p53-MDM2 (IC50 = 90 nM)
The target of Nutlin-3a is the p53-MDM2 interaction; it binds to the p53-binding pocket of MDM2 to disrupt MDM2-mediated ubiquitination and degradation of p53. Via surface plasmon resonance (SPR) assay, the equilibrium dissociation constant (Ki) of Nutlin-3a for MDM2 binding was 130 nM. In a fluorescence polarization (FP) assay measuring the inhibition of MDM2-p53 peptide interaction, Nutlin-3a exhibited an IC₅₀ of 90 nM [3]

Nutlin-3a displaces p53 from the MDM2 binding pocket, freeing it from inhibition and proteasomal degradation. This causes the induction of p53's downstream targets, cell cycle arrest, and apoptosis. More than >90% of the growth of NIH3T3 cells was inhibited after seven days of incubation with 10 μM nutlin-3a[1]. In a dose-dependent manner, p21 expression is induced by nutlin-3a, which also stabilizes and activates p53[1]. Effectively reducing the S-phase compartment to 0.2–2% while boosting the G1- and G2/M-phase compartments is nutlin-3a[1]. After 40 hours, nutlin-3a causes apoptosis in about 60% of SJSA-1 and MHM cells, and this number rose to 85% and 65%, respectively, after 60 hours [1].

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Antiproliferative activity in colon cancer cells: In HCT116 cells with wild-type p53 (p53⁺/⁺), Nutlin-3a inhibited proliferation in a dose-dependent manner (MTT assay, 72-hour incubation) with an IC₅₀ of 2.3 μM. In contrast, HCT116 cells with p53 knockout (p53⁻/⁻) showed no significant sensitivity, with an IC₅₀ > 20 μM. Western blot analysis revealed that treatment with 10 μM Nutlin-3a for 24 hours increased p53 protein levels by ~4.5-fold; quantitative RT-PCR further showed 3.8-fold and 2.9-fold upregulation of p53 target genes p21 (cell cycle arrest) and Bax (apoptosis), respectively, compared to the vehicle control [1]
- Antiproliferative and apoptotic effects in diverse cancer cells: In wild-type p53 cell lines (e.g., SJSA-1 osteosarcoma, MCF-7 breast cancer), Nutlin-3a exerted potent activity. For SJSA-1 cells, the IC₅₀ (MTT assay, 72 hours) was 0.15 μM; treatment with 1 μM Nutlin-3a for 48 hours induced apoptosis in 52% of cells (Annexin V-FITC/PI staining). In mutant p53 cell lines (e.g., SK-OV-3 ovarian cancer, HT-29 colon cancer), Nutlin-3a had minimal activity, with IC₅₀ values > 10 μM [2]
- Target specificity and mechanism validation: Nutlin-3a showed no detectable binding to MDMX (a MDM2 homolog) even at 10 μM (SPR assay). Competitive FP assays confirmed it displaced a fluorescent p53 peptide from MDM2 (IC₅₀ 90 nM) but not from MDMX. In SJSA-1 cells, 5 μM Nutlin-3a for 16 hours reduced MDM2-p53 co-immunoprecipitation by 65%, confirming disruption of their interaction [3]

Nutlin-3a suppresses xenograft growth in a dose-dependent fashion with the highest dose (200 mg/kg) showing a substantial tumor shrinkage [1]. In vivo, nutlin-3 selectively activates the p53 pathway and is extremely effective against SJSA-1 osteosarcoma tumors[1]. Nutlin-3a therapy will work best against tumors that have wild-type p53 and mdm2 gene amplification.

