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Continuing our studies on polyamine-based compounds of potential interest in the field of Alzheimer's disease therapeutics, we investigated the structure-activity relationships (SAR) of a lead compound (caproctamine, 3) identified in a previous work. In particular, we varied the substituents on the phenyl ring and on the nitrogen functions (both the amine and the amide), and studied the effects of such modifications on the inhibitory potency against isolated acetyl- and butyryl-cholinesterase (AChE and BChE). Moreover, the ability of selected compounds to reverse the d-tubocurarine-induced neuromuscular blockade and their antagonism toward muscarinic M(2) receptors in guinea pig left atrium were assayed. The most interesting SAR result was the identification of a relationship between the electronic characteristics of 2-substituents (measured by pK(a)) and the AChE inhibitory potency (pIC(50)) of tertiary amine compounds 6-12, which was confirmed by the invariance of the pIC(50) values of the corresponding methiodide derivatives 14-20. With regard to the biological profile, the most interesting compound was the N-ethyl-analogue of caproctamine (9), that showed pIC(50) values of 7.73 (+/-0.02) and 5.65 (+/-0.03) against AChE and BChE, respectively. The ability to increase the acetylcholine level was maintained in the functional assay (pAI(50) for reversing the neuromuscular blockade was 6.45 (+/-0.07)), as well as the ability to antagonize the M(2) receptors (pK(b) = 5.65 (+/-0.06)). Moreover, 9 showed a long duration of action as AChE inhibitor, an useful property in view of a possible development of this compound as a therapeutic agent.

WB4101 (1)-related compounds 5-10 were synthesized, and their biological profile at alpha(1)-adrenoreceptor (AR) subtypes and 5-HT(1A) serotoninergic receptors was assessed by binding assays in Chinese hamster ovary and HeLa cell membranes expressing the human cloned receptors. Moreover, their receptor selectivity was further determined in functional experiments in isolated rat prostate (alpha(1A)), vas deferens (alpha(1A)), aorta (alpha(1D)), and spleen (alpha(1B)). In functional assays, compound 5 was the most potent at alpha(1D)-ARs with a reversed selectivity profile (alpha(1D)alpha(1A)alpha(1B)) relative to both prototype 1 and phendioxan (2) (alpha(1A)alpha(1D)alpha(1B)), whereas compound 8, bearing a carbonyl moiety at position 1, was the most potent at alpha(1A)-ARs with a selectivity profile similar to that of prototypes. The least potent of the series was the trans isomer 6, suggesting that optimum alpha(1)-AR blocking activity in this series is associated with a cis relationship between the 2-side chain and the 4-phenyl ring rather than a trans relationship as previously observed for the 2-side chain and the 3-phenyl ring in 2 and related compounds. Binding affinity results were not in complete agreement with the selectivity profiles deriving from functional experiments. Although a firm explanation was not available, neutral and negative antagonism and receptor dimerization were considered as two possibilities to account for the difference between binding and functional affinities. Finally, compound 5 was selected for a modeling study in comparison with 1, mephendioxan (3), and open phendioxan (4) to achieve information on the physicochemical interactions that account for its high affinity toward alpha(1d/D)-ARs.

A series of muscarinic agonists, straight chained, branched, cyclic alkyl and aromatic derivatives of the oxime 1 (demox) was designed with the aim of investigating their activity on muscarinic receptor subtypes. Effects on M1 receptor were assessed functionally by a microphysiometer apparatus, while M2, M3, and M4 receptor potency and affinity were studied on isolated preparations of guinea pig heart, ileum, and lung, respectively. The results suggest that the substitution of a hydrogen with a long side-chain or bulky group generally induces a decrease in potency at M1 and M3 subtypes, while a general increase in this parameter is obtained at M2 subtype. Among the agonists 2-18, compound 4 behaves as a full agonist with a preference for M3 subtype. Moreover, compound 12 is inactive at M1 and M4 receptors while it displays a full agonist activity at M2 and M3 subtypes. Since demox displays a variable response on cardiac M2 receptors regulating heart force, an in-depth inquiry of the functional behaviour of this compound was carried out at M2 receptors. In presence of 10(-11) and 10(-10) M demox, the binding of [3H]-NMS was increased by approximately 30% as a consequence of an increase of the association of [3H]-NMS to membranes; this effect was not observed in presence of a higher concentration of [3H]-NMS. Higher concentrations of demox decreased the binding of [3H]-NMS to heart atrial membranes but significantly retarded the dissociation of this radioligand. Our results suggest that demox may interact with orthosteric and allosteric sites of atrial M2 muscarinic receptor.

