Structure-activity relationships of methoctramine-related polyamines as muscular nicotinic receptor noncompetitive antagonists. 3. Effect of inserting the tetraamine backbone into a macrocyclic structure

Article abstract of DOI:10.1021/jm020835f

The present article expands on the study of another aspect of structure-activity relationships of the polymethylene tetraamines, namely, the effect of inserting the tetraamine backbone into a macrocyclic structure. To this end, compounds 8-12 were designed by linking the two terminal nitrogen atoms of prototype methoctramine 2 to an aryl moiety. Alternatively, 2 was first modified to achieve compounds 6 and 7, which in turn were cyclized by linking the two terminal primary amine functions to a polyphenyl spacer, affording 13-20. All the compounds were tested on muscle-type nAChRs and most of them as well on AChE. Furthermore, selected compounds were tested also on peripheral M₂ and M₃ mAChRs. All these cyclic derivatives, like prototypes, were potent noncompetitive antagonists at both frog and Torpedo nAChRs, suggesting that polyamines do not need to be linear or in extended conformation to optimally interact with the nicotinic channel; rather, they may bind in a U-shaped conformation. Relative to muscarinic activity, macrocyclic compounds 10, 13, 14, and 20, in contrast with the profile displayed by 2, were almost devoid of affinity. It is derived that an aryl spacer is detrimental to the interaction of polyamines with mAChRs. Finally, all the diamine diamides investigated in this study were much less potent in inhibiting AChE activity than prototype 3, suggesting that a macrocyclic structure may not be suitable for AChE inhibition.

Full text of DOI:10.1021/jm020835f

3286
J . Med. Chem. 2002, 45, 3286-3295
Str u ctu r e-Activity Rela tion sh ip s of Meth octr a m in e-Rela ted P olya m in es a s
Mu scu la r Nicotin ic Recep tor Non com p etitive An ta gon ists. 3.1 Effect of In ser tin g
th e Tetr a a m in e Ba ck bon e in to a Ma cr ocyclic Str u ctu r e
Maria L. Bolognesi,† M. Gabriele Bixel,‡ Gabriella Marucci,§ Manuela Bartolini,† Michael Krauss,‡ Piero Angeli,§
Alessandra Antonello,† Michela Rosini,† Vincenzo Tumiatti,† Ferdinand Hucho,‡ and Carlo Melchiorre*,†
Department of Pharmaceutical Sciences, University of Bologna, Via Belmeloro 6, 40126 Bologna, Italy, Institut fu¨r
Chemie-Biochemie, Freie Universita¨t Berlin, Thielallee 63, 14195 Berlin, Germany, and Department of Chemical Sciences,
University of Camerino, Via S. Agostino 1, 62032 Camerino (MC), Italy
Received J anuary 29, 2002
The present article expands on the study of another aspect of structure-activity relationships
of the polymethylene tetraamines, namely, the effect of inserting the tetraamine backbone
into a macrocyclic structure. To this end, compounds 8-12 were designed by linking the two
terminal nitrogen atoms of prototype methoctramine 2 to an aryl moiety. Alternatively, 2 was
first modified to achieve compounds 6 and 7, which in turn were cyclized by linking the two
terminal primary amine functions to a polyphenyl spacer, affording 13-20. All the compounds
were tested on muscle-type nAChRs and most of them as well on AChE. Furthermore, selected
compounds were tested also on peripheral M2 and M3 mAChRs. All these cyclic derivatives,
like prototypes, were potent noncompetitive antagonists at both frog and Torpedo nAChRs,
suggesting that polyamines do not need to be linear or in extended conformation to optimally
interact with the nicotinic channel; rather, they may bind in a U-shaped conformation. Relative
to muscarinic activity, macrocyclic compounds 10, 13, 14, and 20, in contrast with the profile
displayed by 2, were almost devoid of affinity. It is derived that an aryl spacer is detrimental
to the interaction of polyamines with mAChRs. Finally, all the diamine diamides investigated
in this study were much less potent in inhibiting AChE activity than prototype 3, suggesting
that a macrocyclic structure may not be suitable for AChE inhibition.
In tr od u ction
We have demonstrated that polymethylene tet-
raamines are a versatile tool for the characterization
of different receptor systems.2-6 For example, benex-
tramine7 (1, Figure 1), the prototype tetraamine disul-
fide for irreversible antagonism of R1-adrenoreceptors,
was shown to antagonize several receptors, including
nicotinic receptors (nAChRs),8 muscarinic receptors
(mAChRs),2,5 neuropeptide Y receptors,9 and acetylcho-
linesterase (AChE).10 The pharmacological profile of 1
was taken as a starting point to develop the universal
template approach to the design of polyamines as
selective ligands for different biological targets.4,6,10
Structural modifications performed on the structure of
1 led to the discovery of methoctramine11 (2, Figure 1),
a prototype tetraamine for antagonism of mAChRs, and
of caproctamine (3, Table 1),10 a prototype diamine
diamide for noncompetitive inhibition of AChE. More
recently, we have modified the structure of 2 to produce
polyamines that have high affinity and selectivity for
muscle-type nAChRs.8,12 Following these structural
modifications, a most intriguing finding was that the
affinity of polyamines for muscle-type nAChRs is de-
pendent on the type and length of the spacer between
the nitrogen atoms and on the substituents on the
terminal amine functions as well. The higher homologue
F igu r e 1. Chemical structure of benextramine (1, X ) S),
methoctramine [2, X ) (CH2)2], and homologue 4 [X ) (CH2)4].
4 (Figure 1) of 2 was significantly more potent at the
frog rectus nAChR than 2, while retaining, however,
most of the affinity of 2 for M2 and M3 mAChRs.8 The
replacement of the flexible 1,12-diaminododecane unit
of 4 with a (4′′-aminomethyl-[1,1′;4′,1′′]terphenyl-4-yl)-
methylamine fragment led to 5 (Table 1), which dis-
played most of the affinity of 4 for nAChRs but lost
almost completely the affinity for both M2 and M3
mAChRs. Thus, it was demonstrated that flexibility of
the spacer between the inner nitrogen atoms of tet-
raamines is an important determinant of potency with
respect to both nAChRs and mAChRs. Consequently,
the selectivity for muscle-type nAChRs relative to
mAChRs could be achieved by replacing a flexible spacer
with a rigid one. Following this observation, we ad-
vanced that the inner nitrogen atoms of 5 are unlikely
to be less than 15 Å apart because, owing to the rigidity
of the terphenyl moiety, the only possibility to alter the
distance between the two inner amine functions is
restricted to the rotation along the axis of the two bonds
between the inner nitrogen atoms and the 4′′,4-carbon
atoms. It follows that this distance may be important
for the interaction with two anionic sites of the channel
* To whom correspondence should be addressed. Phone: +39-051-
2099706. Fax: +39-051-2099734. E-mail: camelch@kaiser.alma.unibo.it.
† University of Bologna.
‡ Freie Universita¨t Berlin.
§ University of Camerino.
