RG7388 (also known as Idasanutlin; RG-7388; RO5503781) is a novel, potent and highly selective antagonist of the p53-MDM2 protein-protein interaction with potential antitumor activity. With an IC50 of 6 nM, it prevents the interaction of p53 and MDM2. Inhibiting the interaction between p53 and MDM2 in order to restore p53 activity has been viewed as a promising method for treating cancer. To create small-molecule inhibitors with desirable pharmacological profiles, the hydrophobic protein-protein interaction surface poses a significant challenge. MDM2 (mouse double minute 2; Mdm2 p53 binding protein homolog) is a tumor suppressor protein that interacts with p53. By binding to MDM2, RG7388 blocks this interaction and has an effect. When the MDM2-p53 interaction is broken, p53 is not enzymatically degraded and its transcriptional activity is reinstated, which leads to the p53-mediated induction of tumor cell apoptosis. MDM2 is a p53 pathway negative regulator that is frequently overexpressed in tumor cells. As an MDM2 antagonist, RG7388 may have anticancer properties.

Physicochemical Properties

Molecular Formula	C31H29CL2F2N3O4
Molecular Weight	616.48
Exact Mass	615.15
Elemental Analysis	C, 60.40; H, 4.74; Cl, 11.50; F, 6.16; N, 6.82; O, 10.38
CAS #	1229705-06-9
Related CAS #	Idasanutlin-d3-1;Idasanutlin (enantiomer)
PubChem CID	53358942
Appearance	White to off-white solid powder
Density	1.4±0.1 g/cm3
Boiling Point	737.3±60.0 °C at 760 mmHg
Flash Point	399.7±32.9 °C
Vapour Pressure	0.0±2.5 mmHg at 25°C
Index of Refraction	1.623
LogP	7.09
Hydrogen Bond Donor Count	3
Hydrogen Bond Acceptor Count	8
Rotatable Bond Count	8
Heavy Atom Count	42
Complexity	1040
Defined Atom Stereocenter Count	4
SMILES	ClC1=C([H])C([H])=C([H])C(=C1F)[C@@]1([H])[C@]([H])(C(N([H])C2C([H])=C([H])C(C(=O)O[H])=C([H])C=2OC([H])([H])[H])=O)N([H])[C@@]([H])(C([H])([H])C(C([H])([H])[H])(C([H])([H])[H])C([H])([H])[H])[C@]1(C#N)C1C([H])=C([H])C(=C([H])C=1F)Cl
InChi Key	TVTXCJFHQKSQQM-LJQIRTBHSA-N
InChi Code	InChI=1S/C31H29Cl2F2N3O4/c1-30(2,3)14-24-31(15-36,19-10-9-17(32)13-21(19)34)25(18-6-5-7-20(33)26(18)35)27(38-24)28(39)37-22-11-8-16(29(40)41)12-23(22)42-4/h5-13,24-25,27,38H,14H2,1-4H3,(H,37,39)(H,40,41)/t24-,25-,27+,31-/m0/s1
Chemical Name	4-[[(2R,3S,4R,5S)-3-(3-chloro-2-fluorophenyl)-4-(4-chloro-2-fluorophenyl)-4-cyano-5-(2,2-dimethylpropyl)pyrrolidine-2-carbonyl]amino]-3-methoxybenzoic acid
Synonyms	Idasanutlin; RG-7388; RO-5503781; RG 7388; RO5503781; 1229705-06-9; RG7388; RG-7388; Idasanutlin (RG-7388); Idasanutlin (RG7388); RO5503781; 4-((2R,3S,4R,5S)-3-(3-chloro-2-fluorophenyl)-4-(4-chloro-2-fluorophenyl)-4-cyano-5-neopentylpyrrolidine-2-carboxamido)-3-methoxybenzoic acid; RG7388; RO 5503781
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-MDM2 (IC50 = 6 nM)
ln Vitro	In cancer cells expressing wild-type p53, isasanutlin inhibits cell proliferation with an IC50 of 30 nM and induces dose-dependent p53 stabilization, cell cycle arrest, as well as cell apoptosis.[1] RG7388 displayed improved in vitro binding as well as cellular potency/selectivity. Thus, compound 12 was chosen for further studies. In cell-based mechanistic studies), RG7388 induced dose-dependent p53 stabilization, cell cycle arrest, and apoptosis in cancer cells expressing wild-type p53, consistent with a nongenotoxic p53 activation mechanism.[1] RG7388-induced apoptosis in SJSA osteosarcoma cells is delayed relative to drug exposure but does not require continuous treatment[2].
