Design, synthesis, and biological evaluation of symmetrically and unsymmetrically substituted methoctramine-related polyamines as muscular nicotinic receptor noncompetitive antagonists

Article abstract of DOI:10.1021/jm991110n

The universal template approach to drug design foresees that a polyamine can be modified in such a way to recognize any neurotransmitter receptor. Thus, hybrids of polymethylene tetraamines and philanthotoxins, exemplified by methoctramine (1) and PhTX-343 (2), respectively, were synthesized to produce novel inhibitors of muscular nicotinic acetylcholine receptors. Polyamines 3-25 were synthesized and their biological profiles were evaluated at frog rectus abdominis muscle nicotinic receptors and guinea pig left atria (M₂) and ileum longitudinal muscle (M₃) muscarinic acetylcholine receptors. All of the compounds, like prototypes 1 and 2, were noncompetitive antagonists of nicotinic receptors while being, like 1, competitive antagonists at muscarinic M₂ and M₃ receptor subtypes. Interestingly, polyamines bearing a low number of methylenes between the nitrogen atoms, as in 3, 6, and 7, displayed a biological profile similar to that of 2: a noncompetitive antagonism at nicotinic receptors in the 7-25 μM range while not showing any antagonism for muscarinic receptors up to 10 μM. Increasing the number of methylenes separating these nitrogen atoms in methoctramine- related tetraamines resulted in a significant improvement in potency at nicotinic receptors. The most potent tetraamine was 19, bearing a 12 methylene spacer between the nitrogen atoms, which was 12-fold and 250-fold more potent than prototypes 1 and 2, respectively. Tetraamines 9-11, bearing a rather rigid spacer between the nitrogen atoms instead of the very flexible polymethylene chain, displayed a profile similar to that of 1 at nicotinic receptors, whereas a significant decrease in potency was observed at muscarinic M₂ receptors. This finding may have relevance in understanding the mode of interaction with these receptors. Similarly, the constrained analogue 12 of methoctramine showed a decrease in potency at nicotinic and muscarinic M₂ receptors, revealing that the tricyclic system, which incorporates the 2-methoxybenzylamine moiety of 1, does not represent a good pharmacophore for activity at these sites. A most intriguing finding was the observation that the photolabile tetraamine 22 was more potent than methoctramine at nicotinic receptors and, what is more important, it inhibited a closed state of the receptor.

Full text of DOI:10.1021/jm991110n

5212
J . Med. Chem. 1999, 42, 5212-5223
Design , Syn th esis, a n d Biologica l Eva lu a tion of Sym m etr ica lly a n d
Un sym m etr ica lly Su bstitu ted 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
Michela Rosini,† Roberta Budriesi,† M. Gabriele Bixel,‡ Maria L. Bolognesi,† Alberto Chiarini,†
Ferdinand Hucho,‡ Povl Krogsgaard-Larsen,§ Ian R. Mellor,| Anna Minarini,† Vincenzo Tumiatti,†
Peter N. R. Usherwood,| and Carlo Melchiorre*,†
Department of Pharmaceutical Sciences, University of Bologna, Via Belmeloro 6, 40126 Bologna, Italy, Institut fu¨r Biochemie,
Freie Universita¨t Berlin, Thielallee 63, 14195 Berlin, Germany, Department of Medicinal Chemistry, Royal Danish School of
Pharmacy, 2 Universitetsparken, DK 2100 Copenhagen, Denmark, and Division of Molecular Toxicology, School of Biological
Sciences, University of Nottingham, University Park, Nottingham NG7 2RD, United Kingdom
Received J une 25, 1999
The universal template approach to drug design foresees that a polyamine can be modified in
such a way to recognize any neurotransmitter receptor. Thus, hybrids of polymethylene
tetraamines and philanthotoxins, exemplified by methoctramine (1) and PhTX-343 (2),
respectively, were synthesized to produce novel inhibitors of muscular nicotinic acetylcholine
receptors. Polyamines 3-25 were synthesized and their biological profiles were evaluated at
frog rectus abdominis muscle nicotinic receptors and guinea pig left atria (M2) and ileum
longitudinal muscle (M3) muscarinic acetylcholine receptors. All of the compounds, like
prototypes 1 and 2, were noncompetitive antagonists of nicotinic receptors while being, like 1,
competitive antagonists at muscarinic M2 and M3 receptor subtypes. Interestingly, polyamines
bearing a low number of methylenes between the nitrogen atoms, as in 3, 6, and 7, displayed
a biological profile similar to that of 2: a noncompetitive antagonism at nicotinic receptors in
the 7-25 µM range while not showing any antagonism for muscarinic receptors up to 10 µM.
Increasing the number of methylenes separating these nitrogen atoms in methoctramine-related
tetraamines resulted in a significant improvement in potency at nicotinic receptors. The most
potent tetraamine was 19, bearing a 12 methylene spacer between the nitrogen atoms, which
was 12-fold and 250-fold more potent than prototypes 1 and 2, respectively. Tetraamines 9-11,
bearing a rather rigid spacer between the nitrogen atoms instead of the very flexible
polymethylene chain, displayed a profile similar to that of 1 at nicotinic receptors, whereas a
significant decrease in potency was observed at muscarinic M2 receptors. This finding may
have relevance in understanding the mode of interaction with these receptors. Similarly, the
constrained analogue 12 of methoctramine showed a decrease in potency at nicotinic and
muscarinic M2 receptors, revealing that the tricyclic system, which incorporates the 2-meth-
oxybenzylamine moiety of 1, does not represent a good pharmacophore for activity at these
sites. A most intriguing finding was the observation that the photolabile tetraamine 22 was
more potent than methoctramine at nicotinic receptors and, what is more important, it inhibited
a closed state of the receptor.
In tr od u ction
suitable pharmacophores can be inserted to achieve
selectivity for any given receptor.6 The discovery that
1, at micromolar concentrations, antagonizes nicotinic
acetylcholine receptors (nAChR) has provided the op-
portunity to apply the universal template approach to
the design of polyamines as nAChR ligands.
Methoctramine (1, Figure 1) is the prototype polym-
ethylene tetraamine for antagonism of muscarinic ace-
tylcholine receptors (mAChR).1 The development of
polymethylene tetraamines as antagonists of mAChR
has been the subject of several review articles.2 Ap-
propriate modifications of the chain lengths separating
the nitrogen atoms and of the substituents on the
terminal nitrogen atoms modulate both affinity and
selectivity (specificity) for different receptors.3,4 On the
basis of these observations, it was suggested that a
polyamine backbone represents a special feature in the
polymethylene tetraamine-mAChR recognition pro-
cess.5 More recently, it has been suggested that such a
structure may represent a universal template on which
Philanthotoxins, as exemplified by philanthotoxin-343
(PhTX-343) (2, Figure 1), a synthetic analogue of a wasp
(Philanthus triangulum) toxin PhTX-433,7 are noncom-
petitive antagonists of muscle8,9 and neuronal nAChR,10
although at submillimolar concentrations they may also
competitively antagonize these receptors.11 However,
the mode of action of 2 on nAChR has not yet been fully
evaluated. The affinity of 2 for nAChR is not high.
However, by combining structural features of 2 and
polymethylene tetraamines it may be possible to obtain
ligands that have high affinities and selectivities for
nAChR. The synthesis and pharmacological assay of
these hybrid molecules is the subject of this report.
† University of Bologna.
‡ Freie Universita¨t Berlin.
§ Royal Danish School of Pharmacy.
| University of Nottingham.
10.1021/jm991110n CCC: $18.00 © 1999 American Chemical Society
Published on Web 11/23/1999

Methoctramines as Receptor Antagonists
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 25 5213
Sch em e 1
F igu r e 1. Chemical structure of methoctramine (1) and
PhTX-343 (2), prototypes of polymethylene tetraamines and
polyamine amides, respectively. The numerals in PhTX-343
refers to the number of methylenes between the polyamine
nitrogen atoms.
Structure-activity relationship (SAR) studies on the
action of philanthotoxins on quisqualate-sensitive iono-
tropic glutamate receptors (qGluR) of insect skeletal
muscle12-17 and competition binding studies of these
compounds on nAChR of Torpedo electroplax11,18 have
shown that an aromatic moiety at one terminus and a
primary amine function at the other terminus are
important for antagonism. Thus, a suitable starting
point for the design of novel ligands for nAChR might
be a tetraamine backbone to which may be attached
either one or both terminal groups of 1 and 2. To this
end, compounds 3-8 were synthesized to verify the
influence on potency of the butyryltyrosyl moiety of 2
and of the 2-methoxybenzyl group of 1. Tetraamine 1
can assume many low-energy conformations in an
aqueous environment because of its flexible polymeth-
ylene chain. Therefore, we have designed tetraamines
9-11 with less flexible chains to determine whether
flexibility is an important determinant of potency with
respect to nAChR antagonism. Also, the terminal 2-meth-
oxybenzyl groups of 1 have been included into a tricyclic
system (12) to determine whether the spatial relation-
ship of the methoxy moiety relative to the amine
function differently affects affinity for nAChR and
mAChR. Previously, homologues of 1 have been inves-
tigated with the aim of assessing the structural require-
ments for optimum antagonism of mAChR.1,19 In this
study we have tested a number of these compounds
(13-21) on nAChR in which the chain length between
the inner nitrogen atoms varies between 4 and 14
methylenes. Finally, to determine which amino acid
residues are involved in the binding of polymethylene
tetraamines to nAChR, we have designed the photo-
labile analogue MR44 (22). During synthesis of the
linear pentaamine required for 22, the branched iso-
meric pentaamine was obtained, which gave the op-
portunity to synthesize the polyamines 23-25. All of
the compounds synthesized in this study were tested
on peripheral M2 and M3 mAChR as well as on muscle-
type nAChR.
N-butyryl-O-benzyl-L-tyrosine p-nitrophenyl ester16 (26)
was allowed to react with tetraamines spermine (27),
28,1 and 8. Intermediates 29-31 were debenzylated by
catalytic hydrogenolysis to give 2,7 4 and 5, respectively.
The condensation of 2 with 2-methoxybenzaldehyde and
subsequent reduction with NaBH4 of the intermediate
Schiff base afforded 3 (Scheme 1).
Mono- and disubstituted spermine derivatives 6 and
7 were easily obtained through the condensation of
spermine in a 1:2 or 10:1 molar ratio with 2-methoxy-
benzaldehyde and subsequent reduction with NaBH4 of
the intermediate Schiff bases.
Although monosubstituted tetraamine 8 was already
known,20 it was synthesized following the procedure
shown in Scheme 2. N-(tert-Butoxycarbonyl)-6-aminoca-
proic acid was amidated with N-[(benzyloxy)carbonyl]-
1,8-octanediamine21 to give 32. Removal of the N-(ben-
zyloxy)carbonyl group was achieved by catalytic hydroge-
nation over 10% palladium on charcoal. Thus, hydro-
genolysis of 32 gave 33, which was reacted with N-[(ben-
zyloxy)carbonyl]-6-aminocaproic acid to afford diamine
diamide 34, having the two terminal amine functions
protected with different moieties. Removal of the N-
(benzyloxy)carbonyl group of 34 by catalytic hydro-
genolysis gave 35, which was treated with 2-methoxy-
benzaldehyde followed by reduction with NaBH4 of the
intermediate Schiff base and subsequent hydrolysis with
3 N HCl to remove the N-tert-butoxycarbonyl group to
afford the monosubstituted diamine diamide 36. Reduc-
tion of 36 with borane-methyl sulfide complex gave 8
(Scheme 2).
Ch em istr y
All the compounds were synthesized by standard
procedures (Schemes 1-7) and were characterized by
1
IR, H NMR, mass spectra, and elemental analysis.
Triamine amides 4 and 5 were synthesized by follow-
Suberic acid and 4,4′-biphenyldicarboxylic acid were
amidated with benzyl N-[4-(aminomethyl)benzyl]car-
ing an adapted procedure described for 2.7,16 Thus,

