Properly substituted 1,4-dioxane nucleus favours the selective M3 muscarinic receptor activation

Article abstract of DOI:10.1016/j.bmc.2009.10.027

Novel analogues of cis-N,N,N-trimethyl-(6-methyl-1,4-dioxan-2-yl)methanaminium iodide (2a) were synthesized by inserting methyl groups alternatively or simultaneously in positions 5 and 6 of the 1,4-dioxane nucleus in all combinations. Their biological pr

Full text of DOI:10.1016/j.bmc.2009.10.027

Bioorganic & Medicinal Chemistry 17 (2009) 8174–8185
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Properly substituted 1,4-dioxane nucleus favours the selective M3
muscarinic receptor activation
a,*
a
a
a
a
Alessandro Piergentili , Wilma Quaglia , Fabio Del Bello , Mario Giannella , Maria Pigini ,
b
b
c
c
d
e
Elisabetta Barocelli , Simona Bertoni , Rosanna Matucci , Marta Nesi , Bruno Bruni , Massimo Di Vaira
Dipartimento di Scienze Chimiche, Università di Camerino, Via S. Agostino 1, 62032 Camerino, Italy
Dipartimento di Scienze Farmacologiche, Biologiche e Chimiche Applicate, Università degli Studi di Parma, V.le G.P. Usberti 27/A, 43100 Parma, Italy
Dipartimento di Farmacologia, Università di Firenze, Viale G. Pieraccini 6, 50139 Firenze, Italy
Dipartimento di Scienze Farmaceutiche, Università di Firenze, Via U. Schiff 6, 50019 Sesto Fiorentino, Firenze, Italy
Dipartimento di Chimica, Università di Firenze, Via della Lastruccia 3, 50019 Sesto Fiorentino, Firenze, Italy
a
b
c
d
e
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel analogues of cis-N,N,N-trimethyl-(6-methyl-1,4-dioxan-2-yl)methanaminium iodide (2a) were
synthesized by inserting methyl groups alternatively or simultaneously in positions 5 and 6 of the 1,4-
dioxane nucleus in all combinations. Their biological profile was assessed by receptor binding assays
Received 2 September 2009
Revised 7 October 2009
Accepted 15 October 2009
Available online 20 October 2009
1 5
at human muscarinic M –M receptors stably expressed in CHO cells and by functional studies performed
on classical isolated organ preparations, namely, rabbit electrically stimulated vas deferens, and guinea
pig electrically stimulated left atrium, ileum, and lung strips. The results showed that the simultaneous
presence of one methyl group in both positions 5 and 6 with a trans stereochemical relationship with
each other (diastereomers 4 and 5) or the geminal dimethylation in position 6 (compound 8) favour
Keywords:
Muscarinic agonists
Subtype-selectivity
the selective activation of M
ization of the M receptor, as well as provide useful information for the design and development of novel
selective M antagonists.
3
receptors. Compounds 4, 5, and 8 might be valuable tools in the character-
1
,4-Dioxane nucleus
3
3
Ó 2009 Elsevier Ltd. All rights reserved.
1
. Introduction
Muscarinic receptors belong to membrane-bound acethylcho-
that ACh is synthesized and released in lung and other multiple
cancers and acts as an autocrine growth factor, suggesting that this
cholinergic autocrine loop may provide new significant therapeutic
1
2
line (ACh) receptors and are members of the family of heterotri-
meric G protein-coupled receptors which, upon activation by
ACh, initiate an intracellular chemical pathway to transduce, prop-
opportunities.
Though several muscarinic agonists and antagonists have been
synthesized, their therapeutic utility is limited by various side ef-
fects due to the lack of a marked subtype-discrimination.
1
agate, and amplify the received signals. Their characteristic com-
mon structural elements are the seven transmembrane (7TM)
helical segments. On the basis of genetic and pharmacological
The modest results obtained with muscarinic agonists might be
due to the high sequence identity of the TM regions among the five
1
3
characterizations they have been classified into five subtypes,
human muscarinic receptor subtypes and to the ‘particularly sen-
sitive’ structure of the muscarinic agonists, whose small chemical
modifications often cause a decrease in activity, albeit not a total
loss. In both cases, the major problem has been the insufficient
selectivity of the molecules studied to date. Therefore, the discov-
ery of novel selective agonists might improve the characterization
of muscarinic receptors and facilitate identification of therapeutic
agents potentially useful in different pathological states.
2
termed M
1
5
–M .
Muscarinic receptors are widely distributed in
both the central and the peripheral nervous systems, in parasym-
pathetic innervated organs and in several non-innervated tissues,
and play a critical role in a wide range of diseases, including cogni-
3
4
tive function disorders such as Alzheimer’s disease, schizophre-
5
6
7
nia, and Parkinson’s disease, as well as visceral disorders such
as overactive bladder, irritable bowel syndrome,9 and chronic
obstructive pulmonary disease.10 Moreover, the muscarinic system
regulates glucose-induced insulin secretion; in particular, func-
8
Muscarine is the specific agonist for which muscarinic receptors
have been named (Fig. 1). Other pentatomic cyclic compounds,
such as cis-N,N,N-trimethyl-(2-methyl-1,3-dioxolan-4-yl)methan-
aminium iodide (1a), are potent non-selective muscarinic ago-
tional studies have revealed that the M
3
subtype is involved in
the insulinotropic effect of ACh.11 Recently, it has been reported
1
4
nists. Recently, we have reported that the enlargement of the
,3-dioxolane nucleus of 1a by inserting a methylene group be-
tween the 1-oxygen atom and the 2-carbon atom afforded the
1
*
Corresponding author. Tel.: +39 0737402237; fax: +39 0737637345.
E-mail address: alessandro.piergentili@unicam.it (A. Piergentili).
0
968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.10.027

A. Piergentili et al. / Bioorg. Med. Chem. 17 (2009) 8174–8185
8175
HO
The trans stereochemical relationship between the 2-side chain
and the 5-methyl group of 3b was assigned through single-crystal
X-ray diffraction analysis (Fig. 3).
N(CH ) I
3
3
H C
O
3
The synthetic procedure described for compounds 3a and 3b,
through the opening of the intermediate 4,5-dimethyl-3,6,8-triox-
Muscarine
1
8
O
O
abicyclo[3.2.1]octane, proved poorly suited to synthesize the
stereoisomers 4–7, because it was very difficult to separate the
mixture of diastereomers and to obtain sufficient amounts of each
of them. Therefore, for compounds 4–7 we followed the same
method we recently described for the synthesis of the enantiomers
O
N(CH ) I
3
3
N(CH ) I
3 3
H C
O
1
3
H C
3
a
2a
1
9
Figure 1. Chemical structures of muscarine and compounds 1a and 2a.
of 2a and 2b (Scheme 2). Two parallel divergent synthetic proce-
dures were carried out starting from (2R*,3S*)- and (2R*,3R*)-2,3-
dimethyloxirane to prepare the pairs of diastereomers 4/5, and 6/
higher homologue cis-N,N,N-trimethyl-(6-methyl-1,4-dioxan-2-
yl)methanaminium iodide (2a), which in functional assays showed
the same cholinergic profile as 1a.
7
, respectively. The preparation of (2R*,3R*)- (18a) and (2R*,3S*)-
3
-allyloxy-butan-2-ol (18b) was readily accomplished in basic
15
conditions by treating (2R*,3S*)- and (2R*,3R*)-2,3-dimethyl-oxi-
rane, respectively, with Na and allyl alcohol. The structure of b-hy-
droxy allyl ethers 18a and 18b was assigned by the chemical shift
The hexacyclic 1,4-dioxane structure of compound 2a, com-
pared to the 1,3-dioxolane one of its lower homologue 1a, has
the advantage of offering the carbon 5 as an additional position
where it is possible to introduce chemical functions potentially
able to modulate the selectivity of the ligand. Therefore, in the
present study, methyl groups have alternatively or simultaneously
been inserted in positions 5 and 6 of the 1,4-dioxane nucleus in all
combinations, in order to investigate their role on muscarinic po-
tency and subtype selectivity (compounds 3–12) (Fig. 2).
1
20
of the OH proton observed in the relative H NMR spectra. The
intermediate alcohol 18a was subjected to oxymercuration–
demercuration reaction with mercury(II) acetate followed by an
aqueous solution of iodine and potassium iodide which stereose-
lectively afforded
a mixture of the diastereomeric forms
(
2S*,3S*,5S*)- and (2S*,3S*,5R*)-5-iodomethyl-2,3-dimethyl-[1,4]-
dioxane (19a and 19b, respectively), which were separated by col-
umn chromatography. The amination of 19a and 19b with dimeth-
ylamine followed by treatment with an ethereal solution of methyl
iodide yielded the methiodides 4 and 5, respectively. Analogously,
diastereomers 6 and 7 were obtained from (2R*,3S*)-3-allyloxy-bu-
tan-2-ol (18b).
The muscarinic profile of the novel compounds has been evalu-
ated through radioligand binding and functional assays.
2
. Chemistry
Diastereomers 3a and 3b were prepared following the synthetic
The structure of the compounds 4–7 was determined at the le-
procedure reported in Scheme 1. The reaction of glycerine with 2-
chloro-1,1-dimethoxypropane gave the corresponding diastereo-
meric mixture of [2-(1-chloro-ethyl)-[1,3]dioxolan-4-yl]-methanol
13) (cis/trans ratio = 1:1), whose treatment with KOH afforded
the corresponding 3,6,8-trioxa-bicyclo[3.2.1]octane 14. The subse-
quent regiospecific opening17 with LiAlH
and AlCl yielded a mix-
ture of (2R*,5R*)-(5-methyl-[1,4]dioxan-2-yl)-methanol and
2R*,5S*)-(5-methyl-[1,4]dioxan-2-yl)-methanol (15a and 15b,
respectively), whose diastereomers were separated by column
chromatography. Finally, reaction of the alcohols 15a and 15b with
tosyl chloride and subsequent amination with dimethylamine pro-
vided the corresponding amines 17a and 17b, which were treated
with methyl iodide to give the methiodides 3a and 3b,
respectively.
1
vel of the iododerivative intermediates by H NMR spectral studies.
It is known that in six-membered rings, the chemical shift differ-
ence between the axial and equatorial protons is of about
1
6
(
21
0
.5 ppm; the same behaviour occurs for axial and equatorial
22
methyl groups.
4
3
The stereoselective cyclization of 18a produced only two (19a
and 19b, ratio 7:3) of the four 5-iodomethyl-2,3-dimethyl-[1,4]-
(
dioxane diastereomers with a trans stereochemical relationship be-
tween the methyl groups in positions 2 and 3. From the 1H NMR
spectra of 19a and 19b it can be deduced that, due to their similar
chemical shift (d 1.08 ppm and 1.14 ppm), the two methyl groups
1
are in trans equatorial position. In the H NMR spectrum of 19a,
the precursor of 4, the axial C6–H at d 3.28 ppm shows two large
coupling constants (J = 10.26 Hz and J = 11.11 Hz), one with the
equatorial geminal proton at d 3.98 ppm and the other with the ax-
ial C5–H at d 3.64 ppm. Moreover, an evident nuclear Overhouse
effect (NOE) between the axial protons C5–H at d 3.64 ppm and
C3–H at d 3.34 ppm was observed, and the equatorial position of
the two methyl groups in positions 2 and 3 was confirmed. In con-
clusion, all three substituents of compound 19a are in equatorial
position.
R'''
R''
R'
O
4
5
6
3
2
1
N(CH ) I
3 3
O
R
1
3
3
4
5
6
7
8
9
1
1
1
1
1
a, R = R' = R''' = H, R'' = CH
b, R = R' = R'' = H, R''' = CH
, R = R''' = CH , R' = R'' = H
, R = R''' = H, R' = R'' = CH
, R = R'' = H, R' = R''' = CH
, R = R'' = CH
, R = R' = CH
, R = R' = H, R'' = R''' = CH
0a, R = R'' = R''' = CH , R' = H
0b, R = H, R' = R'' = R''' = CH
1a, R = R' = R'' = CH , R''' = H
1b, R = R' = R''' = CH , R'' = H
2, R = R' = R'' = R''' = CH
3
In the H NMR spectrum of compound 19b, the proton in posi-
3
tion 5 is equatorial because none of the protons in position 6 shows
a large coupling constant with it. Also the two methyl groups are
equatorial because they show the same chemical shift values as
in compound 19a (d 1.08 ppm and 1.14 ppm) and because an evi-
dent NOE effect between the axial protons C6–H at d 3.80 ppm and
C2–H at d 3.20 ppm was observed. Therefore, the precursors of the
two diastereomers 4 and 5 were assigned the structures reported
in Figure 4.
3
3
3
3
, R' = R''' = H
, R'' = R''' = H
3
3
3
3
By stereoselective cyclization of (2R*,3S*)-3-allyloxy-butan-2-ol
3
(18b) only the two diastereomers 19c and 19d with a cis stereo-
3
chemical relationship between the methyl groups in positions 2
and 3 were obtained. In this case, one methyl group is in axial po-
3
1
Figure 2. Chemical structures of compounds 3–12.
sition and the other is in equatorial position. In fact, in the H NMR

