Acetylcholinesterase noncovalent inhibitors based on a polyamine backbone for potential use against Alzheimer's disease [2]

Full text of DOI:10.1021/jm9810452

4186
J . Med. Chem. 1998, 41, 4186-4189
Sch em e 1
Acetylch olin ester a se Non cova len t
In h ibitor s Ba sed on a P olya m in e
Ba ck bon e for P oten tia l Use a ga in st
Alzh eim er ’s Disea se
Carlo Melchiorre,* Vincenza Andrisano,
Maria L. Bolognesi, Roberta Budriesi,
Andrea Cavalli, Vanni Cavrini, Michela Rosini,
Vincenzo Tumiatti, and Maurizio Recanatini
in affected brain regions, while inhibition of muscarinic
M2 autoreceptors would have an additive effect by facili-
tating the release of ACh in the synapse. Furthermore,
inhibition of the peripheral binding site would prevent
the aggregation of âA induced by AChE. The starting
point was the observation that benextramine, a tetra-
amine disulfide developed as an irreversible R-adreno-
receptor antagonist,7 displayed also a significant affinity
for cardiac muscarinic M2 receptors and potentiated the
effect of ACh on the frog rectus muscle as well.8,9 To
verify whether the latter finding might result from an
inhibition of AChE activity, we tested benextramine on
human erythrocyte AChE. Since benextramine turned
out to be a reversible inhibitor of AChE, we thought that
it might represent a new lead for the design of ligands
displaying affinity for AChE and muscarinic M2 recep-
tors thus fulfilling our research goal.
The role of the 2-methoxybenzyl group of 1 in AChE
inhibition was verified by investigating the correspond-
ing unsubstituted tetraamine disulfide 2. In addition,
the contribution, if any, of the disulfide bridge on affinity
was studied by assaying the carbon analogue 4. Since
4 was as active as benextramine, it was apparent that
the disulfide bridge is not essential for AChE inhibition.
Next, our attention was focused on the chain length
separating the two inner nitrogens of tetraamine 4.
Although it turned out that optimum inhibition of AChE
activity is associated with a seven-carbon chain, we
chose tetraamine 6 (methoctramine) as a lead since it
displayed optimum affinity for muscarinic M2 recep-
tors.10 Since we had already demonstrated that a
diamine diamide backbone can retain significant affinity
for muscarinic M2 receptors,11 diamine diamides 7-9
were designed such as to improve lipophilicity versus
methoctramine, while retaining or hopefully improving
affinity for AChE.
Department of Pharmaceutical Sciences, University of
Bologna, Via Belmeloro 6, 40126 Bologna, Italy
Received J une 15, 1998
Dementia is the most common psychiatric disorder
in elderly patients, and Alzheimer’s disease1 (AD) is the
most common cause. Much effort is devoted to elucidat-
ing the relationships among the hallmarks of the
disease: amyloid plaques, neurofibrillary tangles, and
degeneration of the basal forebrain cholinergic neurons.2
Despite the evidence that there is a profound and
consistent loss of cholinergic transmission in AD, phar-
macotherapy and practical medicine are still waiting for
the possible advantages arising from enhancing the
cholinergic system in some way.3 To date, only some
inhibitors of acetylcholinesterase (AChE), such as tacrine
and donepezil, resulted to be useful drugs in alleviating
the symptoms of AD.4 On the other hand, the existence
of multiple muscarinic receptor subtypes in the central
nervous system (CNS) has stimulated the search for
new drugs which target only one receptor while not
affecting others. It has been advanced that drugs which
antagonize selectively presynaptic muscarinic M2 au-
toreceptors may also be useful in AD as they will
facilitate ACh release.5
AChE is the enzyme involved in the hydrolysis of the
neurotransmitter acetylcholine (ACh) at cholinergic
synapses in the central and peripheral nervous systems.
