In Vivo Ester Hydrolysis as a New Approach in Development of Positron Emission Tomography Tracers for Imaging Hypoxia

Article abstract of DOI:10.1021/acs.molpharmaceut.8b01131

Hypoxia is an important biochemical and physiological condition associated with uncontrolled growth of tumor. Measurement of hypoxia in tumor tissue may be useful in characterization of tumor progression and monitoring drug treatment. [¹⁸F]FMISO is the most widely employed radiotracer for imaging of hypoxic tissue with positron emission tomography (PET). However, it showed relatively low uptake in hypoxic tissues, which led to low target-to-background contrast in PET images. To overcome these shortcomings, two novel 2-fluoroproprioic acid esters, nitroimidazole derivatives 2-fluoropropionic acid 2-(2-nitro-imidazol-1-yl)-ethyl ester (FNPFT, [¹⁹F]5) and 2-fluoropropionic acid 2-(2-methyl-5-nitro-imidazol-1-yl)-ethyl ester (FMNPFT, [¹⁹F]8), were prepared and tested. Radiolabeling of [¹⁸F]5 and [¹⁸F]8 was accomplished in 45 min (radiochemical purity >95%, the decay-corrected radiochemical yield of [¹⁸F]5 was 11 ± 2%, and that of [¹⁸F]8 was 13 ± 2%, n = 5). In vitro cell uptake studies using EMT-6 tumor cells showed that both radiotracers [¹⁸F]5 and [¹⁸F]8 displayed significantly higher uptake in hypoxic cells than those under normoxic condition, while 2-[¹⁸F]fluoropropionic acid (2-[¹⁸F]FPA) displayed no difference. Biodistribution studies in mice bearing EMT-6 tumor showed that [¹⁸F]5, [¹⁸F]8, and 2-[¹⁸F]FPA displayed similar tumor and major organ uptakes. Tumor uptake values for all three agents were higher than those of [¹⁸F]FMISO, respectively (P < 0.05). This is likely due to a rapid in vivo hydrolysis of [¹⁸F]5 and [¹⁸F]8 to their metabolite, 2-[¹⁸F]FPA. Micro PET imaging studies in the same EMT-6 implanted mice tumor model also demonstrated that both [¹⁸F]5 and [¹⁸F]8 displayed similar tumor uptake comparable to that of 2-[¹⁸F]FPA. In conclusion, two new fluorine-18 labeled nitroimidazole derivatives, [¹⁸F]5 and [¹⁸F]8, showed good tumor uptakes in mice bearing EMT-6 tumor. However, in vivo biodistribution results suggested that they were more likely reflect the predominance of in vivo produced metabolite, 2-[¹⁸F]FPA, which may not be related to tumor hypoxic condition.

Full text of DOI:10.1021/acs.molpharmaceut.8b01131

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Article
In vivo ester hydrolysis as a new approach in
development of PET tracers for imaging hypoxia
Lifang Zhang, Xinyue Yao, Jianhua Cao, Haiyan Hong, Aili Zhang, Ruiyue Zhao,
Yan Zhang, Zhihao Zha, Yajing Liu, Jinping Qiao, Lin Zhu, and Hank F. Kung
Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b01131 • Publication Date (Web): 24 Jan 2019
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In vivo ester hydrolysis as a new approach in development of PET
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Lifang Zhang , Xinyue Yao , Jianhua Cao , Haiyan Hong , Aili Zhang , Ruiyue Zhao ,
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Yan Zhang , Zhihao Zha , Yajing Liu , Jinping Qiao , Lin Zhua,b*, and Hank F Kungb,c*
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Key Laboratory of Radiopharmaceuticals, Ministry of Education, College of Chemistry,
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Beijing Institute for Brain Disorders, Capital Medical University, Beijing, 100069, P. R.
China
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Department of Radiology, University of Pennsylvania, Philadelphia, PA 19014, USA
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Corresponding author contact information:
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Hank F. Kung, Ph.D., Department of Radiology University of Pennsylvania, 3700
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Market Street, Room 305 Philadelphia, PA 19104, USA
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Tel: +1 215 662 3989; Fax: +1 215 349 5035; E-mail: kunghf@gmail.com
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Lin Zhu, Ph.D., Key Laboratory of Radiopharmaceuticals, Ministry of Education,
College of Chemistry, Beijing Normal University, No.19, XinJieKouWai Street, Haidian
District, Beijing, 100875, P.R. China
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Abstract:
Hypoxia is an important biochemical and physiological condition associated with
uncontrolled growth of tumor. Measurement of hypoxia in tumor tissue may be useful in
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characterization of tumor progression and monitoring drug treatment. [ F]FMISO is the
most widely employed radiotracer for imaging of hypoxic tissue with positron emission
tomography (PET). However, it showed relatively low uptake in hypoxic tissues, which
led to low target-to-background contrast in PET images. To overcome these shortcomings,
two novel 2-fluoroproprioic acid esters, nitroimidazole derivatives, 2-fluoropropionic
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acid 2-(2-nitro-imidazol-1-yl)-ethyl ester (FNPFT, [ F]5) and 2-fluoropropionic acid
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2-(2-methyl-5-nitro-imidazol-1-yl)-ethyl ester (FMNPFT, [ F]8), were prepared and
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tested. Radiolabeling of [ F]5 and [ F]8 were accomplished in 45 min (radiochemical
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purity > 95%, the decay-corrected radiochemical yield of [ F]5 was 11 ± 2%, and that of
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[ F]8 was 13 ± 2%, n = 5). In vitro cell uptake studies using EMT-6 tumor cells showed
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that both radiotracers [ F]5 and [ F]8 displayed significantly higher uptake in hypoxic
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cells than those under normoxic condition, while 2-[ F]fluoropropionic acid
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(2-[ F]FPA) displayed no difference. Biodistribution studies in mice bearing EMT-6
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tumor showed that [ F]5, [ F]8, and 2-[ F]FPA displayed similar tumor and major
organ uptakes. Tumor uptake values for all three agents were higher than those of
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[ F]5 and [ F]8 to their metabolite, 2-[ F]FPA. Micro PET imaging studies in the same
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EMT-6 implanted mice tumor model also demonstrated that both [ F]5 and [ F]8
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displayed similar tumor uptake comparable to that of 2-[ F]FPA.
