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Methyl ester of 1-O-(4-(2-[18F]fluoroethyl-carbamoyloxymethyl)-2-nitrophenyl)-O-β-d-glucopyronuronate

, PhD
National Center for Biotechnology Information, NLM, Bethesda, MD 20894

Created: ; Last Update: April 27, 2012.

Chemical name:Methyl ester of 1-O-(4-(2-[18F]fluoroethyl-carbamoyloxymethyl)-2-nitrophenyl)-O-β-d-glucopyronuronate
image 135631863 in the ncbi pubchem database
Abbreviated name:[18F]FEAnGA-Me
Agent Category:Compound
Target:β-Glucuronidase (β-GUS; βG)
Target Category:Enzyme
Method of detection:Positron emission tomography (PET)
Source of signal / contrast:18F
  • Checkbox In vitro
  • Checkbox Rodents
Click on above structure for more information in PubChem.



The β-glucuronidase (β-GUS; EC 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 in situ 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-O-(4-(2-[18F]fluoroethyl-carbamoyloxymethyl)-2-nitrophenyl)-O-β-d-glucopyronuronate ([18F]-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 in vitro/in vivo activation of [18F]-FEAnGA is described elsewhere (4). Although [18F]-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 [18F]-fluoride-labeled methyl ester of FEAnGA was synthesized ([18F]-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 [18F]-FEAnGA-Me to [18F]-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 [18F]-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-[18F]fluoroethylamine ([18F]-FEA). Subsequently, [18F]-FEA can be detected with PET imaging because it accumulates in the cells by passive diffusion (10).



The synthesis of FEAnGA-Me and its labeling with [18F]-fluoride have been described by Antunes et al. (4, 12). The radiochemical yield (RCY) and radiochemical purity (RCP) of the labeled compound were reported to be 10 ± 5% (based on [18F]-fluoride) and 95%, respectively. The total time required for the synthesis and purification of the final labeled product was 120 min. [18F]-FEAnGA-Me was reported to have a specific activity of 185 ± 71 GBq/μmol (4.9 ± 1.9 Ci/μmol).

[18F]-FEAnGA was synthesized for use in studies, and the RCY and the RCP of this tracer were reported to be 10%–15% and >95%, respectively. The specific activity of [18F]-FEAnGA was 149 ± 33 GBq/μmol (4.0 ± 0.89 Ci/μmol) (12).

In Vitro Studies: Testing in Cells and Tissues


At least >95% of [18F]-FEAnGA-Me was intact in phosphate-buffered saline (pH not reported) after incubation for 1 h at 37°C as determined with radio-thin-layer chromatography (radio-TLC; the Rf values for FEA, FEAnGA, and FEAnGA-Me were 0.1, 0.4, and 1.0, respectively) (4, 12).

The lipophilicity (Log D) of [18F]-FEAnGA-Me in an n-octanol/water mixture was determined to be −0.58 ± 0.0002 at pH 7.4 (12) compared with −1.61 ± 0.01 for [18F]-FEAnGA at the same pH (4).

To study the in vitro hydrolysis of non-radioactive FEAnGA-Me in the presence of commercially available Escherichia coli β-GUS (74 U) and porcine liver carboxylesterase (15 U), the compound (200 μM) was incubated with the enzymes for 1 h at 37°C (12). Measurement of the amount of HNBA generated from the reaction showed that 80% of the compound was completely hydrolyzed at the end of the incubation. When [18F]-FEAnGA-Me was incubated with rat plasma, 99% of the radiolabeled compound was converted to [18F]-FEAnGA within 30 min (12). In another study, [18F]-FEAnGA-Me was incubated with rat plasma in the presence of E. coli β-GUS, and 50% of the tracer was reported to be hydrolyzed to [18F]-FEA within 30 min (12).

