Receptor-interacting protein kinase 1 (RIP1), receptor-interacting protein kinase 3 (RIP3), and mixed lineage kinase domain-like protein (MLKL) [1]

Compound 6i exhibited potent antiproliferative activity against U251 human glioma cells with an IC50 value of 0.94 μM, which was approximately 4.7-fold more potent than celastrol (IC50 = 4.43 μM). It also showed activity against A172 (IC50 = 3.03 μM), LN229 (IC50 = 1.78 μM), and U87 (IC50 = 1.22 μM) cell lines. [1]
In normal human liver L02 cells, compound 6i showed an IC50 value of 2.14 μM, compared to celastrol (IC50 = 0.83 μM). [1]
Compound 6i time- and concentration-dependently inhibited U251 cell growth. At 0.5 μM for 72 h, the inhibition rate was approximately 49%. [1]
In a colony formation assay, compound 6i at 0.5 μM and 1.0 μM for 24-72 h remarkably decreased the number of U251 cell colonies, with no clones formed at 0.5 μM for 48 h. [1]
In a wound-healing assay, compound 6i (0.5 and 1.0 μM) concentration-dependently decreased the migration rate of U251 cells compared to control groups. [1]
Hoechst 33342 staining showed that compound 6i (1.0 μM, 48 h) did not increase the proportion of apoptotic cells (chromatin condensation and nuclear fragment) compared to control. [1]
Annexin V-FITC/PI flow cytometry analysis showed that compound 6i (0.5, 1.0, and 2.0 μM, 48 h) did not increase the percentage of early apoptotic cells (AV+/PI-). [1]
Pre-treatment with the pan-caspase inhibitor z-VAD-FMK (20, 40, 80 μM) could not prevent cell death induced by compound 6i, indicating that the antiproliferative activity was not mainly mediated by apoptosis. [1]
Transmission electron microscopy of U251 cells treated with compound 6i revealed necroptotic morphological characteristics: loss of plasma membrane integrity, swollen mitochondria, cytoplasmic vacuolation, and autophagosome formation. No apoptotic features (apoptotic bodies, membrane blebbing, chromatin condensation) were observed. [1]
Compound 6i (0.5, 1.0, and 2.0 μM, 48 h) concentration-dependently increased cell membrane permeability as measured by PI staining and flow cytometry. [1]
Pre-treatment with the RIP1 inhibitor Nec-1 (10 μM) significantly prevented compound 6i-induced viability reduction in U251 cells. [1]
Compound 6i (0.5, 1.0, and 2.0 μM, 48 h) induced mitochondrial depolarization in a concentration-dependent manner, as measured by JC-1 staining and flow cytometry. The percentage of cells with depolarized mitochondrial membrane was reduced from 164.5% of control to 1.6% at 2 μM. [1]
Pre-treatment with RIP1 inhibitor GSK2982772 (2.5 μM), RIP3 inhibitor GSK872 (2.5 μM), and MLKL inhibitor necrosulfonamide (1.0 μM) effectively rescued compound 6i (1.0 μM)-triggered U251 cell death. [1]
Western blot analysis showed that compound 6i (0.5, 1.0, 2.0, and 4.0 μM, 24 or 48 h) concentration-dependently upregulated the expression of p-RIP1, RIP1, p-RIP3, RIP3, p-MLKL, and MLKL in U251 cells. [1]
Compared to celastrol, compound 6i had a stronger ability to induce necroptosis in U251 cells, as demonstrated by the greater viability rescue effect of necroptosis inhibitors (Nec-1, GSK872, necrosulfonamide) in compound 6i-treated cells versus celastrol-treated cells. [1]

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In human glioma cell lines, RIP1/RIP3/MLKL activator 1 (compound 6i) (96 hours) demonstrated antiproliferative action [1]. On U251 cells, RIP1/RIP3/MLKL activator 1 (0–4 µM, 0-72 hours) demonstrates strong anti-proliferative activity in a concentration- and time-dependent way [1]. Acceptable stability is demonstrated by RIP1/RIP3/MLKL activator 1 (10 µM, 0-72 hours) [1]. It has been observed that RIP1/RIP3/MLKL activator 1 (0–2 µM, 24 hours) efficiently prevents U251 cell migration [1]. In U251 cells, RIP1/RIP3/MLKL activator 1 causes mitochondrial depolarization and necroptosis via the RIP1/RIP3/MLKL pathway [1]. In U251 cells, RIP1/RIP3/MLKL activator 1 is unable to cause apoptosis [1].

