Evofosfamide (TH-302) is potent and selective that has an IC50 of 19 nM. It exhibits 270-fold increased cytotoxicity under hypoxia compared to their potency under aerobic conditions and is stable to cytochrome P450 metabolism. Under hypoxia, TH-302 is selectively potent and liver microsomes tolerate it well. With a high hypoxic selectivity [Hypoxia cytotoxicity ratio (HCR) = 270], replacing the chlorine with bromine in the phosphorus mustard in 3b results in a 10-fold increase in potency. Under N2, TH-302 exhibits strong cytotoxicity against human lung cancer H460 cells and human colon cancer HT29 cells. With an IC90 of 0.1 μM and 0.2 μM, respectively, TH-302 inhibits HT29 cells and H460 cells.

Physicochemical Properties

Molecular Formula	C9H16BR2N5O4P
Molecular Weight	449.036200523376
Exact Mass	446.93
Elemental Analysis	C, 24.07; H, 3.59; Br, 35.59; N, 15.60; O, 14.25; P, 6.90
CAS #	918633-87-1
Related CAS #	918633-87-1
PubChem CID	11984561
Appearance	Solid powder
LogP	0.6
Hydrogen Bond Donor Count	2
Hydrogen Bond Acceptor Count	7
Rotatable Bond Count	9
Heavy Atom Count	21
Complexity	374
Defined Atom Stereocenter Count	0
InChi Key	UGJWRPJDTDGERK-UHFFFAOYSA-N
InChi Code	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)
Chemical Name	2-bromo-N-[(2-bromoethylamino)-[(3-methyl-2-nitroimidazol-4-yl)methoxy]phosphoryl]ethanamine
Synonyms	Evofosfamide; TH302; TH 302; TH-302
HS Tariff Code	2934.99.9001
Storage	Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month
Shipping Condition	Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Biological Activity

Targets	Hypoxia-activated prodrug
ln Vitro	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].
ln Vivo	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]
Cell Assay	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].
Animal Protocol	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
References	[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.
Additional Infomation	TH-302 is a novel cancer therapeutic specifically activated under the low oxygen or "hypoxic" conditions typical of solid tumor cancer cells. TH-302 is a nitroimidazole-linked prodrug of a brominated derivative of an isophosphoramide mustard previously used in cancer drugs such as ifosfamide, cyclophosphamide, and glufosfamide. TH-302 has been shown, in preclinical studies, to be both efficacious and well tolerated. Evofosfamide is a hypoxia-activated prodrug of the cytotoxin bromo-isophosphoramide mustard (Br-IPM) conjugated with 2-nitroimidazole, with potential antineoplastic activity. When exposed to hypoxic conditions, such as those found in hypoxic tumors, the 2-nitroimidazole moiety of evofosfamide is reduced. This releases the DNA-alkylating Br-IPM moiety, which introduces intra- and inter-strand DNA crosslinks in nearby cells; the crosslinks inhibit both DNA replication and cell division, and may lead to apoptosis of cells in the tumor. The inactive form of the prodrug is stable under normoxic conditions, which may limit systemic toxicity. Drug Indication Investigated for use/treatment in solid tumors. Mechanism of Action TH-302 combines a 2-nitroimidazole oxygen-sensing trigger with a masked DNA crosslinker. Upon activation in oxygen deficient zones, TH-302 is converted selectively to the drug's active form, dibromo isophosphoramide mustard, a potent alkylator. TH-302 targets levels of severe hypoxia that are found in tumors but are rare in normal tissues - this is how selective targeting of the tumor occurs. After conversion to the active form of the drug, the hypoxic cells are exposed to high concentrations of released cytotoxic agent, which can also diffuse into the adjacent regions of the tumor.

Solubility Data

Solubility (In Vitro)

DMSO:90 mg/mL (200.4 mM) Water:10 mg/mL (22.3 mM) Ethanol:90 mg/mL (200.4 mM)

Solubility (In Vivo)

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. 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. 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. Solubility in Formulation 4: 30% Propylene glycol , 5% Tween 80 , 65% D5W: 30 mg/mL  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	2.2270 mL	11.1349 mL	22.2697 mL
	5 mM	0.4454 mL	2.2270 mL	4.4539 mL
	10 mM	0.2227 mL	1.1135 mL	2.2270 mL

*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.