One of the strategies, which can lead to the discovery of novel biologically active compounds and over the last 20 years has emerged as a fruitful approach, is the identification and use of molecular scaffolds with versatile binding properties. For such structures, bearing appropriate molecular decoration and, therefore, able to recognize with high affinity more unrelated targets, the term ''privileged structure'' has been proposed. In this study it has been demonstrated that the properly substituted 1,4-dioxane nucleus has proven to be a suitable scaffold for: a) potent muscarinic receptor (mAChRs) agonists and antagonists; b) selective alpha2A-adrenoreceptor (alpha2A-ARs) antagonists; c) selective alpha1D-adrenoreceptor (alpha1D-ARs) antagonists; d) potent 5-HT1A serotoninergic receptor full agonists; e) potential anticancer agents. It is well known that the (1,3-dioxolan-4-yl)-N,N,N-trimethylmethanaminium iodide nucleus has been used for many years as a scaffold for its ability to give potent muscarinic agonists or antagonists, depending on the size of the substituent in position 2 (methyl or bulkier group, respectively). Inspired by this scaffold, I designed, prepared and studied at all five muscarinic receptor subtypes two series of 1,4-dioxane analogues substituted in positions 5 and 6 with methyl or bulkier groups to obtain agonists and antagonists, respectively. Concerning agonists the most interesting compound was the cis-trimethyl-(6-methyl-1,4-dioxan-2-ylmethyl)-ammonium iodide. Such a compound behaved as a full agonist at M1-M3 subtypes (pD2 = 5.65, 7.57, 7.34, respectively) and as a partial agonist at M4 subtype (pD2 = 5.87). As expected, the replacement of the methyl group in positions 5 or 6 of the agonist compounds with bulkier aryl or cycloalkyl rings modulated their muscarinic profile from agonists to antagonists. The most productive modification for affinity was the insertion of two phenyl groups in position 6, such a derivative proving to be a potent antagonist at all muscarinic receptor subtypes. The biological evaluation of the enantiomers of the most interesting both agonist or antagonist compounds demonstrated that the muscarinic effect was closely related to the stereochemistry of the ligands. It is well known that the benzodioxane nucleus bearing an appropriate substituent at position 2 can discriminate markedly among alpha-AR adrenoreceptor subtypes. In fact, WB 4101 and Idazoxan, both carrying a 1,4-benzodioxan-2-yl moiety as a basic feature but having a different 2-substituent [(2,6-dimethoxyphenoxy)ethylamine, or imidazoline] are highly selective for alpha1- and alpha2-ARs, respectively. A variety of their analogues have been studied involving a modification of the dehydrodioxane ring to develop high-affinity, site-selective ligands for each of the three alpha 1- and alpha2-AR subtypes (alpha1a/A, alpha1b/B,B alpha1d/D; alpha2A, alpha2B,B alpha2C). I have expanded on the structure-activity relationship study by investigating the possibility that the quite planar 1,4- benzodioxane privileged structure of WB 4101 and Idazoxan might be replaced by the less conformationally constrained 1,4-dioxane ring. The increased flexibility of 1,4-dioxane moiety might allow a better interaction with alpha-AR subtypes or favour the interaction with only one of them, leading hopefully to selectivity. Therefore, WB 4101- and Idazoxan-related derivatives obtained by inserting one or two phenyl groups in 5 or 6 position of 1,4-dioxane nucleus were designed and studied by binding and functional experiments. The pharmacological profiles of alpha2-AR antagonists related to Idazoxan were investigated using CHO cell lines stably expressing cDNAs encoding human alpha2A-, alpha2B-, and alpha2C-AR subtypes by cytosensor microphysiometer. Among the new compounds, only trans-2-(5-phenyl-1,4-dioxan-2-yl)-4,5-dihydro-1H-imidazole behaved as a competitive antagonist at alpha2A-AR subtype with a pKb value of 7.14 similar to that of Idazoxan (pKb = 7.73). Interestingly, unlike Idazoxan, this compound was not active at alpha2B-, and alpha2C-AR subtypes. Therefore, for its particular alpha2A selective antagonism, this compound might be of interest for antinociceptive drug development, according to the observation that alpha2A-AR selective antagonists mightincrease the opioid analgesia. Concerning the alpha1-AR antagonists the replacement of the