Fluorescent polycyclic ligands : strategies towards the synthesis and evaluation of fluorescently labelled receptor and enzyme ligands

Abstract

Neurodegenerative disorders, including Alzheimer's and Parkinson's disease, and the development of neuroprotective agents have received significant research attention in recent years. Development of novel imaging techniques to study the biological mechanisms involved in the progression of these disorders have become an area of research interest. The design of novel small molecule imaging probes in combination with modem imaging techniques may provide information on neuroprotective binding site interactions and would assist in the design of novel biological assay methods. Techniques to visualize physiological or pathophysiological changes in proteins and living cells have become increasingly important in biomedical sciences, especially fluorescent techniques. Fluorescent ligands in combination with sophisticated fluorescent imaging technologies are useful tools to analyze and clarify the roles of biomolecules in living cells, affording high spatial and temporal resolution. This study is based on the development of polycyclic fluorescent ligands, which may be used in the study of receptor-ligand and/or enzyme-ligand interactions, utilizing these fluorescently labeled ligands in combination with fluorescent imaging techniques. Fluorescent conjugates with high affinity for the N-methyl-D-aspartate (NMDA) receptor, voltage gated calcium channels (VGCC) and/or the nitric oxide synthase (NOS) enzyme were designed and synthesised with the aim to directly measure binding of these novel molecules to receptors and/or enzymes. The first goal was to develop fluorescent ligands that exhibit similar inhibitory activity on NOS compared to the well-known selective neuronal NOS inhibitor 7-nitroindazole (7-NI). Polycyclic compounds, including amantadine and pentacycloundecane derivatives, were conjugated to fluorescent moieties that resemble the structure of 7-NI. It was thought that the lipophilic nature of the polycyclic compounds would increase the activity of the fluorescent moieties by facilitating increased blood brain barrier permeability and penetration through cell membranes. This would also potentially increase the selectivity of the novel conjugated compounds as selective neuronal NOS inhibitors, similar to 7-NI. The results from the NOS inhibition studies indicated that the novel fluorescent conjugates (5-14) inhibited the NOS enzyme at micromolar concentrations. Although none of the novel fluorescent polycyclic compounds were found to be more potent than 7-NI (IC50 = 0.11 11M), the indazole pentacyclorindecane (5), the coumarin-adamantane (7), the dansyl-adamantane (8), and the cyanoisoindole-adamantane (11) conjugates, exhibited IC5o values below 1 µM. These compounds could possibly be used as molecular probes in the development of high-throughput screening or competitive NOS displacement assays. Further studies on isoform selectivity will elaborate on the potential of these compounds as fluorescent molecular probes. The aforementioned fluorescent derivatives were further developed resulting in a series of novel fluorescent polycyclic conjugates with potent NOS inhibition indicating the potential of these compounds as neuroprotective agents. Due to the polycyclic structure's inherent inhibitory activity towards the NMDA receptor and VGCC we evaluated these derivatives as possible multifunctional neuroprotective agents acting on various neuroprotective targets. In the biological studies it was observed that four adamantane fluorescent compounds (7, 8, 10, 11) exhibited a high degree of inhibitory activity against the NOS enzyme and NMDA receptor and blocked VGCC. The fluorescent compounds were further able to scavange detrimental neurodegenerative free radicals. In silica studies also predicted a high degree of oral bioavailability and that these novel compounds should be effectively transported across the blood brain barrier. Taking the positive findings on the inhibition of the NMDA receptor and VGCC activity of the novel fluorescent polycyclic ligands into account we focused on the expansion of this series. This resulted in the synthesis of a series of fluorescent derivatives utilizing adamantane-3-aminopropanol as an intermediate to extend the chain length between the adamantyl and fluorescent moieties, to potentially reduce sterical hindrance and increase activity. These novel adamantane-3-aminopropanol fluorescent ligands were also evaluated for inhibition of the NMDA receptor and VGCC. The coumarin-, dansyl- and cyanoisoindole adamantane-3-aminopropanol fluorescent conjugates (15, 16, 19) displayed significant VGCC inhibition, with the dansyl (16) and di-nitrobenzene (20) fluorescent derivatives exhibiting NMDA receptor antagonistic activity. All these compounds showed improved activity when compared to known NMDA receptor and VGCC inhibitors in this class. Generally it was observed that the increased chain length analogues had improved VGCC inhibition and NMDA receptor activity when compared to their directly• conjugated counterparts. This led to the conclusion that an increase in chain length might indicate deeper immersion into the NMDA receptor and VGCC which may be necessary for stronger interaction with their putative binding sites. The dansyl analogue, N-[3-(1-adamantylamino)propyl]-5- dimethylaminonaphthalene-1-sulfonamide (16), was further used as a fluorescent NMDA receptor ligand in a fluorescent competition assay, utilizing known NMDA receptor inhibitors to demonstrate the possible applications of these novel fluorescent analogues and their benefit over the use of hazardous and expensive radioligand binding studies. Further investigation on the application of these derivatives, especially on the NOS enzyme and the NMDA receptor, will develop their potential as fluorescent ligands in the study of neurodegeneration and may also yield novel therapeutic agents against neurodegenerative disorders.