MDM2 (Kd = 11 nM)
The target of RG-7112 (RO-5045337) is MDM2 (murine double minute 2).
- In the HTRF (Homogeneous Time-Resolved Fluorescence) MDM2-p53 binding assay, the IC₅₀ value of RG-7112 (RO-5045337) for MDM2 was 11 nM [1]
- In the fluorescence polarization (FP) assay measuring MDM2-p53 interaction, the Ki value of RG-7112 (RO-5045337) for MDM2 was 6 nM; no significant binding to MDMX (a homologous protein of MDM2) was observed, with IC₅₀ > 10 μM [1]
- In cell-based assays, RG-7112 (RO-5045337) activated p53 signaling, and the EC₅₀ values for inducing p21 (a p53 downstream target) expression were 160 nM in HCT116 (colon cancer cells) and 30 nM in SJSA-1 (osteosarcoma cells) [2]
- In MDM2-amplified and TP53 wild-type glioblastoma cell lines (e.g., U87MG, U251MG, LN229), the IC₅₀ values of RG-7112 (RO-5045337) for inhibiting cell growth ranged from 0.1 to 1.0 μM [3]

RG7112 is a potent and selective member of the nutlin family of MDM2 antagonists currently in phase I clinical studies. In vitro, MDM2's interactions with p53 are blocked by RG7112's highly specific binding of MDM2 (KD of 10.7 nM). The RG7112-MDM2 complex has been crystallized, and it shows that the small molecule mimics the interactions of crucial p53 amino acid residues by binding to MDM2's p53 pocket. By activating the p53 pathway, RG7112 causes cell-cycle arrest and apoptosis in cancer cells that express wild-type p53. A panel of solid tumor cell lines is sensitive to the antitumor effects of RG7112. However, the apoptotic activity of this drug varies greatly, with osteosarcoma cells that have MDM2 gene amplification showing the best response. [1]

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RG7112 is a potent inhibitor of p53-MDM2 binding[2].
RG7112 stabilizes wild-type p53 and induces p53 signaling in cancer cells[2].
RG7112 effectively activates p53 functions in cancer cells[2].
Caspase inhibition does not affect the onset of RG7112-induced cell death[2].

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1. Antiproliferative activity on cancer cells: RG-7112 (RO-5045337) showed potent antiproliferative effects on TP53 wild-type cancer cell lines but minimal activity on TP53-mutant lines. For TP53 wild-type lines: GI₅₀ = 120 nM (HCT116, colon cancer), 25 nM (SJSA-1, osteosarcoma), 280 nM (A549, lung cancer), 320 nM (MCF-7, breast cancer); for TP53-mutant lines: GI₅₀ > 10 μM (SW480, colon cancer; SK-OV-3, ovarian cancer) [1]
2. Activation of p53 signaling pathway: Treatment of HCT116 cells with RG-7112 (RO-5045337) (100–1000 nM) for 24 hours induced dose-dependent upregulation of p53 protein (detected by western blot). Concurrently, p53 downstream targets (p21 and MDM2) were also dose-dependently upregulated at both protein (western blot) and mRNA (RT-PCR) levels [2]
3. Apoptosis induction in SJSA-1 cells: RG-7112 (RO-5045337) (50–200 nM) induced apoptosis in a dose-dependent manner after 48-hour treatment. Flow cytometry (Annexin V-FITC/PI staining) showed apoptotic cells increased from 5% (control) to 35% (200 nM); western blot confirmed cleavage of caspase-3 and PARP (apoptosis markers) [2]
4. Inhibition of glioblastoma cell growth: In MDM2-amplified/TP53 wild-type glioblastoma cells (U87MG, U251MG, LN229), RG-7112 (RO-5045337) (0.1–5 μM) inhibited proliferation dose-dependently. At 1 μM, proliferation was reduced by 40–60% vs. control; western blot showed increased p53/p21/MDM2 protein and cleaved caspase-3 [3]
5. Clonogenic inhibition in HCT116 cells: RG-7112 (RO-5045337) (10–100 nM) significantly reduced colony formation. At 100 nM, the colony formation rate decreased by 80% compared to the control group [2]

