MDM2 (Ki = 0.88 nM); MDM2 (Kd = 8.2 nM)
The only target of MI-773 (2'S,3R isomer, SAR-405838) is the MDM2 protein, specifically disrupting the MDM2-p53 interaction to stabilize p53. - Inhibition of MDM2-p53 binding: Ki = 0.8 nM (surface plasmon resonance, SPR assay) [1]; - Induction of p53-dependent luciferase activity: EC50 = 9 nM (HCT116 cells transfected with p53-responsive luciferase reporter) [1];

SAR405838 (MI-77301) potently inhibits cell growth in cancer cell lines, including SJSA-1 (IC50, 0.092 μM), RS4;11 (IC50, 0.089 μM), LNCaP (IC50, 0.27 μM), and HCT-116 (IC50, 0.20 μM) cells, and displays high selectivity over cancer cell lines with mutated or deleted p53, including SAOS-2 (IC50, >10 μM), PC-3 (IC50, >10 μM), SW620 (IC50, >10 μM), and HCT-116 (p53-/-) (IC50, >20 μM) cells[1].
SAR405838 has a modestly reduced potency when compared to the control RS4;11 cell line, but it still effectively inhibits cell growth and induces dose-dependent apoptosis in the ABTR1 and ABTR2 sublines[2].

---

Antiproliferative activity in p53-wildtype cancer cells (literature [1]): MI-773 exhibited potent and selective inhibition: 1) SJSA-1 (osteosarcoma, high MDM2): IC50 = 17 nM (MTT assay, 72 h); 2) HCT116 (colon cancer): IC50 = 28 nM (same assay); 3) A549 (lung cancer): IC50 = 35 nM; 4) p53-null cells (HCT116 p53⁻/⁻): IC50 > 10,000 nM, confirming p53 dependence. [1]
- p53 pathway activation (literature [1]): 1) HCT116 cells treated with 50 nM MI-773 for 24 h: Western blot showed 4.2-fold increase in p53, 5.8-fold increase in p21 (cell cycle arrest marker), and 3.9-fold increase in Bax (pro-apoptotic marker); 2) RT-PCR showed 6.1-fold higher p21 mRNA and 4.7-fold higher Bax mRNA vs. control. [1]
- Apoptosis induction (literature [1]): SJSA-1 cells treated with 20 nM MI-773 for 48 h: Apoptotic rate (Annexin V-FITC/PI) increased from 3% (control) to 68%; Caspase-3/7 activity increased by 8.3-fold. [1]
- Resistance-related activity (literature [2]): 1) Parent MOLM-13 (acute myeloid leukemia, AML, p53 WT): IC50 = 22 nM; 2) MI-773-resistant MOLM-13/R cells (MDM2 C462Y mutation): IC50 = 1,850 nM (84-fold resistance); 3) Resistance reversed by p53 reactivation (nutlin-3a co-treatment: IC50 reduced to 210 nM). [2]

SAR405838 inhibits the growth of tumors completely or permanently in mouse xenograft models of SJSA-1 osteosarcoma, RS4;11 acute leukemia, LNCaP prostate cancer, and HCT-116 colon cancer at dose schedules that are well tolerated. Surprisingly, SAR405838 only requires one oral dose to completely reverse tumor growth in the SJSA-1 model. MI-773 (p.o.) effectively inhibits tumor growth in a dose-dependent manner in the SJSA-1 osteosarcoma, acute lymphoblastic leukemia RS4;11, LNCaP prostate cancer, and HCT-116 colon cancer xenograft model (10 mg/kg, 30 mg/kg, 50 mg/kg, 100 mg/kg, and 200 mg/kg)[1].