10.1021/jm020835f CCC: $22.00 © 2002 American Chemical Society
Published on Web 06/14/2002

Tetraamine Backbone in Macrocyclic Structure
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 15 3287
Ta ble 1. Antagonist Affinities, Expressed as pIC50, Kapp, or pKB Values, at Nicotinic Acetylcholine Receptors (nAChRs) of the Isolated
Frog Rectus Abdominis Muscle (FRA) and Torpedo and at Muscarinic Acetylcholine Receptors (mAChRs) of Isolated Guinea Pig Left
Atrium (M2) and Longitudinal Ileum (M3), Respectively, and Inhibitory Activity, Expressed as pIC50 Values, on Acetylcholinesterase
(AChE) from Human Erythrocytes of the Compounds Studied
mAChR
nAChR
pKapp (Torpedo)
AChE
pIC50
compda
n
X
R
pKB (M2)
pKB (M3)
pIC50 (FRA)
2
1
1
5
CH2
CO
H
CH3
H
7.91 ( 0.03b
6.39 ( 0.23c
7.35 ( 0.09b
6.14 ( 0.06b
5.55 ( 0.12c
5.98 ( 0.07b
5.27 ( 0.03c
5.93 ( 0.03b
6.21 ( 0.10b
4.99 ( 0.02
5.66 ( 0.05b
6.35 ( 0.06b
6.46 ( 0.02
6.40 ( 0.01
6.43 ( 0.06
6.35 ( 0.01
6.21 ( 0.01
5.10 ( 0.01
3
6.77 ( 0.01c
4
CH2
7.02 ( 0.04b
6.30 ( 0.02b
6.80 ( 0.01
6.48 ( 0.01
5.46 ( 0.03
6.35 ( 0.01
6.64 ( 0.01
6.28 ( 0.01
5.37 ( 0.02
6.77 ( 0.03
6.23 ( 0.02
6.62 ( 0.02
5
<5d
<5d
6
CO
4.48 ( 0.01
4.27 ( 0.08
7
CH2
CH2
CH2
8
1
2
9
10
<5d
<5d
11
2
CO
12
<4e
13
1
2
CO
H
H
<5d
<5d
<5d
<5d
5.85 ( 0.03
5.47 ( 0.04
5.21 ( 0.02
4.80 ( 0.03
3.98 ( 0.04
6.47 ( 0.01
6.64 ( 0.03
6.70 ( 0.01
6.03 ( 0.01
6.01 ( 0.02
6.01 ( 0.01
5.48 ( 0.02
6.26 ( 0.01
5.70 ( 0.12b
14
CO
15
CO
16
3
2
1
2
CO
H
17
CO
CH3
H
6.15 ( 0.01
6.49 ( 0.02
5.92 ( 0.03
6.72 ( 0.01
6.05 ( 0.09b
18
CH2
CH2
CH2
19
H
20
<5
<5
TPMP+ f
a
b
1, 4, 5, 7, 8, 12, 18-20, tetrahydrochlorides; 3, 11, 13-17, dihydrochlorides; 6, dioxalate; 9, 10, tetraoxalates. Data from ref 12.
c Data from ref 10. Not active up to a concentration of 5 µM. e Not able to displace bound ethidium up to a concentration of 100 µM.
d
f Triphenylmethylphosphonium bromide.
located most likely at a distance comparable with that
between the inner nitrogen atoms of 5.
end, we designed macrocyclic polyamines 8-20. Com-
pounds 8-12 were obtained by linking the two terminal
nitrogen atoms of 2 to an aryl moiety. Alternatively, to
link to each other the two terminal nitrogen atoms to
an octamethylene spacer and the two inner nitrogen
atoms to an aryl moiety, prototype 2 was first modified
to achieve compounds 6 and 7, which in turn were
cyclized by linking the two terminal primary amine
functions to an aryl spacer, affording 13-20. The design
strategy for our compounds is shown in Figure 2.
All of the compounds synthesized in this study were
tested on muscle-type nAChRs, and most of them were
tested on AChE as well. Furthermore, selected com-
pounds were tested also on peripheral M2 and M3
mAChRs.
The present article expands on the study of another
aspect of structure-activity relationships of prototype
2, namely, the effect of inserting the tetraamine back-
bone into a macrocyclic structure. The rationale for this
structural modification stands on the need to gain
information on the active conformation of polyamines
in the interaction with muscle-type nAChRs. Photolabile
compounds have been used to gain insight into the
mode of interaction of polymethylene tetraamines with
nAChRs. As a most interesting finding, we observed
that a radioactive photolabile compound, bearing two
identical azido groups on the terminal N-aryl-substi-
tuted nitrogen atoms, photolabeled nAChR R-subunits,
suggesting that each of the two nAChRs R-subunits
interacts with one of the two terminal N-aryl groups of
this compound, resulting in a U-shaped conformation
when bound to the lumen of the receptor ion channel.12
Thus, a cyclic structure should give an answer as to
whether a tetraamine interacts with the receptor in an
extended or in a folded (U-shaped) conformation. To this
Ch em istr y
All the newly synthesized compounds were character-
ized by IR, 1H NMR, mass spectra, and elemental
analysis.
The synthesis of diamine diamide 6 was accomplished
using our previously established procedures, as shown

3288 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 15
Bolognesi et al.
F igu r e 2. Design strategy for the synthesis of macrocyclic polyamines of the present investigation. First, methoctramine (2,
structure I, R1 ) R2 ) H, X ) CH2) has been modified to achieve structure II by moving the two methoxybenzyl groups from the
terminal to the inner nitrogen atoms (a). Second, the outer nitrogen atoms of structures I and II have been linked by a suitable
spacer to give structures III (b) and IV (c), respectively.
Sch em e 1a
a
Boc ) Me3COCO-.
in Scheme 1. Thus, N-(tert-butoxycarbonyl)-6-aminoca-
proic acid was amidated with diamine 21, synthesized
in turn from 1,8-diaminooctane and 2-methoxybenzal-
dehyde to give 22. Removal of the N-tert-butoxycarbonyl
groups of 22 was achieved by acidic hydrolysis in CF3-
COOH.
Reduction of the carbonyl functions of diamine dia-
mide 6 to obtain tetraamine 7 was next attempted.
Unfortunately, this transformation, using different
reducing agents, led to a complex mixture and to
significant amounts of decomposition products. Thus,
another synthetic pathway to achieve 7 was then
followed (Scheme 2). Alkylation of 21 with 24, obtained
in two steps starting from 6-amino-1-hexanol, afforded
25. Removal of the protecting groups of 25 by hydrolysis
with 6 N HCl gave 7.
All the polyazamacrocycles listed in Table 1 were
obtained in good yields (37-65%), following a one-step
synthetic method that includes a first [1 + 1] condensa-
tion of the terminal primary amino groups of polyamines
with the corresponding dialdehydes, followed by hydro-
genation of the intermediate Schiff bases with NaBH4.

Tetraamine Backbone in Macrocyclic Structure
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 15 3289
Sch em e 2a
nAChRs were determined by using the fluorescent
noncompetitive inhibitor ethidium in a displacement
assay.23
Methoctramine (2), caproctamine (3), analogues 4 and
5, and triphenylmethylphosphonium bromide (TPMP+)
were used as controls, and their pIC50, pKapp, and pKB
values were within the error of previous determinations.