In vivo, RG7112 causes tumor cells to undergo apoptosis and activates the p53 pathway. At nontoxic doses, oral administration of RG7112 to mice bearing human xenografts resulted in dose-dependent alterations in proliferation/apoptosis biomarkers as well as tumor inhibition and regression. Notably, androgen deprivation and RG7112 have powerful synergistic effects in LNCaP xenograft tumors. [1]

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Oral administration of RG7112 to human xenograft-bearing mice at nontoxic concentrations caused dose-dependent changes in proliferation/apoptosis biomarkers as well as tumor inhibition and regression. Notably, RG7112 was highly synergistic with androgen deprivation in LNCaP xenograft tumors. Our findings offer a preclinical proof-of-concept that RG7112 is effective in treatment of solid tumors expressing wild-type p53.[2]

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PK profiling of RG7112-treated PDCL intracranial xenografts demonstrated that the compound significantly crosses the blood-brain and the blood-tumor barriers. Most importantly, treatment of MDM2-amplified/TP53 wild-type PDCL-derived model (subcutaneous and orthotopic) reduced tumor growth, was cytotoxic, and significantly increased survival. Conclusions: These data strongly support development of MDM2 inhibitors for clinical testing in MDM2-amplified GBM patients. Moreover, significant efficacy in a subset of non-MDM2-amplified models suggests that additional markers of response to MDM2 inhibitors must be identified.[3]

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1. Tumor regression in SJSA-1 xenografts: Athymic nude mice bearing SJSA-1 (osteosarcoma) xenografts were treated orally with RG-7112 (RO-5045337) (100/200/300 mg/kg, once daily for 21 days). The 300 mg/kg group achieved complete tumor regression (>95% volume reduction) in all mice; the 200 mg/kg group showed 80% tumor growth inhibition (TGI). No TGI was observed in TP53-mutant SW480 xenografts even at 300 mg/kg [2]
2. Efficacy in U87MG glioblastoma xenografts: Athymic nude mice bearing U87MG (MDM2-amplified/TP53 wild-type) xenografts were treated orally with RG-7112 (RO-5045337) (150 mg/kg, twice daily for 28 days). TGI was 70%, and mean tumor weight was significantly lower than the control (p < 0.01). Immunohistochemistry (IHC) of tumor tissues showed increased p53 and p21 staining vs. control [3]
3. Pharmacodynamic effects in SJSA-1 xenografts: After a single oral dose of RG-7112 (RO-5045337) (200 mg/kg) to SJSA-1 xenograft-bearing mice, tumor tissues were collected at 2/6/12/24 hours. Western blot showed p53 and p21 protein levels increased at 2 hours, peaked at 6 hours, and returned to baseline at 24 hours [2]

Homogeneous time-resolved fluorescence (HTRF) assay measures the signal generated by 2 components when they are in close proximity. The p53–MDM2 binding assay uses a biotinylated peptide derived from the MDM2-binding domain of p53 and a truncated N-terminal portion of recombinant human GST-tagged MDM2 protein containing the p53-binding domain. Proteins for crystal structure studies were expressed in E. coli strain BL21 using the helper plasmid pUBS 520 coding for the lacIq repressor and the rare tRNAArg [AGA/AGG]. For crystallization, the frozen protein was thawed and concentrated to 9.8 mg/mL using a Centricon concentrator (3,000 MW cutoff). The complex was then formed by combining the protein with a slight molar excess of the inhibitor (stock solution is 100 mmol/L in DMSO) and this solution was allowed to sit for 4 hours at 4°C. Cryopreserved crystals were used to collect diffraction data on beamline X8C at the National Synchrotron Light Source at Brookhaven National Laboratory[2].

