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Milademetan (DS-3032b; DS3032; DS-3032) is a potent, selective and orally bioactive MDM2 inhibitor with potential antitumor activity. DS-3032b binds to, and prevents the binding of MDM2 protein to the transcriptional activation domain of the tumor suppressor protein p53. By preventing this MDM2-p53 interaction, the proteosome-mediated enzymatic degradation of p53 is inhibited and the transcriptional activity of p53 is restored. This results in the restoration of p53 signaling and leads to the p53-mediated induction of tumor cell apoptosis.
Physicochemical Properties
| Molecular Formula | C₃₀H₃₄CL₂FN₅O₄ |
| Molecular Weight | 618.526468753815 |
| Exact Mass | 617.197 |
| CAS # | 1398568-47-2 |
| Related CAS # | Milademetan tosylate hydrate;2095625-97-9 |
| PubChem CID | 73297272 |
| Appearance | White to light yellow solid powder |
| Density | 1.4±0.1 g/cm3 |
| Boiling Point | 840.7±65.0 °C at 760 mmHg |
| Flash Point | 462.2±34.3 °C |
| Vapour Pressure | 0.0±3.1 mmHg at 25°C |
| Index of Refraction | 1.646 |
| LogP | 3.54 |
| Hydrogen Bond Donor Count | 4 |
| Hydrogen Bond Acceptor Count | 7 |
| Rotatable Bond Count | 4 |
| Heavy Atom Count | 42 |
| Complexity | 1090 |
| Defined Atom Stereocenter Count | 5 |
| SMILES | CC1(CCC2(CC1)[C@@]3([C@H]([C@@H](N2)C(=O)N[C@@H]4CC[C@H](OC4)C(=O)N)C5=C(C(=NC=C5)Cl)F)C6=C(C=C(C=C6)Cl)NC3=O)C |
| InChi Key | RYAYYVTWKAOAJF-QISPRATLSA-N |
| InChi Code | InChI=1S/C30H34Cl2FN5O4/c1-28(2)8-10-29(11-9-28)30(18-5-3-15(31)13-19(18)37-27(30)41)21(17-7-12-35-24(32)22(17)33)23(38-29)26(40)36-16-4-6-20(25(34)39)42-14-16/h3,5,7,12-13,16,20-21,23,38H,4,6,8-11,14H2,1-2H3,(H2,34,39)(H,36,40)(H,37,41)/t16-,20+,21+,23-,30-/m1/s1 |
| Chemical Name | (3'R,4'S,5'R)-N-[(3R,6S)-6-carbamoyloxan-3-yl]-6''-chloro-4'-(2-chloro-3-fluoropyridin-4-yl)-4,4-dimethyl-2''-oxo-1'',2''-dihydrodispiro[cyclohexane-1,2'-pyrrolidine-3',3''-indole]-5'-carboxamide |
| Synonyms | DS-3032B DS 3032B DS3032B DS-3032 DS 3032 DS3032 Milademetan free base |
| HS Tariff Code | 2934.99.9001 |
| 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) |
Biological Activity
| Targets | Milademetan (DS-3032b; DS-3032) is a small molecule inhibitor of MDM2, which disrupts the binding of MDM2 to the transcriptional activation domain of TP53, thereby reactivating TP53 signaling[4]. |
| ln Vitro |
In wild-type TP53 neuroblastoma cells, meledemetan (DS-3032) selectively induces the expression of CDKNA1, BAX, and MDM2 while stabilizing TP53 [3]. Treatment with meledemetan (DS-3032b) increases the expression of the TP53 target gene and causes apoptosis, senescence, and G1 cell cycle arrest [3]. Regardless of MYCN status, treatment with meledemetan (DS-3032b, 0-2000 nM) specifically inhibits the viability, proliferation, and migration of neuroblastoma cells harboring wild-type TP53 [4]. Treatment with Milademetan (DS-3032b) reduced viability in neuroblastoma cell lines (SK-N-SH, SH-SY5Y, IMR32, IMR5, LAN5) with wildtype TP53 in a dose- and time-dependent manner, while the TP53-mutant cell line Kelly was significantly less sensitive[4]. DS-3032b treatment (48 h) reduced bromodeoxyuridine incorporation in all wildtype TP53 cell lines in a dose-dependent manner[4]. DS-3032b (250 nM, 24 h) reduced scratch healing (migration) in all wildtype TP53 cell lines except LAN5[4]. DS-3032b (125 and 250 nM, 48 h) induced G1 cell cycle arrest and increased senescence-associated beta-galactosidase activity in wildtype TP53 cell lines[4]. DS-3032b upregulated MDM2, CDKN1A, and BAX protein expression in wildtype TP53 cell lines, and increased TP53 protein levels[4]. DS-3032b induced apoptosis in wildtype TP53 cell lines as shown by