8
176
A. Piergentili et al. / Bioorg. Med. Chem. 17 (2009) 8174–8185
O
CH OH
O
H C
OCH3
OCH3
2
H C
3
3
a
b
+
CHOH
OH
O
O
O
Cl
Cl
H
3
C
CH OH
2
13
14
c
H C
O
O
H C
O
3
3
OH
OH
O
1
5a
15b
d
d
H C
O
H
3
C
O
3
OTs
OTs
O
O
16a
16b
e
e
H C
O
H
3
C
O
3
N(CH3)2
N(CH )
3 2
O
O
17a
17b
f
f
H C
O
H
3
C
O
3
N(CH ) I
N(CH
3
)
3
I
3
3
O
O
3a
3b
Scheme 1. Reagents: (a) TsOH, toluene; (b) KOH powder; (c) LiAlH
4
, AlCl
3
2 3
, diethyl ether; (d) p-TsCl, pyridine; (e) Me NH, benzene; (f) CH I, diethyl ether.
3
-allyloxy-2-methyl-butan-2-ol (24). The reaction of 21–25 with
mercury(II) acetate followed by treatment with potassium iodide
and iodine gave the intermediate iodo-derivatives 26–30, whose
amination with dimethylamine and subsequent treatment with
methyl iodide afforded compounds 8–12.
The structures of diastereomers 10a, 10b, 11a, and 11b were as-
1
signed by comparing their H NMR spectra with those of the corre-
sponding 6- and 5-methyl analogues 2a, 2b and 3a, 3b,
respectively, whose structures were assigned by X-ray crystallog-
raphy. Similarly to the case of the tertiary amines of compounds
1
5
2
a and 2b, the chemical shift value of the methylene group of
Figure 3. A view of the cation in the X-ray structure of 3b; 30% probability
ellipsoids are shown.
the 2-side chain in the cis diastereomer 33a (d 2.14–2.43 ppm) is
at higher fields than those of the trans diastereomer 33b (d 2.39–
2
.69 ppm). In the 11a/11b pair, the chemical shift of the axial
methyl group in position 5 of diastereomer 11a (d 1.21 ppm) is
at lower fields than those of the equatorial methyl group of trans
analogue 11b (d 0.99 ppm), analogously to the 3a/3b pair (d 1.10
and 0.99 ppm, respectively). Moreover, during the separation of
the mixture of 6-methyl substituted stereoisomers by column
chromatography, the cis diastereomers eluted first, whereas the
opposite occurred for the mixture of 5-methyl substituted
stereoisomers.
spectra, a difference of about 0.30 ppm in the chemical shift of the
two methyl groups was observed. The trans stereochemical rela-
tionship between the chain in 5 and the methyl substituents in
positions 2 and 3 of 19c was confirmed by an evident NOE effect
between the axial C5–H at d 3.78 ppm and the 3-methyl protons
at d 1.29 ppm; no NOE effect was observed between the same pro-
tons of the diastereomer 19d. Moreover, in compound 19d an evi-
dent NOE effect between the axial proton C6–H at d 3.47 ppm and
the 2-methyl protons at d 1.40 ppm was also observed. Therefore,
the precursors of the two diastereomers 6 and 7 were assigned
the structures reported in Figure 5.
3. Results and discussion
Compounds 8–12 were obtained following the synthetic proce-
dure shown in Scheme 3. The opening of 2,2-dimethyloxirane in
the presence of Na afforded 1-allyloxy-2-methyl-propan-2-ol
The muscarinic pharmacological profile of all methiodides was
evaluated by receptor binding assays following a previously de-
2
5
scribed protocol. (2-Hydroxyethyl)trimethylammonium chloride
carbamate (carbachol) and 4-[N-(3-chlorophenyl)carbamoyloxy]-
2-butynyltrimethylammonium chloride (McN-A-343) were used
2
3
(
21). Intermediates 22, 23, and 25 were obtained by opening
the appropriate oxirane in the presence of HClO . Treatment of
-allyloxy-propionic acid methyl ester24 with CH
MgI furnished
4
3
3
2
3
as reference compounds. [ H]N-methylscopolamine ([ H]NMS)

A. Piergentili et al. / Bioorg. Med. Chem. 17 (2009) 8174–8185
8177
H C
3
CH3
H C
3
CH3
O
O
a
a
HO
CH3
O
HO
CH3
O
H C
3
H C
3
1
8a
b, c
H C
18b
b, c
H C
O
O
O
H C
3
O
H C
3
O
3
3
I
I
I
I
H C
3
H C
3
O
H C
3
O
H C
3
O
19d
1
9a
19b
19c
d
d
d
d
H C
O
O
H C
O
H C
O
H C
3
O
3
3
3
N(CH3)2
N(CH3)2
N(CH3)2
N(CH3)2
H C
H C
O
20b
H C
O
H C
3
O
20d
3
3
3
20a
20c
e
e
e
e
H C
3
O
H C
3
O
H C
3
O
H C
3
O
N(CH ) I
N(CH ) I
3 3
3
3
N(CH ) I
N(CH ) I
3
3
3 3
H C
3
O
H C
3
O
H C
3
O
H C
3
O
4
5
6
7
3 2 2 2 3
Scheme 2. Reagents: (a) Na, allyl alcohol; (b) (CH COO) Hg; (c) KI, I ; (d) Me NH, benzene; (e) CH I, diethyl ether.
muscarinic receptors,30 and that the contractions of isolated strips
of ileum are primarily mediated by M
receptor activation.31 In-
stead, the inhibition of electrically induced contractions of rabbit
H
H
3
H
H
2
H C
1
O
6
H
H
H C
O
3
vas deferens has for long been considered an effect mediated by
3
O
O
4
H C
H
26
H C
CH2I
3
3
1
M -receptor subtypes, whereas more recent studies attribute this
3
H
5
receptor stimulation.32 Similarly, the validity of the gui-
4
H
CH2I
effect to M
nea pig lung strips as an M
H
3
3
34
4
model has been questioned. There-
fore, in the present paper we will use theterms vas deferens and lung
muscarinic receptor models for the latter two preparations. The
functional data obtained for the new compounds, reported in
1
9 a
19 b
Figure 4. NOE correlations of compounds 19a and 19b.
Table 2, are expressed as pD
2
(ꢀlog ED50, agonist potency), or as
pK , antagonist potency), and as a
B
(ꢀlog K
B
(intrinsic activity). Carb-
amyl-b-methylcholine chloride (bethanechol) and McN-A-343 were
included in the study as reference compounds.
H
H
H
CH
3
H
From an analysis of the binding data of compounds 3–12, re-
ported in Table 1, it can be observed that all tested compounds
show low affinity values for all muscarinic subtypes. However,
compounds 3a, 5, and 7 are particularly interesting, because, anal-
ogously to the leads 2a and 2b, they show significant selectivity for
H
H
H
H
H C
O
H
H
O
3
O
O
CH2I
3
C
CH I
2
CH3
H
1
9 c
19 d
the M
2
subtype compared to the other muscarinic subtypes.
19,35
Based on our previous experience and on literature data,
Figure 5. NOE correlations of compounds 19c and 19d.
which showed that poor binding affinity would not prevent good
functional activity, we tested the compounds on functional models
of muscarinic receptors, since discrepancies between binding stud-
ies and functional activity of agonists are quite common.36 The
apparent discrepancy between binding and functional data is ac-
counted for by the species difference in receptor targets and by
the type of information collected in each experimental model, that
is, on the one hand, affinity data to human receptors, collected in
binding assays, and, on the other, evaluation of the agonistic po-
tency (which depends on the intrinsic activity of the compounds
in addition to their affinity) in isolated animal tissues.
was the radioligand used to label cloned human muscarinic hM
hM receptors, expressed in Chinese hamster ovary (CHO) cells.
Affinity values, expressed as pK , are reported in Table 1.
1
–
5
i
Moreover, the muscarinic activity of compounds 3–12 was eval-
uated by functional studies performed on classical isolated organ
2
6
preparations, namely, rabbit electrically stimulated vas deferens,
27
28
and guinea pig electrically stimulated left atrium, ileum, and
2
9
lung strips. It is known that negative inotropic responses to mus-
carinic agonists in guinea pig atria are directly mediated by M
2

