Molecular and Stereochemical Requirements of 1,4-Dioxane Ligands for the Interaction with Different Receptor Systems

A strategy that 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. The 1,4-dioxane nucleus is a versatile scaffold to obtain drugs directed to mAChRs, α1-ARs, and 5-HT1A receptors. The aim of this PhD thesis was to extend the previous structure-activity relationship (SAR) studies about ligands interacting with the above receptors and to investigate the possibility to address novel ligands toward other receptor systems. In particular, it has been confirmed that the properly substituted 1,4-dioxane nucleus is a suitable scaffold for: • potent muscarinic receptor (mAChRs) agonists and antagonists; • selective α1d-adrenoreceptor (α1d-ARs) antagonists; • potent 5-HT1A serotoninergic receptor full agonists. Moreover, it has proven to be a versatile scaffold to obtain: • selective α2A-adrenoreceptor (α2A-ARs) antagonists/I2 imidazoline binding site (I2-IBS) ligands; • blockers of the PCP binding site of NMDA receptor; • selective σ1 or σ2 ligands; • dopamine D2-like receptor ligands. A series of mAChRs agonists, obtained by moving the methyl substituent in position 6 of the 1,4-dioxane of the potent M2/M3 agonist cis-N,N,N-trimethyl-(6-methyl-1,4-dioxan-2- yl)methanaminium iodide to position 2 or 3, was prepared to complete a previous SAR study. To verify whether the methyl group in position 6 of 1,4-dioxane was essential for its potent mAChR activity, its desmethyl analogue was also prepared and pharmacologically characterized. To rationalize the experimental observations and to get information about the mode of interaction of the already published and novel 1,4-dioxane agonists, a retrospective computational study was performed. Afterwards, docking studies, using the recently published crystal structures of human M2 and rat M3 receptors were conducted to investigate relationships between the biological behaviour of 1,4-dioxane compounds and 1,3-dioxolane analogues and their functional groups involved in the interaction with these protein receptors. Moreover, the modulation of the pharmacological profile from agonist to antagonist was successfully obtained by replacing the methyl group in position 6 of the 1,4-dioxane scaffold of the potent M2/M3 mAChR agonist cis-N,N,N-trimethyl-(6-methyl-1,4-dioxan-2-yl)methanaminium iodide with bulkier groups. In particular, the 6,6-diphenyl substitution provided a selective M3 antagonist, whose racemic form and its (S) eutomer proved to be effective in reducing the volume-induced contractions of rat urinary bladder and were devoid of cardiovascular effects in an in vivo study. It is well known that the benzodioxane nucleus bearing an appropriate substituent at position 2 can discriminate markedly among α-AR subtypes. Indeed, Idazoxan and WB 4101, both carrying a 1,4-benzodioxan-2-yl moiety as a basic feature but a different 2-substituent [imidazoline or (2,6- dimethoxyphenoxy)ethylamine], are highly selective for α2- and α1-ARs, respectively. To investigate the possibility that the quite planar 1,4-benzodioxane privileged structure of Idazoxan and WB 4101 might be replaced by the less conformationally constrained 1,4-dioxane ring, two series of ligands were prepared and pharmacologically characterized. With the Idazoxan-related analogues, we demonstrated that α2A-AR antagonism might favourably contribute to I2-IBSmediated morphine analgesia enhancement. Therefore, the most interesting compound and its eutomer, which showed such a biological profile and lacked sedative and hypotensive side effects due to their selective α2A-AR antagonism, might be novel promising tools therapeutically useful in pain management in combination with opioids. WB 4101-related analogues allowed us to demonstrate that 5-HT1A receptor and α1-AR binding sites recognized by the 1,4-dioxane compounds display reversed stereochemical requirements: the (R) configuration favoured high affinity and selectivity for the α1d-AR subtype with respect to α1a and α1b-AR subtypes, while the (S) configuration was associated to higher affinity and potency for 5-HT1A receptor with high selectivity ratios. Moreover, eutomers at the α1d-AR subtype also displayed the best antiproliferative and cytotoxic effects in human prostate cancer cells (PC-3), indicating the involvement of the α1d-AR subtype in their anticancer properties, which was confirmed evaluating the most interesting compounds in α1d-AR silenced PC-3 cells. We also demonstrated that 1,4-dioxane nucleus proved to be a suitable scaffold for the design of ligands directed to other receptor systems, such as sigma, NMDA and dopamine (DA) receptors. Recently, it was demonstrated that the properly substituted 4-(2-aminoethyl)-1,3-dioxane and the Nbenzyl-4-(2-aminoethyl)-1,3-dioxane derivatives, ring and side chain homologues of dexoxadrol and etoxadrol, resulted in compounds showing high NMDA or σ1 receptor affinity, respectively. Therefore, two series of regioisomers, bearing the 1,4-dioxane nucleus were prepared and evaluated in vitro for their affinities at σ1, σ2, and PCP binding site of NMDA receptor. Within the aminomethyl series, trans-(6-cyclohexyl-6-phenyl-1,4-dioxan-2-yl)methanamine, which showed binding affinity and functional activity not significantly different from those of (S)-(+)-ketamine, due to its promising therapeutic potential, deserves to be pharmacologically studied in depth. The N-benzyl-aminomethyl derivative N-Benzyl-1-(5,5-diphenyl-1,4-dioxan-2-yl)methanamine showed the highest affinity and selectivity for σ1 with respect to both σ2 and the PCP binding site. Some compounds, already published or previously synthesized by the members of the research group, were selected to be evaluated for their binding affinity at human dopamine D2, D3 or D4 receptor subtypes, expressed on HEK293 cells, which I carried out during my experience at the National Institute on Drug Abuse. Encouraged by the preliminary results, during the six months I spent at NIDA in Baltimore (U.S.A.), I designed, synthesized and biologically evaluated a novel series of hybrid molecules, obtained by coupling 5,5-, 6,6-diphenyl-substituted 1,4-dioxane nucleus or the more conformationally constrained 1,4-benzodioxane nucleus with the N-(4-(4-(2,3-dichloro)-piperazine-1-yl)butyl)carboxamide or N-(3-fluoro or 3-hydroxy-4-(4-(2,3-dichloro)-piperazine-1-yl)butyl)carboxamides fragments, which had previously been shown to play a crucial role in increasing selectivity toward the dopamine D3 receptor subtype compared to the D2 and D4 subtypes. From the SAR study some interesting D3-selective compounds emerged, for which functional studies are in progress. Moreover, during my research experience in Germany, at Westfa'¤lische Wilhelm-Universita'¤t Ma'¼nster, I optimized and validated a cell-based functional assay for GluN2A and GluN2B subunitcontaining NMDA receptors, which was performed to test the antagonist potency of 1,4-dioxane NMDA channel blockers and a novel series of selective GluN2B subunit antagonists.