Resu lts a n d Discu ssion
The newly synthesized compounds 6-20 were as-
sayed on nAChRs of isolated frog rectus abdominis
muscle and Torpedo. Furthermore, selected compounds
were evaluated also for their muscarinic M2 and M3
antagonist potency (10, 13, 14, 20) and for their inhibi-
tion of AChE from human erythrocytes (6, 11, 13-17).
Polyamines 2-5 and TPMP+ were used as standards
for a comparison. The results are summarized in Table
1.
a
Boc ) Me3COCO-.
Dropwise slow addition of 1 equiv of dialdehydes 28-
3013,14 to a dilute solution containing 1 equiv of polyamine
2715 in the presence of NaBH4 afforded intermediate
macrocycles 31, 12, and 32, which were subsequently
converted to the desired macrocycles 8-10 through
reductive amination of the secondary amine functions
with 2-methoxybenzaldehyde (Scheme 3).
It was observed that over the concentration range
investigated all of the compounds were noncompetitive
antagonists of muscle-type nAChRs. The maximum
response to carbachol was reduced, and the magnitude
of this reduction was dependent on the concentration
of antagonist. In all cases, washing the muscle in drug-
free saline reversed the antagonism. To determine the
binding affinities of the various polyamine derivatives
at nAChRs, we have used the well-characterized lumi-
nal noncompetitive inhibitor ethidium in a fluorescent
displacement assay.23 Ethidium, when bound to the
high-affinity site for noncompetitive antagonists of the
nAChR in its desensitized state, shows an intensive
emission maximum at 590 nm upon excitation at 480
nm.23,24 Since bound ethidium is displaceable by well-
characterized luminal noncompetitive antagonists, ethid-
ium can be used as a reference fluorophor to charac-
terize new ligands of this binding site. It turned out that
all of the polyamines investigated in this study, with
the exception of compound 12, compete with bound
ethidium, indicating that these compounds also overlap
with the high-affinity binding site for noncompetitive
antagonists. This observation is in agreement with the
noncompetitive mechanism of action observed at the
frog rectus muscle nAChR.
Similarly, cyclization of diamine diamide 2615 with
29 provided the required precursor 33, which was
alkylated with 2-methoxybenzyl chloride16 to give the
macrocycle 11, as illustrated in Scheme 4.
Under the same cyclization conditions, reaction of
polyamines 6 and 7 with the different dialdehydes 28-
30 or 3417 provided the final compounds 13-16 (Scheme
5) and 18-20 (Scheme 6). Alkylation of 14 with form-
aldehyde and formic acid afforded the N,N′-dimethyl
macrocycle 17 (Scheme 5). While compounds 28-30 are
soluble in EtOH, p-terphenyl-4,4′′-dicarboxyaldehyde 34
is highly insoluble, and the use of a different solvent
rather than an alcohol then became necessary, affecting
the outcome of cyclization, as indicated by the low yield
of 16 (22%), which was synthesized using CHCl3 as
cosolvent.
Biology
The effects of compounds 10, 13, 14, and 20 on M2
mAChRs were determined using guinea pig left atria
electrically stimulated at 1 Hz.18,19 The guinea pig ileum
longitudinal muscle was used to study their effects on
M3 mAChRs.18,19 In both cases the agonist was arecai-
dine propargyl ester (APE). The biological data are
expressed as the negative logarithm of the apparent
dissociation constants (pKB).20
The inhibitory activity against AChE of compounds
6, 11, and 13-17 was studied using the method of
Ellman et al.21 The inhibitory activity was expressed
as pIC50 values that represent the concentration of
inhibitor required to decrease enzyme activity by 50%.
The effects of compounds 6-15 and 17-20 on muscle-
type nAChRs were studied using the frog rectus abdo-
minis muscle and carbachol-induced contractions as the
measured parameter.22 The results are expressed as
pIC50 values, i.e., the negative logarithm of the concen-
trations required to inhibit the maximal response to
carbachol by 50%.
Interestingly, analysis of the results reported in Table
1 reveals that the pIC50 values obtained at frog rectus
nAChRs are comparable with pKapp values calculated
at Torpedo nAChRs because the difference was within
(0.5 log units with the exception of 4, 8, 11, and 12,
whose pIC50 and pKapp values differed to a larger extent.
However, the observed discrepancy between pIC50 and
pKapp values is not surprising because it was already
noticed with noncyclic polyamines. We argued that the
different potencies displayed by some compounds at the
frog rectus relative to the Torpedo nAChR might be the
result of subtle differences in the mode of interaction
of polyamines with the two receptors, owing to struc-
tural differences in their ion channel, or simply of a
different bioavailability of the compounds at the recep-
tor site.12
Taking 2 and 3 as reference compounds, it can be
observed how their potency at nAChRs, mAChRs, and
AChE can be modified by introducing modifications into
their structure.
Apparent binding affinities (pKapp) of compounds
6-20 at the noncompetitive binding site of Torpedo
Moving the two 2-methoxybenzyl groups from the two
terminal nitrogen atoms to the inner nitrogen atoms of

3290 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 15
Bolognesi et al.
Sch em e 3
2 afforded polyamines 6 and 7. These compounds were
more potent than prototype 2 at both frog and Torpedo
nAChRs while being equipotent to each other at Torpedo
nAChRs, and diamine diamide 6 was more potent than
the corresponding tetraamine 7 at frog nAChRs.
for nAChRs that was comparable to that of open
prototypes 6 and 7. N-Methylation of the two amide
functions did not improve the affinity for nAChRs
because 14 was more potent than the corresponding
N,N′-dimethyl analogue 17 at both frog and Torpedo
nAChRs. A di-p-tolylmethane spacer, as in 15 and 20,
appears to confer optimum interaction with the nAChR,
while a longer spacer, as in 16, caused a significant
decrease in affinity at Torpedo nAChRs.
Cyclization of the N,N′-dimethyl analogue of proto-
type 2 or of prototype 3 by linking the two terminal
amine functions to an aryl spacer gave macrocyclic
polyamines 8-11. Again, these compounds displayed
affinity for nAChRs that was higher or comparable to
that exhibited by prototypes 2 and 3. However, in this
case, diamine diamide 11 was not more potent than the
corresponding tetraamine 9. This finding suggests that
the two sets of compounds, that is, 8-11 and 13-20,
Cyclization of 6 and 7 by linking together the two
terminal amine functions to an aryl spacer gave 13-
20. All these cyclic compounds were potent noncompeti-
tive antagonists at both frog and Torpedo nAChRs,
suggesting that polyamines do not need to be linear or
in extended conformation to optimally interact with the
nAChR; rather, they bind in a U-shaped conformation
as hypothesized earlier.12 Analysis of the results reveals
that diamine diamides 13-15 were more potent than
or as active as the corresponding tetraamines 18-20
at nAChRs, in line with the results observed for diamine
diamide 6 and the corresponding tetraamine 7. Inter-
estingly, these cyclic compounds displayed an affinity

Tetraamine Backbone in Macrocyclic Structure
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 15 3291
Sch em e 4
Sch em e 5
may interact differently with the nAChR. Compound 10,
bearing a di-p-tolylmethane spacer, was more potent
than both 8 and 9 at frog nAChRs, in line with the
results observed for 15 and 20 relative to 13, 14, 18,
and 19. However, 10 was slightly less potent than 8 and
9 at Torpedo nAChR. The markedly lower activity of
12 relative to the corresponding 2-methoxybenzyl-
bearing analogue 9 suggests clearly that the 2-meth-
oxybenzyl group had a relevant role in receptor binding
mechanism.
