- EF5 is not a drug with a conventional pharmacological target. It is a substrate for one-electron reductases under hypoxia, primarily NADPH:cytochrome P450 oxidoreductase (CYPOR). The document does not provide IC50, Ki, or EC50 values for EF5 against specific enzymes. [1]
- EF5 binds covalently to cellular macromolecules (proteins, thiols) following hypoxia-dependent bioreduction, forming adducts recognized by the ELK3-51 monoclonal antibody. [2]

- CYPOR Substrate: In SiHa and HCT116 human tumor cell lines, overexpression of CYPOR induced a 2- to 4-fold increase in hypoxic [¹⁴C]-EF5 covalent binding compared to parental lines. Aerobic binding was minimal. [1]
- Correlation with Bioreductive Prodrug Metabolism: Across a panel of 14 human tumor cell lines, one-electron reduction (hypoxic minus aerobic) of EF5 showed a strong correlation with metabolic reduction of the hypoxia-activated prodrugs tirapazamine (TPZ) and CEN-209 (SN30000). For CEN-209, R² = 0.64 (P = 0.0005); for TPZ, R² = 0.65 (P = 0.0005). EF5 binding was a better predictor of prodrug reduction than CYPOR activity alone. [1]
- Oxygen-Dependent Binding: Binding of [¹⁴C]-EF5 to cells is highly dependent on oxygen concentration, with negligible binding under aerobic conditions and increasing binding under hypoxia. The rate of binding is inversely proportional to pO₂. [1][2]

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Overexpression of CYPOR in SiHa and HCT116 cell lines resulted in a 2- to 4-fold increase in EF-5 binding, decreased metabolism of tirapazamine and CEN-209, and increased production of γH2AX. In 14 hypoxic tumor cell lines, reduced EF-5 binding and prodrug metabolism are significantly linked [1].

- Tumor Hypoxia Imaging (9L Glioma Model): In rats bearing epigastric 9L gliomas, intravenous injection of EF5 (100 µmol/kg, approx. 60 mg/kg) resulted in heterogeneous binding within the tumor. Fluorescence microscopy of frozen sections stained with Cy-3-conjugated ELK3-51 antibody revealed patterns consistent with chronic hypoxia (diffusion-limited, ~100-250 µm from vessels) and acute hypoxia (larger, homogeneous regions). Photometric analysis showed up to a 17-fold contrast between bright (hypoxic) and dim (oxic) regions within a single tumor section. [2]
- Correlation with Radiation Resistance: In the same 9L model, tumors with high EF5 binding showed significant radiation resistance (oxygen enhancement ratio of 2.9 for 1% survival). The in situ radiation response of air-breathing rats was more resistant than that of euthanized rats (which have no oxygenated blood flow), and this resistance correlated with EF5-positive hypoxic cells. [2]
- Correlation with Reductase Overexpression (HCT116 Xenografts): In HCT116 tumors with CYPOR overexpression, EF5 binding (mean fluorescence intensity of EF5-positive cells) was significantly higher than in wild-type tumors, even though the hypoxic fraction (percentage of EF5-positive cells) was similar. Breathing 10% oxygen increased EF5 binding, while hyperbaric oxygen (HBO, 100% O₂ at 2.25 atm) decreased it. [1]
- Correlation with CEN-209 DNA Damage: In HCT116 xenografts, EF5 binding (EF5-positive cells) showed a strong correlation with γH2AX induction (a marker of DNA damage) by the hypoxia-activated prodrug CEN-209 (R² = 0.68, P < 0.0001) at the individual tumor level. CEN-209-induced γH2AX was selectively increased in EF5-positive cells. [1]

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A potential stratification biomarker for benzotriazine-N-oxide bioreducible prodrugs is EF-5 binding. There is a strong correlation between CEN209-induced DNA damage at the individual tumor level and CYPOR overexpression, which also significantly increased EF-5 binding and decreased CEN-209 in HCT116 xenografts. Additionally, altering tumor hypoxia led to similar changes in bioreductive activation of both drugs, resulting in EF-5 binding. After intravenous injection of EF-5, monoclonal antibodies specifically bind to and identify 9L gliomas; this process is oxygen-dependent. On frozen tissue, binding can be identified by fluorescence microscopy. For light microscopic examination, tissue sections can be counterstained with hematoxylin and eosin. As an alternative, flow cytometry methods can be used to analyze individual tumor cells in order to infer the distribution of hypoxia inside tumors [2].

CYPOR enzyme activity was measured in S9 fractions of cell lysates using a cyanide-resistant, NADPH-dependent cytochrome c reduction assay (absorbance at 550 nm). [1]

- [¹⁴C]-EF5 Covalent Binding Assay (96-well plate): Cells (10⁵ per well) were incubated with [¹⁴C]-EF5 (20-60 µM) under oxic (air/5% CO₂) or hypoxic (90% N₂/5% H₂/5% CO₂) conditions for 3-5 hours at 37°C. After incubation, medium was removed, cells were trypsinized, and proteins were precipitated with cold 15% trichloroacetic acid (TCA). Precipitates were collected onto glass fiber filters using a Harvester 96 system, washed with 1% TCA, and radioactivity was measured by liquid scintillation counting. [1]
- Flow Cytometric Analysis of EF5 Binding: Cells or dissociated tumor tissues were fixed in 70% ethanol, stained with a Cy5-conjugated mouse monoclonal antibody (ELK3-51) against EF5 adducts (75 µg/mL, overnight at 4°C), and counterstained with DAPI. For dual staining, cells were also stained with mouse monoclonal anti-γH2AX antibody (1:4000) followed by Alexa-488 goat anti-mouse IgG. Analysis was performed on a BD LSR II flow cytometer. [1]
- Hypoxia-Modulated Irradiation Studies: 9L tumor cells were dissociated and irradiated as single-cell suspensions (in air) or as solid tumors (in situ) in air-breathing or euthanized rats. Clonogenic survival was assessed via plating efficiency assay (cells plated with 50,000 irradiated feeder cells, incubated 10-12 days, colonies counted). [2]