In a zebrafish xenograft model, compound 6i (2.50 ng/tail, vein circulation microinjection) significantly reduced U251 xenograft fluorescence intensity compared to the control group, indicating inhibition of U251 cell proliferation in vivo. The positive control 5-FU was used at 50.0 ng/tail. [1]
Blood-brain barrier permeability study in Kunming mice: compound 6i was administered via tail vein injection at dosages of 1.0, 3.0, and 9.0 mg/kg. Brain tissue analysis at 0.5 and 1.0 h post-injection showed that compound 6i could cross the BBB, and the concentration in brain tissue increased in a concentration-dependent manner. [1]

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Compound 6i, RIP1/RIP3/MLKL activator 1 (2.50 ng/tail; intravenous injection; 48 hours), exhibits acceptable BBB permeability while inhibiting U251 cell proliferation in vivo [1].

Cell viability assay (MTT): Cells were seeded in 96-well plates at 2000 cells/well for 12 h. Different concentrations of compound 6i were added and incubated for 96 h. MTT (5 mg/mL, 20 μL) was added and incubated for 3-4 h at 37°C. Supernatant was discarded, DMSO (150 μL/well) was added, and absorbance at 490 nm was read. IC50 values were calculated. [1]
Colony formation assay: U251 cells (800/well) were seeded in 6-well plates and treated with compound 6i (0, 0.5, 1.0 μM) for 24-72 h, then cultured in drug-free medium for 11 days. Colonies were fixed with 4% paraformaldehyde, stained with 0.1% crystal violet for 30 min, and colonies with >50 cells were counted. [1]
Wound-healing assay: U251 cells (1 × 10⁵/well) were cultured to >90% confluence. Scratches were made with a 200 μL pipette tip. Cells were treated with compound 6i (0.5 and 1.0 μM). Images were captured at 0 and 24 h, and migration rate was calculated. [1]
Hoechst 33342 staining: U251 cells were treated with compound 6i (1.0 μM) or celastrol (1.0 μM) for 48 h. Hoechst 33342 buffer was added for 20 min in the dark, and apoptotic cells were observed by fluorescence microscope. [1]
Apoptosis analysis (Annexin V-FITC/PI): U251 cells were treated with different concentrations of compound 6i for 48 h. Cells were harvested, incubated with Annexin V-FITC and PI, and analyzed by flow cytometry. [1]
Mitochondrial membrane potential measurement (JC-1): U251 cells were treated with compound 6i for 48 h, stained with JC-1 for 30 min, and analyzed by flow cytometry. [1]
Western blot analysis: U251 cells were treated with compound 6i (0.5, 1.0, 2.0, 4.0 μM) for 24 or 48 h. Cells were lysed, and protein concentrations were determined by BCA kit. Proteins were separated by SDS-PAGE, transferred to PVDF membranes, blocked with 5% BSA, probed with primary antibodies against GAPDH, p-RIP1, RIP1, p-RIP3, RIP3, p-MLKL, and MLKL at 4°C for 12 h, then with HRP-conjugated secondary antibodies for 2 h. Protein bands were detected using ImageLab software. [1]
PI staining for membrane permeability: U251 cells were treated with compound 6i (0.5, 1.0, 2.0 μM) for 48 h, stained with PI, and analyzed by flow cytometry. [1]
Transmission electron microscopy: U251 cells were treated with compound 6i, fixed, and examined under a transmission electron microscope to observe morphological changes. [1]

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Cell proliferation assay[1]
Cell Types: A172, LN229, U87, U251 and L02 Cell lines
Tested Concentrations: 0-4 µM for U251 cells
Incubation Duration: 96 hrs (hours); 24, 48 and 72 hrs (hours) for U251 cells
Experimental Results: For A172, LN229, U87, U251 and L02 demonstrated antiproliferative activity with IC50 values of 3.03 ± 0.70, 1.78 ± 0.79, 1.22 ± 0.89, 0.94 ± 0.45 and 0.99 ± 0.46 µM cells, respectively. Inhibits the growth of U251 cells in a time- and concentration-dependent manner.

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Western Blot Analysis[1]
Cell Types: U251
Tested Concentrations: 0, 0.5, 1, 2 and 4 µM
Incubation Duration: 24 or 48 hrs (hours)
Experimental Results: Concentration-dependent upregulation of the expression of p-RIP1, RIP1, p-RIP3, RIP3, p -MLKL and MLKL at 24 or 48 hrs (hours).

