Synthesis and radiolabeling of a nitrofuran derivate with 18F for identification of areas of hypoxia in the tumor microenvironment

doi:10.21203/rs.3.rs-3918714/v1

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Synthesis and radiolabeling of a nitrofuran derivate with 18F for identification of areas of hypoxia in the tumor microenvironment

https://doi.org/10.21203/rs.3.rs-3918714/v1

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Background

Positron Emission Tomography (PET) is a non-invasive molecular imaging technique widely known for studying hypoxia mostly employing 2-nitroimidazole-based radiotracers. These probes are based on the oxygen-mimetic chemical sensitizers of hypoxic cells developed for cancer therapy during the 1970s. 5-nitrofuran derivates are more electron affinic than nitroimidazoles, therefore, higher specificity for hypoxic regions is expected for the formers, and new radiotracer probes bearing a 5-nitrofuran ring could be used for imaging hypoxia.

Results

A nitrofuran-based radiotracer for detection of hypoxic areas in the tumor microenvironment, (E)-1-(4-[¹⁸F]-fluorophenyl)-3-(5-nitrofuran-2-yl)prop-2-en-1-one, baptized as [¹⁸F]FNFP, was obtained. Two copper-mediated nucleophilic radiofluorination procedures were tested and compared using the same pinacol-derived aryl boronic ester precursor: method 1, using K₂₂₂/K₂CO₃ and [Cu(OTf)₂(py)₄] afforded the product in 56 ± 8% (n = 5) RCY after HPLC analysis of the crude reaction mixture; method 2: an azeotropic drying-free [¹⁸F]-labelling procedure, using Cu(OTf)₂ as [¹⁸F]-elution agent and copper source, yielded [¹⁸F]FNFP in 88 ± 4% (n = 5) RCY. Method 2 was chosen as the standard for the synthesis of the radiotracer, obtaining the product with an overall radiochemical yield of 38,4 ± 3% (n = 5), high radiochemical purity (> 99%), total synthesis time of 85 minutes and a molar activity of 41.56 GBq/µmol. [¹⁸F]FNFP was found to be stable in serum and Phosphate-buffered saline for up to 6h, and lipophilicity measurements concluded that it is more hydrophilic than [¹⁸F]FMISO (log10𝑃=2.6), with log10𝑃=1.05.

Conclusion:

The first nitrofuran-based radiotracer to be used as a PET hypoxia imaging agent was efficiently radiolabeled with 18F. In vitro and in vivo studies are being lined up to compare [18F]FNFP with [18F]FMISO and [18F]FAZA.

(E)-1-(4-[18F]-fluorophenyl)-3-(5-nitrofuran-2-yl)prop-2-en-1-one

[18F]FNFP

hypoxia

Positron Emission Tomography

5-nitrofuran

oxygen-mimetic chemical sensitizers

Hypoxia, one of the hallmarks of cancer (1, 2), is a condition where oxygen levels are below the normal physiological concentration in tissues that results from an imbalance between oxygen supply and oxygen consumption. Solid tumors are likely to develop hypoxic regions and are associated with poor prognosis, an increased risk of invasion and metastasis, and resistance to chemo and radiation therapies (1–4).

Tumor hypoxia has been studied using several different techniques such as extrinsic (e.g., pimonidazole) and intrinsic (e.g., carbonic anhydrase IX, CAIX) biomarkers; blood oxygen level-dependent (BOLD) and tissue oxygen level-dependent (TOLD); molecular imaging through Single Photon Emission Computed Tomography (SPECT) and Positron Emission Tomography (PET), magnetic resonance imaging (MRI) and oxygen electrodes (5), often referred as the “gold standard” (6). Among them, SPECT and PET as non-invasive modalities rely on radiolabeled probes that are selectively accumulated within cells under hypoxic conditions after undergoing enzymatic-mediated reductions. Many of these SPECT/PET-used radiolabeled probes are based on nitroaromatic hypoxic-cell sensitizers, oxygen-mimetic compounds developed to improve the radiosensitivity of hypoxic cells within solid tumors in radiotherapy (7). In the 1970s nitroaromatic compounds such as the antibiotic and antibacterial agents nitrofurans (e.g. nitrofurazone, nifurtimox, nitrofurantoin nifuroxime) and specially nitroimidazoles were identified as sensitizers; Misonidazole and Metronidazole (Fig. 1) were two of the earliest developed ones, with the former been more effective due to a higher electron affinity as measured by one-electron reduction potential (\({E}_{7}^{1}\), first electron reduction potential at neutral pH), which is a key factor in their efficacy (8). Subsequent attempts to demonstrate hypoxic cell sensitization by these compounds in vivo unfortunately failed in most cases due to neurotoxicity, compromised stability and water low water solubility in case of nitrofurans, and in case of misonidazole the maximum clinical doses that could be achieved fell well short of those required for maximum sensitizing effectiveness (9, 10).

