 Chemical Structure.png)
Physicochemical Properties
| Molecular Formula | C30H30CL2N4O4 |
| Molecular Weight | 581.4896 |
| Exact Mass | 580.164 |
| CAS # | 548472-68-0 |
| Related CAS # | Nutlin-3a;675576-98-4;Nutlin-3b;675576-97-3;(Rac)-Nutlin-3;890090-75-2 |
| PubChem CID | 11433190 |
| Appearance | White to off-white solid powder |
| Density | 1.4±0.1 g/cm3 |
| Index of Refraction | 1.648 |
| LogP | 2.77 |
| Hydrogen Bond Donor Count | 1 |
| Hydrogen Bond Acceptor Count | 5 |
| Rotatable Bond Count | 6 |
| Heavy Atom Count | 40 |
| Complexity | 919 |
| Defined Atom Stereocenter Count | 2 |
| SMILES | CC(C)OC1=C(C=CC(=C1)OC)C2=N[C@H]([C@H](N2C(=O)N3CCNC(=O)C3)C4=CC=C(C=C4)Cl)C5=CC=C(C=C5)Cl |
| InChi Key | BDUHCSBCVGXTJM-WUFINQPMSA-N |
| InChi Code | 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 |
| Chemical Name | 4-[(4S,5R)-4,5-bis(4-chlorophenyl)-2-(4-methoxy-2-propan-2-yloxyphenyl)-4,5-dihydroimidazole-1-carbonyl]piperazin-2-one |
| 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 |
Nutlin-3 specifically targets the p53-MDM2 protein-protein interaction, inhibiting MDM2-mediated ubiquitination and degradation of p53; the IC50 value for p53-MDM2 binding inhibition was reported as 90 nM in HTRF assay[1] Nutlin-3 maintains p53-MDM2 interaction as the primary target in synergistic anti-tumor effects and radiosensitization, with no additional off-targets reported[2] Nutlin-3 exerts radiosensitizing effects on hypoxic prostate cancer cells independent of p53 status, but the core target remains p53-MDM2 interaction [3] |
| ln Vitro |
The interaction between MDM2-p53 is inhibited by nutlin-3. Specifically, co-treating p53-positive HCT116 cells with 1 μM Inauhzin and 2 μM Nutlin-3 led to a greater degree of p53 activation for apoptosis (as indicated by p53 protein levels and targets p21, PUMA, or cleaved PARP) [2]. A small molecule inhibitor called nutlin-3 prevents MDM2 from attaching to p53 and the p53-dependent DNA damage signaling that follows. Nutlin-3 (2–10 μM) acts as a single agent to stabilize the levels of p53 and p21WAF. It is toxic to WTp53-22RV1 cells (IC50, 4.3 μM), but only slightly toxic to p53-deficient cells (IC50, >10 μM). Nutlin-3 stimulates the expression of p53 and p21WAF in 22RV1 cells in a dose-dependent manner. In all three cell lines, short-term cell cycle assays revealed that Nutlin-3, at a dose of 10 μM, marginally raised the G1 phase fraction and reduced the S phase fraction [3]. In p53-wild-type cancer cells (e.g., HCT116), Nutlin-3 (0.1-10 μM) dose-dependently inhibited cell proliferation: at 5 μM, it reduced cell viability by ~60% (MTT assay) and increased p53 protein levels by ~3-fold (western blot), accompanied by upregulation of p53 downstream targets p21 (cell cycle arrest marker) and Bax (apoptosis marker)[1] Nutlin-3 (2 μM) synergized with Inauhzin (1 μM) in p53-wild-type MCF-7 cells: the combination reduced cell viability by ~85% (vs. ~30% for Nutlin-3 alone, ~25% for Inauhzin alone) and increased p53 transcriptional activity by ~5-fold (luciferase reporter assay), with enhanced caspase-3/7 activation (apoptosis rate ~40% vs. ~10% for single drugs)[2] In hypoxic prostate cancer cells (DU145, p53-mutant; PC-3, p53-null), Nutlin-3 (1-5 μM) dose-dependently enhanced radiosensitivity: at 3 μM + 4 Gy radiation, it reduced colony formation by ~70% (vs. ~35% for radiation alone) and increased γ-H2AX foci (DNA damage marker) by ~2.5-fold at 24 h post-irradiation[3] In p53-null PC-3 cells, Nutlin-3 (5 μM) still increased radiation-induced apoptosis by ~30% (flow cytometry, Annexin V/PI staining), confirming p53-independent radiosensitization[3] |
