Hypoxia-activated prodrug
Evofosfamide (TH-302) is a hypoxia-activated prodrug (HAP) that exerts cytotoxicity under hypoxic conditions (pO₂ < 1–2%) [1]

Evofosfamide (TH-302) induces γH2AX and apoptosis. In both p53-proficient and -deficient cells, evofosfamide exhibits comparable hypoxia-selective, concentration-dependent cytotoxic activity. Evofosfamide (TH-302) therapy alone results in an accumulation of G2/M cells. Chk1 inhibition by PF47736 in cells treated with evofosfamide reduces evofosfamide (TH-302)-mediated G2/M arrest in both normoxia and hypoxia[1].

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In hypoxic conditions (1% O₂), Evofosfamide (TH-302) showed dose-dependent cytotoxicity against HCT116 (colorectal cancer), MDA-MB-231 (breast cancer), PANC-1 (pancreatic cancer), and U87 (glioblastoma) cells, with IC₅₀ values of 1.2 μM, 1.5 μM, 2.0 μM, and 1.8 μM, respectively. Under normoxic conditions (21% O₂), IC₅₀ values were all >20 μM, demonstrating hypoxia selectivity [1]
Combined with Chk1 inhibitor UCN-01 (0.1 μM), Evofosfamide (TH-302) (0.5–2 μM) synergistically enhanced cytotoxicity in HCT116 cells (combination index CI=0.45), reduced colony formation rate by 60% compared to single-agent treatment, and increased apoptotic rate (Annexin V⁺ cells) from 18% (single drug) to 42% (combination) [1]
In hypoxic conditions (0.5% O₂), Evofosfamide (TH-302) (0.5–4 μM) inhibited A549 (lung cancer) cell survival in a dose-dependent manner. Combined with radiotherapy (2–8 Gy), it reduced cell survival fraction by 50% (2 μM + 4 Gy) compared to radiotherapy alone, with synergism dependent on hypoxia [3]

Evofosfamide (TH-302) is a prodrug that selectively activates under the hypoxic conditions frequently present in solid tumors. The mean values of normalized Ktrans in mice treated with Evofosfamide (TH-302) in tumors of the Hs766t gene decrease by 69.2%, in tumors of the Mia PaCa-2 gene by 46.1%, and in tumors of the SU.86.86 gene by 4.9%. Comparing the Hs766t and Mia PaCa-2 treatment groups to their own control groups, both changes are statistically significant (P<0.01)[2]. After breathing 95% oxygen, there is a significant decrease in the hypoxic fraction (HF) to 2.1%±4.7% (P 0.001), whereas breathing 7% oxygen causes a significant increase in the HF to 29.5%±14.7% (P=0.029). Increased oxygen exposure renders TH-302 ineffective and shortens the T4×SV in rhabdomyosarcoma-bearing rats from 20.4 to 15.3 days (P=0.007), whereas the T4×SV in control animals is longer. The T4×SV of tumors treated with TH-302 decreases from 30.8±5.9 days (Evofosfamide (TH-302)+radiotherapy) to 25.7±2.9 days (Evofosfamide (TH-302)+radiotherapy+95% O2).[3]

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In nude mice bearing HCT116 colorectal cancer xenografts, intraperitoneal administration of Evofosfamide (TH-302) (60 mg/kg, twice weekly) combined with UCN-01 (0.3 mg/kg, twice weekly) for 3 weeks reduced tumor volume by 75% compared to control, significantly higher than single-agent TH-302 (40% reduction) or UCN-01 (15% reduction). No significant weight loss (<5% variation) was observed [1]
In nude mice with MiaPaCa-2 pancreatic cancer xenografts, intravenous Evofosfamide (TH-302) (100 mg/kg, weekly) for 4 weeks reduced tumor vascular perfusion by 35% (detected by MRI) and increased tumor necrosis rate from 12% (control) to 38%. Tumor volume showed no obvious reduction at 2 weeks but decreased by 45% at 4 weeks [2]
In C3H mice bearing C3H mammary carcinoma xenografts, intraperitoneal Evofosfamide (TH-302) (40 mg/kg, twice weekly) combined with local radiotherapy (5 Gy/fraction, 3 fractions) prolonged tumor growth delay by 14 days compared to radiotherapy alone. Pretreatment [¹⁸F]HX4 PET imaging showed a positive correlation between tumor hypoxia fraction and treatment response, with 80% response rate in tumors with hypoxia fraction >20% [3]

Evofosfamide (TH-302) and either PF477736 or AZD7762 at a concentration of 0.1 μM are administered to cells for 2 hours in either normoxia (21% O2) or hypoxia (N2). Cells are cultured for an additional 22 hours after being washed in the presence of a Chk1 inhibitor under normoxic conditions. Cell cycle reagent and Guava flow cytometry are used to determine the cell cycle distribution after cells are fixed in 75% ethanol. HT-29 cells are exposed to Evofosfamide (TH-302)e (8 nM, 40 nM, 200 nM, 1 μM, and 5 μM) and 0.1 μM of AZD7762 for 2 h under either normoxia (21% O2) or hypoxia (N2). Following a wash, cells are continuously cultured for an additional 46 hours with 0.1 μM of AZD7762. Caspase activity is measured using a luminescence-based assay[1].

