| Size | Price | |
|---|---|---|
| 500mg | ||
| 1g | ||
| Other Sizes |
| Targets |
MDM2 (mouse double minute 2) – a potent inhibitor of the MDM2/p53 protein-protein interaction. Also exhibits moderate inhibitory activity against MDM4/p53 interaction [1].
IC50: 39.6 nM (HCT-116 cells); 4.28μM (HEK-293 cells)[1]. |
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| ln Vitro |
In Vitro: JN122 is a highly potent MDM2 inhibitor with a binding Ki value of 0.7 ± 0.1 nM in fluorescence polarization (FP) assays. It also inhibits MDM4/p53 binding with a Ki value of 527 ± 14 nM [1].
In cell growth inhibition assays, JN122 exhibits potent antiproliferative activity against various cancer cell lines harboring wild-type p53: HCT-116 (colorectal) with IC50 < 1.0 nM, RKO (colorectal, MDM4-overexpressing) with IC50 = 43.2 ± 7.7 nM, U2-OS (osteosarcoma) with IC50 = 10.8 ± 5.4 nM, A549 (lung) with IC50 = 2.9 ± 1.6 nM, MSTO-211H (mesothelioma) with IC50 = 9.48 ± 9.42 nM, HepG2 (liver) with IC50 = 0.32 ± 0.21 nM, H460 (lung) with IC50 = 0.26 ± 0.10 μM, and MOLM-13 (AML) with IC50 = 6.4 ± 4.3 nM (2-day treatment). In contrast, in p53-mutant SW480 cells and p53-undetectable HeLa cells, JN122 showed much weaker activity (IC50 = 7.10 ± 0.24 μM and 12.24 ± 1.71 μM, respectively), indicating p53-dependent antiproliferative activity [1]. In HCT-116 cells, JN122 dose-dependently increased p53, p21, and MDM2 protein levels at 24 and 48 h, and induced PARP cleavage (apoptosis marker) at 5-10 μM. At 48 h, PUMA and BAX upregulation was observed, and MDM2 and MDM4 decreased at higher concentrations due to cell death. In RKO cells, JN122 (78.1 nM) showed comparable p53 activation to RG7388 at 312.5 nM (4-fold improvement). JN122 also induced MDM4 degradation in H460, A549, U2-OS, MSTO-211H, and RKO cells [1]. Flow cytometry analysis showed that JN122 dose-dependently increased the proportion of G1 cells at low concentrations and G2 cells at high concentrations in HCT-116 cells. It also dose-dependently induced apoptosis at 12.5 and 25 μM after 48 h treatment [1]. |
| ln Vivo |
In Vivo: In a systemic MOLM-13 xenograft model (AML) in female NOD.CB17-PrkdcscidIl2rgtm1/Bcgen (B-NDG) mice, JN122 was administered orally at doses of 25, 50, and 100 mg/kg once daily for 21 consecutive days. The median overall survival time of vehicle-treated mice was 20 days (range 18-26 days). JN122 significantly extended median survival to 25.5 days at 25 mg/kg (range 20-27 days, p < 0.01), 25.5 days at 50 mg/kg (range 17-28 days, p < 0.001), and 31 days at 100 mg/kg (range 26-33 days, p < 0.001). For comparison, RG7388 at 50 mg/kg extended survival to 27.5 days (range 26-30 days, p < 0.001) [1].
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| Enzyme Assay |
Enzyme Assay: For MDM2 and MDM4 binding affinity determination, fluorescence polarization (FP) assays were used. Recombinant GST-MDM2 (1-118) or GST-MDM4 (1-123) proteins were incubated with a fluorescently labeled p53 peptide (FAM-labeled) and serially diluted test compounds. After incubation at room temperature for 30 min, polarization values were measured using a microplate reader. Ki values were calculated using the Cheng-Prusoff equation [1].
For computational docking, the structures of MDM2 (PDB: 4LWU) and MDM4 (PDB: 6Q9W) were obtained from the Protein Data Bank. The MDM2 structure was optimized using the Protein Preparation Wizard module. A grid box of 20 × 20 × 20 ų centered on the active site was generated. Conformations of JN122 were prepared and their ionized states (pH 7.4 ± 1.0) were generated. Docking was performed using the Glide module, and the best binding modes were selected based on glide docking scores [1]. |
| Cell Assay |
Cell Assay: For cell growth inhibition, cells were seeded in 96-well plates at densities of 3,000-20,000 cells per well. After 4 days of treatment (2 days for MOLM-13), cell viability was measured using CCK-8 reagent or CellTiter-Glo 2.0 assay. Absorbance at 450 nm was detected, and IC50 values were calculated by nonlinear regression curve fitting in GraphPad Prism [1].