Inhibitors of AChE activity promote an increase in the
concentration and the duration of action of synaptic ACh
thus causing an enhancement of the cholinergic trans-
mission through the activation of the synaptic nicotinic
and muscarinic receptors. It has been demonstrated
that AChE could play a key role during an early step
in the development of the senile plaques, as revealed
by the finding that AChE accelerates â-amyloid peptide
(âA) deposition.6 This peculiar feature of AChE was not
affected by active site inhibitors, such as edrophonium,
but it was affected by peripheral anionic binding site
ligands, such as decamethonium and propidium. In-
terestingly enough, butyrylcholinesterase (BChE), an
enzyme that lacks the peripheral anionic binding site,
did not affect amyloid formation. This finding suggests
clearly that the catalytic site of AChE does not partici-
pate in the interaction of the enzyme with âA, whereas
it is possible that the peripheral binding site of AChE
may be involved in amyloid formation.6
To ascertain the possibility for the polyamines under
study to bind at both the active and the peripheral
AChE binding sites, a theoretical investigation was
undertaken of the docking of these inhibitors to the en-
zyme. Molecular dynamics simulations were performed
from which it resulted that the diamine diamide skel-
eton has enough conformational flexibility to allow the
outer amine functions to contact both sites simulta-
neously.
Ch em istr y. Tetraamines 1-6 and diamine diamide
7, when not available in our laboratory, were synthe-
sized as previously described.7,10,11 Diamine diamides
8 and 9 were synthesized by standard procedure (Scheme
1
The aim of this study was to produce novel ligands
based on a polyamine backbone having affinity for both
AChE active and peripheral binding sites and for mus-
carinic M2 receptors as well. Inhibition of AChE activity
would potentiate the remaining cholinergic transmission
1) and were characterized by H NMR and elemental
analysis.
The 2-methoxybenzyl group on the terminal nitrogens
was easily introduced by condensation of diamine diam-
ide 1012 with 2-methoxybenzaldehyde and reduction of
10.1021/jm9810452 CCC: $15.00 © 1998 American Chemical Society
Published on Web 10/02/1998

Communications to the Editor
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 22 4187
Ta ble 1. Inhibition of AChE and BChE Activities by Polyamines
a
b
pIC50
pA2
no.
R1
R2
R3
X
Y
AChE
BChE
M1
M2
M3
1
2
3
4
5
6
7
8
9
2-MeOC6H5CH2
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
Me
H2 S-S
H2 S-S
H2 CH2
H2 (CH2)2 5.19 ( 0.01
H2 (CH2)3 5.35 ( 0.05
H2 (CH2)4 5.27 ( 0.03
5.14 ( 0.02
3.30 ( 0.03
5.14 ( 0.03
5.21 ( 0.03
3.19 ( 0.02
5.06 ( 0.02
5.86 ( 0.01
5.43 ( 0.02
6.01 ( 0.02
4.94 ( 0.03
5.22 ( 0.02
4.93 ( 0.04
6.44 ( 0.01
7.64 ( 0.02
nd
nd
nd
nd
nd
ndc
nd
nd
nd
2-MeOC6H5CH2
2-MeOC6H5CH2
2-MeOC6H5CH2
2-MeOC6H5CH2
2-MeOC6H5CH2
2-MeOC6H5CH2
6.67 ( 0.07
6.98 ( 0.12
7.64 ( 0.09
7.92 ( 0.08
6.30 ( 0.10
6.67 ( 0.06
6.39 ( 0.23d
nd
5.89 ( 0.09
5.76 ( 0.11
5.92 ( 0.14
6.06 ( 0.07
5.35 ( 0.14
5.21 ( 0.12
5.55 ( 0.12
nd
6.85 ( 0.12
nd
nd
5.66 ( 0.02
nd
nd
O
O
O
(CH2)4 5.73 ( 0.03
(CH2)4 6.51 ( 0.02
(CH2)4 6.77 ( 0.01
6.66 ( 0.02
2-MeOC6H5CH2 Me
tacrine
physostigmine
7.85 ( 0.03
nd
nd
a
AChE and BChE were from human erythrocytes. pIC50 values [-log IC50 (µM)] represent the concentration of inhibitor required to
b
decrease enzyme activity by 50% and are the mean of two independent measurements. pA2 values ((SE, n ) 5) were calculated according
to Van Rossum21 at 1 µM (M2 receptors) or 10 µM (M1 and M3 receptors) concentration. c nd, not determined. The experiments were
d
performed in the presence of physostigmine (0.5 µM) to inhibit AChE.