ln Vivo	In initial efficacy testing, daily dosing at 30 mg/kg and twice a week dosing at 50 mg/kg of RG7388 were statistically equivalent in our tumor model. In addition, weekly dosing of 50 mg/kg was equivalent to 10 mg/kg given daily. The implementation of modeling and simulation on these data suggested several possible intermittent clinical dosing schedules. Further preclinical analyses confirmed these schedules as viable options.[2] RG7388 also achieved impressive in vivo efficacy against established human SJSA1 osterosarcoma xenografts in nude mice at significantly lower doses and exposures compared to RG7112[1].
Enzyme Assay	The 50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 1 mM DTT, and 0.02 or 0.2 mg/ml BSA buffer is used for the p53-MDM2 HTRF assay. Aliquots of small-molecule inhibitors are kept at 4°C in 96-deep-well plates as stock solutions of 10 mM DMSO. Just before testing, it is thawed and blended. A biotinylated p53 peptide and GST-MDM2 are incubated with the substance for an hour at 37°C. Following the addition of Eu-8044-streptavidin and Phycolink goat anti-GST (Type 1) allophycocyanin, an hour-long incubation at room temperature is required. Using the Envision fluorescence reader, plates are read. Data sets in duplicate or triplicate between plates are used to calculate IC50 values. Data is analyzed by XLfit4 (Microsoft) using a Sigmoidal Dose-Response Model with 4 Parameter Logistic Model and the equation Y= (A+ ((B-A)/ (1+ ((C/x)^D)))), where A and B are enzyme activity in the absence or presence of infinite inhibitor compound, respectively, C is the IC50, and D is the Hill coefficient.[1]
Cell Assay	Tetrazolium dye assay is used to assess cell proliferation. The linear regression of a plot of the concentration versus percent inhibition's logarithm yields the concentration at which cell proliferation is 50% (IC50) or 90% (IC90) inhibited.[1] In vitro testing in cancer cell lines[2] RG7388 was prepared at concentrations of 1 and 10 mmol/L in DMSO and stored in aliquots at −20°C. SJSA, RKO, HCT116, H460, A375, SK-MEL-5, SW480, MDA435, and HeLa cells were obtained from the ATCC. Cell lines were authenticated by short tandem repeat analysis through Promega authentication services. For in vitro studies, cells were cultured in their ATCC-designated media. Medium was supplemented with 10% FBS and 1% 200 nmol/L l-glutamine. To assess cell viability, cells were seeded at densities identified for best growth for a 5-day assay in 96-well plates in normal growth media. Serial dilutions of RG7388 (1–3 in fresh media) starting at 300 μmol/L were applied to wells (1–10) in triplicate for a final concentration range of 0.01 to 30 μmol/L and control wells were treated with 0.3% DMSO equivalent to DMSO at the highest RG7388 concentration. Cell respiration, as an indicator of cell viability, was measured by the reduction of MTT to formazan as previously described. Percent apoptosis was determined as described in Tovar and colleagues. For Western blot analysis, cells were cultured in T-75 flasks (4 mL total volume at 5 × 105 cells/well) and incubated overnight at 37°C, 5% CO2. Cells were treated with 0.3 or 1.8 μmol/L of RG7388 or 0.1% DMSO as control. Treatment duration was 16 hours, and lysates were prepared before washout and at 4, 8, 24, and 48 hours after RG7388 washout.
