Technical note
A new 18F-labelled derivative of the MMP inhibitor CGS 27023A for PET: Radiosynthesis and initial small-animal PET studies

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Abstract

The CGS 27023A derivative (R)-2-(N-((6-fluoropyridin-3-yl)methyl)-4-methoxyphenyl-sulphonamido)-N-hydroxy-3-methylbutanamide 1a was identified as a very potent matrix metalloproteinase inhibitor. Here, we describe a one-step radiosynthesis of the target compound [18F]1a. The syntheses of [18F]1a resulted in a radiochemical yield of 12.1±5.9% (decay-corrected), a radiochemical purity of 98.8±0.6%, and a specific activity of 39±27 GBq/μmol at the end of synthesis within 160±18 min from the end of radionuclide production (n=5). Initial small-animal PET studies in wild-type mice (C57/BL6) showed no unfavourable tissue accumulation of [18F]1a.

Introduction

Matrix metalloproteinases (MMPs) are a family of zinc- and calcium-dependent endopeptidases that are able to degrade all protein components of the extracellular matrix (ECM) with overlapping substrate specificities (Skiles et al., 2004; Tayebjee et al., 2005; Beaudeux et al., 2004; Whittaker and Ayscough, 2001). To date at least 26 human MMPs are known (Verma and Hansch, 2007). Based on their specificity the MMPs are classified into collagenases, gelatinases, stromelysins, and matrilysins. Another subclass of MMPs is represented by the membrane-type MMPs (MT-MMPs) that additionally contain a transmembrane and intracellular domain, a membrane linker domain or are membrane associated (Overall and López-Otín, 2002). Several endogenous inhibitors, e.g. the four tissue inhibitors of metalloproteinases (TIMPs), regulate MMP activity (Stamenkovic, 2003; Rhee and Coussens, 2002).

Extracellular proteolysis, in which the MMPs and TIMPs are involved, plays a key role in many biological processes, e.g. in physiological connective tissue development, morphogenesis and wound healing. On the other hand the important role of upregulated MMP expression and activity has been demonstrated in numerous diseases including inflammation, tumour cell metastasis and atherosclerosis (Galis and Khatri, 2002). Consequently, there has been a great deal of effort to design and prepare different synthetic matrix metalloproteinase inhibitors (MMPIs) for therapeutic applications that inhibit activated enzymes by chelating the active-site zinc ion of the MMP with a metal complexing moiety (e.g. carboxylic or hydroxamic acids) (Brinckerhoff and Matrisian, 2002).

A potential diagnostic application of MMPIs is offered by modern scintigraphic techniques (e.g. single photon emission computed tomography, SPECT and positron emission tomography, PET). Here, radiolabelled analogues of MMPIs might be useful as radiotracers for the detection of MMP activity. The most common approach for imaging of activated MMPs is based on the application of radiolabelled small molecule non-peptide MMPIs (Wagner et al., 2006).

The N-sulphonyl amino acid hydroxamates CGS 25966 and CGS 27023A represent prominent lead structures for potential radiolabelled tracers (Fig. 1). Both are non-selective inhibitors that inhibit MMP-1 (interstitial collagenase, Ki=43 and 33 nM, respectively), MMP-2 (gelatinase A, Ki=11 and 20 nM, respectively), MMP-3 (stromelysin-1, Ki=34 and 43 nM, respectively) and MMP-9 (gelatinase B, Ki=27 and 8 nM, respectively) by chelating the zinc ion of the enzyme active-site with the hydroxamic acid moiety (MacPherson et al., 1997; Scozzafava and Supuran, 2000).

Our group developed a SPECT-compatible 123I-labelled derivate of CGS 27023A (HO-[123I]I-CGS 27023A) for the assessment of MMPs in vitro and in vivo (Fig. 1). This compound was successfully used to specifically image activated MMPs in vivo in vascular lesions developing after carotid artery ligation in apolipoprotein E-deficient (ApoE−/−) mice (Kopka et al., 2004; Schäfers et al., 2004).

Recently, our group and others focused on the development and radiosynthesis of complementary PET-compatible 11C- and 18F-labelled CGS 27023A derivatives as well as derivatives of CGS 25966 where the picolyl substituent is exchanged for a benzyl group. Among these compounds, only [11C]CGS 25966 (Fig. 1) has been studied in two animal models of breast cancer, but due to unpromising results arising from these studies [11C]CGS 25966 was excluded from further in vivo evaluations (Zheng et al., 2004). Our group realised the synthesis of two 18F-labelled CGS 27023A/25966 analogues with a 2-fluoroethoxy-subunit ([18F]1c and [18F]1d, Fig. 1) (Breyholz et al., 2007; Wagner et al., 2007), which are currently evaluated in vivo.

The present work focuses on the development of a radiosynthesis for (R)-2-(N-((6-[18F]fluoropyridin-3-yl)methyl)-4-methoxyphenyl-sulphonamido)-N-hydroxy-3-methylbutanamide [18F]1a, whose non-radioactive counterpart 1a was found to be a very potent inhibitor of the prominent MMPs gelatinases A and B (MMP-2 and -9) and collagenases 2 and 3 (MMP-8 and -13) in a series of compounds introduced by Wagner et al. (2007). Furthermore, small-animal PET studies were performed to initially evaluate the potency of [18F]1a for PET.

Section snippets

General methods

All chemicals, reagents and solvents for the syntheses were analytical grade and purchased from commercial sources (Acros Organics, Geel, Belgium; B. Braun Melsungen AG, Melsungen, Germany; Honeywell Seelze GmbH, Seelze, Germany; Merck KGaA, Darmstadt, Germany; Rotem GmbH Leipzig, Leipzig, Germany). Radiosyntheses were partly carried out using an automated PET tracer synthesiser (TRACERLab FxFDG Synthesiser; GE Functional Imaging GmbH). The recorded data were processed by the TRACERLab Fx

Results and discussion

A reliable radiosynthesis for the potential MMP tracer [18F]1a, that showed no unfavourable behaviour in initial small-animal PET studies with wild-type mice (C57/BL6), was developed.

In detail, in previous work of our group the CGS 27023A derivative 1a was identified as a potent MMPI. In fluorogenic in vitro inhibition assays 1a showed IC50-values in the nanomolar to subnanomolar range for MMP-2, MMP-8, MMP-9 and MMP-13. Compound 1a possesses similar IC50-values for MMP-2, MMP-8 and MMP-9 and a

Acknowledgements

This work was supported by grants from the Deutsche Forschungsgemeinschaft (DFG), Sonderforschungsbereich 656, Münster, Germany (projects A1, A2, A3, B1 and Z2) and the Interdisciplinary Center of Clinical Research (IZKF) Münster (project ZPG 4b). We gratefully acknowledge Dr. Sven Hermann for coordination of the small-animal PET scans and Christine Bätza, Anne Kanzog, Sandra Schröer, Daniel Burkert and Sven Fatum for technical assistance.

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    These authors contributed equally to this work.

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