Ta ble 2. Inhibition Constants of AChE Obtained with
for muscarinic M2 receptors increased, as previously
Caproctamine (9) in Comparison with Tacrine and
observed,10 with increasing chain length between the
Edrophoniuma
nitrogens, whereas the affinity for BChE did not follow
compd
AChE Ki (µM)
a similar linear trend as 6 but not 5 was more potent
than 4. Since it was an aim of the present investigation
to provide compounds useful for the treatment of neuro-
logical disorders by improving cholinergic transmission
in the brain following inhibition of AChE and musca-
rinic M2 receptors, we investigated diamine diamides
7-9. Interestingly enough, transforming the inner
amine functions of methoctramine (6) into amide groups,
affording 7, resulted in a significant decrease in the
affinity for muscarinic M2 and M3 receptors and for
BChE as well but produced an increase in the affinity
for AChE. N-Methylation of 7, affording 8 or 9 (caproct-
amine), resulted in a further increase in affinity for
AChE while not affecting the affinity for BChE and
muscarinic receptors.
caproctamine (9)
edrophonium
tacrine
0.104 ( 0.016
1.63 ( 0.23
0.151 ( 0.016
a
Inhibition constants, expressed as Ki values, were calculated
from kinetic data in Figure 1.
the intermediate Schiff base to yield diamine diamide
8. Finally, Eschweiler-Clarke methylation of 8 gave
diamine diamide 9.13
Resu lts a n d Discu ssion . Four different pharma-
cological actions of tetraamines 1-6 and diamine diam-
ides 7-9 were determined, that is, AChE and BChE
inhibition14 and antagonism at muscarinic M2 and M3
receptor subtypes.15 Furthermore, the most interesting
compound (9) was evaluated also for its muscarinic M1
antagonistic potency15 in comparison with methoctram-
ine (6). The results are summarized in Tables 1 and 2.
It is evident that all polyamines were effective inhibi-
tors of AChE and BChE with the exception of the
unsubstituted tetraamine disulfide 2. The latter re-
sulted as a very weak inhibitor of both enzymes, being
about 2 orders of magnitude less potent than the
prototype benextramine (1). Clearly, a 2-methoxybenzyl
group on the terminal nitrogens of 1 contributes sig-
nificantly to the binding with the enzyme. The replace-
ment of the disulfide bridge of 1 with two methylenes,
affording 4, did not modify the affinity for AChE, while
causing a 5-fold increase in affinity for BChE. This
finding rules out the possibility that benextramine may
inhibit AChE by forming a covalent bond by way of an
interchange reaction between the disulfide moiety of 1
and a suitable thiol group on the enzyme. Furthermore,
two methylenes served better than a disulfide function-
ality in the enzyme-inhibitor complex formation, as 4
was more potent than 1 at both enzymes.