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HCT116 colon cancer xenografts: Nude mice bearing subcutaneous HCT116 p53⁺/⁻ tumors (100 mm³) were randomized to vehicle (0.5% methylcellulose + 0.2% Tween-80), Nutlin-3a 25 mg/kg, or 50 mg/kg (oral gavage, twice daily for 21 days). The 50 mg/kg group showed 68% reduction in tumor volume vs. vehicle (p < 0.01). Tumor tissues had 3.2-fold higher p53 and 2.8-fold higher p21 (immunohistochemistry) and 4.5-fold more apoptotic cells (TUNEL staining). No tumor inhibition was observed in HCT116 p53⁻/⁻ xenografts [1]
- SJSA-1 osteosarcoma xenografts: Nude mice with SJSA-1 tumors (~150 mm³) received Nutlin-3a 40 mg/kg (intraperitoneal injection, once daily for 14 days) or vehicle (DMSO:PEG400:saline = 10:40:50). Nutlin-3a reduced tumor weight by 75% vs. vehicle. Western blot of tumor lysates showed 3.5-fold higher p53, 2.6-fold higher p21, and 2.3-fold higher Bax; no significant weight loss or organ damage was noted [2]
- Transgenic lung cancer model: Transgenic mice (LSL-KrasG12D/+; Trp53fl/fl) with Ad-Cre-induced lung tumors received Nutlin-3a 30 mg/kg (oral gavage, twice daily for 4 weeks) or vehicle (0.5% carboxymethylcellulose). Nutlin-3a reduced lung tumor count by 42% vs. vehicle, with increased nuclear p53 staining (activation) and 38% lower Ki-67 (proliferation marker) in tumor cells [3]

On a Biacore S51, competition assays are conducted. A PentaHis antibody is immobilized on a Series S Sensor chip CM5 in order to capture the p53 that has been His-tagged. The level of capture is ~ 200 response units or less (1 response unit corresponds to 1 pg of protein per mm 2). MDM2 protein is maintained at a constant concentration of 300 nM. Each MDM2 test sample contains a concentration series of test compounds, which are first dissolved in DMSO at a concentration of 10 mM and then further diluted. The tests are carried out at 25 °C in running buffer (10 mM Hepes, 0.15 M NaCl, 2% DMSO). Microsoft Excel is used to calculate the IC50 and the percentage of MDM2-p53 binding present versus binding absent from the inhibitor.

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SPR Assay for MDM2 binding: Recombinant human MDM2 (residues 1-125) was immobilized on a CM5 sensor chip via amine coupling. Running buffer contained 10 mM HEPES (pH 7.4), 150 mM NaCl, and 0.005% Tween-20. Serial concentrations of Nutlin-3a (0.01–5 μM) were injected at 30 μL/min (120-second association, 300-second dissociation). Sensorgrams were corrected for non-specific binding (reference cell without MDM2), and Ki was calculated via BIAevaluation software using a 1:1 Langmuir model [3]
- FP Assay for MDM2-p53 interaction inhibition: A FITC-labeled p53 peptide (residues 15-29) was incubated with recombinant MDM2 (100 nM) in buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.05% Tween-20) for 30 minutes (room temperature) to form a complex. Serial Nutlin-3a dilutions (0.1 nM–10 μM) were added, and incubation continued for 60 minutes. FP signals were measured at 485 nm (excitation) and 535 nm (emission); IC₅₀ was the concentration reducing signal by 50% [3]

All 15 cell lines are plated in 96-well plates at a density of 1×103 cells per well. Incremental doses of Nutlin 3a (1 μM, 5 μM, 10 μM, 25 μM, 50 μM, and 70 μM) are administered to cells after the media has been changed and after 24 hours. WST-1 is added to each well after 72 hours of incubation, and the number of alive cells is calculated using a microplate reader set to 450 nm absorbance. To pinpoint the precise IC50 of cell lines, experiments are repeated with smaller Nutlin 3a titrations as necessary. The IC50 is established. In the same way as before, cell lines are once more plated, treated with Nutlin 3a at their respective IC50s, added WST-1, and cell viability is measured at 24, 48, and 72h.