Prototype 2 was a potent and selective M2 mAChR
antagonist relative to M3 mAChRs, muscle-type nAChRs,
and AChE. In contrast, the higher homologue 4 had high
affinity for frog nAChRs that was only slightly lower
than the affinity for M2 mAChRs, whereas analogue 5
displayed significant antagonism at nAChRs while being
almost devoid of affinity, at least up to 5 mM concentra-
tion, for M2 and M3 mAChRs. It is derived that an aryl
spacer is detrimental to the interaction of polyamines
with mAChRs. This observation was confirmed by the
results obtained with cyclic compounds 10, 13, 14, and
20 that were, like 5, devoid of affinity, up to 5 µM
concentration, for M2 and M3 mAChRs.
Prototype 3, which bears two amide functions, dis-
played higher affinity for AChE relative to mAChRs and
Torpedo nAChRs. For this reason, we tested at AChE
all diamine diamides synthesized in the present study.
It turned out that all diamine diamides investigated,
unlike 3, were much less potent in inhibiting AChE
activity than muscle-type nAChRs, suggesting that a
cyclic structure may not be suitable for AChE inhibition.
Exp er im en ta l Section
Ch em istr y. Melting points were taken in glass capillary
tubes on a Bu¨chi SMP-20 apparatus and are uncorrected. IR,
MALDI-TOF-MS, and 1H NMR spectra were recorded on
Perkin-Elmer 297, Bruker Biflex III, and Varian VXR 300
instruments, respectively. Chemical shifts are reported in
parts per million (ppm) relative to tetramethylsilane (TMS),
and spin multiplicities are given as s (singlet), br s (broad
singlet), d (doublet), t (triplet), or m (multiplet). Although the
IR spectra data are not included (because of the lack of unusual
features), they were obtained for all compounds reported and
were consistent with the assigned structures. The elemental
compositions of the compounds agreed to within (0.4% of the
calculated value. When the elemental analysis is not included,
crude compounds were used in the next step without further
purification. Chromatographic separations were performed on
silica gel columns by flash (Kieselgel 40, 0.040-0.063 mm;
Merck) or gravity column (Kieselgel 60, 0.063-0.200 mm;
Merck) chromatography. Compounds were named following
IUPAC rules as applied by Beilstein-Institut AutoNom (version
In conclusion, we have demonstrated that inserting
the tetraamine or the diamine diamide backbone of 2
and 3, respectively, into a cyclic structure did not
negatively affect affinity for muscle-type nAChRs be-
cause the newly synthesized polyamines were more
potent than or as active as the prototypes, confirming
our previous observation that a tetraamine is unlikely
to interact with the receptor in an extended conforma-
tion. It is derived that the active conformation of
polyamines may be a folded one.

3292 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 15
Bolognesi et al.
1.60-1.83 (m, 4), 3.01-3.32 (m, 8), 3.79-3.82 (m, 6), 4.44-
4.59 (m, 4), 4.70 (br s, 2, exchangeable with D2O), 6.79-6.99
(m, 6), 7.09-7.28 (m, 2).
Sch em e 6
6-Am in oh exa n oic Acid {8-[(6-a m in oh exa n oyl)(2-m eth -
oxyben zyl)a m in o]octyl}(2-m eth oxyben zyl)a m id e Diox-
a la te (6). A solution of 22 (5.73 g, 7.03 mmol) in TFA (40 mL)
was stirred at room temperature for 12 h. The solvent was
evaporated, yielding a solid that was dissolved in water (100
mL). The obtained solution was then washed with ether (3 ×
30 mL), made basic with NaOH pellets, and extracted with
CHCl3 (3 × 30 mL). The organic phase was evaporated to give
6 (95% yield) as the free base that was transformed into the
dioxalate salt as a white solid: mp 54-56 °C (EtOH/Et2O);
1H NMR (free base, CDCl3) δ 1.25-1.78 (m, 24 + 4 exchange-
able with D2O), 2.25-2.39 (m, 4), 2.64-2.75 (m, 4), 3.28-3.36
(m, 4), 3.83 (s, 6), 4.47-4.61 (m, 4), 6.83-7.28 (m, 8). Anal.
(C40H62N4O12) C, H, N.
6-Ch lor oh exyla m in e Hyd r och lor id e (23). A solution of
6-amino-1-hexanol (15.5 g, 0.13 mol) and SOCl2 (43 mL, 0.59
mol) in toluene (100 mL) was refluxed for 1 h. Removal of the
solvent under reduced pressure afforded crude 23 in quantita-
tive yield as a hygroscopic solid: 1H NMR (CDCl3) δ 1.44-
1.54 (m, 4), 1.74-1.83 (m, 4), 3.01-3.04 (m, 2), 3.55 (t, 2), 8.31
(br s, 3, exchangeable with D2O).
(6-Ch lor oh exyl)ca r ba m ic Acid ter t-Bu tyl Ester (24). To
a solution of 23 (10.3 g, 59.85 mmol) in 30 mL of water was
added Et3N (6.6 mL, 89.8 mmol), and the resulting mixture
was stirred at room temperature for 15 min. After dilution
with THF (60 mL) di-tert-butyl dicarbonate (14.54 g, 66.6
mmol) was added portionwise and the reaction mixture was
stirred for further 12 h. Separation between the two phases
is favored by addition of EtOAc (100 mL) and water (50 mL).
The dried organic phase is evaporated to dryness to give a
residue that was purified by gravity chromatography. Eluting
with petroleum ether/ethyl acetate (8.25:1.75) afforded 24:
1
57% yield; H NMR (CDCl3) δ 1.31-1.54 (m, 15), 1.75-1.83
(m, 2), 3.12-3.14 (m, 2), 3.55 (t, 2), 4.52 (br s, 1, exchangeable
with D2O).
{6-[{8-[(6-ter t-Bu toxyca r bon yla m in oh exyl)(2-m eth oxy-
b en zyl)a m in o]oct yl}(2-m et h oxyb en zyl)a m in o]h exyl}-
ca r ba m ic Acid ter t-Bu tyl Ester (25). A mixture of 24 (4.9
g, 20.8 mmol), 21 (2 g, 5.2 mmol), K2CO3 (2.87 g, 20.8 mmol),
and KI (1.41 g, 8.5 mmol) in absolute ethanol (100 mL) was
refluxed for 78 h. Removal of the solvent gave a residue that
was purified by flash chromatography. Eluting with chloroform/
petroleum ether/ethanol/aqueous 30% ammonia (6:3.4:0.6:0.01)
afforded 25 as transparent oil: 13% yield; 1H NMR (CDCl3) δ
1.23-1.50 (m, 46), 2.46 (t, 8), 3.05-3.10 (m, 4), 3.64 (s, 4), 3.82
(s, 6), 4.53 (br s, 2, exchangeable with D2O), 6.82-6.98 (m, 4),
7.23 (t, 2), 7.42 (d, 2).
