Resibufogenin targets receptor-interacting protein kinase 3 (RIP3) and triggers necrosis by up-regulating RIP3 and phosphorylating Ser358 of mixed lineage kinase domain-like protein (MLKL). It is an anticancer component of huachansu (cinobufogenin).

Resibufogenin showed cytotoxicity against Hela cells with an IC50 of 4.5 × 10⁻² μM (0.045 μM, 45 nM) as determined by MTT assay [1].
Resibufogenin triggers necrosis by up-regulating RIP3 and phosphorylating MLKL at Ser358. It has anti-cancer activity through induction of necroptotic cell death. The compound also exhibits anti-angiogenic properties. Detailed IC50 values for its anti-proliferative effects have been characterized in various cancer cell lines.

In vivo, Resibufogenin has shown anti-tumor activity in animal models. Its anti-angiogenic properties set it apart from similar compounds, making it a candidate for further research and clinical applications. The compound is one of the most significant bufadienolide compounds found in the Bufonidae family.

Cell-free assays for Resibufogenin include RIP3 kinase activity assays using purified recombinant RIP3 and MLKL as substrate. The kinase reaction is performed in the presence of ATP and increasing concentrations of the compound. Phosphorylation of MLKL at Ser358 is quantified using phospho-specific antibodies or radiometric detection. Alternatively, direct binding to RIP3 can be assessed.

Cytotoxicity assay (MTT method): The cytotoxicities of Resibufogenin and its transformed products against Hela cells were determined by the MTT method. The IC50 value of Resibufogenin (compound 1) was 4.5 × 10⁻² μM [1].
Cancer cell lines (e.g., hepatocellular carcinoma, lung cancer, breast cancer cells) are treated with Resibufogenin at various concentrations. Cell viability is assessed by MTT or CellTiter-Glo assays to determine IC50 values. Necroptosis is confirmed by assessment of RIP3 and phosphorylated MLKL levels by Western blot, and by using necroptosis inhibitors (e.g., necrostatin-1) to block cell death.

In vivo animal studies for Resibufogenin are conducted in mouse xenograft models of various cancers. The compound is administered via intraperitoneal or oral routes. Tumor growth is measured over time. Angiogenesis is assessed by CD31 immunohistochemistry or other vascular markers. Pharmacodynamic markers (RIP3 and p-MLKL levels in tumor tissue) are assessed.

Pharmacokinetic properties of Resibufogenin have been characterized in preclinical studies. As a small molecule (MW 384.52, formula C24H32O4) with moderate lipophilicity, it is expected to have reasonable oral bioavailability. PK parameters including Tmax, half-life, clearance, and bioavailability have been studied in animal models.

Toxicity data for Resibufogenin are limited. As a natural product with potent biological activity, standard toxicological assessments would be required for development. The compound's mechanism of action—inducing necroptosis—suggests that it may have a narrow therapeutic window. Cardiotoxicity is a known concern with bufadienolide compounds.

:J Asian Nat Prod Res. 2011 Sep;13(9):831-7.;Nat Prod Commun. 2011 Nov;6(11):1581-4.

Bufogenin is a steroidal lactone found in toad venom (a traditional Chinese medicine extracted from the venom glands of toad skin). It is a specific Na/K-ATPase protein inhibitor and can be used as a cardiotonic, central nervous system (CNS) and respiratory drug, analgesic, anesthetic, and ulcer treatment. It is an EC 3.6.3.9 (Na(+)/K(+)-transfer ATPase) inhibitor. It is a steroidal lactone and an epoxysteroid. Its function is similar to that of bufotalin. Bufogenin has also been reported in the African toad (Phrynoidis asper), the Chinese toad (Bufo gargarizans), and other organisms with relevant data. Bufogenin is a bufodiene lactone toxin, initially isolated from the venom of the Chinese toad (Bufo gargarizans). It is also a glycoside in the traditional Chinese medicine toad venom, with potential cardiotonic effects. Although the mechanism of action of bufotoxin is still under investigation, it is a specific Na+/K+-ATPase inhibitor and has been shown to lower blood pressure in a rat model of preeclampsia.

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Resibufogenin is a major bufadienolide in Chan Su, with reported inhibitory activities against Hela and Bel7402 cells (IC50 1-10 nM). It has blood pressure-stimulating, strong cardiac, antiviral, and local anesthetic activities. Poor water solubility limits its clinical use. The microbial transformation of Resibufogenin by Fusarium solani AS 3.1829 produced five transformed products including 3-ketone-resibufogenin (2), 3-one-cyclic-3-(1,2-dimethyl-1,2-ethanediylacetal)-resibufogenin (3), 3-dimethoxyl-resibufogenin (4), 3-epi-resibufogenin (5), and 3-epi-15α-hydroxy-7βH-bufalin (6). Among these, 3, 4, and 6 are new compounds. The biotransformation reactions such as 3-isomerization, 3-dioxidation, and hydrolysis of C-14 and C-15 decrease the cytotoxic activities of Resibufogenin [1].
Resibufogenin (molecular formula C24H32O4, MW 384.52) is also known as bufogenin and recibufogenin. It is a naturally occurring bufadienolide found in toad venom that has been used in traditional Chinese medicine. The compound triggers necrosis by up-regulating RIP3 and phosphorylating MLKL. It has anticancer and anti-angiogenic properties.

C24H32O4

384.5085

384.23

465-39-4

6917974

White to off-white solid powder

1.3±0.1 g/cm3

554.9±50.0 °C at 760 mmHg

155ºC

190.7±23.6 °C

0.0±3.4 mmHg at 25°C

1.603

2.89

1

4

1

28

786

9

O1[C@]2([H])C([H])([H])[C@]([H])(C3=C([H])OC(C([H])=C3[H])=O)[C@@]3(C([H])([H])[H])C([H])([H])C([H])([H])[C@]4([H])[C@@]5(C([H])([H])[H])C([H])([H])C([H])([H])[C@@]([H])(C([H])([H])[C@@]5([H])C([H])([H])C([H])([H])[C@@]4([H])[C@]132)O[H]

ATLJNLYIJOCWJE-CWMZOUAVSA-N

InChI=1S/C24H32O4/c1-22-9-7-16(25)11-15(22)4-5-18-17(22)8-10-23(2)19(12-20-24(18,23)28-20)14-3-6-21(26)27-13-14/h3,6,13,15-20,25H,4-5,7-12H2,1-2H3/t15-,16+,17+,18-,19-,20-,22+,23-,24-/m1/s1

5-[(1R,2S,4R,6R,7R,10S,11S,14S,16R)-14-hydroxy-7,11-dimethyl-3-oxapentacyclo[8.8.0.02,4.02,7.011,16]octadecan-6-yl]pyran-2-one

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 (~260.07 mM)

Solubility in Formulation 1: ≥ 2.75 mg/mL (7.15 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.

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Solubility in Formulation 2: ≥ 2.75 mg/mL (7.15 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.

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Solubility in Formulation 3: ≥ 2.75 mg/mL (7.15 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.

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