Those same properties that allow radiosensitizers to accumulate under hypoxic conditions are used to determine the location of hypoxic cells and measure tumor hypoxia, approach that was first carried out with ¹⁴C-radiolabelled misonidazole (11) and was the breakthrough that led to PET and SPECT imaging probes for detection of hypoxia. In this regard, [¹⁸F]fluoromisonidazole ([¹⁸F]FMISO, 1H-1-(3-[¹⁸F]fluoro-2-hydroxypropyl)-2-nitroimidazole) first synthetized in 1986 (12) is the most widely used hypoxia imaging radiopharmaceutical, but its high lipophilicity causes slow tracer accumulation, slow plasma clearance, and low tumor-to-background contrast. Most of the other radiotracers developed to solve [¹⁸F]FMISO pharmacokinetic issues, such as [¹⁸F]Fluoroazomycin Arabinoside ([¹⁸F]FAZA), [18F]Fluoroerythronitroimidazole ([¹⁸F]FETNIM), [¹⁸F]fluoroetanidazole ([¹⁸F]FETA) and [¹⁸F]-Flortanidazole ([¹⁸F]FHX4), rely on the 2-nitroimidazole moiety (Fig. 1), or even 4- or 5-nitroimidazole derivates have been tested (13, 14). The literature shows that each radiotracer for detection of hypoxic regions in tumors has its strengths and weaknesses depending on the study and the information required a different one can be put forward in every situation.

The nitroimidazole moiety is not considered to be essential for an “ideal” nitroaromatic radiosensitizer and therefore neither for a hypoxia imaging agent. This compound should have high membrane permeability (measured by partition coefficient) to ensure a feasible cell-membrane penetration and accumulation in the target tissue, but also has to be sufficiently soluble in water to deliver appreciable amounts of the drug (15). Additionally, the \({E}_{7}^{1}\) must be between that of O₂ (-155 mV) and the cytochromes, which transport these reducing equivalents (approximately − 450 mV) (15): a compound with a \({E}_{7}^{1}\) close to oxygen would be too electron affinic therefore toxic, and a weakly-affinic one would lose its sensitivity and selectivity for hypoxia. According to Martin Brown, Paul Workman (16) the best balance between toxicity and sensitivity occurs for radiosensitizers approximately at -390 mV, which is where the \({E}_{7}^{1}\) of most 2-nitroimidazoles is (-380 to -400 mV range). However, the actual \({E}_{7}^{1}\) values depend on the side chain and the inductive effects of the substituents present on it.

Based on the idea that, in general, the therapeutic benefit or efficiency of hypoxic cell sensitizers could be improved by increasing radiosensitizing potency, fact that could be achieved by raising the redox potential of the nitroheterocycle, Naylor, M A et al. (17) synthetized a group of 5-nitrofuran derivates in order to test their efficiency as sensitizers based on the premise that these nitroaromatic compounds are slightly more electron affinic than 2-nitroimidazoles. The 5-nitrofurans investigated showed high radiosensitizing activity in vitro and all of them were more efficient sensitizers than misonidazole, with \({E}_{7}^{1}\) values ranging from − 211 to approximately − 327 mV. The major issue was their toxicity, at least 1 order of magnitude greater than are the toxicities of similar nitroimidazoles (17).

A radiotracer bearing a 5-nitrofuran moiety instead of a nitroimidazole one for imaging hypoxia could be an interesting solution to finding the “ideal” imaging agent. Theoretically, the higher reduction potential of the nitrofuran ring would not only allow this probe to maintain its selectivity for hypoxia, but this selectivity would be, at least, equal to that of 2-nitroimidazole derivatives. Then its side chain will domain its lipophilicity and pharmacokinetic properties.

In this work, and for the first time, a nitrofuran-based radiotracer (E)-1-(4-[¹⁸F]fluorophenyl)-3-(5-nitrofuran-2-yl)prop-2-en-1-one, baptized as [¹⁸F]FNFP was radiolabeled with fluorine-18 in order to be tested as a hypoxia imaging agent. The chalcone-based structure used would increase even more its electron affinity as result of electron delocalization throughout the whole structure, idea supported by several authors (18, 19).

Chemical synthesis

The synthesis path to obtain (E)-1-(4-fluorophenyl)-3-(5-nitrofuran-2-yl)prop-2-en-1-one, [¹⁹F]FNFP (compound 4), and (E)-(4-(3-(5-nitrofuran-2-yl)acryloyl)phenyl)boronic acid pinacol ester, [¹⁸F]FNFP precursor (compound 6) is shown in scheme 1.

First, nitration of furfural in acetic anhydride in the presence of HNO₃ and catalytic H₂SO₄ yielded 5-nitro-2-furaldehyde diacetate (compound 2) as a white crystalline solid in 60% yield after recrystallization from ethanol. The 1,1-diacetate (acylal) protecting group was removed in refluxing acetonitrile in the presence of catalytic SnCl₂xH₂O to yield 5-nitro-2-furaldehyde (compound 3) in 90% yield as a yellow solid after column chromatography in silica gel. Claisen-Schmidt condensation of 5-nitrofuraldehyde and 4-fluoroacetophenone in acidic media yielded cold standard (E)-1-(4-fluorophenyl)-3-(5-nitrofuran-2-yl)prop-2-en-1-one (compound 4) as yellow needles in 75% yield after recrystallization from ethanol.