8
178
A. Piergentili et al. / Bioorg. Med. Chem. 17 (2009) 8174–8185
O
R
R''
R'
R'''
a
b
OH
OH
O
R
R
for 24
O
O
O
O
R'
R'
c
R''
R''
R'''
R'''
CH3
2
1, R=R'= CH , R''=R'''= H
22, R=R'= H, R''=R'''= CH
3
3
2
2
2
3, R= H, R'=R''=R'''= CH
3
4, R=R'=R''= CH , R'''= H
3
5, R=R'=R''=R'''= CH
3
d, e
d, e
2
2
6, R=R'= CH , R''=R'''= H
3
R'''
O
7, R=R'= H, R''=R'''= CH
3
R''
28a, R=R''=R'''= CH , R'= H
3
2
8b, R= H, R'=R''=R'''= CH
R'
O
R
I
3
29a, R=R'=R''= CH
, R'''= H
3
2
9b, R=R'=R'''= CH , R''= H
3
3
0, R=R'=R''=R'''= CH
3
f
3
1, R=R'= CH , R''=R'''= H
3
R'''
O
3
2, R=R'= H, R''=R'''= CH
3
R''
33a, R=R''=R'''= CH , R'= H
3
3
3b, R= H, R'=R''=R'''= CH
R'
O
R
N(CH3)2
3
34a, R=R'=R''= CH
, R'''= H
3
3
3
4b, R=R'=R'''= CH , R''= H
3
5, R=R'=R''=R'''= CH
3
g
8
, R=R'= CH , R''=R'''= H
R'''
O
3
9, R=R'= H, R''=R'''= CH
3
R''
1
0a, R=R''=R'''= CH , R'= H
3
R'
O
R
N(CH ) I 10b, R= H, R'=R''=R'''= CH
3
3
3
1
1
1
1a, R=R'=R''= CH , R'''= H
3
3
1b, R=R'=R'''= CH , R''= H
2, R=R'=R''=R'''= CH
3
Scheme 3. Reagents: (a) Na, allyl alcohol; (b) HClO
4
, allyl alcohol; (c) CH
3
MgBr; (d) (CH
3
COO)
2
Hg; (e) KI, I
2 2 3
; (f) Me NH, benzene; (g) CH I, diethyl ether.
An analysis of the functional data reported in Table 2 shows
that, unlike the lead compounds 2a and 2b, none of the compounds
tested on rabbit vas deferens activates the muscarinic receptors of
ists between the same substituents. Interestingly, the two com-
pounds 4 and 5, which exhibit pD values at M receptors (6.95
and 6.70, respectively) significantly higher than those at M
(4.93 and 5.01, respectively) display good selectivity for the M
type (M /M
and 5, showing respectively 74- and 141-fold lower potency in the
guinea pig lung, prove to be selective for M sites also in comparison
with the muscarinic receptors of this tissue. On the contrary, no dis-
crimination between M and M subtypes is observed in the case of
compound 7, while derivative 6 combines a full agonist activity at
ileal tissue with only a weak efficacy at M receptors. In the lung
2
3
2
ones
sub-
this tissue. Concerning M
2
and M
3
subtypes and the guinea pig
3
lung model, all the compounds behave as full or partial agonists
with the exception of compound 10b, which is unable to bind
3
2
= 105 and 49, respectively). In addition, compounds 4
the muscarinic receptors at concentrations up to 30
lM, and of
3
compound 11a, which behaves as a weak antagonist at guinea
pig lung muscarinic sites. The shift of the methyl group from posi-
tion 6 to 5 of the 1,4-dioxane nucleus is detrimental to muscarinic
activity: compounds 3a and 3b are less potent than the corre-
2
3
2
sponding 6-methyl derivatives 2a and 2b at M
and less efficacious in the lung tissue. Moreover, unlike the 2a/2b
pair, where the cis diastereomer 2a is more potent than the trans
2
and M
3
receptors
model, the diastereomers 4, 5, and 7 behave as partial agonists; dia-
stereomer 6 can neither activate nor block the muscarinic receptors
of this tissue at concentrations up to 10 lM.
2
b, the stereochemical relationship between the 2-side chain and
Of note is the effect produced by the geminal dimethylation; in
the 5-methyl group does not influence the activity at any musca-
rinic preparation, the diastereomers 3a and 3b showing similar po-
tency profiles.
fact, the 5,5-dimethyl derivative 9 behaves as a weak partial ago-
nist at both M
4.85, respectively) and is inactive at the other subtypes, while,
interestingly, the 6,6-dimethyl analogue 8 fully activates the M
subtype, being selective for this subtype with respect to M , lung
3 2
and lung muscarinic receptors (pD = 4.91 and
The simultaneous presence of one methyl group in both positions
3
5
and 6 furnishes the diastereomers 4–7. All four diastereomers
show similar potency values at M , whereas the diastereomers 4
and 5, with a trans stereochemical relationship between the methyl
groups in positions 5 and 6, are more active at M receptors than the
diastereomers 6 and 7, in which a cis stereochemical relationship ex-
2
2
and vas deferens muscarinic receptors. This observation indicates
that, unlike the case of the dimethyl analogue of the dioxolane
3
7
3
1a, the presence of two methyl groups in position 6 is still well
tolerated by the corresponding M receptor site, compound 8
3

A. Piergentili et al. / Bioorg. Med. Chem. 17 (2009) 8174–8185
8179
Table 1
a
Affinity constants, expressed as pK
i
of compounds 2–12, carbachol, and McN-A-343 for human cloned muscarinic receptors, expressed in CHO cells
R'''
O
R''
R'
R
N(CH ) I
3 3
O
0
00
000
R
Compd
R
R
R
hM
1
hM
2
hM
3
hM
4
hM
5
a
pK
i
2
2
3
3
4
5
6
7
8
9
1
1
1
1
1
a
b
a
b
CH
H
H
H
CH
H
3
3
H
CH
H
H
H
CH
CH
H
CH
H
H
H
CH
H
H
CH
H
CH
H
CH
CH
CH
CH
H
H
H
H
CH
CH
H
CH
H
4.52 ± 0.07
<4
<4
5.80 ± 0.10
5.31 ± 0.10
4.95 ± 0.10
4.76 ± 0.11
4.86 ± 0.11
4.78 ± 0.10
4.05 ± 0.10
4.70 ± 0.10
4.74 ± 0.11
4.02 ± 0.09
4.22 ± 0.08
4.14 ± 0.10
4.56 ± 0.09
4.47 ± 0.06
4.33 ± 0.08
5.91 ± 0.06
5.49 ± 0.07
5.12 ± 0.11
4.45 ± 0.09
4.18 ± 0.10
4.35 ± 0.12
4.96 ± 0.11
<4
4.94 ± 0.10
4.06 ± 0.07
4.03 ± 0.05
<4
4.51 ± 0.08
<4
5.11 ± 0.07
4.17 ± 0.06
4.03 ± 0.06
4.20 ± 0.08
4.81 ± 0.07
<4
3
3
3
3
<4
4.61 ± 0.08
<4
<4
3
3
H
3
3
<4
<4
<4
<4
<4
CH
CH
H
CH
H
CH
CH
CH
3
3
3
<4
4.01 ± 0.08
4.55 ± 0.08
<4
4.28 ± 0.09
4.08 ± 0.07
4.49 ± 0.09
4.78 ± 0.06
4.18 ± 0.06
4.16 ± 0.07
5.42 ± 0.06
3
H
4.32 ± 0.07
<4
4.67 ± 0.11
4.11 ± 0.11
4.49 ± 0.09
4.33 ± 0.08
4.79 ± 0.09
4.98 ± 0.08
4.39 ± 0.08
4.36 ± 0.10
5.62 ± 0.09
4.35 ± 0.07
<4
3
3
3
3
CH
CH
CH
H
CH
CH
3
3
3
0a
0b
1a
1b
2
3
H
4.17 ± 0.08
4.02 ± 0.08
4.70 ± 0.07
4.42 ± 0.07
<4
4.24 ± 0.10
4.06 ± 0.08
4.59 ± 0.09
4.52 ± 0.07
4.27 ± 0.07
5.20 ± 0.07
5.04 ± 0.07
CH
CH
CH
CH
3
3
3
3
3
3
3
3
3
CH
3
Carbachol
McN-A-343
4.42 ± 0.09
5.71 ± 0.09
a
The values represent the arithmetic mean ± SEM of at least three experiments.
showing a pD
of compound 2a (pD
mental for M and the other two muscarinic receptor models, mak-
ing compound 8 an interesting novel selective M subtype agonist
/vas deferens >275; M /M = 81; M /lung = 123). The decline of
2
value (pD
2
= 6.94) of the same order of magnitude
2 3
fact, the functional responses at M and M receptors are only scar-
2
= 7.34). Instead, this modification is detri-
cely ameliorated when the methyl group is in cis stereochemical
relationship with the 2-side chain (compound 10a), and they are
completely absent when the methyl group is in trans stereochem-
ical relationship (compound 10b).
2
3
(
M
3
3
2
3
muscarinic profile produced by 5,5-dimethyl substitution is not
improved by the introduction of a methyl group in position 6. In
When a third methyl group is inserted into position 5 of the 6,6-
dimethyl substituted derivative, affording diastereomers 11a and
Table 2
Potency values, expressed as pD
guinea pig left atrium (M
2
(ꢀlog ED50),a intrinsic activity (
a
)b and dissociation constant (pK
B
)c of compounds 2–12, and bethanechol in the isolated rabbit vas deferens,
d
2
), longitudinal ileum (M
3
), and lung muscarinic receptors
R'''
O
R''
R'
N(CH ) I
3
3
O
R
0
00
000
R
Compd
R
R
R
Rabbit vas deferens
Guinea pig atrium (M
2
)
Guinea pig ileum (M
3
)
Guinea pig lung
B
pD (pK )
a
pD
2
(pK
B
)
a
pD
2
(pK
B
)
a
2
pD (pK
B
)
a
2
2
2
3
3
4
5
6
7
8
9
1
1
1
1
1
a
b
a
b
CH
H
H
3
H
CH
H
H
H
CH
CH
H
CH
H
H
H
CH
H
H
CH
H
CH
H
CH
CH
CH
CH
H
H
H
H
CH
CH
H
CH
H
1.00 ± 0.01
1.00 ± 0.01
5.65 ± 0.06
1.00 ± 0.01
1.00 ± 0.01
1.00 ± 0.01
0.95 ± 0.06
0.80 ± 0.08
0.89 ± 0.02
0.27 ± 0.09
0.91 ± 0.01
0.73 ± 0.01
7.57 ± 0.11
6.66 ± 0.19
5.44 ± 0.16
5.75 ± 0.02
4.93 ± 0.01
5.01 ± 0.01
5.04 ± 0.01
5.18 ± 0.10
1.00 ± 0.01
1.00 ± 0.01
1.00 ± 0.01
1.00 ± 0.01
0.88 ± 0.02
1.18 ± 0.23
0.85 ± 0.11
1.00 ± 0.01
1.16 ± 0.05
0.68 ± 0.01
0.47 ± 0.14
7.34 ± 0.13
6.02 ± 0.16
5.71 ± 0.18
5.55 ± 0.07
6.95 ± 0.11
6.70 ± 0.17
4.91 ± 0.04
5.27 ± 0.01
6.94 ± 0.03
4.91 ± 0.02
0.81 ± 0.07
1.00 ± 0.01
0.55 ± 0.26
0.57 ± 0.07
0.35 ± 0.15
0.65 ± 0.15
5.90 ± 0.14
6.01 ± 0.03
5.13 ± 0.22
5.08 ± 0.01
5.08 ± 0.30
3
5.08 ± 0.24
g
3
g
f
H
3
CH
H
3
3
e
f
3
3
3
4.59 ± 0.18
g
H
3
e
f
CH
CH
H
3
3
0.50 ± 0.10
0.18±0.16
0.22 ± 0.20
5.00 ± 0.25
4.85 ± 0.30
3
3
H
5.03 ± 0.04
f
g
3
3
3
3
CH
CH
CH
H
CH
CH
3
3
3
4.85 ± 0.30
g
0a
0b
1a
1b
2
CH
H
3
H
0
(4.61 ± 0.20)
0.33 ± 0.07
4.92 ± 0.17
5.12 ± 0.08
f
f
f
g
CH
CH
CH
CH
3
3
3
3
f
g
CH
3
CH
3
CH
3
0.47 ± 0.11
0.66 ± 0.17
0.93 ± 0.07
5.25 ± 0.08
5.70 ± 0.04
5.09 ± 0.16
0
(5.15 ± 0.15)
e
f
g
3
0.46 ± 0.16
0.71 ± 0.14
4.99 ± 0.01
5.12 ± 0.01
g
CH
3
3
McN-A-343
Bethanechol
1.00 ± 0.01
6.40 ± 0.16
1.00 ± 0.01
5.88 ± 0.05
1.00 ± 0.01
6.16 ± 0.07
1.00 ± 0.01
5.01 ± 0.13
a
pD
2
values are the negative logarithm of the agonist concentration that caused 50% of the maximum response attainable in that tissue.
b
Intrinsic activity was measured by the ratio between the maximum response of the agonist and the maximum response of McN-A-343 at rabbit vas deferens, and
bethanechol at guinea pig atrium, ileum and lung receptors.
c
49
Dissociation constants were calculated according to Furchgott.
The results are the means ± SEM of four to six independent experiments.
Not tested.
This compound showed no agonist activity and, when tested as antagonist, proved inactive up to 30
This compound showed no agonist activity and, when tested as antagonist, proved inactive up to 10
d
e
f
l
l
M.
M.
g

