Synthesis of a 2-nitroimidazole derivative N-(4-[18F]fluorobenzyl)-2-(2-nitro-1H-imidazol-1-yl)-acetamide ([18 F]FBNA) as PET radiotracer for imaging tumor hypoxia

Abstract

Background: Tissue hypoxia is a pathological condition characterized by reducing oxygen supply. Hypoxia is a hallmark of tumor environment and is commonly observed in many solid tumors. Non-invasive imaging techniques like positron emission tomography (PET) are at the forefront of detecting and monitoring tissue hypoxia changes in vivo.

Results: We have developed a novel ¹⁸F-labeled radiotracer for hypoxia PET imaging based on cytotoxic agent benznidazole. Radiotracer N-(4-[¹⁸F]fluorobenzyl)-2-(2-nitro-1H-imidazol-1-yl)acetamide ([¹⁸F]FBNA) was synthesized through acylation chemistry with readily available 4-[¹⁸F]fluorobenzyl amine. Radiotracer [¹⁸F]FBNA was obtained in good radiochemical yields (47.4 ± 5.3%) and high radiochemical purity (> 95%). The total synthesis time was 100 min, including HPLC purification and the molar activity was greater than 40 GBq/µmol. Radiotracer [¹⁸F]FBNA was stable in saline and mouse serum for 6 h. [¹⁸F]FBNA partition coefficient (logP = 1.05) was found to be more lipophilic than [¹⁸F]EF-5 (logP = 0.75), [¹⁸F]FMISO (logP = 0.4) and [¹⁸F]FAZA (logP = - 0.4). In vitro studies showed that [¹⁸F]FBNA accumulates in gastric cancer cell lines AGS and MKN45 under hypoxic conditions.

Conclusions: Hence, [¹⁸F]FBNA represents a novel and easy-to-prepare PET radioligand for imaging hypoxia.

Keywords: Hypoxia; N-(4-[18F]fluorobenzyl)-2-(2-nitro-1H-imidazol-1-yl)acetamide ([18F]FBNA); Positron emission tomography (PET).

Fig. 1

Chemical structures of selected ¹⁸F-labeled 2-nitroimidazoles used for PET imaging of tissue hypoxia

Fig. 2

Chemical structures of benznidazole and [¹⁸F]FBNA

Scheme 1

Synthesis of the labeling precursor 3 and reference compound 4. Reagents and conditions: (a) CH₃CO₂CH₂CH₂Br, K₂CO₃, CH₃CN, rt, 21 h; (b) 4.0 N NaOH, H₂O/MeOH, rt, 3 h; (c) 4-Fluorobenzylamine, trifluoroacetic acid anhydride, THF, 0 °C, 1 h

Scheme 2

Radiosynthesis of [¹⁸F]FBNA from its precursor, 2-nitroimidazole acetic acid (3)

Fig. 3

Radio-HPLC traces of the reaction ([¹⁸F]FBNA: t_R. = 15.1 ± 0.3 min) performed at room temperature (A), 65 °C (B), 85 °C (C) and 100 °C (D)

Fig. 4

HPLC traces of [¹⁸F]FBNA (left) ([¹⁸F]FBNA: t_R. = 15.1 ± 0.3 min) and reference compound FBNA (right) (FBNA: t_R. = 15.0 ± 0.3 min). UV detector was installed ahead of radioactivity detector

Fig. 5

Radio-HPLC traces obtained after the incubation of [¹⁸F]FBNA (t_R: 15.2 min) at (A) the initial time (t = 0 min), (B) physiological saline solution (NaCl 0.9%) after 6 h or (C) mouse serum after 6 h

Fig. 6

In vitro uptake assay. AGS and MKN45 cells were seeded in a 6-well plate and cultured for 48 h under normoxic (21% O₂) or hypoxic (1% of O₂) for 48 h. After this period, 0.37 MBq of (A) [¹⁸F]FBNA or (B) [¹⁸F]FAZA was incubated for 1 h. Bars show the mean ± standard deviation (SD) of the uptake of the radiotracer (cpm)/2 × 10⁵ cells of three independent experiments (n = 3). Data were analyzed by one unpaired t-test (multiple t tests); *p < 0.5, ****p < 0.0001 (Holm-Sidak method)

Fig. 7

Characteristic of hypoxic PET tracers under development and number of clinical trials