The synthesis of (E)-(4-(3-(5-nitrofuran-2-yl)acryloyl)phenyl)boronic acid pinacol ester, compound 6, started with a metal-free borylation reaction using 4-aminoacetophenone as starting material to obtain 4-acetyl phenylboronic acid pinacol ester (compound 5) as a white-off crystalline solid in 65% yield after flash chromatography in silica gel. Finally, another acidic Claisen-Schmidt condensation now between 5-nitro-2-furaldehyde and 4-acetyl phenylboronic acid pinacol ester yielded (E)-(4-(3-(5-nitrofuran-2-yl)acryloyl)phenyl)boronic acid pinacol ester as yellow needles in 72% yield after recrystallization from ethanol.

Radiochemistry

Two different procedures were studied for the synthesis of [¹⁸F]FNFP. Scheme 2 shows the reaction details:

Following the radiosynthesis procedure described in method 1 in the experimental section, [¹⁸F]FNFP was obtained with a radiochemical yield (RCY) of 56 ± 8% (n = 5) after HPLC analysis of the crude reaction mixture using [Cu(OTf)₂(py)₄] as copper source. “Cold” standard [¹⁹F]FNFP was used as a reference for the comparison during quality control of the radioactive peak during the radio-HPLC run (Fig. 2) shows a representative chromatogram of a crude reaction mixture sample.

Now following the radiosynthesis procedure described in method 2 (scheme 2), [¹⁸F]FNFP was obtained with a radiochemical yield of 88 ± 4 (n = 5) after radio-HPLC analysis of the crude reaction mixture, also using [¹⁹F]FNFP “cold” standard as reference (Fig. 3).

Comparing both methods 1 and 2 for the synthesis of [¹⁸F]FNFP, the results showed that radiochemical yields are higher for method 2. Then, this protocol was chosen as the standard for the synthesis of our radiotracer.

After the synthesis following method 2, semipreparative HPLC purification afforded > 99% radiochemically pure [¹⁸F]FNFP (Fig. 4), with an overall radiochemical yield of 38,4 ± 3% (n = 5), total synthesis time of 85 minutes and a molar activity of 41.56 GBq/µmol.

In vitro stability

The stability of the new radiopharmaceutical [¹⁸F]FNFP in 0.1 mol/L PBS at pH = 7.2 and in serum was evaluated for a period of up to 6 hours. Aliquots were taken immediately after the synthesis, 1, 3 and 6 hours after it, and the radiochemical purity of the compound was assessed by HPLC following the same conditions as described earlier for its quality control.

As demonstrated in Fig. 5, no significant metabolites derived from [¹⁸F]FNFP were observed after 6h of incubation either in PBS or serum.

Determination of partition coefficient

The logarithm of [¹⁸F]FNFP partition coefficient calculated according to the methodology described was \({\text{log}}_{10}P\)=1.05 ± 0.06 (n = 7).

Historically, nitroimidazole-based compounds have been used as radiotracers for detection of hypoxia based on their previous introduction as radiosensitizers of hypoxic cells and their good pharmacokinetic properties, specially 2-nitroimidazole ones, due to their higher electron affinity compared to 4- and 5-nitroimidazoles derivates. In this regard, [¹⁸F]FMISO is the most widely used radiopharmaceutical for imaging tumor hypoxia using PET (20), but because of its high lipophilicity pharmacokinetic issues are associated to it. Second and third-generation radiotracers have been developed (20), and even some of them have undergone preclinical trials in the past recent years (21). Nevertheless, none of these radiotracers have shown to be vastly superior to [¹⁸F]FMISO so the latter remains as the radiolabeled imaging agent that has been more used for investigating tumor hypoxia.

Nitrofurans, as one of the most important and studied nitroaromatic compounds, were also tested as sensitizers with promising results related to their higher sensitizing potency (attributable to their higher reduction potential) compared to 2-nitroimidazole derivates. The issue associated with them was their toxicity, so more attention was paid to nitroimidazoles. The mechanism of action of these nitroaromatic compounds involves metabolic changes related to the nitro group accepting electrons from reducing enzymes (nitroreductases) hence generating intermediates that react with DNA to exert toxic and mutagenic effects (22, 23). These sensitizers get into intracellular space by diffusion and under hypoxic condition the process described above takes place, but under normal oxygen concentrations the compound is re-oxidized back to the original one and diffuses out of the cell (20).