8
180
A. Piergentili et al. / Bioorg. Med. Chem. 17 (2009) 8174–8185
1
1b, the cis compound 11a behaves as a partial agonist at M
receptor with a pD value about 17-fold lower than that of the cor-
responding 6,6-dimethyl derivative 8, and behaves as a weak
antagonist on lung muscarinic receptors. No interaction with M
and vas deferens muscarinic receptors is observed. The trans iso-
mer 11b partially activates both M and M receptors and is inac-
3
2 4
dried over Na SO . Evaporation of the solvent gave a residue, which
was purified by distillation under reduced pressure: bp 135 °C
2
(10 mmHg). A mixture of two diastereomers was obtained (ratio
1
2
1:1, 12.5 g, 75% yield). H NMR (CDCl
3
): d 1.45–1.50 (two d, 6,
and cycle).
CH
3
), 2.85 (br s, 2, OH), 3.60–5.15 (m, 14, CH, CH
2
2
3
tive at the other tissues. Finally, the tetramethyl derivative 12, as
well, fails to activate vas deferens and lung muscarinic receptors,
4
(
.1.2. (4R*)/(4S*)-4-Methyl-3,6,8-trioxa-bicyclo[3.2.1]octane
14)
A diastereomeric mixture of 13 (10.5 g; 63 mmol) and pow-
dered KOH (5.3 g; 94 mmol) was heated until an exothermic reac-
tion developed. After cooling, the residue was distilled under
but behaves as a weak full and partial agonist at M
3
2
and M recep-
tors, respectively, with the same potency value (pD
2
= 5.09 and
5
1
.12, respectively).
In conclusion, this study strengthens the effectiveness of the
,4-dioxane nucleus as a suitable scaffold for designing active mus-
reduced pressure: bp 80 °C (25 mmHg). A mixture of two diaste-
1
reomers 14 was obtained (5 g, 61% yield). H NMR (CDCl
3
): d
carinic agonists endowed, in some cases, with subtype-selectivity.
It confirms the importance of the stereochemical relationship be-
tween the methyl substituent and the side chain, with the cis 6-
methyl derivative 2a adhering quite well to the rule of the fourth
N-substituent governing the high efficacy of muscarinic agonists.
Moreover, it highlights that the substituent in position 5 may be
involved in the receptor interaction modulating the subtype-selec-
tivity. In fact, among the polymethyl compounds, the presence of
one methyl group in both positions 5 and 6 with a trans stereo-
chemical relationship with each other (diastereomers 4 and 5) fa-
1
.10 (d, 3, CH
3 3
), 1.29 (d, 3, CH ), 3.39–5.15 (m, 14, cycle).
4
.1.3. (2R*,5R*)-(5-Methyl-[1,4]dioxan-2-yl)-methanol (15a)
and (2R*,5S*)-(5-methyl-[1,4]dioxan-2-yl)-methanol (15b)
A solution of AlCl (5.1 g, 38 mmol) in Et O (30 mL) was added
dropwise over 3 min to a stirred solution of 14 (5.0 g, 38 mmol)
and LiAlH (1.5 g, 39 mmol) in Et O (70 mL). The reaction mixture
was refluxed vigorously until the starting material disappeared.
Then it was cooled to 0 °C and quenched cautiously by the drop-
wise addition of Na
off and the filtrate was dried over Na
gave an oil, which was purified by column chromatography elut-
ing with cyclohexane/EtOAc (7:3). The trans isomer 15b eluted
3
2
4
2
2
SO
4
saturated solution. The solid was filtered
vours the selective activation of the
3
M receptor subtype.
2
SO . Removal of the solvent
4
Interestingly and surprisingly, the same subtype preference is pro-
duced by the geminal dimethylation in position 6 (compound 8).
Finally, it is well known that the replacement of the methyl
group of muscarinic agonists with bulkier groups modulates their
profile from agonists to antagonists.38 Therefore, compounds 4, 5,
and 8 not only are valuable tools in the characterization of the
1
first: 1.6 g (32% yield). H NMR (CDCl
3
): d 1.08 (d, 3, CH
3
), 2.21
(
br s, 1, OH), 3.25–3.80 (m, 8, CH
was the cis isomer 15a: 0.3 g (6% yield); H NMR (CDCl
d, 3, CH ), 1.92 (br s, 1, OH), 3.50–4.01 (m, 8, CH O and cycle).
2
O and cycle). The second fraction
1
3
): d 1.20
(
3
2
M
3
receptor but also provide useful information for the design
and development of novel selective M antagonists.
3
4
2
.1.4. Toluene-4-sulfonic acid (2S*,5R*)-5-methyl-[1,4]dioxan-
-ylmethyl ester (16a)
Tosyl chloride (0.75 g, 3.9 mmol) was added to a stirred solution
of 15a (0.35 g, 2.6 mmol) in pyridine (4 mL) at 0 °C over 30 min.
After being stirred for 3 h at 0 °C, the mixture was left for 20 h at
4
4
. Experimental
.1. Chemistry
4
°C in the freezer. Then it was poured into ice and concentrated
HCl (4 mL) and extracted with CHCl . The organic layers were
washed with HCl 2 N (15 mL), NaHCO saturated solution
15 mL), and H O (15 mL) and then dried over Na SO . The solvent
Melting points were taken in glass capillary tubes on a Büchi
SMP-20 apparatus and are uncorrected. IR and 1H NMR spectra
were recorded on Perkin–Elmer 297 and Varian EM-390 instru-
ments, respectively. Chemical shifts are reported in parts per mil-
lion (ppm) relative to tetramethylsilane (TMS), and spin
multiplicities are given as s (singlet), br s (broad singlet), d (dou-
blet), t (triplet), q (quartet), or m (multiplet). Although the IR spec-
tra data are not included because of the lack of unusual features,
they were obtained for all compounds reported and were consis-
tent with the assigned structures. The elemental compositions of
the compounds were performed by the Microanalytical Laboratory
of our department and agreed to within ±0.4% of the calculated va-
lue. When the elemental analysis was not included, crude com-
pounds were used in the next step without further purification.
Chromatographic separations were performed on silica gel col-
umns (Kieselgel 40, 0.040–0.063 mm, Merck) by flash chromatog-
raphy. Compounds were named following IUPAC rules as applied
by Beilstein-Institut AutoNom (version 2.1), a software for system-
atic names in organic chemistry. Carbachol, McN-A-343, and
bethanechol chloride were commercially available (Sigma).
3
3
(
2
2
4
was concentrated in vacuo to give a residue, which was purified by
column chromatography. Eluting with cyclohexane/EtOAc (9:1)
afforded compound 16a: 0.35 g (47% yield). H NMR (CDCl
1
1
3
): d
.10 (d, 3, CH
3
), 2.43 (s, 3, ArCH
3
), 3.21–4.41 (m, 8, CH
2
O and cycle),
7
.37 (d, 2, ArH), 7.82 (d, 2, ArH).
4.1.5. Toluene-4-sulfonic acid (2S*,5S*)-5-methyl-[1,4]dioxan-
2-ylmethyl ester (16b)
This was prepared as described for compound 16a starting from
1
15b: 75% yield. H NMR (CDCl
ArCH
(d, 2, ArH).
3 3
): d 1.05 (d, 3, CH ), 2.46 (s, 3,
3
), 3.19–4.00 (m, 8, CH
2
O and cycle), 7.35 (d, 2, ArH), 7.79
4
.1.6. Dimethyl-((2R*,5R*)-5-methyl-[1,4]dioxan-2-ylmethyl)-
amine (17a)
A solution of 16a (0.58 g, 2 mmol) and dimethylamine (5 mL) in
dry benzene (15 mL) was heated in a sealed tube for 60 h at 120 °C.
After evaporation of the solvent, the residue was dissolved in
4
.1.1. (2R*,4R*)/(2S*,4R*)-[2-(1-Chloro-ethyl)-[1,3]dioxolan-4-
yl]-methanol (13)
CHCl
The solvent was concentrated in vacuo to give a residue, which
was purified by column chromatography. Eluting with CHCl
CH
OH (9:1) afforded 17a as the free base: 0.3 g (94% yield). 1
NMR (CDCl ): d 1.19 (d, 3, CH ), 2.28 (m, 2, CH N), 2.30 (s, 6,
N(CH ), 2.70–2.82 (m, 2, cycle), 3.48–3.79 (m, 4, cycle).
3 2 4
, which was washed with NaOH 2 N and dried over Na SO .
A solution of 2-chloro-1,1-dimethoxypropane (13.86 g, 100
mmol), propane-1,2,3-triol (12 g, 130 mmol), and p-toluenesul-
fonic acid (0.25 g, 1.5 mmol) in toluene (100 mL) was refluxed with
vigorous stirring and in a Stark apparatus for 5 h. After cooling, the
3
/
3
H
3
3
2
mixture was washed with K
2
CO
3
saturated solution (50 mL) and
3 2
)

