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].
The primary target of milademetan is MDM2 (murine double minute 2), an E3 ubiquitin ligase that negatively regulates the tumor suppressor p53. Milademetan is a potent and selective inhibitor of the MDM2-p53 protein-protein interaction .

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].

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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].

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Milademetan demonstrates potent in vitro activity across various cancer cell lines. It reduces cell viability specifically in TP53 wild-type (WT) cell lines. In Merkel cell carcinoma models, milademetan triggered a rapid and sustained p53 response and showed dose-dependent inhibition of tumor cell growth . Preclinical testing revealed that cell lines with CDKN2A homozygous loss in the context of wild-type TP53 exhibit significantly higher sensitivity, with a median IC50 of 79.5 nM compared to 10,000 nM in cell lines with mutant TP53 or non-homozygous loss of CDKN2A . The compound also showed potent activity against MDM2-amplified, TP53-wildtype laboratory models .

Milademetan (DS-3032b, 50 mg/kg, orally administered) xenografts neuroblastoma cells with functioning TP53, delaying tumor growth and enhancing survival [4].

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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].

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In vivo, milademetan demonstrated significant anti-tumor activity. It showed dose-dependent inhibition of tumor growth in MKL-1 xenograft and patient-derived xenograft (PDX) models of Merkel cell carcinoma . Xenograft models with CDKN2A homozygous loss and wild-type TP53 all demonstrated tumor growth inhibition with milademetan. Furthermore, combination with an anti-PD1 antibody in a colon-26 syngeneic model (with CDKN2A homozygous loss) showed significant enhancement in tumor growth inhibition compared to either agent alone .

While detailed assay protocols are not publicly available in the provided documents, milademetan is characterized as a selective inhibitor of the MDM2-p53 protein-protein interaction. The compound works by binding to MDM2 and preventing its interaction with the transcriptional activation domain of p53, thereby inhibiting the proteasome-mediated enzymatic degradation of p53 .

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.

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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].

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In vitro cell assays were performed using cancer cell lines treated with milademetan to assess viability and p53 pathway activation. Cell viability was measured using standard assays, and p53 response was evaluated by assessing downstream target gene expression or protein stabilization. The sensitivity of 215 cancer cell lines to milademetan treatment was evaluated to determine IC50 values based on TP53 and CDKN2A status .

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.

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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].

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Preclinical efficacy was evaluated using xenograft models. Immunocompromised mice were implanted with human tumor cell lines (such as MKL-1) or patient-derived tumor tissue. Milademetan was administered orally, typically once daily, using various intermittent or continuous dosing schedules. Tumor growth inhibition was monitored over time. In combination studies, anti-PD1 antibody was administered via intraperitoneal injection .

Milademetan is an orally bioavailable small molecule. In the first-in-human phase I study, pharmacokinetic analyses were conducted to characterize its absorption and exposure profile. The recommended phase II dose and schedule (260 mg once daily on days 1-3 and 15-17 every 28 days, i.e., a 3/14 day intermittent schedule) was selected based on PK/PD data to mitigate dose-limiting toxicities while maintaining efficacy .

In mouse xenograft studies, the DS-3032b treatment regimen was well tolerated, with no observed weight loss, changes in physical condition, or significant signs of toxicity.[4] Preliminary results from an ongoing Phase I clinical trial in adult patients with hematologic malignancies indicate that clinical side effects are acceptable, including myelosuppression, renal and gastrointestinal symptoms.[4]

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The most common grade 3/4 treatment-emergent adverse events observed in clinical trials include thrombocytopenia (29.0%), neutropenia (15.0%), and anemia (13.1%). An intermittent dosing schedule (3/14 days) significantly reduced the rates of these high-grade hematologic abnormalities (thrombocytopenia: 15.0%, neutropenia: 5.0%, anemia: 0%) while maintaining efficacy. Other reported adverse events included gastrointestinal issues, fatigue, diarrhea, leukopenia, and renal/electrolyte abnormalities .

[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.

Milademetan is currently being investigated in the clinical trial NCT02319369 (Safety, tolerability, and pharmacokinetics of milademetan monotherapy and in combination with 5-azacytidine (AZA) for the treatment of acute myeloid leukemia (AML) or high-risk myelodysplastic syndrome (MDS)). Milademetan is an orally potent MDM2 (mouse double microsome 2) antagonist with potential antitumor activity. After oral administration, milademetan binds to the MDM2 protein and prevents MDM2 from binding to the transcriptional activation domain of the tumor suppressor protein p53. By inhibiting the MDM2-p53 interaction, proteasome-mediated p53 enzymatic degradation is inhibited, thereby restoring p53 transcriptional activity. This ultimately leads to the restoration of the p53 signaling pathway and induces p53-mediated tumor cell apoptosis. MDM2 is a zinc finger protein and a negative regulator of the p53 pathway, overexpressed in cancer cells; it is closely related to cancer cell proliferation and survival.

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Milademestan (DS-3032b) is an orally administered, nonproportional MDM2 inhibitor[4].
High expression of MDM2 in primary neuroblastoma is associated with poor patient survival and aggressive disease characteristics[4].
DS-3032b reactivates the TP53 signaling pathway in neuroblastoma cells with wild-type TP53, regardless of MYCN amplification status[4].
This study supports preclinical evaluation of DS-3032b for the treatment of high-risk, refractory, or recurrent neuroblastoma[4].

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Milademetan has been evaluated in multiple clinical trials including MANTRA (phase III in liposarcoma), MANTRA-2 (phase II in MDM2-amplified solid tumors), and MANTRA-4 (planned for combination with atezolizumab). In a phase II trial of MDM2-amplified, TP53-wildtype solid tumors, the objective response rate was 19.4% (6/31), including a patient with endometrial stromal sarcoma who achieved 100% target lesion reduction. The median progression-free survival was 3.5 months . In dedifferentiated liposarcoma, the disease control rate was 58.5% with median PFS of 7.2 months . In myeloid malignancies, clinical efficacy was minimal despite being relatively well-tolerated .

C₃₀H₃₄CL₂FN₅O₄

618.526468753815

617.197

C, 58.26; H, 5.54; Cl, 11.46; F, 3.07; N, 11.32; O, 10.35

1398568-47-2

Milademetan tosylate hydrate;2095625-97-9; 1398568-47-2; 1398569-75-9

73297272

White to light yellow solid powder

1.4±0.1 g/cm3

840.7±65.0 °C at 760 mmHg

462.2±34.3 °C

0.0±3.1 mmHg at 25°C

1.646

3.54

4

7

4

42

1090

5

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

RYAYYVTWKAOAJF-QISPRATLSA-N

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

(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

DS-3032B; DS 3032B; Milademetan; 1398568-47-2; MDM2 inhibitor DS-3032; R3I80TLN7S; DS3032B; DS-3032; DS 3032; DS3032; Milademetan free base

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 : ~16.67 mg/mL (~26.95 mM)

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.

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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.

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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.

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