Animal Protocol	Efficacy of Oral MDM2 Inhibitor RG7388 in the SJSA Human Osteosarcoma Xenograft Model [1] Female SJSA tumor-bearing nude mice were orally administered daily doses of RG7388 and blood samples were collected in EDTA tubes from 2 animals/group/time point (1, 2, 4, 8 and 24 hours) after the first (acute PK) or last (chronic PK) dose. Plasma and tumor samples were prepared and analyzed for RG7388 by LC/MS/MS. Mean plasma concentrations were calculated from 2 animals/group/time point. S-9 Efficacy of Oral MDM2 Inhibitor RG7388 in the SJSA Human Osteosarcoma Xenograft Model The anti-tumor efficacy of RG7388 in the SJSA1 model was determined in female nude mice as reported in Tovar et al.1 In the current study, mice were randomized into treatment groups of 10 mice with similar mean tumor volumes of 190-230 mm3 . RG7388 was administered by gavage at doses from 12.5-50 mg/kg daily for two weeks. Efficacy of Oral MDM2 Inhibitor RG7388 in the SJSA Human Osteosarcoma Xenograft Model [1] The anti-tumor efficacy of RG7388 in the SJSA1 model was determined in female nude mice as reported in Tovar et al.1 In the current study, mice were randomized into treatment groups of 10 mice with similar mean tumor volumes of 190-230 mm3 . RG7388 was administered by gavage at doses from 12.5-50 mg/kg daily for two weeks. Pharmacokinetic analysis[2] To determine RG7388 plasma concentrations, blood samples were collected from female mice during in vivo antitumor studies. In each treatment group, on the first and/or last dosing day, blood samples (generally n = 2/time point) were collected at various predetermined time points ranging from 0.5 to 24 hours after the last dose. Pharmacokinetic assessment was performed via noncompartmental analysis using Watson v7.4, where parameters were calculated on the basis of the composite concentration–time data from each treatment group and sampling day. Sampling times were reported as nominal time, with concentrations below the limit of quantitation excluded. Parameters reported include plasma half-life (t1/2), Cmax, Tmax, and area under the plasma concentration–time curve from end of dosing to the last bleeding time point (AUC0–24 hours). The AUCs were calculated using the linear trapezoidal rule. The Cmax and Tmax values were taken directly from the plasma concentration–time profiles without extrapolations. In vivo activity studies in xenograft tumor models[2] Athymic female nude mice (Crl:NU-Foxn1nu) were obtained from Charles River Laboratories. The health of all animals was monitored daily by gross observation and analyses of blood samples of sentinel animals. All animal experiments were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee in our Association for Assessment and Accreditation of Laboratory Animal Care-accredited facility. At 10 to 12 weeks of age, mice were implanted with a 1:1 mixture of human SJSA osteosarcoma cells (ATCC) suspended in phenol-free Matrigel and PBS. Mice were implanted in the right flank at a concentration of 5 × 106 cells in 0.2 mL total volume. At approximately day 10, animals were randomized according to tumor volume, so that all groups of 10 randomized mice had similar starting mean tumor volumes of 100 to 250 mm3. Tumor measurements and weights were taken two to three times per week. TGI was calculated from percent change in mean tumor volume compared with the control group. Average percent weight change was used as a surrogate endpoint for tolerability in the experiment. Animals in each group were continuously followed beyond the last day of treatment to see whether tumor regrowth would occur. In this second phase of analysis, survival was calculated using a cutoff individual tumor volume of 1,500 mm3 as a surrogate for mortality. The increase in lifespan (ILS) was calculated as a percentage using the formula: [(median day of death in treated tumor-bearing mice) − (median day of death in control tumor-bearing mice)]/median day of death in control tumor-bearing mice × 100. Statistical analysis was performed as previously described. RG7388 was administered as an amorphous solid dispersion microbulk precipitate powder containing 30% drug substance and 70% hydroxypropyl methylcellulose acetate succinate polymer that was reconstituted immediately before administration as a suspension in Klucel/Tween, and remaining suspension was discarded after dosing. Formulated in 30% drug substance and 70% hydroxypropyl methylcellulose acetate succinate polymer; 25 mg/kg; p.o. Athymic female nude mice (Crl:NU-Foxn1nu) bearing SJSA human osteosarcoma xenograft model
References	[1]. Discovery of RG7388, a potent and selective p53-MDM2 inhibitor in clinical development. J Med Chem. 2013 Jul 25;56(14):5979-83. [2]. Preclinical optimization of MDM2 antagonist scheduling for cancer treatment by using a model-based approach. Clin Cancer Res. 2014, 20(14), 3742-3752.
