QSAR studies on BK channel activators.

QSAR studies were developed on the basis of a dataset comprising BK channel activators previously synthesized and biologically assayed in our laboratory, in order to obtain highly accurate models enabling prediction of affinity toward the channel for New Chemical Entities (NCEs). Many molecular descriptors were computed by the CODESSA software. They were initially exploited in order to rationally split the available dataset into training and test set pairs, which supplied the basis for the development of QSAR models. Models were subjected to rigorous validation analysis based on the estimate of several statistical parameters, for the seek of the most accurate and simplest model enabling prediction of BK channel affinity.

1,2,3-Triazol-carboxanilides and 1,2,3-triazol-(N-benzyl)-carboxamides as BK-potassium channel activators. XII.

The chemical structures of many synthetic activators of large-conductance calcium-activated potassium channels (BK channels) satisfy a simple pharmacophore model, consisting of two appropriately substituted phenyl rings connected by a linker of a heterogeneous nature. In this paper, a series of new compounds with modifications of the linker portion of the above pharmacophore are described. In particular, in these new derivatives, the linker portion is represented by a 1,2,3-triazole-carboxamide group, which can be viewed as a combination of two different kinds of linker, independently used in previous series of BK-openers: the amide function and the 1,2,3-triazole ring. The overall finding of this study indicated that the triazole-carboxamide derivatives were generally poorly effective and that this structural modification of the linker is deleterious for activity on BK channels. Therefore, it can be hypothesized that the increase of the steric hindrance of the linker and/or the increase of the distance between the two aromatic portions are negative for the interaction with the biological target.

N6-1,3-diphenylurea derivatives of 2-phenyl-9-benzyladenines and 8-azaadenines: synthesis and biological evaluation as allosteric modulators of A2A adenosine receptors.

Some 1-[4-(9-benzyl-2-phenyl-9H-purin-6-ylamino)-phenyl]-3-phenyl-urea derivatives and some 1-[4-(9-benzyl-2-phenyl-9H-8-azapurin-6-ylamino)-phenyl]-3-phenyl-urea derivatives were synthesised and evaluated for their interaction with adenosine receptors. It was found that some of these compounds can act as positive enhancers of agonist and antagonist radioligands for the A(2A) adenosine receptors. This evidence was also strengthened by functional data. Other compounds can act as negative modulators. Furthermore these compounds show inhibitory properties for A(1) and A(3) adenosine receptors.

New amido derivatives as potential BKCa potassium channel activators. XI.

The vasorelaxing effects of exogenous activators of large-conductance calcium-activated potassium channels (BK channels) can furnish the pharmacological rational basis for the treatment of hypertension and/or other diseases related with an impaired contractility of vessels. Since in previous works some benzanilide derivatives showed BK channel-induced vasorelaxing activity, in this paper we have taken into consideration the introduction of methylene spacer(s) between the amide linker and one or both the aromatic substituents, to evaluate the pharmacological effect caused by these lengthenings and to obtain possible useful information about structure-activity relationships. Overall, the main findings of this work suggest that the introduction of one or two methylene group(s) in the amide linker exerts a negative influence on the BK-opening properties, which can be due to an excessive lengthening of the spacer between the two aromatic rings and/or to further degrees of conformational freedom.

Synthesis of new 2-phenyladenines and 2-phenylpteridines and biological evaluation as adenosine receptor ligands.

Synthesis and biological assays of a series of 2-phenylpteridine derivatives are described to compare their affinities to adenosine receptors with those of the corresponding adenines, purposely prepared, and 8-azaadenines previously described. This study demonstrates that the enlargement of the five-membered ring of the adenine nucleus to a six-membered one is a modification that does not allow the molecules to maintain high activity towards adenosine receptors; in fact, pteridine derivatives did not show themselves to be good adenosine receptor ligands. On the contrary, N(6)-cycloalkyl- or N(6)-alkyl-2-phenyladenines showed a very high affinity and selectivity for A(1) adenosine receptors. We demonstrate also that the 9-benzyl substituent is crucial for conferring high affinity for A(3) receptors to molecules having a 2-phenyladenine-like nucleus.

Synthesis, biological activity and molecular modelling of new trisubstituted 8-azaadenines with high affinity for A1 adenosine receptors.