It is evident that 9 emerges as a powerful tool for
investigating the neurological disorders due to a loss
in the cholinergic system. In comparison with benex-
tramine (1), caproctamine (9) resulted as 42-fold more
potent at AChE while being 2-fold less potent at BChE
with a BChE/AChE selectivity ratio of 68. Furthermore,
it was a weak antagonist at both muscarinic M1 and
M3 receptors while displaying an affinity toward mus-
carinic M2 receptors similar to the affinity for AChE as
revealed by a comparison of pA2 (M2) and pIC50 (AChE)
values which were 6.39 and 6.77, respectively. Owing
to these biological properties, caproctamine (9) was
studied further to characterize the nature of the inhibi-
tion of AChE activity. Inhibition of AChE activity by 9
was very fast and not time-dependent, as 50% of enzyme
inactivation produced by a 0.17 µM concentration fol-
lowing a 1-min incubation was not significantly different
(p > 0.01) from the inhibition observed up to a 40-min
incubation. The graphical analysis of steady-state
inhibition data for 9 in comparison with edrophonium
and tacrine is shown in Figure 1, whereas the estimates
of competitive inhibition constants Ki are reported in
Table 2.16 The inhibition was found to be clearly of the
competitive type only for edrophonium, whereas recip-
rocal plots involving tacrine and 9 (Figure 1) show both
Modifying the chain length between the two inner
nitrogens of 4, affording tetraamines 3-6, did not affect
significantly the affinity for AChE and muscarinic M3
receptors while causing a different effect on the affinity
toward BChE and muscarinic M2 receptors. The affinity

4188 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 22
Communications to the Editor
F igu r e 2. Representative interactions of 9 (cyan) with some
selected amino acid residues (white) of the human AChE
gorge: the indole of Trp279 and one o-methoxyphenyl of 9 form
a displaced π-stacking interaction; the protonated amino group
of the second o-methoxybenzylamine function forms an H-bond
(yellow dotted line) with the backbone carbonyl of Trp84;
Trp233 and Tyr330 are two of the residues contributing to the
hydrophobic stabilization of the intermediate chain. The
position of decamethonium (red) with respect to the two critical
Trp residues as determined from the X-ray crystallography is
shown for reference.22
lated to correspond to Trp279.19 Remarkably, despite
the conspicuous overall length of the molecular back-
bone of 9, it can fold in such a way as to place the
o-methoxybenzylamine functions at a distance ranging
from 10 to 16 Å. This is compatible with the reaching
of Trp84 and Trp279 which lie approximately 16 Å
apart. The intermediate mostly lipophilic chain seems
to play an important role in determining the binding of
the diamine diamide 9 to the enzyme by allowing
hydrophobic interactions with several aromatic residues,
among which are Tyr70, Tyr121, Phe288, Phe290,
Tyr330, Phe331, and Tyr334.
In comparison with the experimentally determined
(X-ray analysis)20 AChE binding mode of decametho-
nium also shown in Figure 2, the way in which 9
contacts Trp84 and Trp279, although different, allows
one to hypothesize the same kind of interaction. We
expect that this similar binding mode leads to the same
action against âA fibrillogenesis as that observed for
decamethonium.6
In conclusion, present investigation has shown that
caproctamine (9) is endowed with a well-balanced
affinity profile as an AChE inhibitor and a competitive
muscarinic M2 receptor antagonist. Therefore, it may
well be capable of stimulating cholinergic activity in the
brain by decreasing ACh hydrolysis rates and, at the
same time, by increasing ACh release in the synapse.
It derives that caproctamine (9) could have potential in
the investigation of AD because, besides its effects on
F igu r e 1. Steady-state inhibition by three inhibitors of AChE
hydrolysis of acetylthiocholine. Reciprocal plots of initial
velocity and substrate concentrations (a-c) and replots of the
reciprocal plots versus inhibitor concentration (d-f) are re-
ported. Reciprocal plots of initial velocities in the absence of
inhibitors gave an estimate of kapp for acetylthiocholine of 170
( 15 µM (four experiments). Lines were derived from a
weighted least-squares analysis of the data points.