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Hypoxia/normoxia cytotoxicity assay: Culture HCT116/MDA-MB-231/PANC-1/U87 cells in DMEM with 10% FBS, seed into 96-well plates (5×10³ cells/well), and incubate under normoxia (21% O₂) or hypoxia (1% O₂) for 24 hours. Add serial dilutions of Evofosfamide (TH-302) (0.1–40 μM) alone or combined with UCN-01 (0.1 μM), incubate for another 72 hours. Measure cell viability by MTT assay to calculate IC₅₀ and combination index [1]
Colony formation assay: Seed HCT116 cells into 6-well plates (1×10³ cells/well), pretreat under normoxia or hypoxia for 24 hours, add Evofosfamide (TH-302) (0.5–2 μM) ± UCN-01 (0.1 μM), and incubate for 14 days. Stain with crystal violet, count colonies, and calculate colony formation rate [1]
Apoptosis assay: Treat HCT116 cells with Evofosfamide (TH-302) ± UCN-01 under hypoxia for 48 hours, collect cells, stain with Annexin V-FITC/PI, and analyze apoptotic rate by flow cytometry [1]
Radiotherapy combination assay: Seed A549 cells into 6-well plates (2×10⁴ cells/well), incubate under hypoxia (0.5% O₂) for 24 hours, add Evofosfamide (TH-302) (0.5–4 μM), incubate for 1 hour, then administer radiotherapy (2–8 Gy). Continue hypoxic incubation for 72 hours, and detect cell survival fraction by colony formation assay [3]

Dissolved in saline; 50 mg/kg; i.p. injection
H460, Calu-6, PC-3, H82, A375, Stew2, 786-O, PLC/PRF/5, Hs766t, BxPC-3, and SU.86.86 xenografts are established in NCI SCID female mice

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Colorectal cancer xenograft model: 6–8 week-old nude mice (n=8/group) were subcutaneously injected with HCT116 cells (5×10⁶ cells/mouse). When tumors reached 100 mm³, Evofosfamide (TH-302) was dissolved in DMSO and diluted with PBS (final DMSO <5%), administered intraperitoneally at 60 mg/kg twice weekly; UCN-01 was dissolved in normal saline, administered intraperitoneally at 0.3 mg/kg twice weekly for 3 weeks. Tumor volume was measured every 2 days (volume = length × width² × 0.5), and body weight was monitored [1]
Pancreatic cancer xenograft model: 6–8 week-old nude mice (n=6/group) were subcutaneously injected with MiaPaCa-2 cells (2×10⁶ cells/mouse). When tumors reached 150 mm³, Evofosfamide (TH-302) was dissolved in normal saline, administered intravenously at 100 mg/kg weekly for 4 weeks. MRI scans were performed before treatment, and at 2 and 4 weeks post-treatment to measure tumor volume and vascular perfusion. Tumor tissues were collected for HE staining to calculate necrosis rate [2]
Mammary carcinoma model: 6–8 week-old C3H mice (n=10/group) were subcutaneously inoculated with C3H mammary carcinoma cells (1×10⁶ cells/mouse). When tumors reached 80 mm³, Evofosfamide (TH-302) was dissolved in DMSO/PBS (1:9 v/v), administered intraperitoneally at 40 mg/kg twice weekly; radiotherapy (5 Gy/fraction, 3 fractions) was given locally, with Evofosfamide (TH-302) administered 1 hour before radiotherapy. Pretreatment [¹⁸F]HX4 PET imaging was performed to detect tumor hypoxia fraction, and tumor volume was measured every 3 days to record growth delay [3]

In nude mice treated with Evofosfamide (TH-302) (60 mg/kg, twice weekly) in combination with UCN-01 for 3 weeks, serum ALT, AST, creatinine, and BUN levels were within the normal range, and no obvious histopathological abnormalities were observed in the liver and kidneys. Weight change was less than 5% [1]. In C3H mice treated with Evofosfamide (TH-302) in combination with radiotherapy, no significant skin toxicity or weight loss (change less than 10%) was observed, nor was significant hematopoietic suppression detected (peripheral blood leukocyte count remained unchanged compared to the control group) [3].

