Synthesis and antagonistic activity at muscarinic receptor subtypes of some derivatives of diphenidol

Article abstract of DOI:10.1016/S0014-827X(03)00100-9

A series of new derivatives, related to diphenidol and to its 2-carbonyl analogue, were designed as antimuscarinic agents. The synthesized compounds were evaluated both as hydrochlorides and as methiodides by functional tests at guinea-pig heart (M₂), guinea-pig ileum (M₃) and rabbit vas deferens (putative M₄). Two derivatives (3a and 5a) showed an M₃-selective profile similar to that of the reference compounds, though they resulted less potent.

Full text of DOI:10.1016/S0014-827X(03)00100-9

Il Farmaco 58 (2003) 651Á657
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www.elsevier.com/locate/farmac
Synthesis and antagonistic activity at muscarinic receptor subtypes of
some derivatives of diphenidol
Lucilla Varoli a,*, Piero Angeli b, Michela Buccioni b, Silvia Burnelli a, Andrea Cavalli a,
Gabriella Marucci b, Maurizio Recanatini a
a Department of Pharmaceutical Sciences, University of Bologna, Via Belmeloro 6, I-40126 Bologna, Italy
b Department of Chemical Sciences, University of Camerino, Via S. Agostino 1, I-62032 Camerino (MC), Italy
Received 14 September 2001; accepted 18 March 2003
Dedicated to the memory of Prof. Piero Pratesi
Abstract
A series of new derivatives, related to diphenidol and to its 2-carbonyl analogue, were designed as antimuscarinic agents. The
synthesized compounds were evaluated both as hydrochlorides and as methiodides by functional tests at guinea-pig heart (M2),
guinea-pig ileum (M3) and rabbit vas deferens (putative M4). Two derivatives (3a and 5a) showed an M3-selective profile similar to
that of the reference compounds, though they resulted less potent.
´
# 2003 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
Keywords: Receptor; Muscarinic activity; Antagonists; SAR
1. Introduction
However, the unambiguous identification of muscari-
nic receptor M1ÁM5 subtypes mediating many impor-
/
Muscarinic receptors are present both in parasympa-
thetic and central nervous systems. In the periphery,
muscarinic receptors mediate smooth muscle contrac-
tion, glandular secretion, and modulation of cardiac rate
and force. In the central nervous system there is evidence
that muscarinic receptors are involved in motor control,
temperature and cardiovascular regulation, and mem-
ory. Interest in the classification of muscarinic receptors
involved in functions at different locations has been
heightened by the potential therapeutic application of
selective ligands. Currently, selective compounds may
emerge as therapeutics in several diseases: agonists may
be used to treat Alzheimer’s disease, nociceptive pain,
schizophrenia, while antagonists may be used for
Parkinson’s disease, urinary incontinence, irritable bo-
wel syndrome, gastric ulceration and chronic obstructive
tant responses has been impeded by the lack of any
selective agonist and the paucity of highly selective
antagonists.
As a part of our studies on diphenidol (1,1-diphenyl-
4-piperidin-1-yl-butan-1-ol, 1, Fig. 1) [5Á7], we synthe-
/
sized some analogues in which the intermediate chain
connecting the lipophilic head of the molecule to the
cationic center was modified, rendering it more polar or
less flexible [6]. The introduction of a carbonyl group in
position 2 of the butyl chain of diphenidol 1 led to
derivative 2a with enhanced affinity for both the M2 and
the M3 receptor subtypes and a better M3/M2 selectivity
ratio.
In order to check our hypothesis that the carbonyl
group on the butyl chain might reinforce the ligand-
receptor interaction, or affect the conformational beha-
vior of the molecule in such a way as the pharmaco-
phoric groups are exposed to the receptor in a favorable
spatial arrangement, we synthesized some derivatives
pulmonary disease [1Á4].
/
with an additional carbon atom in the alkyl chain (3Á7,
/
Fig. 1). In particular, we examined the effects of the
distance between the active functions (lipophilic moiety
* Corresponding author.
E-mail address: luvaroli@alma.unibo.it (L. Varoli).
´
0014-827X/03/$ - see front matter # 2003 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
doi:10.1016/S0014-827X(03)00100-9

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L. Varoli et al. / Il Farmaco 58 (2003) 651Á657
/
Fig. 1. Structure of diphenidol (1), 2-carbonyl analogue (2) and target compounds (3Á7).