5214 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 25
Rosini et al.
Sch em e 2a
Sch em e 3a
Sch em e 4
bamate21 and N-[(benzyloxy)carbonyl]-1,6-diamine,22
respectively, to give 37 or 38. Removal of the N-
(benzyloxy)carbonyl group of 37 and 38 by hydrolysis
with HBr gave 39 and 40, respectively, which were
treated with 2-methoxybenzaldehyde followed by the
reduction of the formed Schiff bases to amine amides
41 and 42 that, in turn, were reduced with borane to 9
and 11, respectively (Scheme 3).
Alkylation of 44, obtained through the reaction of 4320
with mesitylenesulfonyl chloride, with di[(p-bromo-
methyl)phenyl]methane23 afforded 45. Removal of the
protecting groups of 45 by hydrolysis with 30% HBr
gave 10 (Scheme 4).
The tricyclic compound 46 required for the synthesis
of the constrained analogue 12 of methoctramine was
synthesized by following an adapted procedure reported
for its N-methyl analogue24 starting from N-benzylgly-
cine and 2-allyloxybenzaldehyde. Removal of the benzyl
group of 46 by hydrogenolysis gave 47, which was
treated with N-[(benzyloxy)carbonyl]-6-aminocaproic
acid to afford 48. Hydrogenolysis of 48 gave 49, which,
upon reduction with borane-methyl sulfide complex,
was transformed into 50 that was treated with suberic

Methoctramines as Receptor Antagonists
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 25 5215
Sch em e 5
cally at 1 Hz.4 The guinea pig ileum longitudinal muscle
was used to study their effects on M3 mAChR. In both
cases the agonist was arecaidine propargyl ester (APE).
The biological data are expressed in apparent dissocia-
tion constants (KB) according to Furchgott.27
The effects of compounds 1-25 on muscle-type nAChR
were studied on the frog rectus abdominis muscle with
carbachol-induced contractions as the measured param-
eter.28 The results are expressed as IC50 values, i.e., the
concentrations required to inhibit the maximal response
to carbachol by 50%.
Sch em e 6
In all of the experiments, 1 and 2 were used as
standards.
Resu lts a n d Discu ssion
Summaries of the results are presented in Table 1
and Figure 2. Over the concentration range investi-
gated, all of the compounds were noncompetitive an-
tagonists of muscle-type nAChR. The maximum re-
sponse to carbachol was reduced and the magnitude of
this reduction was dependent on the concentration of
antagonist. In all cases, the antagonism was reversed
by washing the muscle in drug-free saline. In contrast
to the data for nAChR, compounds 1-25 competitively
antagonized mAChR, as revealed by the parallelism of
dose-response data for APE obtained in the presence
and absence of the compounds.
Methoctramine (1) was a potent and selective antago-
nist of M2 mAChR, whereas 10 µM PhTX-343 (2) was
inactive on both M2 and M3 mAChR. Tetraamine 1 was
20-fold more potent than polyamine amide 2 at nAChR.
As a consequence, it was necessary to determine whether
the higher potency of 1 is due to the presence in the
molecule of methoxybenzyl groups or to the different
polyamine backbones of 1 and 2. To this end, a series
of methoctramine analogues bearing either one or both
of the aromatic features of the two prototypes was
investigated. Compound 8, which lacks one of the
methoxybenzyl groups of 1, was a less potent antagonist
of nAChR than 1 and, as reported previously,20 a less
potent antagonist of mAChR. This suggests that the
methoxybenzyl group plays a role in the binding of 1 to
nAChR and mAChR. Attachment of the butyryltyrosyl
residue of 2 onto the unsubstituted tetraamine backbone
of 1, to give compound 4, resulted in an increase in
potency (to that of 8) at nAChR. Again, the inclusion of
a methoxybenzyl group on the terminal nitrogen atom
acid to afford diamine diamide 51. Reduction of amide
functions of 51 gave 12 (Scheme 5).
Tetraamine 13 was synthesized by following an
adapted procedure described for methoctramine.25 Thus,
N-[(benzyloxy)carbonyl]-6-aminocaproic acid was ami-
dated with 1,4-diaminobutane to give 52 that, upon
hydrolysis with HBr in acetic acid, afforded 53, which
was treated with 2-methoxybenzaldehyde followed by
the reduction of the formed 54 into 13 (Scheme 6).
The addition of tetraamine 281 to acrylonitrile gave
a mixture of compounds: two of them (55 and 56) were
isolated and characterized also through their transfor-
mation into 57 and 58. Linear and branched nitriles 55
and 56 were transformed into 59 and 60 followed by
reduction with LiAlH4 or Raney Ni into the correspond-
ing amines 61 and 62. 4-Azidosalicylic acid N-hydroxy-
succinimide ester26 was amidate with 61 to give 63,
which, upon treatment with CF3COOH to remove the
protecting groups, afforded the photolabile compound
MR44 (22). Condensation of 62 with 2-methoxybenzal-
dehyde followed by the reduction of the intermediate
Schiff base and subsequent hydrolysis with HCl gave
23. Finally, amidation of 2621 with 62 gave 64, which,
upon hydrolysis with CF3COOH to 65, was transformed
into 24 through removal of the benzyl group. Condensa-
tion of 24 with 2-methoxybenzaldehyde afforded 25
(Scheme 7).
Biology
The effects of compounds 1-25 on M2 mAChR were
determined on guinea pig left atria stimulated electri-

5216 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 25
Rosini et al.
Sch em e 7a
of 4, giving compound 5, resulted in activities at nAChR
and mAChR which were qualitatively similar to that of
1. Analogously, methoxybenzyl and butyryltyrosyl moi-
eties have been introduced onto the spermine backbone
of 2, affording 3, 6, and 7. The inclusion of a methoxy-
benzylamine function in place of the terminal primary
amino group of 2 and of 6 affording, respectively, 3 and
7 did not lead to the same improvement in activity
observed for the methoctramine backbone at both
nAChR and mAChR. This suggests the importance of
the chain length separating the nitrogen atoms of a
polyamine ligand in binding nicotinic and muscarinic
receptors.
The influence on activity at nAChR of the distance
between the two inner nitrogen atoms of 1 was inves-
tigated with compounds 13-21. The number of carbon
atoms in the alkyl chain separating these nitrogens has
previously been shown to influence activity at M2 but
not at M3 mAChR.1,19 Apparently, the chain length effect
was also important for the activity at nAChR (Table 1).
Figure 2 shows graphically that optimum activity at
nAChR occurred with a chain length of 12 carbon atoms
as in 19, whereas optimum activity at M2 mAChR was
obtained with 1, which has a chain of eight carbon
atoms. The pIC50 value for 19 was 250-fold higher than
that for 2 and 12-fold higher than that for 1.
The effect of reductions in the flexiblity of polymeth-
ylene tetraamines was studied with compounds 9-11.
In 9 the hexamethylene chain between the inner and
outer nitrogen atoms of 1 was replaced with a p-xylene
moiety. This did not influence activity at nAChR and
M3 mAChR (9 was almost as potent as 1), but there was
a dramatic loss of potency at M2 mAChR. It follows from
these data that changes in flexibility may enable one
to design polyamine-containing compounds with speci-
fity for nAChR over mAChR. The tetraamines 10 and
11 contain a bis(4-methylphenyl)methane moiety or a
4,4′ -dimethylbiphenyl moiety, respectively, between the
inner nitrogen atoms. Both compounds were similarly
potent to 1 at nAChR and M3 mAChR, but significantly
less potent (39-fold and 24-fold, respectively) than 1 at
M2 mAChR. In 10 and 11, the inner nitrogens are
unlikely to be less than 11 Å apart. Since the biphenyl
moiety of 11 can only rotate along the axis of the 4,4′-
bond without altering the distance between the two
amine functions it follows that this distance is important
for its interaction with nAChR. Interestingly, this
distance corresponds to that separating the inner ni-
trogens of 1 when it is in its fully extended comforma-
tion. This finding, in addition to the observed increase
in potency at nAChR when the polyamine chain length
is increased up to 12 methylenes, suggests that optimum

Methoctramines as Receptor Antagonists
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 25 5217
Ta ble 1. Antagonist Affinities, Expressed as pIC50 or pKB Values, in the Isolated Frog Rectus Abdominis Muscle (FRA) and Guinea
Pig Left Atrium (M2) and Longitudinal Ileum (M3) unless Otherwise Specified
pKB
no.a
R1
R2
R3
m
n
pIC50 (FRA)
M2
M3
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
8
5.93 ( 0.03
4.62 ( 0.03
5.14 ( 0.04
5.27 ( 0.06
5.82 ( 0.05
4.60 ( 0.02
4.64 ( 0.01
5.27 ( 0.04
5.75 ( 0.03
6.23 ( 0.01
5.79 ( 0.01
5.28 ( 0.03
5.19 ( 0.07
5.22 ( 0.06
5.64 ( 0.02
5.61 ( 0.01
6.10 ( 0.01
6.35 ( 0.02
7.02 ( 0.04
6.69 ( 0.02
5.96 ( 0.01
6.39 ( 0.02
5.60 ( 0.05
4.92 ( 0.04
5.90 ( 0.01
7.91 ( 0.03
6.14 ( 0.06
H
A
H
A
A
A
A
4
4
8
8
4
4
8
3
3
6
6
3
3
6
<5
<5
6.01 ( 0.02
7.49 ( 0.09
<5
<5
6.76 ( 0.01
<5
<5
5.81 ( 0.10
6.07 ( 0.03
<5
<5
5.81 ( 0.01
H
H
H
H
A
H
5.44 ( 0.01
6.53 ( 0.08
6.32 ( 0.04
6.12 ( 0.05
6.01 ( 0.04
6.71 ( 0.15b
6.87 ( 0.18b
7.80 ( 0.08b
7.71 ( 0.20b
7.43 ( 0.08d
7.35 ( 0.09d
7.02 ( 0.14d
6.87 ( 0.11d
7.01 ( 0.09
8.61 ( 0.01
7.18 ( 0.02
8.24 ( 0.04
5.90 ( 0.02
5.97 ( 0.09
5.63 ( 0.09
6.11 ( 0.02
5.49 ( 0.01
5.42 ( 0.07b,c
5.29 ( 0.10b,c
5.37 ( 0.07b,c
5.73 ( 0.06b,c
5.64 ( 0.04c,d
5.98 ( 0.07
5.91 ( 0.04c,d
5.85 ( 0.04c,d
7.35 ( 0.03
7.61 ( 0.04
6.34 ( 0.06
7.45 ( 0.03
0
1
4
5
6
7
10
11
12
13
14
8
8
8
8
B
H
H
A
H
C
D
D
H
H
H
A
6
6
6
6
a
Compounds 1, 6-11, 13-21, and 25, tetrahydrochlorides; 2-4, trihydrochlorides; 5, trioxalate; 12, tetraoxalate; 22 and 24,
tetratrifluoroacetates; 23, pentahydrochloride. Data from ref 1. c Rat Ileum. Data from ref 19.
b
d
potency is reached when two positively charged nitro-
gens match the distance between two anionic sites on
the receptor surface rather than as a result of increasing
hydrophobicity.
more potent than 1 and 2, respectively at nAChR.
Studies by Mellor et al.8 on nAChR of TE671 cells
(expressing human, muscle-type nAChR), in which it
was possible to determine the voltage dependence of
antagonism and, thereby, whether the open channel
state of nAChR was antagonized, have shown that 22
interacts exclusively with the closed channel conforma-
tion of this receptor. Compound 22 has been used to
label nAChR of Torpedo electroplax; details of this study
are published elsewhere.29
The tetraamine 12, which has two terminal 2-meth-
oxybenzylamine moieties incorporated in a rather rigid
tricyclic system, was a weaker antagonist than 1 at both
nAChR and M2 mAChR while being similarly potent to
1 at M3 mAChR. This indicates that the 2-methoxy
group and the amine function are forced to assume a
position that does not allow them to interact optimally
with the receptor or, alternatively, the tricyclic system
sterically hinders binding of the ligand to nAChR and
M2 mAChR.
Finally, the potencies of branched polyamines (23-
25) at nAChR depended on the substituent on their
N-propylamino moiety. A comparison of 23 and 24
shows that a derivative containing a butyryltyrosyl
substituent was less potent at nAChR and at both M2
and M3 mAChR as well. The inclusion of 2-methoxy-
benzyl groups on the terminal amino functions of 24,
The photoaffinity label MR44 (22) proved to be a most
promising tool for studying the binding of a polyamine-
containing ligand to nAChR. It was 3-fold and 59-fold