In this work, the first use of a lipophilic nitrofuran-based radiotracer ([¹⁸F]-FNFP) labeled with ¹⁸F intended to become a hypoxia imaging agent is described. In order to improve its electron affinity, a side chain based on a chalcone structure was built according to the following ideas: A) in a study reported by Valdez et al. (2009) (18), where the authors synthetized several 5-nitroimidazole derivates and tested their antimicrobial activity against Giardia lamblia, compounds having olefins with a conjugated bridge connecting the nitroheterocycle and a substituted phenyl or heterocyclic ring showed greatly increased antigiardial activity without toxicity (Fig. 6, A). Electrochemical studies carried out in that work (determination of the half-wave potential of the initial one-electron transfer by cyclic voltammetry) showed that the electron delocalization in these compounds was associated with an easier redox activation and reduction, therefore correlated with greater antigiardial activity. B) In another work published by Tawari, Nilesh R. et al. (2010) (24) the authors synthetized a series of potent nitrofuran-based antitubercular agents coupling electronegative α,β-unsaturated carbonyl bridge to the nitrofuran ring and substituted phenyl or heterocyclic rings (Fig. 6, B). Based on density functional theory (DFT) and computational studies, these compounds were selected as strong candidates to become antitubercular agents because all of them possessed localized negative potential regions near the oxygen atom of the carbonyl group and both the oxygen atoms of the nitro group that extend laterally to the furan ring oxygen, due to electron delocalization, an idea that was previously reported by the same authors in an attempt to highlight structural features required for potent antitubercular activity (19). One more time, the electron delocalization was linked to an improved electron affinity and therefore good antitubercular activity and low toxicity.

Two different radiofluorination procedures were followed to efficiently obtain [¹⁸F]FNFP. The “traditional” radiofluorination of pinacol-derived aryl boronic esters using K₂₂₂/K₂CO₃ and [Cu(OTf)₂(py)₄] first reported by the Gouverneur group (25) afforded [¹⁸F]FNFP in a much lower radiochemical yield (according to a crude reaction mixture analysis) than the azeotropic drying-free [¹⁸F]-labelling of aryl boronic acids and pinacol esters reported by Lahdenpohja, Salla Orvokki et al. (26). Many issues associated with the use of high quantities of base (K₂CO₃) in radiofluorination reactions have been reported in the past few years (27, 28). Low radiochemical yields and the impossibility of using base-sensitive precursors limit the scope of the late-stage radiofluorination approach, especially for radiofluorination of aryl boronic esters and derivates because of the high temperatures (110–130ºC) necessary for this copper-mediated ¹⁸F-fluorination to be carried out. In the second method used for the synthesis of [¹⁸F]FNFP only copper II triflate (a cheaper alternative to [Cu(OTf)₂(py)₄]) was used as [¹⁸F]-elution agent from the anion exchange cartridge, also diminishing the total number of steps compared to method 1.

Chalcone and its derivates (and in our case here FNFP), α,β-unsaturated precursors of flavonoid and isoflavonoid-based compounds, either natural or synthetic exhibit various biological activities, such as anti-inflammatory, analgesic, antioxidant, antibacterial, antifungal, anti-tuberculosis, antimalarial, antiviral, and antitumor (29). These compounds’ conjugated structure is characterized by a high delocalization of the electrons and a low redox potential that provide remarkable chances of undergoing electron transfer reactions (30). The presence of the α,β-unsaturated carbonyl functional group, that acts as a potential Michael acceptor, allows the chalcone (or its derivate) molecule to interact with sulfhydryl of cysteine residue or other thiol groups (30, 31). Stability tests executed with [¹⁸F]FNFP showed that this probe is stable both in serum and Phosphate-buffered saline (PBS), but further in vivo tests need to be carried out in order to evaluate [¹⁸F]FNFP’s possible binding to proteins due to the possible reaction of Michael donors with the α,β-unsaturated carbonyl group present on its structure.

Lipophilicity measurement afforded a \({\text{log}}_{10}P\) value of 1.05 for [¹⁸F]FNFP, which is lower than that of [¹⁸F]FMISO (\({\text{log}}_{10}P\)=2.6) (32) and the same for another 2-nitroimidazole-based hypoxia imaging probe developed by our group in another work, N-(4-[¹⁸F]fluorobenzyl)-2-(2-nitro-1H-imidazol-1-yl)-acetamide [¹⁸F]FBNA (33).

Now it is necessary to evaluate the uptake of this probe under hypoxic/normoxic conditions. Our group is already planning those experiments, and also a comparison in vitro and in vivo with two other stablished radiotracers for studying hypoxia, [¹⁸F]FMISO and [¹⁸F]FAZA.

General methods and instruments

Reagents and solvents were purchased from Sigma Aldrich LTDA, Brazil, and used without further purification. Reactions were monitored by thin-layer chromatography (TLC) using silica gel 60 UV-254 pre-coated silica gel plates from MERCK®. Detection was by means of a UV lamp. Flash column chromatography was performed on 300–400 mesh silica gel. Nuclear Magnetic Resonance (NMR), one-dimensional and two-dimensional analyzes of ¹H and ¹³C nuclei were acquired with Bruker Avance spectrometers (USA) model DPX-300 MHz and DRX-600 MHZ and Bruker Ultra-Shield 300 spectrometer FT-NMR. Chemical shifts are given in ppm referenced to internal standards (s = singlet, singlet, d = doublet, t = triplet, m = multiplet); the coupling constant (J), described in Hertz (Hz); and the number of hydrogens deduced from the relative integral.