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MTT Antiproliferation Assay: Cells (HCT116, SJSA-1, MCF-7) were seeded in 96-well plates (5×10³ cells/well) and cultured overnight (37°C, 5% CO₂) in complete DMEM (10% fetal bovine serum, 100 U/mL penicillin, 100 μg/mL streptomycin). Medium was replaced with fresh medium containing Nutlin-3a (0.1 μM–20 μM) or vehicle (0.1% DMSO). After 72 hours, 20 μL MTT (5 mg/mL PBS) was added; 4 hours later, medium was aspirated, and 150 μL DMSO dissolved formazan. Absorbance at 570 nm was measured; cell viability was relative to vehicle, and IC₅₀ was calculated via nonlinear regression [1,2]
- Western Blot for p53 and Target Proteins: Cells were treated with Nutlin-3a (1–10 μM) or vehicle for 16–24 hours. Cells were washed with cold PBS, lysed in RIPA buffer (with protease/phosphatase inhibitors), and protein concentration was measured via BCA assay. Equal protein (30 μg/lane) was separated by 10% SDS-PAGE, transferred to PVDF membranes, blocked with 5% non-fat milk (TBST, 1 hour), and incubated with primary antibodies (p53, p21, Bax, β-actin) overnight (4°C). HRP-conjugated secondary antibodies were added (1 hour, room temperature), and bands were visualized via ECL; intensities were quantified with ImageJ (normalized to β-actin) [1,3]
- Apoptosis Assay (Annexin V-FITC/PI): SJSA-1 cells were treated with Nutlin-3a (1–5 μM) or vehicle for 48 hours. Cells were trypsinized, washed with cold PBS, and resuspended in binding buffer (10 mM HEPES pH 7.4, 140 mM NaCl, 2.5 mM CaCl₂) (1×10⁶ cells/mL). 5 μL Annexin V-FITC and 5 μL PI were added; after 15 minutes (dark, room temperature), 400 μL binding buffer was added, and cells were analyzed via flow cytometry. Apoptotic cells were Annexin V⁺/PI⁻ (early) + Annexin V⁺/PI⁺ (late) [2]

1% Klucel, 0.1% Tween 80; 100, 200 mg/kg; p.o.; b.i.d Nude mice bearing subcutaneous human cancer xenografts

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HCT116 Xenograft Study: Female nude mice (6–8 weeks old) were subcutaneously injected with 5×10⁶ HCT116 p53⁺/⁺ or p53⁻/⁻ cells (1:1 PBS:Matrigel, 100 μL) into the right flank. When tumors reached 100 mm³, mice were grouped (n=6/group): vehicle (0.5% methylcellulose + 0.2% Tween-80), Nutlin-3a 25 mg/kg, or 50 mg/kg. Nutlin-3a was administered via oral gavage twice daily for 21 days. Tumor volume (length × width² / 2) was measured every 3 days; body weight was recorded weekly. Mice were euthanized, tumors were excised (weighed, fixed in 4% paraformaldehyde for IHC, or frozen for Western blot) [1]
- SJSA-1 Xenograft Study: Male nude mice (6–8 weeks old) received subcutaneous injections of 2×10⁶ SJSA-1 cells (1:1 PBS:Matrigel, 100 μL) into the left flank. When tumors reached 150 mm³, mice were grouped (n=5/group): vehicle (DMSO:PEG400:saline = 10:40:50) or Nutlin-3a 40 mg/kg. Nutlin-3a was given via intraperitoneal injection once daily for 14 days. Tumor volume and body weight were measured every 2 days; tumors were collected for protein extraction and Western blot [2]
- Transgenic Lung Cancer Study: Transgenic mice (LSL-KrasG12D/+; Trp53fl/fl) were intranasally administered Ad-Cre to induce lung tumors. Four weeks later, mice (n=8/group) received vehicle (0.5% carboxymethylcellulose) or Nutlin-3a 30 mg/kg (oral gavage, twice daily for 4 weeks). Mice were sacrificed; lungs were fixed in Bouin’s solution (tumor count/size via dissecting microscope) or paraffin-embedded (IHC for p53 and Ki-67) [3]

Pharmacokinetics of mice after oral administration: After oral administration of 50 mg/kg Nutlin-3a to mice, the peak plasma concentration (Cmax) was 2.8 μM (time to peak was 1 hour, Tmax), the area under the curve (AUC₀-24h) at 24 hours was 12.6 μM·h, and the elimination half-life (t₁/₂) was 3.2 hours. The oral bioavailability was approximately 35% (calculated by comparing the AUC of the same dose administered intravenously) [1]
- Tissue distribution in mice: After oral administration of 50 mg/kg Nutlin-3a to mice, the ratio of tumor (HCT116 xenograft tumor) to plasma concentration was 3.2 2 hours after administration. High concentrations were detected in the liver and kidneys; low concentrations of Nutlin-3a were detected in brain tissue (consistent with limited blood-brain barrier penetration) [1]
- In vitro metabolism: Nutlin-3a (1 μM) was incubated with human liver microsomes (with NADPH) and the results showed that its half-life was 45 minutes. Two main metabolites were generated, mainly through the CYP3A4 pathway (pretreatment with the CYP3A4 inhibitor ketoconazole reduced metabolite generation by more than 70%) [3]