5218 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 25
Rosini et al.
and NMR spectra were recorded on Perkin-Elmer 297 and
Varian VXR 300 instruments, respectively. Chemical shifts are
reported in parts per million (ppm) relative to tetramethylsi-
lane (TMS), and spin multiplicities are given as s (singlet), br
s (broad singlet), d (doublet), dd (double doublet), t (triplet),
dt (double triplet), q (quartet), or m (multiplet). Although 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. Fast atom
bombardment (FAB) and electron impact (EI) mass spectra
were obtained on VG707EH-F and VG7070E spectrometers,
respectively. 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.
Reactions were followed by thin-layer chromatography (TLC)
on Merck (0.25 mm) glass-packed precoated silica gel plates
(60 F254) that were visualized in an iodine chamber. The term
“dried” refers to the use of anhydrous sodium sulfate.
N1-{(1S)-2-{[6-({8-[(6-Am in oh exyl)a m in o]octyl}a m in o)-
h e xyl]a m in o}-1-[4-(b e n zyloxy)b e n zyl]-2-oxoe t h yl}b u -
ta n a m id e (30). A solution of 281 (0.32 g, 0.94 mmol) in
methanol (10 mL) was added dropwise with stirring at room
temperature to a suspension of 2616 (0.36 g, 0.78 mmol) in
methanol (10 mL). After 1 h, the reaction mixture was
evaporated to give a residue that was purified by flash
chromatography. Eluting with CHCl3-MeOH-aqueous 28%
ammonia (5:4.5:0.3) gave 30 as an oil: 31% yield; 1H NMR
(CDCl3) δ 0.85 (t, 3), 1.19-1.82 (complex m, 30), 2.02-2.38
(m, 2 + 4 exchangeable with D2O), 2.53-2.71 (m, 10), 2.96 (t,
2), 3.14 (q, 2), 4.57 (q, 1), 5.05 (s, 2), 6.16 (br s, 1, exchangeable
with D2O), 6.44 (d, 1, exchangeable with D2O), 6.91 (d, 2), 7.15
(d, 2), 7.28-7.46 (m, 5).
N1-[(1S)-2-{[6-({8-[(6-Am in oh exyl)a m in o]octyl}a m in o)-
h exyl]a m in o}-1-(4-h yd r oxyb en zyl)-2-oxoet h yl]b u t a n a -
m id e Tr ih yd r och lor id e (4). A solution of 30 (0.16 g, 0.24
mmol) in methanol (15 mL) was hydrogenated over 10% Pd
on charcoal (wet, Degussa type E101 NE/W) (16 mg) for 3 h.
Following catalyst removal, the solvent was evaporated, yield-
ing 4 as free base, which was converted into the trihydrochlo-
ride salt: 75% yield; mp 159-161 °C; 1H NMR (CD3OD) δ 0.84
(t, 3), 1.19-1.69 (complex m, 30), 2.15 (t, 2), 2.79-2.99 (m,
10), 3.07-3.14 (m, 2), 3.55-3.67 (m, 2), 4.44 (t, 1), 6.68 (d, 2),
7.04 (d, 2); MS (FAB) calcd for C33H61N5O3 576.5 [M + H+],
found 576.5.
N 1-{(1S )-1-[4-(B e n zy lo x y )b e n zy l]-2-[(6-{[8-({6-[(2-
m eth oxyben zyl)a m in o]h exyl}a m in o)octyl]a m in o}h exyl)-
a m in o]-2-oxoeth yl}bu ta n a m id e (31). It was synthesized
from 26 (0.2 g, 0.43 mmol) and 8 (0.2 g, 0.48 mmol) following
the procedure described for 30 and purified by flash chroma-
tography. Eluting with CHCl3-MeOH-aqueous 28% ammonia
(8.5:1.5:0.15) gave 31 as an oil: 18% yield; 1H NMR (CDCl3) δ
0.85 (t, 3), 1.23-1.90 (complex m, 30), 2.15 (t, 2), 2.52-2.67
(complex m, 10), 2.97 (d, 2), 3.15 (q, 2), 3.66 (br s, 3,
exchangeable with D2O), 3.79 (s, 2), 3.84 (s, 3), 4.64 (q, 1), 5.02
(s, 2), 6.71-6.94 (m, 6), 7.25 (d, 2), 7.29-7.43 (m, 5).
F igu r e 2. Effect of the carbon chain length of tetraamines 1
and 13-21 (n ) 4-8 or 10-14) on blocking activity of frog
rectus abdominis muscle nicotinic receptors. Data are from
Table 1.
to give 25, resulted, as expected, in a significant
improvement in potency at nAChR and M2 mAChR.
However, rather surprisingly, this structural modifica-
tion resulted in a significant increase in activity also
for M3 mAChR, as 25 was 13-fold and 20-fold more
potent than 24 and 1, respectively. Furthermore, 25
turned out to be almost as potent as 23 at nAChR and
at both M2 and M3 mAChR as well. The high potency of
23 for M2 mAChR deserves comment. In previous
studies1-6 tetraamines were shown to display a potency
higher than that of 1 only when an appropriate sub-
stituent was present on at least one of the two terminal
nitrogen atoms of the tetraamine backbone. Now we
have demonstrated that this may not be necessary
provided that one of the two inner nitrogen atoms
carries an appropriate substituent as in 23. It follows
that the affinity for mAChR can be tuned by an
appropriate substituent when it is present on any one
of the four nitrogen atoms of the tetraamine template.
Philanthotoxins or polyamine amides, like 2, have
more than one antagonistic mode of action on muscle
nAChR.8,9 Polyamine amide 2 acts noncompetitively on
the closed channel state of this receptor, although at
high concentrations it may also cause open channel
block and exhibit competitive antagonism.11 Further-
more, a recent report from one of our laboratories shows
that 2 potentiates nAChR by binding to a high-affinity
site on this receptor.8 This implies that the effect of 2
on this receptor is a balance between potentiation and
antagonism. Interestingly, noncompetitive antagonism
of neuronal nAChR by 2 is voltage-dependent, suggest-
ing that it is the open channel state of this receptor that
is targeted by the toxin.10 The interactions of polym-
ethylene tetraamines with nAChR may be similarly
complex. The question of whether 1 and 2 act at the
same site or sites on nAChR can only be addressed by
ligand binding studies.
N 1 -{(1 S )-1 -(4 -H y d r o x y b e n z y l )-2 -[(6 -{[8 -({6 -[(2 -
m eth oxyben zyl)a m in o]h exyl}a m in o)octyl]a m in o}h exyl)-
a m in o]-2-oxoeth yl}bu ta n a m id e Tr ioxa la te (5). It was
obtained from 31 (0.05 g, 0.064 mmol) following the procedure
described for 4 and purified by flash chromatography. Eluting
with CH2Cl2-EtOH-aqueous 28% ammonia (7.5:2.5:0.2) gave
the free base that was converted into the trioxalate salt: 68%
1
yield; mp 212-214 °C; H NMR (CD3OD) δ 0.85 (t, 3), 1.27-
1.90 (complex m, 30), 2.17 (t, 2), 2.71-3.22 (complex m, 14),
3.94 (s, 3), 4.22 (s, 2), 4.41-4.51 (m, 1), 6.71 (d, 2), 6.92-7.13
(m, 4), 7.40-7.51 (m, 2), 7.88-8.06 (m, 1, exchangeable with
D2O). Anal. (C47H75N5O16) C, H, N.
Exp er im en ta l Section
N 1 -{(1 S )-1 -(4 -H y d r o x y b e n z y l )-2 -[(3 -{[4 -({3 -[(2 -
m e t h oxyb e n zyl)a m in o]p r op yl}a m in o)b u t yl]a m in o}-
p r op yl)a m in o]-2-oxoet h yl}b u t a n a m id e Tr ih yd r och lo-
Ch em istr y. Melting points were taken in glass capillary
tubes on a Bu¨chi SMP-20 apparatus and are uncorrected. IR