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In conclusion, two new fluorine-18 labeled nitroimidazole derivatives, [ F]5 and
[18F]8, showed good tumor uptakes in mice bearing EMT-6 tumor. However, in vivo
biodistribution results suggested that they were more likely reflect the predominance of in
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vivo produced metabolite, 2-[ F]FPA, which may not be related to tumor hypoxic
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Keywords: Micro PET imaging; in vivo metabolism; EMT-6 tumor; nitroimidazole;
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hypoxia
12 Abbreviations
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PET: Positron Emission Tomography; HPLC: High performance liquid chromatography;
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TLC: Thin-layer chromatography; [ F]FMISO: [ F]Fluoromisonidazole; [ F]FAZA:
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[ F]fluoroazomycin arabinofuranoside; [ F]FETNIM: [ F]fluoroerythronitroimidazole;
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[ F]FETA: [ F]fluoroetanidazole; [ F]EF5: 2-(2-nitro-1H-imidazol-1-yl)-N-(2, 2, 3, 3,
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3-[ F]pentafluoro-propyl)acetamide; 2-[ F]FPA: 2-[ F]fluoropropionic acid; NITTP:
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1-(2-nitro-10-imidazolyl)-2-O-tetra-hydropyranyl-3-O-toluenesulfonylpropane-diol;
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[ F]EF3: 2-(2-nitroimidazol-1-yl)-N-(3, 3, 3-[ F]trifluoropropyl)-acetamide; RCY:
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Radiochemical yield; RCP: Radiochemical purity; OTBDMS: tert-butyldimethylsilyloxy
group.
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Introduction
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Hypoxia in tumor tissue is an important pathological process that commonly
associated with uncontrolled growth of tumor. It is considered to be a factor determining
the curability of radiotherapy and chemotherapy.1-4 The effect of hypoxia on biological
processes depends on tumor type, as well as the degree and duration of oxygen
deprivation.5, 6 Thus, determining the extent of tumor hypoxia is an important factor for
planning of cancer therapy.7, 8 There is an urgent need to develop effective approaches for
measuring hypoxia in tumor tissue. Currently, oxygen sensitive electrode and
immunohistochemistry are gold standards for detecting hypoxic areas in tumor. However,
they are not suitable for routine use due to their technical difficulty and invasiveness.9-11
Positron emission tomography (PET) imaging may have certain advantages, which may
overcome the difficulties in evaluation of hypoxia tumor tissue non-invasively. In
addition, PET imaging has superior global sensitivity and quantification capability
making it as one of the most promising technologies for detecting tumor hypoxia.7
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, 11-13
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Originally, 2-nitroimidazole compounds, for example, [ F]fluoromisonidazole
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([ F]FMISO, Figure 1), were developed for detecting tumor hypoxia in 1979. Currently,
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[ F]FMISO is still the most commonly used PET agent for imaging hypoxia and it is
considered as a gold standard for studying hypoxia by in vivo PET imaging.15-20 However,
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wide acceptance for routine clinical application. This is due to its relatively low uptake in
hypoxic tumor, slow clearance from the blood and normal tissues, which contribute to
low target-to-background contrast in PET images. In the past few decades, significant
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efforts have been reported in developing better hypoxia PET imaging agents.12, 21-25 New
nitroimidazole PET tracers with different side chains were produced and evaluated
successively for PET imaging of hypoxic tumors, including the introduction of sugars
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([ F]fluoroazomycin arabinofuranoside, [ F]FAZA),
adding hydroxyl groups
and adding amide bonds
(Figure 1) However, these 18F-labeled
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([ F]fluoroerythronitroimidazole, [ F]FETNIM),
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([ F]fluoroetanidazole, [ F]FETA).
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2-nitroimidazoles shared a common feature: they showed a higher hydrophilicity, thus,
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reduced lipophilicity for cell membrane penetration than that of [ F]FMISO. These
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radiotracers displayed either low signal/noise ratios due to lower uptake in hypoxic
lesions or slower clearance from normal tissues.10, 11, 32 It has been shown that
hydrophilicity facilitates clearance in well oxygenated tissues, while lipophilicity
enhances accumulation in hypoxic tissues. In search of optimal hypoxia imaging agents
achieving a delicate balance between these contrasting properties is critically important.
In recent years, a new series of highly lipophilic derivatives have been studied, such as
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2-(2-nitro-1H-imidazol-1-yl)-N-(2, 2, 3, 3, 3-[ F]pentafluoropropyl)-acetamide
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([ F]EF5) and 2-(2-nitroimidazol-1-yl)-N-(3, 3, 3-[ F]trifluoropropyl)-acetamide
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(Figure 1) In addition, several hypoxia radiotracers based on
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metronidazole derivatives (i.e. 5-nitroimidazole derivatives) have been reported, some of
which demonstrated very favorable in vivo biodistribution properties.38-40
Earlier reports have pointed out that more lipophilic nitroimidazole analogs would
be expected to cross cellular membranes and the blood–brain barrier (BBB) more readily
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and resulted in a greater initial uptake in the targeted hypoxic tissue than [ F]FMISO,
although there is a concomitant increase of nonspecific uptake.21, 41, 42 This is a necessary
trade-off between a higher lipophilicity (promoting cellular uptake through diffusion) and
a higher liver uptake (due to a higher lipophilicity). The requirement of optimal
lipophilicity leading to favorable biodistribution and pharmacokinetics of the radiotracer
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must be carefully balanced in the course of design an ideal hypoxia imaging agent.
Recently, 18F-acetyl has been conjugated to 2-nitroimidazole to form a 18F-labeled
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fluoroacetate of 2-nitroimidazole.43 Results of LC-MS data showed that the hypoxia
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tracer was hydrolyzed in vivo to hydrophilic F-fluoroacetic acid. Results suggested that
it was possible to enhance tumor uptake and speed up non-target clearance in vivo by
introducing 18F-aliphatic ester into nitroimidazole analogues. Additionally, previous
studies reported that hydrolysable nitroimidazole aliphatic esters might be useful as
radiosensitizers for cancer therapy.44, 45 The esters were subject to enzyme-catalyzed
hydrolysis in human plasma and in pure buffer solution. The length of the linear carbon
chain in the aliphatic esters influences the rate of enzyme catalyzed degradation.