/
and cationic head), and of the presence of the carbonyl
group at different positions in the pentyl chain. The
contribution to the affinity and/or selectivity of the
benzylic hydroxy group was also studied.
Considering that Lambrecht et al. [8] observed that
the methylation of the basic nitrogen atom of hexahy-
drodiphenidol and hexahydrosiladiphenidol affects dif-
ferently the affinity for the M2 and M3 receptor
subtypes, the designed compounds were prepared and
tested as both methiodide and hydrochloride salts.
In the present paper, we report on the synthesis and
the pharmacological evaluation by functional studies of
and 5a and methiodides 3b, 4b and 5b with a hydro-
chloric acid saturated solution in ether and methyl
iodide in acetone, respectively (Scheme 1). Reaction of
silyl enol ether 8 (Scheme 2), prepared from 1-piper-
idinobutan-3-one and chlorotrimethylsilane, with ben-
zophenone activated by titanium tetrachloride, afforded
a mixture of compounds 6 and 7, which were separated
by column chromatography and converted to the
hydrochlorides 6a and 7a and methiodide 6b, as
described previously. Methiodide 7b resulted unstable
and could not be isolated.
compounds 3a,bÁ
/
7a and we discuss their structureÁ
/
activity relationships.
3. Results and discussion
All the synthesized compounds were tested on M2
(guinea-pig heart), M3 (guinea-pig ileum) and putative
M4 (rabbit vas deferens) muscarinic receptor subtypes
for their antimuscarinic activity, using arecaidine pro-
2. Chemistry
The designed compounds 3a,bÁ
/
7a were synthesized
by standard procedures as reported in Schemes 1 and 2.
Treatment in dry conditions and under nitrogen of d-
piperidinovalerophenone with phenyl lithium, prepared
by addition of a stoichiometric amount of tert-butyl
lithium to bromobenzene, afforded the pentanol 3. This
compound was dehydrated with 37% hydrochloric acid
in glacial acetic acid giving the corresponding pentene
derivative 4. Oxidation of 4 with potassium permanga-
nate in diluted acetic acid and acetone led to the
hydroxy-pentanone 5. Compounds 3, 4 and 5 were
converted into the corresponding hydrochlorides 3a, 4a
pargyl ester (APE) at M2 and M3, and p-ClÁ
/
McNÁA-
/
343 at M4 receptor, as reference agonists. The antag-
onistic potency was expressed as dissociation constant
(pKb); the results are reported in Table 1, together with
the corresponding data at M2 and M3 receptor subtypes
of the diphenidol 1 and of the previously published
diphenidol analogue 2 [6]. Although many authors use
rabbit vas deferens for the putative muscarinic M1
receptors, recently Melchiorre and co-workers [9]
showed that a series of polymethylene tetra-amines are
able to significantly differentiate muscarinic M1 and M4

L. Varoli et al. / Il Farmaco 58 (2003) 651Á
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657
653
Scheme 1. (a) C6H5Br, t-BuLi, ether; (b) HCl, AcOH; (c) KMnO4, AcOH/H2O/acetone; (d) HCl/ether; (e) CH3I, acetone. *Directly obtained from
work up of the reaction mixture.
receptors. In fact, these authors report that affinity
values (pA2) obtained in functional studies on the
prostatic portion of rabbit vas deferens for some of
these tetra-amines, namely Dipitramine and AM172,
most closely correlate with the affinity values (pKi)
obtained at native and human recombinant muscarinic
M4 receptors, suggesting that the muscarinic receptor
mediating inhibition of neurogenic contractions of
rabbit vas deferens could belong to the M4 subtype.
For this reason in this paper we prefer to appoint the
term ‘putative M4 muscarinic receptor subtype’ to the
receptors active on rabbit vas deferens.
Examination of the pharmacological results reveals
that, generally, all the studied compounds present a
decrease in affinity with regard to the reference struc-
tural analogues 1a,b, 2a,b [6]. Considering the hydro-
chloride salts, the derivative 3a, bearing an additional
methylene group with respect to diphenidol (1a), shows
a five and twofold fall in the affinity for the M2 and M3
receptor subtypes, respectively, but a slightly improved
Scheme 2. (a) (CH3)3SiCl, NaI, (C2H5)3N, CH3CN; (b) (C6H5)2CO, TiCl4, CH2Cl2; (c) HCl/ether; (d) CH3I; acetone.

