Effect of the rigidification of propranolol, a mixed ß-adrenoceptor and 5-HT1AR antagonist.

Propranolol is a popular ß adrenergic antagonists that, together with pindolol, binds also to serotoninergic receptors, namely 5-HT1A/B. In this work the rigidification of the propranolol structure by locking its hydroxyl group within a 1,3-dioxolane ring was investigated. Constrained derivatives of propranolol were synthesized, fully characterized and tested for their affinity at ß-adrenoreceptors and 5-HT1A/B/C receptors using radioligand binding assay. The constrained derivatives were inactive, as expected, at ß1/2/3 adrenergic receptors. Although less expected, these derivatives failed to bind also to 5-HT1A/B/C receptors. The rigidification of propranolol is detrimental for 5-HT1AR activity.

Enantioselectivity of muscarinic antagonists. 2,2-Dicyclohexyl-5-[(dimethylamino)methyl]-1,3-oxathiolane methiodides and related 3-oxides.

The enantiomers of three chiral muscarinic antagonists carrying a 1,3-oxathiolane nucleus were prepared and their absolute configuration established. The enantioselectivity and tissue selectivity of such compounds were studied on rat bladder and guinea pig ileum and heart. The results show that introduction of a sulfoxide function brings about a small but definite enantioselectivity in the 1,3-oxathiolane compound (2), which in itself does not show enantioselectivity among the tissues studied. The results obtained point to differences among cardiac and ileal muscarinic receptors. Comparison of the absolute configuration related agonists shows that the most potent isomers of both series share the same absolute stereochemistry.

Enantioselectivity of muscarinic antagonists. Isomeric 2-cyclohexyl-2-phenyl-5-[(dimethylamino)methyl]-1,3-oxathiolane methiodides.

The four isomers of 2-cyclohexyl-2-phenyl-5-[(dimethylamino)methyl] -1,3-oxathiolane methiodide were prepared. Their absolute configuration was attributed by means of X-ray crystallography and circular dichroism. The compounds were tested on rat bladder and guinea pig ileum and heart, and their antimuscarinic potency was evaluated and expressed as pA2. The results show that the introduction of a chiral center into position 2 brings about a small but definite enantioselectivity on rat bladder and guinea pig ileum which is not seen for guinea pig heart. This supports the view that differences exist among the muscarinic receptors of these tissues (M2 receptors). Comparison of the absolute configuration of the antagonists studied in this and in the preceding paper2 and that of strictly related agonists supports the hypothesis of a common binding site for agonists and antagonists of this kind.

Resolution, absolute configuration, and cholinergic enantioselectivity of (-)- and (+)-c-2-methyl-r-5-[(dimethylamino)methyl]-1,3-oxathiolane t-3-oxide methiodide and related sulfones.

As a continuation of previous studies on chiral cholinergic agonists carrying a 1,3-oxathiolane nucleus, the enantiomers of c-2-methyl-r-5-[(dimethylamino)methyl]-1,3-oxathiolane t-3-oxide methiodide ((+)- and (-)-1) and of cis-2-methyl-5-[(dimethylamino)methyl]-1,3-oxathiolane 3,3-dioxide ((+)- and (-)-2) were obtained and their absolute configurations established by synthesis. The cholinergic potency of the four isomers was evaluated in vitro on guinea pig ileum and frog rectus abdominis models, and the results show that (-)-1, which has the same absolute configuration as L-(+)-muscarine, is a selective and potent muscarinic agent. The (+)-1 enantiomer is some hundred times less potent than (-)-1 on the muscarinic guinea pig ileum while, on the same tissue, the corresponding sulfone derivatives ((+)- and (-)-2) show no enantioselectivity.

Structure-activity relationships for prazosin and WB 4101 analogues as alpha 1-adrenoreceptor antagonists.