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stability of the nitroimidazole aliphatic ester derivatives in aqueous solution and in
plasma. The hydrolysis rate of the esters was remarkably increased in human plasma, but
the rate of hydrolysis was significantly decreased, or none, in buffer solution with
increased length of the acyl side chain from the methyl to the pentyl. Thus, it offers
flexibility to design hypoxia tracers with optimized physicochemical and
pharmacokinetic characteristics using 18F-labeled nitroimidazole aliphatic esters by
controlling the hydrolysis rate via regulating length of the acyl side chain. This might
maintain a balance between an ester acyl chain length and its hydrolysis rate, which may
lead to optimal tracers for hypoxia imaging. In an attempt to control the in vivo
hydrolysis and maintain the hypoxic uptake towards tumor cells envision new
nitroimidazole ester derivatives may be a reasonable starting point for designing better
hypoxic imaging agents.
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In this work, we have developed two new nitroimidazole derivatives,
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2-fluoropropionic acid 2-(2-nitro-imidazol-1-yl)-ethyl ester (FNPFT, [ F]5), and
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2-fluoropropionic acid 2-(2-methyl-5-nitro-imidazol-1-yl)-ethyl ester (FMNPFT, [ F]8),
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which containing 2-fluoropropionyl ester group without changing the hypoxia-targeting
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nitroimidazole group. Reported herein is the synthesis of [ F]NPFT ([ F]5) and
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[ F]MNPFT ([ F]8) (Figure 1), and evaluation of their potentiality as hypoxia imaging
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agents by measuring their stability in vitro, partition coefficient, tumor cell uptake, and
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biodistribution/Micro
PET
imaging
in
EMT-6
tumor-bearing
mice.
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Experimental Section
General
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Reagents and solvents
1
8
4
5
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0
The
precursor
used
for
preparation
of
[ F]FMISO,
1-(2-nitro-10-imidazolyl)-2-O-tetra-hydropyranyl-3-O-toluenesulfonylpropane-diol
(NITTP), was purchased from Huayi Chemicals Co., Ltd. (Changshu, Jiang Su, China).
Other reagents and solvents were of analytical reagent grade and purchased from
commercial sources (Aldrich, Acros, or Alfa Inc.), and were used without further
purification unless otherwise specified.
1
1
Instrument and consumables materials
1
Thin-layer chromatography (TLC) was run on pre-coated plates of silica gel 60
1
12
F254 (Merck, Darmstadt, Germany). H NMR spectra were recorded on a 400 MHz
1
1
1
3
4
5
Bruker Advance spectrophotometer (Bruker Co., Germany), 13C NMR spectra were
recorded on a 600 MHz JMTC-600/54/JJ (Japan Superconductor Technology, Inc.,
Japan), and the chemical shifts were reported as δ values (parts per million) relative to
residual protons of deuterated solvent. Coupling constants are reported in Hertz (Hz). The
multiplicity is defined by s (singlet), d (doublet), t (triplet), br (broad), and m (multiplet).
Melting points were measured on an X-5 melting point apparatus. Mass spectrometric
detection was performed in the positive ion mode on a Micromass Quattro micro API
16
1
1
7
8
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mass spectrometer (Waters, Milford, MA) with an electrospray ionization (ESI) source
and high-resolution mass spectrometry (HRMS) data was registered by an Agilent (Santa
Clara, CA) G3250AA LC/MSD TOF system. Solid-phase extraction cartridges (Oasis
HLB (3cc) cartridge, Sep-Pak light QMA cartridge, neutral Al O cartridge and Sep-pak
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
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9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
3
1
8
C-18 cartridge) were obtained from Waters (Milford, MA, USA). The [ F]fluoride ion
was produced by Peking University Cancer Hospital.
Animals
8
9
0
1
2
Animal studies were performed in BALB/c female mice (weight, 18 ± 2 g)
bearing EMT-6 tumors (purchased from Cancer Hospital Chinese Academy of Medical
Sciences, Beijing, China), which grew to right upper limb diameter of 10–15 mm. Male
Sprague–Dawley rats, 6–7 weeks old, 180–220 g, were obtained from Vital River
Laboratory Animal Co. Ltd (Beijing, China). All the animals were maintained according
as the Chinese government guidelines for care and use of laboratory animals.
Synthesis
1
1
1
13
1
4
15
16
1-(2-(tert-Butyldimethylsilyloxy)ethyl)-2-nitro-1H-imidazole (2)
A mixture of 1 (313 mg, 2.77 mmol), (2-bromoethoxy)(tert-butyl)dimethylsi -lane
1
1
7
8
(892 mg, 3.73 mmol) and potassium carbonate (K CO ) (404 mg, 2.92 mmol) in 5 mL N,
2
3
N-dimethylformamide (DMF) was stirred at 100°C for 4 h. The mixture was then diluted
with 15 mL ethyl acetate (EtOAc) and washed by saturated solution of brine (10 mL × 3).
19
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2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
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4
4
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5
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1
2
3
4
5
6
The organic layers were dried by anhydrous Na SO and filtered. The filtrate was
2 4
concentrated, and the residual crude product was purified by flash chromatography
1
(EtOAc/hexane: 3/7) to give 696 mg white solid 2 (yield: 97%): H NMR (400 MHz,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
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9
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1
2
3
4
5
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2
3
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0
CDCl ) δ: 7.21 (s, 1 H), 7.20 (s, 1 H), 4.61 (t, J = 4.9 Hz, 2 H), 4.01 (t, J = 4.9 Hz, 2 H),
3
0.89 (s, 9 H), 0.10 (s, 6 H).
2-(2-Nitro-1H-imidazol-1-yl)-ethanol (3)
7
8
9
0
1
2
To a solution of 2 (796 mg, 2.93 mmol) in 5 mL tetrahydrofuran (THF) was
added 5 mL HCl solution (0.5 mL 1% HCl in 4.5 mL ethanol) dropwise at room
temperature. After 3 h, the mixture was cooled to 0°C and neutralized with 10% Na CO .