Hybrid tetraaamine disulfides 4-9 were synthesized by combining the structural features of prazosin (1), a competitive alpha1-adrenoreceptor antagonist, and benextramine (2), an irreversible alpha1/alpha2-adrenoreceptor antagonist, and their biological profiles at alpha1-adrenoreceptor subtypes were assessed by functional experiments in isolated rat vas deferens (alpha1A), spleen (alpha1B), and aorta (alpha1D). To verify the role of the disulfide moiety on the interaction with alpha1-adrenoreceptor subtypes, carbon analogues 10-15 were included in this study. All quinazolines lacking the disulfide bridge behaved, like 1, as competitive antagonists, whereas all polyamine disulfides displayed a nonhomogeneous mechanism of inhibition at the three subtypes since they were, like 2, noncompetitive antagonists at the alpha1A and alpha1B subtypes while being, unlike 2, competitive antagonists at the alpha1D. In particular, the blocking effects were characterized by a decrease of the maximal response to noradrenaline that was affected only slightly by washings. Probably the alpha1A and alpha1B subtypes bear in the binding pocket a suitable thiol function that would suffer an interchange reaction with the disulfide moiety of the antagonist and which is missing, or not accessible, in the alpha1D subtype. Polyamines 8, 9, and 14, among others, emerged as promising tools for the characterization of alpha1-adrenoreceptors, owing to their receptor subtype selectivity. Finally, the effect of nonbasic substituents on the phenyl ring of prazosin analogues 16-28 on potency and selectivity for the different subtypes can hardly be rationalized.

Novel derivatives of natural muscarine and allo -muscarine, i.e. the benzyl ethers (−)- 10 and (−)- 12 and the benzoate (−)- 13 , were synthesized in very high enantiomeric excess. Target compounds were tested in vitro on guinea pig tissues, and their muscarinic potency was evaluated at M 2 (heart force and rate) and M 3 (ileum and bladder) receptor subtypes. The derivatives under study were also assayed in vivo on pithed rat. In addition, muscarinic receptor heterogeneity was investigated by determining the affinity and the relative efficacy of compounds (−)- 10 , (−)- 12 and (−)- 13 at M 2 (heart force and rate) and M 3 (ileum and bladder) receptor subtypes.

Methylation of the carbon atom C of compound 1, a potent and not selective muscarinic antagonist, was carried out. The resulting diastereomers were separated and the corresponding racemate further resolved to give four enantiomers, which were tested both as hydrogen oxalate and methiodide salts. The pharmacological results obtained at M1, M2 and M3 muscarinic receptor subtypes, show that methylation at C1, depending on the stereochemistry, increases antagonist potency, having thus the same effect of nitrogen quaternization. These results may well lead to the development of new potent antimuscarinic drugs lacking a cationic head.

Several analogues of cirazoline (2), a selective alpha1-adrenoreceptor agonist, were prepared and their pharmacological profiles studied. Although at the alpha1-adrenoreceptor all the compounds displayed a significant agonist activity, at the alpha2-adrenoreceptor they showed either agonist or antagonist activity depending on the nature of the phenyl substituent. The qualitative structure-activity relationship led us to the conclusion that the oxygen atom in the side-chain is essential for alpha1-agonist activity, while the cyclopropyl ring is not, and may be replaced by several groups. Of the groups studied, isopropoxy appears to be the best. Instead, the same substitution (i.e., isopropoxy for the cyclopropyl ring) at alpha2-adrenoreceptors causes a reversal of activity. On the other hand, the cyclopropyl ring seems to be important for alpha1-selectivity. Compound 20 is the most potent alpha1-agonist of the series, being equiactive with cirazoline on rat vas deferens and in pithed rat.

WB 4101-related benzodioxans 3-9 were synthesized, and their biological profiles at alpha(1)-adrenoreceptor subtypes and 5-HT(1A) serotoninergic receptors were assessed by binding assays in CHO and HeLa cells membranes expressing the human cloned receptors. Furthermore, receptor selectivity of selected benzodioxan derivatives was further determined in functional experiments in isolated rat vas deferens (alpha(1A)) and aorta (alpha(1D)) and guinea pig spleen (alpha(1B)), in additional receptor binding assays in rat cortex membranes containing alpha(2)-adrenoreceptors and 5-HT(2) serotoninergic receptors, and in rat striatum membranes containing D(2) dopaminergic receptors. An analysis of the results of receptor binding experiments for benzodioxan-modified derivatives 3-9 showed high affinity and selectivity toward the alpha(1a)-adrenoreceptor subtype for compounds 3-5 and 7 and a reversed selectivity profile for 9, which was a selective alpha(1d) antagonist. Furthermore, the majority of structural modifications performed on the prototype 1 (WB 4101) led to a marked decrease in the affinity for 5-HT(1A) serotoninergic receptors, which may have relevance in the design of selective alpha(1A)-adrenoreceptor antagonists. The exception to these findings was the chromene derivative 8, which exhibited a 5-HT(1A) partial agonist profile.