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L. Varoli et al. / Il Farmaco 58 (2003) 651Á657
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Table 1
Affinity values (pKb) and selectivity ratios for muscarinic antagonists 3a,bÁ
deferens (putative M4)
/
7a, at guinea-pig heart (force) (M2), guinea-pig ileum (M3), and rabbit vas
Selectivity ratios b
a
No.
pKb9
/
SEM
M2
M3
M4
M2/M3
M2/M4
M3/M4
1a c
1b c
2a c
2b c
3a
6.729
7.479
7.489
6.229
5.999
6.599
6.519
7.009
6.929
7.499
5.949
4.589
5.579
/
0.02
0.04
0.05
0.09
0.11
0.05
0.05
0.08
0.06
0.21
0.15
0.08
0.02
7.029
7.269
8.129
7.409
6.729
6.569
6.309
7.029
7.679
7.309
/
0.04
0.02
0.03
0.02
0.19
0.11
0.16
0.11
0.02
0.06
0.5
1.6
0.2
0.07
0.2
1
/
/
/
/
/
/
/
/
6.109
7.349
6.339
6.749
6.699
7.559
5.889
/
0.21
0.15
0.11
0.04
0.13
0.07
0.17
0.8
0.2
1.5
2
4
3b
4a
/
/
/
0.2
1
/
/
/
2
4b
/
/
/
1
2
5a
5b
/
/
/
0.2
1.5
2
9.5
0.6
/
/
/
0.9
1
6a
6b
/
B
B
/5.52
/
ꢀ
ꢀ
/3
ꢀ
ꢀ
/0.4
/
/5.52
B/5
/
0.1
0.4
ꢀ
/
0.4
2
/3
7a
/
5.979
/
0.25
5.299
/
0.12
5
a
Affinity constants calculated from the equation log(DR-1)ꢀ
/
log[ant]ꢁlog Kb for a single concentration of the antagonist, according to van
/
Rossum [10], are reported.
b
Antilog of the difference between the pKb values for M2, M3 and putative M4 muscarinic receptor subtypes.
Ref. [6].
c
M3 selectivity. The introduction of a double bond in
position 1 (4a) affects in a different way the affinity for
the M2 and M3 receptor subtypes, if compared with
compound 3a: it causes an about threefold increase of
affinity at M2 subtype and an equivalent decrease at the
M3 subtype. Compound 5a, the most potent M3
antagonist of the new series, compared with the lower
homologue 2a, shows a behaviour similar to that of
compound 3a, with a decreased affinity at both M2 and
M3 receptor subtypes, four and threefold, respectively.
The introduction of the carbonyl group in b-position
(7a) instead of a-position (5a) with regard to the
hydroxy group, leads to a more important fall in the
affinity at both M2 and M3 receptor subtypes (22- and
50-fold, respectively). Likewise, the introduction of a b-
carbonyl group is detrimental for the affinity also in the
case of the pentene derivative 6a when compared with
4a.
duction of a b-carbonyl group (6b) affords a decrease in
the affinity at both M2 and M3 receptor subtypes when
compared with 4b.
Compound 3b proves to be the only antagonist
showing a higher affinity (five times) at putative M4
respect to M2 and M3 receptors; moreover, this com-
pound and the a-carbonyl analogue 5a show the highest
selectivity ratios between M3 and putative M4 receptors
(0.2 and 9.5, respectively).
In conclusion, the results of the present study confirm
our hypothesis that the carbonyl group a to the benzylic
carbon is the most important feature of this class of
diphenidol derivatives. Furthermore, the polar benzylic
OH group seems to play a critical role for the affinity
and the selectivity at M3 receptor subtype, on condition
that it is correctly oriented [6]. The lack of the hydroxy
benzylic group influences the affinity at M2 and M3
receptor subtypes in such a way that it causes a loss in
selectivity.
With regard to the methiodide series, a similar trend is
evident for compound 3b when compared with dipheni-
dol methiodide 1b, with an eight and fivefold lower
affinity at M2 and M3 receptor subtypes, respectively.