Infrared (IR) spectra were obtained on a Fourier Transform Infrared Spectrometer (FTIR) Fourier Transform Infrared Spectrophotometer, IRTracer-100 (Shimadzu), in the spectral region from 4000 to 500 cm^− 1. High resolution mass spectroscopy (HRMS) data were obtained on a Bruker Daltonics mass spectrometer, model microOTOF-Q II - ESI-Qq-TOF and 4000 QTRAP AB SCIEX. Melting points were determined on a Buchi M-560 melting point apparatus BUCHI Brazil Ltda

Measurement of [¹⁸F] radioactivity was performed using a Carpintec dose calibrator (CRC-15R, New Jersey, USA). The Agilent High Performance Liquid Chromatography (HPLC) system (USA) was used for quality control of compounds labeled with [¹⁸F]. The system is equipped with a model 1260 quaternary pump, a model 1260 UV absorbance detector and a radioactivity detector (Raytest, Germany). Agilent Chem Station software was used to operate Agilent HPLC systems. The HPLC equipment with UV and radioactivity detector, as well as the column used for analysis (ZORBAX Eclipse Plus C18 Analytical 4.6 x 250 mm, 5 µm) are from Agilent Technologies, Brazil.

Chemical synthesis

Synthesis of 5-nitro-2-furaldehyde diacetate

Furfural was nitrated following the procedure reported by Taimoory, S. Maryamdokht et al. with some modifications (34). Briefly, Acetic anhydride (180 mL) was placed in a dried 500-mL three-necked, round-bottomed flask equipped with a mechanical stirrer and a thermometer, and afterwards the mixture was then cooled to -10ºC in a benzyl alcohol/dry ice bath. 17.2 mL of concentrated HNO₃ and 0.15 mL of concentrated H₂SO₄ were mixed in a small beaker, and this solution was slowly and carefully added into the chilled acetic anhydride while stirring between − 10ºC and − 5ºC. Afterwards, 20.8 mL (0.251 mol) of freshly distilled furfural was added dropwise over 20 minutes into the cold acid mixture with stirring at the same temperature, and the resulting solution was left to stir for 1 h between − 10ºC and − 5ºC. After this time 160 mL of water were added, and the mixture was left to stir at room temperature for 30 min, over which a white precipitate formed. A 10% NaOH solution was added to the mixture until the pH rose to 2.5–2.7, and it was warmed on a water bath at 55ºC for 1h. The mixture was poured into crushed ice, stirred for 10 min and the white precipitate formed filtered, washed with water, recrystallized twice from ethanol, and air-dried to provide 36.63 g of the product as a white crystalline solid in 60% yield. ¹H NMR (300 MHz, Chloroform-d) δ 7.73 (s, 1H), 7.30 (d, J = 3.6 Hz, 1H), 6.75 (d, J = 3.6 Hz, 1H), 2.19 (s, 6H) (Supplementary file 1). m.p. 90–91 ºC.

Synthesis of 5-nitro-2-furaldehyde

5-nitro-2-furaldehyde was obtained following a procedure reported by Mohammadpoor-Baltork, I. and Aliyan, H. (35). In short, to a solution of 5-Nitro-2-furaldehyde diacetate (4.864 g, 20 mmol) in acetonitrile (100 mL), was added SnCI₂.2H₂0 (902.52 mg, 4 mmol, 20% mol) and the clear mixture was refluxed for 1h (turned dark after 20 min of reaction) when TLC (100% toluene) indicated completion. The solvent was evaporated, and the residue was purified by flash column chromatography eluting with chloroform to give 2.54 g of 5-nitro-2- furaldehyde as a yellow solid in 90% yield. ¹H-NMR (600 MHz, Chloroform-d) δ 9.85 (s, 1H), 7.44 (dd, J = 3.8, 0.4 Hz, 1H), 7.38 (d, J = 3.8 Hz, 1H) (Supplementary file 2). m.p. 38-38.7ºC.

Synthesis of (E)-1-(4-fluorophenyl)-3-(5-nitrofuran-2-yl)prop-2-en-1-one, [¹⁹F]-FNFP

Following the methodology reported by Tawari, Nilesh R et al. (24), 5-nitro-2-furaldehyde (988 mg, 7 mmol) was dissolved in 10 mL of glacial acetic acid at room temperature. 400 µL of concentrated sulfuric acid and 4-fluoroacetophenone (7 mmol, 846 µL) were added in sequence, and the mixture was stirred at room temperature overnight. Ice-cold water (50 mL) was added, the solid was recovered by filtration and washed carefully with ice cold ethanol. The product was then recrystallized from ethanol to yield 1.37 g of (E)-1-(4-fluorophenyl)-3-(5-nitrofuran- 2-yl)prop-2-en-1-one as yellow needles in 75% yield. ¹H-NMR (600 MHz, DMSO-d6) δ 8.26–8.20 (m, 2H), 7.89 (d, J = 15.6 Hz, 1H), 7.83 (d, J = 3.9 Hz, 1H), 7.61 (d, J = 15.6 Hz, 1H), 7.47 (d, J = 3.9 Hz, 1H), 7.45–7.41 (m, 2H) (Supplementary file 3). m.p. 183–184ºC.