2
3
1
1
1
The aqueous layer was extracted with dichloromethane (20 mL × 3). The organic layers
were dried by anhydrous Na SO and filtered. The filtrate was concentrated, and the
2
4
residual crude product was purified by flash chromatography (EtOAc/hexane: 3/7) to
1
13
give 356.4 mg yellow solid 3 (yield: 70%): H NMR (400 MHz, DMSO) δ: 7.61 (s, 1 H),
1
1
4
5
7.16 (s, 1 H), 5.01 (t, J = 5.4 Hz, 2 H), 3.70 (t, J = 5.24 Hz, 2 H), 3.33 (s, 1 H).
19
2-Fluoropropionic acid 2-(2-nitro-imidazol-1-yl)-ethyl ester ([ F]5)
1
6
2-Fluoropropionic acid (0.1 mL, 1.32 mmol) was dissolved in anhydrous DMF (5
17
mL),
N,N-Diisopropylethylamine
(DIPEA,
0.25
mL,
1.43
mmol),
1
1
8
9
1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCl, 250 mg, 1.30
mmol), and 1-Hydroxybenzotriazole hydrate (HOBt, 180 mg, 1.33 mmol) were added
20
under ice-water bath conditions, then dropwise with the compound 3 (105 mg, 0.67
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1
mmol). After 2 h, the ice-water bath was removed. After stirring overnight, the mixture
was diluted with 20 mL dichloromethane (DCM) and washed by saturated solution of
brine (10 mL × 3). The organic layers were dried by anhydrous Na SO and filtered. The
2
4
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
filtrate was concentrated, and the residual crude product was purified by flash
1
9
chromatography (DCM/Methanol: 100/1) to give 139 mg yellow solid [ F]5 (yield:
1
89.7%): Mp: 66.9–67.2°C. H NMR (400 MHz, CDCl ) δ: 7.18 (s, 1 H), 7.11 (s, 1 H),
3
4.90-5.08 (dq, 1 H), 4.77 (t, J = 5.06 Hz, 2 H), 4.60 (t, J = 5.04 Hz, 2 H), 1.50-1.58 (dd, 3
1
3
H). C NMR (600 MHz, CDCl ) δ: 170.04, 169.89, 128.70, 126.79, 86.18, 84.96, 63.32,
3
+
48.77, 18.39, 18.24. HRMS (ESI) calculated for C H FN O (M+H ), 232.0734; found,
8
11
3
4
1
1
232.0732.
2-Bromo-propionic acid 2-(2-nitro-imidazol-1-yl)-ethyl ester (4)
1
2
To a mixture of compound 3 (162 mg, 1.03 mmol) and triethylamine (0.4 mL,
2.88 mmol) in anhydrous DCM, bromopropionyl bromide (0.2 mL, 2.00 mmol) in
anhydrous DCM (2 mL) was added dropwise in 1 h. The reaction solution was then
13
1
1
4
5
stirred at room temperature overnight. H O (10 mL) was added and extracted with DCM
2
16
(10 mL × 2), and the organic layers were combined and dried over anhydrous Na SO ,
2
4
1
1
1
7
8
9
filtered, the filtrate was evaporated and purified by flash chromatography (EtOAc/hexane:
1
2/3) to give product 4 (116 mg, yield: 38.7%) as a yellow oily liquid. H NMR (400MHz,
CDCl ) δ: 7.19 (s, 2 H), 4.75 (t, J = 7.44 Hz, 2 H), 4.34 (t, J = 6.91 Hz, 2 H), 4.10–4.15
3
1
3
20
(m, 1 H), 1.79-1.80 (d, J = 6.92 Hz, 3 H). C NMR (600 MHz, CDCl ) δ: 169.81, 128.68,
3
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1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
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5
5
5
5
5
5
5
5
5
6
+
1
2
3
127.07, 63.81, 48.85, 39.32, 21.55. HRMS (ESI) calculated for C H BrN O (M+H ),
8
11
3
4
291.9933; found, 291.9926.
2-Fluoropropionic acid 2-(2-methyl-5-nitro-imidazol-1-yl)-ethyl ester ([ F]8)
1
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
9
4
5
6
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8
9
0
1
2
Compound [ F]8 was prepared from 2-Methyl-5-nitroimidazole-1-ethanol (171
mg, 1 mmol), DIPEA (0.35 mL, 2 mmol), HOBt (270 mg, 2 mmol) and 2-fluoropropionic
1
9
acid (0.15 mL, 2 mmol), with the same procedure described for compound [ F]5.
1
9
1
Compound [ F]8: 146 mg yellow solid (yield: 59.6%): Mp: 71.0–71.5°C. H NMR (400
MHz, CDCl ) δ: 7.98 (s, 1 H), 4.90-5.07 (dq, 1 H), 4.66 (t, J = 5.04 Hz, 2 H), 4.55 (t, J =
3
1
3
3.49 Hz, 2 H), 2.54 (s, 3 H), 1.49–1.57 (dd, 3 H). C NMR (600 MHz, CDCl ) δ: 170.12,
3
1
1
1
169.97, 133.41, 86.29, 85.07, 63.71, 45.06, 18.39, 18.24, 14.45. HRMS (ESI) calculated
+
for C H FN O (M+H ), 246.0890; found, 246.0885.
9
13
3
4
2-Bromo-propionic acid 2-(2-methyl-5-nitro-imidazol-1-yl)-ethyl ester (7)
13
To a mixture of 2-Methyl-5-nitroimidazole-1-ethanol (177 mg, 1.01 mmol) and
triethylamine (0.4 mL, 2.88 mmol) in anhydrous DCM, bromopropionyl bromide (0.2 mL,
2.00 mmol) in anhydrous DCM (2 mL) was added dropwise in 1 h. The reaction solution
1
1
1
4
5
6
was then stirred at room temperature overnight. H O (10 mL) was added and extracted
2
17
with DCM (10 mL × 2), and the organic layers were combined and dried over anhydrous
Na SO , filtered, the filtrate was evaporated and purified by flash chromatography
1
1
8
9
2
4
(EtOAc/Petroleum ether: 2/1) to give product 7 (169 mg, yield: 55.4%) as a yellow oily
1
20
liquid. H NMR (400 MHz, CDCl ) δ: 7.91 (s, 1 H), 4.57 (t, J = 3.12 Hz, 2 H), 4.45 (t, J
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4
= 4.52 Hz, 2 H), 4.20–4.26 (q, 1 H), 2.50 (s, 3 H), 1.71 (d, J = 6.92 Hz, 3 H). C NMR
(600 MHz, CDCl ) δ: 169.88, 151.21, 133.45, 64.09, 44.99, 39.12, 21.58, 14.72. HRMS
3
+
(ESI) calculated for C H BrN O (M+H ), 306.0089; found, 306.0085.
