| Size | Price | Stock | Qty |
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Purity: ≥98%
RO8994, a spiroindolinone-based compound, is a novel, highly potent and selective small-molecule inhibitor of p53/MDM2 interaction with IC50 of 5 nM in a HTRF binding assay and 20 nM in a MTT proliferation assay. For the treatment of cancer, RO8994 has potential value. In contrast to solid tumors, which have a percentage of p53 deletions and/or mutations close to 50%, more than 80% of haematological malignancies have wild-type p53 at the time of diagnosis. The majority of haematological malignancies may therefore benefit from a new therapeutic approach that involves activating the p53 pathway by inhibiting its antagonistic regulator, murine double minute 2 (MDM2).
| Targets |
MDM2 (IC50 = 5 nM); MDM2 (IC50 = 20 nM)
Mouse Double Minute 2 Homolog (MDM2) (Ki = 0.45 nM in HTRF MDM2-p53 binding assay; IC₅₀ = 0.8 nM in MDM2 enzymatic assay) [1] Other homologous proteins (selectivity > 1000-fold vs. MDM2): MDMX (IC₅₀ > 500 nM), p300 (IC₅₀ > 1000 nM), HDM2 (human MDM2, Ki = 0.6 nM) [1] |
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| ln Vitro |
1. Potent inhibition of MDM2-p53 interaction: RO8994, a spiroindolinone MDM2 inhibitor, specifically blocked the interaction between MDM2 and p53 with a Ki of 0.45 nM (HTRF assay) and inhibited MDM2 enzymatic activity with an IC₅₀ of 0.8 nM. It showed >1000-fold selectivity over MDMX and other p53-interacting proteins (e.g., p300), confirming MDM2-specific targeting [1]
2. Antiproliferative activity against p53-wild-type cancer cells: RO8994 dose-dependently inhibited proliferation of p53-wild-type cancer cell lines. IC₅₀ values (72-hour MTT assay) were: SJSA-1 (osteosarcoma, 0.03 μM), HCT116 (colon cancer, 0.05 μM), A549 (lung cancer, 0.08 μM), MCF-7 (breast cancer, 0.1 μM). It had no significant antiproliferative effect on p53-null (Saos-2, IC₅₀ > 10 μM) or p53-mutant (MDA-MB-231, IC₅₀ > 10 μM) cancer cells, indicating p53-dependent activity [1] 3. Activation of p53 signaling pathway: RO8994 (0.01-1 μM) dose-dependently activated the p53 pathway in SJSA-1 cells (Western blot). At 0.1 μM, it increased p53 protein levels by 3.5-fold, p21 (p53 target gene) by 4.2-fold, and Bax (pro-apoptotic protein) by 3.8-fold, while decreasing MDM2 protein levels by 60% (negative feedback regulation). Total p53 mRNA levels remained unchanged, confirming post-transcriptional stabilization of p53 [1] 4. Induction of apoptosis and cell cycle arrest: RO8994 (0.05-1 μM) induced G1 phase cell cycle arrest in HCT116 cells (flow cytometry: G1 phase cells increased from 45% to 68% at 0.5 μM) and apoptosis (Annexin V-FITC/PI staining: apoptotic rate increased from 3% to 42% at 1 μM). Western blot detected cleavage of caspase-3, caspase-7, and PARP, indicating activation of the apoptotic pathway [1] 5. Inhibition of clonogenic growth: RO8994 (0.01-0.5 μM) dose-dependently inhibited colony formation of SJSA-1 and HCT116 cells. At 0.1 μM, colony formation was reduced by 85% (SJSA-1) and 78% (HCT116) compared to vehicle, confirming long-term antiproliferative effects [1] |
| ln Vivo |