annexin V/PI staining and caspase-3 activity assays[4]. DS-3032b also induced apoptosis in primary neuroblastoma cells (OHC-NB-1) with wildtype TP53[4]. Stable expression of a dominant-negative TP53 mutant attenuated DS-3032b-mediated reduction in viability, apoptosis induction, and TP53 target gene upregulation[4]. CRISPR-mediated MDM2 knockout mimicked DS-3032b treatment by reducing cell viability[4]. Microarray analysis showed DS-3032b treatment upregulated TP53 signaling genes and downregulated cell cycle progression genes in SH-SY5Y and IMR5 cells[4]. |
| ln Vivo |
Milademetan (DS-3032b, 50 mg/kg, orally administered) xenografts neuroblastoma cells with functioning TP53, delaying tumor growth and enhancing survival [4]. Oral administration of Milademetan (DS-3032b) (50 mg/kg, 4 days on/2 days off for 30 days) significantly suppressed SH-SY5Y xenograft tumor growth in nude mice compared to vehicle control[4]. DS-3032b treatment prolonged survival in tumor-bearing mice[4]. DS-3032b treatment increased TP53, CDKN1A, and BAX protein expression in tumor tissues[4]. Immunohistochemistry showed increased cleaved caspase-3 (apoptosis) and decreased Ki-67 (proliferation) in tumors from DS-3032b-treated mice[4]. |
| Cell Assay |
Cell viability assay[4] Cell Types: SK-N-SH, SH-SY5Y, IMR32, IMR5 and LAN5 cell lines. Tested Concentrations: 0-2000 nM. Incubation Duration: 24-72 hrs (hours). Experimental Results: Vitality diminished in a dose- and time-dependent manner. In SK-N-SH, SH-SY5Y, IMR32, IMR5 and LAN5 cell lines (72 hrs (hours)), the IC50 values were 21.9 nM, 17.7 nM, 52.63 nM, 25.7 nM and 44.1 nM, respectively. Cell viability assay: Cells were seeded in 96-well plates, allowed to adhere for 24 h, then treated with 0–2000 nM DS-3032b for 24, 48, or 72 h[4]. Viability was assessed using the XTT assay[4]. Proliferation assay: Cell proliferation was measured using a bromodeoxyuridine ELISA assay after 48 h of DS-3032b treatment[4]. Senescence assay: Senescence was measured using a fluorometric senescence-associated beta-galactosidase activity assay after DS-3032b treatment, normalized to cell viability[4]. Migration assay: A scratch assay was performed[4]. Cells were starved in low-serum medium, scratched, and treated with 0 or 250 nM DS-3032b for 24 h[4]. The scratched area was photographed and quantified[4]. Cell cycle analysis: Cells were treated with DS-3032b for 48 h, stained with propidium iodide and RNase, and analyzed by flow cytometry[4]. Apoptosis analysis: Cells were treated with DS-3032b for 48 h, stained with annexin V-FITC and propidium iodide, and analyzed by flow cytometry[4]. Caspase-3 activity assay: Cells were treated with DS-3032b for 48 h, lysed, and incubated with Ac-DEVD-AFC substrate[4]. Caspase-3 activity was measured fluorometrically[4]. Western blot analysis: Cells were treated with DS-3032b for 48 h, lysed, and proteins were separated by SDS-PAGE, transferred to membranes, and probed with antibodies against TP53, MDM2, CDKN1A, BAX, and Actin[4]. Microarray analysis: SH-SY5Y and IMR5 cells were treated with 125 nM DS-3032b for 24 h, and gene expression was analyzed using Affymetrix GeneChip Human Gene 2.0 ST Array[4]. |
| Animal Protocol |