- Rat 9L Glioma Model: 9L tumors were grown as tissue-isolated implants on the epigastric artery and vein in rats. EF5 was administered as a single intravenous injection (100 µmol/kg body weight, prepared in 0.9% saline, volume = 1% of body weight). Three hours post-injection, tumors were excised, rapidly cooled, and half was frozen for histology, half processed for flow cytometry and plating efficiency. For radiation studies, tumors were irradiated with 0-30 Gy (orthovoltage X-ray, 225 kVp, dose rate 4.0 Gy/min) in air-breathing or euthanized rats. [2]
- Mouse HCT116 Xenograft Model: Nude mice bearing HCT116 (wild-type or CYPOR-overexpressing) xenografts were dosed with EF5 (60 mg/kg, i.p.) with or without CEN-209 (200 mg/kg, i.p.). Mice were placed in ventilated boxes breathing air, 10% O₂, or hyperbaric oxygen (100% O₂ at 2.25 atm) for 90-120 minutes. Tumors and liver were excised and frozen for LC/MS-MS analysis, or dissociated for flow cytometry and clonogenic assay. [1]

- Serum Half-Life: The serum half-life of EF5 in rats is approximately 150 minutes, requiring rapid cooling of excised tissues to prevent post-excision binding. [2]
- Biodistribution (Mice): Whole-body distribution of EF5 (using ¹⁴C-labeled EF5) at 0.5 hours post-injection was very uniform (as noted for mice; rat data not given). [2]
- Dose: Typical intravenous or intraperitoneal dose for in vivo studies is 60 mg/kg (approximately 100 µmol/kg). [1][2]

- The provided documents do not describe acute or chronic toxicity of EF5 (e.g., LD50, organ toxicity). No significant adverse effects were reported at the administered doses (60 mg/kg, i.p. or i.v.) in mice or rats. [1][2]

[1]. The 2-nitroimidazole EF5 is a biomarker for oxidoreductases that activate the bioreductive prodrug CEN-209 under hypoxia. Clin Cancer Res. 2012 Mar 15;18(6):1684-95.
[2]. Identification of hypoxia in cells and tissues of epigastric 9L rat glioma using EF5 [2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl) acetamide]. Br J Cancer. 1995 Oct;72(4):875-82

- Mechanism of Action (as a Hypoxia Probe): Under normoxia, the nitro group of EF5 is rapidly reoxidized, preventing covalent binding. Under hypoxia, one-electron reduction generates a reactive radical intermediate (nitro radical anion) that undergoes further reduction to form amine or hydroxylamine derivatives, which then covalently bind to thiols and other cellular nucleophiles. This binding is irreversible and proportional to the degree of hypoxia. [1][2]
- Detection Methods: EF5 adducts can be detected with the ELK3-51 monoclonal antibody (specific for EF5) conjugated to fluorochromes (e.g., Cy3, Cy5) for flow cytometry and fluorescence microscopy, or by PET imaging when EF5 is labeled with ¹⁸F ([¹⁸F]-EF5). [1][2]
- Dual Reporter Function: EF5 binding reflects both hypoxia and the expression of one-electron reductases (e.g., CYPOR) that activate bioreductive prodrugs. This makes EF5 a potential companion diagnostic for hypoxia-activated prodrugs like CEN-209 (SN30000) and tirapazamine. [1]
- Structural Features: The pentafluorinated propyl group on EF5 enhances its lipophilicity and improves its ability to penetrate tissues. It also allows for spectroscopic distinction from other 2-nitroimidazoles. [1][2]
- Correlation with Prognosis: In head and neck cancer patients, EF5 binding (by immunostaining) has been shown to correlate with outcome after radiotherapy. [1]

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EF5 is a fluorinated derivative of 2-nitroimidazole etonitrile. Under hypoxic conditions, EF5 can effectively increase oxygen levels in tumor tissues by forming adducts with intracellular macromolecules. Various enzymes in the cytoplasm, microsomes, and mitochondria can reduce this substance. EF5 has been reported to be used to detect tissue hypoxia in various cancers, including cervical squamous cell carcinoma, head and neck squamous cell carcinoma, and sarcoma.

C8H7N4O3F5

302.15818

302.044

C, 31.80; H, 2.34; F, 31.44; N, 18.54; O, 15.88

152721-37-4

389053

White to off-white solid powder

2.468

1

9

4

20

383

0

O=C(NCC(F)(F)C(F)(F)F)CN1C=CN=C1[N+]([O-])=O

JGGDSDPOPRWSCX-UHFFFAOYSA-N

InChI=1S/C8H7F5N4O3/c9-7(10,8(11,12)13)4-15-5(18)3-16-2-1-14-6(16)17(19)20/h1-2H,3-4H2,(H,15,18)

2-(2-nitroimidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)acetamide

EF-5; 383HJ2T87O; EF5; 2-(2-nitro-1h-imidazol-1-yl)-n-(2,2,3,3,3-pentafluoropropyl)acetamide; RefChem:909166; 152721-37-4;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~125 mg/mL (~413.69 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (6.88 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (6.88 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (6.88 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)