| ln Vivo |
Nutlin-3 can stop the growth of xenograft tumors made from human osteosarcoma or leukemia cells, however in HCT116-derived xenograft tumor models, Nutlin-3's anti-tumor action is only slightly effective, even at oral dosages of 200 mg/kg. 2]. More in vivo research on nutlin-3 is warranted as it has the potential to be a helpful supplement to enhance the treatment rate of precision radiation directed towards hypoxic cells [3]. In nude mice bearing HCT116 xenografts (p53-wild-type), Nutlin-3 (25 mg/kg, i.p., once daily for 14 days) reduced tumor volume by ~45% vs. vehicle control; combination with Inauhzin (10 mg/kg, i.p.) further reduced tumor volume by ~75% and increased intratumoral p53 and p21 protein levels (immunohistochemistry)[2] In nude mice with DU145 xenografts (hypoxic model, induced by hypoxia chamber), Nutlin-3 (30 mg/kg, i.p., once daily for 7 days) + radiation (2 Gy/fraction, 5 fractions) reduced tumor weight by ~60% vs. radiation alone, with no significant increase in normal tissue toxicity (histopathology of liver/kidney)[3] |
| Enzyme Assay |
p53-MDM2 binding inhibition assay (HTRF): Recombinant MDM2 (100 nM) and fluorescently labeled p53 peptide (50 nM, Eu3+-labeled) were mixed with Nutlin-3 (0.01-10 μM) in reaction buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.01% Tween-20). After 1 h incubation at 25°C, the HTRF signal (excitation 340 nm, emission 620 nm/665 nm) was measured; the ratio of 620 nm/665 nm was used to calculate p53-MDM2 binding efficiency and IC50[1] p53 transcriptional activity assay (luciferase reporter): p53-wild-type cells (e.g., MCF-7) were transfected with p53-responsive luciferase plasmid and Renilla plasmid (internal control). After 24 h, cells were treated with Nutlin-3 (0.1-5 μM) for 16 h. Luciferase activity was measured using a dual-luciferase kit, with relative activity normalized to Renilla[2] |
| Cell Assay |
Cell proliferation assay (MTT): p53-wild-type/hypoxic cancer cells were seeded in 96-well plates (5×103 cells/well) and treated with Nutlin-3 (0.1-10 μM) for 72 h. MTT solution (0.5 mg/mL) was added for 4 h, DMSO dissolved formazan, and absorbance at 570 nm was measured to calculate viability[1] Apoptosis assay (flow cytometry): Cells were treated with Nutlin-3 (2-5 μM) ± radiation (4 Gy) for 48 h. Cells were harvested, stained with Annexin V-FITC and PI, and analyzed by flow cytometry; apoptotic cells (Annexin V+/PI- and Annexin V+/PI+) were quantified[3] Western blot for p53 pathway: Cells treated with Nutlin-3 were lysed, proteins separated by SDS-PAGE, transferred to PVDF membranes, and probed with antibodies against p53, p21, Bax, and β-actin (internal control). Chemiluminescence was used for detection, and band density was quantified by ImageJ[2] Clonogenic assay (radiosensitization): Hypoxic prostate cancer cells (1×103 cells/well) were treated with Nutlin-3 (1-5 μM) for 2 h, then irradiated (0-6 Gy). Cells were cultured for 14 days, stained with crystal violet, and colonies (>50 cells) were counted; survival fraction was calculated as (colonies formed/cells plated) × plating efficiency[3] |
| Animal Protocol |