N,N-Bis(6-a m in oh exyl)-N,N-b is(2-m et h oxyb en zyl)oc-
ta n e-1,8-d ia m in e Tetr a h yd r och lor id e (7). A solution of 25
(200 mg, 0.26 mmol) in 6 N HCl (20 mL) was stirred at room
temperature for 2 h. The reaction mixture was then washed
with ether (3 × 20 mL), made basic with NaOH pellets, and
extracted with chloroform (3 × 20 mL). The organic phase was
evaporated to give 7 (90% yield) as the free base that was
transformed into the tetrahydrochloride salt as a hygroscopic
solid: 1H NMR (free base, CDCl3) δ 1.26-1.70 (m, 28 + 4
exchangeable with D2O), 2.44 (t, 8), 2.70 (t, 4), 3.59 (s, 4), 3.83
(s, 6), 6.83-6.98 (m, 4), 7.18-7.22 (m, 2), 7.41-7.45 (m, 2).
Anal. (C36H66Cl4N4O2) C, H, N.
10,19-Dim e t h y l-3,10,19,26-t e t r a a za b ic yc lo [26.2.2]-
d otr ia con ta -1(31),28(32),29-tr ien e (31). A solution of 28
(45.6 mg, 0.34 mmol) in 100 mL of dry ethanol was added
dropwise to a mixture of 2715 (130 mg, 0.34 mmol) and
molecular sieves (3 Å) over a period of 72 h at room temper-
ature. NaBH4 (29 mg, 0.70 mmol) was then added, and the
stirring was continued for further 12 h. Following removal of
molecular sieves, the solution was made acidic with 6 N HCl
(2 mL). Removal of the solvent gave a residue, which was
dissolved in water (40 mL). The solution was washed with
ether (3 × 20 mL) to remove nonbasic materials and then was
made basic with 5 N NaOH and finally extracted with
2.1), a PC integrated software package for systematic names
in organic chemistry with the exception of compound 16, which
was named by applying ACD/NAME (Advanced Chemistry
Development, Inc.).
N,N′-Bis(2-m eth oxyben zyl)octa n e-1,8-d ia m in e Dih y-
d r och lor id e (21). A solution of 1,8-diaminooctane (3.6 g, 25.0
mmol) and 2-methoxybenzaldehyde (7.49 g, 55.0 mmol) in
toluene (100 mL) was refluxed, and the water formed was
continuously removed for 3 h. The cooled mixture was filtered
and the filtrate evaporated to give the corresponding Schiff
base that was dissolved in EtOH (100 mL) and treated with
NaBH4 (2.08 g, 55.0 mmol). The mixture was stirred at room
temperature for 12 h, cautiously acidified with 6 N HCl, made
basic with 2 N NaOH, and finally extracted with chloroform
(3 × 50 mL). Removal of dried (Na2SO4) solvents afforded, in
quantitative yield, 21 as the free base that was converted into
the dihydrochloride salt: mp 200-205 °C (from EtOH/Et2O);
1H NMR (free base, CDCl3) δ 1.28-1.49 (m, 12), 1.75 (br s, 2,
exchangeable with D2O), 2.58 (t, 4), 3.78 (s, 4), 3.84 (s, 6), 6.84-
6.95 (m, 4), 7.20-7.26 (m, 4).
{5-[{8-[(6-ter t-Bu toxyca r bon yla m in oh exa n oyl)(2-m eth -
oxyb en zyl)a m in o]oct yl}(2-m et h oxyb en zyl)ca r b a m oyl]-
p en tyl}ca r ba m ic Acid ter t-Bu tyl Ester (22). Ethyl chlo-
rocarbonate (2.63 mL, 27.5 mmol) in dry dioxane (5 mL) was
added dropwise to a stirred and cooled (5 °C) solution of N-(tert-
butoxycarbonyl)-6-aminocaproic acid (6.35 g, 27.5 mmol) and
triethylamine (3.83 mL, 27.5 mmol) in dioxane (120 mL),
followed after standing for 30 min by the addition of 21 (5.28
g, 13.7 mmol) in dioxane (30 mL). After being stirred at room
temperature overnight, the mixture was evaporated, affording
a
residue that was purified by gravity chromatography.
Eluting with petroleum ether/ethyl acetate/ethanol (5:5:0.1)
afforded 22: 55% yield; 1H NMR (CDCl3) δ 1.19-1.49 (m, 42),

Tetraamine Backbone in Macrocyclic Structure
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 15 3293
chloroform (3 × 20 mL). Removal of the solvent gave a residue
that was purified by chromatography. Eluting with methylene
chloride/methanol/aqueous 30% ammonia (9:1:0.1) afforded
31: 60% yield; 1H NMR (CDCl3) δ 1.09-1.66 (m, 28 + 2
exchangeable with D2O), 2.32 (s, 6), 2.41-2.48 (m, 8), 2.64 (t,
4), 3.82 (s, 4), 7.33 (s, 4).
1.55 (m, 24), 1.75 (br s, 2, exchangeable with D2O), 2.17-2.23
(m, 4), 2.50-2.57 (m, 4), 2.86 (s, 6), 3.04-3.16 (m, 4), 3.82-
3.83 (m, 4), 7.33-7.38 (m, 4), 7.52-7.57 (m, 4).
7,30-Bis(2-m et h oxyb en zyl)-14,23-d im et h yl-7,14,23,30-
tetr a a za tr icyclo[30.2.2.20,0]octa tr ia con ta -1(35),2(37),3,5-
(38),32(36),33-h exa en e-13,24-d ion e Dih yd r och lor id e (11).
A mixture of 30 (110 mg, 0.19 mmol), 2-methoxybenzyl
chloride16 (119.5 mg, 0.76 mmol), and N,N-diisopropylethy-
lamine (0.13 mL, 0.76 mmol) in MeOH (5 mL) was refluxed
for 24 h and then stirred at room temperature for further 48
h. Removal of the solvent gave a residue that was purified by
chromatography. Eluting with methylene chloride/petroleum
ether/ethanol/aqueous 30% ammonia (4.5:5:0.5:0.05) afforded
11 (41% yield) as the free base that was transformed into the
dihydrochloride: mp 200-205 °C (EtOH/ether); 1H NMR (free
base, CDCl3) δ 1.20-1.56 (m, 24), 2.15-2.27 (m, 4), 2.30-2.45
(m, 4), 2.82-2.86 (m, 6), 3.14-3.29 (m, 4), 3.55-3.57 (m, 4),
3,26-Bis(2-m et h oxyb en zyl)-10,19-d im et h yl-3,10,19,26-
tetr aazabicyclo[26.2.2]dotr iacon ta-1(31),28(32)-tr ien e Tet-
r a h yd r och lor id e (8). A mixture of 31 (130 mg, 0.27 mmol),
NaCNBH3 (34 mg, 0.54 mmol), and CH3COOH (15.4 mL) in
absolute ethanol (10 mL) was stirred for 10 min at room
temperature, and then 2-methoxybenzaldehyde (150 mg, 1.1
mmol) was added and the stirring was continued overnight.
