Radiolabelled cathepsin inhibitors as potential PET tracers for tumour imaging

Objectives:

Thiol-dependent cathepsins are a class of cysteine proteases that have been shown to be linked to the progression of cancer in multiple ways. Particularly crucial is their involvement in proteolytic pathways that are related to tumour invasion and metastasis [1]. The aim of this study was the design of cathepsin inhibitors based on the structure of the azadipeptide nitrile chemotype [2] that permit the labelling with the positron emitters fluorine-18 and carbon-11 and to evaluate the
potential of this inhibitor class for functional tumour imaging by PET. Additionally, a first insight into the pharmacokinetic behaviour of these inhibitors should be gained.
Methods:
The fluorine atom was linked by an ethylene bridge to the inhibitor core structure. Labelling with fluorine-18 was achieved by fluoroethylation of 1 with different substituted [¹⁸F]2-fluoroethyl benzenesulfonates [3]. In addition, the introduction of radiofluorine into 2 was attempted by direct fluorination of the corresponding tosyl and nosyl precursors. The stability of the tracer against chemical and enzymatic degradation as well as its metabolic fate in rat blood was investigated and its biodistribution was studied in vivo by small animal PET. To obtain an ¹¹C-based tracer, the fluoroethyl group of 2 was replaced by methyl. Introduction of carbon-11 was done by conversion of the phenolic precursor 1 with [¹¹C]methyl iodide generated by the gas-phase method. The affinities of the compounds 1-3 to their targets were determined in kinetic enzyme assays.
Results:
The azadipeptide nitriles 1-3 exhibited inhibition constants in the single-digit to subnanomolar range against the oncologically relevant cathepsins L, S, and B. Among the various ¹⁸F-fluoroethylating agents tested, [¹⁸F]2-fluoroethyl nosylate revealed as the most efficient one. This enabled the two-step radiosynthesis of [¹⁸F]2 in an average RCY (d.c.) of 27±5% (n = 13). The direct radiofluorination of the corresponding sulfonate precursors could provide [¹⁸F]2 in only low labelling yields. PET studies in rats together with in vitro investigations indicated the trapping of the tracer in erythrocytes, which could be attributed to its inherent thiol reactivity. The tracer [¹¹C]3 could be obtained in labelling yields of 35-42% depending on the employed base. Its radiopharmacological behaviour is under current investigation.
Conclusions:
With the azadipeptide nitriles 2 and 3 highly potent cathepsin inhibitors were found and their labelling with fluorine-18 and carbon-11 could be successfully established. The compounds’ suitability as PET tracers for functional tumour imaging seems to be limited due to their thiol reactivity. The radiolabelling of further cathepsin inhibitors is underway.
References:
[1] Mohamed & Sloane (2006) Nat. Rev. Cancer 6, 764-775, [2] Löser et al. (2008) Angew. Chem.
Int. Ed. 47, 4331-4334, [3] Musachio et al. (2005) J. Label. Compd. Radiopharm. 48, 735-747