Nutlin-3b, a less active/inactive isomer of nutlin-3, is a novel antagonist or inhibitor of p53/MDM2 (murine double minute 2) protein protein interaction with IC50 value of 13.6 μM. Compared to Nutlin-3a, it is 150 times less potent.

Physicochemical Properties

Molecular Formula	C30H30CL2N4O4
Molecular Weight	581.49
Exact Mass	580.164
Elemental Analysis	C, 61.97; H, 5.20; Cl, 12.19; N, 9.64; O, 11.01
CAS #	675576-97-3
Related CAS #	Nutlin-3a;675576-98-4;Nutlin-3;548472-68-0
PubChem CID	16755649
Appearance	Off-white to yellow 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	O=C1NCCN(C(N2[C@@H](C3=CC=C(Cl)C=C3)[C@@H](C4=CC=C(Cl)C=C4)N=C2C5=CC=C(OC)C=C5OC(C)C)=O)C1
InChi Key	BDUHCSBCVGXTJM-IZLXSDGUSA-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+/m1/s1
Chemical Name	4-[(4R,5S)-4,5-bis(4-chlorophenyl)-2-(4-methoxy-2-propan-2-yloxyphenyl)-4,5-dihydroimidazole-1-carbonyl]piperazin-2-one
Synonyms	(+)-Nutlin-3; Nutlin 3b; Nutlin-3b; Nutlin3b
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	MDM2/p53 (IC50 = 13.6 μg/mL)
ln Vitro	Nutlin-3b is useful as a negative control s for non-MDM2-related cellular activities.Only in cells that have p53 that is in the wild-type form does nutlin-3a induce the expression of MDM2 and p21 (but not p53). Regardless of whether the cells have p53 or not, nutlin-3b has no impact. Only the active enantiomer Nutlin-3a distinguishes between cells harboring mutant and wild-type p53 and exhibits strong antiproliferative activity. Nutlin-3b's potency against mutant p53 cells is almost identical to that of Nutlin-3a, while it is significantly less potent against wild-type p53 cells. After 48 hours of exposure to Nutlin-3a, 45% of the cell population became TUNEL positive, but cells treated with Nutlin-3b are identical to the untreated controls. [1]
ln Vivo
Cell Assay	On a Biacore S51, competition assay is conducted. An immobilized PentaHis antibody is used to capture the His-tagged p53 on a Series S Sensor chip CM5. A response unit equals one pg of protein per millimeter 2, so the level of capture is around 200. MDM2 protein is kept at a constant 300 nM concentration. Each MDM2 test sample contains a concentration series of Nutlin-3 after it has been dissolved in DMSO at a 10 mM concentration and further diluted. Running buffer (10 mM Hepes, 0.15 M NaCl, 2% DMSO) is used for the assay, which is carried out at 25 °C. Calculating IC50 involves calculating the percentage of MDM2-p53 binding in the presence of Nutlin-3 compared to binding in the absence of Nutlin-3.
Animal Protocol	HCT116, RKO, SJSA-1, SW480, and MDA-MB-435 ~30 μM; 48 h MTT assay
References	[1]. In vivo activation of the p53 pathway by small-molecule antagonists of MDM2. Science. 2004 Feb 6;303(5659):844-8.
Additional Infomation	Nutlin-3 is a Nutlin and a piperazinone. It has a role as an anticoronaviral agent. Nutlin-3 is a small molecule inhibitor that targets p53-Mdm2 interaction.

Solubility Data

Solubility (In Vitro)

DMSO: ~100 mg/mL (~172.0 mM) Water: <1 mg/mL (slightly soluble or insoluble) Ethanol: ~100 mg/mL (~172.0 mM)

Solubility (In Vivo)

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. Solubility in Formulation 2: ≥ 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.  (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

*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.