9
13
3
4
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
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9
0
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2
3
4
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8
9
0
Radiolabeling
1
8
18
5
Preparation of [ F]1-(2-nitro-1-imidazolyl)-3-fluoro-2-propanol ([ F]FMISO)
1
8
6
7
8
9
0
1
2
3
4
The aqueous solution of the [ F]fluoride ion produced by the cyclotron was
passed through a Sep-Pak light QMA cartridge, The cartridge was previously activated
1
8
with 10 mL 1 M NaHCO and 10 mL water, as well as dried with argon. Then the F
3
activity was eluted with 1.1 mL of Kryptofix 2.2.2 (K2.2.2)/K CO solution (11.0 mg
2
3
1
1
1
1
1
K2.2.2 and 2.0 mg K CO in 0.93 mL acetonitrile and 0.17 mL H O). The eluent was
2 3 2
then evaporated at 100°C under an argon stream. Additionally, the residue was
azeotropically dried twice with 1.0 mL anhydrous acetonitrile at 100°C under an argon
stream. A solution of 1 mg precursor (NITTP) in 1 mL anhydrous acetonitrile was added
to the evaporation residue, and the mixture was heated at 100°C for 10 min, followed by
hydrolysis with 2 M HCl (1 mL) at 100°C for 10 min. After cooling down to room
temperature, 2 M NaOH (1 mL) was added to neutralize. The crude reaction mixture was
subsequently delivered to a neutral Al O cartridge and a Sep-pak C-18 cartridge for
15
16
17
18
2
3
1
8
purification. [ F]FMISO was trapped on the cartridge and eluted with 1 mL ethanol. The
19
pure product was diluted with saline to 10% ethanol solution, and then sterilized by
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2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
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5
5
5
5
5
5
5
5
5
5
6
1
2
3
4
5
6
7
passing through a 0.22 μm sterile membrane. The structure of the purified product was
confirmed by HPLC with a gamma ray radio-detector and a UV detector at 320 nm, as
well as the radiochemical purity (RCP). The HPLC condition was as follow: Phenomenex
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Gemini C18 (4.6 mm × 250 mm, 5 μm), mobile phase: acetonitrile/H O 3/7 (V/V), 1
2
mL/min. The RCP was also measured by Radio-TLC, with a mobile phase
acetonitrile/H O 85/15.
2
1
8
18
Preparation of [ F]5 and [ F]8
8
9
0
1
2
[18F]KF/18-crown-6 complex was prepared as previously described for
1
8
[ F]FMISO. A solution of precursor 4 (2 mg) or precursor 7 (2 mg) in 1 mL acetonitrile
1
8
1
1
1
was added to the dried [ F]KF/18-crown-6 complex and heated at 80°C for 10 min. After
cooling down to room temperature, the reaction mixture was quickly diluted with 5 mL
buffer solution (pH = 6.40) containing 0.2 M disodium hydrogen phosphate and 0.1 M
citric acid, and subsequently passed through an Oasis HLB cartridge. The cartridge was
13
1
4
5
rinsed with 2 mL 10% ethanol/H O (V/V) and then eluted with 1 mL ethanol to obtain
2
18
18
1
[ F]5 or [ F]8. The pure product was diluted with saline to 10% ethanol solution, and
then sterilized by passing through a 0.22 μm sterile membrane. The structure and RCP of
the product was confirmed by HPLC with a gamma ray radio-detector and a UV detector
at 320 nm. The HPLC condition was as follow: Column was Phenomenex Gemini C18
(4.6 mm × 250 mm, 5 μm), mobile phase: 0.1% Formic acid solution(A), acetonitrile (B),
16
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1
1
7
8
9
20
1 mL/min with a gradient as shown below: from 0 to 3 min, A 99%, B 1%; from 3 to 10
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New PET tracers for imaging hypoxia
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min, gradient A 99-5%, B 1-95%; from 10 to 15 min, gradient A 5-80%, B 95-20%; from
15 to 18 min, gradient A 80-99%, B 20-1%. The RCP was also measured by Radio-TLC,
with a mobile phase CH Cl /CH OH = 9/1.
2
2
3
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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6
7
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9
0
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2
3
4
5
6
7
8
9
0
18
Preparation of 2-[ F]FPA
1
8
46
5
6
2-[ F]FPA was prepared according to the method previously described.
In vitro stability study
1
8
18
7
8
9
0
The in vitro stability of [ F]5 and [ F]8 were examined by incubating in 1 mL
saline for 2 h at room temperature, and the RCP was measured by Radio-TLC and
Radio-HPLC using chromatographic conditions as previously described.
In vitro metabolism studies in rat serum
1
1
1
1
1
1
2
3
4
Rat blood was freshly drawn from an anesthetized SD rat into tubes.
Centrifugation was performed at 5000 g for 5 min, and the supernatant was taken as rat
serum. Incubations were carried out at 37 C in a water bath. The hydrolysis reactions
18
18
were initiated by adding [ F]5 or [ F]8 (~3 MBq), in 100 μL 10% ethanol solution to
preheated blood samples (900 μL) or 10% ethanol solution (as control). Aliquots (100 μL)
were taken from blood at 2, 5 and 10 min and were deproteinized by mixing with
acetonitrile (200 μL). After centrifuging for 5 min at 14,000 rpm, 10 μL of the
supernatant layer was analyzed by analytical HPLC using Phenomenex Gemini C18
column (4.6 mm × 250 mm, 5 μm) with mobile phase consistent of 20 mM NaH PO (A)
15
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4
and acetonitrile (B), a flow rate of 1 mL/min with a gradient as shown below: from 0 to 3
min, A 99%, B 1%; from 3 to 10 min, gradient A 99-5%, B 1-95%; from 10 to 15 min,
gradient A 5-80%, B 95-20%; from 15 to 18 min, gradient A 80-99%, B 20-1%.