Several alpha-adrenoreceptor antagonists were prepared by coupling one of the two moieties of WB 4101 (1) with one of the two moieties of prazosin (2). Their blocking activity and relative selectivity on alpha 1- and alpha 2-adrenoreceptors were evaluated in the isolated rat vas deferens. Although retaining a significant selectivity toward alpha 1-adrenoreceptors, all the drugs were weaker antagonists than the parent compounds 1 and 2. Opening the piperazine ring of 2 gave 3, which displayed a very high activity and selectivity toward alpha 1-adrenoreceptors (alpha 1/alpha 2 = 3890). This may have relevance in understanding the mode of action of prazosin. In addition, 3 may represent a valuable tool in the characterization of alpha-adrenoreceptor subtypes.

Structure-activity relationships in 1,4-benzodioxan-related compounds. Investigation on the role of the dehydrodioxane ring on alpha 1-adrenoreceptor blocking activity.

Several analogues of 2-[[[2-(2,6-dimethoxyphenoxy)ethyl]amino]methyl]-1,4-benzodioxa n (WB 4101, 1) were prepared and evaluated for their blocking activity on alpha 1- and alpha 2-adrenoreceptors in the isolated rat vas deferens. The results were compared with those obtained for 1 and benoxathian (2). It was shown that the two oxygens at positions 1 and 4 may have a different role in receptor binding. It seems that the oxygen at position 4 as such does not contribute to the binding while it is important in stabilizing an optimal conformation for drug-receptor interaction mechanism. On the other hand, the oxygen at position 1 might interact with a receptor polar pocket of reduced size by way of a donor-acceptor dipolar interaction. Furthermore, it was shown that replacement of the dehydrodioxane ring of 1 by a phenyl or a pyrrole nucleus causes a significant decrease in activity.

Structure-activity relationships among benextramine-related tetraamine disulfides. Chain length effect on alpha-adrenoreceptor blocking activity.

Several N'-substituted N,N''-(dithiodi-2,1-ethanediyl)bis(1, omega-alkanediamines) were prepared and evaluated for their blocking activity on alpha-adrenoreceptors in the isolated rat vas deferens and human blood platelets. The results were compared with those obtained for benextramine (N,N''-(dithiodi-2,1-ethanediyl)bis[N'-[(2-methoxyphenyl)-methyl]- 1 ,6- hexanediamine], 10). Bendotramine (N,N''-(dithiodi-2,1-ethanediyl)bis[N'-[(2-methoxyphenyl)- methyl]-1,12-dodecanediamine], 16) proved to be as active as 10 on alpha 1-adrenoreceptors, showing that optimum activity is associated with two carbon chain lengths separating inner from outer nitrogens of tetraamine disulfides. On the other hand, 16 had no activity up to 20 microM at alpha 2-adrenoreceptors. The optimum activity at this receptor subtype was associated with a six to eight carbon chain (10-12). Furthermore, 10 proved to be more selective toward alpha 2-adrenoreceptors whereas 16 was a selective alpha 1-antagonist. The tetraamine disulfides were shown also to be potent inhibitors of human platelet aggregation induced by ADP or epinephrine. The potency increased with the carbon chain length. However, the results on platelets did not parallel those found in the rat vas deferens, indicating that differences exist between the alpha-adrenoreceptor subtypes investigated. In conclusion, 10 may be a useful tool in characterizing alpha 2-adrenoreceptors whereas 16 might help in investigating alpha 1-adrenoreceptors.

Resolution, absolute configuration, and cholinergic enantioselectivity of (+)- and (-)-cis-2-methyl-5-[(dimethylamino)methyl]-1,3-oxathiolane methiodide.

The potent cholinergic agonist (+/-)-cis-2-methyl-5-[(dimethylamino)methyl]-1,3-oxathiolane methiodide (+/-)-1] was resolved into enantiomeric forms. Their absolute configurations were established by a synthetic pathway that also allowed the synthesis of the corresponding diastereomeric (+)- and (-)-trans-2-methyl-5-[(dimethylamino)-methyl]-1,3-oxathiolane methiodide [(+)- and (-)-10]. Compound (+)-1, which is the most potent of the four isomers, showed the same absolute configuration as L-(+)-muscarine and (+)-cis-dioxolane. The four isomers were tested on guinea pig ileum and frog rectus abdominis, and their muscarinic and nicotinic potency (EPMR) and selectivity were determined. The relationships between stereoisomerism and potency are discussed.

