Eprenetapopt (APR-246; PRIMA-1MET), a methylated PRIMA-1 derivative and mutant p53 reactivator, is a brand-new and powerful small molecule drug that can restore mutant p53's wild-type conformation and function while also inducing apoptosis in tumor cells. It has the potential to treat different p53-dependent human tumor types. While having no effect on cell lines expressing wild-type P53, PRIMA-1 inhibited the growth of Saos-2-His-273 osteosarcoma cells. According to a gel shift assay, PRIMA-1 directly binds to mutant p53. Hsp70, PML, CBP, and p53 were all nucleolarly translocated in MCF 7 cells by PRIMA-1MET. Eprenetapopt was recently described as a ferroptosis inducer with anticancer properties.

Physicochemical Properties

Molecular Formula	C10H17NO3
Molecular Weight	199.25
Exact Mass	199.12
Elemental Analysis	C, 60.28; H, 8.60; N, 7.03; O, 24.09
CAS #	5291-32-7
Related CAS #	PRIMA-1;5608-24-2
PubChem CID	52918385
Appearance	White to off-white solid powder
Density	1.2±0.1 g/cm3
Boiling Point	313.8±17.0 °C at 760 mmHg
Flash Point	143.6±20.9 °C
Vapour Pressure	0.0±1.5 mmHg at 25°C
Index of Refraction	1.537
LogP	0.61
Hydrogen Bond Donor Count	1
Hydrogen Bond Acceptor Count	4
Rotatable Bond Count	3
Heavy Atom Count	14
Complexity	236
Defined Atom Stereocenter Count	0
SMILES	O=C1C2([H])C([H])([H])C([H])([H])N(C([H])([H])C2([H])[H])C1(C([H])([H])O[H])C([H])([H])OC([H])([H])[H]
InChi Key	BGBNULCRKBVAKL-UHFFFAOYSA-N
InChi Code	InChI=1S/C10H17NO3/c1-14-7-10(6-12)9(13)8-2-4-11(10)5-3-8/h8,12H,2-7H2,1H3
Chemical Name	2-(hydroxymethyl)-2-(methoxymethyl)-1-azabicyclo[2.2.2]octan-3-one
Synonyms	APR-246; Eprenetapopt; APR246; APR 246; PRIMA-1Met
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	p53 activator; TrxR1 inhibitor
ln Vitro	APR-246 (PRIMA-1MET) is the first clinical-stage compound that reactivates mutant p53 and induces apoptosis. APR-246 is a prodrug that is transformed into the active ingredient methylene quinuclidinone (MQ), a Michael acceptor that binds to cysteine residues in mutant p53 and returns it to its wild-type conformation. Drug-resistant ovarian cancer cells with p53 mutations are completely restored to cisplatin and doxorubicin sensitivity by APR-246. Along with reactivating p53, it also dose-dependently lowers intracellular glutathione levels. By increasing ROS and ER stress, inhibiting thioredoxin reductase 1 (TrxR1), and inducing ROS and ER stress, APR-246 can cause apoptosis in a p53-independent manner. Additionally, it was noted that APR-246 alters the GSH/ROS balance in myeloma cells, causing cell death regardless of p53 status[1]. In vitro, PRIMA-1Met/APR-246 effectively stopped the growth of SCLC cell lines expressing mutant p53. It also caused apoptosis, which is characterized by an increase in the proportion of DNA-fragmented cells, caspase-3 activation, PARP cleavage, upregulation of Bax and Noxa, and downregulation of Bcl-2 in the cells[2].
ln Vivo	APR-246 demonstrated a favorable safety profile in a Phase I/II clinical dose-finding study on prostate cancer and hematological malignancies, and both clinical and p53-dependent biological responses were noted. APR-246 is well tolerated in studies on animals. When mice with the fast-growing A2780-CP20 tumor xenografts are given just one dose of APR-246, the tumor size is reduced by 21%[1].
Enzyme Assay	Cells are plated in six-well plates at a density of 15 000 cells per cm2. Next day, cells are treated with different concentrations of APR-246 (0, 25, 50, 75 and 100 μM) and harvested after 4, 12 and 24 h. The cells are lysed, and the clarified supernatants are used for either analysis of TrxR enzymatic activities or western blot. Total protein concentrations are determined with a Bradford reagent kit. Cellular TrxR activity is measured using an adapted Trx-dependent end point insulin reduction assay for microwell plates.
Cell Assay	In 12-well plates, 3 ml of medium and 75 000 OVCAR-3 cells were plated per well. The cells were treated for 20 hours with cisplatin, APR-246, or both the following day after 2.5 ml of the medium had been removed. The following day, cells were collected by trypsinization, twice-washed, and stained with Annexin V and propidium iodine (PI). The samples were stained, and then the LSRII flow cytometer was used to analyze them.
Animal Protocol	Dissolved in PBS; 400 mg/kg/day; i.v. injection CD-1 Nu/Nu mice
References	[1]. Cell Death Dis . 2015 Jun 18;6(6):e1794. [2]. Clin Cancer Res . 2011 May 1;17(9):2830-41.
Additional Infomation	Eprenetapopt has been used in trials studying the treatment of Prostatic Neoplasms, Hematologic Neoplasms, and Platinum Sensitive Recurrent High-grade Serous Ovarian Cancer With Mutated p53. Eprenetapopt is a methylated derivative and structural analog of PRIMA-1 (p53 re-activation and induction of massive apoptosis), with potential antineoplastic activity. Upon administration, eprenetapopt covalently modifies the core domain of mutated forms of cellular tumor antigen p53 (p53) through the alkylation of thiol groups. These modifications restore both the wild-type conformation and function to mutant p53, which reconstitutes endogenous p53 activity, leading to cell cycle arrest and apoptosis in tumor cells. This agent may work synergistically with other antineoplastic agents. p53, a tumor suppressor and transcription factor normally activated upon DNA damage, is frequently mutated and overexpressed in cancer cells; it plays a key role in both DNA repair and the induction of apoptosis.

Solubility Data

Solubility (In Vitro)

DMSO: >10mg/mL Water: <1 mg/mL Ethanol: <1 mg/mL

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.5 mg/mL (12.55 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 (12.55 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 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: ≥ 2.5 mg/mL (12.55 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. Solubility in Formulation 4: 100 mg/mL (501.88 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	5.0188 mL	25.0941 mL	50.1882 mL
	5 mM	1.0038 mL	5.0188 mL	10.0376 mL
	10 mM	0.5019 mL	2.5094 mL	5.0188 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.