A. Piergentili et al. / Bioorg. Med. Chem. 17 (2009) 8174–8185
8181
4
.1.7. Dimethyl-((2R*,5S*)-5-methyl-[1,4]dioxan-2-ylmethyl)-
2
J = 5.98, 10.68 Hz, 1, CH I), 3.17 (dq, J = 6.41, 8.55 Hz, 1, 2-CH), 3.28
amine (17b)
(dd, J = 10.26, 11.11 Hz, 1, 6-CH), 3.34 (dq, J = 5.98, 8.55 Hz, 1,
This was prepared as described for 17a starting from 16b: 76%
3-CH), 3.64 (m, 1, 5-CH), 3.98 (dd, J = 2.56, 11.11 Hz, 1, 6-CH). The
1
1
3 3 2
yield. H NMR (CDCl ): d 1.08 (d, 3, CH ), 2.15 (m, 1, CH N), 2.28
second fraction was 19b: 0.7 g (7% yield). H NMR (CDCl
3
): d 1.08
), 3.20 (dq,
J = 6.41, 8.97 Hz, 1, 2-CH), 3.48 (dq, J = 5.99, 8.97 Hz, 1, 3-CH), 3.56
m, 2, CH I), 3.58 (d, J = 11.97 Hz, 1, 6-CH), 3.80 (dd, J = 0.85,
11.97 Hz, 1, 6-CH), 3.86 (m, 1, 5-CH).
(
(
s, 6, N(CH
3
)
2
), 2.37 (m, 1, CH N), 3.31 (m, 2, cycle), 3.53–3.81
2
3 3
(d, J = 6.41 Hz, 3, CH ), 1.14 (d, J = 5.99 Hz, 3, CH
m, 4, cycle).
(
2
4
.1.8. (2R*,5R*)-N,N,N-Trimethyl-(5-methyl-1,4-dioxan-2-
yl)methanaminium iodide (3a)
2
A solution of 17a (0.3 g, 1.9 mmol) in Et O (10 mL) was treated
with an excess of methyl iodide and left at rt in the dark for 24 h.
4
(
[
.1.13. (2S*,3R*,5S*)-5-Iodomethyl-2,3-dimethyl-[1,4]dioxane
19c) and (2R*,3S*,5S*)-5-iodomethyl-2,3-dimethyl-
1,4]dioxane (19d)
These compounds were prepared following the procedure de-
scribed for 19a and 19b starting from 18b. The mixture of diaste-
reomers 19c and 19d (ratio 8:2) was separated by column
The solid was filtered and recrystallized from EtOH; mp 236–
1
2
3
(
4
37 °C. H NMR (DMSO): d 1.10 (d, 3, CH
3
), 3.12 (s, 9, N(CH
N), 3.52–3.75 (m, 4, cycle), 3.98 (m, 1, cycle), 4.23
20INO : C, 35.89; H, 6.69; N,
.65. Found: C, 35.92; H, 6.72; N, 4.58.
3 3
) ),
.35 (m, 2, CH
m, 1, cycle). Anal. Calcd for C
2
9
H
2
chromatography eluting with petroleum ether/EtOAc (9.9:0.1).
1
1
9c eluted first: (21% yield).
H
NMR (CDCl
), 3.02 (d, 2, CH
.30 (dd, J = 10.25, 11.11 Hz, 1, 6-CH), 3.74 (m, 1, 3-CH), 3.78 (m,
, 5-CH), 3.83 (m, 1, 2-CH), 3.95 (dd, J = 2.99, 11.54 Hz, 1, 6-CH).
3
):
d
1.02 (d,
4
.1.9. (2R*,5S*)-N,N,N-Trimethyl-(5-methyl-1,4-dioxan-2-
yl)methanaminium iodide (3b)
J = 6.84 Hz, 3, CH
3
), 1.29 (d, J = 6.84 Hz, 3, CH
3
2
I),
3
1
This was prepared as described for 3a starting from 17b. The so-
1
lid was filtered and recrystallized from EtOH; mp 273 °C. H NMR
1
The second fraction was 19d: 0.7 g (16% yield). H NMR (CDCl
d 1.10 (d, J = 6.84 Hz, 3, CH ), 1.40 (d, J = 6.84 Hz, 3, CH ), 3.07
dd, J = 2.99, 5.55 Hz, 2, CH I), 3.47 (dd, J = 10.69, 10.90 Hz, 1, 6-
CH), 3.55–3.66 (m, 3, cycle), 3.92 (m, 1, 3-CH).
3
):
(
3
3 3 3 2
DMSO): d 0.99 (d, 3, CH ), 3.10 (s, 9, N(CH ) ), 3.25 (m, 2, CH N),
3
3
.30 (m, 2, cycle), 3.51 (m, 1, cycle), 3.68 (m, 2, cycle), 4.10 (m, 1,
cycle). Anal. Calcd for C H20INO : C, 35.89; H, 6.69; N, 4.65. Found:
9 2
C, 36.02; H, 6.93; N, 4.42.
(
2
4
.1.14. ((2R*,5S*,6S*)-5,6-Dimethyl-[1,4]dioxan-2-ylmethyl)-
dimethyl-amine (20a)
A solution of 19a (0.8 g, 3.1 mmol) and dimethylamine (10 mL)
in dry benzene (30 mL) was heated in a sealed tube for 60 h at
4
.1.10. (2R*,3R*)-3-Allyloxy-butan-2-ol (18a)
2R*,3S*)-2,3-Dimethyl-oxirane (5.0 g, 69.3 mmol) was added
dropwise to a stirred solution of freshly cut sodium (0.5 g,
1.7 mmol) in allyl alcohol (49 mL) at rt. After 1 h at rt the reaction
(
2
1
20 °C. After evaporation of the solvent, the residue was dissolved
in CHCl , which was washed with 2 N NaOH and dried over Na SO
The solvent was concentrated in vacuo to give a residue, which was
purified by column chromatography. Eluting with CHCl /CH OH
9.5:0.5) afforded 20a as the free base: 0.4 g (74% yield). H NMR
(CDCl ): d 1.10 (d, 3, CH ), 1.14 (d, 3, CH ), 2.19 (dd, 1, CH N),
.24 (s, 6, N(CH ), 2.37 (dd, 1, CH N), 3.17–3.39 (m, 3, cycle),
.75–3.84 (m, 2, cycle).
mixture was refluxed for 3 h. Most of the unreacted allyl alcohol
was then separated by distillation at atmospheric pressure. After
cooling to rt 6 N aqueous sulphuric acid (5 mL) was added to the
residual solution to neutralize the sodium alloxide, and solvent re-
moval was continued to afford an oil which was distilled under re-
3
2
4
.
3
1
3
(
1
3
3
3
2
duced pressure: bp 65 °C (20 mmHg); 5.10 g (56% yield). H NMR
2
3
)
3 2
2
(
CDCl
3
): d 1.07 (d, 3, CH
3
), 1.15 (d, 3, CH
3
), 2.68 (br s, 1, OH),
3
.20 (m, 1, CH), 3.58 (m, 1, CH), 3.91 (m, 1, CH
2
), 4.15 (m, 1,
2 2
CH ), 5.18–5.26 (m, 2, C@CH ), 5.90 (m, 1, CH@C).
4
.1.15. ((2S*,5S*,6S*)-5,6-Dimethyl-[1,4]dioxan-2-ylmethyl)-
dimethyl-amine (20b)
4
.1.11. (2R*,3S*)-3-Allyloxy-butan-2-ol (18b)
This was prepared as described for 20a starting from 19b: 86%
This was prepared as described for (2R*,3R*)-3-(allyloxy)butan-
1
yield. H NMR (CDCl
3
): d 1.07 (d, 3, CH
N), 2.29 (s, 6, N(CH ), 2.63 (m, 1, cycle), 2.73 (m, 1, cy-
cle), 3.23 (m, 1, cycle), 3.49 (m, 1, cycle), 3.72 (m, 1, cycle).
3 3
), 1.12 (d, 3, CH ), 2.24
2
(
(
-ol starting from (2R*,3R*)-2,3-dimethyl-oxirane: bp 70 °C
1
(
m, 2, CH
2
3 2
)
20 mmHg); 37% yield. H NMR (CDCl
3 3
): d 1.10 (d, 3, CH ), 1.14
3
d, 3, CH ), 2.21 (br s, 1, OH), 3.43 (m, 1, CH), 3.89 (m, 1, CH),
4
.03 (m, 2, OCH ), 5.10–5.28 (m, 2, C@CH ), 5.92 (m, 1, CH@C).
2
2
4
.1.16. ((2R*,5S*,6R*)-5,6-Dimethyl-[1,4]dioxan-2-ylmethyl)-
4
.1.12. (2S*,3S*,5S*)-5-Iodomethyl-2,3-dimethyl-[1,4]dioxane (19a)
dimethyl-amine (20c)
and (2S*,3S*,5R*)-5-iodomethyl-2,3-dimethyl-[1,4]dioxane (19b)
This was prepared as described for 20a starting from 19c: 82%
1
A solution of mercury(II) acetate (12.1 g, 37.9 mmol) in H
2
O
3
yield. H NMR (CDCl ): d 1.02 (d, 3, CH
3 3
), 1.29 (d, 3, CH ), 2.10
(
75 mL) and acetic acid (0.1 mL) was added to a stirred solution of
(m, 2, CH
2
N), 2.24 (s, 6, N(CH ), 2.33 (m, 1, cycle), 3.31 (m, 1, cy-
3 2
)
1
8a (5.1 g, 39.2 mmol). The reaction mixture was heated to reflux
cle), 3.78 (m, 2, cycle), 3.95 (m, 1, cycle).
for 45 min, then allowed to stand overnight at rt. After the reaction
mixture was filtered a solution of KI (6.5 g, 39.2 mmol) in H
2
O
4.1.17. ((2R*,5R*,6S*)-5,6-Dimethyl-[1,4]dioxan-2-ylmethyl)-
(
(
40 mL) was added to the filtrate and a mixture of (2S*,5S*,6S*)-/
2R*,5S*,6S*)-((5,6-dimethyl-1,4-dioxan-2-yl)methyl)mercury(II)
(25 mL). A
(170 mL) was added and
the reaction mixturewas heated to boilingand then allowed to stand
at rt for 18 h. The organic phase was washed with 10% Na SO , 10%
KI, and dried over Na SO . The evaporation of the solvent in vacuo
afforded a residue. The mixture of diastereomers 19a and 19b (ratio
:3) was separated by column chromatography eluting with petro-
leum ether/EtOAc (99.5:0.5). Compound 19a eluted first: 2.0 g
dimethyl-amine (20d)
This was prepared as described for 20a starting from 19d: 86%
1
iodide separated as an oil, which was dissolved in CHCl
solution of I (7.5 g, 29.5 mmol) in CHCl
3
yield. H NMR (CDCl
3
): d 1.02 (d, 3, CH
3 3
), 1.21 (d, 3, CH ), 2.18
(m, 2, CH
2
N), 2.25 (s, 6, N(CH ), 2.39 (m, 1, cycle), 3.46 (m, 1, cy-
3 2
)
2
3
cle), 3.65 (m, 2, cycle), 3.90 (m, 1, cycle).
2
3
4.1.18. ((2R*,5S*,6S*)-5,6-Dimethyl-1,4-dioxan-2-yl)-N,N,N-
trimethylmethanaminium iodide (4)
This was prepared as described for 3b starting from 20a. The so-
lid was filtered and recrystallized from 2-PrOH; mp 160–161 °C. H
2
4
7
1
1
(
3 3
20% yield). H NMR (CDCl ): d 1.08 (d, J = 6.41 Hz, 3, CH ), 1.14 (d,
NMR (DMSO): d 0.98 (d, 3, CH
3 3
), 1.08 (d, 3, CH ), 3.05–3.25 (m, 11,
J = 5.98 Hz, 3, CH ), 3.02 (dd, J = 6.41, 10.68 Hz, 1, CH
3
2
I), 3.07 (dd,
CH N, N(CH ), 3.27–3.43 (m, 3, cycle), 3.65 (m, 1, cycle), 4.19 (m,
2
3 3
)

