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 uM. 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. / PhD (Pharmaceutical Chemistry), North-West University, Potchefstroom Campus, 2012
| Identifer | oai:union.ndltd.org:NWUBOLOKA1/oai:dspace.nwu.ac.za:10394/10408 |
| Date | January 2012 |
| Creators | Joubert, Jacques |
| Publisher | North-West University |
| Source Sets | North-West University |
| Language | English |
| Detected Language | English |
| Type | Thesis |
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