Methyl ester of 1--(4-(2-[F]fluoroethyl-carbamoyloxymethyl)-2-nitrophenyl)--β-d-glucopyronuronate

The β-glucuronidase (β-GUS; EC 3.2.1.31) is a lysosomal enzyme that catalyzes the hydrolysis of β-glucuronic acid residues from the cell-surface glycosaminoglycans for normal reconstruction of the extracellular matrix (ECM) (1), and the enzyme is believed to participate in the processes of angiogenesis, cancer metastasis, and inflammation (2). The β-GUS is known to activate prodrugs (PDs) for the treatment of cancer. β-GUS also has been used to track the path of gene delivery vehicles, and there is evidence that it can be used as a biomarker to detect cancerous tumors (3). Normal tissues have low levels of β-GUS in the ECM, but tissues under pathological stress, such as bacterial infection, fibrosis, and malignancy, show elevated levels of the enzyme (4). As a result of cell lysis, intracellular β-GUS is released from the necrotic parts of neoplastic tumors, and its activity in these lesions has been utilized for the activation of anti-cancer PDs to treat cancers (1). Because chemotherapeutic anti-cancer drugs are non-selectively toxic to healthy cells, they are generally of limited efficacy to the patient due to their undesirable side effects on the normal biological systems. The conversion of a toxic drug into a non-toxic PD that can be activated only under specific conditions (e.g., by enzyme catalysis or chemical hydrolysis) would facilitate drug activation only in tissues that provide the specialized microenvironment and improve its concentration and efficacy at the desired location in the body (5, 6). For example, glucuronide PDs (drugs that are linked to a glucuronic acid moiety with or without a linker) have been shown to have superior anti-tumor activity compared with the parent drugs because the activated drugs are released from the PDs by the β-GUS activity in a site-specific manner (7, 8). β-GUS activity varies among individuals, and its expression or accumulation in tumor tissues may change depending on the type of neoplasm or the location in the body (1, 9). Fluorescent or bioluminescent substrates were developed to determine the expression of β-GUS with optical imaging in the various tissues of mice (3). However, this imaging modality is suitable for the detection of fluorescence or bioluminescence signals generated only in the superficial tissues of small animals such as rodents; the low depth of light penetration in tissues is a limitation for its application in large animals and humans (1, 4, 10). Imaging modalities such as positron emission tomography (PET) and single-photon emission computed tomography (SPECT), which use radionuclides to generate tracer signals, can be used to detect and determine the activity of enzymes such as the β-GUS because signals generated by radiolabeled probes can be detected even in deep locations in the body (11). In general, PET imaging has a higher sensitivity than SPECT and has been used to investigate drug kinetics in preclinical and clinical settings (11). Antunes et al. synthesized 1--(4-(2-[F]fluoroethyl-carbamoyloxymethyl)-2-nitrophenyl)--β-d-glucopyronuronate ([F]-FEAnGA) as a PD in an effort to develop a probe that could be used with PET to detect and visualize β-GUS activity in tumors (1, 4) and inflamed tissues (1, 10) in rodents. The mechanism of / activation of [F]-FEAnGA is described elsewhere (4). Although [F]-FEAnGA could be used to detect β-GUS activity and distinguish cancerous tumors or inflamed tissues from the surrounding normal tissues, only low levels of radioactivity were detected in the tumorous or inflamed areas compared with the healthy tissues. The investigators concluded that the low accumulation of label in the lesions was due to the high hydrophilicity of the PD and its rapid clearance from the body through the kidneys (1). It has been shown that conversion of anti-cancer PDs to their methyl esters renders them considerably less hydrophilic and increases the circulation half-life of the PD because the methylated-PD first has to be demethylated by an esterase (to produce the PD) while in circulation before the active drug could be released from the PD by another enzyme(s) such as the β-GUS at the desired site(s) in the body (12). On the basis of these observations, a [F]-fluoride-labeled methyl ester of FEAnGA was synthesized ([F]-FEAnGA-Me) and evaluated for the imaging of β-GUS activity in a rodent tumor/inflammation model (12). Briefly, a carboxylesterase in the plasma hydrolyzes [F]-FEAnGA-Me to [F]-FEAnGA, which is a substrate for the β-GUS, and its subsequent hydrolysis by the enzyme results in the production of glucuronic acid, 4-hydroxy-3-nitrobenzyl alcohol (HNBA; acts as a spacer moiety in the intact [F]-FEAnGA, and the concentration of this molecule in the reaction mixture can be measured with ultraviolet (UV) spectroscopy at 402 nm after the hydrolysis of FEAnGA), and 2-[F]fluoroethylamine ([F]-FEA). Subsequently, [F]-FEA can be detected with PET imaging because it accumulates in the cells by passive diffusion (10).