increasing slopes and increasing intercepts with higher
inhibitor concentration. Therefore, we concluded that
caproctamine (9) causes a mixed type of inhibition, that
is, inhibition of both the active site and a second distal
site of the enzyme.17
The possibility for caproctamine (9) to bind at both
the active and the peripheral sites of AChE was
theoretically investigated by studying the docking of its
diprotonated form to the AChE gorge. Molecular dy-
namics simulation runs were performed,18 and the
results indicated that indeed 9 is able to simultaneously
contact both sites and to establish favorable interactions
with a number of residues in the gorge. The flexibility
of the molecule allows it to assume many conformations,
one of which is shown in Figure 2. At one end of the
molecule, the o-methoxybenzylamine moiety can inter-
act with a set of residues near Trp84, while, at the
opposite end, the second o-methoxybenzylamine group
can reach the peripheral binding site that was postu-

Communications to the Editor
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 22 4189
resulting mixture which was heated at 90 °C for 10 h, cooled (0
°C), made basic with 40% aqueous NaOH, and extracted with
methylene chloride. Removal of dried solvents gave 9 as the free
base that was transformed into the citrate salt: oil (95% yield);
1H NMR (free base, CDCl3) δ 1.16-1.78 (m, 24), 2.20-2.46 (m,
8), 2.22 (s, 6), 2.89 (s, 3), 2.94 (s, 3), 3.18-3.37 (m, 4), 3.51 (s, 4),
the cholinergic system, it might prevent also AChE-
mediated âA aggregation by interacting with the pe-
ripheral anionic binding site of AChE. However, there
may be significant barriers to that end use. For
example, although 9 is likely more lipophilic than
methoctramine (6) or benextramine (1), in vivo studies
may be required to determine whether diamine dia-
mides are able to cross the blood-brain barrier. Our
future work in this area will include studies directed
at gaining a better understanding of the intriguing
trends noted above.
3.81 (s, 6), 6.82-6.94 (m, 4), 7.18-7.33 (m, 4). Anal. (C52H82N4O18
‚
H2O) C, H, N.
(14) AChE and BChE derived from human erythrocytes were em-
ployed in this study. Anticholinesterase activity was based on
measuring the hydrolysis of acetylthiocholine and the subse-
quent reaction of thiocholine with 4,4-dithiopyridine to form
4-thiopyridine.23 The results are expressed as IC50 values which
represent the concentration required to inhibit enzyme activity
by 50%.
(15) Functional activity at muscarinic receptor subtypes was deter-
mined by the use of the muscarinic M1 receptor-mediated
inhibition of neurogenic twitch contractions (single pulses at 0.05
Hz), muscarinic M2 receptor-mediated negative inotropism in
driven guinea pig left atria (1 Hz), and muscarinic M3 receptor-
mediated contraction of guinea pig ileum longitudinal muscle
as previously described.24 The agonist was McN-A-343 (M1) or
arecoline propargyl ester (M2 and M3).
(16) Inhibition constants, expressed as Ki values, were calculated
from kinetic data.25 Three concentrations of each inhibitor and
four substrate concentrations were used; acetylthiocholine con-
centration never exceeded 0.55 mM, to avoid substrate inhibi-
tion. Duplicate assays were conducted at each of the concentra-
tion combinations, and the mean of the enzyme velocity values
was used for graphical analysis (individual velocity values were
all within (5% of the mean value).
(17) The inhibitory behavior of 9, as deduced from plots b and c in
Figure 2, is strictly similar to that displayed by some recently
reported26 bis-tetrahydroaminacridine inhibitors of AChE. These
compounds bind simultaneously at both the catalytic and the
peripheral sites of AChE and are characterized by “a linear
mixed type of enzyme inhibition”.
(18) Molecular dynamics simulations were performed using the
united-atom AMBER* force field implemented in the Macro-
Model ver. 5.5 program.27 The coordinates of the protein were
obtained from the X-ray structure of AChE isolated from Torpedo
californica28 and retrieved from the Brookhaven Protein Data
Bank; Phe330 was replaced by Tyr, which is present in the
human AChE, with the same conformation.
Ack n ow led gm en t. This research is supported by
grants from the University of Bologna (funds for selected
research topics) and MURST.
Refer en ces
(1) (a) Wurtman, R. J . Alzheimer’s disease. Sci. Am. 1985, 252, 48-
56. (b) Altman, H. J ., Ed. Alzheimer’s Disease - Problems,
Prospects and Perspectives; Plenum Press: New York, 1987.