Synthesis of 4-acetyl phenylboronic acid pinacol ester

Following the procedure reported by Mo, Fanyang et al. (36), 4-aminobenzophenone (1.352 g, 10 mmol), bis(pinacolato)diboron (2.667 g, 10.5 mmol, 1.05 equiv.) and benzoyl peroxide (20.4 mg, 0.08426 mmol, 2% mol) were weighted in a 50 mL round-bottom flask. To this mixture, anhydrous acetonitrile (15 mL) was added, and then tert-butyl nitrite (1.785 mL, 15 mmol, 1.5 equiv.) in 5 mL of anhydrous acetonitrile was added dropwise at room temperature with stirring. The resulting mixture was stirred at room temperature overnight, and the solvent was removed under reduced pressure. The residue was purified by flash column chromatography eluting with hexanes/ethyl acetate 1/50 to 1/20 to give 1.6 g of 4-acetyl phenylboronic acid pinacol ester as a white-off crystalline solid in 65% yield. ¹H-NMR (600 MHz, Chloroform-d) δ 7.85–7.77 (m, 4H), 2.51 (s, 3H), 1.26 (s, 12H) (Supplementary file 4). m.p. 59–60ºC.

Synthesis of (E)-(4-(3-(5-nitrofuran-2-yl)acryloyl)phenyl)boronic acid pinacol ester: 5-nitro-2-furaldehyde (168 mg, 1.2 mmol) was dissolved in 1.5 mL of glacial acetic acid at room temperature. 120 µL of concentrated sulfuric acid and 4-acetyl phenylboronic acid pinacol ester (295.3 mg, 1.2 mmol) were added in sequence, and the mixture was stirred at room temperature overnight. Ice-cold water (5 mL) was added, the solid was recovered by filtration and washed carefully with ice cold ethanol. The product was then recrystallized from ethanol to yield 319 mg of (E)-(4-(3-(5- nitrofuran-2-yl)acryloyl)phenyl)boronic acid pinacol as yellow needles in 72% yield. ¹H-NMR (600 MHz, DMSO-d6) δ 8.12–8.10 (m, 2H), 7.89–7.84 (m, 3H), 7.83 (d, J = 3.9 Hz, 1H), 7.62 (d, J = 15.6 Hz, 1H), 7.47 (d, J = 3.9 Hz, 1H), 1.33 (s, 12H) (Supplementary file 5). ¹³C-NMR DEPTQ (151 MHz, DMSO) δ 153.60, 152.50, 139.30, 135.30, 129.41, 128.28, 125.31, 118.52, 115.26, 84.62, 25.15 (Supplementary file 6). m.p. 170–171ºC. ESI-mass spectrometry. [M + Na]⁺=392,1274 expected MW = 369,18 g/mol (Supplementary file 7). Infrared spectra: ʋ (= C-H) aromatic 3118 cm^− 1; ʋ (C-H) 2929 cm^− 1; carbonyl stretching vibration for the enone (= C-C = O) 1661 cm^− 1; N–O asymmetric stretch 1479 cm^− 1, N–O symmetric stretch 1365 cm^− 1 (Supplementary file 8).

Radiochemistry

No-carrier-added [¹⁸F]F⁻ was produced by ¹⁸O(p,n)¹⁸F reaction in an 18-MeV cyclotron (IBA, Belgium). Sep-Pak Light QMA cartridges (Waters, Brazil) were pre-conditioned using two different methods: 1) 10 mL of 0.5 M K₂CO₃ solution, followed by 20 mL of Milli-Q water and purged with 5 mL of air; 2) 10 mL of 0.5M potassium triflate (KOTf), followed by 20 mL of Milli-Q water and purged with 5 mL of air.

Radiosynthesis of (E)-1-(4-[¹⁸F]fluorophenyl)-3-(5-nitrofuran-2-yl)prop-2-en-1-one [¹⁸F]FNFP

Two different methodologies were studied to prepare [¹⁸F]FNFP manually.

Method 1, following the procedure reported by Tredwell, Matthew et al. (25). In short, the fluoride ion [¹⁸F]⁻ solution was trapped in an anionic Sep Pak light QMA cartridge, previously conditioned with K₂CO₃ 0.5 M and water, and the [¹⁸F]⁻ was then eluted into the reaction vial with a mixture of K₂CO₃ (2.8 mg; 20 µmol) and Kryptofix 2.2.2 (15 mg; 40 µmol) in 1 mL of acetonitrile/water (8:2). Then, the solution was subjected to three cycles of azeotropic distillation with anhydrous acetonitrile (1mL) at 100°C under a nitrogen flow. Afterwards, (E)-(4-(3-(5-nitrofuran-2-yl)acryloyl)phenyl)boronic acid pinacol ester (4.8 mg, 13 µmol) in 150 µL of anhydrous DMA and [Cu(OTf)₂(py)₄] (8.8 mg, 13 µmol) in 150 µL of DMA were added and the resulting solution was stirred at 120ºC for 20 min under air, quenched with water (300 µL) and an aliquot was removed for analysis by radio-HPLC to calculate the RCY and identify the product, respectively.

