THE SYNTHESIS AND EVALUATION OF SMALL MOLECULE INHIBITORS AS MOLECULAR IMAGING AGENTS FOR UROKINASE PLASMINOGEN ACTIVATOR

Abstract

Urokinase-type plasminogen activator (uPA) protein is a serine protease of the trypsin family that is overexpressed by tumors cells seeking to metastasize. Molecular imaging methods using molecular imaging probe designed to target uPA could provide a method for the detection of aggressive cancers and monitoring response to treatment. Four classes of high affinity uPA inhibitors, three which were reversible and one irreversible, were used as platforms to develop radiolabeled probes for uPA. Based on structure-activity relationships, lead compounds were modified to allow for the introduction of a radiohalogen (radioiodine) at different sites in the corresponding molecules. Suitable synthetic strategies were developed to create libraries of iodinated phenyl guanidine, peptide, naphtamidine and phosphonate derivatives. For the phenylguanidines colorimetric assays showed the product had micromolar affinity while for the peptide derivatives low nanomolar affinity for the iodinated analogue was observed (1.4 nM to 2.53 nM). Unfortunately quantitative biodistribution studies showed low tumour uptake (<0.5% ID/g). More promising results were obtained for the irreversible iodinated phosphonated derivative which had an affinity of 2.1 nM. This reagent showed 1.95% ID/g tumour uptake and lower blood uptake in vivo which demonstrates advantageous properties over existing uPA probes in terms of tumour-to-blood ratios. A complementary development was also achieved in that the first example of a 125I-labelled tetrazine was prepared. This new reagent can be used in pre-targeted strategies that utilize bioorthogonal coupling between stained trans-cyclooctene (TCO) and tetrazines. The product was prepared using a concomitant oxidation iodo-destannylation reaction and the product isolated in 80% radiochemical yield. The reaction with transcycloctene proceeded rapidly to produce various isomers which were fully characterized through NMR analysis of the non-radioactive analogues.