Structure-activity relationships in prazosin-related compounds. Effect of replacing a piperazine ring with an alkanediamine moiety on alpha 1-adrenoreceptor blocking activity.

Several prazosin-related compounds were synthesized in which the piperazine ring of prazosin (1) was replaced by an alkanediamine chain and were evaluated for their blocking activity on alpha 1- and alpha 2-adrenoreceptors in isolated rat vas deferens. All the compounds investigated proved highly selective toward the alpha 1-adrenoreceptor owing to a very low affinity for alpha 2-adrenoreceptors. Furthermore, compounds 2, 9, and 13 were also investigated in vivo to determine their hypotensive effect on anesthetized rats, which were compared with that of prazosin (1). It was confirmed that the piperazine moiety of 1 is not essential for potency. However, optimum activity depends on two parameters: carbon-chain length of the alkanediamine moiety and N-methylation of both the amide and the 2-amino functions. In the desmethyl series, optimum activity was associated with the lower homologues (2-4) bearing a chain of two to four methylenes whereas in the N,N'-dimethyl series peak potency was observed with a six-carbon chain as in 13. Compound 13 proved the most active of the series and was more potent than prazosin (1) in both in vivo and in vitro assays. It is hypothesized that the alpha 1-adrenoreceptor incorporates a lipophilic area that is located between the binding sites for the quinazoline and the furoyl moieties and is able to accommodate a polymethylene chain.

Structure-activity relationships among methoctramine-related polymethylene tetraamines. Chain-length and substituent effects on M-2 muscarinic receptor blocking activity.

Several polymethylene tetraamines related to methoctramine (1) were prepared and evaluated for their blocking activity on M-2 muscarinic receptors in guinea pig atria and ileum. It turned out that antimuscarinic potency depends on the following parameters: (a) nature of the substituent on both inner and outer nitrogens and (b) carbon chain length separating the inner nitrogens as well as the inner and outer nitrogens. Optimum activity at cardiac M-2 muscarinic receptors was associated with the chain lengths present in 1, that is, eight methylenes between the inner nitrogens and six methylenes between the inner and outer nitrogens. With regard to the substituents, replacement of the benzylic moiety of 1 by a 2-furyl or a 5-methyl-2-furyl nucleus resulted in enhanced potency toward cardiac M-2 muscarinic receptors. In fact, furtramine (18) and mefurtramine (19) proved to be more potent and more selective than 1. Moreover, N-methylation of the four nitrogens of 1 gave different effects: methylation of the outer nitrogens, giving 22, caused a significant decrease in activity whereas methylation of the inner nitrogens, yielding 23, resulted in an increase in activity in both atria and ileum.

Structure-activity relationships among benextramine-related tetraamine disulfides at peripheral alpha-adrenoreceptors.

Several N,N''-(dithiodi-2,1-ethanediyl)bis[N'-(arylmethyl)-1,6-hex anediamines] were prepared and evaluated for their blocking activity on postsynaptic alpha 1-adrenoreceptors in the isolated rat vas deferens. The results were compared with those obtained for benextramine (1). N,N''-(Dithiodi-2,1-ethanediyl)bis[N'-(pyrrol-2-ylmethyl)-1, 6 -hexanediamine] (pyrextramine, 29) was the most potent among the tetraamine disulfides investigated. Thus, it was selected for further pharmacological evaluation to assess its receptor specificity. At a concentration of 10 microM it did not affect the responses elicited by 5-hydroxytryptamine and histamine in guinea pig ileum and by isoproterenol in guinea pig atria and tracheal chain. Furthermore, it was more specific than benextramine (1) toward the muscarinic receptor, being significantly less potent in inhibiting the carbachol-induced response in rat jejunum. These results show that pyrextramine (29) is an irreversible alpha-blocking agent that is more potent and specific than benextramine (1). In conclusion, 29 may be a useful tool in the elucidation and characterization of the peripheral alpha 1-adrenoreceptor.