Method 2, according to a procedure reported by Lahdenpohja, S.O., Rajala, N.A., Rajander, J. et al. (26). Succinctly, the fluoride ion [¹⁸F]⁻ solution (80–100 mCi, approximately 1–2 mL) was diluted in 8–10 mL of fresh miliQ water in a flask. This solution was taken up in a 20 mL syringe and passed through a pre-conditioned anionic QMA cartridge, previously conditioned with KOTf 0.5 M and water. The cartridge was slowly washed with 5 mL of anhydrous DMA, and the [¹⁸F]⁻ was then eluted with a solution of Cu(OTf)₂ (34.7 mg, 96 µmol) in anhydrous DMA (0.5 mL) into the reaction vial already containing (E)-(4-(3-(5-nitrofuran-2-yl)acryloyl)phenyl)boronic acid pinacol ester (5.5 mg, 15 µmol) diluted in 50 µL of anhydrous DMA and 50 µL of anhydrous pyridine. The QMA cartridge was washed with 0.5 mL of anhydrous DMA into the reaction vial, giving a total volume of 1.1 mL, and the resulting blue solution was stirred at 120ºC for 20 min under air. The reaction was quenched with water (500 µL) and an aliquot was removed for analysis by radio-HPLC to calculate the RCY and identify the product, respectively.

After HPLC identification of the product, [¹⁸F]FNFP crude reaction mixture was then taken up in water (20 mL) and passed through a pre-conditioned C18 plus cartridge. The product was then eluted from the C18 with 1 mL of ethanol and purified by semipreparative HPLC using MiliQ water (0.1% trifluoroacetic acid), channel A, and acetonitrile, channel B, with a gradient run using 10%-100% of B in 20 minutes, with a flux of 3 mL/min, retention time 14–15 min.

Quality control

Aliquots of the final formulation of labeled [¹⁸F]-FNFP and standard (cold) compound [¹⁹F]-FNFP were analyzed by HPLC to determine chemical purity, radiochemical purity, molar activity and confirm chemical identity. The compounds were analyzed with the following analysis conditions:

[¹⁸F]-FNFP and [¹⁹F]-FNFP: Stationary phase: ZORBAX Eclipse Plus C18 Analytical column 4.6 x 250 mm (5 µm). Mobile phase: Solvent (A) Milli Q water 0.1%TFA, Solvent (B) 100% Acetonitrile. Run: Gradient, 90%(A) : 10% B to 100% B for 20 min. Injection: 20 µCi for [¹⁸F]-FNFP and 5 µg for [¹⁹F]-FNFP, Flow: 1 mL/min. Wavelength: 350 nm.

Determination of molar activity

Molar activity was determined by a building a standard curve from the HPLC trace of the corresponding fluorinated “cold” standard compound [¹⁹F]-FNFP [Y axis = UV area, X axis = mole number (µmol)] (Supplementary files 9 and 10). After semipreparative HPLC purification, 20 µCi (0.74 MBq) of [¹⁸F]-FNFP was injected onto the HPLC and the UV area overlapping with the radio peak was recorded and the standard curve was used to calculate mole number. Dividing the product decay corrected activity by the mole number gives the molar activity in GBq/µmol.

In vitro stability

[¹⁸F]-FNFP stability was assessed at room temperature in 0.1 mol/L PBS at pH = 7.2. The samples had an activity of 3.7 MBq (1 mCi) of [¹⁸F]-FNFP in a final volume of 1 mL. The stability of the compound was monitored for 6 hours (immediately after the synthesis, 1, 3 and 6 hours after synthesis) and the results analyzed by HPLC as described in the previous section. The stability of the final formulation was also evaluated in Nude serum. In this case, an aliquot of 800 µL (1mCi) was added to 500 µL of freshly separated serum, and the mixture was incubated at 37°C for up to 6 hours. Aliquots of 100 µL were withdrawn at different times (immediately after the synthesis, 1, 3 and 6 hours after synthesis) and 100 µL of acetonitrile were added to precipitate serum proteins. Then the solution was centrifuged (5000 rpm, for 10 min) and the supernatant was analyzed by HPLC.

Determination of lipophilicity

The lipophilicity of [¹⁸F]FNFP was determined by measuring the octanol-water partition coefficient as described by Barthel, H., Wilson, H., Collingridge, D. et al. (37) with some modifications.. Shortly, 80 − 100 µCi (2.96–3.70 MBq) of [¹⁸F]-FNFP were dissolved in distilled H₂O in a 10 mL falcon tube to a final volume of 0.5 mL and 0.5 mL of octan-1-ol (Sigma-Aldrich Brasil LTDA) was added. The tube was capped and shaken vigorously for 2–3 min and subsequently centrifuged for 30 min (5000 rpm; Rotina 35R, Hettich Zentrifugen, Tuttlingen, Germany). Aliquots (100 µL) of the resulting top layer (representing [¹⁸F]-FNFP dissolved in octanol), and bottom layer ([¹⁸F]-FNFP dissolved in H₂O) were taken and the radioactivity in these samples was measured using a dose calibrator, CRC – 10BC, Carpintec Inc., USA. Eight octanol–H₂O mixtures were analyzed (n = 8), and the octanol –water partition coefficient was calculated by dividing the octanol-containing radioactivity by the water-containing radioactivity.