We describe here the synthesis and biological activity of new 8-azaadenines bearing both a phenyl group on C(2) and a 9-benzyl group substituted in the ortho position with a Cl or a F atom or a CF(3) group, to verify the synergistic effect of a combination of these substitution patterns on binding with the A(1) adenosine receptors. In position N(6) aliphatic and cycloaliphatic substituents were chosen which had been shown to bind well with the A(1) receptors. Because of the high lipophilicity of these kinds of molecules, we also introduced a hydroxyalkyl substituent in the same position. The compounds obtained generally showed a very good affinity and selectivity for A(1) receptors. Some of the compounds showed K(i) in the nanomolar range, one even in the subnanomolar range (0.6 M). Molecular docking calculations were performed in order to evaluate the interaction energies between the bovine A(1) receptor model and the selected ligands, and then to correlate these energies with biological activities of the ligands as obtained from the experiments. Molecular docking analysis suggests different binding modes towards A(1) receptors that are plausible for these ligands.

Structural modifications of benzanilide derivatives, effective potassium channel openers. X.

Large-conductance calcium-activated potassium (BK) channels are involved in many fundamental cell functions. Consistently, the ability to activate BK channels by exogenous compounds is considered as a promising pharmacodynamic pattern for the potential treatment of several pathologies. In this perspective, the development of new and selective BK-openers can be considered as an actual field of research. This paper reports the synthesis and pharmacological evaluation of new benzanilides, useful for deepening the comprehension of the structure-activity relationships, emerged in previous studies on this class of BK-activators. From a structural point of view, these benzanilides belong to a general class of BK-activators, showing a common pharmacophoric model, consisting of two aryl groups linked through an appropriate "spacer" and the almost obligatory presence of a phenolic hydroxyl. In particular, a new series of benzanilides, in which the phenyl rings have been widely changed both on the acidic portion and the basic one of the amide spacer, were synthesised. Their vasorelaxing effects, induced through the activation of BK channels, were also evaluated. Although many compounds exhibited effects which could not be attributed to the activation of BK channels, two derivatives showed a clear profile of BK-activators with vasodilator activity comparable to or slightly lower than that recorded for the reference benzimidazolone NS1619. A further molecular modelling approach allowed us to obtain a molecular electrostatic potential feature which suggests a suitable interaction with the receptor site of the BK channel, from a tri-dimensional point of view. This approach seems to represent a further contribution for the development of new BK-activators, designed on the basis of the pharmacophoric model above-mentioned.

Heterocyclic analogs of benzanilide derivatives as potassium channel activators. IX.

On the basis of our previous works, addressed to synthesise new activators of BK potassium channels, and of many suggestions from the international literature, a simple pharmacophoric model, consisting of two suitably substituted phenyl rings bound to various kinds of linkers, was hypothesised. In particular, the effectiveness of the amidic linker was demonstrated, since several benzanilide derivatives showed interesting BK-opener properties. As a development of these benzanilides, in this work we introduced heterocyclic substituents, replacing the aryl ring on the acid side or on the basic one of the amide linker of the above pharmacophore. The pharmacological results indicated some relevant remarks about the structural requirements, needed for a satisfactory BK-opener activity. In particular, the presence of a phenolic function, with a possible H-bond donor role, has been confirmed. Furthermore, the presence of nitrogen heterocycles on the acid side of the amide linker seems to be a negative requirement, while furan and thiophene were well tolerated. On the contrary, the introduction of insaturated heterocyclic rings (pyridine and thiazole) on the basic side of the amide linker, led to satisfactory biological activity, while the presence of aliphatic heterocycles lowered the pharmacological effect.

Benzoyl and/or benzyl substituted 1,2,3-triazoles as potassium channel activators. VIII.

This paper reports the preparation of new benzoyl and/or benzyl substituted 1,2,3-triazole derivatives and their pharmacological evaluation as potential BK channel openers, as a part of a research program which hypothesized a pharmacophoric structure containing the 1,2,3-triazole ring. The synthetic procedures consist essentially with the 1,3-dipolar cycloaddition of aryl or benzyl azides to the asymmetric alkyne benzoylacetylene to give the wished 4-benzoyl-1,2,3-triazole isomers in larger amount. The pharmacological results show that the 1-(2-hydroxybenzyl)-4-benzyl-1H-1,2,3-triazole possesses high vasorelaxing activity involving the opening of the BK channels. Therefore the structure-activity relationships concerning this pharmacophoric structure confirm the usefulness of a phenolic function in the ortho position of the aromatic ring and would suggest a 1,2,3-triazole model bearing benzyl substituents. In addition such substituents appear more flexible and able to take different conformations with respect to phenyl groups which have higher trend to coplanar conformations.

1,4- and 2,4-substituted-1,2,3-triazoles as potential potassium channel activators. VII.

