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J. Med. Chem. 1999, 42, 5212-5223
Design, Synthesis, and Biological Evaluation of Symmetrically and Unsymmetrically Substituted Methoctramine-Related Polyamines as Muscular Nicotinic Receptor Noncompetitive Antagonists 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 June 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-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. Introduction Methoctramine (1, Figure 1) is the prototype polymethylene tetraamine for antagonism of muscarinic acetylcholine receptors (mAChR).1 The development of polymethylene tetraamines as antagonists of mAChR has been the subject of several review articles.2 Appropriate 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 process.5 More recently, it has been suggested that such a structure may represent a universal template on which †
University of Bologna. Freie Universita¨t Berlin. Royal Danish School of Pharmacy. | University of Nottingham. ‡ §
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 opportunity to apply the universal template approach to the design of polyamines as nAChR ligands. Philanthotoxins, as exemplified by philanthotoxin-343 (PhTX-343) (2, Figure 1), a synthetic analogue of a wasp (Philanthus triangulum) toxin PhTX-433,7 are noncompetitive 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.
10.1021/jm991110n CCC: $18.00 © 1999 American Chemical Society Published on Web 11/23/1999
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Journal of Medicinal Chemistry, 1999, Vol. 42, No. 25 5213
Scheme 1
Figure 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 ionotropic 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 polymethylene 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-methoxybenzyl groups of 1 have been included into a tricyclic system (12) to determine whether the spatial relationship of the methoxy moiety relative to the amine function differently affects affinity for nAChR and mAChR. Previously, homologues of 1 have been investigated with the aim of assessing the structural requirements 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 photolabile analogue MR44 (22). During synthesis of the linear pentaamine required for 22, the branched isomeric pentaamine was obtained, which gave the opportunity 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 muscletype nAChR. Chemistry All the compounds were synthesized by standard procedures (Schemes 1-7) and were characterized by IR, 1H NMR, mass spectra, and elemental analysis. Triamine amides 4 and 5 were synthesized by following an adapted procedure described for 2.7,16 Thus,
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-methoxybenzaldehyde 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-aminocaproic acid was amidated with N-[(benzyloxy)carbonyl]1,8-octanediamine21 to give 32. Removal of the N-(benzyloxy)carbonyl group was achieved by catalytic hydrogenation over 10% palladium on charcoal. Thus, hydrogenolysis of 32 gave 33, which was reacted with N-[(benzyloxy)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 hydrogenolysis gave 35, which was treated with 2-methoxybenzaldehyde 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. Reduction of 36 with borane-methyl sulfide complex gave 8 (Scheme 2). Suberic acid and 4,4′-biphenyldicarboxylic acid were amidated with benzyl N-[4-(aminomethyl)benzyl]car-
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Rosini et al.
Scheme 2a
Scheme 3a
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-bromomethyl)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-benzylglycine and 2-allyloxybenzaldehyde. Removal of the benzyl group of 46 by hydrogenolysis gave 47, which was
Scheme 4
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
Journal of Medicinal Chemistry, 1999, Vol. 42, No. 25 5215
Scheme 5
Scheme 6
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 dissociation 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 parameter.28 The results are expressed as IC50 values, i.e., the concentrations required to inhibit the maximal response to carbachol by 50%. In all of the experiments, 1 and 2 were used as standards. Results and Discussion
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 amidated 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 transformation 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 corresponding amines 61 and 62. 4-Azidosalicylic acid N-hydroxysuccinimide 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-methoxybenzaldehyde 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. Condensation 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-
Summaries of the results are presented in Table 1 and Figure 2. Over the concentration range investigated, all of the compounds were noncompetitive antagonists of muscle-type nAChR. The maximum response 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 antagonist 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
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Rosini et al.
Scheme 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 moieties have been introduced onto the spermine backbone of 2, affording 3, 6, and 7. The inclusion of a methoxybenzylamine 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 investigated 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 polymethylene 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 specifity 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 nitrogens of 1 when it is in its fully extended comformation. 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
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Journal of Medicinal Chemistry, 1999, Vol. 42, No. 25 5217
Table 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 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
R1 H A H A A A A
R2
H H H
R3
m
H A H
8 4 4 8 8 4 4 8
n 3 3 6 6 3 3 6
0 1
B H H A
H C D D
H H H A
4 5 6 7 10 11 12 13 14 8 8 8 8
6 6 6 6
pIC50 (FRA)
M2
M3
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