Methoctramines as Receptor Antagonists
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 25 5219
r id e (3). A mixture of 216 (0.03 g, 0.07 mmol), molecular sieves
(3 Å), and 2-methoxybenzaldehyde (10.5 mg, 0.07 mmol) in
ethanol (10 mL) was stirred for 30 min at room temperature,
and then NaBH4 (2.9 mg, 0.07 mmol) was added and the
stirring was continued overnight. Following removal of mo-
lecular sieves, the solution was made acidic with 6 N HCl (2
mL). Removal of the solvent gave a residue, which was
dissolved in water (20 mL). The solution was washed with
ether (3 × 20 mL) to remove nonbasic materials and then was
made basic with 2 N NaOH, and finally extracted with
chloroform (3 × 20 mL). Removal of washed (brine) and dried
solvent gave 3 as the free base that was converted into the
trihydrochloride salt: quantitative yield; 1H NMR (CD3OD) δ
0.85 (t, 3), 1.29-1.80 (complex m, 10), 2.16 (t, 2), 2.40-2.72
(complex m, 10), 2.75-3.01 (m, 2), 3.15 (q, 2), 3.77-3.85 (m,
5), 4.45 (t, 1), 6.68 (d, 2), 6.81-7.09 (m, 4), 7.20-7.29 (m, 2).
Anal. (C31H52Cl3N5O4) C, H, N.
N1-(3-Am in op r op yl)-N4-{3-[(2-m eth oxyben zyl)a m in o]-
p r op yl}-1,4-bu ta n ed ia m in e Tetr a h yd r och lor id e (6). It
was obtained from 27 (1.5 g, 7.41 mmol) and 2-methoxyben-
zaldehyde (0.1 g, 0.74 mmol) following the procedure described
for 3. The free base was transformed into the tetrahydrocloride
salt: 30% yield; mp 233-235 °C (from EtOH); 1H NMR (D2O)
δ 1.67-1.76 (m, 4), 1.93-2.15 (m, 4), 2.96-3.12 (m, 12), 3.81
(s, 3), 4.21 (s, 2), 6.97-7.12 (m, 2), 7.30-7.52 (m, 2). Anal.
(C18H38Cl4N4O) C, H, N.
described for 33: 1H NMR (CDCl3) δ 1.20-1.69 (complex m,
33), 1.88 (br s, 2, exchangeable with D2O), 2.13-2.16 (m, 4),
2.69 (t, 2), 3.06-3.22 (m, 6), 4.68 (br s, 1, exchangeable with
D2O), 5.82 (br s, 2, exchangeable with D2O).
N 1-(2-Me t h o x y b e n zy l)-6,17-d io x o -7,16-d ia za -1,22-
d ocosa n ed ia m in e (36). A solution of 35 (1.41 g, 3.0 mmol)
and 2-methoxybenzaldehyde (0.45 g, 3.33 mmol) in toluene (80
mL) was refluxed and the water formed was continuously
removed for 3 h. The cooled mixture was filtered and the
filtrate was evaporated to give the corresponding Schiff base
that was dissolved in ethanol (50 mL) and treated with NaBH4
(0.125 g, 3.33 mmol). The mixture was stirred at room
temperature overnight, made acidic with 3 N HCl, and stirred
for a further 5 h. The solution was washed with ether (2 × 50
mL) and chloroform (2 × 50 mL), and then it was made basic
with 40% NaOH and finally extracted with chloroform. Re-
moval of dried solvents gave 36 as an oil: 67% yield; 1H NMR
(CDCl3) δ 1.30-1.63 (complex m, 27), 2.14-2.20 (m, 4), 2.59
(t, 2), 2.69 (t, 2), 3.18-3.27 (m, 4), 3.78 (s, 2), 3.84 (s, 3), 5.61
(br s, 2, exchangeable with D2O), 6.86-6.93 (m, 2), 7.22-7.28
(m, 2).
N 1-(2-Me t h oxyb e n zyl)-7,16-d ia za -1,22-d ocosa n e d i-
a m in e Tet r a h yd r och lor id e (8). A solution of 10 M BH3‚
CH3SCH3 (0.09 mL) in dry diglyme (4 mL) was added dropwise
at room temperature to a solution of 36 (0.85 g, 1.73 mmol) in
dry diglyme (40 mL) with stirring under a stream of dry
nitrogen. When the addition was completed, the reaction
mixture was heated at 120 °C for 18 h. After cooling at 0 °C,
excess borane was destroyed by cautious dropwise addition of
MeOH (7 mL). The resulting mixture was left to stand for 4 h
at room temperature, cooled at 0 °C, treated with HCl gas for
10 min, and then heated at 120 °C for 4 h. After cooling, ether
was added and the resulting mixture was stirred overnight
at room temperature, yielding a solid that was filtered: 55%
yield; mp 221-223 °C (from EtOH); 1H NMR (DMSO-d6) δ
1.29-1.64 (complex m, 28), 2.70-2.81 (complex m, 12), 3.84
(s, 3), 4.07 (m, 2), 6.96-7.11 (m, 2), 7.37-7.54 (m, 2), 8.06 (br
s, 2, exchangeable with D2O), 9.04 (br s, 3, exchangeable with
D2O).
N1,N8-Di[4-{[N-(b en zyloxy)ca r b on yl]a m in om et h yl}-
ben zyl]octa n ed ia m id e (37). It was obtained as a white solid
from suberic acid (0.52 g, 2.9 mmol) and benzyl N-[4-(ami-
nomethyl)benzyl]carbamate21 (1.6 g, 5.9 mmol) following the
procedure described for 32: 75% yield; mp 225-226 °C; 1H
NMR (DMSO-d6) δ 1.18-1.24 (m, 4), 1.41-1.48 (m, 4), 2.08
(t, 4), 4.14 (dd, 4), 5.0 (s, 4), 7.05-7.16 (m, 10), 7.21-7.33 (m,
8), 7.80 (t, 1, exchangeable with D2O), 8.22 (t, 1, exchangeable
with D2O).
N,N′-Di[6-{[(ben zyloxy)ca r bon yl]a m in o}h exyl]-1,1′-bi-
p h en yl-4,4′-d iyld ia m id e (38). It was obtained as a white
solid from 4,4′-biphenylcarboxylic acid (0.1 g, 0.42 mmol) and
N-[(benzyloxy)carbonyl]-1,6-hexanediamine21 (0.21 g, 0.84 mmol)
following the procedure described for 32: 12% yield; mp 244-
246 °C (from DMSO); 1H NMR (DMSO-d6) δ 1.20-1.68 (m,
16), 2.90 (dt, 4), 3.25 (dt, 4), 4.98 (s, 4), 7.23 (t, 2), 7.28-7.40
(m, 10), 7.81 (d, 4), 7.91 (d, 4), 8.52 (t, 2).
N1,N4-Di{3-[(2-m eth oxyben zyl)a m in o]p r op yl}-1,4-bu -
ta n ed ia m in e Tetr a h yd r och lor id e (7). It was obtained in
a quantitative yield from 27 (0.5 g, 2.47 mmol) and 2-meth-
oxybenzaldehyde (0.74 g, 5.43 mmol) following the procedure
described for 3. The free base was transformed into the
tetrahydrocloride salt: mp 205-208 °C; 1H NMR (D2O) δ
1.67-1.76 (m, 4), 2.03-2.12 (m, 4), 2.96-3.12 (m, 12), 3.85-
3.95 (m, 6), 4.18-4.26 (m, 4), 7.02-7.18 (m, 4), 7.32-7.52 (m,
4). Anal. (C26H46Cl4N4O2) C, H, N.
N1-(ter t-Bu toxyca r bon yl)-N15-[(ben zyloxy)ca r bon yl]-
6-oxo-7-a za -1,15-p en ta d eca n ed ia m in e (32) a n d N1-(ter t-
Bu toxyca r bon yl)-N22-[(ben zyloxy)ca r bon yl]-6,17-d ioxo-
7,16-d ia za -1,22-d ocosa n ed ia m in e (34). The procedure used
for the synthesis of 32 is described. Ethyl chlorocarbonate (1.72
mL, 18.0 mmol) in dry dioxane (5 mL) was added dropwise to
a stirred and cooled (5 °C) solution of N-(tert-butoxycarbonyl)-
6-aminocaproic acid (4.16 g, 18.0 mmol) and triethylamine (2.5
mL, 18.0 mmol) in dioxane (100 mL), followed after standing
for 30 min by the addition of N1-[(benzyloxy)carbonyl]-1,8-
octanediamine21 (4.9 g, 18.0 mmol) in dioxane (20 mL). After
being stirred at room temperature overnight, the mixture was
evaporated, affording a residue that was suspended in water
(150 mL). The precipitate was filtered and washed with 2 N
KHSO4, aqueous NaHCO3 satured solution, and water to give
1
32: 80% yield; mp 97-99 °C; H NMR (CDCl3) δ 1.29-1.70
(complex m, 27), 2.12-2.19 (t, 2), 3.08-3.24 (m, 6), 4.54 (br s,
1, exchangeable with D2O), 4.78 (br s, 1, exchangeable with
D2O), 5.09 (s, 1), 5.49 (br s, 1, exchangeable with D2O), 7.26-
7.36 (m, 5).
Similarly, 34 was obtained from N-[(benzyloxy)carbonyl]-
6-aminocaproic acid (3.32 g, 12.5 mmol) and 33 (4.70 g, 13.0
mmol): 64% yield; mp 107-109 °C (from THF); 1H NMR
(CDCl3) δ 1.29-1.69 (complex m, 33), 2.15 (t, 4), 3.06-3.25
(m, 8), 4.60 (br s, 1, exchangeable with D2O), 4.91 (br s, 1,
exchangeable with D2O), 5.08 (s, 2), 5.60 (br s, 2, exchangeable
with D2O), 7.30-7.35 (m, 5).
N1,N8-Di[4-(a m in om eth yl)ben zyl]octa n ed ia m id e (39).
A solution of 37 (1.4 g, 2.1 mmol) and 30% HBr in acetic acid
(12 mL) in CF3COOH (30 mL) was stirred for 1 h. Ether (30
mL) was then added, yielding a solid that was dissolved in
water. The solution was made basic with NaOH pellets and
extracted with chloroform (400 mL) for 10 h. The organic phase
was evaporated to give in a quantitative yield 39 as a white
N1-(ter t-Bu t oxyca r b on yl)-6-oxo-7-a za -1,15-p en t a d e-
ca n ed ia m in e (33). A solution of 32 (7.10 g, 14 mmol) in
methanol (110 mL) was hydrogenated over 10% Pd on charcoal
(0.7 g) for 45 min at room temperature and a pressure of 15
psi. Following catalyst removal, the solvent was evaporated,
1
solid: mp 240-243 °C; H NMR (DMSO-d6) δ 1.18-1.28 (m,
4), 1.41-1.52 (m, 4), 2.12 (t, 4), 3.66 (s, 4), 4.22 (s, 4), 7.05-
7.16 (m, 10), 7.12-7.28 (m, 8).
N,N′-Di(6-a m in oh exyl)-1,1′-b ip h en yl-4,4′-d iyld ia m id e
(40). It was obtained in a quantitative yield as a foam solid
from 38 (0.01 g, 0.10 mmol) as described for 39. It was not
possible to obtain an NMR spectrum owing to the insolubility
of the compound. Anal. (C26H38N4O2) C, H, N.
1
affording 33 in a quantitative yield: mp 69-71 °C; H NMR
(CDCl3) δ 1.29-1.68 (complex m, 29), 2.15 (t, 2), 2.66 (t, 2),
3.09-3.28 (m, 4), 4.61 (br s, 1, exchangeable with D2O), 5.58
(br s, 1, exchangeable with D2O).
N1-(ter t-Bu t oxyca r b on yl)-6,17-d ioxo-7,16-d ia za -1,22-
d ocosa n ed ia m in e (35). It was obtained from 34 (1.85 g, 3.1
mmol) as an oil in a quantitative yield following the procedure
N1,N8-Di(4-{[(2-m eth oxyben zyl)am in o]m eth yl}ben zyl)-
octa n ed ia m id e (41). A mixture of 39 (0.28 g, 0.68 mmol),
2-methoxybenzaldehyde (0.2 g, 1.5 mmol), NaBH4 (56 mg, 1.5

