Ribonucleotide reductase
Ribonucleotide reductase (RR) inhibitor (through iron deprivation mechanism).
NF-κB (p65) activation inhibitor (inhibits nuclear translocation).
p38α (MAPK14) expression inhibitor.
iNOS, COX-2, IL-6, TNF-α expression inhibitor.
SOD1 and catalase expression enhancer. [1]

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].

---

Didox inhibits LPS-induced nitric oxide production in RAW264.7 macrophages, with significant inhibition starting at 6.25 μM and maximal inhibition at 100 μM.
Didox suppresses LPS-induced mRNA expression of iNOS, IL-6, IL-1β, TNF-α, NF-κB (p65), and p38α after 24 h treatment.
Didox reduces secretion of nitric oxide, IL-6, and IL-10 in LPS-stimulated macrophages.
Didox decreases intracellular ROS levels induced by LPS, PMA, and BSO.
Didox inhibits nuclear translocation of NF-κB (p65) in response to LPS stimulation.
Didox enhances mRNA expression of antioxidant genes SOD1 and catalase, especially in combination with LPS.
Didox reduces protein expression of iNOS and COX-2 induced by LPS.
Didox is not cytotoxic up to 200 μM in RAW264.7 macrophages, either alone or with LPS. [1]

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.

---

In syngeneic, therapy-resistant AML mouse models (expressing MLL-ENL with either NrasG12D or Flt3 ITD), daily intraperitoneal administration of Didox (425 mg/kg for 5 days) significantly reduced leukemic burden (as measured by bioluminescence imaging) and provided a significant survival benefit compared to vehicle-treated controls. [2]
Didox treatment at the effective dose (425 mg/kg for 5 days) was well tolerated in normal C57Bl/6 mice, with no morphological differences observed in bone marrow or gastrointestinal tissues upon blinded histopathological review. [2]
Bone marrow cells from Didox-treated normal donors engrafted at least as well as cells from control donors when transplanted into lethally irradiated syngeneic recipients, indicating no impairment of normal hematopoietic stem cell (HSC) function. [2]

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].

---

Cytotoxicity assay: RAW264.7 cells were plated in 96-well plates and treated with Didox (up to 200 μM) with or without LPS for 24 h. Mitochondrial dehydrogenase activity was measured using MTT assay to assess cell viability.
Nitric oxide production assay: Cells were treated with Didox and LPS for 24 h. Nitrite accumulation was measured using Griess reagent. A fluorescent probe (DAF-2 DA) was also used to detect NO radicals.
qRT-PCR array: Total RNA was extracted from macrophages treated with Didox and/or LPS for 24 h. cDNA was synthesized and used in a pre-designed Stress & Toxicity Profiler array for 96 genes.
qRT-PCR validation: RNA was extracted and cDNA synthesized from treated cells. Gene expression was analyzed using SYBR Green-based qPCR with primers for iNOS, IL-6, TNF-α, COX-2, p65, p38α, SOD1, and catalase.
Cytokine secretion assay: IL-6 and IL-10 levels in supernatants were measured using ELISA kits after 24 h treatment.
Western blotting: Cells were lysed and proteins separated by SDS-PAGE, transferred to nitrocellulose membranes, and probed with antibodies against iNOS, COX-2, and β-actin.
Intracellular ROS assay: Cells were treated with Didox, BSO, PMA, and/or LPS for 24 h, then incubated with dihydroethidium (DHE) to detect superoxide radicals by fluorescence.
NF-κB translocation assay: Cytosolic and nuclear proteins were extracted after 3 h treatment. p65 protein levels were detected by Western blot and immunofluorescence using anti-p65 antibody. [1]

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].

---

For in vivo efficacy studies in AML models, luciferase-tagged murine AML cells were injected via tail vein into sublethally irradiated (4.5 Gy) C57Bl/6 mice. Engraftment was monitored by bioluminescence imaging after injection of luciferin. Upon confirmation of engraftment, mice received daily intraperitoneal injections of Didox (425 mg/kg) dissolved in 5% dextrose water, or vehicle control (5% dextrose water), for 5 consecutive days. Tumor burden was monitored by repeat imaging, and survival was tracked. [2]
For toxicology studies, normal C57Bl/6 mice received the same dosing regimen (425 mg/kg Didox IP daily for 5 days). Seventy-two hours after the last dose, mice were sacrificed for tissue collection and histopathological examination. [2]
For bone marrow engraftment studies, normal donor mice (Ly5.2+) were treated with Didox or vehicle as above. Seventy-two hours post-treatment, bone marrow was harvested and transplanted via tail vein injection into lethally irradiated (8 Gy) recipient mice (Ly5.1+). Engraftment of donor-derived cells (Ly5.2+) in recipient bone marrow was assessed by flow cytometry three weeks post-transplant. [2]

A phase I clinical trial in patients with metastatic cancer determined the maximum tolerated dose (MTD) of Didox to be 6 g/m², with a peak plasma concentration of 300 μM. [2]

In normal C57Bl/6 mice, Didox was well tolerated at a dose of 425 mg/kg daily for 5 consecutive days, and blinded histopathological evaluation showed no significant tissue toxicity in the bone marrow, small intestine, or other organs. [2]
In vitro experiments showed that normal hematopoietic stem cells exposed to Didox (at a concentration of up to 200 μM for 24 hours) showed only a slight and statistically insignificant decrease in colony-forming ability. [2]
In vivo experiments showed that Didox treatment did not impair the long-term engraftment capacity of normal hematopoietic stem cells in competitive transplantation trials. [2]

[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.

Didox is a synthetic ribonucleotide reductase inhibitor derived from polyhydroxy-substituted benzo[a]hydroxyxamic acid and was initially developed as an anticancer drug. Didox inhibits ribonucleotide reductase (RR) by chelating iron (a cofactor of the R2 subunit of ribonucleotide reductase). Didox exhibits anti-inflammatory and antioxidant properties by inhibiting pro-inflammatory cytokines, iNOS, COX-2, and ROS, while enhancing the activity of antioxidant enzymes SOD1 and catalase. Didox may have the potential to treat acute and chronic inflammatory diseases as well as oxidative stress-related diseases. Studies have shown that Didox is better tolerated than conventional nonsteroidal anti-inflammatory drugs (NSAIDs) and has no gastrointestinal side effects. [1]

C7H7NO4

169.1348

169.037

C, 49.71; H, 4.17; N, 8.28; O, 37.84

69839-83-4

3045

Light yellow to khaki solid powder

1.6±0.1 g/cm3

1.677

-0.32

4

4

1

12

173

0

O([H])C1=C(C([H])=C([H])C(C(N([H])O[H])=O)=C1[H])O[H]

QJMCKEPOKRERLN-UHFFFAOYSA-N

InChI=1S/C7H7NO4/c9-5-2-1-4(3-6(5)10)7(11)8-12/h1-3,9-10,12H,(H,8,11)

N,3,4-trihydroxybenzamide

NSC-324360; NSC 324360; NSC324360; Didox

2934.99.03.00

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)

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

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.

---

View More

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.)