In this work, the first nitrofuran-based imaging probe to be used as PET hypoxia imaging agent was synthetized and radiolabeled with ¹⁸F. An azeotropic drying-free copper-mediated radiofluorination of a pinacol-derived aryl boronic ester precursor allowed the radiosynthesis to be carried out in 85 minutes obtaining the product [¹⁸F]FNFP with a radiochemical yield of 38,4 ± 3% (n = 5). Subsequent in vitro cellular uptake studies under hypoxic conditions and in vivo in tumor bearing mice are already being planned up, also including a comparison of [¹⁸F]FNFP with [¹⁸F]FMISO and [¹⁸F]FAZA.

B₂pin₂: bis(pinacolato)diboron; BPO: benzoyl peroxide; DMA: dimethyl acetamide; [¹⁸F]F^-: ¹⁸F-fluoride; HPLC: high performance liquid chromatography; K₂₂₂: Kryptofix 2.2.2; K₂CO₃: potassium carbonate; m/z: mass-to-charge ratio; MeCN: acetonitrile; PET: positron emission tomography; RCY: radiochemical yield; QMA: quaternary methyl ammonium; SPECT: Single Photon Emission Computed Tomography; TFA: trifluoroacetic acid; TLC: thin layer chromatography; [Cu(OTf)₂(py)₄]: tetrakis(pyridine)copper(ii) triflate.

Ethics approval and consent to participate

Not applicable

Consent for publication

Not applicable

Competing interests

The authors declare that they have no competing interests

Availability of data and material

Contact the corresponding author

Funding

This research was funded by CNPq – Brazilian Federal Agency for Support and Evaluation of Graduate Education within the Ministry of Education of Brazil - Finance Code 142245/2018-6.

Authors' contributions

All the authors read and approved the final manuscript. YBA performed the experiments and contributed to the design and analysis of data, reviewed the literature, and wrote the manuscript. MAPC, FFAS and ALL contributed to the experiments performed and help revising the manuscript. Methodology, YBA, ALL and ESB. Funding acquisition, ESB.

Acknowledgments

The authors would like to thank to the staff of Cyclotron Facilities and Radiopharmacy Center at IPEN, São Paulo, Brazil.

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Scheme 1 and 2 are available in the Supplementary Files section.

Supplementaryfile1.tiff
Supplementaryfile10.tif
Supplementaryfile2.tiff
Supplementaryfile3.tiff
Supplementaryfile4.tiff
Supplementaryfile5.tiff
Supplementaryfile6.tiff
Supplementaryfile7.pdf
Supplementaryfile8.tif
Supplementaryfile9.docx
scheme1.png
Scheme 1. a) HNO₃, H₂SO₄, Ac₂O, 60%; b) SnCl₂x2H₂O, MeCN, 1h, reflux, 90%; c) 4-fluoroacetophenone, AcOH, H₂SO₄, r.t, overnight, 75%; d) t-BuONO, B₂pin₂, BPO, MeCN, r.t, overnight, 65%; e) 4-acetylboronic acid, pinacol ester, AcOH, H₂SO₄, r.t, overnight, 72%.
scheme2.png
Scheme 2. Synthesis of [¹⁸F]FNFP following method 1 (above) and method 2 (below).

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Synthesis and radiolabeling of a nitrofuran derivate with 18F for identification of areas of hypoxia in the tumor microenvironment

Status:

Version 1

Abstract

Figures

Background

Results

Chemical synthesis

Radiochemistry

Determination of partition coefficient

Discussion

Methods

General methods and instruments

Chemical synthesis

Radiochemistry

Radiosynthesis of (E)-1-(4-[¹⁸F]fluorophenyl)-3-(5-nitrofuran-2-yl)prop-2-en-1-one [¹⁸F]FNFP

Quality control

Determination of molar activity

Determination of lipophilicity

Conclusions

Abbreviations

Declarations

References

Scheme

Supplementary Files

Status:

Version 1

Synthesis and radiolabeling of a nitrofuran derivate with 18F for identification of areas of hypoxia in the tumor microenvironment

Status:

Version 1

Abstract

Figures

Background

Results

Chemical synthesis

Radiochemistry

Determination of partition coefficient

Discussion

Methods

General methods and instruments

Chemical synthesis

Radiochemistry

Radiosynthesis of (E)-1-(4-[18F]fluorophenyl)-3-(5-nitrofuran-2-yl)prop-2-en-1-one [18F]FNFP

Quality control

Determination of molar activity

Determination of lipophilicity

Conclusions

Abbreviations

Declarations

References

Scheme

Supplementary Files

Status:

Version 1

Radiosynthesis of (E)-1-(4-[¹⁸F]fluorophenyl)-3-(5-nitrofuran-2-yl)prop-2-en-1-one [¹⁸F]FNFP