Determination of the partition coefficient (log P)
0
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8
18
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0
Log P was assessed by mixing radiotracer [ F]5, [ F]8 or [ F]FMISO with
1-octanol and phosphate buffer (0.1 M, pH 7.4) system in a centrifuge tube. The tube was
shaken uniformity on a Vortex oscillator (Vortex-Genie 2) for 3 min and then centrifuged
at 5000 g for 5 min. Three samples (0.2 mL each) from the 1-octanol and buffer layers
were then measured for radioactivity. The partition coefficient was calculated as the
mean value of counts per minute in 1-octanol divided by that of the buffer. The
measurement was performed for three times, and the final partition coefficient value was
expressed as log P.
1
11
1
1
2
3
In vitro cell uptake
1
1
1
4
5
6
Cell uptake studies were evaluated using the murine mammary carcinoma cells
EMT6 cell lines, which were purchased from Cancer Hospital Chinese Academy of
o
Medical Sciences, Beijing, China. The cell was maintained (5% CO incubator at 37 C)
2
17
in RPMI 1640 medium (purchased from Corning) containing 10% fetal bovine serum and
1% antibiotics mixture. The cells were dispersed in 20 mL fresh RPMI medium to
1
1
8
9
6
achieve final concentration of approximately 2 × 10 cells/mL. Aliquots of 10 mL were
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added into glass vials and incubated at 37 C with gentle magnetic stirring under
normoxic (5% CO plus 95% air) or hypoxic (5% CO plus 95% N ) atmosphere. In
2
2
2
addition, the cells were equilibrated for 60 min in either normoxic or hypoxic conditions,
1
1
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1
1
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2
2
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3
3
3
3
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4
4
4
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5
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5
5
5
5
5
5
5
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0
1
2
3
4
5
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1
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5
6
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0
1
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3
4
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then the radiotracer ([ F]FMISO, 2-[ F]FPA, [ F]5 or [ F]8, 0.37 MBq/100 µL) was
added to each glass vial and incubated for 30, 60, 120 and 240 min. 1 mL samples were
removed at each time point. From the samples, four aliquots (200 µL) were centrifuged at
1500 rpm for 5 min to separate the cells from the supernatant. 90 µL of the supernatant
was removed for counting as A, and 110 µL of residue medium containing cells was
6
6
counted as B. The cell uptake was calculated as C /C /10 cells (the uptake in 10 cells),
in out
1
1
C /C = (B – A)/A. The cell viability was detected by trypan blue exclusion assay
in out
during the whole procedure, and the viability of cells was more than 90%. Statistical
analysis was performed using the Student’s t-test (n = 3 at each time point).
Biodistribution studies
12
1
3
14
15
16
17
Biodistribution studies were assessed in BALB/c female mice bearing EMT-6
1
8
18
tumor. 0.1 mL of radiotracer ([ F]5 or [ F]8, ~1.11 MBq) was intravenously injected
into each mouse. Five mice for each time were sacrificed at 30, 90, and 120 min
post-injection. The organs or tissues (blood, brain, heart, lung, liver, spleen, kidney,
stomach, bone, muscle, skin, and tumor) were collected, weighed and the corresponding
radio-activities were assayed with a gamma counter. The results were expressed as the
18
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percent uptake of injected dose per gram of tissue (%ID/g) and presented as mean ± SD.
1
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18
[ F]FMISO and 2-[ F]FPA were evaluated in the same animal model and used for
comparison.
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Micro PET/CT imaging
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PET imaging was performed on a Micro PET/CT scanner (Siemens Invenon MM)
18
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using BALB/c female mice bearing EMT-6 tumor. [ F]5, [ F]8, [ F]FMISO or
1
8
2-[ F]FPA (5.55–11.1 MBq/0.1 mL) was intravenously administered to mice via tail
vein. At 30, 60, 90 and 120 min post injection, mice were anesthetized with 2%
isoflurane inhalation, and fixed on the machine in a prone position. At each time point,
static imaging was obtained with 5 min CT scanning followed by 10 min PET. PET/CT
Image was reconstructed with OSEM3D. Image analysis was carried out using Invenon
Research Workplace software.
1
11
1
1
2
3
Statistical Analysis
1
1
1
4
5
6
Data were presented as mean ± standard deviation (SD). All statistical tests were
conducted using the IBM SPSS Statistics Version 20.0 for Windows (SPSS, Inc, IBM
Company). The independent samples nonparametric test was used to compare the
difference of 2 quantitative groups. Pearson product-moment correlation coefficient (r)
was used for correlation analysis between continuous variables. A P value of less than
0.05 was considered statistically significant.
17
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Results and discussion
Synthesis
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1
The radiolabeling precursor 4 and non-radioactive standard [ F]5 were prepared
by reactions shown in the Scheme 1. Starting material 2 was synthesized by N-alkylation
of commercially available 2-nitroimidazole. The protecting group OTBDMS of
compound 2 was removed by treating with 1% HCl to obtain 3. The acylation of
compound 3 with bromopropionyl bromide and 2-flupropionic acid, respectively,
1
9
produced the radiolabeling precursor 4 and non-radioactive standard [ F]5. The
1
9
radiolabeling precursor 7 and non-radioactive standard [ F]8 were prepared by reactions
shown in the Scheme 2. Starting material 6 was purchased directly from commercial
sources. The acylation of compound 6 with 2-bromopropionyl bromide and
2-flupropionic acid, respectively, produced the radiolabeling precursor 7 and
1
1
12
1
9
1
1
1
3
4
5
non-radioactive standard [ F]8. These procedures yielded the desired products after
1
13
chromatographic isolation. All compounds were characterized by H NMR, C NMR
and HRMS (Compound 2 and 3 were synthesized according to the previously reported
4
3
1
16
methods, only H NMR data were included). Results were consistent with proposed
1
1
7
8
structures.