4-(4-Fluorobenzoyl)-1-[2-(4-iodo-2,5-dimethoxyphenyl)ethyl]piperidine ( 7 ) and its derivatives modified at the carbonyl group of the fluorobenzoyl moiety were prepared and evaluated for affinity at 5-HT 2A , 5-HT 2C (rat cortex) and 5-HT 2B (rat stomach fundus) serotonin receptors. Compound 7 bound the 5-HT 2A sites with higher affinity (K i = 8.2 nM) than the 5-HT 2B (K b = 1 290 nM) and 5-HT 2C ones (K i = 54.2 nM). Modification of the benzoyl carbonyl group decreased the 5-HT 2A and 5-HT 2C affinities but did not significantly influence 5-HT 2B affinity. This suggests that the carbonyl group is the determinant for the interaction with 5-HT 2A and 5-HT 2C receptor subtypes. Compound 7 was found to be a 5-HT 2A receptor antagonist.

A series of 2-carbonyl analogues of the muscarinic antagonist diphenidol bearing 1-substituents of different lipophilic, electronic, and steric properties was synthesized and their affinity for the M2 and M3 muscarinic receptor subtypes was evaluated by functional tests. Two derivatives ( Figure 1 , Figure 2 Download : Download high-res image (40KB) Download : Download full-size image Figure 1 . Schild plots obtained for the antagonists Figure 1 , Figure 2 on guinea-pig atria force (M2). The points are the means of five to seven experiments. S.E.M.s are not reported for clarity and are less than 10%. Ordinate: log(DR-1) where DR=EC50 ratio measured from the displacement of the agonist concentration-response curves by the antagonist. Abscissa: −log[antagonist]. Schild correlations, based on three points, do not deviate from linearity, suggesting a competitive behavior of the two antagonists. Download : Download high-res image (59KB) Download : Download full-size image Figure 2 . Experimental dose–response curves of APE on guinea-pig ileum (M3) in the absence (□) and in the presence of 10 μM of Figure 1 , Figure 2 (♦) and 2g (■). The decrease of the maximum effect of APE suggests a non-competitive behavior of the two antagonists. ) showed an M2-selective profile which was confirmed by functional tests on the M1 and M4 receptors. A possible relationship between M2 selectivity and lipophilicity of the 1-substituent was suggested by structure–activity analysis. This work showed that appropriate structural modification of diphenidol can lead to M2-selective muscarinic antagonists of possible interest in the field of Alzheimer's disease.

A series of 1,3-dioxolane-based ligands, bearing ether, thioether and related sulfoxide and sulfone functionalities, were synthesised and tested as potential muscarinic antagonists. The compounds display moderate to low affinity for the three receptor subtypes M1-M3, with some of them showing a significant selectivity for the M1-M3 over the M2 subtype.

To develop ligands that may be useful in exploring muscarinic receptor heterogeneity, we synthesized a series of analogues of 2,2-diphenyl-[1,3]-dioxolan-4-ylmethyl-dimethylamine oxalate and methiodide bearing a modified cationic head. These compounds, when tested on tissues containing the three subtypes M1, M2, and M3, behaved as muscarinic antagonists whose results showed that different substituents on the quaternary and tertiary nitrogen affect affinity and selectivity in different ways. In particular, comparison of the affinities of these ligands with those of the reference compounds points out that compounds bearing an ethyl substituent improve the affinity of the molecule at the three subtypes, while compounds bearing a phenethyl substituent are more selective for the M3 sites.

The concept that polyamines may represent a passe-partout (Master key) for G—protein—coupled receptors recognition is illustrated. Using a linear tetraamine as the focus, appropriate structural modifications have allowed to obtain novel tetraamines displaying selectivity for different receptor systems. It is shown that tetraamines have receptor subtype selectivity since they are site-directed, owing to different chain lengths separating the protonable nitrogens and to the presence of those peculiar structural elements which make them able of discriminating at the binding stage.