Derivative 4b shows a little increment in the affinity at
both M2 and M3 subtypes with respect to the hydroxy
analogue 3b and a more relevant increment when
referred to the butene analogue (pKb values of 6.39
and 6.17 at M2 and M3 receptor subtypes, respectively)
[6]. In the case of compound 5b, the presence of an
additional methylene group compared with compound
2b displays an increased affinity only at M2 receptor
subtype, without any influence at M3 one: this causes a
complete loss in the selectivity. Finally, as for the
corresponding hydrochloride derivative 6a, the intro-
Finally, the changes in the length of the intermediate
chain connecting the active functions (lipophilic moiety
and cationic heads) and in the position of the carbonyl
group (a or b), and the presence of the double bond,
negatively influence the affinity of compounds 3Á7 at
/
M2 and M3 receptor subtypes.
4. Experimental
4.1. Chemistry
Melting points were taken on Electrothermal open
capillary apparatus and are uncorrected. Elemental

L. Varoli et al. / Il Farmaco 58 (2003) 651Á
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657
655
analysis was performed for compounds 3a,bÁ
results (not shown) were within 90.4% of the theoretical
values. Infrared spectra (IR) were recorded on a
PerkinÁElmer 683 instrument for all compounds and
were consistent with the assigned structures; because of
1
the lack of unusual features, they are not included. H
/
7a and the
hydrochloric acid saturated ethereal solution. A white
solid separated, which was collected and recrystallized
/
from CHCl3/ether, m.p.: 223Á
212 8C).
1H NMR (DMSO-d6): d 1.27 (1H, m, piperidine
proton), 1.74Á1.87 (7H, m, 5ꢃpiperidine protonsꢂ2-
CH2), 2.07 (2H, q, Jꢀ6 Hz, 3-CH2), 2.78 (2H, m,
piperidine protons), 2.91 (2H, t, 1-CH2), 3.30 (2H, m,
piperidine protons), 6.10 (1H, t, Jꢀ6 Hz, 4-CH), 7.12Á
7.44 (10H, m, ArH), 10.42 (1H, brs, NH exch. D2O). 13
NMR (DMSO-d6): d 21.33 (CÁpiperidine), 22.25 (2ꢃ
CÁpiperidine), 23.24 (C2), 26.43 (C3), 51.67 (2ꢃCÁ
piperidine), 55.10 (C1), 126.59 (2ꢃCÁAr), 126.87
(C4), 126.97 (C5), 127.53 (CÁAr), 127.99 (2ꢃ
128.23 (2ꢃCÁAr), 129.16 (2ꢃCÁAr), 139.06 (CÁ
141.54 (CÁAr), 141.69 (CÁAr).
/
225 8C (lit. [12] m.p.: 210Á
/
/
/
/
/
and 13C NMR spectra were registered on a Varian VXR
300 spectrometer, peak positions are given in parts per
million (d) relative to the standard chemical shift of the
/
/
/
solvent. Merck silica gel 60 (230Á/400 mesh) was used for
C
column chromatography. Thin-layer chromatography
(Merck silica gel 60 F254 analytical plates) was used to
monitor reactions. Sodium iodide was dried 24 h under
atmospheric pressure at 140 8C and stored under nitro-
gen. Triethylamine was refluxed over potassium hydrox-
ide pellets and then distilled. Methylene chloride was
/
/
/
/
/
/
/
/
/
CÁ/Ar),
/
/
/
/
/Ar),
/
/
˚
dried over molecular sieves 4 A. Anhydrous ether and
acetonitrile were purchased from Aldrich. The term
‘dried’ refers to the use of anhydrous sodium sulfate.
4.1.3. 1-Hydroxy-1,1-diphenyl-5-piperidin-1-yl-pentan-2-
one hydrochloride (5a)
To a stirred solution of 4 (0.305 g, 1 mmol) in acetone
(7 ml), water (1.5 ml) and glacial acetic acid (0.13 ml)
was added, at 0 8C, a solution of powdered potassium
permanganate (0.210 g, 1.3 mmol) in acetone containing
20% water (4.5 ml). The stirring was continued for 30
min at room temperature, the formed manganese
dioxide was filtered, the solution was concentrated,
4.1.1. 1,1-Diphenyl-5-piperidin-1-yl-pentan-1-ol
hydrochloride (3a)
A solution of t-BuLi (1.7 M in pentane) (11.8 ml) was
added (10 min) under nitrogen to a solution of
bromobenzene (1.57 g, 10 mmol) in anhydrous ether
(60 ml), dropwise and at a temperature of ꢁ
stirring at ꢁ15 8C for 1 h, 1-phenyl-5-piperidino-pen-
tan-1-one [11] (2.45 g, 10 mmol) in anhydrous ether (10
ml) was added dropwise, keeping the temperature at ꢁ
15 8C. The mixture was stirred for a further 4 h, then
allowed to warm to ꢂ10 8C, put into iced 2 N HCl (5
ml) and extracted with chloroform (3ꢃ50 ml). The
/
15 8C. After
/
made alkaline (NaOH) and extracted with ether (3ꢃ
/
20 ml). The combined organic layers were washed, dried
and evaporated. The residue was purified on a silica gel
column by flash chromatography eluting first with
chloroform and then with CHCl3/MeOH 95:5, giving 5
as pure oil (yield: 25%). Conversion to the HCl salt
/
/
/
combined organic layers were washed with brine, dried
and evaporated to give a solid product, which was
treated with activated carbon and recrystallized from
afforded a white solid, m.p.: 166Á
ether (lit. [13] m.p.: 176 8C).