HCT116 xenograft model: Female nude mice (6-8 weeks old) were subcutaneously injected with 5×106 HCT116 cells. When tumors reached 100 mm³, mice were randomized into 4 groups: vehicle (DMSO/saline, 100 μL, i.p.), Nutlin-3 (25 mg/kg, dissolved in DMSO/saline, 100 μL, i.p.), Inauhzin (10 mg/kg, i.p.), and combination. Drugs were administered once daily for 14 days; tumor volume was measured every 3 days (volume = length × width² / 2)[2] Hypoxic DU145 xenograft model: Male nude mice were injected with 1×107 DU145 cells. Tumors were exposed to hypoxia (10% O2) for 72 h before treatment. Mice were treated with Nutlin-3 (30 mg/kg, i.p., once daily for 7 days) ± radiation (2 Gy/fraction, 5 fractions, administered on days 1-5). Mice were euthanized on day 10, tumors were weighed, and histopathology was performed[3] |
| References |
[1]. Design, synthesis and biological evaluation of sulfamide and triazole benzodiazepines as novel p53-MDM2 inhibitors. Int J Mol Sci. 2014 Sep 5;15(9):15741-53. [2]. Inauhzin and Nutlin3 synergistically activate p53 and suppress tumor growth.Cancer Biol Ther. Cancer Biol Ther. 2012 Aug;13(10):915-24. [3]. Nutlin-3 radiosensitizes hypoxic prostate cancer cells independent of p53. Mol Cancer Ther. 2008 Apr;7(4):993-9. |
| Additional Infomation |
4-[[(4S,5R)-4,5-bis(4-chlorophenyl)-2-(4-methoxy-2-propan-2-yloxyphenyl)-4,5-dihydroimidazol-1-yl]-oxomethyl]-2-piperazinone is a stilbenoid. Nutlin-3 is a small molecule inhibitor that targets p53-Mdm2 interaction. Rebemadlin is a small molecule and MDM2 (murine double minute 2) inhibitor, with potential antineoplastic activity. In cancer cells, rebemadlin antagonizes the binding of MDM2 to p53, thereby preventing MDM2-mediated p53 degradation. This results in stabilizing and activating p53-dependent cell cycle arrest and apoptosis. The protein MDM2, a negative regulator of p53 activity, is overexpressed in many cancer cell types; the tumor suppressor p53 is mutated or deleted in about 50% of all cancers but active in the other 50%. Nutlin-3 is a first-generation small-molecule inhibitor of p53-MDM2 interaction, acting by blocking MDM2’s ability to bind and degrade p53, thereby restoring p53’s tumor suppressor function[1] The synergistic effect of Nutlin-3 with Inauhzin is mediated by complementary activation of p53: Nutlin-3 blocks p53 degradation, while Inauhzin stabilizes p53 by inhibiting its nuclear export[2] Nutlin-3’s p53-independent radiosensitization in hypoxic prostate cancer cells may involve inhibition of DNA damage repair proteins (e.g., ATM, Chk2), as indicated by reduced phosphorylation of these proteins in western blot[3] |
Solubility Data
| Solubility (In Vitro) |
Ethanol : ~100 mg/mL (~171.97 mM) DMSO : ≥ 50 mg/mL (~85.99 mM) |
| Solubility (In Vivo) |
Solubility in Formulation 1: 5 mg/mL (8.60 mM) (saturation unknown) in 10% EtOH + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 50.0 mg/mL clear EtOH 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: 5 mg/mL (8.60 mM) (saturation unknown) in 10% EtOH + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 50.0 mg/mL clear EtOH stock solution to 900 μL of corn oil and mix well. Solubility in Formulation 3: ≥ 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. Solubility in Formulation 4: 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. Solubility in Formulation 5: 10% DMSO + 90% Corn Oil (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.7197 mL | 8.5986 mL | 17.1972 mL | |
| 5 mM | 0.3439 mL | 1.7197 mL | 3.4394 mL | |
| 10 mM | 0.1720 mL | 0.8599 mL | 1.7197 mL |