Removal of the solvent gave a residue that was purified by
flash chromatography. Eluting with methylene chloride/etha-
nol/aqueous 30% ammonia (9.5:0.5:0.1) afforded 8 (31% yield)
as the free base that was transformed into the tetrahydro-
chloride salt: mp 74-76 °C (EtOH/Et2O); 1H NMR (free base,
CDCl3) δ 1.28-1.51 (m, 28), 2.22 (s, 6), 2.26-2.47 (m, 12), 3.57
(s, 4), 3.62 (s, 4), 3.81 (s, 6), 6.83-6.99 (m, 4), 7.16-7.32 (m,
6), 7.54-7.57 (m, 2). Anal. (C46H76Cl4N4O2) C, H, N.
3.66-3.68 (m, 4), 3.80 (s, 6), 6.83-7.58 (m, 16). Anal. (C52H74
Cl2N4O4) C, H, N.
-
10,19-Bis-(2-m eth oxyben zyl)-3,10,19,26-tetr aazabicyclo-
[26.2.2]d otr ia con ta -1(31),28(32),29-tr ien e-9,20-d ion e Di-
h yd r och lor id e (13). 13 was obtained from 6 (480 mg, 0.88
mmol) and 28 (118 mg, 0.88 mmol) following the procedure
described for 31 and purified by flash chromatography. Eluting
with methylene chloride/petroleum ether/ethanol/aqueous 30%
ammonia (8:1:1:0.1) gave 13 as the free base that was
converted into the dihydrochloride salt: 38% yield; mp 185-
187 °C (EtOH/Et2O); 1H NMR (free base, CDCl3,) δ 1.23-1.45
(m, 24 + 2 exchangeable D2O), 2.36-2.40 (m, 8), 2.57 (t, 4),
3.55 (t, 4), 3.77 (s, 4), 3.79 (s, 6), 6.81-6.93 (m, 4), 7.16-7.27
(m, 6), 7.39-7.41 (m, 2). MALDI-MS calcd for C44H65N4O4
713.49 (M + H)+, found 713.51. Anal. (C44H66Cl2N4O4) C, H,
N.
14,23-Dim eth yl-7,14,23,30-tetr a a za tr icyclo[30.2.2.20,0]-
octa tr ia con ta -1(35),2,4,32(36),33,37-h exa en e Tetr a h yd r o-
ch lor id e (12). 12 was obtained from 2715 (130 mg, 0.34 mmol)
and 2913 (71.5 mg, 0.34 mmol) following the procedure de-
scribed for 31 and purified by flash chromatography. Eluting
with methylene chloride/ethanol/aqueous 30% ammonia (9:1:
0.1) gave 12 as the free base that was converted into the
tetrahydrochloride salt: 64% yield; mp 212-215 °C (EtOH/
1
Et2O); H NMR (free base, CDCl3) δ 1.28-1.50 (m, 28), 1.91
(br s, 2, exchangeable with D2O), 2.21-2.33 (m, 14), 2.58 (t,
4), 3.87 (s, 4), 7.39 (d, 4), 7.60 (d, 4). Anal. (C36H64Cl4N4) C, H,
N.
7,30-Bis(2-m et h oxyb en zyl)-14,23-d im et h yl-7,14,23,30-
tetr a a za tr icyclo[30.2.2.20,0]octa tr ia con ta -1(35),2,4,32(36),-
33,37-h exa en e Tetr a oxa la te (9). 9 was obtained from 12 (90
mg, 0.16 mmol) and 2-methoxybenzaldehyde (98 mg, 0.72
mmol) following the procedure described for 8 and purified by
flash chromatography. Eluting with methylene chloride/etha-
nol/aqueous 30% ammonia (9:1:0.1) gave 9 as the free base
that was converted into the tetraoxalate salt: 48% yield; mp
105 °C; 1H NMR (free base, CDCl3) δ 1.16-1.50 (m, 28), 2.16-
2.33 (m, 14), 2.43 (t, 4), 3.55 (s, 4), 3.68 (s, 4), 3.82 (s, 6), 6.83-
6.87 (m. 2), 6.96 (t, 2), 7.21 (t, 2), 7.40-7.59 (m, 10). Anal.
(C60H84N4O18) C, H, N.
14,23-Bis(2-m eth oxyben zyl)-7,14,23,30-tetr aazatr icyclo-
[30.2.2.20,0]oct a t r ia con t a -1(35),2,4,32(36),33,37-h exa en e-
13,24-d ion e Dih yd r och lor id e (14). 14 was obtained from 6
(210 mg, 0.34 mmol) and 2913 (71.5 mg, 0.34 mmol) following
the procedure described for 31 and purified by flash chroma-
tography. Eluting with methylene chloride/petroleum ether/
ethanol/aqueous 30% ammonia (8:1:1:0.1) gave 14 as the free
base that was converted into the dihydrochloride salt: 56%
yield; mp 280-285 °C (EtOH/Et2O); 1H NMR (free base, CDCl3)
δ 1.04-1.57 (m, 24 + 2 exchangeable D2O), 2.24-2.31 (m, 4),
2.39-2.69 (m, 4), 3.22-3.30 (m, 4), 3.79-3.84 (m, 10), 4.43-
4.57 (m, 4), 6.83-7.56 (m, 16). MALDI-MS calcd for C50H69N4O4
789.52 (M + H)+, found 789.62. Anal. (C50H70Cl2N4O4) C, H,
N.
15,24-Dim eth yl-8,15,24,31-tetr a a za tr icyclo[31.2.2.20,0]-
n on a tr ia con ta -1(36),3,5,33(37),34,38-h exa en e (32). 32 was
obtained from 2715 (130 mg, 0.34 mmol) and 3014(76.3 mg, 0.34
mmol) following the procedure described for 31 and purified
by flash chromatography. Eluting with methylene chloride/
methanol/aqueous 30% ammonia (9:1:0.1) gave 32 as the free
15,24-Bis(2-m eth oxyben zyl)-8,15,24,31-tetr aazatr icyclo-
[31.2.2.20,0]n on a tr ia con ta -1(36),3,5,33(37),34,38-h exa en e-
14,25-d ion e Dih yd r och lor id e (15). 15 was obtained from 6
(230 mg, 0.375 mmol) and 3014 (84 mg, 0.375 mmol) following
the procedure described for 31 and purified by flash chroma-
tography. Eluting with methylene chloride/petroleum ether/
ethanol/aqueous 30% ammonia (8:1:1:0.08) gave 15 as the free
base that was converted into the dihydrochloride salt: 49%
1
base: 57% yield; H NMR (CDCl3) δ 1.24-1.47 (m, 28), 2.01
(br s, 2, exchangeable with D2O), 2.23 (s, 6), 2.32-2.38 (m, 8),
2.59 (t, 4), 3.75 (s, 4), 3.93 (s, 2), 7.11-7.14 (m, 4), 7.22-7.25
(m, 4).