For Western blot analysis, cells were seeded in 6-well plates and treated with compounds for 24 or 48 h. Cells were lysed in RIPA buffer with PMSF, and lysates were centrifuged. Proteins (20-40 μg/lane) were separated by SDS-PAGE, transferred to nitrocellulose membranes, blocked with 5% non-fat milk, and incubated with primary antibodies (p53, MDM2, p21, MDM4, PUMA, BAX, PARP, etc.) overnight at 4°C, followed by HRP-conjugated secondary antibodies. Proteins were visualized using enhanced chemiluminescence [1]. For qPCR analysis, HCT-116 cells were treated with compounds for 48 h. Total RNA was extracted using a kit, and cDNA was synthesized. Quantitative PCR was performed using SYBR Green with primers for MDM2, p21, PUMA, BAX, MDM4, and TP53. Expression was normalized to GAPDH [1]. For cell cycle analysis, HCT-116 cells were treated for 24 h, fixed in 70% ethanol, stained with propidium iodide, and analyzed by flow cytometry [1]. For apoptosis analysis, HCT-116 cells were treated for 48 h, stained with FITC Annexin V and propidium iodide, and analyzed by flow cytometry [1]. |
| Animal Protocol |
Animal Protocol: For the MOLM-13 xenograft efficacy study, 50 female NOD.CB17-PrkdcscidIl2rgtm1/Bcgen (B-NDG) mice were intravenously injected with 2 × 10⁴ MOLM-13 cells in 200 μL PBS on day 1. On day 6, mice were orally administered vehicle (5% DMSO + 10% Cremophor EL + 85% normal saline), JN122 at 25, 50, or 100 mg/kg, or RG7388 at 50 mg/kg once daily for 21 consecutive days. Mouse body weight was monitored, and survival was recorded. Kaplan-Meier survival curves were generated [1].
For PK studies in ICR mice, compounds were administered intravenously at 5 mg/kg or orally at 15 mg/kg. Blood samples were collected at various time points, and plasma drug concentrations were analyzed by LC-MS/MS [1]. |
| ADME/Pharmacokinetics |
ADME/Pharmacokinetics: Upon oral administration of JN122 at 15 mg/kg in ICR mice, the compound exhibited a T1/2 of 7.42 ± 0.35 h, Cmax of 4151 ± 1049 ng/mL, AUC(0-∞) of 58429 ± 14155 h·ng/mL, and oral bioavailability of 30.3 ± 7.3%. Upon intravenous administration at 5 mg/kg, T1/2 was 5.35 ± 0.47 h, Cmax was 23760 ± 18476 ng/mL, AUC was 4251 ± 4098 h·ng/mL, and clearance was 1.21 ± 0.10 mL/min/kg [1].
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| Toxicity/Toxicokinetics |
Toxicity/Toxicokinetics: In the MOLM-13 xenograft efficacy study, JN122 at all three doses (25, 50, 100 mg/kg) caused body weight loss of less than 2 g (<10%) from days 1-15 post first treatment, indicating it was well tolerated with low in vivo toxicity. On day 22, greater than 10% weight loss (>2 g) was observed for vehicle and 25 and 50 mg/kg groups due to disease progression, but not for the 100 mg/kg group [1].
In HEK-293 normal kidney cells, JN122 inhibited cell growth with an IC50 of 4.28 μM, which is >100-fold higher than its antiproliferative IC50 values in p53 wild-type cancer cell lines, suggesting a potentially good safety profile. Microsomal stability studies showed that JN122 was stable in human, dog, and monkey microsomes, but relatively less stable in rat and mouse microsomes [1]. |
| References | |
| Additional Infomation |
JN122 is a spiroindoline-containing MDM2 inhibitor discovered through structure-based optimization of a RG7388-derived compound. It features a novel spiro-ring moiety, distinguishing it from known spiro-oxindole MDM2 inhibitors. JN122 efficiently promotes activation of p53 and its target genes (p21, MDM2, PUMA, BAX), inhibits cell cycle progression, and induces apoptosis in wild-type p53 cancer cells. Interestingly, JN122 also promotes degradation of MDM4 in multiple cancer cell lines. The active enantiomer is (-)-60, which is >100-fold more potent than its (+)-enantiomer in MDM2 binding and cellular assays. The compound exhibits good oral PK properties and robust in vivo antitumor efficacy in a systemic AML xenograft model. JN122 is stable in aqueous solution and does not undergo spontaneous ring-opening-closing isomerization like spiro-oxindole MDM2 inhibitors [1].
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| Molecular Formula |
C33H37CL2N3O4
|
|---|---|
| Molecular Weight |
610.57
|
| Exact Mass |
609.2200
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| Elemental Analysis |
C, 64.92; H, 6.11; Cl, 11.61; N, 6.88; O, 10.48
|
| Appearance |
Typically exists as solid at room temperature
|
| SMILES |
CC(C)(C)C[C@H](N(CC)[C@H]1C(NC2=C(OC)C=C(C(O)=O)C=C2)=O)[C@]3(C4=CC=C(Cl)C=C4NC3)[C@H]1C5=CC(Cl)=CC=C5
|
| InChi Key |
AJQDQXYTMUFRRF-MKYOLNSUSA-N
|
| InChi Code |
InChI=1S/C33H37Cl2N3O4/c1-6-38-27(17-32(2,3)4)33(18-36-25-16-22(35)11-12-23(25)33)28(19-8-7-9-21(34)14-19)29(38)30(39)37-24-13-10-20(31(40)41)15-26(24)42-5/h7-16,27-29,36H,6,17-18H2,1-5H3,(H,37,39)(H,40,41)/t27-,28-,29+,33-/m0/s1
|
| Chemical Name |
4-((2'S,3S,4'R,5'R)-6-chloro-4'-(3-chlorophenyl)-1'-ethyl-2'-neopentylspiro[indoline-3,3'-pyrrolidine]-5'-carboxamido)-3-methoxybenzoic acid
|
| Synonyms |
JN-122; JN 122
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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) |
Typically soluble in DMSO (e.g. 10 mM)
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| Solubility (In Vivo) |
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.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *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). View More
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)] Oral Formulations
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). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.6378 mL | 8.1891 mL | 16.3781 mL | |
| 5 mM | 0.3276 mL | 1.6378 mL | 3.2756 mL | |
| 10 mM | 0.1638 mL | 0.8189 mL | 1.6378 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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