The synthesis and pharmacological activity as muscarinic antagonists of a number of 2-alkylthio-2,2-diphenylacetic acid esters are reported. The compounds studied are potent muscarinic antagonists and many of them show from moderate to high selectivity toward M2 or toward M1 and M2 receptors when tested on tissues but lack selectivity on five muscarinic human receptors (m1–m5) cloned and expressed in CHO-K1 cells. As a consequence, the compounds behave as functionally selective antagonists. Those showing M2 selectivity appear to be good drug candidates for the treatment of cognitive disorders connected with central cholinergic deficit.

The synthesis of a series of 5-(4-piperazinyl)-3(2 H )-pyridazinone has been reported. The blocking activity of these compounds was determined on the pre- and postsynaptic α-adrenoreceptors of isolated rat vas deferens.

The agonistic potencies of (+/-)muscarine, (+/-)cis-2-methyl-5- [(dimethylamino)methyl]-1,3-oxathiolane methiodide (cis-oxathiolane) and its two enantiomers were determined at muscarinic M1 and M2 receptors in the pithed rat. In non-pretreated animals, i.v. administration of these agents produced bradycardic effects mediated by cardiac M2 receptors followed by increases in heart rate mediated by M1 receptors in sympathetic ganglia. As these responses have been shown to partly overlap, "true" M1 and M2 potencies were determined after selective blockade of M1 and M2 receptors by pirenzepine and methoctramine, respectively. A similar rank order of agonist potencies was obtained at M1 and M2 receptors: (+)cis-oxathiolane greater than (+/-)cis-oxathiolane greater than (+/-)muscarine greater than (-)cis-oxathiolane. At both receptor subtypes, (+)cis-oxathiolane was considerably more potent (ca. 30-fold) than its corresponding (-) enantiomer indicating that the agonist binding sites of the two receptor subtypes may have similar stereochemical properties. While (+/-)muscarine showed similar potencies at M1 and M2 receptors, racemic cis-oxathiolane and its two enantiomers showed a slight selectivity (3-7 fold) for M1 receptors indicating the potential usefulness of these compounds in the development of selective M1 receptor agonists.

The application of frontal affinity chromatography-mass spectrometry (FAC-MS), along with molecular modeling studies, to the screening of potential drug candidates toward the recently deorphanized G-protein-coupled receptor (GPCR) GPR17 is shown. GPR17 is dually activated by uracil nucleotides and cysteinyl-leukotrienes, and is expressed in organs typically undergoing ischemic damage (i.e., brain, heart and kidney), thus representing a new pharmacological target for acute and chronic neurodegeneration. GPR17 was entrapped on an immobilized artificial membrane (IAM), and this stationary phase was used to screen a library of nucleotide derivatives by FAC-MS to select high affinity ligands. The chromatographic results have been validated with a reference functional assay ([(35)S]GTPgammaS binding assay). The receptor nucleotide-binding site was studied by setting up a column where a mutated GPR17 receptor (Arg255Ile) has been immobilized. The chromatographic behavior of the tested nucleotide derivatives together with in silico studies have been used to gain insights into the structure requirement of GPR17 ligands.

Four isomers of [(4-fluoro-5-methyl-tetrahydrofuran-2-yl)methyl]trimethylammonium iodide (4-deoxy-4-fluoro-muscarines) were prepared in enantiomerically and diastereomerically pure form from (S)-(-)-methyl 4-methylphenyl sulfoxide, ethyl fluoroacetate, and allyl bromide. Their absolute configurations were assigned by 1H NMR analyses. The four optically pure compounds were tested in vitro on guinea pig and their muscarinic potency was evaluated at M3 (ileum and bladder) and M2 (heart) muscarinic receptor subtypes. Compound 1a, the most potent isomer of the series, was also tested in vivo on pithed rat and its muscarinic activity at the M1 receptor subtype was compared with that of muscarine. Moreover, affinity and relative efficacy were calculated in vitro for this compound at M2 (heart force and rate) and M3 (ileum and bladder) receptors in order to investigate muscarinic receptor heterogeneity. The 4-deoxy-4-fluoromuscarines display a similar trend of potency as the corresponding muscarines and compound 1a shows differences in the affinity constants among the studied tissues. Replacement of a hydroxyl group for a fluorine atom in the 4 position of muscarine produces 1 order of magnitude increase in affinity for cardiac M2 muscarinic receptors controlling rate, while the affinity at cardiac M2 muscarinic receptors controlling force is unchanged, opening the possibility of a further classification of cardiac muscarinic receptors.