1H NMR (DMSO-d6): d 1.35 (1H, m, piperidine
/
167 8C from CHCl3/
CHCl3/ether, m.p.: 194Á
1H NMR (DMSO-d6): d 1.18Á
piperidine protonꢂ3-CH2), 1.67Á1.73 (7H, m, 5ꢃ
piperidine protonsꢂ4-CH2), 2.25 (2H, t, 2-CH2), 2.75
(2H, m, piperidine protons), 2.89 (2H, t, 5-CH2), 3.37
(2H, m, piperidine protons), 5.55 (1H, s, OH exch.
/
195 8C (yield: 55%).
proton), 1.62Á
CH2), 2.78Á2.85 (6H, m, 2ꢃ
CH2ꢂ
(1H, s, OH exch. D2O), 7.27Á
(1H, brs, NH exch. D2O). 13C NMR (DMSO-d6): d
17.50 (C4), 21.19 (CÁpiperidine), 22.06 (2ꢃCÁpiper-
idine), 34.69 (C3), 51.56 (2ꢃCÁpiperidine), 54.81 (C5),
84.30 (C1), 126.87 (4ꢃCÁAr), 127.04 (2ꢃCÁAr),
127.53 (4ꢃCÁAr), 142.53 (2ꢃCÁAr), 210.41 (C2).
/
1.85 (7H, m, 5ꢃ
/
piperidine protonsꢂ
/
4-
/
1.41 (3H, m, 1ꢃ
/
/
/
piperidine protonsꢂ
/3-
/
/
/
/
5-CH2), 3.30 (2H, m, piperidine protons), 6.89
7.35 (10H, m, ArH), 10.42
/
/
/
/
/
D2O), 7.12Á
(4H, d, ArH), 10.15 (1H, brs, NH exch. D2O). 13C NMR
(DMSO-d6): d 20.75 (C3), 21.41 (CÁpiperidine), 22.14
(2ꢃCÁpiperidine), 23.16 (C4), 40.28 (C2), 51.61 (2ꢃ
CÁpiperidine), 55.45 (C5), 76.19 (C1), 125.56 (4ꢃCÁ
Ar), 125.83 (2ꢃCÁAr), 127.55 (4ꢃCÁAr), 148.10 (2ꢃ
CÁAr).
/7.16 (2H, m, ArH), 7.26 (4H, t, ArH), 7.43
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
4.1.4. 1-(3-Trimethylsilanyloxy-but-3-enyl)-piperidine
(8)
/
/
/
/
/
/
Sodium iodide (2.25 g, 15 mmol) in anhydrous
acetonitrile (15 ml) was added dropwise, under nitrogen,
at room temperature, to a solution of 4-piperidin-1-yl-
butan-2-one [14] (2.75 g, 10 mmol), triethylamine (1.52
g, 15 mmol) and trimethylchlorosilane (1.63 g, 15
mmol), successively introduced in the reaction flask.