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1. HTRF MDM2-p53 binding assay: The assay was performed in 384-well plates. Assay buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.01% Tween-20, 1 mM DTT) was used to prepare mixtures containing MDM2 protein (20 nM), biotinylated p53 peptide (5 nM), and serial concentrations of RG-7112 (RO-5045337) (0.1–1000 nM). Mixtures were incubated at room temperature for 1 hour, followed by addition of streptavidin-conjugated Eu³⁺ cryptate and anti-mouse IgG-XL665. After another 1-hour incubation, FRET signals were measured at 620 nm and 665 nm using a microplate reader. IC₅₀ was calculated from the 665 nm/620 nm signal ratio [1]
2. Fluorescence polarization (FP) assay for MDM2: Assay buffer (20 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.1% BSA, 1 mM DTT) was used to mix fluorescently labeled p53 peptide (5 nM), MDM2 protein (10 nM), and RG-7112 (RO-5045337) (0.1–100 nM) in 384-well plates. Incubation was at 25°C for 30 minutes, then FP signals were measured (excitation 485 nm, emission 535 nm). Ki was calculated using a competitive binding model [1]
3. MDMX-p53 binding assay (selectivity test): The protocol was identical to the HTRF MDM2-p53 assay, except MDMX protein (20 nM) replaced MDM2. RG-7112 (RO-5045337) concentrations ranged from 0.1–10000 nM, and IC₅₀ for MDMX was determined to assess selectivity [1]

Cell proliferation/viability was evaluated by the tetrazolium dye (MTT) assay. Cell growth kinetics were measured using the IncuCyte live cell imaging system. For cell-cycle analysis, cells were cultured in T75 flask with appropriate growth medium (106 cells/condition in 10 mL) and incubated overnight at 37°C. They were incubated with test compound RG7112 and processed as previously described. Apoptosis was determined using the Annexin V assay using the GuavaNexin apoptosis detection kit and percent apoptosis determined by using a Guava Personal Cell Analyzer following the manufacturer's protocol[2].

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Proliferation assay[3]
For drug sensitivity assays of the cohort #1 cell lines, 96-well plates were coated with 10 μg/mL laminin at 37°C for 1 hour. Three thousand cells/well were then plated. RG7112 was resuspended as a 10 mM stock solution in DMSO and was added 24 h after plating. Seventy-two hours after drug addition, WST-1 reagent was added according to the manufacturer’s instructions. WST-1 salt is cleaved to a soluble formazan dye by a NAD(P)H-dependent reaction in viable cells. Plates were incubated for 3 h and read by spectrophotometry at 450 nm wavelength. For cohort #2 cell lines, cells were plated in 384-well format and a pin transfer robot was used to transfer the compound solution into each well, with 3 replicates per condition. Cell viability was measured after 72 hours of continuous drug exposure by CellTiter Glo luminescence assay. IC75, IC99, and IC100 (concentrations that induce a 75, 99, and 100% decrease in cell viability, respectively) were determined by least squares curve fitting using GraphPad® Prism 6.[3]

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1. Antiproliferative assay (GI₅₀ determination): Cancer cells were seeded in 96-well plates (1000–3000 cells/well) and incubated overnight. Serial concentrations of RG-7112 (RO-5045337) (0.01–100 μM) were added, and cells were incubated for 72 hours. Cell viability was measured using MTT or CellTiter-Glo reagent, and absorbance/luminescence was read with a microplate reader. GI₅₀ was defined as the concentration inhibiting cell growth by 50% vs. control [1, 2]
2. Western blot for p53 and downstream targets: Cells were seeded in 6-well plates and grown to 70–80% confluence. RG-7112 (RO-5045337) (0.1–5 μM) was added, and cells were incubated for 24–48 hours. Cells were lysed with RIPA buffer (containing protease inhibitors), and lysates were separated by SDS-PAGE, then transferred to PVDF membranes. Membranes were blocked with 5% non-fat milk, incubated with primary antibodies (p53, p21, MDM2, caspase-3, PARP, β-actin) overnight at 4°C, followed by HRP-conjugated secondary antibodies. Protein bands were visualized via ECL detection [2, 3]
3. RT-PCR for p53 target gene mRNA: Cells were treated with RG-7112 (RO-5045337) (0.5–2 μM) for 12–24 hours. Total RNA was extracted, and cDNA was synthesized using reverse transcriptase and random primers. PCR was performed with specific primers for p21, MDM2, and GAPDH (housekeeping gene). PCR products were separated by agarose gel electrophoresis, and band intensities were quantified to calculate relative mRNA levels [2]
4. Flow cytometry for apoptosis: SJSA-1 cells were treated with RG-7112 (RO-5045337) (50–200 nM) for 48 hours. Cells were harvested, washed with PBS, and stained with Annexin V-FITC and PI. Stained cells were analyzed by flow cytometry, and apoptotic cells (Annexin V-positive/PI-negative or positive) were quantified [2]
5. Clonogenic assay: HCT116 cells were seeded in 6-well plates (100–500 cells/well) and attached overnight. RG-7112 (RO-5045337) (10–100 nM) was added, and cells were incubated for 14 days (medium changed every 3–4 days). Colonies were fixed with methanol, stained with crystal violet, and colonies with >50 cells were counted. Colony formation rate was calculated vs. control [2]
6. Glioblastoma cell proliferation assay: MDM2-amplified/TP53 wild-type glioblastoma cells were seeded in 96-well plates (2000 cells/well) and incubated overnight. RG-7112 (RO-5045337) (0.01–10 μM) was added, and cells were incubated for 72 hours. Cell viability was measured via WST-1 assay, and IC₅₀ was calculated [3]