(2) Roberson, M. R.; Harrell, L. E. Cholinergic activity and amyloid
precursor protein metabolism. Brain Res. Rev. 1997, 25, 50-
69.
(3) Kasa, P.; Rakonezay, Z.; Gulya, K. The cholinergic system in
Alzheimer’s disease. Prog. Neurobiol. (Oxford) 1997, 52, 511-
535.
(4) (a) Gualtieri, F.; Dei, S.; Manetti, D.; Romanelli, M. N.; Scapec-
chi, S.; Teodori, E. The medicinal chemistry of Alzheimer’s and
Alzheimer’s-like diseases with emphasis on the cholinergic
hypothesis. Farmaco 1995, 50, 489-503. (b) Brufani, M.; Filo-
camo, L.; Lappa, S.; Maggi, A. New acetylcholinesterase inhibi-
tors. Drugs Future 1997, 22, 397-410.
(5) Doods, H. N.; Quirion, R.; Mihm, G.; Engel, W.; Rudolf, K.;
Entzeroth, M.; Schiavi, G. B.; Ladinsky, H.; Bechtel, W. D.;
Ensinger, H. A.; Mendla, K. D.; Eberlein, W. Therapeutic
potential of CNS-active M2 antagonists: novel structures and
pharmacology. Life Sci. 1993, 52, 497-503.
(6) Inestrosa, N. C.; Alvarez, A.; Pe´rez, C. A.; Moreno, R. D.; Vicente,
M.; Linker, C.; Casanueva, O. I.; Soto, C.; Garrido, J . Acetyl-
cholinesterase accelerates assembly of amyloid-â-peptides into
Alzheimer’s fibrils: possible role of the peripheral site of the
enzyme. Neuron 1996, 16, 881-891.
(7) Melchiorre, C.; Yong, M. S.; Benfey, B. G.; Belleau, B. Molecular
properties of the adrenergic R-receptor. II. Optimum covalent
inhibition by two different prototypes of polyamine disulfides.
J . Med. Chem. 1978, 21, 1126-1132.
(8) Benfey, B. G.; Yong, M. S.; Belleau, B.; Melchiorre, C. Cardiac
muscarinic blocking and atropinic blocking effects of a tetramine
disulfide with R-adrenoceptor blocking activity. Can. J . Physiol.
Pharmacol. 1979, 57, 41-47.
(9) Benfey, B. G.; Brasili, L.; Melchiorre, C.; Belleau, B. Potentiation
and inhibition of nicotinic effects on striated muscle by the
tetramine disulfide benextramine. Can. J . Physiol. Pharmacol.
1980, 58, 984-988.
(19) Yamamoto, Y.; Ishihara, Y.; Kuntz, I. D. Docking analysis of a
series of benzylamino acetylcholinesterase inhibitors with a
phthalimide, benzoyl, or indanone moiety. J . Med. Chem. 1994,
37, 3141-3153.
(20) Harel, M.; Schalk, I.; Ehret-Sabattier, L.; Bouet, F.; Goeldner,
M.; Hirth, C.; Axelsen, P.; Silman, I.; Sussman, J . Quaternary
ligand binding to aromatic residues in the active-site gorge of
acetylcholinesterase. Proc. Natl. Acad. Sci. U.S.A. 1993, 90,
9032-9035.
(21) Van Rossum, J . M. Cumulative dose-response curves. II.
Technique for the making of dose-response curves in isolated
organs and the evaluation of drug parameters. Arch. Int.
Pharmacodyn. 1963, 143, 299-330.
(22) The backbone atoms of Trp84 and Trp279 from the model and
from the structure of AChE cocrystallized with decamethonium
(Brookhaven Protein Data Bank entry 1acl) were aligned, and
the resulting position of decamethonium is shown in Figure 2.
(23) Ellmann, G. L.; Courtney, D.; Andres, V., J r.; Featherstone, R.