Affinity and efficacy correlate with chemical structure more than potency does in a series of pentatomic cyclic muscarinic agonists.

The efficacy and affinity of nine pentatomic cyclic muscarinic agonists were determined on the guinea-pig ileum, according to the method of Furchgott & Bursztyn (1967). The efficacy and affinity of these agonists are affected differently by structural modifications. Our results suggest that a strong dipole oriented in the same direction as that of the hydroxy group of muscarine, or the presence of a polarizable atom in the same position, are important for efficacy.

N,N''-(dithio-2,1-ethanediyl)bis[N'-(pyrrol-2-ylmethyl)-1, 6,- hexanediamine] (pyrextramine), a new irreversible alpha 1-adrenoreceptor blocking agent of the tetramine disulfide class.

In an attempt to further improve the alpha-adrenoreceptor blocking activity of benextramine, the effect of heteroaromatic substituents on the terminal nitrogens was studied. In this communication we report on the newly synthetized N,N''-(dithio-2,1-ethanediyl)bis[N'-(pyrrol-2-ylmethyl)-1,6- hexanediamine] (pyrextramine) that displayed higher affinity (5-10-fold) and higher selectivity (about 12-fold) than benextramine. These preliminary results show that pyrextramine can replace benextramine in the elucidation and characterization of the peripheral alpha 1-adrenoreceptors.

Fluoronorepinephrines: further pharmacological evaluation in vitro and in pithed rats.

The effect of 6F-, 5F- and 2F-norepinephrine (6F-, 5F- and 2F-NE) in rat vas deferens, guinea-pig ileum and pithed rats was compared to that of norepinephrine (NE). The rank order of potency on postsynaptic alpha 1-adrenoreceptors, determined from the isometric contraction of vas deferens, was 6F-NE = 5F-NE = NE greater than 2F-NE. A similar pattern was found for presynaptic alpha 2-adrenoreceptor activity in both noradrenergic nerve terminals of vas deferens and cholinergic nerve terminals of the ileum, determined from the inhibition of contraction elicited by electrical field stimulation. The only exception was the 5F isomer which was 7 times less active than NE to activate the alpha 2-adrenoreceptors of vas deferens. Thus, ring fluorination markedly alters both alpha 1- and alpha 2-agonist properties of NE. Moreover, alpha 1/alpha 2 selectivity, at least as far as rat vas deferens is concerned, is not significantly influenced by the introduction of a fluorine atom in the NE molecule. 6F-NE was about 3-4 times more active than NE in pithed rats. In turn, NE was equiactive with 5F-NE. 2F-NE was the least active isomer, being 30- and 100-fold less active than NE and 6F-NE, respectively.

Alpha-adrenoceptor occupancy by N,N-dimethyl-2-bromo-2-phenethylamine hydrobromide (DMPEA) in rat vas deferens.

The effects of N,N-dimethyl-2-bromo-2-phenethylamine hydrobromide (DMPEA) on alpha 1- and alpha 2-adrenoceptors were examined in the isolated rat vas deferens. The active species of DMPEA was the corresponding ethyleniminium ion, which forms in the biophase, since pretreatment of tissues with sodium thiosulphate completely prevented the DMPEA-induced inhibition at alpha 1- and alpha 2-adrenoceptors. DMPEA was approximately 42-fold more potent in inhibiting alpha 2-adrenoceptors than it was in inhibiting alpha 1-adrenoceptors. The antagonism of both receptor types was reversible since washing of the tissues after incubation with DMPEA brought the agonist dose-response curve back to the control value. At alpha 1-adrenoceptors, DMPEA displaced the noradrenaline dose-response curve to the right and concomitantly depressed the maximum response, effects which are consistent with a non-competitive mechanism of action. At alpha 2-adrenoceptors, DMPEA caused a parallel shift of the clonidine or noradrenaline dose-response curve to the right in field-stimulated prostatic portions of the rat vas deferens. The antagonism appeared to be competitive at low concentrations, whereas the shift of the clonidine dose-response curve at higher concentrations became overproportional to the DMPEA concentration. A combination of DMPEA with idazoxan produced a less-than-additive shift of the dose-response curve for clonidine, indicating that these antagonists do not bind to the same site.