8
182
A. Piergentili et al. / Bioorg. Med. Chem. 17 (2009) 8174–8185
1
, cycle). Anal. Calcd for C10
2
H22INO : C, 38.11; H, 7.04; N, 4.44.
2 2
(m, 1, CH), 3.88–4.21 (m, 2, OCH ), 5.12–5.26 (m, 2, C@CH ), 5.91
Found: C, 38.01; H, 6.89; N, 4.60.
(m, 1, CH@C).
4
.1.19. ((2S*,5S*,6S*)-5,6-Dimethyl-1,4-dioxan-2-yl)-N,N,N-
trimethylmethanaminium iodide (5)
4.1.26. 3-Allyloxy-2,3-dimethyl-butan-2-ol (25)
This was prepared as described for 22 starting from 2,2,3,3-
tetramethyloxirane. After evaporation of the solvent, the residue
3
9
This was prepared as described for 3b starting from 20b. The so-
1
lid was filtered and recrystallized from EtOH; mp 214–216 °C.
NMR (DMSO): d 1.03 (d, 3, CH ), 1.12 (d, 3, CH
CH N), 3.57 (s, 9, N(CH ), 3.78–4.00 (m, 2, cycle), 4.30 (m, 2, cy-
cle), 4.60 (m, 1, cycle). Anal. Calcd for C10 22INO : C, 38.11; H,
.04; N, 4.44. Found: C, 38.22; H, 7.22; N, 4.61.
H
was purified by column chromatography eluting with cyclohex-
1
3
3
), 3.25 (m, 2,
ane/EtOAc (9.2:0.8) 73% yield. H NMR (CDCl
3
): d 1.20 (m, 12,
2
3
)
3
CH
3
), 2.52 (br s, 1, OH), 3.99 (m, 2, CH
2
), 5.11–5.35 (m, 2, C@CH ),
2
H
2
5.91 (m, 1, CH@C).
7
4
.1.27. 6-Iodomethyl-2,2-dimethyl-[1,4]dioxane (26)
This was prepared as described for 19a starting from 21: 59%
4
.1.20. ((2R*,5S*,6R*)-5,6-Dimethyl-1,4-dioxan-2-yl)-N,N,N-
1
trimethylmethanaminium iodide (6)
yield. H NMR (CDCl
3 3 3
): d 1.18 (s, 3, CH ), 1.32 (s, 3, CH ), 3.02 (d,
This was prepared as described for 3b starting from 20c. The so-
2, CH I), 3.15 (dd, 2, cycle), 3.42 (d, 1, cycle), 3.91 (m, 1, cycle),
4.01 (dd, 1, cycle).
2
1
lid was filtered and recrystallized from EtOH; mp 183–184 °C.
NMR (DMSO): d 0.94 (d, 3, CH ), 1.21 (d, 3, CH
N(CH ), 3.20 (m, 2, CH N), 3.32 (m, 2, cycle), 3.65 (m, 1, cycle),
.80 (m, 1, cycle), 4.29 (m, 1, cycle). Anal. Calcd for C10 22INO
C, 38.11; H, 7.04; N, 4.44. Found: C, 38.41; H, 7.01; N, 4.60.
H
3
3
), 3.10 (s, 9,
)
3 3
2
4.1.28. 5-Iodomethyl-2,2-dimethyl-[1,4]dioxane (27)
3
H
2
:
This was prepared as described for 19a starting from 22: 20%
1
yield. H NMR (CDCl
3
3
): d 1.12 (s, 3, CH ), 1.32 (s, 3, CH
3
), 3.14 (d,
2
2
, CH I), 3.41–3.76 (m, 5, cycle).
4
.1.21. ((2R*,5R*,6S*)-5,6-Dimethyl-1,4-dioxan-2-yl)-N,N,N-
trimethylmethanaminium iodide (7)
This was prepared as described for 3b starting from 20d. The so-
lid was filtered and recrystallized from 2-PrOH; mp 190–191 °C. H
4.1.29. (3S*,5S*)/(3R*,5S*)-5-Iodomethyl-2,2,3-trimethyl-
[1,4]dioxane (28a and 28b)
1
This was prepared as described for 19a starting from 23. A mix-
NMR (DMSO): d 0.95 (d, 3, CH
N(CH ), 3.33 (m, 2, CH N), 3.36 (m, 2, cycle), 3.70 (m, 1, cycle),
.95 (m, 1, cycle), 4.20 (m, 1, cycle). Anal. Calcd for C10 22INO
C, 38.11; H, 7.04; N, 4.44. Found: C, 38.40; H, 7.28; N, 4.51.
3 3
), 1.12 (d, 3, CH ), 3.11 (s, 9,
ture of the two diastereomers was obtained: 53% yield. 1H NMR
)
3 3
2
3 3 2
(CDCl ): d 1.12 (m, 18, CH ), 3.11 (m, 4, CH I), 3.41–3.99 (m, 8, cycle).
3
H
2
:
4.1.30. (3R*,6S*)-6-Iodomethyl-2,2,3-trimethyl-[1,4]dioxane
29a) and (3S*,6S*)-6-iodomethyl-2,2,3-trimethyl-[1,4]dioxane
(29b)
These were prepared as described for 19a starting from 24. The
(
4
.1.22. 1-Allyloxy-2-methyl-propan-2-ol (21)
This was prepared as described for 18a starting from 2,2-dime-
1
thyloxirane: 44% yield. H NMR (CDCl
3
): d 1.21 (s, 6, CH
3
), 2.22 (s, 1,
),
two diastereomers were separated by column chromatography
OH), 3.28 (s, 2, CH O), 4.05 (m, 2, OCH
2
2
), 5.18–5.36 (m, 2, C@CH
2
eluting with cyclohexane/EtOAc (9.9:0.1). 29b eluted first: 17%
1
5
.90 (m, 1, CH@C).
yield. H NMR (CDCl
3 3 3
): d 1.02 (d, 3, CH ), 1.11 (s, 3, CH ), 1.21 (s,
3
, CH
4.01 (m, 1, cycle). The second fraction was 29a: 2% yield. H NMR
(CDCl ): d 1.03 (s, 3, CH ), 1.20 (d, 3, CH ), 1.28 (s, 3, CH ), 3.11
(m, 2, CH I), 3.35–3.81 (m, 4, cycle).
3
), 3.00 (m, 2, CH
2
I), 3.18–3.40 (m, 2, cycle), 3.88 (m, 1, cycle),
1
4
.1.23. 2-Allyloxy-2-methyl-propan-1-ol (22)
Perchloric acid (70%, 3.4 mL) was added to a stirred solution of
,2-dimethyloxirane (5.0 g, 69.3 mmol) in allyl alcohol (40 mL) at
°C. After 0.5 h at room temperature the reaction mixture was
3
3
3
3
2
0
2
2 2
poured in H O (75 mL) and extracted with Et O. The organic phase
4.1.31. 5-Iodomethyl-2,2,3,3-tetramethyl-[1,4]dioxane (30)
was washed with NaHCO
solvents gave a residue, which was purified by column chromatog-
3
and dried over Na
2
SO . Removal of dried
4
This was prepared as described for 19a starting from 25: 30%
1
3 3 3
yield. H NMR (CDCl ): d 1.12 (s, 6, CH ), 1.39 (s, 6, CH ), 3.09
raphy, eluting with cyclohexane/EtOAc (9.5:0.5): 3.0 g (33% yield).
2
(m, 2, CH I), 3.45–3.86 (m, 3, cycle).
1
H NMR (CDCl
3
): d 1.20 (s, 6, CH
3
), 2.02 (t, 1, OH), 3.43 (d, 2, CH
2
O),
3
.98 (m, 2, OCH ), 5.10–5.35 (m, 2, C@CH
2
2
), 5.93 (m, 1, CH@C).
4.1.32. (6,6-Dimethyl-[1,4]dioxan-2-ylmethyl)-dimethyl-amine
31)
This was prepared as described for 20a starting from 26: 94%
(
4
.1.24. 3-Allyloxy-3-methyl-butan-2-ol (23)
This was prepared as described for 22 starting from 2,2,3-trime-
1
yield. H NMR (CDCl
3
): d 1.10 (s, 3, CH
3
3
), 1.35 (s, 3, CH ), 2.18
thyloxirane. After evaporation of the solvent, the residue was puri-
fied by column chromatography eluting with petroleum ether/
(m, 1, CH N), 2.24 (s, 6, N(CH
CH), 3.25 (dd, 1, cycle), 3.48 (d, 1, cycle), 3.80 (dd, 1, cycle), 4.00
(m, 1, cycle).
2
3
)
2
), 2.30 (m, 1, CH
2
N), 3.20 (dd, 1,
EtOAc (9:1) (73% yield). 1H NMR (CDCl
): d 1.15 (m, 9, CH
d, 1, OH), 3.63 (m, 1, CH), 3.98 (m, 2, OCH ), 5.10–5.31 (m, 2,
C@CH ), 5.90 (m, 1, CH@C).
3
3
), 2.60
(
2
2
4.1.33. (5,5-Dimethyl-[1,4]dioxan-2-ylmethyl)-dimethyl-amine
(
32)
This was prepared as described for 20a starting from 27: 86%
4
.1.25. 3-Allyloxy-2-methyl-butan-2-ol (24)
A solution of 3 M CH MgI in Et O (13.9 mL, 41.7 mmol) in Et
10 mL) was added dropwise to a stirred solution of 2-allyloxy-pro-
1
3
2
2
O
yield. H NMR (CDCl
1, CH N), 2.24 (s, 6, N(CH
3.51–3.62 (m, 4, cycle).
3 3 3
): d 1.08 (s, 3, CH ), 1.30 (s, 3, CH ), 2.15 (m,
(
2
3
)
2
), 2.43 (m, 1, CH
2
N), 3.39 (d, 1, CH),
2
4
pionic acid methyl ester (2.4 g, 16.6 mmol) in Et
stirring at rt for 2 h the reaction mixture was cooled to 0 °C and
quenched cautiously by the dropwise addition of Na SO saturated
solution. The solid was filtered off and the filtrate was dried over
Na SO . Removal of the solvent afforded a residue, which was puri-
fied by column chromatography eluting with cyclohexane/EtOAc
99:1) to give an oil: 1.8 g (75% yield). 1H NMR (CDCl
): 1.12 (d,
, CH ), 1.15 (s, 3, CH ), 1.18 (s, 3, CH ), 2.50 (br s 1, OH), 3.23
2
O (20 mL). After
2
4
4.1.34. Dimethyl-((2R*,6S*)-5,5,6-trimethyl-[1,4]dioxan-2-yl-
methyl)-amine (33a) and dimethyl-((2R*,6R*)-5,5,6-trimethyl-
[1,4]dioxan-2-ylmethyl)-amine (33b)
These were prepared as described for 20a starting from a mix-
ture of 28a and 28b. The two diastereomers were separated by col-
2
4
(
3
3
3
3
3
3
umn chromatography eluting with petroleum ether/EtOAc/CH OH/