Animal/Disease Models: SH-SY5Y nude mouse xenograft tumor [4]. Doses: 50 mg/kg. Dosing: po (oral gavage) for 30 days, alternating with 4 days of po (oral gavage) treatment and then 2 days of no treatment (4+2). Experimental Results: The survival rate of the mouse group was Dramatically prolonged. diminished neuroblastoma xenograft tumor growth through activation of TP53 signaling. Xenograft tumor model: SH-SY5Y cells were subcutaneously inoculated into the right flank of female athymic NCr (nu/nu) mice[4]. When tumors reached 150–250 mm³, mice were treated orally with Milademetan (DS-3032b) (50 mg/kg in 0.5% methylcellulose) or vehicle on a schedule of 4 days of daily treatment followed by 2 days off, for 30 consecutive days[4]. TP53 signaling activation study: Mice with established SH-SY5Y xenografts were treated orally with 4 doses of DS-3032b (100 mg/kg) over 36 h (0, 12, 24, 36 h)[4]. Tumors were harvested 4 h after the last dose for molecular analysis[4]. |
| Toxicity/Toxicokinetics |
In the mouse xenograft study, the DS-3032b treatment regimen was well tolerated with no observed weight loss, physical status changes, or obvious signs of toxicity[4]. Preliminary results from an ongoing phase I trial in adults with hematological malignancies indicated acceptable clinical side effects including myelosuppression, nephrological, and gastrointestinal symptoms[4]. |
| References |
[1]. ARYL SULFONOHYDRAZIDES. WO 2017069289 A1. [2]. Milademetan, an oral MDM2 inhibitor, in well-differentiated/dedifferentiated liposarcoma: results from a phase 1 study in patients with solid tumors or lymphomas. European Journal of Cancer 138S2 (2020) S1–S62. [3]. Development of novel PROTAC Small-Molecule Degraders of MDM2 Protein and Peptidomimetic Inhibitors Targeting WDR5-MLL1 Protein-Protein Interaction. [4]. Reactivating TP53 signaling by the novel MDM2 inhibitor DS-3032b as a therapeutic option for high-risk neuroblastoma. ncotarget. 2018 Jan 5; 9(2): 2304–2319. |
| Additional Infomation |
Milademetan is under investigation in clinical trial NCT02319369 (Safety, Tolerability and Pharmacokinetics of Milademetan Alone and With 5-Azacitidine (AZA) in Acute Myelogenous Leukemia (AML) or High-Risk Myelodysplastic Syndrome (MDS)). Milademetan is an orally available MDM2 (murine double minute 2) antagonist with potential antineoplastic activity. Upon oral administration, milademetan binds to, and prevents the binding of MDM2 protein to the transcriptional activation domain of the tumor suppressor protein p53. By preventing this MDM2-p53 interaction, the proteasome-mediated enzymatic degradation of p53 is inhibited and the transcriptional activity of p53 is restored. This results in the restoration of p53 signaling and leads to the p53-mediated induction of tumor cell apoptosis. MDM2, a zinc finger protein and a negative regulator of the p53 pathway, is overexpressed in cancer cells; it has been implicated in cancer cell proliferation and survival. Milademetan (DS-3032b) is an orally available, disproportionate-based MDM2 inhibitor[4]. High MDM2 expression in primary neuroblastomas correlates with poor patient survival and aggressive disease features[4]. DS-3032b reactivates TP53 signaling in neuroblastoma cells with wildtype TP53, independent of MYCN amplification status[4]. The study supports the preclinical evaluation of DS-3032b for high-risk, refractory, or relapsed neuroblastoma[4]. |
Solubility Data
| Solubility (In Vitro) | DMSO : ~16.67 mg/mL (~26.95 mM) |
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 1.67 mg/mL (2.70 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 16.7 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: 1.67 mg/mL (2.70 mM) 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; with ultrasonication. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 16.7 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. Solubility in Formulation 3: ≥ 1.67 mg/mL (2.70 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 16.7 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 | 1.6167 mL | 8.0837 mL | 16.1674 mL | |
| 5 mM | 0.3233 mL | 1.6167 mL | 3.2335 mL | |
| 10 mM | 0.1617 mL | 0.8084 mL | 1.6167 mL |