When C6 rat glioma cells were incubated with [18F]-FEAnGA-Me in the presence of E. coli β-GUS and porcine liver carboxylesterase for 1 h, 60% of the radioactivity was determined to be associated with the cells at the end of the incubation (12). Radio-TLC analysis of the C6 cell growth medium revealed that 99% of [18F]-FEAnGA-Me was converted to [18F]-FEA in the presence of both the enzymes; however, in the presence of the β-GUS alone, only 7% of the radiochemical substrate was recovered as [18F]-FEA (12).

Animal Studies



The use of [18F]-FEAnGA-Me and [18F]-FEAnGA was compared for the PET imaging of C6 cell glioma tumors (right shoulder) and inflammation (induced with turpentine oil in the thigh of left hind leg) in the same rats (12). Anesthetized animals were intravenously injected with either [18F]-FEAnGA-Me (n = 6 rats) or [18F]-FEAnGA (n = 8 rats) through the penile vein, and 60-min PET scans of the tumors were initiated. PET scans of inflammation in the legs could not be acquired because of equipment limitations, and the presence of high levels of radioactivity in the bladder interfered with acquiring precise images of the inflamed tissues. In the PET images, [18F]-FEAnGA-Me was observed to generate a high background compared to [18F]-FEAnGA. Both tracers had similar time-activity curves and showed peak accumulation of label in the viable parts of the tumors at 1.5 min postinjection (p.i.), followed by an exponential decrease over time. The half-lives of [18F]-FEAnGA-Me and [18F]-FEAnGA in the viable parts of the tumor were 16 ± 2 min and 24 ± 9 min, respectively. In addition, the area under the curve values for the tumors were 23 ± 6 and 20 ± 4 (P = 0.65) with [18F]-FEAnGA-Me and [18F]-FEAnGA, respectively.

After completion of the PET scans, the ex vivo biodistribution of [18F]-FEAnGA-Me and [18F]-FEAnGA was compared at 60 min p.i. by calculating the standardized uptake values (SUV) for radioactivity accumulated in the various tissues of the rodents as described by Antunes et al. (12). Both tracers were reported to have similar SUVs for the peripheral organs of the animals. With [18F]-FEAnGA-Me, the SUVs for the liver and kidneys were significantly (P < 0.05) higher (0.97 ± 0.29 and 3.13 ± 0.67, respectively) than with [18F]-FEAnGA (0.34 ± 0.09 and 1.03 ± 0.48, respectively). The SUVs obtained with [18F]-FEAnGA-Me and [18F]-FEAnGA for the tumor, inflamed muscle, and normal muscle in the rats are presented in Table 1.

Table 1: Uptake of radioactivity from [18F]-FEAnGA-Me and [18F]-FEAnGA in tumor, inflamed muscle, and normal muscle (12).

TracerTissue SUVT/N ratioI/N ratio
[18F]-FEAnGA-Me0.13 ± 0.020.13 ± 0.050.08 ± 0.021.85 ± 0.231.48 ± 0.22
[18F]-FEAnGA0.13 ± 0.030.07 ± 0.020.04 ± 0.013.53 ± 0.52*1.59 ± 0.19

SUV, standardized uptake value; T/N, tumor/normal tissue; I/N, inflamed tissue/normal tissue.

*P = 0.02.

Although the tumor SUVs with [18F]-FEAnGA-Me and [18F]-FEAnGA were similar (Table 1), the tumor/normal muscle ratio (T/N ratio) was significantly lower (P = 0.02) with [18F]-FEAnGA-Me (1.85 ± 0.23) than with [18F]-FEAnGA (3.53 ± 0.52). The lower T/N ratio observed with [18F]-FEAnGA-Me was due to the higher uptake of radioactivity in the normal muscle with this tracer compared with [18F]-FEAnGA (Table 1), and this was attributed to the nonspecific uptake of the tracer in the muscle tissue.

From these studies, the investigators concluded that the enzyme-based, two-step PD activation strategy they used with [18F]-FEAnGA-Me was not suitable for the visualization of tumors or inflammation with PET in rodents (12).

Other Non-Primate Mammals


No publication is currently available.

Non-Human Primates


No publication is currently available.

Human Studies


No publication is currently available.

Supplemental Information


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