An abundant white precipitate formed, while the
acetonitrile solution became yellow. The stirring was
maintained for 10 min, cold hexane (30 ml) and then ice-
4.1.2. 1-(5,5-Diphenyl-pent-4-eny1)-piperidine
hydrochloride (4a)
Compound 3 was treated with conc. hydrochloric acid
in glacial acetic acid, at reflux for 2 h, according to a
standard method. From the basified (NaOH) and
worked up reaction mixture, 4 was obtained in 33%
yield. A sample was solubilized in ether and treated with

656
L. Varoli et al. / Il Farmaco 58 (2003) 651Á657
/
water (30 ml) were added. After decantation, the
aqueous layer was rapidly extracted with cold hexane
3b: m.p. 185 8C from abs.ethanol. 1H NMR (DMSO-
d6): d 1.21 (2H, m, 3-CH2), 1.54 (2H, m, piperidine
(2ꢃ
/
30 ml) and the combined organic layers were
washed with ice-water, dried and evaporated. 1H
protons), 1.64Á
CH2), 2.27 (2H, m, 4-CH2), 2.92 (3H, s, NCH3), 3.19Á
3.23 (6H, m, 4ꢃpiperidine protonsꢂ1-CH2), 5.55 (1H,
s, OH exch. D2O), 7.15Á7.17 (2H, m, ArH), 7.27 (4H, t,
Jꢀ6 Hz, ArH), 7.42 (4H, d, Jꢀ6 Hz, ArH).
4b: m.p. 187Á
188 8C from abs.ethanol. 1H NMR
(DMSO-d6): d 1.53 (2H, m, piperidine protons), 1.75Á
1.86 (6H, m, 4ꢃpiperidine protonsꢂ2-CH2), 2.10 (2H,
q, Jꢀ7.2 Hz, 3-CH2), 2.98 (3H, s, NCH3), 3.24Á3.29
(6H, m, 4ꢃpiperidine protonsꢂ1-CH2), 6.14 (1H, t,
Jꢀ7.2 Hz, 4-CH), 7.14Á7.45 (10H, m, ArH).
5b: m.p. 173-174 8C from abs.ethanol. 1H NMR
(DMSO-d6): d 1.51 (2H, m, piperidine protons), 1.72Á
1.82 (6H, m, 4ꢃpiperidine protonsꢂ2ÁCH2), 2.82 (2H,
t, 3-CH2), 2.96 (3H, s, NCH3), 3.14Á3.27 (6H, m, 4ꢃ
piperidine protonsꢂ1-CH2), 6.85 (1H, s, OH exch.
D2O), 7.28Á7.37 (10H, m, ArH).
6b: m.p. 140Á
141 8C from abs.ethanol/ether. 1H
NMR (DMSO-d6): d 1.50 (2H, m, piperidine protons),
1.72Á1.75 (4H, m, piperidine protons), 2.90 (3H, s,
NCH3), 3.24Á3.27 (6H, m, 4ꢃpiperidine protonsꢂ1-
CH2), 3.49 (2H, t, 2-CH2), 6.84 (1H, s, 4-CH), 7.16 (2H,
m, ArH), 7.31 (2H, m, ArH), 7.40Á7.43 (6H, m, ArH).
/
1.74 (6H, m, 4ꢃ
/
piperidine protonsꢂ
/
2-
/
NMR spectroscopy of the crude product showed a
signal corresponding to enoxysilane (dꢀ
(CH3)3Si) while the signal corresponding to trimethyl-
chlorosilane was not detectable. The crude product was
immediately submitted to the next reaction without
further characterization to prevent hydrolysis.
/
/
/
0.19, s,
/
/
/
/
/
/
/
/
/
/
/
4.1.5. 1,1-Diphenyl-5-piperidin-1-yl-pent-1-en-3-one
hydrochloride (6a) and 1-hydroxy-1,1-diphenyl-5-
piperidin-1-yl-pentan-3-one hydrochloride (7a)
/
/
/
To a mixture of benzophenone (1.82 g, 10 mmol) and
titanium tetrachloride (1.90 g, 10 mmol) in dry CH2Cl2
(50 ml) was added dropwise a solution of 8 (2.04 g, 9
mmol) in dry CH2Cl2 (50 ml), carrying out the reaction
under nitrogen at room temperature. The reaction
mixture was stirred at room temperature for not more
than 10 min. Longer stirring gave only derivative 6.
