DNA (via intercalation), leading to activation of Z-DNA binding protein 1 (ZBP1), receptor-interacting protein kinase 3 (RIP3), and mixed lineage kinase domain-like protein (MLKL) pathways [1]

Compound 3a exhibited DNA intercalation properties with a Stern-Volmer quenching constant (Ksv) of 4.5 × 10⁴ M⁻¹ in an ethidium bromide displacement assay. [1]
In an agarose gel electrophoresis assay using pBR322 plasmid DNA, compound 3a induced significant DNA conformational changes at a [compound:DNA base-pair] ratio of 2:1, with complete structural alteration and DNA condensation/precipitation observed at a ratio of 2.5:1. [1]
Compound 3a demonstrated potent anticancer activity against various cancer cell lines with the following IC50 values: HCT-116 (1.55 ± 0.17 μM), HeLa (3.33 ± 0.45 μM), HT-29 (4.0 ± 0.9 μM), Huh7 (6.9 ± 0.15 μM). It showed selectivity towards cancer cells over normal cells with IC50 values of 14.2 ± 0.85 μM in HEK293T and >40 μM in HS-5. [1]
In a clonogenic assay, compound 3a effectively suppressed colony formation in a dose-dependent manner (1-5 μM) in HCT-116, HeLa, and HT-29 cell lines. Treatment at 5 μM significantly inhibited colony growth across all cell lines. [1]
Compound 3a induced histone eviction in an in vitro assembled mononucleosome-based assay, with significant reduction in band intensity for both DNA and nucleosome at concentrations of 25, 50, and 75 μM, and complete histone eviction at 150 μM. [1]
In an alkaline comet assay, compound 3a treatment led to detectable tail formation in HeLa (4 μM), HCT116 (2 μM), and HT-29 (6 μM), indicating substantial DNA strand breaks. [1]
Western blot analysis showed that compound 3a increased expression of phosphorylated γ-H2AX and cleaved caspase-3 in a dose-dependent manner (0-6 μM) across HCT-116, HeLa, and HT-29 cell lines. [1]
Flow cytometric analysis with annexin V-FITC and PI staining showed that compound 3a induced apoptotic cell death in a dose-dependent manner (0-8 μM) in tested cell lines. [1]
Compound 3a increased cellular reactive oxygen species (ROS) levels in a dose-dependent manner in HCT116, HeLa, and HT-29 cell lines as measured by DCFDA staining and flow cytometry. [1]
Compound 3a induced mitochondrial dysfunction as evidenced by clumping of mitochondria (visualized by mitotracker red CMXROS staining) and mitochondrial depolarization (measured by JC-1 staining and flow cytometry). [1]
Compound 3a increased ZBP1 expression in HCT-116, HT-29, and HeLa cell lines in a dose-dependent manner (0-6 μM), as shown by immunoblotting and immunofluorescence microscopy. [1]
In HT-29 cells (RIP3-expressing), treatment with compound 3a (0-10 μM) in the presence of the pan-caspase inhibitor Z-VAD (20 μM) resulted in dose-dependent reduction in cell viability, indicating induction of necroptosis. [1]
In HT-29 cells treated with compound 3a (0-8 μM) plus Z-VAD (20 μM), immunoblotting revealed increased expression of necroptotic markers p-RIP3 and p-MLKL. Immunofluorescence microscopy further confirmed elevated p-RIP3 expression. [1]
In RIP3-silenced cell lines (HeLa and HCT116), pre-treatment with the DNA hypomethylating agent 5-aza-2'-deoxycytidine (5-AD, 2 μM) restored RIP3 expression, and subsequent treatment with compound 3a (3 μM) induced p-RIP3 and p-MLKL expression, indicating necroptosis through the ZBP1-RIP3-MLKL pathway. [1]