A. Piergentili et al. / Bioorg. Med. Chem. 17 (2009) 8174–8185
8183
NH
4
OH (3:1.8:0.1:0.01). The cis diastereomer 33a eluted first: 0.6 g
4.1.42. N,N,N-Trimethyl((2R*,5R*)-5,6,6-trimethyl-1,4-dioxan-
2-yl)methanaminium iodide (11a)
1
(
17% yield). H NMR (CDCl
3 3 3
): d 1.06 (d, 3, CH ), 1.14 (s, 3, CH ), 1.36
(
s, 3, CH ), 2.14–2.43 (m, 2, CH
3
2
N), 2.22 (s, 6, N(CH
3
)
2
), 3.50 (m, 3,
This was prepared as described for 3b starting from 34a. The so-
lid was filtered and recrystallized from i-PrOH; mp 226–228 °C. H
1
cycle), 3.72 (m, 1, cycle). The second fraction was the trans diaste-
1
reomer 33b: 0.5 g (14% yield); H NMR (CDCl
.19 (d, 3, CH ), 1.27 (s, 3, CH ), 2.31 (s, 6, N(CH
m, 2, CH N), 3.49–3.91 (m, 4, cycle).
3
): d 1.16 (s, 3, CH
3
),
NMR (DMSO): d 1.02 (s, 3, CH
3.09 (m, 11, CH N, N(CH ), 3.29–3.57 (m, 3, cycle), 4.29 (m, 1, cy-
cle). Anal. Calcd for C11 24INO : C, 40.13; H, 7.35; N, 4.25. Found: C,
3 3 3
), 1.21 (d, 3, CH ), 1.40 (s, 3, CH ),
1
(
3
3
3
)
2
), 2.39–2.69
2
3 3
)
2
H
2
3
9.87; H, 7.02; N, 3.89.
4
.1.35. Dimethyl-((2R*,5R*)-5,6,6-trimethyl-[1,4]dioxan-2-
4
.1.43. N,N,N-Trimethyl((2R*,5S*)-5,6,6-trimethyl-1,4-dioxan-2-
yl)methanaminium iodide (11b)
ylmethyl)-amine (34a)
This was prepared as described for 20a starting from 29a: 97%
1
This was prepared as described for 3b starting from 34b. The so-
yield. H NMR (CDCl
, CH ), 2.21–2.41 (m, 2, CH
cle), 3.99 (m, 1, cycle).
3
): d 1.08 (s, 3, CH
3
), 1.25 (d, 3, CH
3
), 1.39 (s,
1
lid was filtered and recrystallized from i-PrOH; mp 168–169 °C. H
3
3
2
N), 2.22 (s, 6, N(CH )
3 2
), 3.51 (m, 3, cy-
NMR (DMSO): d 0.99 (d, 3, CH
.09 (m, 12, CH N, N(CH , cycle), 3.68 (m, 1, cycle), 4.29 (m, 1, cy-
cle). Anal. Calcd for C11 24INO : C, 40.13; H, 7.35; N, 4.25. Found: C,
3 3 3
), 1.04 (s, 3, CH ), 1.22 (s, 3, CH ),
3
2
3 3
)
H
2
4
.1.36. Dimethyl-((2R*,5S*)-5,6,6-trimethyl-[1,4]dioxan-2-
ylmethyl)-amine (34b)
4
0.42; H, 7.59; N, 4.38.
This was prepared as described for 20a starting from 29b: 91%
4
.1.44. N,N,N-Trimethyl(5,5,6,6-tetramethyl-1,4-dioxan-2-
1
yield. H NMR (CDCl
3
): d 1.05 (d, 3, CH
N), 2.22 (s, 6, N(CH
.25 (m, 2, cycle), 3.79 (m, 1, cycle), 3.95 (m, 1, cycle).
3
), 1.11 (s, 3, CH
3
), 1.26 (s,
N),
yl)methanaminium iodide (12)
3
3
, CH
3
), 2.12 (m, 1, CH
2
3
)
2
), 2.29 (m, 1, CH
2
This was prepared as described for 3b starting from 35. The so-
1
lid was filtered and recrystallized from i-PrOH; mp 252–253 °C. H
NMR (DMSO): d 0.99 (s, 3, CH
3
), 1.00 (s, 3, CH
), 3.10 (s, 9, N(CH ), 3.19–3.45 (m, 4, CH
.29 (m, 1, cycle). Anal. Calcd for C12 26INO : C, 41.99; H, 7.63; N,
.08. Found: C, 41.79; H, 7.41; N, 4.00.
3
), 1.23 (s, 3, CH
3
),
4
.1.37. Dimethyl-(5,5,6,6-tetramethyl-[1,4]dioxan-2-ylmethyl)-
1
4
4
.36 (s, 3, CH
3
3
)
3
2
N, cycle),
amine (35)
This was prepared as described for 20a starting from 30: 83%
yield. H NMR (CDCl
3
3
H
2
1
3
): d 1.07 (s, 6, CH
), 2.15 (m, 1, CH N), 2.22 (s, 6, N(CH
.50 (m, 2, cycle), 4.00 (m, 1, cycle).
3
), 1.31 (s, 3, CH
3
), 1.35 (s,
N),
, CH
3
2
3
)
2
), 2.30 (m, 1, CH
2
4
.2. X-ray crystal structure analysis of (2R*,5S*)-N,N,N-trimeth-
yl-(5-methyl-1,4-dioxan-2-yl)methanaminium iodide (3b)
4
.1.38. (6,6-Dimethyl-1,4-dioxan-2-yl)-N,N,N-trimethylmethan-
Crystals of 3b were obtained from methanol/2-propanol/buta-
nol solutions in 1:1:2 volume ratios. In spite of repeated attempts,
only crystals of limited quality could be obtained. X-ray data
collection was performed at room temperature with an Oxford Dif-
fraction Xcalibur 3 CCD diffractometer and graphite-monochro-
aminium iodide (8)
This was prepared as described for 3b starting from 31. The so-
1
lid was filtered and recrystallized from 2-PrOH; mp 180–181 °C. H
NMR (DMSO): d 1.04 (s, 3, CH
.09 (s, 9, N(CH ), 3.15 (m, 1, CH
cycle), 3.65 (dd, 1, cycle), 4.35 (m, 1, cycle). Anal. Calcd for
22INO : C, 38.11; H, 7.04; N, 4.44. Found: C, 38.32; H, 7.22;
N, 4.55.
3
3 2
), 1.31 (s, 3, CH ), 3.01 (m, 1, CH N),
3
3
)
3
2
N), 3.30 (m, 2, cycle), 3.50 (d, 1,
mated Mo K
a radiation (k = 0.71069 Å). Intensity data were
4
0
corrected for absorption by a multi-scan procedure. The structure
was solved by direct methods, with SIR-97, and refined by full-
4
1
C
10
H
2
4
2
matrix least-squares with SHELXL-97. In the final refinement cycles
all non-hydrogen atoms were refined anisotropically and hydro-
gens were in geometrically calculated positions, riding on the
respective carrier atoms, each with an isotropic temperature factor
linked to that of its carrier atom. The absolute structure could not
4
.1.39. (5,5-Dimethyl-1,4-dioxan-2-yl)-N,N,N-trimethylmethan-
aminium iodide (9)
This was prepared as described for 3b starting from 32. The so-
1
43
lid was filtered and recrystallized from EtOH; mp 215–216 °C.
NMR (DMSO): d 1.05 (s, 3, CH ), 1.32 (s, 3, CH
N(CH ), 3.28–3.52 (m, 6, CH N, cycle), 4.05 (m, 1, cycle). Anal.
Calcd for C10 22INO : C, 38.11; H, 7.04; N, 4.44. Found: C, 38.01;
H, 6.92; N, 4.36.
H
be determined unambiguously, presumably due to limited qual-
3
3
), 3.11 (s, 9,
ity of the data and to the presence of a centrosymmetric pattern
formed by the heavy iodine atoms; nevertheless, the aspects of
interest for the present study are unambiguously clarified by the
structural model. For graphics ORTEP-3 was employed.44 Crystal
data, data collection parameters and analysis statistics are summa-
rised in Table 3, as Supplementary data, and values of bond dis-
tances and selected values of bond angles and torsion angles are
listed in Table 4. Crystallographic data have been deposited with
the Cambridge Crystallographic Data Centre, 12 Union Road, Cam-
bridge CB2 1EZ, UK (Fax: +44 1223 336033; e-mail: deposit@ccdc.-
cam.ac.uk), and are available on request quoting the deposition
number CCDC 734905.
3
)
3
2
H
2
4
.1.40. N,N,N-Trimethyl((2R*,6S*)-5,5,6-trimethyl-1,4-dioxan-2-
yl)methanaminium iodide (10a)
This was prepared as described for 3b starting from 33a. The so-
1
lid was filtered and recrystallized from i-PrOH; mp 176–178 °C. H
NMR (DMSO): d 0.98 (d, 3, CH
.08 (s, 9, N(CH ), 3.22–3.59 (m, 5, CH
Anal. Calcd for C11 24INO : C, 40.13; H, 7.35; N, 4.25. Found: C,
0.40; H, 7.09; N, 4.07.
3
3 3
), 1.04 (s, 3, CH ), 1.15 (s, 3, CH ),
3
3
)
3
2
N, cycle), 4.16 (m, 1, cycle).
H
2
4
4
.3. Pharmacology
4
2
.1.41. N,N,N-Trimethyl((2R*,6R*)-5,5,6-trimethyl-1,4-dioxan-
-yl)methanaminium iodide (10b)
4.3.1. Binding studies
This was prepared as described for 3b starting from 33b. The so-
lid was filtered and recrystallized from i-PrOH; mp 205–206 °C. H
4.3.1.1. Cell culture and membrane preparation. Chinese ham-
ster ovary cells stably expressing cDNA encoding human musca-
rinic hM –hM receptors were generously provided by Professor
R. Maggio (Department of Experimental Medicine, University of
L’Aquila, Italy). Growth medium consisted in Dulbecco’s modified
Eagle’s medium supplemented with 10% fetal bovine serum (Gibco,
1
NMR (DMSO): d 0.99 (s, 3, CH
3
), 1.07 (s, 3, CH
), 3.20–3.41 (m, 3, CH N, cycle), 3.88 (m, 1, cycle),
.01 (m, 1, cycle), 4.31 (m, 1, cycle). Anal. Calcd for C11 24INO : C,
0.13; H, 7.35; N, 4.25. Found: C, 39.89; H, 7.48; N, 4.60.
3 3
), 1.16 (d, 3, CH ),
1
5
3
4
4
.12 (s, 9, N(CH
3
)
3
2
H
2