8,31-Bis(2-m et h oxyb en zyl)-15,24-d im et h yl-8,15,24,31-
t et r a a za t r icyclo[31.2.2.20,0]n on a t r ia con t a -1(36),3,5,33-
(37),34,38-h exa en e Tetr a oxa la te (10). 10 was obtained from
32 (110 mg, 0.20 mmol) and 2-methoxybenzaldehyde (106 mg,
0.78 mmol) following the procedure described for 8 and purified
by flash chromatography. Eluting with methylene chloride/
ethanol/aqueous 30% ammonia (9.4:0.6:0.05) gave 10 as the
free base that was converted into the tetraoxalate salt: 50%
1
yield; mp 121 °C (EtOH/Et2O); H NMR (free base, CDCl3) δ
1.23-1.58 (m, 24), 2.10 (br s, 2, exchangeable with D2O), 2.22-
2.40 (m, 4), 2.55-2.68 (m, 4), 3.21 (t, 2), 3.32 (t, 2), 3.76-3.82
(m, 10), 3.95 (s, 2), 4.45 (s, 2), 4.59 (s, 2), 6.84-7.27 (m, 16).
MALDI-MS calcd for C51H71N4O4 803.54 (M + H)+, found
803.59. Anal. (C51H72Cl2N4O4) C, H, N.
18,27-Di(2-m etossifen il)-11,18,27,34-tetr a a za tetr a cyclo-
[34.2.2.22,5.26,9]t et r a t et r a con t a -1(38),2,4,6,8,36,39,41,43-
n on a en e-17,28-d ion e Dih yd r och lor id e (16). 16 was ob-
tained from 6 (171 mg, 0.28 mmol) and 3417 (80 mg, 0.28 mmol)
following the procedure described for 31 and purified by flash
chromatography. In this case, the aldehyde was dissolved in
150 mL of CHCl3. Eluting with methylene chloride/petroleum
ether/ethanol/aqueous 30% ammonia (8:1:1:0.05) gave 16 as
the free base that was converted into the dihydrochloride
salt: 22% yield; mp 185-187 °C (EtOH/Et2O); 1H NMR
(CDCl3, free base) δ 1.26-1.46 (m, 24), 2.01 (br s, 2, exchange-
able with D2O), 2.29-2.33 (m, 4), 2.49-2.63 (m, 4), 3.08-3.31
1
yield; mp 110 °C (EtOH/Et2O); H NMR (free base, CDCl3) δ
1.29-1.93 (m, 28), 2.23 (s, 6), 2.26-2.47 (m, 12), 3.59 (s, 4),
3.63 (s, 4), 3.82 (s, 6), 3.96 (s, 2), 6.84-7.00 (m, 4), 7.12-7.33
(m, 10), 7.53-7.57 (m, 2). Anal. (C61H86N4O18) C, H, N.
14,23-Dim et h yl-7,14,23,30-t et r a za t r icyclo[30.2.2.20,0]-
oct a t r ia con t a -1(35),2,4,32(36),33,37-h e xa e n e -13,24-d i-
on e (33). 33 was obtained from 2615 (140 mg, 0.35 mmol) and
2913 (72 mg, 0.34 mmol) following the procedure described for
31 and purified by flash chromatography. Eluting with me-
thylene chloride/petroleum ether/ethanol/aqueous 30% am-
monia (8:1:1:0.1) gave 33: 55% yield; 1H NMR (CDCl3) δ 1.20-

3294 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 15
Bolognesi et al.
(m, 4), 3.78-3.84 (m, 10), 4.42-4.58 (m, 4), 6.86-7.60 (m, 20).
ileo-caecal junction. The tissue was cleaned, and the ileum
longitudinal muscle was separated from the underlying cir-
cular muscle and set up at 37 °C in organ baths containing
PSS of the following composition (mM): NaCl, 118; KCl, 4.7;
CaCl2, 2.52; MgSO4‚7H2O, 1.18; KH2PO4, 1.18; NaHCO3, 23.8;
glucose, 11.7. Tension changes were recorded isotonically.
Tissues were allowed to equilibrate for at least 30 min, during
which time the bathing solution was changed every 10 min.
Concentration-response curves to arecaidine propargyl ester
(APE) (0.01-0.5 µM) were obtained at 30 min intervals, the
first one being discarded and the second one taken as the
control. Following incubation with the antagonist for 60 min,
Anal. (C56H74Cl2N4O4) C, H, N.
14,23-Bis(2-m et h oxyb en zyl)-7,30-d im et h yl-7,14,23,30-
tetr a a za tr icyclo[30.2.2.20,0]octa tr ia con ta -1(35),2,4,32(36),
33,37-h exa en e-13,24-d ion e Dih yd r och lor id e (17). Formic
acid (96%, 1.4 mL, 37 mmol) was added dropwise to 14 (200
mg, 0.25 mmol), and then 40% formaldehyde (1.2 mL, 17.4
mmol) was added to the resulting mixture, which was heated
at 100 °C for 18 h, cooled (5 °C), made basic with 40% aqueous
NaOH, and extracted with CHCl3 (3 × 30 mL). Removal of
dried solvents gave a residue that was purified by gravity
chromatography. Eluting with methylene chloride/petroleum
ether/ethanol/aqueous 30% ammonia (8:1:1:0.1) gave 17 as the
free base that was converted into the dihydrochloride salt: 30%
yield; mp 205-215 °C (EtOH/Et2O); 1H NMR (free base, CDCl3)
δ 1.12-1.32 (m, 12), 1.32-1.56 (m, 12), 2.16-2.23 (m, 14),
2.40-2.42 (m, 4), 3.14-3.30 (m, 4), 3.55 (s, 4), 3.82 (s, 6), 6.85-
7.59 (m, 16). Anal. (C52H74Cl2N4O4) C, H, N.
10,19-Bis(2-m eth oxyben zyl)-3,10,19,26-tetr a a za bicyclo-
[26.2.2]d ot r ia con t a -1(31),28(32),29-t r ien e Tet r a h yd r o-
ch lor id e (18). 18 was obtained from 7 (200 mg, 0.34 mmol)
and 28 (46.6 mg, 0.34 mmol) following the procedure described
for 31 and purified by gravity chromatography. Eluting with
methanol/aqueous 30% ammonia (10:0.1) gave 18 as the free
base that was converted into the tetrahydrochloride salt: 37%
yield; mp 80-84 °C (EtOH/Et2O); 1H NMR (free base, CDCl3)
δ 1.23-1.45 (m, 28), 1.75 (br s, 2, exchangeable with D2O),
2.36-2.42 (m, 8), 2.55-2.60 (m, 4), 3.55 (s, 4), 3.77 (s, 4), 3.80
(s, 6), 6.81-6.93 (m, 4), 7.16-7.27 (m, 6), 7.40 (d, 2). Anal.
(C44H72Cl4N4O2) C, H, N.
a
new concentration-response curve to the agonist was
obtained.
