Design and Synthesis of Potent and Selective Anticancer Compounds Targeting Tubulin Polymerisation and Aminopeptidase N

Abstract

This thesis describes the synthesis and evaluation of novel drug conjugates targeting aminopeptidase N (APN) (E.C. 3.4.11.2) and tubulin dynamics. Chapter 1 opens with a thorough review of the existing literature on targeted anticancer agents that contain a tubulin targeting moiety. In this context, particular emphasis has been placed on attaching polysaccharides, peptides, proteins and antibodies that exploit the overexpression of receptors for these substances. The antibody targeted approach has been the most successful to date with six such examples having gained clinical approval. Additionally, conjugates targeting the folate receptor have shown promising efficacy and toxicity profiles in pre-clinical models and in early-stage clinical studies. This section of the thesis has been published in full in Current Medicinal Chemistry, Collyer, S.E., Stack, G.D. and Walsh, J.J., 2022., 29(31), pp.5179-5211. The chapter continues with a discussion on APN, a membrane-bound ectopeptidase found in a variety of tissues and cell types within the body. In the context of tumour targeting, APN is expressed on activated endothelial cells of angiogenic vasculature (i.e. tumour vasculature) but not by quiescent vasculature. Furthermore, APN is overexpressed by certain tumour cells. The functions of APN in these locations facilitate progression of disease by promoting angiogenesis, tumour growth and metastasis. Thus the exclusive expression of APN on tumour vasculature as opposed to normal vasculature can be exploited for the selective delivery of anticancer agents to the tumour site. This is the subject matter of this thesis. Chapter 1 closes with an overview of the principal objectives of the work described in the remaining chapters. Chapter 2 opens with a description of the synthetic methodology that was optimised to scale-up the synthesis of the tubulin binding agent (TBA), ADR269. It then follows on with a description of the methodology used to conjugate APN-targeting peptides to it, based on the APN inhibitors bestatin and probestin. Synthetic pathways of up to 18 linear steps is described for these. Emphasis was placed on the use of 2-D NMR techniques for structural elucidation purposes. In addition to the use of HSQC and HMBC spectra, particular emphasis was placed on the generation of variable temperature, NOESY and ROESY spectra for the identification of cis/trans conformers and rotamers in some of the synthesised conjugates of the probestin type. This chapter concludes with the evaluation of the binding affinity (Ki) of the conjugates to APN, studies on their antiproliferative activity against PC3 and DU-145 prostate cancer cells and HUVECs, their inhibition of APN at a cellular level using PC3 cells, and their anti-migratory (i.e., anti-angiogenic) properties against HUVECs using the scratch assay. Chapter 3 describes the scale-up of a novel synthetic method that converts essentially ADR269 into a different class of TBA, namely the 4-arylcoumarin type via a ring contraction reaction. The mechanism involved was investigated by monitoring the ring contraction reaction visually (as evolution of gases, thought to be nitrogen and hydrogen cyanide) and by NMR. APN-targeted conjugates with this TBA were then synthesised by coupling APN-targeting peptides to it. These conjugates were biologically evaluated using the identical set of assays for those conjugates described in Chapter 2. Chapter 4 outlines the synthesis of an analogous series of conjugates based on having doubly functionalised attachment points on phenstatin-based TBAs. In essence, two targeting moieties can couple to these TBAs. This scaffold type allows for exploration of the effect of increasing the number of targeting moieties (e.g. additive or synergistic effects). Hybrid conjugates with both inhibitor and enzyme substrate moieties are also described. Structure-activity relationship information from hybrid conjugates and those comprising two targeting moieties is then described based on data from biological studies. Overall, several conjugates displayed low nM Ki to APN, with 4.14 displaying a Ki of 6.62 +/- 1.12 nM. Of those based on the bestatin moiety, 4.12 displayed a 13.6-fold increase in binding affinity compared to bestatin. In the cell-based APN assay, 4.16 was 12.6 times more potent than probestin with virtually all conjugates displaying higher binding relative to probestin in this assay. In the antiproliferative cell-based assay, 3.08 was 2.4-fold more selective for APN-expressing PC3 cells versus non-APN-expressing DU-145 cells, with the unconjugated TBA, GJH140, displaying a 1.2-fold selectivity. In the migration assay, several conjugates displayed inhibition of cellular migration at 50 nM, with 3.08 and 4.12 being particularly effective. Further studies are required before the true potential of these conjugates can be realised in cancer therapy. In this regard, the antiproliferative activity and selectivity of the conjugates in other APN expressing cancer cell lines should be carried out. The invasion assay, ex vivo aortic ring assay of angiogenesis/vasculature and in vivo tumour efficacy studies should form part of future studies. Profiling the rate of release, by LCMS, of the APN binding motif of the conjugate(s) should be determined in both in vitro and in vivo settings.