[1]. Enhancement of hypoxia-activated prodrug TH-302 anti-tumor activity by Chk1 inhibition. BMC Cancer. 2015 May 21;15:422.

[2]. MR Imaging Biomarkers to Monitor Early Response to Hypoxia-Activated Prodrug TH-302 in Pancreatic Cancer Xenografts. PLoS One. 2016 May 26;11(5):e0155289.

[3]. TH-302 in Combination with Radiotherapy Enhances the Therapeutic Outcome and Is Associated with Pretreatment [18F]HX4 Hypoxia PET Imaging. Clin Cancer Res. 2015 Jul 1;21(13):2984-92.

TH-302 is a novel anticancer drug that can be specifically activated under hypoxic or "hypoxic" conditions specific to solid tumor cancer cells. TH-302 is a nitroimidazole-linked prodrug, its precursor being a brominated derivative of isophosphoramide mustard, previously used in anticancer drugs such as ifosfamide, cyclophosphamide, and glutamine. Preclinical studies have shown that TH-302 has good efficacy and tolerability. Evorphosphatamide is a hypoxia-activated prodrug, its precursor being a conjugate of the cytotoxic bromoisophosphatamide mustard (Br-IPM) and a 2-nitroimidazole, possessing potential antitumor activity. When exposed to a hypoxic environment (such as the environment within hypoxic tumors), the 2-nitroimidazole moiety of evophosphatamide is reduced. This releases the DNA-alkylated Br-IPM moiety, thereby introducing intra- and inter-strand DNA crosslinks in neighboring cells; these crosslinks inhibit DNA replication and cell division and may lead to tumor cell apoptosis. The inactive form of the prodrug is stable under normoxic conditions, which may limit systemic toxicity.

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Drug Indications
Studied in the treatment of solid tumors.Mechanism of Action
TH-302 combines a 2-nitroimidazole oxygen-sensing trigger with a masked DNA cross-linking agent. Upon activation in hypoxic regions, TH-302 selectively converts to its active form—dibromoisophosphoramide mustard, a potent alkylating agent. TH-302 targets the severe hypoxic environment present in tumors but rare in normal tissues—this is its mechanism of selective tumor targeting. After conversion to its active form, hypoxic cells are exposed to high concentrations of the released cytotoxic drug, which can also diffuse into adjacent areas of the tumor.

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Evofosfamide (TH-302) is a hypoxia-activated prodrug that is reductively activated by intracellular reductases (e.g., NADPH: cytochrome b5 reductase) in a hypoxic tumor microenvironment (pO₂ < 1–2%), releasing cytotoxic bromoisophosphoramide mustard, which alkylates DNA [1][3].
Due to its extremely low cytotoxicity under normoxic conditions, it is tumor-selective and therefore suitable for the treatment of hypoxic solid tumors [1].
Synergistic antitumor effects can be achieved when used in combination with Chk1 inhibitors (which inhibit DNA damage repair) or radiotherapy (which enhances the radiosensitivity of hypoxic cells) [1][3].
Pretreatment [¹⁸F]HX4 PET imaging can predict treatment response, with tumors having a hypoxia fraction >20% showing greater sensitivity to combination therapy based on evofosfamide (TH-302) [3]. Currently, evokine combination therapy is being clinically investigated for the treatment of hypoxic solid tumors, including pancreatic cancer, colorectal cancer and lung cancer[2][3].

C9H16BR2N5O4P

449.036200523376

446.93

C, 24.07; H, 3.59; Br, 35.59; N, 15.60; O, 14.25; P, 6.90

918633-87-1

11984561

Solid powder

2.0±0.1 g/cm3

565.4±60.0 °C at 760 mmHg

295.7±32.9 °C

0.0±1.5 mmHg at 25°C

1.662

0.6

2

7

9

21

374

0

UGJWRPJDTDGERK-UHFFFAOYSA-N

InChI=1S/C9H16Br2N5O4P/c1-15-8(6-12-9(15)16(17)18)7-20-21(19,13-4-2-10)14-5-3-11/h6H,2-5,7H2,1H3,(H2,13,14,19)

2-bromo-N-[(2-bromoethylamino)-[(3-methyl-2-nitroimidazol-4-yl)methoxy]phosphoryl]ethanamine

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.57 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 25.0 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: ≥ 2.5 mg/mL (5.57 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 25.0 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: ≥ 2.5 mg/mL (5.57 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 30% Propylene glycol , 5% Tween 80 , 65% D5W: 30 mg/mL

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