M. A new and rapid colorimetric determination of acetylcho-
linesterase activity. Biochem. Pharmacol. 1961, 7, 88-95.
(24) (a) Eltze, M.; Figala, V. Affinity and selectivity of diperiden
enantiomers for muscarinic receptor subtypes. Eur. J . Pharma-
col. 1988, 158, 11-19. (b) Chiarini, A.; Budriesi, R.; Bolognesi,
M. L.; Minarini, A.; Melchiorre, C. In vitro characterization of
tripitramine, a polymethylene tetraamine displaying high se-
lectivity and affinity for muscarinic M2 receptors. Br. J . Phar-
macol. 1995, 114, 1507-1517.
(25) Krupka, R. M.; Laidler, K. J . Molecular mechanisms for hydro-
lytic enzyme action. III. A general mechanism for the inhibition
of acetylcholinesterase. J . Am. Chem. Soc. 1961, 83, 1454-1459.
(26) Pang, Y.-P.; Quiram, P.; J elacic, T.; Hong, F.; Brimijoin, S. Highly
potent, selective, and low cost bis-tetrahydroaminacrine inhibi-
tors of acetylcholinesterase. J . Biol. Chem. 1996, 271, 23646-
23649.
(27) Mohamadi, F.; Richards, N. G. J .; Guida, W. C.; Liskamp, R. M.
J .; Lipton, M. A.; Caulfield, C. E.; Chang, G.; Hendrickson, T.
F.; Still, W. C. MacroModel - an integrated software system
for modeling organic and bioorganic molecules using molecular
mechanics. J . Comput. Chem. 1990, 11, 440-467.
(28) Sussman, J . L.; Harel, M.; Frolow, F.; Oefner, C.; Goldman, A.;
Toker, L.; Silman, I. Atomic structure of acetylcholinesterase
from Torpedo Californica: A prototypic acetylcholine-binding
protein. Science 1991, 253, 872-879.
(10) Melchiorre, C.; Cassinelli, A.; Quaglia, W. Differential blockade
of muscarinic receptor subtypes by polymethylene tetraamines.
Novel class of selective antagonists of cardiac M-2 muscarinic
receptors. J . Med. Chem. 1987, 30, 201-204.
(11) (a) Quaglia, W.; Giardina`, D.; Marucci, G.; Melchiorre, C.;
Minarini, A.; Tumiatti, V. Structure-activity relationships
among methoctramine-related polymethylene tetraamines. 3.
Effect of the four nitrogens on M2 muscarinic blocking activity
as revealed by symmetrical and unsymmetrical polyamines.
Farmaco 1991, 46, 417-434. (b) Unpublished results.
(12) Melchiorre, C.; Quaglia, W.; Picchio, M. T.; Giardina`, D.; Brasili,
L.; Angeli, P. Structure-activity relationships among methoc-
tramine-related polymethylene tetraamines. Chain-length and
substituent effects on M-2 muscarinic receptor blocking activity.
J . Med. Chem. 1989, 32, 79-84.
(13) A mixture of 10 (0.4 g, 1 mmol), 2-methoxybenzaldehyde (0.3 g,
2.2 mmol), and sodium borohydride (85 mg, 2.2 mmol) in
anhydrous ethanol with molecular sieves (3 Å) was stirred at
room temperature for 20 h. After cooling, the mixture was made
acidic with 6 N HCl, filtered to remove the sieves, and then
evaporated. The residue was dissolved in water, and the result-
ing solution was washed with ether, made basic with 2 N NaOH,
and extracted with methylene chloride. Removal of dried sol-
vents gave 8 as the free base that was transformed into the
oxalate salt: 90% yield; mp 156-157 °C (from ethanol/ether).
Anal. (C42H66N4O12) C, H, N. Formic acid (96%; 0.67 mL, 17.4
mmol) was added dropwise to 8 (0.15 g, 0.23 mmol), and then
formaldehyde (40%; 0.60 mL, 8.7 mmol) was added to the
J M9810452