RRx-001 (RRx001; ABDNAZ) is a novel hypoxia-selective epigenetic modulator with potential radiosensitizing and anticancer activity. It binds to hemoglobin, inhibits glucose 6-phosphate dehydrogenase (G6PD) in human tumor cells, and promotes RBC-mediated redox reactions in hypoxic conditions. A novel, non-toxic epigenetic anticancer drug for a variety of tumor types, RRx-001 works by increasing the production of nitric oxide (NO) and inhibiting PPP. In several cell lines and tumor models, the novel, nonexplosive molecule RRx-001, which was derived from a class of solid rocket propellants, has shown promise as a novel cancer therapeutic agent.

Physicochemical Properties

Molecular Formula	C5H6BRN3O5
Molecular Weight	268.02
Exact Mass	266.949
Elemental Analysis	C, 22.41; H, 2.26; Br, 29.81; N, 15.68; O, 29.85
CAS #	925206-65-1
Related CAS #	925206-65-1
PubChem CID	15950826
Appearance	Solid powder
LogP	0.457
Hydrogen Bond Donor Count	0
Hydrogen Bond Acceptor Count	5
Rotatable Bond Count	1
Heavy Atom Count	14
Complexity	273
Defined Atom Stereocenter Count	0
SMILES	BrC([H])([H])C(N1C([H])([H])C(C1([H])[H])([N+](=O)[O-])[N+](=O)[O-])=O
InChi Key	JODKFOVZURLVTG-UHFFFAOYSA-N
InChi Code	InChI=1S/C5H6BrN3O5/c6-1-4(10)7-2-5(3-7,8(11)12)9(13)14/h1-3H2
Chemical Name	2-bromo-1-(3,3-dinitroazetidin-1-yl)ethanone
Synonyms	RRX-001; RRX 001; RRx001; ABDNAZ
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	G6PD; Nrf2-ARE
ln Vitro	Glucose 6 phosphate dehydrogenase (G6PD), a crucial enzyme in the pentose phosphate pathway, is in charge of ensuring that adequate levels of the main cellular reductant, NADPH, are kept in the body. This is how RRx-001 exerts its anti-proliferative effect, at least in part. In three distinct cancer cell lines, Hep G2, CACO-2, and HT-29, RRx-001 alters the activity of the glucose and G6PD enzymes. The concentration-dependent effects of RRx-001 resulted in G6PD inhibition and increased glucose consumption. By producing ROS/RNS, RRx-001 activates p53 and PARP-1. It exerts anticancer activity, at least in part, by interfering with 3 crucial metabolic demands of rapidly proliferating cells: bioenergetics, macromolecular biosynthesis, and control of cytosolic redox homeostasis in living cells[1]. HO-1 and NQO1—two of Nrf2's downstream enzymes—are activated and translocated to the nucleus in tumor cells as a result of RRx-001. Other than epigenetic changes, RRx-001 works through pleiotropic mechanisms that involve redox signaling and redox-induced dysregulation of numerous signal pathways, including Nrf2, p53, PARP cleavage, HIF1 alpha, and G6PD activity. Along with deregulating cancer cell energetics and metabolism, it also activates p53 and p21 in response to double-stranded DNA breaks. By producing ROS and RNS, the drug RRx-001 effectively activates the Nrf2-ARE signaling pathway[3].
ln Vivo	RRx-001 shows promise for short-term blood flow redistribution in tumors with a pericyte- and α-SMA-rich vasculature[2]. RRx-001 monotherapy is well tolerated and free of toxicities that would limit its dosage. It not only helps Nrf2 move into the nucleus, but it also increases endogenous Nrf2 expression in mouse SCC VII tumors[3].
Cell Assay	Cell growth and proliferation is assessed using the MTT proliferation assay kit. In a nutshell, 96-well plates with 5×103 cells in each well are used to treat or leave untreated cells. Up to 72 hours can pass during cell culture. Each well is given 10 μL of MTT reagent before being left to sit for 3 hours. Following incubation, the culture medium is taken out, 100 μL of Crystal Dissolving Solution is poured into each well, and the solution's absorbance at 570 nm is gauged. In triplicate, MTT assays are carried out on samples from the same conditions. Adding MTT solution (0.5 mg/mL), allowing cells to incubate for 4 hours at 37 degrees Celsius, and then measuring absorption at 540 nm with a microplate reader are the steps in short order.
Animal Protocol	5 mg/kg or 10 mg/kg; i.v. CB-17 SCID-mice
References	[1]. Discov Med . 2016 Apr;21(116):251-65. [2]. Clin Epigenetics . 2016 May 11:8:53. [1]. Oncotarget . 2015 Aug 28;6(25):21547-56.
Additional Infomation	RRx-001 has been used in trials studying the treatment of Lymphomas, Brain Metastases, Cholangiocarcinoma, Colorectal Neoplasms, and Malignant Solid Tumor, among others. Nibrozetone is a dinitroazetidine derivative with potential radiosensitizing activity. Upon administration, nibrozetone is able to dilate blood vessels, thereby increasing tumor blood flow and thus improving oxygenation to the tumor site. By increasing oxygen levels, these tumor cells may be more susceptible to radiation therapy. Tumor hypoxia is correlated with tumor aggressiveness, metastasis and resistance to radiotherapy.

Solubility Data

Solubility (In Vitro)

DMSO: ~53 mg/mL (~197.7 mM) Water: <1 mg/mL Ethanol: ~23 mg/mL ( ~85.8 mM)

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.5 mg/mL (9.33 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 (9.33 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 (9.33 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.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	3.7311 mL	18.6553 mL	37.3106 mL
	5 mM	0.7462 mL	3.7311 mL	7.4621 mL
	10 mM	0.3731 mL	1.8655 mL	3.7311 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.