Roffel, A. F.; Elzinga, C. R. S.; Zaagsma, J. Cholinergic
contraction of the guinea pig lung strip is mediated by muscarinic M2-
like receptors. Eur. J. Pharmacol. 1993, 250, 267−279.
AUTHOR INFORMATION
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Corresponding Author
*Phone: +390737402237. Fax: +390737637345. E-mail:
alessandro.piergentili@unicam.it.
(15) Eltze, M. Muscarinic M1- and M2-receptors mediating opposite
effects on neuromuscular transmission in rabbit vas deferens. Eur. J.
Pharmacol. 1988, 151, 205−221.
ACKNOWLEDGMENTS
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(16) Choppin, A. Muscarinic receptors in isolated urinary bladder
smooth muscle from different mouse strains. Br. J. Pharmacol. 2002,
137, 522−528.
This work was supported by grants from the MIUR (Rome),
the University of Camerino, Italy, and the Monte dei Paschi di
Siena Foundation.
(17) Caulfield, M. P.; Birdsall, N. J. M. International Union of
Pharmacology. XVII. Classification of muscarinic acetylcholine
receptors. Pharm. Rev. 1998, 50, 279−290.
ABBREVIATIONS USED
■
(18) Roffel, A. F.; Davis, J. H.; Elzinga, C. R. S.; Wolf, D.; Zaagsma,
J.; Kilbinger, H. Characterization of the muscarinic receptor
subtype(s) mediating contraction of the guinea-pig lung strip and
inhibition of acetylcholine release in the guinea-pig trachea with the
selective muscarinic receptor antagonist tripitramine. Br. J. Pharmacol.
1997, 122, 133−141.
mAChR, muscarinic acetylcholine receptor; COPD, chronic
obstructive pulmonary disease; OAB, overactive bladder; IBS,
irritable bowel syndrome; [3H]NMS, [3H]N-methylscopol-
amine; CHO, Chinese hamster ovary; TMS, tetramethylsilane;
MAP, mean arterial pressure; HR, heart rate; AUC, area under
the curve; VIBC, volume-induced bladder contraction
(19) Hegde, S. S.; Choppin, A.; Bonhaus, D.; Briaud, S.; Loeb, M.;
Moy, T. M.; Loury, D.; Eglen, R. M. Functional role of M2 and M3
muscarinic receptors in the urinary bladder of rats in vitro and in vivo.
Br. J. Pharmacol. 1997, 120, 1409−1418.
(20) 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, 882−898.
(21) Caulfield, M. P. Muscarinic receptors: characterization, coupling
and function. Pharmacol. Ther. 1993, 58, 319−379.
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