Tretazicar (CB-1954; CB1954), a dinitrobenzamide analog and prodrug, is a potent and highly selective DNA alkylating agent that generates highly cytotoxic interstrand crosslinks in DNA.It is transformed, in the presence of the co-substrate caricotamide (EP-0152R) (EP) and the enzyme NQO2, into a strong cytotoxic bifunctional DNA-alkylating agent. CB1954 has been suggested for use with the Escherichia coli enzyme nitroreductase (Ntr) in enzyme-prodrug gene therapy systems. After CB1954 is converted by Ntr to 2- and 4-hydroxylamino analogues, the 4-hydroxylamino analogue reacts non-enzymatically with cellular thio-esters to produce a strong cytotoxic bifunctional alkylating agent that can cross-link DNA.

Physicochemical Properties

Molecular Formula	C9H8N4O5
Molecular Weight	252.1836
Exact Mass	252.049
Elemental Analysis	C, 42.86; H, 3.20; N, 22.22; O, 31.72
CAS #	21919-05-1
PubChem CID	89105
Appearance	White solid powder
Density	1.7±0.1 g/cm3
Boiling Point	427.2±45.0 °C at 760 mmHg
Melting Point	173 °C
Flash Point	212.2±28.7 °C
Vapour Pressure	0.0±1.0 mmHg at 25°C
Index of Refraction	1.715
LogP	1.28
Hydrogen Bond Donor Count	1
Hydrogen Bond Acceptor Count	6
Rotatable Bond Count	2
Heavy Atom Count	18
Complexity	385
Defined Atom Stereocenter Count	0
SMILES	[O-][N+](C1C([H])=C(C(C(N([H])[H])=O)=C([H])C=1N1C([H])([H])C1([H])[H])[N+](=O)[O-])=O
InChi Key	WOCXQMCIOTUMJV-UHFFFAOYSA-N
InChi Code	InChI=1S/C9H8N4O5/c10-9(14)5-3-7(11-1-2-11)8(13(17)18)4-6(5)12(15)16/h3-4H,1-2H2,(H2,10,14)
Chemical Name	5-(aziridin-1-yl)-2,4-dinitrobenzamide
Synonyms	CB-1954; CB1954; CB 1954
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

ln Vitro	The monofunctional alkylating agent CB1954 can be transformed into a toxic form by overexpressing nitroreductase oxidored nitro domain containing protein 1 (NOR1), which reduces the potent cytotoxin CB1954's 4 nitro group. When used on the NPC cell line CNE1, toxic CB1954 increases cell death. Grb2 expression is upregulated and MAPK signal transduction is activated in the HepG2 cell line by the NOR1 gene, which increases CB1954-mediated cell cytotoxicity [1].
ln Vivo	In vivo, the NTR/CB1954 system is used to specifically ablate cells. This inducible ablation system has a dose-dependent effect[3]. Cell proliferation is not necessary for CB1954-mediated NTR-mediated cell killing. DNA cross-linking caused by the activated CB1954 likely starts the apoptosis cascade and swiftly kills off cells. Functional p53 is not necessary for NTR-CB1954 to kill cells in a targeted and efficient manner[2].
Cell Assay	The human hepatocellular carcinoma cells, HepG2, are kept in a humidified culture incubator at 37˚C with 5% CO2 and 95% air, supplemented with 10% fetal calf serum (FCS). Cell cytotoxicity tests are carried out in accordance with earlier guidelines. After reaching approximately 80% confluence, HepG2 cells are treated with CB1954 or a signal transduction inhibitor after being rinsed with PBS. In each experiment, measurements are taken from ten to twelve different microscopic fields, and data are compiled from three to five experiments.
Animal Protocol	RED 40 female mice expressing high levels of BLG-NTR transgene in the mammary gland and nontransgenic control mice on lactation day 6 50 mg/kg i.p.
References	[1]. Oncol Lett . 2012 Sep;4(3):566-570. [2]. Gene Ther . 1999 May;6(5):764-70. [3]. J Endocrinol . 2002 Nov;175(2):487-98.
Additional Infomation	Tretazicar is under investigation in clinical trial NCT00746590 (Study of Anti-tumour Effects and Safety of Prolarix™ in Hepatocellular Carcinoma). Tretazicar is a prodrug of a bifunctional alkylating, dinitrobenzamide derivative with antineoplastic activity. Tretazicar can be activated by the human enzyme quinone oxidoreductase 2 (NQO2) in the presence of the cosubstrate caricotamide, an analogue of the natural cosubstrate dihydronicotinamide riboside (NRH), which acts as an electron donor. The resulting active, but short-lived metabolite, dinitrobenzamide, leads to DNA replication inhibition and the induction of apoptosis in NQO2 expressing cancer cells. Due to the lack of the natural cosubstrate NRH, NQO2 expression is normally latent but is upregulated in certain types of tumor cells.

Solubility Data

Solubility (In Vitro)

DMSO: 50~125 mg/mL (198.3~495.7 mM)

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.08 mg/mL (8.25 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. Solubility in Formulation 2: ≥ 2.08 mg/mL (8.25 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.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	3.9654 mL	19.8271 mL	39.6542 mL
	5 mM	0.7931 mL	3.9654 mL	7.9308 mL
	10 mM	0.3965 mL	1.9827 mL	3.9654 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.