Additional Infomation	Idasanutlin has been used in trials studying the treatment of Neoplasms, Non-Hodgkin's Lymphoma, Leukemia, Myeloid, Acute, Recurrent Plasma Cell Myeloma, and Neoplasms, Leukemia, Acute Myeloid Leukemia. Idasanutlin is an orally available, small molecule, antagonist of MDM2 (mouse double minute 2; Mdm2 p53 binding protein homolog), with potential antineoplastic activity. Idasanutlin binds to MDM2 blocking the interaction between the MDM2 protein and the transcriptional activation domain of the tumor suppressor protein p53. By preventing the MDM2-p53 interaction, p53 is not enzymatically degraded and the transcriptional activity of p53 is restored. This may lead to p53-mediated induction of tumor cell apoptosis. MDM2, a zinc finger nuclear phosphoprotein and negative regulator of the p53 pathway, is often overexpressed in cancer cells and has been implicated in cancer cell proliferation and survival. Drug Indication Treatment of all conditions included in the category of malignant neoplasms (except nervous system, haematopoietic and lymphoid tissue) Treatment of acute lymphoblastic leukaemia, Treatment of acute myeloid leukaemia Restoration of p53 activity by inhibition of the p53-MDM2 interaction has been considered an attractive approach for cancer treatment. However, the hydrophobic protein-protein interaction surface represents a significant challenge for the development of small-molecule inhibitors with desirable pharmacological profiles. RG7112 was the first small-molecule p53-MDM2 inhibitor in clinical development. Here, we report the discovery and characterization of a second generation clinical MDM2 inhibitor, RG7388, with superior potency and selectivity.[1] Purpose: Antitumor clinical activity has been demonstrated for the MDM2 antagonist RG7112, but patient tolerability for the necessary daily dosing was poor. Here, utilizing RG7388, a second-generation nutlin with superior selectivity and potency, we determine the feasibility of intermittent dosing to guide the selection of initial phase I scheduling regimens. Experimental design: A pharmacokinetic-pharmacodynamic (PKPD) model was developed on the basis of preclinical data to determine alternative dosing schedule requirements for optimal RG7388-induced antitumor activity. This PKPD model was used to investigate the pharmacokinetics of RG7388 linked to the time-course of the antitumor effect in an osteosarcoma xenograft model in mice. These data were used to prospectively predict intermittent and continuous dosing regimens, resulting in tumor stasis in the same model system. Results: RG7388-induced apoptosis was delayed relative to drug exposure with continuous treatment not required. In initial efficacy testing, daily dosing at 30 mg/kg and twice a week dosing at 50 mg/kg of RG7388 were statistically equivalent in our tumor model. In addition, weekly dosing of 50 mg/kg was equivalent to 10 mg/kg given daily. The implementation of modeling and simulation on these data suggested several possible intermittent clinical dosing schedules. Further preclinical analyses confirmed these schedules as viable options. Conclusion: Besides chronic administration, antitumor activity can be achieved with intermittent schedules of RG7388, as predicted through modeling and simulation. These alternative regimens may potentially ameliorate tolerability issues seen with chronic administration of RG7112, while providing clinical benefit. Thus, both weekly (qw) and daily for five days (5 d on/23 off, qd) schedules were selected for RG7388 clinical testing.[2]

Solubility Data

Solubility (In Vitro)

DMSO: ≥ 45 mg/mL Water: <1mg/mL Ethanol: <1mg/mL

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.06 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 2: 5% DMSO+40% PEG 300+5% Tween 80+ddH2O: 1.25mg/mL Solubility in Formulation 3: 10 mg/mL (16.22 mM) in 0.5%HPMC 1%Tween80 (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	1.6221 mL	8.1106 mL	16.2211 mL
	5 mM	0.3244 mL	1.6221 mL	3.2442 mL
	10 mM	0.1622 mL	0.8111 mL	1.6221 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.