5220 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 25
Rosini et al.
mmol), and molecular sieve (3 Å) in dry methanol (60 mL) was
stirred at room temperature for 18 h. After cooling, the mixture
was cautiously made acidic with 6 N HCl, filtered, and
evaporated. The residue was dissolved in water, and the
resulting solution was washed with ether, made basic with 2
N NaOH, and extracted with CH2Cl2. Removal of the solvent
gave a residue that was purified by flash chromatography.
Eluting with CH2Cl2-EtOH-aqueous 28% ammonia (9:1:0.1)
Water was added, followed by extraction with ether. The
aqueous layer was made basic with 2 N NaOH and extracted
with CH2Cl2. Removal of the dried solvent gave crude 10 that
was transformed into the tetrahydrochloride salt: 70% yield;
mp 187-191 °C (from EtOH/MeOH); 1H NMR (CDCl3) δ 1.05-
1.60 (complex m, 16), 2.17 (br s, 4, exchangeable with D2O),
2.58 (t, 8), 3.73 (s, 6), 3.81 (s, 8), 3.95 (s, 2), 6.89 (q, 4), 7.08-
7.40 (m, 12). Anal. (C48H74Cl4N4O2) C, H, N.
cis-1-Ben zyl-1,2,3,3a ,4,9b-h exa h yd r och r om en o[4,3-b]-
p yr r ole Hyd r och lor id e (46). A mixture of N-benzylglycine
hydrochloride (2.4 g, 11.9 mmol) and chlorotrimethylsilane (15
mL, 118 mmol) was gently refluxed under nitrogen for 3 h.
After cooling, the formed solid was collected by filtration and
washed with dry ether. To a suspension of this solid in dry
toluene (70 mL) were added diisopropylethylamine (2.77 mL,
15.9 mmol) and 2-allyloxybenzaldehyde (0.41 mL, 2.7 mmol),
and the reaction mixture was heated under reflux and the
water formed was continuously removed for 18 h. Removal of
the solvent gave an oil that was partitioned between aqueous
NaHCO3-CH2Cl2. The organic phase was dried and evapo-
rated to give a residue that was purified by flash chromatog-
1
afforded 41: 75% yield; mp 152-155 °C; H NMR (CDCl3) δ
1.18-1.35 (m, 4), 1.53-1.66 (m, 4), 2.05 (br s, 2, exchangeable
with D2O), 2.16 (t, 4), 3.73-3.82 (d + s, 14), 4.38 (d, 4), 6.00
(t, 2, exchangeable with D2O), 6.90 (q, 4), 7.19-7.32 (m, 12).
N ,N ′-Di{6-[(2-m e t h oxyb e n zyl)a m in o]h e xyl}-1,1′-b i-
p h en yl-4,4′-d iyld ia m id e (42). It was obtained as a solid from
40 (0.05 g, 0.11 mmol) and 2-methoxybenzaldehyde (0.05 g,
0.36 mmol) in refluxing MeOH following the procedure de-
scribed for 41: 26% yield; mp 164-166 °C; 1H NMR (CD3OD)
δ 1.28-1.77 (m, 16), 2.19 (br s, 2, exchangeable with D2O),
2.52 (t, 4), 3.45-3.55 (m, 4), 3.82 (s, 4), 3.90 (s, 6), 6.42 (t, 2),
6.88-7.02 (m, 4), 7.23-7.40 (m, 4), 7.69 (d, 4), 7.89 (d, 4).
N1,N8-Di(4-{[(2-m eth oxyben zyl)am in o]m eth yl}ben zyl)-
1,8-octa n ed ia m in e Tetr a h yd r och lor id e (9). This com-
pound was obtained by reduction of 41 with BH3‚MeSMe as
described for 8: 60% yield; mp > 280 °C (from MeOH/2-PrOH);
1H NMR (DMSO-d6) δ 1.19-1.38 (m, 4), 1.55-1.75 (m, 4),
2.71-2.93 (m, 4), 3.80 (s, 6), 3.91-4.23 (m, 14), 6.93-7.11 (m,
4), 7.37-7.49 (m, 4) 7.57-7.71 (m, 8), 9.50 (br s, 8, exchange-
able with D2O). Anal. (C40H58Cl4N4O2) C, H, N.
raphy. Elution with
a gradient of 0-2% MeOH-CH2Cl2
afforded 46 as the free base that was transformed in a
quantitative yield into the hydrochloride salt: mp 175-176
°C (from 2-PrOH/ether); 1H NMR (CD3OD) δ 1.95-2.15 (m,
1), 2.44-2.56 (m, 1), 3.00-3.19 (m, 1), 3.30-3.52 (m, 2), 4.10-
4.14 (m, 2), 4.50-4.57 (m, 2), 4.80-4.97 (m, 2), 6.93-7.10 (m,
2), 7.23-7.42 (m, 2), 7.45-7.60 (m, 5).
cis-1,2,3,3a,4,9b-Hexah ydr och r om en o[4,3-b]pyr r ole (47).
A solution of 46 (0.31 g, 1.03 mmol) in MeOH (15 mL) was
hydrogenated over 10% Pd on charcoal (wet, Degussa type
E101 NE/W) (250 mg) overnight at 40 °C and a pressure of 75
psi. Following catalyst removal, the solvent was evaporated
to give a residue that was purified by flash chromatography.
Eluting with CH2Cl2-EtOH-aqueous 28% ammonia (9.1:1.0:
0.01) afforded 47: 85% yield; mp 210-215 °C (HCl salt from
EtOH/ether); 1H NMR (CDCl3) δ 1.40-1.60 (m, 1), 2.00-2.20
(m, 1), 2.27 (br s, 1, exchangeable with D2O) 2.38-2.58 (m, 1),
2.82-3.15 (m, 2), 3.52 (t, 1), 3.95 (d, 1) 4.03-4.11 (m, 1), 4.80-
4.97 (m, 2), 6.83-6.95 (m, 2), 7.15 (t, 1), 7.36 (d, 1).
Ben zyl N-{6-[cis-1,2,3,3a ,4,9b -H exa h yd r och r om en o-
[4,3-b]p yr r ol-1-yl]-6-oxoh exyl}ca r ba m a te (48). It was syn-
thesized from 47 (0.45 g, 2.57 mmol) and N-[(benzyloxy)-
carbonyl]-6-aminocaproic acid following the procedure reported
for 32 and purified by flash chromatography. Elution with a
step gradient system of EtOAc-cyclohexane (5:5) and CH2-
Cl2-EtOAc-EtOH (8.8:1:0.2) gave 48 as a clear oil: 70% yield;
1H NMR (CDCl3) δ 1.38-1.72 (complex m, 6), 1.93-2.59
(complex m, 5), 3.14 (q, 2) 3.35-3.52 (m, 2), 4.06-4.19 (m, 2),
5.04 (br s, 2 + 1 exchangeable with D2O), 5.52 (d, 1), 6.71 (d,
1), 6.83 (t, 1), 7.04 (t, 1), 7.16-7.35 (m, 5), 7.64 (d, 1).
1-[cis-1,2,3,3a ,4,9b-Hexa h yd r och r om en o[4,3-b]p yr r ol-
1-yl]-6-a m in o-1-h exa n on e (49). It was synthesized from 48
(0.76 g, 1.8 mmol) following the procedure described for 33.
The crude compound was taken up in 2 N HCl, and the
resulting solution was washed with ether, made basic with 2
N NaOH, and extracted with CH2Cl2. Removal of the dried
solvents gave 49 as a clear oil: 93% yield; 1H NMR (CDCl3) δ
1.32-1.74 (complex m, 6 + 2 exchangeable with D2O), 1.93-
2.68 (complex m, 7), 3.39-3.50 (m, 2), 4.06-4.21 (m, 2), 5.54
(d, 1), 6.71 (d, 1), 6.82 (t, 1), 7.07 (t, 1), 7.63 (d, 1).
N ,N ′-Di{6-[(2-m e t h oxyb e n zyl)a m in o]h e xyl}-1,1′-b i-
p h en yl-4,4′-d iyld im eth yla m in e Tetr a h yd r och lor id e (11).
This compound was obtained by reduction of 42 with BH3‚
MeSMe as described for 8: 47% yield; mp 245-250 °C (from
1
EtOH/ether); H NMR (DMSO-d6) δ 1.38-1.52 (m, 8), 1.65-
1.87 (m, 8), 2.95-3.15 (m, 8), 3.91 (s, 6), 4.20 (s, 4), 4.25 (s, 4),
6.98-7.12 (m, 4), 7.34-7.51 (m, 4), 7.62 (d, 4), 7.76 (d, 4). Anal.
(C42H62Cl4N4O2) C, H, N.
N1-{6-[(Mesitylsu lfon yl)a m in o]h exyl}-N1-(2-m eth oxy-
ben zyl)-2,4,6-tr im eth yl-1-ben zen esu lfon a m id e (44). A so-
lution of mesitylenesulfonyl chloride (0.41 g, 1.9 mmol) in
CH2Cl2 (6 mL) was added dropwise to a vigorously stirred
solution of 4320 dihydrochloride (0.31 g; 1.0 mmol) in 1 N
NaOH (6 mL). After the addition was complete, the biphasic
mixture was stirred for 24 h (0 °C to room temperature). The
layers were separated and the aqueous phase was extracted
with CH2Cl2 (2 × 10 mL). The combined organic extracts were
washed with 0.5 N HCl and then water and evaporated to give
44: 95% yield; mp 125-126 °C; 1H NMR (CDCl3) δ 0.92-1.52
(complex m, 8), 2.33 (s, 6), 2.65 (s, 6), 2.66 (s, 6), 2.84 (q, 2),
3.09 (t, 2), 3.73 (s, 3), 4.38 (s, 2), 4.57 (t, 1, exchangeable with
D2O), 6.81-6.92 (m, 2), 6.97 (d, 4), 7.09 (d, 1), 7.25 (t, 1).
N 1-{6-[(Me sit ylsu lfon yl)(4-{4-[((m e sit ylsu lfon yl){6-
[(m esitylsu lfon yl)(2-m eth oxyben zyl)am in o]h exyl}am in o)-
m eth yl]ben zyl}ben zyl)a m in o]h exyl}-N1-(2-m eth oxyben -
zyl)-2,4,6-tr im eth yl-1-ben zen esu lfon a m id e (45). Sodium
hydride (60% in mineral oil, 16 mg, 0.38 mmol) was added to
a solution of 44 (0.16 g, 0.27 mmol) in dry DMF (15 mL). The
resulting suspension was stirred for 1 h at room temperature
under nitrogen. A solution of di[(p-bromomethyl)phenyl]-
methane23 (46 mg, 0.13 mmol) in dry DMF (5 mL) was added
dropwise and the reaction mixture was heated at 85 °C for 20
h. After cooling to 0 °C, H2O (30 mL) was cautiously added,
followed by extraction with CH2Cl2 (3 × 30 mL). Removal of
the solvent gave a residue that was purified by flash chroma-
tography. Eluting with cyclohexanes-EtOAc (8:2) afforded 45
6-[cis-1,2,3,3a ,4,9b-Hexa h yd r och r om en o[4,3-b]p yr r ol-
1-yl]-1-h exa n a m in e (50). It was synthesized from 49 (0.48
g, 1.66 mmol) following the procedure described for 8: 75%
1
1
as an oil: 70% yield; H NMR (CDCl3) δ 0.92-1.52 (complex
yield; mp 192-195 °C (HCl salt from EtOH/ether); H NMR
m, 8), 2.33 (s, 6), 2.65 (s, 6), 2.66 (s, 6), 2.84 (q, 2), 3.09 (t, 2),
3.73 (s, 3), 4.38 (s, 2), 4.57 (t, 1, exchangeable with D2O), 6.81-
6.92 (m, 2), 6.97 (d, 4), 7.09 (d, 1), 7.25 (t, 1).
N1-(2-Meth oxyben zyl)-N6-(4-{4-[({6-[(2-m eth oxyben zyl)-
am in o]h exyl}am in o)m eth yl]ben zyl}ben zyl)-1,6-h exan edi-
a m in e Tetr a h yd r och lor id e (10). HBr in acetic acid (30%;
4 mL) was added to a solution of 45 (72 mg, 0.09 mmol) and
phenol (0.3 g, 0.4 mmol) in CH2Cl2 (4 mL) at 0 °C. The reaction
mixture was stirred overnight (0 °C to room temperature).
(CDCl3) δ 1.20-1.56 (complex m, 8 + 2 exchangeable with
D2O), 1.90-2.45 (complex m, 7), 2.63 (t, 2), 2.91-3.19 (m, 3),
3.50-3.75 (m, 1), 3.92-4.04 (m, 2), 6.81-6.93 (m, 2), 7.10-
7.24 (m, 2).
N1,N8-Di[6-(cis-1,2,3,3a ,4,9b-h exa h yd r och r om en o[4,3-
b]p yr r ol-1-yl)-1-h exyl]octa n ed ia m id e (51). It was synthe-
sized from 50 (0.34 g, 1.24 mmol) and suberic acid (0.11 g,
0.62 mmol) following the procedure described for 32 and
purified by gravity chromatography. Elution with CH2Cl2-