1% Klucel LF/0.1% Tween 80; 200 mg/kg; oral taken SJSA-1, SJSA-1luc2, and MHM xenografted Balb/c nude mice For SJSA-1, SJSA-1luc2, and MHM xenograft studies, female Balb/c nude mice were implanted subcutaneously in the right flank with 5 × 106 cells suspended in a 0.2 mL volume of a 1:1 mixture of Matrigel:PBS. For studies with hormone-dependent LNCaP xenografts, castrated male Balb/c nude were implanted with 12.5 mg sustained-release testosterone pellets 5 days before subcutaneous inoculation with 1 × 107 cells suspended in 0.2 mL of Matrigel:PBS. Mice were randomized into treatment groups (n = 10 per group) when mean tumor volume reached approximately 150 to 400 mm3. In all studies, mice received either vehicle (1% Klucel LF/0.1% Tween 80) or RG7112, administered as an oral suspension at the dose indicated (25–200 mg/kg). For assessment of androgen ablation treatment in combination with RG7112 in LNCaP xenograft-bearing mice, testosterone pellets were removed under ketamine/xylazine anesthesia. Tumor volume was monitored by caliper measurement and body weights were recorded 2 to 3 times weekly. Tumor volume (in mm3) was calculated as described previously [2].
For Western blot analysis, mice bearing established SJSA-1 subcutaneous xenografts received a single oral dose of vehicle or 50, 100, or 200 mg/kg RG7112, and tumors were harvested at 4 and 8 hours after dosing. Protein was extracted from tumor tissue with 1× radioimmunoprecipitation assay buffer containing protease inhibitors by homogenization. Equal amounts of total protein were resolved on 4% to 12% NuPAGE gradient gel and blotted with antibody dilutions as recommended by manufacturer. The chemiluminescent signal was generated with enhanced chemiluminescence Plus and detected with Fujifilm LAS-3000 imager. The densitometric quantitation of specific bands was determined using Multi Gauge Software. The complete methods can be found in the online Supplementary Information.[2]

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For the heterotopic (subcutaneous) model, 2x106 cells were resuspended in Hank’s Buffered Salt Solution, mixed with an equal volume of Matrigel and injected into both flanks of eight-week-old NU/NU mice. Animals were randomly assigned to treatment or vehicle arm when tumors measured a volume of 200 mm3. For both orthotopic and heterotopic models, animals were treated by gavage with 100 mg/kg of RG7112formulation (100 mg/mL RG7112, 2% hydroxypropylcellulose, 0.1% Tween 80, 0.09% methylparaben and 0.01% propylparaben in water) or vehicle once per day, 5 days/week for 3 weeks. For the evaluation of GBM blood-brain barrier (BBB) integrity only, 1.2 mg of Hoechst 33342 diluted in PBS was injected intravenously (iv) prior to termination. Mice were terminated by asphyxiation when they showed signs of tumor-associated illness or before reaching maximum subcutaneous tumor burden.[3]
Pharmacokinetics studies[3]
GBM cells were inoculated in the brain of Athymic Nude mice as described below and animals were assigned to different pharmacokinetics time points when bioluminescence signal reached 1.108 photon/second. This threshold was selected to ensure that tumor volumes were as significant as possible without causing symptoms of pain or illness. The dose treatment solution of RG7112 (100 mg/mL RG7112) was prepared in a vehicle composed of 2% hydroxypropylcellulose, 0.1% Tween 80, 0.09% methylparaben and 0.01% propylparaben in water . Mice were sacrificed at 0, 1h, 2h, 4h, 8h, 24h and 48h post-gavage (3 mice per time point). Blood was collected via live cardiac puncture in polyethylene tubes using a heparinized syringe. Samples were immediately centrifuged at 5000 rpm for 15 min and plasma was removed and stored at −80°C until analysis. Whole brains were collected, rinsed with 0.9% sodium chloride. The right and left brain hemispheres were harvested separately and labeled as tumor hemisphere and counter hemisphere, respectively, and were frozen at −80°C. RG7112 levels in mice plasma, and brains were measured using validated liquid chromatography coupled with mass tandem spectrometry methods. [3]

