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

Polymethylene tetraamine methoctramine (1) is a prototypical antimuscarinic ligand endowed with a significant affinity for muscular nAChRs. Thus, according to the universal template approach, structural modifications were performed on 1 in order to improve affinity and selectivity for the muscle-type nAChR. The polyamine derivatives synthesized were tested at both frog rectus and Torpedo nAChRs and at guinea pig left atria (M(2)) and ileum longitudinal muscle (M(3)) mAChRs. All of the compounds, like prototype 1, were noncompetitive antagonists of nicotinic receptors while being competitive antagonists at M(2) and M(3) mAChRs. The biological profile of polyamines 4-7 revealed that increasing the number of amine functions and the chain length separating these nitrogen atoms led to a significant improvement in potency at nAChRs. Moreover, the role of the number and type of amine functions in the interaction with nAChRs was further investigated through the synthesis of compounds 9 and 10. Tetraamines 8 and 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 nAChRs, whereas a significant decrease in potency was observed at mAChRs. Tetraamine 12, bearing a 2-methoxyphenethyl group, was less potent than 1, whereas tetraamine 13, carrying a diphenylethyl moiety, was more potent than 1, confirming that an increase in size of the hydrophobic group on the terminal nitrogen atoms increases significantly the binding affinity for nAChRs. Tetraamines 14-17 were significantly more potent than prototype 2 at both frog rectus and Torpedo nAChRs, confirming that an increase in the distance between the amine functions results in a parallel increase in the affinity for nAChRs. To gain insight into the mode of interaction of polymethylene tetraamines with nAChRs, photolabile (19 and 20) and fluorescent (21 and 22) compounds were synthesized. A most intriguing finding was the observation that 19, which bears two identical azido groups on the terminal nitrogen atoms, was found to bind the Torpedo nAChR with a 1:1 stoichiometry, suggesting a U-shaped conformation in the receptor interaction. Moreover, the high potency shown by fluorescent compounds 21 and 22 appears promising for a further characterization of the polymethylene tetraamines binding site with the muscle-type nAChR.

To map the structure of a ligand-gated ion channel, we used the photolabile polyamine-containing toxin MR44 as photoaffinity label. MR44 binds with high affinity to the nicotinic acetylcholine receptor in its closed channel conformation. The binding stoichiometry was two molecules of MR44 per receptor monomer. Upon UV irradiation of the receptor-ligand complex, I-125-MR44 was incorporated into the receptor alpha -subunit. From proteolytic mapping studies, we conclude that the site of I-125-MR44 cross-linking is contained in the sequence alpha His-186 to alpha Leu-199, which is part of the extracellular domain of the receptor. This sequence partially overlaps in its C-terminal region with one of the three leaps that form the agonist-binding site. The agonist carbachol and the competitive antagonist alpha -bungarotoxin had only minor influence on the photocross-linking of I-125-MR44. The site where the hydrophobic head group of I-125-MR44 binds must therefore be located outside the zone that is sterically influenced by agonist bound at the nicotinic acetylcholine receptor. In binding and photocross-linking experiments, the luminal noncompetitive inhibitors ethidium and triphenylmethylphosphonium were found to compete with I-125-MR44. We conclude that the polyamine moiety of I-125-MR44 interacts with the high affinity noncompetitive inhibitor site deep in the channel of the nicotinic acetylcholine receptor, while the aromatic ring of this compound binds in the upper part of the ion channel (i.e. in the vestibule) to a hydrophobic region on the alpha -subunit that is located in close proximity to the agonist binding site. The region of the alpha -subunit labeled by I-125-MR44 should therefore be accessible from the luminal side of the vestibule.

Several wasp venoms contain philanthotoxins (PhTXs) that act as noncompetitive inhibitors (NCIs) on cation-selective ion channels including the nicotinic acetylcholine receptor (nAChR). In the search for a ligand with high affinity and specificity for the nAChR we tested a series of newly developed PhTX analogues. Modulation of the structural elements of PhTXs can significantly influence their binding affinities. This approach resulted in the development of the photolabile compound MR44. In photoaffinity labelling studies 125I-MR44 was used to map the ligand-binding site at the Torpedo californica nAChR. Upon UV irradiation of the receptor-ligand complex, 125I-MR44 was mainly incorporated into the receptor alpha-subunit. Proteolytic mapping and microsequencing identified the site of 125I-MR44 cross-linking within the sequence alphaHis-186 to alphaLeu-199 that in its C-terminal region partially overlaps with the agonist-binding site. Since bound agonists had only minor influence on 125I-MR44 photocrosslinking, the site where the hydrophobic head group of 125I-MR44 binds must be located outside the zone that is sterically influenced by agonists bound at the nAChR. A possible site of interaction of 125I-MR44 would be the N-terminal region of the labelled sequence, in which aromatic amino-acid residues are accumulated. We suggest that the polyamine moiety of 125I-MR44 interacts with the high affinity non-competitive inhibitor site deep in the ion channel, while the aromatic ring of this compound binds in the vestibule of the nAChR to a hydrophobic region on the alpha-subunit that is located close to the agonist binding site.

Several wasp venoms contain philanthotoxins (PhTXs), natural polyamine amides, which act as noncompetitive inhibitors (NCIs) on the nicotinic acetylcholine receptor (nAChR). Effects of varying the structure of PhTXs and poly(methylene tetramine)s on the binding affinity have been investigated. Using the fluorescent NCI ethidium in a displacement assay Kapp values of these compounds have been determined. We found that an increase in size of the PhTX's hydrophobic head group significantly increased the binding affinity, while inserting positive charge almost completely destroyed it. Elongating the PhTX polyamine chain by introducing an additional aminomethylene group decreased the binding affinity, whereas a terminal lysine improved it. In general, poly(methylene tetramine)s showed higher binding affinities than PhTX analogues. The stoichiometry of PhTX binding was determined to be two PhTX molecules per receptor monomer. PhTXs appeared to bind to a single class of nonallosterically interacting binding sites and bound PhTX was found to be completely displaced by well-characterized luminal NCIs. To elucidate the site of PhTX binding, a photolabile, radioactive PhTX derivative was photocross-linked to the nAChR in its closed channel conformation resulting in labeling yields for the two alpha and the beta, gamma and delta subunits of 10.4, 11.1, 4.0 and 7.4%, respectively. Based on these findings we suggest that PhTXs and poly(methylene tetramine)s enter the receptor's ionic channel from the extracellular side. The hydrophobic head groups most likely bind to the high-affinity NCI site, while the positively charged polyamine chains presumably interact with the negatively charged selectivity filter located deep in the channel lumen.

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 (M(2)) and ileum longitudinal muscle (M(3)) 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 M(2) and M(3) 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 microM range while not showing any antagonism for muscarinic receptors up to 10 microM. 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 M(2) 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 M(2) 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.