Methoctramines as Receptor Antagonists
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 25 5221
MeOH-aqueous 28% ammonia (9.4:0.6:0.06) gave 51 as a clear
oil: 45% yield; mp 175-180 °C (oxalate salt from EtOH/ether);
1H NMR (CDCl3) δ 1.08-1.70 (complex m, 24), 1.83-2.27
(complex m, 12), 2.30-2.44 (m, 2), 2.90-3.25 (m, 10), 3.85-
4.05 (m, 4), 5.45-5.57 (m, 2, exchangeable with D2O), 6.78-
6.93 (m, 4), 7.25-7.50 (m, 4).
N1,N8-Di[6-(cis-1,2,3,3a ,4,9b-h exa h yd r och r om en o[4,3-
b]p yr r ol-1-yl)-1-h exyl]-1,8-octa n ed ia m in e Tetr a oxa la te
(12). It was synthesized from 51 (0.09 g, 0.13 mmol) following
the procedure described for 8 and transformed into the
tetraoxalate salt: 70% yield; mp 192-195 °C (EtOH/MeOH);
1H NMR (CD3OD) δ 1.32-1.45 (complex m, 16), 1.60-1.83 (m,
12),1.93-2.10 (m, 2), 2.40-2.57 (m, 2), 2.94-3.04 (m, 12),
3.20-3.34 (m, 2), 3.47-3.56 (m, 2), 3.61-3.68 (m, 2), 4.00 (dt,
2), 4.15 (dd, 2), 4.65 (d, 2), 6.97 (d, 2), 7.07 (t, 2), 7.36 (t, 2),
7.49 (d, 2). Anal. (C50H74N4O18) C, H, N.
for 3 as an oil: 90% yield; 1H NMR (CDCl3) δ 1.22-1.41
(complex m, 32), 2.40-2.56 (m, 14), 2.83 (t, 2), 3.69 (s, 2), 3.75
(s, 3), 6.77-6.87 (m, 2), 7.12-7.19 (m, 2).
3-{{6-[(2-Me t h o x y b e n z y l)a m i n o ]h e x y l}[8-({6-[(2-
m e t h o x y b e n zy l)a m in o ]h e x y l}a m in o )o c t y l]a m in o }-
p r op a n en itr ile (58). It was obtained from 56 (0.22 g, 0.55
mmol) and 2-methoxybenzaldehyde (0.150 g, 1.21 mmol)
following the procedure described for 3 as an oil: 70% yield;
1H NMR (CDCl3) δ 1.29-1.69 (complex m, 31), 2.35-2.42 (m,
6), 2.53-2.56 (m, 8), 2.75 (t, 2), 3.76 (s, 4), 3.82 (s, 6), 6.82-
6.92 (m, 4), 7.17-7.28 (m, 4).
3-{{6-[(ter t-Bu t oxyca r b on yl)a m in o]h exyl}[8-(ter t-b u -
t oxyca r b on yl)({6-[(ter t-b u t oxyca r b on yl)a m in o]h exyl}-
a m in o)octyl]a m in o}p r op a n en itr ile (60). A solution of 56
as free base (0.1 g, 0.25 mmol) in chloroform (10 mL) and di-
tert-butyl dicarbonate (0.182 g, 0.83 mmol) was stirred over-
night and then washed with aqueous satured NaHCO3 solution
(2 × 30 mL) and brine (2 × 30 mL). Removal of dried solvents
gave in a quantitative yield 60 as a yellow oil: 1H NMR
(CDCl3) δ 1.20-1.51 (complex m, 55), 2.40 (t, 6), 2.76 (t, 2),
3.04-3.10 (m, 8), 4.62 (br s, 2, exchangeable with D2O).
N1,16,N22-Tr i(ter t-bu toxycar bon yl)-7-(3-am in opr opyl)-
7,16-d ia za -1,22-d ocosa n ed ia m in e (62). A suspension of 60
(0.95 g, 1.36 mmol), aqueous 40% NaOH (0.75 mL) and Raney
Ni (nickel sponge; suspension in water) (0.25 g) in EtOH (15
mL) was hydrogenated for 28 h at room temperature and a
pressure of 15 psi. Following catalyst removal, the solvent was
evaporated, yielding a residue that was dissolved in water (50
mL) and extracted with CHCl3 (3 × 20 mL). Removal of dried
solvents gave 62 as an oil: 90% yield; 1H NMR (CDCl3) δ 1.12-
1.43 (complex m, 55), 1.60 (t, 2), 2.31 (t, 4), 2.44 (t, 4), 2.80 (t,
2), 2.98-3.08 (m, 8), 4.70 (br s, 2, exchangeable with D2O).
N1,N8-Di(6-am in oh exyl)-N1-{3-[(2-m eth oxyben zyl)am i-
n o]p r op yl}-1,8-octa n ed ia m in e P en ta h yd r och lor id e (23).
A mixture of 62 (0.1 g, 0.15 mmol), 2-methoxybenzaldehyde
(0.023 g, 0.17 mmol), NaBH4 (7 mg, 0.17 mmol) and molecular
sieve (3 Å) in dry ethanol (15 mL) was stirred at room
temperature for 18 h. After cooling, the mixture was cautiously
made acidic with 3 N HCl and stirred for 2 h at room tem-
perature, then filtered and evaporated. The residue was dis-
solved in water, and the resulting solution was washed with
ether, made basic with 2 N NaOH, and extracted with CH2Cl2.
Removal of the dried solvents gave 23 as an oil: 25% yield;
1H NMR (CDCl3) δ 1.27-1.51 (complex m, 36), 2.32-2.47 (m,
6), 2.54-2.69 (m, 10), 3.77 (s, 2), 3.83 (s, 3), 6.81-6.94 (m, 2),
7.19-7.28 (m, 2). It was transformed into the pentahydrochlo-
ride salt as a hygroscopic solid. Anal. (C31H66Cl5N5O) C, H, N.
N1-{(1S)-2-{[3-((6-[ter t-Bu toxyca r bon yl]a m in oh exyl)-
{8-[(6-(ter t-b u t oxyca r b on yl]a m in oh exyl)a m in o]oct yl}-
am in o)pr opyl]am in o}-1-[4-(ben zyloxy)ben zyl]-2-oxoeth yl}-
bu ta n a m id e (64). It was obtained from 26 (0.28 g, 0.6 mmol)
and 62 (0.42 g, 0.6 mmol) following the procedure described
for 30 and purified by flash chromatography. Elution with CH2-
Cl2-MeOH (9:1) gave 64 as an oil: 65% yield; 1H NMR (CDCl3)
δ 0.86 (t, 3), 1.24-1.45 (complex m, 57), 1.58-1.62 (m, 2), 2.17
(t, 2), 2.38-2.50 (m, 6), 2.96 (t, 2), 3.05-3.16 (m, 8), 3.20-
3.26 (m, 2), 4.52-4.59 (m, 1), 4.75 (br s, 1, exchangeable with
D2O), 5.00 (s, 2), 6.28 (br s, 1, exchangeable with D2O), 6.85
(d, 2), 7.08 (d, 2), 7.23 (br s, 1, exchangeable with D2O), 7.30-
7.38 (m, 5).
N1,N4-Di(6-{[(ben zyloxy)ca r bon yl]a m in o}ca p r oyl)-1,4-
bu ta n ed ia m in e (52). It was synthesized from 1,4-butanedi-
amine (0.30 g, 3.36 mmol) and N-[(benzyloxy)carbonyl]-6-
aminocaproic acid following the procedure described for 32:
1
75% yield; mp 165-166 °C; H NMR (DMSO-d6) δ 1.18-1.24
(m, 4), 1.30-1.48 (m, 12), 1.99 (t, 4), 2.92-3.11 (m, 8), 4.97 (s,
4), 7.18-7.33 (m, 10 + 2 exchangeable with D2O), 7.73 (t, 2,
exchangeable with D2O).
N1,N4-Di(6-a m in oca p r oyl)-1,4-b u t a n ed ia m in e Dih y-
d r obr om id e (53). It was synthesized in quantitative yield
from 52 (1.40 g, 2.4 mmol) and 30% HBr in acetic acid (20
mL) following the procedure described for 39: mp 204-205
°C; 1H NMR (DMSO-d6) δ 1.13-1.60 (m, 16), 2.03 (t, 4), 2.65-
2.75 (m, 4), 2.93-3.12 (m, 4), 7.78 (br s, 8, exchangeable with
D2O).
N1,N4-Di[6-(2-m eth oxyben zyl)a m in o}ca p r oyl]-1,4-bu -
ta n ed ia m in e (54). It was synthesized from 53 (0.69 g, 2.19
mmol) and 2-methoxybenzaldehyde (0.66 g, 4.83 mmol) fol-
lowing the procedure described for 41: 80% yield; mp 78-80
°C; 1H NMR (CDCl3) δ 1.12-1.69 (m, 16), 1.80 (br s, 2,
exchangeable with D2O), 2.13 (t, 4), 2.60 (t, 4), 3.18 (q, 4), 3.74
(s, 4), 3.81 (s, 6), 6.25 (t, 2, exchangeable with D2O), 6.87 (q,
4), 7.17-7.27 (m, 4).
N1,N4-Di[6-{(2-m et h oxyb en zyl)a m in o}h exyl]-1,4-b u -
ta n ed ia m in e Tetr a h yd r och lor id e (13). This compound was
obtained by reduction of 53 with BH3‚MeSMe following the
procedure described for 8: 60% yield; mp 240 °C (from EtOH/
MeOH); 1H NMR (DMSO-d6) δ 1.22-1.40 (m, 8), 1.55-1.79
(m, 12), 2.73-2.95 (m, 12), 3.85 (s, 6), 4.08 (s, 4), 6.93-7.12
(m, 4), 7.35-7.49 (m, 4) 7.57-7.71 (m, 8), 9.10 (br s, 8,
exchangeable with D2O). Anal. (C36H58Cl4N4O2) C, H, N.
3-{[6-({8-[(6-Am in oh exyl)a m in o]oct yl}a m in o)h exyl]-
am in o}pr opan en itr ile Tetr ah ydr och lor ide (55) an d 3-((6-
Am in oh e xyl){8-[(6-a m in oh e xyl)a m in o]oct yl}a m in o)-
p r op a n en itr ile Tetr a h yd r och lor id e (56). A solution of
acrylonitrile (0.58 mL, 8.86 mmol) in MeOH (3 mL) was added
dropwise to a cooled (0 °C) and stirred solution of 28 (3.04 g,
8.86 mmol) in MeOH (10 mL). Stirring and cooling were
continued for 8 h, and then removal of the solvent gave a
complex mixture of compounds that was purified by flash
chromatography. Elution with a step gradient system of
CHCl3-MeOH-aqueous 28% ammonia (5:4.5:0.5 to 5:4.5:0.7)
afforded a main fraction that was transformed into the
tetrahydrochloride salts. They were separated by fractional
crystallization from MeOH. The precipitate was 55: 17% yield;
Rf 0.48 [eluent system CHCl3-MeOH-aqueous 28% ammonia
(5:4.5:1)]; mp 280 °C (dec); 1H NMR (free base) (CDCl3) δ 1.25-
1.63 (complex m, 33), 2.49-2.69 (m, 14), 2.91 (t, 2); EI MS m/z
395 (M+).
N1-{(1S)-2-{[3-((6-Am in oh exyl){8-[(6-am in oh exyl)am in o]-
oct yl}a m in o)p r op yl]a m in o}-1-[4-(b en zyloxy)b en zyl]-2-
oxoeth yl}bu ta n a m id e Tetr a tr iflu or oa ceta te (65). A so-
lution of 64 (0.39 g, 0.39 mmol) and CF3COOH (0.9 mL) in
dry CH2Cl2 (10 mL) was stirred at room temperature for 2 h.
Removal of the solvent gave 65 in a quantitative yield: 1H
NMR (CD3OD) δ 0.83 (t, 3), 1.38-1.85 (complex m, 32), 2.16
(t, 2), 2.80-3.08 (m, 16), 3.14-3.24 (m, 2), 4.42 (t, 1), 5.02 (s,
2), 6.91 (d, 2), 7.16 (d, 2), 7.28-7.41 (m, 5).
N1-[(1S)-2-{[3-((6-Am in oh exyl){8-[(6-am in oh exyl)am in o]-
octyl}a m in o)p r op yl]a m in o}-1-(4-h yd r oxyben zyl)-2-oxo-
eth yl]bu ta n a m id e Tetr a tr iflu or oa ceta te (24). It was ob-
tained in a quantitative yield as a hygroscopic solid from 65
(0.39 g, 0.39 mmol) following the procedure described for 4.
Evaporation of mother liquor afforded 56 as an oil: 15%
yield; Rf 0.42 [eluent system CHCl3-MeOH-aqueous 28%
1
ammonia (5:4.5:1)]; H NMR (free base) (CDCl3) δ 1.27-1.46
(complex m, 33), 2.40-2.45 (m, 6), 2.58-2.82 (complex m, 10);
EI MS m/z 395 (M+).
3-[(6-{[8-({6-[(2-Met h oxyb en zyl)a m in o]h exyl}a m in o)-
octyl]a m in o}h exyl)a m in o]p r op a n en itr ile (57). It was ob-
tained from 55 (0.19 g, 0.48 mmol) and 2-methoxybenzalde-
hyde (0.131 g, 1.06 mmol) following the procedure described