Radiolabeling
1
8
1
9
Preparation of [ F]FMISO
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0
The radiosynthesis of [ F]FMISO was carried out according to the previously
reported methods.23, 40 [18F]FMISO was synthesized from the precursor NITTP by a
nucleophilic substitution fluorination reaction followed by acid hydrolysis of alcohol
0
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protecting group. The pure radiotracer of [ F]FMISO was obtained by using a neutral
Al O cartridge and a Sep-pak C-18 cartridge. The decay-corrected radiochemical yield
2
3
1
8
of [ F]FMISO was 25 ± 5% (n = 5). The preparation took about 60 min. Radio-HPLC
1
8
and radio-TLC analysis showed that the RCP of [ F]FMISO was more than 95%. The
radiochemical identity was confirmed by HPLC (see Supporting information, Figure S1)
1
8
19
([ F]FMISO = 3.9 min, [ F]FMISO = 3.8 min).
1
8
18
1
Preparation of [ F]5 and [ F]8
1
8
18
1
1
1
2
[ F]5 and [ F]8 were synthesized from the corresponding bromo precursors 4
and 7 by the reactions shown in Scheme 3. The bromo precursors, 4 and 7, were reacted
1
8
13
with dried [ F]KF/18-crown-6 complex in acetonitrile at 80°C for 10 min. The
1
1
4
5
radiotracers were purified with Oasis HLB cartridge and reformulated in 10%
18
ethanol/saline. The decay-corrected radiochemical yields of [ F]5 was 11 ± 2% (n = 5),
1
8
16
and [ F]8 was 13 ± 2% (n = 5). The synthesis time was about 45 min. Radio-HPLC and
1
8
18
1
1
1
7
8
9
radio-TLC analysis showed that the RCP of [ F]1 and [ F]2 were more than 95%. The
1
8
18
specific activity of [ F]5 and [ F]8 were 30 ± 2 GBq/μmol (n = 5) and 61 ± 8 GBq/μmol
(n = 5), respectively. The radiochemical identity was confirmed by HPLC (Supporting
1
8
19
18
20
information Figure S2 and Figure S3). ([ F]5 = 13.5 min, [ F]5 = 13.4 min; [ F]8 =
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13.6 min, [ F]8 = 13.4 min).
1
8
Preparation of 2-[ F]FPA
1
8
3
4
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7
The radiosynthesis of 2-[ F]FPA was carried out according to the previously
1
1
1
1
1
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2
2
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3
4
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5
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0
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5
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8
reported methods. The decay-corrected radiochemical yields of 2-[ F]FPA was 37 ±
10% (n = 5). The synthesis time was about 60 min. Radio-HPLC analysis showed that the
1
8
RCP of 2-[ F]FPA was more than 95%.
Determination of the partition coefficient (log P)
8
9
0
1
2
3
The partition coefficient (between octanol and 0.1 M phosphate buffer, pH 7.4)
was conducted by the method described in experimental section. The log P was
1
8
18
1
1
1
1
calculated shown in Table 1. The log P of [ F]5 and [ F]8 were 0.42 and 0.63,
1
8
comparable to that of [ F]FMISO (0.38). The values for all three agents were statistically
similar.
In vitro stability study
14
After incubating with saline up to 2 h after preparation, the radiochemical purities
1
8
18
1
1
5
6
of [ F]5 and [ F]8 were over 95% suggesting their great stability in vitro for further
investigation. These two tracers displayed excellent in vitro stability in saline.
In vitro metabolism studies
17
1
8
18
1
1
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9
In vitro stability of these two tracers, [ F]5 and [ F]8, were evaluated in the
presence of rat serum. The key difference between saline and rat serum is that in rat
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blood the tracers were exposed to various enzymes which might hydrolyze the ester
linkage. Measurement of potential radioactive metabolites and the rate of hydrolysis
kinetics were carried out after incubating the samples in rat blood at 37°C. The
0
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percentages of unchanged [ F]5 and [ F]8 at different time points was shown in Table 2.
1
8
The percentage of intact parent compound [ F]5 was 9.4% after incubating in rat blood
1
8
18
for 2 min, while [ F]8 was 15.2%. After incubating for 10 min, both radiotracers [ F]5
1
8
and [ F]8 were very closed to completely hydrolyzed. Furthermore, the radiolabeled
metabolite was more polar than the original parent radiotracers, which was likely the
1
8
expected product, 2-[ F]FPA, after the cleavage of the ester bond. (See Supporting
1
1
information Figure S4, Figure S5 and Figure S6)
18
18
Results of in vitro hydrolysis of [ F]5 and [ F]8 indicated that the rate of
1
8
18
12
hydrolysis of [ F]5 was faster than that [ F]8 in rat blood. Due to the existence of ester
13
14
15
groups in the radiotracers, it would be reasonable to suspect that esterases in the blood
1
8
might catalyze the hydrolysis of these two tracers. After the hydrolysis 2-[ F]FPA was
expected to be the major labeled compound in the circulating blood. It was known that
1
8
46
18
16
2-[ F]FPA also showed high tumor uptake in tumor models. Therefore, 2-[ F]FPA
might be a major contributor to the observed uptake in EMT-6 tumor tissue in vivo.
In vitro cell uptake
1
1
7
8
1
8
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9
In vitro cellular uptake of [ F]5, [ F]8, [ F]FMISO and 2-[ F]FPA (Figure 2A,
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2B, 2C and 2D, respectively) in EMT-6 cells under hypoxic and normoxic conditions
were determined according to previous reported methods.47-49 This method of in vitro
study has been successfully validated in our laboratory in the evaluation of PEG-modified
1
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50 18
nitroimidazole derivatives. [ F]FMISO was used as a gold standard for comparison,
1
8
18
while 2-[ F]FPA as a negative control. As expected, [ F]FMISO (Figure 2C) showed a
significantly greater accumulation in EMT-6 cells after 1 h in hypoxic conditions (P <
1
8
0.01), while the uptakes of 2-[ F]FPA (Figure 2D) displayed no difference between
hypoxic cells and normoxic cells (P > 0.05). Under similar hypoxia conditions, cell
1
8
18
uptakes of [ F]5 and [ F]8 (Figure. 2A and 2B, respectively) showed initial increase and
1
8
18
18
1
1
follow by a small reduction at 2 h. Similar to that of [ F]FMISO, both [ F]5 and [ F]8
showed significantly higher (P < 0.05) cellular uptakes under the hypoxia condition than
1
8
12
that under the normoxic condition at all time points. In contrast, 2-[ F]FPA (Figure. 2D)
displayed no difference between hypoxic and normoxic conditions. Results in Figure 2
both two new radiotracers displayed highly significant hypoxia selectivity in EMT-6
cells.
