|dc.description.abstract||Adenosine A1 receptor (A1AR), cannabinoid type 1 receptor (CB1R) and cannabinoid type 2 receptor (CB2R) are class A G protein-coupled receptors (GPCRs) and play important roles in human pathophysiological conditions such as cardiovascular, neurological, metabolic and immunological disorders. Fluorescent ligands are powerful tools to investigate processes such as receptor expression, localisation, trafficking and receptor-protein interactions in the native cell environment. Fluorescent ligands can also be used as tracers in pharmacological assays, instead of the commonly used radioligands that carry inherent safety risks. The development of fluorescent ligands with high affinity, selectivity and suitable imaging properties for A1AR, CB1R and CB2R would greatly contribute to an increased understanding of receptor biology and thus facilitate the drug development process. Development of fluorescent ligands with sufficient polarity for cannabinoid receptors (CBRs), which have lipid-based endogenous ligands, is an especially challenging task. This thesis describes the development of small molecule-based fluorescent ligands for A1AR, CB1R and CB2R, via attachment of a linker and fluorophore to a ligand.
(Benzimidazolyl)isoquinolinols, analogues of previously reported high affinity A1AR (benzimidazolyl)isoquinolines, were explored in chapter 2 for the development of A1AR fluorescent ligands. A procedure for 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) mediated aromatisation of tetrahydroisoquinolines and multistep synthesis for the preparation of (benzimidazolyl)isoquinolinols was developed. Based on the previously reported structure–activity relationship (SAR), linkers (and linker plus fluorophore conjugates) were introduced in the C-6 or C-7 position of (benzimidazolyl)isoquinolinols. Unfortunately, these fluorescent ligands did not exhibit any significant affinity for A1AR in a bioluminescence resonance energy transfer (BRET) assay using the NanoLuc luciferase and it was concluded that (benzimidazolyl)isoquinolinols might not be a suitable scaffold for the development of A1AR fluorescent ligands. NMR spectroscopy and reverse phase HPLC studies showed (benzimidazolyl)isoquinolinols exhibit tautomerism.
Chromenopyrazoles, previously reported as high affinity CB1R ligands, were investigated for development into CB1R fluorescent ligands in chapter 3. Based on previous SAR, linkers then fluorophores were introduced at six different chromenopyrazole positions. Disappointingly, fluorescent chromenopyrazoles did not exhibit high affinity for CB1R in a radioligand binding assay. However, several chromenopyrazoles including a linker conjugate 3.22 and a peptide linker conjugate 3.39 exhibited high affinity for CB2R that were promising candidates for development of CB2R fluorescent ligands. All of the chromenopyrazoles that were evaluated in a cyclic adenosine monophosphate (cAMP) functional assay behaved as agonists at CB2R. Docking studies were carried out using a CB2R homology model and showed that high affinity CB2R chromenopyrazoles with linkers attached likely exit via a cavity located between transmembrane helix (TM) 1 and TM7.
In chapter 4, efforts were made to develop high affinity CB2R fluorescent ligands and more polar CB2R linker conjugates that built on the results of chapter 3. The highest affinity CB2R linker conjugate 3.22 (obtained in chapter 3) was conjugated to four different fluorophores (BODIPY-FL, Cy5, TAMRA, BODIPY-630/650) and two high affinity CB2R fluorescent ligands (4.01, 4.02) were obtained. The highest affinity CB2R fluorescent ligand 4.02 (Ki = 41.8 ± 4.5 nM at hCB2R; 5856 ± 1264 nM at hCB1R) behaved as an inverse agonist (4.02, EC50 = 142.0 ± 13.1 nM at hCB2R, 196.7 ± 9.11 % of forskolin response at hCB2R) in the cAMP functional assay and showed CB2R-specific-binding in widefield imaging experiments using CB2R expressing HEK-293 cells. Fluorescent ligand 4.02 exhibited higher affinity for CB2R than any other reported CB2R fluorescent ligands and is the first high affinity CB2R fluorescent ligand for which functional data has been reported (as of July 2018). Fluorescent ligand 4.02 possesses suitable CB2R imaging properties and will be a useful tool for researchers studying CB2R biology in techniques such as fluorescence-based assays, widefield microscopy and flow-cytometry and can be used in resonance energy transfer experiments with other fluorescent partners. In addition, three high-moderate affinity peptide linker conjugates (4.06, 4.07, 4.08) with considerably higher polarity compared to commonly used cannabinoid receptor ligand CP55,940 were obtained, all of which behaved as CB2R agonists.
Development of high affinity CB1R ligands based on a reported pyridyl scaffold was explored in chapter 5. Fluorescent ligands were designed using previously reported SAR coupled with results obtained from CB1R docking using the reported CB1R crystal structure. O-Linker pyridyl-2-carboxamides and corresponding fluorescent conjugates were prepared via a multistep synthesis. Unfortunately, none of the fluorescent ligands exhibited any significant affinity for CB1R, however, three moderate affinity CB1R linker conjugates (5.33, 5.34, 5.35) were obtained. It was therefore concluded that optimised derivatives of pyridyl-2-carboxamide with different O-linkers or with linkers conjugated at different positions of the pyridine could be another strategy for the development of CB1R fluorescent ligands.||