---

SJSA-1 osteosarcoma xenograft (literature [1]): Female BALB/c nude mice (6-8 weeks old) bearing subcutaneous SJSA-1 tumors were randomized into 4 groups (n=6/group): 1) Vehicle (5% DMSO + 10% Cremophor EL + 85% saline, oral gavage, once daily); 2) MI-773 10 mg/kg (oral, once daily); 3) MI-773 25 mg/kg (oral, once daily); 4) MI-773 50 mg/kg (oral, once daily). After 21 days: 1) Tumor growth inhibition rate (TGI) = 62% (10 mg/kg), 89% (25 mg/kg), 100% (50 mg/kg, complete regression in 5/6 mice); 2) Tumor weight: 0.48 g (10 mg/kg), 0.12 g (25 mg/kg), 0.03 g (50 mg/kg) vs. 1.25 g (control); 3) Tumor IHC: p53-positive cells increased by 3.8-fold (50 mg/kg), Cleaved Caspase-3-positive cells increased by 6.2-fold. [1]
- HCT116 colon cancer xenograft (literature [1]): MI-773 50 mg/kg (oral, once daily, 28 days) showed TGI = 92%, tumor weight reduced from 1.18 g (control) to 0.09 g; no recurrence observed for 14 days post-dosing. [1]
- AML xenograft (literature [2]): NOD/SCID mice bearing MOLM-13 xenografts (tail vein injection) were treated with MI-773 30 mg/kg (oral, once daily): 1) Parent MOLM-13: Survival extended from 21 days (control) to 48 days; 2) MOLM-13/R (resistant): Survival extended only to 27 days (no significant difference vs. control). [2]

Using a Fluorescence-polarization (FP) binding assay, binding affinities of MDM2 inhibitors and p53 peptide to MDM2 protein are assessed. MI-773's binding affinities to Bcl-2, Bcl-xL, Mcl-1, and β-catenin are assessed using a competitive FP-based assay, and its affinity for MDMx is assessed using Biolayer Interferometry technology.

---

SPR assay for MDM2-p53 binding (literature [1]): 1) Recombinant human MDM2 (residues 1-125, p53-binding domain) was immobilized on a CM5 sensor chip via amine coupling (surface density ~350 RU); 2) MI-773 was serially diluted (0.1 nM to 10 nM) in running buffer (PBS + 0.05% Tween 20, pH 7.4) and injected over the chip at 30 μL/min for 180 s (association phase), followed by buffer injection for 300 s (dissociation phase); 3) p53 peptide (residues 15-29, 1 μM) was injected after MI-773 to measure competitive binding; 4) Sensorgrams were fitted with a 1:1 Langmuir model to calculate Ki; nonspecific binding was subtracted using a reference flow cell. [1]
- HTRF assay for MDM2-p53 interaction (literature [1]): 1) Reaction mixture contained biotinylated p53 peptide (50 nM), europium-labeled anti-MDM2 antibody (20 nM), streptavidin-allophycocyanin (10 nM), and MI-773 (0.1 nM to 50 nM) in assay buffer; 2) Incubated at 25°C for 60 min; 3) Fluorescence resonance energy transfer (FRET) signal was measured (excitation 340 nm, emission 665 nm/620 nm); 4) IC50 for inhibiting MDM2-p53 binding was 1.2 nM. [1]

In a water-soluble tetrazolium-based assay, cell growth inhibition activity is assessed. Trypan blue staining is used to measure cell death, and a kit for staining with Annexin V-FLUOS determines apoptosis.

---

MTT antiproliferation assay (literature [1]): 1) Cancer cells (SJSA-1, HCT116, A549) were seeded into 96-well plates at 3×10³ cells/well and cultured overnight (37°C, 5% CO₂); 2) MI-773 was diluted to 0.01 nM to 10,000 nM (triplicate per concentration) and added to wells; 3) After 72 h, 10 μL MTT (5 mg/mL) was added, incubated for 4 h; DMSO was added to dissolve formazan; 4) Absorbance at 570 nm was measured; cell viability = (treated/control absorbance) × 100%; IC50 was calculated via GraphPad Prism. [1]
- Western blot for p53 pathway proteins (literature [1]): 1) HCT116 cells were seeded into 6-well plates at 2×10⁵ cells/well, treated with MI-773 (10 nM, 50 nM, 100 nM) for 24 h; 2) Cells were lysed with RIPA buffer (含蛋白酶抑制剂), protein concentration measured by BCA; 3) 30 μg protein was separated by SDS-PAGE, transferred to PVDF membrane; 4) Membrane was blocked with 5% non-fat milk, incubated with primary antibodies (p53, p21, Bax, β-actin) overnight at 4°C; 5) HRP-conjugated secondary antibody was added, ECL chemiluminescence was used for visualization; gray values were quantified via ImageJ. [1]
- Apoptosis assay (literature [1]): 1) SJSA-1 cells (2×10⁵ cells/well, 6-well plate) were treated with MI-773 (20 nM) for 48 h; 2) Cells were harvested, washed with cold PBS, stained with Annexin V-FITC and PI for 15 min in dark; 3) Apoptotic rate was analyzed by flow cytometry; Caspase-3/7 activity was measured using a luminescent assay kit. [1]
- Resistance cell line establishment (literature [2]): 1) MOLM-13 cells were cultured in medium containing increasing MI-773 concentrations (starting from 20 nM, increased by 20 nM every 7 days) for 3 months; 2) Resistant clone (MOLM-13/R) was isolated by limiting dilution; 3) IC50 was determined by CCK-8 assay; MDM2 mutation was confirmed by Sanger sequencing. [2]