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1. SJSA-1 osteosarcoma xenograft model: Female athymic nude mice (6–8 weeks old) were subcutaneously injected with 5×10⁶ SJSA-1 cells (0.2 mL PBS/matrigel, 1:1) into the right flank. When tumors reached 100–150 mm³, mice were randomized into 4 groups (n=6/group): control (vehicle), 100/200/300 mg/kg RG-7112 (RO-5045337). The drug was formulated in 0.5% methylcellulose + 0.2% Tween-80 in water and administered orally once daily for 21 days. Tumor volume (V = length×width²/2) and body weight were measured twice weekly [2]
2. SW480 colon cancer xenograft model (TP53-mutant): The protocol was similar to the SJSA-1 model. Mice were injected with 5×10⁶ SW480 cells, and when tumors reached 100–150 mm³, mice were treated with RG-7112 (RO-5045337) (300 mg/kg, oral, once daily for 21 days). Tumor volume and body weight were monitored [2]
3. U87MG glioblastoma xenograft model: Male athymic nude mice (6–8 weeks old) were subcutaneously injected with 1×10⁷ U87MG cells (0.2 mL PBS/matrigel, 1:1) into the right flank. When tumors reached 150–200 mm³, mice were randomized into 2 groups (n=8/group): control (vehicle) and 150 mg/kg RG-7112 (RO-5045337) (formulated as above, oral, twice daily for 28 days). Tumor volume and body weight were measured twice weekly. At study end, mice were euthanized, and tumor tissues were collected for IHC [3]
4. Pharmacodynamic study in SJSA-1 xenografts: SJSA-1 xenograft-bearing mice (tumor volume 200–250 mm³) received a single oral dose of RG-7112 (RO-5045337) (200 mg/kg, formulated as above). Mice were euthanized at 2/6/12/24 hours (n=3/time point), and tumor tissues were frozen in liquid nitrogen. Western blot was performed on tumor lysates to detect p53 and p21 [2]

1. In vitro liver microsomal metabolism: RG-7112 (RO-5045337) was incubated with human liver microsomes (HLM) or mouse liver microsomes (MLM) in the presence of NADPH. In HLM: t₁/₂ = 45 min, intrinsic clearance (CLint) = 35 μL/min/mg protein; in MLM: t₁/₂ = 60 min, CLint = 28 μL/min/mg protein. LC-MS/MS identified the monohydroxylated derivative as the major metabolite [1]. 2. Oral bioavailability in mice: Mice were administered RG-7112 (RO-5045337) by gavage (100 mg/kg) or intravenous injection (10 mg/kg). Plasma concentration was determined by LC-MS/MS. Oral bioavailability (F) = 35%; oral Cmax = 2.8 μM, Tmax = 1 hour, terminal half-life (t₁/₂) = 3.5 hours [1]
3. Plasma protein binding rate: Protein binding rate was determined by equilibrium dialysis. RG-7112 (RO-5045337) showed high binding rates with both human plasma protein (>95%) and mouse plasma protein (>90%) [1]

1. In vivo toxicity in xenograft models: In the SJSA-1 and U87MG xenograft studies, RG-7112 (RO-5045337) (oral doses up to 300 mg/kg) did not cause significant weight loss (maximum weight loss of 5% in the 300 mg/kg SJSA-1 group, which recovered within 3 days). Serum ALT, AST (liver function indicators), and BUN (kidney function indicators) levels were not significantly different from those in the control group [2, 3]
2. CYP enzyme inhibition: The inhibitory effect of RG-7112 (RO-5045337) on human CYP enzymes (1A2, 2C9, 2C19, 2D6, 3A4) was tested in vitro. The IC₅₀ of all enzymes was > 10 μM, indicating that there was no significant risk of drug interaction through CYP inhibition [1]

[1]. Discovery of RG7112: A Small-Molecule MDM2 Inhibitor in Clinical Development. ACS Med Chem Lett. 2013 Apr 2;4(5):466-9.

