NK-252 targets the Keap1-DC domain (Kelch/double-glycine repeat motif and C-terminal region) which contains the Nrf2-binding site. [1]

The Huh-7.5 cells treated with oltipraz (OPZ) or NK-252 exhibited dose-dependent activation of NAD (P) H quinone oxidoreductase 1 (NQO1)-ARE as evidenced by luciferase activity. Based on the fact that NK-252 exhibits a higher potency for this effect than OPZ, NK-252's EC2 value (concentration induced by two times above background) is 1.36 μM, while OPZ's is 20.8 μM, determined by linearly extrapolating values above and below the induction threshold. NK-252 may function in hepatocytes as a Nrf2 activator. It has been documented that prototypic Nrf2 activators, such as OPZ, shield microglia from the cytotoxicity caused by H2O2. Huh-7 cells were used to assess the antioxidant qualities of OPZ and NK-252 in relation to their ability to protect against H2O2-induced cytotoxicity. When OPZ or NK-252 are applied to cells, they enhance their resistance against H2O2-induced cytotoxicity in comparison to control cells [1].

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NK-252 activates the NQO1-ARE (antioxidant response element) in a dose-dependent manner in Huh-7.5 cells. The EC2 value (concentration for a 2-fold induction above background) for NK-252 is 1.36 μM, whereas OPZ has an EC2 value of 20.8 μM. The luciferase activity induced by NK-252 is almost completely inhibited by Nrf2 siRNA. [1]
NK-252 protects Huh-7 cells against H2O2-induced cytotoxicity. Cells treated with NK-252 (0.3–30 μM for 24 hours) show increased resistance to 1 mM H2O2-induced cytotoxicity compared to control cells, and the protective effect of NK-252 is considerably stronger than that of OPZ. [1]

When compared to CDAA control rats, animals on a choline-deficient L-amino acid-defining (CDAA) diet administered OPZ or NK-252 had lower fibrosis scores, with a median score of 3, indicating bridging fibrosis. When compared to rats fed a normal control diet (naïve), the liver fibrosis area in CDAA control rats was nearly 20 times larger (14.7 vs. 0.72%, respectively). by giving either NK-252 or OPZ (5.80% of NK-252_high, 6.20% of NK-252_low, and 4.97% of OPZ). Dose-dependent effects of NK-252 are observed on fibrosis area and score [1]. P388/S and P388/VCR mice showed no anticancer effect from NK-252 alone. When NK-252 was given orally in addition to Etoposide, the mice that were intraperitoneally injected with P388/S lived much longer when compared to when Etoposide was used alone. When compared to the comparable survival time of Etoposide alone, the combined treatment of NK-252 and Etoposide considerably increased the lifespan of mice intraperitoneally vaccinated with P388/VCR [2].

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In rats fed a choline-deficient L-amino acid-defined (CDAA) diet (a NASH model), NK-252 administered orally at 20 mg/kg or 60 mg/kg once daily for 9 weeks significantly attenuates the progression of histologic abnormalities, especially hepatic fibrosis. The median fibrosis score (modified Brunt’s method) in CDAA control rats is 4 (pseudolobule formation), while NK-252-treated rats show a median score of 3 (bridging fibrosis). The liver fibrosis area (Sirius red-positive area) is 14.7% in CDAA controls, reduced to 6.20% (low dose) and 4.97% (high dose) with NK-252. NK-252 also reduces the number of α-SMA-positive cells (indicating hepatic stellate cell activation) and decreases inflammatory cell infiltration. [1]
NK-252 (60 mg/kg once daily for 9 weeks) significantly reduces plasma ALT and AST levels in CDAA diet-fed rats compared to CDAA controls (ALT: 120.8 ± 3.2 vs. 170.7 ± 9.6; AST: 238.5 ± 9.6 vs. 344.4 ± 17.3). [1]
NK-252 upregulates NQO1 gene expression in the liver of CDAA diet-fed rats as measured by real-time quantitative PCR. It also reduces protein carbonyl content (a marker of protein oxidation) in the liver (from ~4.5 nmol/mg protein in CDAA controls to ~2.5–3.0 nmol/mg with NK-252). [1]
NK-252 downregulates fibrogenic gene expression (TGF-β1, collagen α1(I), TIMP-1) in the liver of CDAA diet-fed rats in a dose-dependent manner. [1]
In a delayed-administration regimen (NK-252 60 mg/kg once daily for 4 weeks starting after 6 weeks of CDAA diet feeding), NK-252 prevents further progression of established fibrosis: fibrosis score median is 3 (vs. 4 in CDAA control), and fibrosis area is 5.20% (vs. 14.7% in CDAA control). Infiltration of inflammatory cells is almost completely inhibited. [1]