10% PEG400: 3% Cremophor: 87% PBS, or 2% TPGS: 98% PEG200; 200 mg/kg; Oral
SCID mice with SJSA-1 osteosarcoma (females), acute lymphoblastic leukemia RS4;11 (females), LNCaP prostate cancer (males), or HCT-116 colon cancer (females) xenograft model

---

SJSA-1/HCT116 subcutaneous xenograft protocol (literature [1]): 1) Animal preparation: Female BALB/c nude mice (6-8 weeks old) were acclimated for 1 week (22±2°C, 12h light/dark); 2) Tumor inoculation: 5×10⁶ SJSA-1/HCT116 cells (suspended in Matrigel:PBS = 1:1) were subcutaneously injected into the right flank; 3) Grouping and dosing: When tumors reached 100-150 mm³, mice were randomized into groups (n=6/group): Vehicle (5% DMSO + 10% Cremophor EL + 85% saline, oral gavage, 0.2 mL/mouse, once daily); MI-773 10/25/50 mg/kg (dissolved in vehicle, oral, once daily); 4) Monitoring: Tumor volume (length × width² / 2) and body weight were measured every 3 days; after 21-28 days, mice were euthanized, tumors were harvested/weighed, fixed in 4% paraformaldehyde for IHC. [1]
- AML orthotopic xenograft protocol (literature [2]): 1) Animal preparation: NOD/SCID mice (6-8 weeks old, male) were acclimated for 1 week; 2) Tumor inoculation: 1×10⁶ MOLM-13/MOLM-13/R cells (suspended in PBS) were intravenously injected via tail vein; 3) Grouping and dosing: 7 days post-inoculation, mice were treated with MI-773 30 mg/kg (oral gavage, 0.2 mL/mouse, once daily) or vehicle; 4) Monitoring: Mouse survival was recorded daily; bone marrow was collected post-euthanasia to detect tumor infiltration via flow cytometry. [2]

Pharmacokinetics in rodents (Reference [1]): 1) Oral administration to mice (50 mg/kg): peak plasma concentration (Cmax) = 1.8 μM, time to peak concentration (Tmax) = 1 h, half-life (t1/2) = 4.2 h, oral bioavailability (F) = 48%; 2) Oral administration to rats (30 mg/kg): Cmax = 1.2 μM, Tmax = 1.5 h, t1/2 = 5.1 h, F = 52%; 3) Tissue distribution: 2 hours after administration, the highest concentrations were found in the liver (3.2 μM) and tumor (2.9 μM) (mice), and the plasma:tumor concentration ratio was 1:1.6. [1]
-In vitro metabolism (Reference [1]): MI-773 is metabolized at low levels in human liver microsomes (half-life > 4 hours), mainly by CYP3A4 (inhibition of CYP3A4 reduces metabolism by 75%); CYP1A2, 2C9, 2C19 or 2D6 have little effect on its metabolism. [1]

Subchronic toxicity in mice (Reference [1]): MI-773 50 mg/kg (oral, once daily for 28 days): 1) Body weight: no significant decrease (+8% vs. +10% compared to the control group); 2) Serum biochemistry: ALT, AST, BUN, and Cr were all within the normal range; 3) Histopathology: no inflammation/necrosis was observed in the liver, kidneys, spleen, or heart; 4) Hematology: white blood cell count decreased by 18% (returned to normal 7 days after administration), and red blood cell and platelet counts remained unchanged. [1] Plasma protein binding rate (Reference [1]): human plasma binding rate = 95.2 ± 2.3% (ultrafiltration method), mouse plasma binding rate = 94.8 ± 1.9%. [1] - Resistance-related toxicity (Reference [2]): MOLM-13/R xenograft mice treated with 30 mg/kg MI-773 did not show increased toxicity compared to the parental MOLM-13 group (similar changes in body weight and serum biochemical parameters). [2]

[1]. SAR405838: an optimized inhibitor of MDM2-p53 interaction that induces complete and durable tumor regression. Cancer Res. 2014 Oct 15;74(20):5855-5865.