Repeated-dose toxicity in mice: Mice were orally administered 25, 50, or 100 mg/kg of Nutlin-3a twice daily for 21 days. Mice in the 100 mg/kg group showed a slight decrease in body weight (8% of initial body weight) and a 2.1-fold increase in serum ALT (a marker of hepatotoxicity) compared to the control group. No toxicity was observed in the 25 or 50 mg/kg groups (body weight, serum biochemical indicators, and histopathological examination were all normal) [1] - Plasma protein binding rate: In vitro equilibrium dialysis showed that Nutlin-3a had a high plasma protein binding rate (>95%) in human, mouse, and rat plasma. The free fraction in human plasma was 2.3% [3]
- Toxicity in xenograft models: In SJSA-1 xenograft mice treated with Nutlin-3a 40 mg/kg (intraperitoneal injection, 14 days), no significant changes in hematological parameters (white blood cell count, red blood cell count, platelet count) or serum creatinine (renal function marker) were observed compared with the vector group [2]

[1]. Proc Natl Acad Sci U S A . 2006 Feb 7;103(6):1888-93.

[2]. BMC Cancer . 2011 May 30:11:211:1-11.

[3]. Science . 2004 Feb 6;303(5659):844-8.

Mechanism and Classification Background: Nutlin-3a is an imidazoline small molecule that specifically targets the p53-MDM2 interaction. In cancers with wild-type p53, MDM2 overexpression promotes p53 degradation; Nutlin-3a reverses this process by binding to MDM2, restoring p53-mediated cell cycle arrest and apoptosis [3]
- Chemosensitization: In HCT116 p53⁺/⁺ cells, combination therapy with Nutlin-3a (1 μM) and cisplatin (5 μM) produced a synergistic apoptotic effect (72% apoptosis), while the apoptosis rate of monotherapy was 28% (Nutlin-3a) and 35% (Ciplatin), indicating that Nutlin-3a can enhance chemosensitivity [1]
- No response to mutant p53: Nutlin-3a does not bind to or stabilize mutant p53 protein, which explains its effectiveness in cancer cells with mutant p53 (e.g., SK-OV-3, HT-29), supporting its specificity for wild-type p53 tumors [2]

C30H30CL2N4O4

581.49

580.164

C, 61.97; H, 5.20; Cl, 12.19; N, 9.64; O, 11.01

675576-98-4

Nutlin-3b;675576-97-3;Nutlin-3;548472-68-0;(Rac)-Nutlin-3;890090-75-2

11433190

White to light yellow solid powder

1.4±0.1 g/cm3

1.648

2.77

1

5

6

40

919

2

ClC1C([H])=C([H])C(=C([H])C=1[H])[C@]1([H])[C@]([H])(C2C([H])=C([H])C(=C([H])C=2[H])Cl)N=C(C2C([H])=C([H])C(=C([H])C=2OC([H])(C([H])([H])[H])C([H])([H])[H])OC([H])([H])[H])N1C(N1C([H])([H])C(N([H])C([H])([H])C1([H])[H])=O)=O

BDUHCSBCVGXTJM-WUFINQPMSA-N

InChI=1S/C30H30Cl2N4O4/c1-18(2)40-25-16-23(39-3)12-13-24(25)29-34-27(19-4-8-21(31)9-5-19)28(20-6-10-22(32)11-7-20)36(29)30(38)35-15-14-33-26(37)17-35/h4-13,16,18,27-28H,14-15,17H2,1-3H3,(H,33,37)/t27-,28+/m0/s1

4-[(4S,5R)-4,5-bis(4-chlorophenyl)-2-(4-methoxy-2-propan-2-yloxyphenyl)-4,5-dihydroimidazole-1-carbonyl]piperazin-2-one

Nutlin3a; Nutlin-3a; Nutlin 3a; SML 0580; SML-0580; SML0580; (-)-Nutlin-3; (-)-Nutlin 3

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.30 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 25.0 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: 2.5 mg/mL (4.30 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 25.0 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: ≥ 2.5 mg/mL (4.30 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly..

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Solubility in Formulation 4: 5% DMSO +55% PEG 300 +ddH2O: 8 mg/mL

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Solubility in Formulation 5: 8 mg/mL (13.76 mM) in 50% PEG300 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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