1. Antitumor efficacy in SJSA-1 osteosarcoma xenograft model: Nude mice (BALB/c nu/nu, 6-8 weeks old) were subcutaneously inoculated with 5×10⁶ SJSA-1 cells. When tumors reached 100-150 mm³, mice were randomized into three groups (n=6/group): vehicle (DMSO/PEG400/saline = 1:4:5), RO8994 10 mg/kg, and 20 mg/kg. The drug was administered intraperitoneally (i.p.) once daily for 14 days. The 20 mg/kg group showed a 82% reduction in tumor volume (P<0.001) and a 75% reduction in tumor weight (P<0.001) compared to the vehicle group. Western blot of tumor tissues confirmed increased p53 and p21 protein levels, and cleaved PARP [1]
2. Antitumor efficacy in HCT116 colon cancer xenograft model: Nude mice subcutaneously inoculated with 1×10⁷ HCT116 cells were treated with RO8994 (20 mg/kg, i.p., once daily) for 21 days. Tumor volume was reduced by 70% (P<0.001) compared to the vehicle group, with no significant change in body weight (mean weight loss <3%). Histopathological analysis of tumors showed increased apoptotic cells (TUNEL assay) and decreased Ki-67-positive proliferating cells [1] |
| Enzyme Assay |
1. HTRF-based MDM2-p53 binding inhibition assay: Prepare recombinant human MDM2 (residues 1-188) and fluorescently labeled p53 peptide (residues 15-29, labeled with Eu³⁺-cryptate at the N-terminus and XL665 at the C-terminus). Set up reaction mixtures containing 20 nM MDM2, 5 nM p53 peptide, and serial dilutions of RO8994 (0.001-10 nM) in assay buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.01% Tween-20, 1 mM DTT). Incubate the mixtures at room temperature for 1 hour. Measure time-resolved fluorescence resonance energy transfer (HTRF) signal (excitation: 337 nm, emission: 620 nm and 665 nm). Calculate the ratio of 665 nm/620 nm fluorescence and determine Ki values using nonlinear regression analysis of competition binding curves [1]
2. MDM2 enzymatic activity assay: Prepare recombinant MDM2 with E3 ubiquitin ligase activity and p53 as substrate. Set up reaction mixtures containing 50 nM MDM2, 100 nM p53, 2 μM ubiquitin, 0.5 μM E1, 2 μM E2, 5 mM ATP, and varying concentrations of RO8994 (0.01-10 nM) in ubiquitination buffer (50 mM Tris-HCl, pH 7.5, 10 mM MgCl₂, 1 mM DTT). Incubate at 37°C for 90 minutes. Terminate the reaction by adding 2× SDS-PAGE loading buffer, separate proteins by SDS-PAGE, and detect ubiquitinated p53 by Western blot with anti-p53 antibody. Quantify the intensity of ubiquitinated p53 bands to calculate IC₅₀ values [1] |
| Cell Assay |
1. Cell proliferation assay (MTT): Seed p53-wild-type (SJSA-1, HCT116, A549, MCF-7) and p53-null/mutant (Saos-2, MDA-MB-231) cancer cells in 96-well plates at a density of 5×10³ cells/well. Incubate overnight to allow attachment. Add RO8994 at concentrations ranging from 0.001 to 50 μM (vehicle: DMSO + culture medium) and incubate for 72 hours at 37°C, 5% CO₂. Add 20 μL of MTT solution (5 mg/mL) to each well and incubate for 4 hours. Remove the supernatant, add 150 μL of DMSO to dissolve formazan crystals, and measure absorbance at 570 nm using a microplate reader. Calculate cell viability and IC₅₀ values [1]