Gu in ea P ig Left Atr ia . The guinea pig heart was rapidly
removed, and right and left atria were separated out. The left
atria were mounted at 30 °C in PSS of the same composition
used for the ileum. Tissues were stimulated through platinum
electrodes by square-wave pulses (1 ms, 1 Hz, 5-10 V) (Tetra
Stimulus, N. Zagnoni). Inotropic activity was recorded iso-
metrically. Tissues were equilibrated for 2 h, and cumulative
concentration-response curves to APE (0.01-1 µM) were
constructed. Following incubation with the antagonist for 60
min, a new concentration-response curve to APE was ob-
tained.
F r og Rectu s Abd om in is Mu scle. The rectus abdominis
muscle of frogs was set up at room temperature in Clark frog
Ringer solution of the following composition (mM): NaCl, 111;
KCl, 1.88; CaCl2, 1.08; NaH2PO4, 0.08; NaHCO3, 2.38; glucose,
11.1. The tissues were equilibrated for 60 min. Two cumulative
concentration-response curves to carbachol (1-100 µM) were
constructed at 1 h intervals, the first one being discarded and
the second one taken as the control. Following incubation with
the antagonist for 60 min, a new concentration-response curve
to the agonist was obtained.
F lu or escen ce Titr a tion . nAChR-rich membranes were
prepared from frozen Torpedo californica electric organ as
described earlier.25 All fluorescence spectra were recorded
using an Aminco Bowman spectrometer series 2 (Rochester).
For fluorescence titration experiments, aliquots of competing
ligand were added stepwise to a solution containing nAChR-
rich membranes (1 µM receptor concentration), ethidium (7
µM), and carbachol (1 mM) in 50 mM NaPi, pH 7.4. Ethidium
fluorescence was measured by employing an excitation wave-
length of 480 nm (slit widths 4 nm/4 nm) while monitoring
the emission from 540 to 740 nm.
In h ibition of Acetylch olin ester a se. AChE (0.5 UI/mg)
derived from human erythrocytes was purchased from Sigma
Chemical (Italy). Buffer components and other chemicals were
of the highest purity commercially available. The method of
Ellman et al. was followed.21 A 0.037 M acetylthiocoline iodide
solution was prepared in water, 0.01 M 5,5′-dithiobis(2-
nitrobenzoic acid) (DTNB, Ellman’s reagent) was dissolved in
pH 7.0 phosphate buffer, and 0.15% (w/v) NaHCO3 was added.
AChE solution was prepared by dissolving 20 units in 5 mL
of 0.2% aqueous gelatine with sonication at 35 °C. A 1:1
dilution with water was performed before use to get the
enzyme activity between 0.13 and 0.100 AU/min. Stock solu-
tions of the test compounds (1 mM) were prepared in water,
as well as caproctamine (3) reference stock solution. The assay
solutions were prepared by diluting the stock solutions in
water. Five different concentrations of each compound were
used in order to obtain inhibition of AChE activity between
20% and 80%. The assay solution consisted of a 0.1 M
phosphate buffer, pH 8.0, with the addition of 340 µM DTNB,
0.035 UI/mL AChE, and 550 µM acetylthiocoline iodide. The
final assay volume was 1 mL. Test compounds were added to
the assay solution and preincubated with the enzyme for 20
min, the addition of substrate following. Initial rate assays
were performed at 37 °C with a J asco Uvidec-610 double-beam
spectrophotometer; the rate of increase in the absorbance at
412 nm was followed for 5 min. Assays were done with a blank
containing all components except AChE in order to account
for nonenzymatic reaction. The reaction rates were compared,
14,23-Bis(2-m eth oxyben zyl)-7,14,23,30-tetr aazatr icyclo-
[30.2.2.20,0]octatr iacon ta-1(35),2,4,32(36),33,37-h exaen e Tet-
r a h yd r och lor id e (19). 19 was obtained from 7 (260 mg, 0.44
mmol) and 2913 (93 mg, 0.44 mmol) following the procedure
described for 31 and purified by flash chromatography. Eluting
with methylene chloride/petroleum ether/ethanol/aqueous 30%
ammonia (8:1:1:0.1) gave 19 as the free base that was
converted into the tetrahydrochloride salt: 40% yield; mp 214-
218 °C (EtOH/Et2O); 1H NMR (free base, CDCl3) δ 1.18-1.50
(m, 28), 1.86 (br s, 2, exchangeable with D2O), 2.37 (t, 8), 2.52
(t, 4), 3.54 (s, 4), 3.78 (s, 6), 3.83 (s, 4), 6.82 (d, 2), 6.90 (t, 2),
7.18 (t, 2), 7.35-7.38 (m, 6), 7.57 (d, 4). MALDI-MS calcd for
C
50H73N4O2 761.51 (M + H)+, found 761.57. Anal. (C50H76
Cl4N4O2) C, H, N.
-
15,24-Bis(2-m eth oxyben zyl)-8,15,24,31-tetr aazatr icyclo-
[31.2.2.20,0]n on a tr ia con ta -1(36),3,5,33(37),34,38-h exa en e
Tetr a h yd r och lor id e (20). 20 was obtained from 7 (200 mg,
0.34 mmol) and 3014 (76 mg, 0.34 mmol) following the
procedure described for 31 and purified by flash chromatog-
raphy. Eluting with methanol/aqueous 30% ammonia (10:0.1)
gave 20 as the free base that was converted into the tetrahy-
drochloride salt as a hygroscopic solid: 38% yield; 1H NMR
(free base, CDCl3) δ 1.25-1.52 (m, 28), 1.72 (br s, 2, exchange-
able with D2O), 2.24 (t, 8), 2.59 (t, 4), 3.58 (s, 4), 3.77 (s, 4),
3.82 (s, 6), 3.97 (s, 2), 6.82-6.96 (m, 4), 7.13-7.27 (m, 10),
7.41-7.44 (m, 2). Anal. (C51H78Cl4N4O2) C, H, N.
Biology. F u n ction a l An ta gon ism . Male guinea pigs
(200-300 g) and frogs (10-20 g) were sacrificed by cervical
dislocation. The organs required were set up rapidly under 1
g of tension in 20 mL organ baths containing physiological salt
solution (PSS) kept at an appropriate temperature (see below)
and aerated with 5% CO2/95% O2. Concentration-response
curves were constructed by cumulative addition of the refer-
ence agonist. The concentration of agonist in the organ bath
was increased approximately 3-fold at each step, with each
addition being made only after the response to the previous
addition had attained a maximal level and remained steady.
Contractions were recorded by means of a force displacement
transducer connected to a MacLab system PowerLab/800. In
all cases, parallel experiments in which tissues did not receive
any antagonist were run in order to check for variations in
sensitivity.
Gu in ea P ig Ileu m Lon gitu d in a l Mu scle. The 2 cm long
portions of terminal ileum were taken at about 5 cm from the

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Ack n ow led gm en t. Grants from the University of
Bologna, the European Community (Grant BMH4-
CT97-2395), MIUR, CNR (Grant 99.01100.CT03), the
Deutsche Forschungsgemeinschaft (Grant DFG, Sfb
449), and the Fonds der Chemischen Industrie sup-
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