8
184
A. Piergentili et al. / Bioorg. Med. Chem. 17 (2009) 8174–8185
Grand Iland, NY), 50 units/mL of penicillin G and 0.05 mg/mL
streptomycin, 2 mM -glutamine (Sigma Aldrich, Milano, Italy),
non essential aminoacids solution 100ꢁ (Sigma Aldrich, Milano,
Italy) and 500 g/mL of geneticin (Gibco, Grand Iland, NY) in a
humidified atmosphere consisting of 5% CO and 95% air.
Confluent CHO cell lines were scraped, washed with phosphate
buffer (25 mM sodium phosphate, 5 mM MgCl , pH 7.4), and
gan baths under 0.5 g tension at 33 °C, immersed in a modified
Krebs–Henseleit solution (mM composition: NaCl 118.9, KCl 4.6,
L
CaCl
2
2.5, KH
2
PO
4
1.2, NaHCO
3
25, MgSO
4
ꢂ7H
2
O 1.2, glucose 11.1)
l
and gassed with a 95% O
2
–5% CO
2
mixture. After a period of stabil-
2
ization of 45 min, tissues were electrically stimulated through plat-
inum electrodes by square-wave submaximal pulses (2 Hz, 5 ms,
5 V) and inotropic activity was recorded isometrically.
2
homogenized for 30 s using an Ultra-Turrax (setting 3). The pellet
was sedimented at 17,000g for 15 min at 4 °C, and the membranes
were resuspended in the same buffer, rehomogenized with Ultra-
4.3.2.3. Guinea pig ileum. Ileal segments 2–3 cm long were set
up under 1.0 g tension at 37 °C in 10 mL organ baths filled with
Krebs–Henseleit solution (see above), bubbled with carbogen. Tis-
sues were allowed to equilibrate for 45 min and afterwards con-
45
Turrax, and stored at ꢀ80 °C. An aliquot was taken for the assess-
4
6
ment of protein content according to the method of Bradford
2
8
using the Bio-Rad protein assay reagent (Bio-Rad Laboratories,
Munchen, West Germany) and bovine serum albumin was used
as the standard.
tractile responses were isometrically recorded.
4.3.2.4. Guinea pig lung strips. Strips of peripheral lung tissue
(
15 ꢁ 2 ꢁ 2 mm) were cut from the peripheral margin of a lower
4
.3.1.2. Binding assays. Radioligand binding assays were run in
lobe and mounted under 0.3 g resting tension at 37 °C in 10 mL or-
polypropylene 96-well plates (Sarstedt, Verona, Italy) and per-
formed for 120 min at room temperature in a final volume of
gan baths filled with Krebs–Henseleit solution (see above), bub-
bled with carbogen. After a period of 45 min of equilibration,
contractile responses were isometrically recorded.
3
5
0
.25 mL in 25 mM sodium phosphate buffer containing 5 mM
MgCl at pH 7.4. Final membrane protein concentrations were
g/mL (hM ), 70 g/mL (hM ), 25 g/mL (hM ), 50 g/mL
), and 25 g/mL (hM ).
2
3
0
l
4
1
l
2
l
3
l
4.3.2.5. Protocols. Agonist concentration–response curves were
(
hM
l
5
constructed in each tissue by cumulative application of concentra-
3
48
In homologous competition curves, [ H]NMS was present at
tions of the test compounds. The agonist potency was expressed
0
.2 nM in wells containing increasing concentration of unlabeled
as pD
2
(ꢀLog ED50) calculated by linear regression analysis using
NMS (0.03–1000 nM) and at 0.075–0.2 nM in wells without
unlabeled ligand. In heterologous competition curves, a fixed
concentration of the tracer (0.2 nM) was displaced by increasing
the least square method. Intrinsic activity (
a
) was calculated as a
fraction of the maximal response to the reference full agonist,
Bethanechol or McN-A-343. When the compounds were tested as
antagonists, a concentration–response curve to the full agonist
Bethanechol or McN-A-343 was repeated after 10 (ileal tissue), 30
or 60 min (lung strips) incubation with the test compounds
concentrations of several unlabeled ligands (0.01–1000 lM); all
measurements were obtained in duplicate. At the end of the binding
reaction, free radioligand was separated from bound ligand by rapid
filtration through UniFilter GF/C plates (Perkin–Elmer Life Science,
Boston, MA) using a FilterMate cell harvester (Perkin–Elmer Life Sci-
ence, Boston, MA). After filtration, the filters were washed several
times with ice-coldbuffer and allowedtodry overnightat roomtem-
(100 nM–30 lM). The potency of the compounds acting as antago-
nists was expressed as pK
B
value according to Furchgott’s method.49
Acknowledgements
perature under air flow, 25 lL of scintillation liquid (Microscint-20,
Perkin–Elmer Life Science, Boston, MA) was added, and the radioac-
tivity was counted by a TopCount NXT microplate scintillation coun-
ter (Perkin–Elmer Life Science, Boston, MA). The binding data were
analyzed by the weighted least-squares iterative curve fitting pro-
This work was supported by grants from the MIUR (Rome), the
University of Camerino, and the Monte dei Paschi di Siena
Foundation.
4
7
i
gram LIGAND to obtain the affinity constant (K ) of the tested agents.
Supplementary data
4
.3.2. Functional studies with isolated organs
General considerations. Male guinea pigs (250–350 g) and New
Crystal data and structure refinement parameters for 3b. Bond
lengths (Å) and selected values of bond angles (°) and torsion an-
gles (°) for 3b are available. Supplementary data associated with
this article can be found, in the online version, at doi:10.1016/
j.bmc.2009.10.027.
Zealand white rabbits (3.0–3.5 kg) (Morini, S. Polo, Italy) were
used. The tissues for in vitro experiments were removed from ani-
mals fasted 24 h before the experiments and killed by CO inhala-
2
tion. Isolated preparations were set up according to the following
techniques.
References and notes
4
.3.2.1. Electrically stimulated rabbit vas deferens. According
1
2
.
.
Ishii, M.; Kurachi, Y. Curr. Pharm. Des. 2006, 12, 3573.
Eglen, R. M. Prog. Med. Chem. 2005, 43, 105.
2
6
to Eltze, the prostatic portion of each vas deferens was mounted
in a 10 mL organ bath, containing a modified Krebs solution (mM
3. van der Zee, E. A.; Luiten, P. G. M. Prog. Neurobiol. 1999, 58, 409.
4
5
6
.
.
.
Clader, J. W.; Wang, Y. Curr. Pharm. Des. 2005, 11, 3353.
Dean, B.; Bymaster, F. P.; Scarr, E. Curr. Mol. Med. 2003, 3, 419.
Calabresi, P.; Centonze, D.; Gubellini, P.; Pisani, A.; Bernardi, G. Trends Neurosci.
2000, 23, 120.
Wallis, R. M.; Napier, C. M. Life Sci. 1999, 64, 395.
Hegde, S. S. Br. J. Pharmacol. 2006, 147, S80.
composition: NaCl 134, KCl 3.4, CaCl
6, MgSO 0.6, glucose 7.7) kept at 31 °C and bubbled with 95%
–5% CO . Yohimbine (1.0 M) was present throughout the
experiment to prevent prejunctional -adrenoceptors stimula-
2 2 4 3
2.8, KH PO 1.3, NaHCO
1
O
4
2
2
l
7.
8.
9.
a
2
tion. For isometric recordings, the tissues were left to equilibrate
for 45 min under a passive load of 0.75 g before electrical field
stimulation through platinum electrodes was applied by square-
wave pulses (0.5 ms, 0.05 Hz, supramaximal intensity 450 mA;
LACE Elettronica Mod. ES3, Ospedaletto PI, Italy).
Dinan, T. G.; Clarke, G.; Quigley, E. M.; Scott, L. V.; Shanahan, F.; Cryan, J.;
Cooney, J.; Keeling, P. W. Am. J. Gastroenterol. 2008, 103, 2570.
10. Gosens, R.; Zaagsma, J.; Meurs, H.; Halayko, A. J. Respir. Res. 2006, 7, 73.
1
1
1
1
1
1. Winzell, M. S.; Ahrén, B. Pharmacol. Ther. 2007, 116, 437.
2. Song, P.; Spindel, E. R. J. Pharmacol. Sci. 2008, 106, 180.
3. Pedretti, A.; Vistoli, G.; Marconi, C.; Testa, B. Chem. Biodiv. 2006, 3, 481.
4. Angeli, P. Farmaco 1995, 50, 565.
5. Piergentili, A.; Quaglia, W.; Giannella, M.; Del Bello, F.; Bruni, B.; Buccioni, M.;
Carrieri, A.; Ciattini, S. Bioorg. Med. Chem. 2007, 15, 886.
6. Hallonquist, E. G.; Hibbert, H. Can. J. Res. 1933, 8, 129.
4
.3.2.2. Electrically stimulated guinea pig left atrium. The
heart was rapidly dissected and right and left atria were separated.
As described by Eglen, the left atria were mounted in 20 mL or-
1
2
7
17. Duclos, R. I., Jr.; Makriyannis, A. J. Org. Chem. 1992, 57, 6156.

A. Piergentili et al. / Bioorg. Med. Chem. 17 (2009) 8174–8185
8185
1
8. Fjeldskaar, I. R.; Grace, D.; Roemming, C.; Skatteboel, L. Acta Chem. Scand. B
988, B42, 280.
9. Piergentili, A.; Quaglia, W.; Giannella, M.; Del Bello, F.; Buccioni, M.; Nesi, M.;
Matucci, R. Bioorg. Med. Chem. Lett. 2008, 18, 614.
35. Scapecchi, S.; Nesi, M.; Matucci, R.; Bellucci, C.; Buccioni, M.; Dei, S.;
Guandalini, L.; Manetti, D.; Martini, E.; Marucci, G.; Romanelli, M. N.;
Teodori, E.; Cirilli, R. J. Med. Chem. 2008, 51, 3905.
36. Scapecchi, S.; Matucci, R.; Bellucci, C.; Buccioni, M.; Dei, S.; Guandalini, L.;
Martelli, C.; Manetti, D.; Martini, E.; Marucci, G.; Nesi, M.; Romanelli, M. N.;
Teodori, E.; Gualtieri, F. J. Med. Chem. 2006, 49, 1925.
37. Chang, K. J.; Deth, R. C.; Triggle, D. J. J. Med. Chem. 1972, 15, 1409.
38. Dei, S.; Bellucci, C.; Buccioni, M.; Ferraroni, M.; Guandalini, L.; Manetti, D.;
Marucci, G.; Matucci, R.; Nesi, M.; Romanelli, M. N.; Scapecchi, S.; Teodori, E. J.
Med. Chem. 2007, 50, 1409.
1
1
2
2
2
2
2
0. Landmann, B.; Hoffman, R. W. Chem. Ber. 1987, 120, 331.
1. Traficante, D. D.; Meadows, M. D. Concepts Magn. Reson. 1997, 9, 359.
2. Gelas, J.; Veyssières-Rambaud, S. Carbohydr. Res. 1974, 37, 293.
3. Beckwith, A. L. J.; Bowry, V. W. J. Org. Chem. 1988, 53, 1632.
4. Gilchrist, T. L.; Wasson, R. C.; King, F. D.; Wootton, G. J. Chem. Soc., Perkin Trans.
1
1987, 11, 2517.
2
5. Dei, S.; Angeli, P.; Bellucci, C.; Buccioni, M.; Gualtieri, F.; Marucci, G.; Manetti,
39. Sawwan, N.; Greer, A. J. Org. Chem. 2006, 71, 5796.
D.; Matucci, R.; Romanelli, M. N.; Scapecchi, S.; Teodori, E. Biochem. Pharmacol.
40. Oxford Diffraction, CrysAlisPro (Version 1.171.29.2). Oxford Diffraction Ltd:
Abingdon, Oxforddhire, England, 2006.
2
005, 69, 1637.
2
2
2
6. Eltze, M. Eur. J. Pharmacol. 1988, 151, 205.
41. Altomare, A.; Burla, M. C.; Camalli, M.; Cascarano, G. L.; Giacovazzo, C.;
Guagliardi, A.; Moliterni, A. G. G.; Polidori, G.; Spagna, R. J. Appl. Crystallogr.
1999, 32, 115.
42. Sheldrick, G. M. SHELXL97, Program for Crystal Structure Refinement; University of
Göttingen, Göttingen: Germany, 1986.
7. Eglen, R. M.; Watson, N. Pharmacol. Toxicol. 1996, 78, 59.
8. Barocelli, E.; Ballabeni, V.; Bertoni, S.; Dallanoce, C.; De Amici, M.; De Micheli,
M.; Impicciatore, M. Life Sci. 2000, 67, 717.
29. Roffel, A. F.; Elzinga, C. R. S.; Zaagsma, J. Eur. J. Pharmacol. 1993, 250, 267.
0. Eglen, R. M.; Montgomery, W. W.; Whiting, R. L. J. Pharmacol. Exp. Ther. 1988,
3
43. Flack, H. D.; Bernardinelli, G. Acta Crystallogr., Sect. A 1999, 55, 908.
44. Farrugia, L. J. J. Appl. Crystallogr. 1997, 30, 565.
247, 911.
3
3
1. Ehlert, F. J.; Sawyer, G. W.; Esqueda, E. E. Life Sci. 1999, 64, 387.
2. Budriesi, R.; Cacciaguerra, S.; Di Toro, R.; Bolognesi, M. L.; Chiarini, A.; Minarini,
A.; Rosini, M.; Spampinato, S.; Tumiatti, V.; Melchiorre, C. Br. J. Pharmacol.
45. Dörje, F.; Wess, J.; Lambrecht, G.; Tacke, R.; Mutschler, E.; Brann, M. R. J.
Pharmacol. Exp. Ther. 1991, 256, 727.
46. Bradford, M. M. Anal. Biochem. 1976, 72, 248.
2
001, 132, 1009.
47. Munson, P. J.; Rodbard, D. Anal. Biochem. 1980, 107, 220.
48. van Rossum, J. M. Arch. Int. Pharmacodyn. Ther. 1963, 143, 299.
49. Furchgott, R. F.; Bursztyn, P. Ann. N.Y. Acad. Sci. 1967, 144, 882.
3
3
3. Haddad, E. B.; Trifilieff, A.; Landry, Y.; Gies, J. P. Pulm. Pharmacol. 1993, 6, 119.
4. Roffel, A. F.; Davids, J. H.; Elzinga, C. R. S.; Wolf, D.; Zaagsma, J.; Kilbinger, H. Br.
J. Pharmacol. 1997, 122, 133.