Didox (also known as NSC-324360) is a novel, potent and synthetic ribonucleotide reductase (RR) inhibitor. It was first created as an anti-cancer agent and is derived from polyhydroxy-substituted benzohydroxamic acid. Didox may have anti-inflammatory and anti-oxidative stress-like effects, according to certain research, while other studies suggest the opposite.

Physicochemical Properties

Molecular Formula	C7H7NO4
Molecular Weight	169.1348
Exact Mass	169.037
Elemental Analysis	C, 49.71; H, 4.17; N, 8.28; O, 37.84
CAS #	69839-83-4
Appearance	Light yellow to khaki solid powder
Density	1.6±0.1 g/cm3
Index of Refraction	1.677
LogP	-0.32
SMILES	O([H])C1=C(C([H])=C([H])C(C(N([H])O[H])=O)=C1[H])O[H]
InChi Key	QJMCKEPOKRERLN-UHFFFAOYSA-N
InChi Code	InChI=1S/C7H7NO4/c9-5-2-1-4(3-6(5)10)7(11)8-12/h1-3,9-10,12H,(H,8,11)
Chemical Name	N,3,4-trihydroxybenzamide
Synonyms	NSC-324360; NSC 324360; NSC324360; Didox
HS Tariff Code	2934.99.03.00
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	Ribonucleotide reductase
ln Vitro	Didox (NSC-324360) reduces the amounts of mRNA that LPS-induced iNOS, IL-6, IL-1, TNF-α, NF-κβ (p65), and p38-α, after 24 h of treatment. Nitric oxide (NO), IL-6, and IL-10 secretion are also inhibited by Didox treatment. The effects of Didox on cellular respiration in RAW264.7 are investigated over a range of concentrations for 24 hours using mitochondrial dehydrogenase activity as a measure of cytotoxicity. Significant cellular toxicity is not observed in cells exposed to Didox at concentrations below 200 μM, either in the presence or absence of LPS [1]. With mean IC50 values in the low micromolar range (mean IC50 37 µM [range 25.89-52.70 µM]), Didox (NSC-324360) exhibits activity against all human and murine acute myeloid leukemia (AML) lines tested[2].
ln Vivo	The animals are administered 425 mg/kg of Didox intraperitoneally (IP) every day for five days, following the establishment of engraftment as determined by bioluminescent imaging. Treatment with didox (NSC-324360) diminishes leukemic burden significantly when compared to vehicle-treated controls (p=0.0026 and p=0.0342). Didox (NSC-324360) offers a noteworthy survival advantage (p<0.0001 and p=0.0094)[2], which is more significant.
Cell Assay	Didox, 0.1 μg/mL LPS, or both are used to treat RAW264.7 macrophages. The MTT assay, which measures mitochondrial dehydrogenase activity, is used to quantify cellular respiration as a sign of cytotoxicity. 100 μL of DMEM medium is used to plating macrophages at 105 cells per well into 96-well Costar plates. The compounds and DMSO carrier control (0.01% final) are added in triplicate over serial dilutions starting with 200 μM per well in a total volume of 200 μL, and the plates are incubated for 24 hours after 4 hours of incubation at 37°C for adherence. Each well is given 20 μL of a 5 mg/mL MTT solution in unsupplemented DMEM four hours prior to the assay's termination. Following centrifugation, the supernatant from each well is disposed of, and 100 μL of acidified isopropanol (containing 4 mM HCl and 0.1% NP-40) is used to solubilize the cells containing reduced MTT. The optical density (O.D.) of each well is measured at 550 nm after a brief period of shaking. Every experiment is run three times, with the data from each triplicate being averaged and then expressed as a percentage of the control O.D. values for every experiment [1].
Animal Protocol	Mice: Transplanted 1.0×106 leukemia cells per mouse, tagged with luciferase, are injected via tail vein into 8-week-old C57Bl/6 mice that have received sublethally irradiation (4.5 Gy). Isoflurane is used to induce unconsciousness in mice, and then they receive a 150 mg/kg D-luciferin injection before being imaged with the IVIS 100 imaging system. When a clear signal is detected, the mice start receiving treatment with Didox. For five days, intraperitoneal injection (IP) of Didox (NSC-324360) at a dose of 425 mg/kg is administered daily to the animals. Animals in the control group are given an intraperitoneal injection of 5% dextrose water. On the day after the last treatment, repeat imaging is carried out[2].
References	[1]. 3,4-Dihydroxy-benzohydroxamic acid (Didox) suppresses pro-inflammatory profiles and oxidative stress in TLR4-activated RAW264.7 murine macrophages. Chem Biol Interact. 2015 May 25;233:95-105. [2]. The efficacy of the ribonucleotide reductase inhibitor Didox in preclinical models of AML. PLoS One. 2014 Nov 17;9(11):e112619.

Solubility Data

Solubility (In Vitro)

DMSO: 34~100 mg/mL (201.0~591.3 mM) Water: ~34 mg/mL Ethanol: ~34 mg/mL

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.75 mg/mL (16.26 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 27.5 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: ≥ 2.75 mg/mL (16.26 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 27.5 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: ≥ 2.75 mg/mL (16.26 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 27.5 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	5.9126 mL	29.5631 mL	59.1261 mL
	5 mM	1.1825 mL	5.9126 mL	11.8252 mL
	10 mM	0.5913 mL	2.9563 mL	5.9126 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.