Determination of muscarinic agonist potencies at M1 and M2 muscarinic receptors in a modified pithed rat preparation.

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.

Chiral muscarinic agonists possessing a 1,3-oxathiolane nucleus: enantio- and tissue-selectivity on isolated preparations of guinea-pig ileum and atria and of rat urinary bladder.

Racemate and corresponding enantiomers of muscarinic agonists carrying a 1,3-oxathiolane nucleus were studied on isolated preparations of guinea-pig ileum and atria and of rat urinary bladder. The efficacy of these agonists were determined according to the method of Furchgott and Bursztyn (1967) and enantio-selectivity and tissue-selectivity were investigated. The enantio-selectivities of the most potent compounds studied (expressed as the ratio of potencies or affinities of the enantiomers) vary significantly from tissue to tissue, supporting the view that M2 receptors are not homogeneous. In particular, the data all indicate that the ileal receptors are different to the atrial and bladder ones.

Thiol group may be involved in the irreversible blockade of presynaptic alpha 2-adrenoreceptors by pyrextramine and benextramine in the isolated guinea pig ileum.

This study, conducted on isolated guinea pig ileum, was designed to establish the mechanism of presynaptic alpha 2-adrenoreceptor blockade by the tetramine disulfides, benextramine and pyrextramine. At 1 microM these drugs irreversibly blocked norepinephrine (NE)-induced inhibition of the twitch response to electrical stimulation. This may be the result of covalent bond formation between the disulfide bridge of the inhibitor and a thiol function at the receptor level through an interchange reaction since the benextramine carbon analogue did not affect NE response under the same conditions. Furthermore, NE (10 microM) failed to protect presynaptic alpha 2-adrenoreceptors from pyrextramine blockade whereas idazoxan (O.T microM) completely abolished the irreversible antagonism of pyrextramine (1 microM). This finding suggests that the tetramine disulfide binding site may coincide with that of idazoxan and is different from the NE binding site.

Molecular requirements of the active site of cholinergic receptors XV: Synthesis and biological activity of 2,3-dehydrodeoxamuscarone and 2,3-dehydrodeoxamuscarines.

To elucidate the molecular requirements of the active sites of cholinergic receptors, 3-methyl-4-oxo-1-(N,N-dimethylaminomethyl)cyclopent-2-ene methiodide (2,3-dehydrodeoxamuscarone) and cis- and trans-3-methyl-4-hydroxy-1-(N,N-dimethylaminomethyl)cyclopent-2-ene methiodides (cis- and trans-dehydrodeoxamuscarines) were synthesized and tested. The results, compared with those of the corresponding oxygenated compounds, seem to indicate the 2,3-dehydrodeoxamuscarines and muscarine bind at the same site while 2,3-dehydrodeoxamuscarone interacts with the site normally occupied by muscarone. Furthermore, the previously suggested hypothesis that the unpolar site might somehow incorporate that of muscarone was considered.

Synthesis and muscarinic receptors affinity of a series of antagonist bivalent ligands.

A series of bivalent ligands (2-8) derived from 2,2-diphenyl-[1,3]-dioxolan-4-ylmethyl-dimethylamine methiodide 1 has been synthesized and tested to evaluate affinity and selectivity for M1, M2 and M3 muscarinic receptor subtypes. In order to study the contribution of the spacer and of a second cationic head to the binding process, unsymmetrical ligands (9,10) have also been prepared. The results, expressed in terms of pA2 values, show that, although the spacer negatively affects the interaction of the bivalent ligands with the three receptor subtypes, affinity and selectivity are modulated by its length; this indicates that the pharmacophore binding sites are organized differently with respect to their mutual proximity and orientation, in each receptor subtype.