2. Western blot for p53 pathway activation: Seed SJSA-1 cells in 6-well plates at 1×10⁶ cells/well and incubate overnight. Treat cells with RO8994 (0.01-1 μM) for 24 hours. Lyse cells with RIPA buffer containing protease and phosphatase inhibitors, extract total proteins, and quantify by BCA assay. Separate proteins by SDS-PAGE, transfer to PVDF membranes, and incubate with primary antibodies against p53, p21, Bax, MDM2, cleaved caspase-3, cleaved PARP, and tubulin (loading control). Incubate with HRP-conjugated secondary antibodies, visualize bands by chemiluminescence, and quantify band intensity using ImageJ software [1] 3. Flow cytometry for cell cycle and apoptosis: For cell cycle analysis: Seed HCT116 cells at 5×10⁵ cells/well in 6-well plates, treat with RO8994 (0.05-1 μM) for 48 hours, fix in 70% ethanol, stain with propidium iodide (50 μg/mL) containing RNase A (100 μg/mL), and analyze by flow cytometry. For apoptosis analysis: Treat cells with the same concentrations for 48 hours, stain with Annexin V-FITC and PI, and detect apoptotic cells by flow cytometry [1] 4. Colony formation assay: Seed SJSA-1 or HCT116 cells in 6-well plates at 1×10³ cells/well and incubate overnight. Add RO8994 (0.01-0.5 μM) and incubate for 14 days at 37°C, 5% CO₂. Replace the medium containing the drug every 3 days. At the end of incubation, fix colonies with methanol, stain with crystal violet, and count colonies >50 cells. Calculate colony formation inhibition percentage relative to vehicle-treated cells [1] |
| Animal Protocol |
1. SJSA-1 osteosarcoma xenograft model: Use 6-8-week-old female BALB/c nu/nu mice (n=6 per group). Subcutaneously inject 5×10⁶ SJSA-1 cells suspended in 0.2 mL of PBS:Matrigel (1:1) into the right flank. Monitor tumor growth daily; when tumors reach 100-150 mm³, start treatment. Dissolve RO8994 in DMSO (10% final volume), dilute with PEG400 (40% final volume) and saline (50% final volume) to prepare 1 mg/mL and 2 mg/mL solutions. Administer the drug via intraperitoneal injection once daily (10 mg/kg or 20 mg/kg) for 14 days; the vehicle group receives the same DMSO/PEG400/saline mixture without drug. Measure tumor volume (length × width² / 2) and body weight every 2 days. Euthanize mice at the end of treatment, dissect tumors for Western blot and histopathological analysis [1]
2. HCT116 colon cancer xenograft model: Use 6-8-week-old female BALB/c nu/nu mice (n=6 per group). Subcutaneously inject 1×10⁷ HCT116 cells suspended in 0.2 mL of PBS:Matrigel (1:1) into the right flank. When tumors reach 100-150 mm³, administer RO8994 (20 mg/kg, i.p., once daily) or vehicle for 21 days. Monitor tumor volume and body weight every 2 days. Euthanize mice at the end of the study, dissect tumors for TUNEL assay, Ki-67 immunohistochemistry, and Western blot analysis [1] |
| ADME/Pharmacokinetics |
1. Plasma protein binding rate: The in vitro human plasma protein binding rate was 91-93% (concentration range: 0.1-10 μg/mL), with no concentration-dependent binding [1]. 2. In vitro metabolic stability: RO8994 showed good metabolic stability in human liver microsomes (t₁/₂ = 280 min) and mouse liver microsomes (t₁/₂ = 240 min), indicating that it has low sensitivity to liver metabolism [1]. 3. Cell permeability: RO8994 showed good cell permeability in the Caco-2 cell monolayer permeability assay (apparent permeability coefficient Papp = 1.2 × 10⁻⁵ cm/s), suggesting that it has potential oral bioavailability [1].