condensed benzene ring of WB 4101 with two phenyl rings inserted in position 6 of the 1,4-dioxane nucleus, along with the simultaneous removal of one methoxy group in the 2,6-dimethoxyphenoxy moiety, favoured alpha 1d subtype selectivity. In fact, the alpha1d affinity value (pKi = 8.94) was significantly higher than those for alpha1a and alpha1b subtypes (pKi =7.56 and 7.25, respectively). Moreover, the WB 4101-related derivatives were also evaluated on HeLa cells, expressing 5-HT1A serotoninergic receptor, since it is well known that this receptor exhibits a high degree of homology to alpha1-ARs. Among all the 5-phenyl, 6-phenyl and 5,5-diphenyl derivatives the presence of a 2-methoxy substituent in the 2-phenoxyethyl moiety was beneficial for high 5-HT1A affinity. When two phenyl rings were inserted in position 6, the methoxy-substitution in the 2-phenoxyethyl moiety did not seem to affect binding to the 5-HT1A receptor, all three derivatives (unsubstituted, 2-methoxy- and 2,6-dimethoxy-substituted) showing similar nM affinity values. Interestingly, the 6,6-diphenyl derivative bearing the 2,6- dimethoxyphenoxy moiety, examined in the [35S]GTP_S binding at the human cloned 5-HT1A receptor, was a potent full agonist with a pD2 value of 8.28. It also showed good selectivity for 5-HT1A towards the alpha1A-, alpha1B- and alpha1D-AR subtypes, representing a new lead in the design of potent full 5-HT1A agonists significantly selective over alpha1-ARs. Moreover, following our recent observation that alpha1D- and alpha1B-ARs are expressed in PC-3 prostate cancer cells and are involved in the modulation of apoptosis and cell proliferation, the cytotoxic effects of the new compounds on this cell line were determined. The 6,6-diphenyl derivative unsubstituted in the 2,6-dimethoxyphenoxy moiety exhibited the highest potency in inhibiting PC-3 cell growth and seemed to activate a mitochondrial-independent apoptotic pathway. In conclusion, the results obtained in my research proved the ability of the 1,4-dioxane nucleus to act as a versatile biological scaffold.

1,4-Dioxane Nucleus as a Suitable Scaffold for the Characterization of Different Receptor Systems

DEL BELLO, FABIO
2009

Abstract

One of the strategies, which can lead to the discovery of novel biologically active compounds and over the last 20 years has emerged as a fruitful approach, is the identification and use of molecular scaffolds with versatile binding properties. For such structures, bearing appropriate molecular decoration and, therefore, able to recognize with high affinity more unrelated targets, the term ''privileged structure'' has been proposed. In this study it has been demonstrated that the properly substituted 1,4-dioxane nucleus has proven to be a suitable scaffold for: a) potent muscarinic receptor (mAChRs) agonists and antagonists; b) selective alpha2A-adrenoreceptor (alpha2A-ARs) antagonists; c) selective alpha1D-adrenoreceptor (alpha1D-ARs) antagonists; d) potent 5-HT1A serotoninergic receptor full agonists; e) potential anticancer agents. It is well known that the (1,3-dioxolan-4-yl)-N,N,N-trimethylmethanaminium iodide nucleus has been used for many years as a scaffold for its ability to give potent muscarinic agonists or antagonists, depending on the size of the substituent in position 2 (methyl or bulkier group, respectively). Inspired by this scaffold, I designed, prepared and studied at all five muscarinic receptor subtypes two series of 1,4-dioxane analogues substituted in positions 5 and 6 with methyl or bulkier groups to obtain agonists and antagonists, respectively. Concerning agonists the most interesting compound was the cis-trimethyl-(6-methyl-1,4-dioxan-2-ylmethyl)-ammonium iodide. Such a compound behaved as a full agonist at M1-M3 subtypes (pD2 = 5.65, 7.57, 7.34, respectively) and as a partial agonist at M4 subtype (pD2 = 5.87). As expected, the replacement of the methyl group in positions 5 or 6 of the agonist compounds with bulkier aryl or cycloalkyl rings modulated their muscarinic profile from agonists to antagonists. The most productive modification for affinity was the insertion of two phenyl groups in position 6, such a derivative proving to be a potent antagonist at all muscarinic receptor subtypes. The biological evaluation of the enantiomers of the most interesting both agonist or antagonist compounds demonstrated that the muscarinic effect was