Fluorescence intercalator displacement (FID) assay: A mixture containing 20 μM calf thymus DNA (CT-DNA) and 10 μM ethidium bromide (EtBr) was prepared in buffer consisting of Na-phosphate (10 mM, pH 7), NaCl (10 mM), and 1% DMSO. The mixture was titrated with increasing concentrations of compound 3a (5-30 μM). Fluorescent measurements were conducted using a spectrophotometer with an excitation wavelength of 480 nm and emission scan ranging from 520 to 700 nm. The Stern-Volmer quenching constant (Ksv) was calculated from the obtained data. [1]
Circular dichroism (CD) spectroscopy: CD spectra of DNA (30 μM) with increasing concentrations of compound 3a were recorded on a CD spectrometer at room temperature with a scan from 240 to 450 nm in buffer containing Na-phosphate (10 mM, pH 7), NaCl (10 mM), and 1% DMSO. The change in the positive B-DNA peak near 275 nm and negative induced CD (ICD) were monitored to assess DNA structural alteration. [1]
UV-vis absorbance study: UV-visible spectra of compound 3a with increasing concentrations of DNA (AT-rich, GC-rich, and mixed sequence CT-DNA) were recorded using a UV-vis spectrophotometer in phosphate-buffered saline (10 mM Na-P, 10 mM NaCl, 1% DMSO) at room temperature. The decrease in absorption maxima at ~390 nm (hypochromic shift) indicated DNA binding capacity. [1]

Cell viability assay (MTT): Cells (5 × 10³) were seeded in 96-well plates and incubated at 37°C with 5% CO₂. Cells were treated with increasing doses of compound 3a for 24 hours. MTT (0.5 mg/mL) was added and incubated for 3 hours. Formazan crystals were dissolved in DMSO, and absorbance was detected at 570 nm using a plate reader. IC50 values were calculated. [1]
Clonogenic assay: Cells (2 × 10³) were seeded in 6-well plates and incubated overnight. Cells were treated with increasing doses of compound 3a (1-5 μM). Medium was changed every three days. After 10 days, colonies were washed with PBS, fixed with methanol and acetic acid (7:1), stained with 0.5% crystal violet, and counted. [1]
Western blot analysis: Cells were seeded in 6-well plates and incubated overnight. After 24 hours of compound 3a treatment (0-6 μM), cells were lysed with RIPA buffer containing PhosSTOP and protease inhibitor cocktail. Protein concentrations were measured by Bradford method. Proteins were separated by 4-20% SDS-PAGE, transferred to PVDF membrane, blocked with 5% skimmed milk, incubated with primary antibodies overnight at 4°C, then with secondary antibodies for 2 hours at room temperature, and developed by ECL substrates. Antibodies used included those against ZBP1, p-γH2AX, cleaved caspase-3, p-RIP3, p-MLKL, RIP3, and GAPDH. [1]
Apoptosis assay (Annexin V-FITC/PI staining): Cells were treated with increasing concentrations of compound 3a (0-8 μM) for 24 hours. Cells were washed with PBS, trypsinized, resuspended in 1X annexin binding buffer, stained with FITC-tagged annexin V (1 μL) for 15 minutes in the dark, and then PI solution (0.5 μL of 1 mg/mL) was added 5 minutes before FACS analysis. [1]
Intracellular ROS measurement: Cells were treated with increasing doses of compound 3a for 24 hours. Cells were trypsinized, washed with PBS, incubated with H2DCFDA for 15 minutes in the dark at room temperature, and analyzed by flow cytometry. [1]
Alkaline comet assay: Cells were treated with compound 3a (HeLa: 4 μM, HCT116: 2 μM, HT-29: 6 μM) for 24 hours. Cells were mixed with 1% low-melting agarose, spread on agarose-coated slides, and placed in alkaline lysis buffer (100 mM Na₂-EDTA, 1.2 M NaCl, 0.1% sodium lauryl sarcosinate, 0.26 M NaOH, pH >13) at 4°C overnight. Electrophoresis was performed at 30 V for 20 minutes. Slides were stained with PI (10 μg/mL) for 10 minutes and visualized by confocal microscope. [1]
Mitochondrial membrane potential measurement (JC-1 staining): Cells were treated with compound 3a for 24 hours. CCCP treatment was done for 5 minutes at 37°C as positive control. Cells were trypsinized, washed with PBS, stained with 2 μM JC-1 for 15 minutes in the dark, and analyzed by flow cytometry. [1]
Immunofluorescence confocal microscopy: Cells were seeded on cover slides in 35 mm plates, treated with compound 3a (HeLa: 4 μM, HT-29: 6 μM) for 24 hours, fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, blocked with 5% BSA, incubated with primary antibody (ZBP1 or p-RIP3), followed by Alexa Fluor 594-tagged secondary antibody. Nuclei were stained with Hoechst 33342 (16.1 mM) for 20 minutes. Images were captured using a confocal microscope. [1]
Histone eviction assay: In vitro assembled nucleosome (using an Epimark nucleosome assembly kit) was treated with compound 3a (25, 50, 75, and 150 μM) in 50 mM Tris-Cl buffer at 37°C for 4 hours. The reaction mixture was run on 6% native gel in TBE buffer for 1.5 hours at constant current of 10 mA. Gels were stained with EtBr (1 μg/mL) followed by silver staining to visualize DNA and nucleosome complex. [1]
MNase digestion assay: HeLa cell nuclei were isolated and treated with compound 3a for 15 minutes followed by MNase treatment. DNA was extracted by phenol-chloroform method, precipitated with ethanol, run on 1.5% agarose gel in TAE buffer for 30 minutes, and stained with EtBr (0.5 μg/mL). [1]