Surface plasmon resonance (SPR) interaction analysis was performed to assess binding of NK-252 to the Keap1-DC domain. A recombinant GST-fused Keap1-DC protein (residues 321–609 of human Keap1) was immobilized onto a sensor chip via amine coupling. A reference flow cell with GST alone was used. NK-252 solutions at concentrations of 12.5, 25, 50, and 100 μM were injected for 60 seconds at a flow rate of 10 μl/min. Binding of NK-252 to Keap1-DC was detected as a dose-dependent increase in resonance units. In contrast, no binding was detected for oltipraz (OPZ) under the same conditions. [1]

For the luciferase reporter gene assay, Huh-7.5 cells stably transfected with an ARE (antioxidant response element)-luciferase reporter (three-tandem repeat of ARE from the NQO1 gene) were used. Cells were transfected with control siRNA or Nrf2 siRNA for 30 hours, then treated with NK-252 (0.3–30 μM) or DMSO (control) for 16 hours. Luciferase activity was measured using a Steady-Glo Luciferase Assay System. NK-252 activated NQO1-ARE in a dose-dependent manner, and this activation was almost completely inhibited by Nrf2 siRNA. [1]
For the H2O2-induced cytotoxicity assay, Huh-7 cells were treated with NK-252 (0.3–30 μM) or DMSO (control) for 24 hours. Cells were then washed and exposed to 1 mM H2O2 for another 24 hours. Cell viability was determined using a Cell Titer 96 AQueous One Solution Reagent. NK-252-treated cells showed increased resistance to H2O2-induced cytotoxicity compared to control cells, with a dose-dependent protective effect. [1]

Male Fischer 344 rats (6 weeks old) were fed a choline-deficient L-amino acid-defined (CDAA) diet to induce NASH. NK-252 was suspended in 0.5% (w/v) methyl cellulose and administered orally once daily. In the preventive regimen, NK-252 was given at doses of 20 mg/kg (low) or 60 mg/kg (high) starting 1 week after CDAA diet initiation, for a total of 9 weeks. In the delayed-administration (therapeutic) regimen, NK-252 was given at 60 mg/kg once daily for 4 weeks starting after 6 weeks of CDAA diet pre-feeding. At the end of the study, rats were euthanized under isoflurane anesthesia, and livers were collected for histopathological examination (H&E, Sirius red, α-SMA immunostaining), gene expression analysis (real-time qPCR), and protein carbonyl measurement. Blood samples were collected from the abdominal aorta for plasma ALT and AST measurement. [1]

NK-252 exhibits relatively low calcium antagonistic activity and low toxicity compared to other dihydropyridines, but no specific toxicity data (e.g., LD50, hepatotoxicity, protein binding) are provided. [2]

[1]. Nrf2 activators attenuate the progression of nonalcoholic steatohepatitis-related fibrosis in a dietary rat model. Mol Pharmacol. 2013 Jul, 84(1):62-70.

[2]. Enhancement of antitumour activity of etoposide by dihydropyridines on drug-sensitive and drug-resistant leukaemia in mice. Br J Cancer. 1991 Aug;64(2):221-6.

See also: NK 252 (Note moved to).

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NK-252 is a novel biaryl urea compound with no thiol-reactive group, designed as a more specific Nrf2 activator compared to prototypical thiol-reactive agents like oltipraz. It is proposed to be the first chemical compound that competitively inhibits the binding of Nrf2 to Keap1, though further structural analysis (e.g., X-ray crystallography) is needed. The antifibrotic effects of NK-252 in the CDAA rat NASH model are attributed to Nrf2 activation, leading to antioxidant, hepatoprotective, anti-inflammatory, and direct hepatic stellate cell-modulating properties. NK-252 may delay the development of liver fibrosis in NASH patients. [1]

C13H11N5O3

285.2581

285.086

1414963-82-8

71618700

White to off-white solid powder

2.51

2

6

4

21

357

0

FNSCFQXZZNCDAI-UHFFFAOYSA-N

InChI=1S/C13H11N5O3/c19-12(15-8-9-4-1-2-6-14-9)16-13-18-17-11(21-13)10-5-3-7-20-10/h1-7H,8H2,(H2,15,16,18,19)

1-[5-(furan-2-yl)-1,3,4-oxadiazol-2-yl]-3-(pyridin-2-ylmethyl)urea

2934.99.9001

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 : ≥ 29 mg/mL (~101.66 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (7.29 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.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (7.29 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.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (7.29 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)