5222 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 25
Rosini et al.
1H NMR (CD3OD) δ 0.85 (t, 3), 1.38-1.85 (complex m, 32), 2.16
(t, 2), 2.80-3.08 (m, 16), 3.18-3.24 (m, 2), 4.38 (t, 1), 6.70 (d,
2), 7.05 (d, 2). Anal. (C44H72F12N6O11) C, H, N.
attained a maximal level and remained steady. Contractions
were recorded by means of a force displacement transducer
(FT.03 Grass and 7003 Basile) connected to a four-channel pen
recorder (Battaglia-Rangoni KV 380). 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 Left Atr ia : The guinea pig heart was rapidly
removed and washed by perfusion through the aorta with
oxygenated physiological salt solution, and right and left atria
were separated out. The left atria were mounted under 0.2-
0.3 g tension at 35 °C in Tyrode solution of the following
composition (mM): NaCl, 136.9; KCl, 5.4; MgSO4‚7H2O, 1.0;
CaCl2, 2.52; NaH2PO4, 0.4; NaHCO3, 11.9; glucose, 5.5. Tissues
were stimulated through platinum electrodes by square-wave
pulses (0.6-0.8 ms, 1 Hz, 1-5 V). Inotropic activity was
recorded isometrically. Tissues were equilibrated for 1 h, and
cumulative concentration-response curves to arecaidine pro-
pargyl ester (APE) (0.01-1 µM) were constructed. Following
incubation with the antagonist for 60 min, a new concentra-
tion-response curve to APE was obtained.
Gu in ea P ig Ileu m Lon gitu d in a l Mu scle: The terminal
portion of the ileum was excised after the 8-10 cm nearest to
the ileo-caecal junction was discarded. The tissue was cleaned,
and segments 2-3 cm long of ileum longitudinal muscle were
separated from the underlying circular muscle and set up
under 1g tension at 37 °C in organ baths containing Tyrode
solution of the following composition (mM): NaCl, 118; KCl,
4.75; CaCl2, 2.54; MgSO4, 1.2; KH2PO4‚2H2O, 1.19; NaHCO3,
25; glucose, 11. 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 APE (0.01-0.5 µM) were
obtained at 30-min intervals, the first one being discarded and
the second one taken as control. Following incubation with the
antagonist for 60 min, a new concentration-response curve
to the agonist was obtained.
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 under 1g tension for 60
min. Two cumulative concentration-response curves to car-
bachol (1-100 µM) were constructed at 45 min intervals, the
first one being discarded and the second one taken as control.
Following incubation with the antagonist for 30 min a new
concentration-response curve to the agonist was obtained.
N1-{(1S)-1-(4-H yd r oxyb en zyl)-2-[(3-{{6-[(2-m et h oxy-
b en zyl)a m in o]h exyl}[8-({6-[(2-m et h oxyb en zyl)a m in o]-
h exyl}a m in o)oct yl]a m in o}p r op yl)a m in o]-2-oxoet h yl}-
bu ta n a m id e Tetr a h yd r och lor id e (25). It was obtained from
24 (free base, 0.14 g, 0.22 mmol) following the procedure
described for 3 and purified by flash chromatography. Elution
with a step gradient system of CHCl3-MeOH-aqueous 28%
ammonia (9:1:0.1 to 8:2:0.17) gave 25 as the free base that
was converted into the tetrahydrochloride salt as a hygroscopic
solid: 72% yield; 1H NMR (CDCl3) δ 0.90 (t, 3), 1.24-1.46
(complex m, 30), 1.61 (q, 2), 2.14 (t, 2), 2.18-2.31 (m, 6), 2.50-
2.59 (m, 8), 2.70-3.05 (m, 2), 3.10-3.30 (m, 2), 3.75-3.81 (m,
10), 4.33-4.48 (m, 1), 6.35 (d, 1, exchangeable with D2O), 6.63
(d, 2), 6.83-7.01 (m, 6), 7.18-7.27 (m, 4). Anal. (C52H88Cl4N6O5)
C, H, N.
3,11,20,N26-Tetr a (ter t-bu toxyca r bon yl)-4,11,20-tr ia za -
1,26-h exa cosa n ed ia m in e (61). Compound 59 was obtained
in a quantitative yield as a yellow oil from 55 as free base
(0.60 g, 1.51 mmol) and di-tert-butyl dicarbonate (1.45 g, 6.64
mmol) following the procedure described for 60: 1H NMR (free
base) (CDCl3) δ 1.08-1.53 (complex m, 64), 2.43-2.59 (m, 2),
2.94-3.27 (complex m, 12), 3.40 (t, 2), 4.58 (br, 1, exchangeable
with D2O).
A solution of 59 (1.15 g, 1.45 mmol) in anhydrous ether (30
mL) was added to a stirred and cooled (0 °C) suspension of
LiAlH4 (0.2 g, 5.33 mmol) in anhydrous ether (100 mL) under
a nitrogen atmosphere. After 30 min of stirring, 1 N NaOH
(30 mL) was added to the mixture to destroy the excess of
LiAlH4. Following filtration through Celite, the organic phase
was separated and the aqueous phase was extracted with ether
(2 × 40 mL). Combined organic layers were washed with 5%
citric acid (2 × 20 mL) and brine (2 × 20 mL). Removal of the
dried solvent gave a residue that was purified by flash column.
Elution with CHCl3-MeOH (9:1) afforded 61 as an oil: 25%
yield; 1H NMR (free base) (CDCl3) δ 1.15-1.67 (complex m,
66), 1.71-1.82 (m, 2), 2.67 (t, 2), 3.01-3.29 (complex m, 14),
4.59 (br s, 1, exchangeable with D2O).
N1-(3-{[6-({8-[(6-Am in oh exyl)am in o]octyl}am in o)h exyl]-
a m in o}p r op yl)-4-a zid o-2-h yd r oxyb en za m id e Tet r a t r i-
flu or oa ceta te (22). A solution of 4-azidosalicylic acid N-hy-
droxysuccinimide ester26 (0.08 g, 0.29 mmol) in dry chloroform
(5 mL) was added dropwise to a stirred solution of 61 (0.21 g,
0.26 mmol) in distilled chloroform (10 mL). After stirring for
2 h at room temperature, the solvent was evaporated to give
a residue that was purified by flash chromatography. Eluting
with petroleum ether-acetone (7.5:2.5) afforded 63 as an oil:
0.1 g (40% yield);1H NMR (free base) (CDCl3) δ 1.24-1.47
(complex m, 64), 1.69-1.77 (m, 2), 3.10-3.18 (m, 12), 3.35-
3.39 (m, 4), 4.61 (br s, 1, exchangeable with D2O), 6.49-6.59
(m, 2), 7.62 (d, 2), 8.33-8.43 (br s, 1, exchangeable with D2O).
CF3COOH (0.25 mL) was added to a stirred solution of 63
(0.1 g, 0.10 mmol) in dry CH2Cl2 (3 mL) under a nitrogen
atmosphere. After stirring for 2 h at room temperature, the
solvent was removed to give an oil that was triturated with
ether (3 × 30 mL) to remove the excess CF3COOH, yielding
in a quantitative yield 22 as a foam solid: 1H NMR (CD3OD)
δ 1.27-1.34 (complex m, 16), 1.53-1.60 (m, 12), 1.82-1.93 (m,
2), 2.80-2.94 (complex m, 14), 3.40 (t, 2), 6.45-6.50 (m, 2),
7.68 (d, 2); MS (FAB) calcd for C30H56N8O2 561.8 [M + H+],
found 561.8.
Biology. F u n ction a l An ta gon ism : Guinea pigs of either
sex (200-400 g) and frogs (10-20 g) were sacrificed under
ketamine or ether anesthesia, respectively, and the organs
required were set up rapidly under a suitable resting tension
in 15-mL organ baths containing physiological salt solution
kept at appropriate temperature (see below) and aerated with
5% CO2-95% O2 at pH 7.4. Concentration-response curves
were constructed by cumulative addition of the agonist. The
concentration of agonist in the organ bath was increased
approximately 5-fold at each step, with each addition being
made only after the response to the previous addition had
Da ta An a lysis: Antagonism of mAChR, expressed as pKB
values, was estimated according to the Furchgott equation:
KB ) [antagonist]/(DR - 1), where DR is the ratio between
individual EC50 values in the presence and in the absence of
antagonists.27 The potency of the agonist, i.e., the concentration
resulting in 50% of the maximum response (EC50), was
estimated graphically from the individual concentration-
response curves after checking for parallelism of the curves.
Antagonism of nAChR was estimated by determining the
concentration of the noncompetitive antagonist that inhibited
50% of the maximum response to the agonist. Three different
antagonist concentrations were used and each concentration
was tested at least four times. Data were analyzed by a
pharmacological computer program.30
Ack n ow led gm en t. This research was supported by
grants from the University of Bologna, the European
Community (BMH4-CT97-2395), and MURST.
Refer en ces
(1) 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.
(2) (a) Melchiorre, C. Polymethylene Tetraamines: a New Genera-
tion of Selective Muscarinic Antagonists. Trends Pharmacol. Sci.
1988, 9, 216-220. (b) Melchiorre, C.; Minarini, A.; Angeli, P.;
Giardina`, D.; Gulini, U.; Quaglia, W. Polymethylene Tet-