Blood-brain barrier permeability study: Male Kunming mice (6-8 weeks, n=4 per group) were randomly divided into six groups. Compound 6i was dissolved in saline containing 6.0% EtOH and 6.0% polyoxyethylene castor oil and administered at dosages of 1.0, 3.0, and 9.0 mg/kg via tail vein injection. Mice were sacrificed at 0.5 or 1.0 h after injection. Brain samples were excised, weighed, mixed with sterile saline (1:5 w/v), homogenized, and extracted with 2-fold volume of methanol/acetonitrile (1:9), then filtered and analyzed by LC-MS. [1]
Zebrafish xenograft model: Wild-type AB strain zebrafish embryos at 2 days post-fertilization (dpf) were microinjected with CM-Dil-labeled U251 cells into the yolk sac. Embryos were placed at 35°C to 3 dpf. Zebrafish embryos were randomly assigned to 6-well plates (30 embryos/well). Compound 6i (2.50 ng/tail) and 5-FU (50 ng/tail, positive control) were administered via vein circulation microinjection and incubated for 48 h. Ten embryos from each well were randomly selected and photographed under a fluorescence microscope. Fluorescence intensity was analyzed using image software. [1]

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Animal/Disease Models: Zebrafish broad AB strain; 200 CM-DiI labeled U251 cells were transplanted into the yolk sac of each wild-type zebrafish embryo at 2 dpf (2 days after fertilization) [1]
Doses: 2.50 ng/tail
Route of Administration: microinjection; 48-hour
Experimental Results: The fluorescence intensity of U251 xenografts was Dramatically diminished.

Stability in cell culture medium: Compound 6i was dissolved in DMEM culture medium to a final concentration of 100 μg/mL and incubated at 37°C. Samples were taken at 0, 6, 12, 24, 48, and 72 h and analyzed by HPLC. [1]

Compound 6i showed lower cytotoxicity against normal human liver L02 cells (IC50 = 2.14 μM) compared to celastrol (IC50 = 0.83 μM), indicating improved selectivity. [1]

[1]. Synthesis and biological evaluation of celastrol derivatives as potential anti-glioma agents by activating RIP1/RIP3/MLKL pathway to induce necroptosis. Eur J Med Chem. 2022 Feb 5;229:114070.

Compound 6i (3-hydroxy-9β,13α-dimethyl-2-oxo-24,25,26-trinoroleana-1(10),3,5,7-tetraen-29-oic-(3-(4-tert-butylbenzyl)-3H-1,2,3-triazole-4-yl)carboxamide) is a celastrol derivative with a 1,2,3-triazole moiety introduced at the C-20 COOH position. [1]
The compound induces necroptosis in U251 glioma cells primarily by activating the RIP1/RIP3/MLKL pathway, leading to phosphorylation of RIP1, RIP3, and MLKL, necrosome formation, and subsequent necroptotic cell death. [1]
Compound 6i does not induce apoptosis in U251 cells, as evidenced by the lack of increased apoptotic cells by Hoechst staining, Annexin V-FITC/PI flow cytometry, and the inability of the pan-caspase inhibitor z-VAD-FMK to prevent cell death. [1]
The ability of compound 6i to induce necroptosis is stronger than that of celastrol, as demonstrated by the greater protective effect of necroptosis inhibitors in compound 6i-treated cells compared to celastrol-treated cells. [1]
Compound 6i exhibits acceptable blood-brain barrier permeability, which is a key factor for anti-glioma agents to ensure in vivo efficacy in the brain. [1]

C43H56N4O3

676.929751396179

676.435

2682850-41-3

163322291

Orange to red solid powder

1.19±0.1 g/cm3(Predicted)

8.3

2

5

6

50

1560

5

CC1=C(C(=O)C=C2C1=CC=C3[C@]2(CC[C@@]4([C@@]3(CC[C@@]5(C4C[C@](CC5)(C)C(=O)NCC6=CN(N=N6)CC7=CC=C(C=C7)C(C)(C)C)C)C)C)C)O

QLFCJGYFBTYJGC-RSWNTZNOSA-N

InChI=1S/C43H56N4O3/c1-27-31-14-15-34-41(7,32(31)22-33(48)36(27)49)19-21-43(9)35-23-40(6,17-16-39(35,5)18-20-42(34,43)8)37(50)44-24-30-26-47(46-45-30)25-28-10-12-29(13-11-28)38(2,3)4/h10-15,22,26,35,49H,16-21,23-25H2,1-9H3,(H,44,50)/t35?,39-,40-,41+,42-,43+/m1/s1

(2R,4aS,6aR,6aS,14aS)-N-[[1-[(4-tert-butylphenyl)methyl]triazol-4-yl]methyl]-10-hydroxy-2,4a,6a,6a,9,14a-hexamethyl-11-oxo-1,3,4,5,6,13,14,14b-octahydropicene-2-carboxamide

RIP1/RIP3/MLKL activator 1; 2682850-41-3; RIP1/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

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture.

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 (~147.73 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (3.69 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), suspension solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 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 2: 2.5 mg/mL (3.69 mM) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 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.)