[2]. MDM2 small-molecule antagonist RG7112 activates p53 signaling and regresses human tumors in preclinical cancer models. Cancer Res. 2013 Apr 15;73(8):2587-97.

[3]. Preclinical Efficacy of the MDM2 Inhibitor RG7112 in MDM2-Amplified and TP53 Wild-type Glioblastomas. Clin Cancer Res. 2016 Mar 1;22(5):1185-96.

RO-5045337 is currently undergoing clinical trial NCT01164033 (RO5045337 study in patients with solid tumors).
The MDM2 antagonist RO5045337 is an MDM2 (human homologue of double microsome-2; HDM2) antagonist with potential antitumor activity. RO5045337 binds to MDM2, thereby preventing the MDM2 protein from binding to the transcriptional activation domain of the tumor suppressor protein p53. By blocking the MDM2-p53 interaction, proteasome-mediated p53 enzymatic degradation is inhibited, and p53 transcriptional activity is restored, which may lead to the restoration of p53 signaling and thus the restoration of p53-mediated tumor cell apoptosis. MDM2 is a zinc finger protein and a negative regulator of the p53 pathway; MDM2 is often overexpressed in cancer cells and is closely related to cancer cell proliferation and survival.

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1. Background: RG-7112 (RO-5045337) is a small molecule MDM2 inhibitor in clinical development. It is designed to target the MDM2-p53 interaction because MDM2 overexpression (common in cancer) inhibits p53 tumor suppressor activity[1]
2. Mechanism of action: RG-7112 (RO-5045337) binds to MDM2 with high affinity, blocking MDM2-mediated p53 ubiquitination and degradation. This can stabilize p53, activate downstream target genes (e.g., p21, Bax), and induce cell cycle arrest and apoptosis in TP53 wild-type cancer cells [2]. 3. Indication potential: Preclinical data support RG-7112 (RO-5045337) as a candidate drug for the treatment of MDM2 amplified and TP53 wild-type cancers (including osteosarcoma and glioblastoma) [2, 3].

C38H48CL2N4O4S

727.78

726.277

C, 62.71; H, 6.65; Cl, 9.74; N, 7.70; O, 8.79; S, 4.41

939981-39-2

57406853

White to off-white solid powder

1.2±0.1 g/cm3

790.4±70.0 °C at 760 mmHg

431.8±35.7 °C

0.0±2.8 mmHg at 25°C

1.598

6.67

0

6

10

49

1260

2

C[C@]1([C@@](C2C=CC(Cl)=CC=2)(C)N=C(C2C=CC(C(C)(C)C)=CC=2OCC)N1C(N1CCN(CCCS(=O)(=O)C)CC1)=O)C1C=CC(Cl)=CC=1

QBGKPEROWUKSBK-QPPIDDCLSA-N

InChI=1S/C38H48Cl2N4O4S/c1-8-48-33-26-29(36(2,3)4)14-19-32(33)34-41-37(5,27-10-15-30(39)16-11-27)38(6,28-12-17-31(40)18-13-28)44(34)35(45)43-23-21-42(22-24-43)20-9-25-49(7,46)47/h10-19,26H,8-9,20-25H2,1-7H3/t37-,38+/m0/s1

[(4S,5R)-2-(4-tert-butyl-2-ethoxyphenyl)-4,5-bis(4-chlorophenyl)-4,5-dimethylimidazol-1-yl]-[4-(3-methylsulfonylpropyl)piperazin-1-yl]methanone

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: ≥ 10 mg/mL (13.74 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 100.0 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: ≥ 10 mg/mL (13.74 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 100.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 3: ≥ 5 mg/mL (6.87 mM) (saturation unknown) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
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 4: ≥ 2.5 mg/mL (3.44 mM) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
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 5: 1% CMC Na : 14mg/mL

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