Abstract
There are increasing numbers of reports that G protein-coupled receptors (GPCRs) can signal
from intracellular locations. Cannabinoid type 2 receptor (CB2R), a Class A GPCR, is a
promising target to treat pain, inflammation, cancer, and rheumatoid arthritis. CB2R is an
example of a GPCR that has considerable intracellular expression which has been suggested
to elicit unique signal transduction pathways in comparison with surface-resident CB2R. This
research aims to design, synthesise and evaluate a series of novel tool compounds which can
specifically target intracellular CB2R.
To realise the specific intracellular CB2R targeting, chemical tools were designed as prodrugs
of a CB2R agonist, which could either be activated by an intracellularly expressed luciferase
(and luciferin) via a bioluminescence resonance energy transfer (BRET) process or could be
activated by an intracellular enzyme such as cathepsin B.
A photo-activated CB2R agonist prodrug (2.2) and two photo-activated model prodrugs (2.4
and 2.5) were designed, synthesised and evaluated. All three photo-activated prodrugs were
tested under LED light and the results were analysed by analytical RP-HPLC and LC-MS.
Although the investigation of photolysis illustrated the reaction mechanisms were different
in organic and aqueous solutions, all three prodrugs showed promising photolysis profiles
under the LED light. The photo-activated CB2R agonist prodrug 2.2 had the fastest cleavage
at 450-455 nm blue LED light, which matched the design specification. The photo-activated
CB2R agonist prodrug 2.2 will likely prove a very useful prodrug to study CB2R in a triggered
way using artificial light. Biological evaluation of prodrug 2.2 is currently being undertaken
in Auckland by collaborators.
Unfortunately, the initial attempt of BRET-triggered cleavage on the photo-activated CB2R
agonist prodrug 2.2 by the untemplated luciferin and luciferase failed. The overall original
idea was to template the prodrug to the luciferin. However, this was not completed due to
time constraints. The results of this thesis show that a templated prodrug is an absolute
requirement for successful BRET and prodrug cleavage.
A series of cathepsin B cleavable CB2R agonist prodrugs were also designed, synthesised
and evaluated as another intracellular CB2R targeting strategy. The cathepsin B enzymatic
cleavage on model prodrugs 4.11, 4.12, 4.13, 4.24 and prodrugs 4.1, 4.17, 4.18, 4.25 were
monitored by analytical RP-HPLC and LC-MS. Although some of the model prodrugs (4.13
and 4.24) showed possible cleavage after incubation with cathepsin B, none of the prodrugs
which released CB2R agonists were successfully cleaved in vitro. The results suggested that
the steric hinderance caused by the CB2R agonist was the problem, which could be solved in
future work by using other less bulky CB2R agonists.