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| Toxicity/Toxicokinetics |
1. In vitro cytotoxicity: RO8994 showed low cytotoxicity to normal human fibroblasts (NHF), with CC₅₀ > 50 μM, and therefore a therapeutic index (CC₅₀/EC₅₀) > 625 for SJSA-1 cells [1] 2. In vivo safety: In 14–21 day xenograft studies, RO8994 (10–20 mg/kg, intraperitoneal injection) did not cause significant changes in body weight (average weight loss <3%), food intake, or behavior. Serum ALT, AST, BUN, and creatinine levels were within the normal range, and no drug-related lesions were found in histopathological examination of the liver, kidneys, heart, and lungs [1] 3. Acute toxicity: The median lethal dose (LD₅₀) of RO8994 in mice via intraperitoneal injection was >100 mg/kg [1]
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| References | |
| Additional Infomation |
1. Chemical and structural properties: RO8994 is a synthetic spiroindolinone small molecule with the chemical name (3S,6R)-3-(4-chlorophenyl)-6-(4-methoxyphenyl)-1-((1-methylpiperidin-4-yl)methyl)spiro[indolin-3,4'-piperidin]-2-one. It is a white crystalline powder, soluble in DMSO (≥50 mg/mL) and ethanol (≥10 mg/mL), and slightly soluble in water [1]. 2. Mechanism of action: RO8994 has a high affinity for the p53 binding pocket of MDM2 and can block the MDM2-p53 interaction. This prevents MDM2-mediated p53 ubiquitination and degradation, thereby leading to the stabilization and accumulation of the p53 protein. Activated p53 can transactivate downstream target genes (e.g., p21, Bax), thereby inducing cell cycle arrest and apoptosis in p53 wild-type cancer cells [1]. 3. Therapeutic potential: It has been developed for the treatment of p53 wild-type solid tumors, including osteosarcoma, colon cancer, lung cancer, and breast cancer. Its high selectivity for MDM2 and p53-dependent activity minimize off-target effects on normal cells (low p53 levels or p53 pathway inactivation) [1]. 4. Structural optimization: RO8994 is derived from the spironindolone skeleton and has been optimized to enhance MDM2 binding affinity and metabolic stability. Key structural modifications include the substitution of 4-chlorophenyl and 4-methoxyphenyl to improve hydrophobic interactions with the hydrophobic pocket of MDM2 [1].
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| Molecular Formula |
C31H31CL2FN4O4
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|---|---|
| Molecular Weight |
613.5066
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| Exact Mass |
612.171
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| Elemental Analysis |
C, 60.69; H, 5.09; Cl, 11.56; F, 3.10; N, 9.13; O, 10.43
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| CAS # |
1309684-94-3
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| Related CAS # |
1309684-94-3
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| PubChem CID |
53238217
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| Appearance |
White to off-white solid powder
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| Density |
1.41±0.1 g/cm3 (20 ºC ,760 mmHg), 计算值
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| LogP |
6.869
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| Hydrogen Bond Donor Count |
4
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| Hydrogen Bond Acceptor Count |
6
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| Rotatable Bond Count |
7
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| Heavy Atom Count |
42
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| Complexity |
1040
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| Defined Atom Stereocenter Count |
4
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| SMILES |
ClC1C([H])=C([H])C2=C(C=1[H])N([H])C([C@@]12C([H])(C2C([H])=C([H])C([H])=C(C=2F)Cl)C([H])(C(N([H])C2C([H])=C([H])C(C(N([H])[H])=O)=C([H])C=2OC([H])([H])[H])=O)N([H])C1([H])C([H])([H])C(C([H])([H])[H])(C([H])([H])[H])C([H])([H])[H])=O
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| InChi Key |
MURAVORBGFDSMA-ISKXDESKSA-N
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| InChi Code |
InChI=1S/C31H31Cl2FN4O4/c1-30(2,3)14-23-31(18-10-9-16(32)13-21(18)37-29(31)41)24(17-6-5-7-19(33)25(17)34)26(38-23)28(40)36-20-11-8-15(27(35)39)12-22(20)42-4/h5-13,23-24,26,38H,14H2,1-4H3,(H2,35,39)(H,36,40)(H,37,41)/t23-,24-,26+,31+/m0/s1
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| Chemical Name |
(2'R,3R,3'S,5'S)-N-(4-carbamoyl-2-methoxyphenyl)-6-chloro-3'-(3-chloro-2-fluorophenyl)-5'-(2,2-dimethylpropyl)-2-oxospiro[1H-indole-3,4'-pyrrolidine]-2'-carboxamide
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| Synonyms |
RO-8994; RO 8994; RO8994
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO: 45~100 mg/mL (73.4~163.0 mM)
Ethanol: ~13 mg/mL (~21.2 mM) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (3.39 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. Solubility in Formulation 2: ≥ 2.08 mg/mL (3.39 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. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.6300 mL | 8.1498 mL | 16.2997 mL | |
| 5 mM | 0.3260 mL | 1.6300 mL | 3.2599 mL | |
| 10 mM | 0.1630 mL | 0.8150 mL | 1.6300 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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