After hydrolysis, the resulting mixture was purified on a
silica gel column by flash chromatography eluting first
with chloroform to separate compound 6 (yield 50%)
and then with CHCl3/MeOH 9:1 to obtain 7 (yield 15%)
as pure oils. Conversion to the HCl salts afforded 6a,
/
/
/
/
/
/
/
/
/
/
/
/
/
4.2. Pharmacology
m.p.: 174Á
175 8C from CHCl3/ether. 1H NMR (DMSO-
/
d6): d 1.34 (1H, m, piperidine proton), 1.73 (5H, m,
piperidine protons), 2.76 (2H, m, 4-CH2), 3.10 (4H, m,
piperidine protons), 3.24 (2H, m, 5-CH2), 6.83 (1H, s, 2-
4.2.1. General considerations
Male guinea pigs (200Á/300 g) and male New Zealand
white rabbits (3.0Á3.5 kg) were killed by cervical
/
CH), 7.14 (2H, m, ArH), 7.30Á
(1H, brs, NH exch. D2O). 13C NMR (DMSO-d6): d
21.26 (CÁpiperidine), 22.23 (2ꢃCÁpiperidine), 37.34
(C4), 50.40 (C5), 51.85 (2ꢃCÁpiperidine), 124.81 (C2),
128.00 (5ꢃCÁAr), 128.45 (2ꢃCÁAr), 128.93 (2ꢃCÁ
Ar), 129.54 (CÁAr), 138.45 (CÁAr), 140.12 (CÁAr),
152.79 (C1), 196.06 (C3).
7a: m.p.: 169Á
170 8C from CHCl3/ether. 1H NMR
(DMSO-d6): d 1.32 (1H, m, piperidine proton), 1.70
(5H, m, piperidine protons), 2.70 (2H, m, 4-CH2), 3.01Á
3.14 (6H, m, 4ꢃpiperidine protonsꢂ5-CH2), 3.54 (2H,
s, 2-CH2), 6.04 (1H, s, OH exch. D2O), 7.16Á7.32 (6H,
m, ArH), 7.44 (4H, d, ArH), 10.37 (1H, brs, NH exch.
D2O). 13C NMR (DMSO-d6): d 21.05 (CÁ
piperidine),
22.13 (2ꢃCÁpiperidine), 37.82 (C4), 49.74 (C5), 51.65
(2ꢃCÁpiperidine), 53.27 (C2), 75.66 (C1), 125.27 (4ꢃ
CÁAr), 126.12 (2ꢃCÁAr), 127.56 (4ꢃCÁAr), 146.96
(2ꢃCÁAr), 205.54 (C3).
/7.40 (8H, m, ArH), 10.47
dislocation. The organs required were set up rapidly
under 1 g of tension in 20 ml organ baths containing
physiological salt solution (PSS) maintained at an
appropriate temperature (see below) and aerated with
/
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5% CO2Á
/
95% O2. DoseÁresponse curves were con-
/
/
/
/
structed by cumulative addition of the reference agonist.
The concentration of agonist in the organ bath was
increased approximately threefold at each step, with
each addition being made only after the response to the
previous addition had attained a maximal level and
remained steady. Following 30 min of washing, tissues
were incubated with the antagonist for 1 h, and a new
dose-response curve to the agonist was obtained. Con-
tractions were recorded by means of a force displace-
ment transducer connected to the MacLab system
PowerLab/800. In addition parallel experiments in
which tissues did not receive any antagonist were run
in order to check any variation in sensitivity.
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4.1.6. General procedure for the synthesis of 1-(5-
hydroxy-5,5-diphenyl-pentyl)-1-methyl-piperidinium
4.2.2. Guinea-pig ileum
Two-centimeter-long portions of terminal ileum were
taken at about 5 cm from the ileum-cecum junction. The
tissue was cleaned and the ileum longitudinal muscle
was separated from the underlying circular muscle, and
mounted in PSS, at 37 8C, of the following composition
iodide (3b) and derivatives 4bÁ
Methiodide derivatives were prepared according to
the general procedure previously described [5] in 40Á
60% yields.
/
6b
/

L. Varoli et al. / Il Farmaco 58 (2003) 651Á
/
657
657
(mM): NaCl (118), NaHCO3 (23.8), KCl (4.7),
MgSO4.7H2O (1.18), KH2PO4 (1.18), CaCl2 (2.52),
and glucose (11.7). Tension changes were recorded
isotonically. Tissues were equilibrated for 30 min, and
dose-response curves to arecaidine propargyl ester
(APE) were obtained at 30-min intervals, the first one
being discarded and the second one being taken as the
control.
Acknowledgements
This work was supported by a grant from MIUR
(Ministero dell’Istruzione, dell’Universita` e della Ricer-
ca).
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4.2.6. Statistical analysis
¨
Values are given as mean9standard error of four or
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/
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/