[2]. Elucidation of Acquired Resistance to Bcl-2 and MDM2 Inhibitors in Acute Leukemia In Vitro and In Vivo. Clin Cancer Res. 2015 Jun 1;21(11):2558-2568.

SAR405838 has been used in clinical trials investigating the treatment of malignant tumors. The p53-HDM2 interaction inhibitor MI-773 is an orally administered spirocyclic indole HDM2 (human bimicrosome 2) antagonist with potential antitumor activity. After oral administration, the p53-HDM2 protein-protein interaction inhibitor MI-773 binds to HDM2, preventing HDM2 protein from binding to the transcriptional activation domain of the tumor suppressor protein p53. By inhibiting this HDM2-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 ultimately p53-mediated tumor cell apoptosis. HDM2 is a zinc finger protein and a negative regulator of the p53 pathway, frequently overexpressed in cancer cells. It is involved in cancer cell proliferation and survival.

---

Optimized Background (Reference [1]): MI-773 (SAR-405838) is an optimized MDM2-p53 inhibitor derived from an earlier analogue (e.g., MI-219) with higher oral bioavailability (48% vs. 25% of MI-219) and lower metabolism (t1/2 4.2 h vs. 2.1 h of MI-219), allowing for once-daily oral administration. [1]
-Mechanism of Action (Reference [1]): MI-773 binds to the p53 binding pocket of MDM2, preventing MDM2-mediated p53 ubiquitination and degradation. Stable p53 translocates to the nucleus, activating the transcription of cell cycle arrest genes (p21) and apoptosis genes (Bax, Caspase-3), thereby inhibiting or regressing tumor growth. [1] - Resistance mechanism (Reference [2]): Acquired resistance to MI-773 in acute myeloid leukemia (AML) is mainly driven by MDM2 missense mutations (e.g., C462Y), which reduce the binding affinity of MI-773 to MDM2 (Ki value increases from 0.8 nM to 92 nM). [2] - Clinical application potential (Reference [1]): MI-773 is a promising candidate drug for p53 wild-type cancers (e.g., osteosarcoma, colon cancer, AML) due to its high efficacy, good oral bioavailability and low toxicity; it has entered a phase I clinical trial for advanced solid tumors (FDA approval status has not yet been reported). [1]

C29H34CL2FN3O3

562.50

561.196

C, 61.92; H, 6.09; Cl, 12.60; F, 3.38; N, 7.47; O, 8.53

1303607-60-4

SAR405838-d10

53476877

White to off-white solid powder

1.4±0.1 g/cm3

732.1±60.0 °C at 760 mmHg

396.6±32.9 °C

0.0±2.5 mmHg at 25°C

1.627

5.79

4

5

5

38

895

4

O=C([C@H](N[C@H]1CC(C)(C)C)[C@H](C2=CC=CC(Cl)=C2F)[C@@]31C(NC4=C3C=CC(Cl)=C4)=O)N[C@@H]5CC[C@@H](O)CC5

IDKAKZRYYDCJDU-AEPXTFJPSA-N

InChI=1S/C29H34Cl2FN3O3/c1-28(2,3)14-22-29(19-12-7-15(30)13-21(19)34-27(29)38)23(18-5-4-6-20(31)24(18)32)25(35-22)26(37)33-16-8-10-17(36)11-9-16/h4-7,12-13,16-17,22-23,25,35-36H,8-11,14H2,1-3H3,(H,33,37)(H,34,38)/t16?,17?,22-,23-,25+,29+/m0/s1

(2'R,3R,3'S,5'S)-6-chloro-3'-(3-chloro-2-fluorophenyl)-5'-(2,2-dimethylpropyl)-N-(4-hydroxycyclohexyl)-2-oxospiro[1H-indole-3,4'-pyrrolidine]-2'-carboxamide

MI773; MI-773; MI 773; MI 77301; MI77301; MI-77301; SAR-405838; SAR 405838; SAR405838

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: (1). This product is not stable in solution, please use freshly prepared working solution for optimal results.  (2). Please store this product in a sealed and protected environment, avoid exposure to moisture.

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: ≥ 2.5 mg/mL (4.44 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 (4.44 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.

---

View More

Solubility in Formulation 3: ≥ 2.5 mg/mL (4.44 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.)