Methoctramines as Receptor Antagonists
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 25 5223
raamines as Muscarinic Receptor Probes. Trends Pharmacol. Sci.
1989, Suppl. IV, 55-59. (c) Melchiorre, C. Polymethylene
Tetraamines: a Novel Class of Cardioselective M2 Antagonists.
Med. Res. Rev. 1990, 3, 327-349.
(16) Goodnow, R., J r.; Konno, K.; Niwa, M.; Kallimopoulos, T.;
Bukonwik, R.; Lenares, D.; Nakanishi, K. Synthesis of Glutamate
Receptor Antagonist Philanthotoxin-433 (PhTX-433) and Its
Analogues. Tetrahedron 1990, 46, 3267-3286.
(17) Choi, S.-K.; Goodnow, R. A.; Kalivretenos, A.; Chiles, G. W.;
Fushiya, S.; Nakanishi, K. Synthesis of Novel and Photolabile
Philanthotoxin Analogues: Glutamate Receptor Antagonists.
Tetrahedron 1992, 48, 4793-4822.
(18) Nakanishi, K.; Huang, X.; J iang, H.; Liu, Y.; Fang, K.; Huang,
D.; Choi, S.-K.; Katz, E.; Eldefrawi, M. Structure-Binding
Relation of Philanthotoxins from Nicotinic Acetylcholine Recep-
tor Binding Assay. Biorg. Med. Chem. 1997, 5, 1969-1988.
(19) Melchiorre, C.; Quaglia, W.; Picchio, M. T.; Giardina`, D.; Brasili,
L.; Angeli, P. Structure-Activity Relationships among Meth-
octramine-Related Polymethylene Tetraamines. Chain-Length
and Substitution Effects on M2 Muscarinic Receptor Blocking
Activity. J . Med. Chem. 1989, 32, 79-84.
(20) Quaglia, W.; Giardina`, D.; Marucci, G.; Melchiorre, C.; Minarini,
A.; Tumiatti, V. Structure-Activity Relationships Among Meth-
octramine-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.
(3) (a) Melchiorre, C. Tetramine Disulfides: a New Tool in R-Adre-
nergic Pharmacology. Trends Pharmacol. Sci. 1981, 2, 209-211.
(b) Melchiorre, C.; Recanatini, M.; Bolognesi, M. L.; Filippi, P.;
Minarini, A. Polyamines:
a Case of Multiple Yet Specific
Receptor Recognition. Curr. Top. Med. Chem. 1993, 1, 43-65.
(4) (a) Melchiorre, C.; Bolognesi, M. L.; Chiarini, A.; Minarini, A.;
Spampinato, S. Synthesis and Biological Activity of Some
Methoctramine-Related Tetraamines Bearing an 11-Acetyl-5,-
11-dihydro-6H-pyrido[2,3-b][1,4]-benzodiazepin-6-one Moiety as
Antimuscarinics: a Second Generation of Highly Selective M2
Muscarinic Receptor Antagonists. J . Med. Chem. 1993, 36,
3734-3737. (b) Minarini, A.; Bolognesi, M. L.; Budriesi, R.;
Canossa, M.; Chiarini, A.; Spampinato, S.; Melchiorre, C. Design,
Synthesis, and Biological Activity of Methoctramine-Related
Tetraamines Bearing an 11-Acetyl-5,11-dihydro-6H-pyrido[2,3-
b][1,4]benzodiazepin-6-one Moiety: Structural Requirements for
Optimum Occupancy of Muscarinic Receptor Subtypes As Re-
vealed by Symmetrical and Unsymmetrical Polyamines. J . Med.
Chem. 1994, 37, 3363-3372.
(5) Melchiorre, C.; Angeli, P.; Brasili, L.; Giardina`, D.; Pigini, M.;
Quaglia, W. Polyamines: a Possible “Passe-Partout” for Receptor
Characterization. Actual. Chim. The´r. - 15e se´rie 1988, 149-
168.
(6) Bolognesi, M. L.; Minarini, A.; Budriesi, R.; Cacciaguerra, S.;
Chiarini, A.; Spampinato, S.; Tumiatti, V.; Melchiorre, C.
Universal Template Approach to Drug Design: Polyamines as
Selective Muscarinic Receptor Antagonists. J . Med. Chem. 1998,
41, 4150-4160.
(7) Eldefrawi, A. T.; Eldefrawi, M. E.; Konno, K.; Mansour, N. A.;
Nakanishi, K.; Oltz, E.; Usherwood, P. N. R. Structure and
Synthesis of a Potent Glutamate Receptor Antagonist in Wasp
Venom. Proc. Natl. Acad. Sci. U.S.A. 1988, 85, 4910-4913.
(8) Mellor, I. R.; et al. Unpublished results.
(9) J ayaraman, V.; Usherwood, P. N. R.; Hess, G. P. Inhibition of
the Nicotinic Acetylcholine Receptor by Philanthotoxin-343:
Kinetic Investigations in the Microsecond Time Region Using a
Laser-Pulse Photolysis Technique. Biochem. J . 1999 (in press).
(10) Liu, M.; Nakazawa, K.; Inoue, K.; Ohno, Y. Potent and Voltage-
Dependent Block by Philanthotoxin-343 of Neuronal Nicotinic
Receptor Channels in PC-12 Cells. Br. J . Pharmacol. 1997, 122,
379-385.
(21) N-[(Benzyloxy)carbonyl]-1,8-octanediamine and benzyl N-[4-
(aminomethyl)benzyl]carbamate were synthesized from 1,8-
octanediamine and p-xylenediamine, respectively, following the
procedure reported in ref 22.
(22) Atwell, G. J .; Denny, W. A. Monoprotection of R,ω-Alkanedi-
amines with the N-Benzyloxycarbonyl Group. Synthesis 1984,
27, 1032-1033.
(23) Steinberg, H.; Cram, D. J . Macro Rings. II. Polynuclear Para-
cyclophanes. J . Org. Chem. 1952, 17, 5388-5391.
(24) Confalone, P. N.; Huie, E. M. The Stabilized Iminium Ylide-
Olefin [3+2] Cycloaddition Reaction. Total Synthesis of Scele-
tium Alkaloid A4. J . Am. Chem. Soc. 1984, 106, 7175-7178.
(25) Minarini, A.; Quaglia, W.; Tumiatti, V.; Melchiorre, C. An
Improved Synthesis of N,N′-Bis[6-o-methoxybenzyl methyl hex-
ylamino]-1,8-octanediamine (Methoctramine). Chem. Ind. 1989,
652-653.
(26) Dupuis, G. An Asymmetrical Disulfide-Containing Photoreactive
Heterobifunctional Reagent Designed to Introduce Radioactive
Labeling Into Biological Receptors. Can. J . Chem. 1987, 65,
2450-2453.
(27) Furchgott, R. F. The Classification of Adrenoceptors (Adrenergic
Receptors). An Evaluation from the Standpoint of Receptor
Theory. In Catecholamines; Blaschko, H., Muscholl, E., Eds.;
Springer-Verlag: Berlin, 1972; pp 283-335.
(28) Teodori, E.; Gualtieri, F.; Angeli, P.; Brasili, L.; Giannella, M.;
Pigini, M. Resolution, Absolute Configuration, and Cholinergic
Enantioselectivity of (+)- and (-)-cis-2-Methyl-5-[(dimethylami-
no)methyl]-1,3-oxathiolane Methiodide. J . Med. Chem. 1986, 29,
1610-1615.
(11) Rozental, R: Giles, G.; Scoble, T.; Albuquerque, E. X.; Idriss,
M.; Sherby, S.; Satelle, D. B.; Nakanishi, K.; Konno, K.; Elde-
frawi, A. T.; Eldefrawi, M. E. Allosteric Inhibition of Nicotinic
Acetylcholine Receptors of Vertebrates and Insects by Philan-
thotoxin. J . Pharmacol. Exp. Ther. 1989, 249, 123-130.
(12) Usherwood, P. N. R.; Blagbrough, I. S. Electrophysiology of
Polyamines and Polyamine Amides. In Neuropharmacology of
Polyamines; Carter, C. J ., Ed.; Academic Press: London, 1994;
pp 185-204.
(13) Usherwood, P. N. R.; Blagbrough, I. S. Spider Toxins Affecting
Glutamate Receptors: Polyamines in Therapeutic Neurochem-
istry. Pharmacol. Ther. 1991, 52, 245-268.
(14) Sudan, H. L.; Kerry, C. J .; Mellor, I. R.; Choi, S.-K.; Huang, D.;
Nakanishi, K.; Usherwood, P. N. R. The Action of Philantho-
toxin-343 and Pholabile Analogues on Locust (Schistocerca
gregaria) Muscle. Invertebrate Neurosci. 1995, 1, 159-172.
(15) Choi, S.-K.; Kalivretenos, A. G.; Usherwood, P. N. R.; Nakanishi,
K. Labeling Studies of Photolabile Philanthotoxins with Nicotinc
Acetylcholine Receptors: Mode of Interaction Between Toxin and
Receptor. Chem. Biol. 1995, 2, 23-32.
(29) Bixel, M. G.; et al. unpublished results.
(30) Tallarida, R. J .; Murray, R. B. Manual of Pharmacological
Calculations with Computer Programs, Version 4.2; Springer-
Verlag: New York, 1991.
J M991110N