Compound 3a exhibited selectivity towards cancer cells over normal cells, with IC50 values of 14.2 ± 0.85 μM in HEK293T (human embryonic kidney cell line) and >40 μM in HS-5 (normal fibroblast stromal cell line), compared to 1.55 ± 0.17 μM in HCT-116 colon cancer cells. [1]

[1]. Mono-quinoxaline-induced DNA structural alteration leads to ZBP1/RIP3/MLKL-driven necroptosis in cancer cells. European Journal of Medicinal Chemistry, 2024, 270: 116377.

Compound 3a (N²-([1,1'-biphenyl]-4-ylmethyl)-N³-(3-(dimethylamino)propyl)-6-nitroquinoxaline-2,3-diamine) is a mono-quinoxaline-based small molecule designed with an extended aromatic π-surface to enhance DNA intercalation capacity. It features a [1,1'-biphenyl]-4-ylmethanamine moiety at the C-2 position and a dimethylaminopropyl tail at the C-3 position of the 6-nitroquinoxaline core. [1]
The mechanism of action involves DNA intercalation-induced structural alteration, leading to histone eviction, DNA damage, ROS generation, and mitochondrial dysfunction. These events upregulate ZBP1 expression, which can trigger both apoptosis and, when apoptosis is blocked (by Z-VAD), necroptosis via the ZBP1/RIP3/MLKL pathway. [1]
In RIP3-silenced cancer cells (HeLa, HCT116), compound 3a can induce necroptosis when combined with DNA hypomethylating agents (such as 5-aza-2'-deoxycytidine) that restore RIP3 expression. [1]
Compound 3a preferentially induces nucleobase de-stacking in GC-rich DNA compared to AT-rich DNA, as evidenced by circular dichroism spectroscopy. [1]

C26H28N6O2

456.54

456.22737

171713829

Yellow to orange solid at room temperature

5.2

2

7

9

34

615

0

CN(C)CCCNC1=NC2=C(C=CC(=C2)[N+](=O)[O-])N=C1NCC3=CC=C(C=C3)C4=CC=CC=C4

PZOYNYJLPZBQFO-UHFFFAOYSA-N

InChI=1S/C26H28N6O2/c1-31(2)16-6-15-27-25-26(29-23-14-13-22(32(33)34)17-24(23)30-25)28-18-19-9-11-21(12-10-19)20-7-4-3-5-8-20/h3-5,7-14,17H,6,15-16,18H2,1-2H3,(H,27,30)(H,28,29)

3-N-[3-(dimethylamino)propyl]-6-nitro-2-N-[(4-phenylphenyl)methyl]quinoxaline-2,3-diamine

CHEMBL5567248; ZBP1/RIP3/MLKL activator 1; CHEMBL-5567248; ZBP1/RIP3/MLKL activator-1;

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: 100 mg/mL (219.0 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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