closely related to the stereochemistry of the ligands. It is well known that the benzodioxane nucleus bearing an appropriate substituent at position 2 can discriminate markedly among alpha-AR adrenoreceptor subtypes. In fact, WB 4101 and Idazoxan, both carrying a 1,4-benzodioxan-2-yl moiety as a basic feature but having a different 2-substituent [(2,6-dimethoxyphenoxy)ethylamine, or imidazoline] are highly selective for alpha1- and alpha2-ARs, respectively. A variety of their analogues have been studied involving a modification of the dehydrodioxane ring to develop high-affinity, site-selective ligands for each of the three alpha 1- and alpha2-AR subtypes (alpha1a/A, alpha1b/B,B alpha1d/D; alpha2A, alpha2B,B alpha2C). I have expanded on the structure-activity relationship study by investigating the possibility that the quite planar 1,4- benzodioxane privileged structure of WB 4101 and Idazoxan might be replaced by the less conformationally constrained 1,4-dioxane ring. The increased flexibility of 1,4-dioxane moiety might allow a better interaction with alpha-AR subtypes or favour the interaction with only one of them, leading hopefully to selectivity. Therefore, WB 4101- and Idazoxan-related derivatives obtained by inserting one or two phenyl groups in 5 or 6 position of 1,4-dioxane nucleus were designed and studied by binding and functional experiments. The pharmacological profiles of alpha2-AR antagonists related to Idazoxan were investigated using CHO cell lines stably expressing cDNAs encoding human alpha2A-, alpha2B-, and alpha2C-AR subtypes by cytosensor microphysiometer. Among the new compounds, only trans-2-(5-phenyl-1,4-dioxan-2-yl)-4,5-dihydro-1H-imidazole behaved as a competitive antagonist at alpha2A-AR subtype with a pKb value of 7.14 similar to that of Idazoxan (pKb = 7.73). Interestingly, unlike Idazoxan, this compound was not active at alpha2B-, and alpha2C-AR subtypes. Therefore, for its particular alpha2A selective antagonism, this compound might be of interest for antinociceptive drug development, according to the observation that alpha2A-AR selective antagonists mightincrease the opioid analgesia. Concerning the alpha1-AR antagonists the replacement of the condensed benzene ring of WB 4101 with two phenyl rings inserted in position 6 of the 1,4-dioxane nucleus, along with the simultaneous removal of one methoxy group in the 2,6-dimethoxyphenoxy moiety, favoured alpha 1d subtype selectivity. In fact, the alpha1d affinity value (pKi = 8.94) was significantly higher than those for alpha1a and alpha1b subtypes (pKi =7.56 and 7.25, respectively). Moreover, the WB 4101-related derivatives were also evaluated on HeLa cells, expressing 5-HT1A serotoninergic receptor, since it is well known that this receptor exhibits a high degree of homology to alpha1-ARs. Among all the 5-phenyl, 6-phenyl and 5,5-diphenyl derivatives the presence of a 2-methoxy substituent in the 2-phenoxyethyl moiety was beneficial for high 5-HT1A affinity. When two phenyl rings were inserted in position 6, the methoxy-substitution in the 2-phenoxyethyl moiety did not seem to affect binding to the 5-HT1A receptor, all three derivatives (unsubstituted, 2-methoxy- and 2,6-dimethoxy-substituted) showing similar nM affinity values. Interestingly, the 6,6-diphenyl derivative bearing the 2,6- dimethoxyphenoxy moiety, examined in the [35S]GTP_S binding at the human cloned 5-HT1A receptor, was a potent full agonist with a pD2 value of 8.28. It also showed good selectivity for 5-HT1A towards the alpha1A-, alpha1B- and alpha1D-AR subtypes, representing a new lead in the design of potent full 5-HT1A agonists significantly selective over alpha1-ARs. Moreover, following our recent observation that alpha1D- and alpha1B-ARs are expressed in PC-3 prostate cancer cells and are involved in the modulation of apoptosis and cell proliferation, the cytotoxic effects of the new compounds on this cell line were determined. The 6,6-diphenyl derivative unsubstituted in the 2,6-dimethoxyphenoxy moiety exhibited the highest potency in inhibiting PC-3 cell growth and seemed to activate a mitochondrial-independent apoptotic pathway. In conclusion, the results obtained in my research proved the ability of the 1,4-dioxane nucleus to act as a versatile biological scaffold.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11581/401936
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