Abstract
Bioorthogonal chemistry refers to a chemical reaction taking place within a living system, without interfering with any native, biochemical processes. Over the last 20 years, research in this area has exploded, with initial applications in protein labelling and cellular imaging expanding to include cell engineering, materials science, protein isolation and drug delivery. Bioorthogonal prodrug activation strategies that make use of the hydrolytic instability of 1,3-dipolar cycloaddition adducts following reaction of azides with TCO provide exciting possibilities for drug delivery. However, drug release in vivo was insufficient meaning further optimisation and amplification strategies are required.
This thesis focuses on the use of bioorthogonally triggered aryl azide prodrugs, activated with trans-cyclooctene (TCO), to deliver highly cytotoxic drugs specifically to a target site. Chapter 2 investigated methoxy substituted aryl azides as a method to simultaneously improve activation rates and the amount of drug released. Electron donating groups have previously been found beneficial for release, however, methoxy substitution of the aryl azide had limited influence on activation rate and negatively impacted prodrug stability. As an alternative and complementary approach, dendrimer scaffolds were developed to release multiple drug molecules per click event, reducing the need for stoichiometric TCO. A primary, 1,6-immolative electron deficient core was designed to facilitate a rapid click with TCO, while a second electron rich core was modified with 1,4-,1,4-immolating or 1,4-,1,6-immolating linkers and N-methyl, methoxy and/or benzylic substitution for improved release. The N-methyl, 1,4-,1,6-immolating scaffold 147d (Figure i) demonstrated enhanced amount (compared to unsubstituted 1,6-immolating linker 144a) and rate of release of a fluorophore.
Chapter 3 investigated 1,3-dipolar cycloaddition between TCO and thiocarbamate prodrugs to observe the influence of self-amplification on the rate of activation. Self-amplifying systems are designed such that after activation, in addition to drug release, a self-propagating molecule (such as COS which induces H2S generation) is produced. The self-propagator can then react with another molecule of prodrug stimulating more release in a domino effect to generate a self-amplification loop which terminates upon complete consumption of the prodrug trigger group. Some tentative evidence of self-amplification was observed, however this was impacted by instability and slow activation. Phenols were not compatible with thiocarbonyl installation, however primary or secondary amines (generated via activation with 1,1-thiocarbonyldiimidazole (TCDI)) facilitated synthesis of thiocarbamate prodrugs. Comparable carbamate analogues investigated in vitro assessed the applicability of prodrugs in biological systems prior to full synthesis of the thiocarbamate analogue series. The toxicity of nitrogen mustards was reduced upon formation of dendrimer prodrugs, relative to monomeric counterparts which was very promising. Unfortunately, limited killing from triggered release of the free drug was observed due to degradation and further thiocarbamates were not investigated.
For successful self-amplification, release of COS and H2S generation before complete azide consumption is imperative. In Chapter 4 modifications to the thiocarbamate prodrugs and assay conditions enabled observation of H2S mediated self-amplification in azido prodrugs. A 1,6-immolating thiocarbamate 175a demonstrated a 1.5-fold faster rate than the carbamate 144a, which was independent of H2S generation, although stability impacted overall release. 1,4-1,6-self-immolating thiocarbamate 175b had a 2-fold faster rate than carbamate 176b at 10 eq. TCO (Figure i), which was reduced when H2S generation could not occur. Higher prodrug concentrations may aid further observation of self-amplification in the dual-core
tetra-fluorinated scaffold 175d/147d.
This thesis demonstrates the importance of linker design and the power of amplification strategies in bioorthogonal prodrug approaches.