New 1,4- and 2,4-substituted 1,2,3-triazole derivatives were synthesized and tested as potential BK(Ca) channel openers, as a part of a research program, which hypothesizes a pharmacophoric structure containing the 1,2,3-triazole ring. The structure-activity relationships were studied introducing some structural changes concerning molecular geometry and the presence of a hydrogen bond donor as a primary amino group and a phenolic or alcoholic hydroxy function. The compounds were prepared by nucleophilic substitution on the 1,2,3-triazole ring and by 1,3-dipolar cycloaddition of azides to selected alkynes and to phenylacetone. The new compounds tested on rat aortic rings did not exhibit any significant vasorelaxing activity.

2,9-disubstituted-N6-(arylcarbamoyl)-8-azaadenines as new selective A3 adenosine receptor antagonists: synthesis, biochemical and molecular modelling studies.

A number of N6-(N-arylcarbamoyl)-2-substituted-9-benzyl-8-azaadenines, obtained by a modification of the synthetic scheme used to prepare selective A1 ligands, by only three or two steps, are described. At first we prepared a series of 2-phenyl-9-benzyl-8-azaadenines having as N6 substituent a variously substituted N-phenylcarbamoyl group. Some of these derivatives demonstrated good affinity towards the A3 subtype but low selectivity. Compounds having p-CF3, p-F and p-OCH3, as substituents on the phenylcarbamoyl group were selected as lead compounds for the second part of this study. Without modifying the N6 substituent, which would assure A3 affinity, we varied the 9 and 2 positions on these molecules to enhance selectivity. Some compounds having a p-methyl group on the 2-phenyl substituent showed a very good affinity and selectivity for the A3 subtype, revealing the first class of A3 adenosine receptor selective antagonists with a bicyclic structure strictly correlated to the adenine nucleus. The molecular modelling work, carried out using the DOCK program, supplied two models which may be useful for a better understanding of the binding modes. Both models highlighted the preferred interacting tautomeric forms of the antagonists for human A1 and A3 receptors.

Synthesis and biological activity of novel substituted benzanilides as potassium channel activators. V.

As part of our program toward designing potassium channel openers, the synthesis of a novel series of substituted benzanilides and their vasodilating activity are presented. The facile synthetic pathway generally involves coupling between the appropriate benzoyl chloride and commercial available anilines, followed by the selective or non-selective cleavage of methyl ether substituent(s), affording the corresponding phenol or bisphenol derivatives. The pharmacological evaluation of these structurally novel potential BK-openers on vascular contractile activity was studied in vitro, using isolated rat aortic rings pre-contracted with KCl 20 mM. Some derivatives were found to be potent smooth muscle relaxants and the vasodilation effects of these compounds were inhibited by tetraethylammonium (TEA) and iberiotoxin (IbTX), suggesting that the opening of BK channels is prevalent in the mechanism of action of these compounds. The best compound of the series was N-(2-hydroxy-5-phenyl)-(2-methoxy-5-chloro)-benzamide (16b) showing a full vasorelaxant efficacy and almost nanomolar potency index.

1,5-Diarylsubstituted 1,2,3-triazoles as potassium channel activators. VI.

As part of our program toward designing potassium channel openers, synthesis of a novel series of 1,5-diphenylsubstituted 1,2,3-triazoles, as potential activators of the large-conductance calcium-activated potassium channels (BK), as well as their vasorelaxant activity are presented. The functional effect of these potential structurally novel BK-openers on vascular contractile function were studied in vitro, using isolated rat aortic rings pre-contracted with KCl 20 mM. Among the target compounds, only 16 showed appreciable effectiveness, exhibiting an efficacy parameter (57%) lower than that of NS1619 and a comparable potency index (pIC50: 5.58).

N6-cycloalkyl-2-phenyl-3-deaza-8-azaadenines: a new class of A1 adenosine receptor ligands. A comparison with the corresponding adenines and 8-azaadenines.

Several 9-benzyl-N6-cycloalkyl-2-phenyladenines, 9-benzyl-N6-cycloalkyl-2-phenyl-8-azaadenines and 4-cycloalkylamino-1-benzyl-6-phenyl-1H-1,2,3-triazolo[4,5-c]pyridines were prepared and assayed as A1 adenosine receptor ligands. The 1H-1,2,3-triazolo[4,5-c]pyridines were obtained starting from N,N-diethyl-1-benzyl-4-carboxyamido-5-methyl-1H-1,2,3-triazole by lithiation in anhydrous tetrahydrofurane in the presence of benzonitrile. The usual work up afforded the isolation of 1-benzyl-6-phenyl-1H-1,2,3-triazolo[4,5-c]pyridin-4-one which was treated with phosphorous oxychloride and cycloalkylamines. Some compounds showed high affinity and selectivity and the trend of Ki values corresponds to the series of 9-benzyl-N6-cycloalkyl-2-phenyladenines and 9-benzyl-N6-cycloalkyl-2-phenyl-8-azaadenines, therefore they can be considered bioisosteres. The affinity data permitted us to ascertain the role and the importance of the N3 in the adenine or 8-azaadenine moiety in the receptor binding and to study the dimension of the receptor lipophilic pocket which is filled by the N6 substituent of adenosine derivatives.