1
1
1
3
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5
16
Biodistribution studies
18
18
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18
1
7
Biodistribution of [ F]5, [ F]8, [ F]FMISO as well as 2-[ F]FPA were evaluated
in BALB/c mice implanted with EMT-6 tumor, which was commonly used as hypoxia
tumor model.51-53 Results of biodistribution expressed as the percentage injected dose per
18
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gram tissue (%ID/g) for tissues of various organs of mice were presented in Tables 3.
1
8
18
Biodistribution study showed that [ F]5 and [ F]8 displayed a high uptake (%ID/g, n =
1
8
5) in all major organs (Table 3A, 3B and 3C). Specifically, the tumor uptakes of [ F]5
0
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were 8.49 ± 0.72, 8.89 ± 0.42 and 8.69 ± 0.78 %ID/g at 30, 60 and 120 min post-injection,
1
8
while [ F]8 were 8.71 ± 0.81, 9.03 ± 1.21, 8.41 ± 0.93 %ID/g. Comparable tumor uptake
1
8
values were also observed for 2-[ F]FPA (tumor uptake was 8.61 ± 0.33, 8.52 ±0.51 and
1
8
7.95 ± 0.37 %ID/g at 30, 60 and 120 min post-injection. All three agents, [ F]5 and
1
8
18
[ F]8 and 2-[ F]FPA showed similar uptake in all major organs and the uptakes at 30,
60 and 120 min were remarkably similar. Lipophilicity (Table 1) appeared not to play a
1
8
1
1
role in tumor uptake. Since in vitro studies in the presence of blood showed that [ F]5
18
18
and [ F]8 decomposed to 2-[ F]FPA vis esterase hydrolysis, similar results of
12
biodistribution for these three agents suggested that the biodistribution in vivo might be
1
8
18
13
14
15
predominantly determined by the 2-[ F]FPA. The tumor uptake for [ F]FMISO was
4.41 ± 0.83, 5.43 ± 0.62 and 3.24 ± 1.58 %ID/g at 30, 60 and 120 min, respectively, using
the same mice tumor model. Similar to the results above, at all three time points the
1
8
18
18
16
tumor uptakes showed no change. The tumor uptake for [ F]5 and [ F]8 and 2-[ F]FPA
1
1
7
8
showed noticeable higher tumor uptake (7.9 to 9.0 %ID/g) at all three time points than
18
18
those for [ F]FMISO (4.4 to 3.2 %ID/g) (P < 0.05); however, the blood levels for [ F]5
1
8
18
19
and [ F]8 and 2-[ F]FPA were also higher leading to lower tumor to blood ration when
1
8
18
2
0
compared to that of [ F]FMISO. Faster in vivo kinetics of [ F]FMISO also led to lower
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uptake in retention in all major organs. However, it is likely that the in vivo
1
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18
biodistribution of [ F]5 and [ F]8 was determined by a rapid in vivo hydrolysis to
1
8
possible metabolite, 2-[ F]FPA; therefore, the higher and selective hypoxic cell uptake
measured by in vitro cell uptake studies (Figure 2) were not observed in the in vivo
biodistribution study in EMT-6 mice model.
1
1
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Micro PET/CT imaging
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Using the same EMT-6 tumor-bearing mice micro PET images were acquired at
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30, 60, 90, 120 min post-injection of [ F]5, [ F]8, 2-[ F]FPA and [ F]FMISO,
respectively (Figure 3 and 4). PET images demonstrated that the EMT-6 tumors were
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clearly visualized using either [ F]5 or [ F]8, and the tumor uptakes for [ F]5, [ F]8
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and 2-[ F]FPA were very comparable at 120 min post-injection (Figure 4). While
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[ F]FMISO displayed a lower tumor uptake and less uptake in all other major organs.
This is similar to the results of biodistribution study measured by dissection method.
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Although results of higher tumor uptake in the PET images of [ F]5 and [ F]8,
the tumors looked very distinctive (Figure 4); however, the tumor uptake observed
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appeared to be associated with the metabolite, 2-[ F]FPA, which was likely a major
contributor in tumor uptake. Additional studies will be needed to see how much of the
original imidazole derivatives, if any, have contributed to the in vivo hypoxia tumor
uptake observed in the EMT-6 tumor model. The ester linkage may not be a useful linker
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Molecular Pharmaceutics
Page 28 of 50
New PET tracers for imaging hypoxia
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for 2-[ F]FPA in developing novel hypoxic targeting agents.
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It has been reported that 2-[ F]FPA accumulated in prostate cancers with high
tumor to background ratios, and it may be useful in the clinical diagnosis of prostate
cancer in humans. In general, 18F labeled fluoro-alkyl fatty acids54 displayed tumor
uptakes. The mechanism(s) of uptake and their relationship with hypoxia conditions has
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not been reported. Although, 2-[ F]FPA did not showed preference for in vitro tumor
cell uptake under hypoxia condition (Figure 2D). There is a hard lesson to be learned that
in vivo metabolism of these nitroimidazole ester derivatives, instead of the tumor hypoxia
condition, may be the driving force determining the outcome of in vivo biodistribution.
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Conclusions
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In summary, two new 2-fluoroproprionic acid esters of nitroimidazole,
2-fluoropropionic acid 2-(2-nitro-imidazol-1-yl) ethyl ester, 5 and 2-fluoropropionic acid
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2-(2-methyl-5-nitro-imidazol-1-yl) ethyl ester, 8 ([ F]5 and [ F]8) have been
synthesized and evaluated as specific PET tracers for hypoxia imaging. In vivo
biodistribution results suggested that they were more likely reflecting predominance of in
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vivo produced metabolite, 2-[ F]FPA, which formed after an iv injection.
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Page 29 of 50
Molecular Pharmaceutics
New PET tracers for imaging hypoxia
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Acknowledgment
This work was supported in part by grants from the National Key Research and
Development Program of China (2016YFC1306300) and Beijing Municipal Science &
Technology Commission (Z151100003915116).
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Supporting Information
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Supporting Information describing the HPLC profiles of [ F]FMISO, [ F]5 and
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[ F]8 for this paper is available in a separate file.
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