New N6- or N(9)-hydroxyalkyl substituted 8-azaadenines or adenines as effective A1 adenosine receptor ligands.

In this paper we describe synthesis and biological assays of some A(1) ligands more water-soluble than the effective, but very lipophilic, 8-azaadenines and adenines discovered in the past and obtained introducing on N(6) or N(9) substituent a hydroxy group. Five of the new N(6)-hydroxyalkyl- and N(6)-hydroxycycloalkyl-2-phenyl-9-benzyl-8-azaadenines showed very high affinity (Ki<40 nM) and selectivity for A(1) adenosine receptors. Among the 2-phenyl-9-(2-hydroxy-3-alkyl)-8-azaadenines or adenines prepared, the compounds with the higher A(1) affinity and selectivity resulted 2-phenyl-9-(2-hydroxy-3-propyl)-N(6)-cyclopentyl- and cyclohexyl-8-azaadenine with Ki 2.2+/-0.2 nM and 2.8+/-0.3 nM respectively. From the point of view of water-solubility, 2-phenyl-9-(2-hydroxy-3-propyl)-8-azaadenine was the most interesting compound, having a CLogP of 1.066991 and a water-solubility of 1.2 mg mL(-1).

Characterization and preliminary use of 1-, 2- and 3-methyl-5-phenyl-7-chloro-1,2,3-triazolo[4,5-d]pyrimidine as reaction intermediates.

This paper reports synthesis and characterization, by spectroscopic methods, of three new N-methyl-5-phenyl-7-chloro-1,2,3-triazolo[4,5-d]pyrimidine isomers 3a, 3b and 3c. For comparison purpose the 3-benzyl-5-phenyl-7-chloro-1,2,3-triazolo[4,5-d]pyrimidine (7) was also prepared. Starting from the isomer mixture 3a-c and the chloroderivative 7, by nucleophilic substitution reaction with ethyl carbazate and subsequent intramolecular cyclization, the new tricyclic derivatives 5a-c and 9 were prepared and tested towards the benzodiazepine and adenosine A1 and A2A receptors.

New 1,2,3-triazolo[1,5-a]quinoxalines: synthesis and binding to benzodiazepine and adenosine receptors. II.

On pursuing research about 1,2,3-triazolo[1,5-a]quinoxalines, in this paper we report synthesis and binding assays toward the benzodiazepine and A(1) and A(2A) adenosine receptors, of a new series of derivatives, bearing some structural changes (introduction of fluorine and trifluoromethyl in the seventh position, amino substituents in the fourth position, benzyl group in the fifth position and aroyl substituents in the third position). The biological tests have shown that only the 7-fluorosubstituted compounds 3a and 4a and the N-benzyl derivative 7 have a good affinity toward the benzodiazepine receptors, while only the 7-trifluoromethyl substituted compound 3b presents a moderate affinity with low selectivity toward the A(1) adenosine receptors. The other structural modifications strongly decreased biological activity.

Erythro- and threo-2-hydroxynonyl substituted 2-phenyladenines and 2-phenyl-8-azaadenines: ligands for A1 adenosine receptors and adenosine deaminase.

erythro-2-Phenyl-9-(2-hydroxy-3-nonyl)adenine and its 8-aza analog were prepared and showed a very high inhibitory activity towards adenosine deaminase (ADA), with Ki 0.55 and 1.67 nM, respectively, and high affinity for A1 adenosine receptors, with Ki 28 and 2.8 nM, respectively. To increase affinity for A1 receptors we introduced a substituent on the N6 position such as alkyl or cycloalkyl groups, which are present in effective agonists or antagonists. Furthermore, for some compounds, we prepared the two diastereoisomers erythro and threo to verify whether the binding with A1 receptors is stereoselective, as in ADA. Results show that some of the synthesised compounds are good inhibitors for ADA and good ligands for A1, and the erythro diastereoisomers are more active than the threo ones. The experimental evidence allows us to hypothesise some similarity in the three dimensional structures of the binding site of the two proteins, ADA and A1 adenosine receptor, in spite of lacking any homologies in the amino acid sequences.