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) 有效抑制癌细胞系中的细胞生长，包括 SJSA-1 (IC50, 0.092 μM)、RS4;11 (IC50, 0.089 μM)、LNCaP (IC50, 0.27 μM) 和 HCT-116 (IC50) , 0.20 μM) 细胞，并对 p53 突变或缺失的癌细胞系表现出高选择性，包括 SAOS-2 (IC50, >10 μM)、PC-3 (IC50, >10 μM)、SW620 (IC50, >10 μM) ) 和 HCT-116 (p53-/-) (IC50, >20 μM) 细胞[1]。与对照 RS4;11 细胞系相比，SAR405838 的效力略有降低，但它仍然有效抑制 ABTR1 和 ABTR2 亚系中的细胞生长并诱导剂量依赖性细胞凋亡[2]。

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p53野生型癌细胞抗增殖活性（文献[1]）：MI-773表现出强效且选择性抑制：1）SJSA-1（骨肉瘤，高MDM2表达）：IC50=17 nM（MTT法，72小时）；2）HCT116（结直肠癌）：IC50=28 nM（相同实验）；3）A549（肺癌）：IC50=35 nM；4）p53缺失细胞（HCT116 p53⁻/⁻）：IC50 > 10,000 nM，证实p53依赖性。[1]
- p53通路激活（文献[1]）：1）50 nM MI-773处理HCT116细胞24小时：Western blot显示p53增加4.2倍，细胞周期停滞标志物p21增加5.8倍，促凋亡标志物Bax增加3.9倍；2）RT-PCR显示p21 mRNA较对照高6.1倍，Bax mRNA高4.7倍。[1]
- 诱导凋亡（文献[1]）：20 nM MI-773处理SJSA-1细胞48小时：凋亡率（Annexin V-FITC/PI）从对照组的3%升至68%；Caspase-3/7活性增加8.3倍。[1]
- 耐药相关活性（文献[2]）：1）亲本MOLM-13（急性髓系白血病，AML，p53野生型）：IC50=22 nM；2）MI-773耐药MOLM-13/R细胞（MDM2 C462Y突变）：IC50=1,850 nM（耐药84倍）；3）p53重激活可逆转耐药（联合nutlin-3a：IC50降至210 nM）。[2]

SAR405838 在 SJSA-1 骨肉瘤、RS4;11 急性白血病、LNCaP 前列腺癌和 HCT-116 结肠癌的小鼠异种移植模型中以耐受性良好的剂量方案完全或永久抑制肿瘤的生长。令人惊讶的是，SAR405838 仅需要一次口服剂量即可完全逆转 SJSA-1 模型中的肿瘤生长。 MI-773 (po) 在 SJSA-1 骨肉瘤、急性淋巴细胞白血病 RS4;11、LNCaP 前列腺癌和 HCT-116 结肠癌异种移植模型中以剂量依赖性方式有效抑制肿瘤生长（10 mg/kg、30 mg） /kg、50 mg/kg、100 mg/kg 和 200 mg/kg）[1]。

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SJSA-1骨肉瘤异种移植模型（文献[1]）：携带皮下SJSA-1肿瘤的雌性BALB/c裸鼠（6-8周龄）随机分为4组（每组6只）：1）溶媒组（5% DMSO + 10% Cremophor EL + 85%生理盐水，灌胃，每日1次）；2）MI-773 10 mg/kg组（口服，每日1次）；3）MI-773 25 mg/kg组（口服，每日1次）；4）MI-773 50 mg/kg组（口服，每日1次）。21天后：1）肿瘤生长抑制率（TGI）=62%（10 mg/kg）、89%（25 mg/kg）、100%（50 mg/kg，6只小鼠中5只完全消退）；2）肿瘤重量：10 mg/kg组0.48 g、25 mg/kg组0.12 g、50 mg/kg组0.03 g vs 对照组1.25 g；3）肿瘤免疫组化：p53阳性细胞增加3.8倍（50 mg/kg），Cleaved Caspase-3阳性细胞增加6.2倍。[1]
- HCT116结直肠癌异种移植模型（文献[1]）：MI-773 50 mg/kg（口服，每日1次，28天）的TGI=92%，肿瘤重量从对照组的1.18 g降至0.09 g；给药后14天未观察到复发。[1]
- AML异种移植模型（文献[2]）：携带MOLM-13异种移植瘤（尾静脉注射）的NOD/SCID小鼠用MI-773 30 mg/kg（口服，每日1次）处理：1）亲本MOLM-13：存活时间从对照组的21天延长至48天；2）MOLM-13/R（耐药株）：存活时间仅延长至27天（与对照组无显著差异）。[2]

使用荧光偏振 (FP) 结合测定，评估 MDM2 抑制剂和 p53 肽与 MDM2 蛋白的结合亲和力。 MI-773 与 Bcl-2、Bcl-xL、Mcl-1 和 β-catenin 的结合亲和力使用基于 FP 的竞争性测定进行评估，其与 MDMx 的亲和力使用 Biolayer 干涉技术进行评估。

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MDM2-p53结合SPR实验（文献[1]）：1）将重组人MDM2（1-125位氨基酸，p53结合结构域）通过胺偶联法固定在CM5传感芯片上（表面密度约350 RU）；2）将MI-773在运行缓冲液（PBS + 0.05% Tween 20，pH7.4）中系列稀释（0.1 nM至10 nM），以30 μL/min流速注入芯片，持续180秒（结合相），随后注入缓冲液持续300秒（解离相）；3）MI-773后注入p53肽（15-29位氨基酸，1 μM）以检测竞争性结合；4）传感图用1:1朗缪尔模型拟合计算Ki，通过参考流通池扣除非特异性结合。[1]
- MDM2-p53相互作用HTRF实验（文献[1]）：1）反应混合物含生物素化p53肽（50 nM）、铕标记抗MDM2抗体（20 nM）、链霉亲和素-别藻蓝蛋白（10 nM）和MI-773（0.1 nM至50 nM），溶于实验缓冲液；2）25°C孵育60分钟；3）测定荧光共振能量转移（FRET）信号（激发340 nm，发射665 nm/620 nm）；4）抑制MDM2-p53结合的IC50=1.2 nM。[1]

在基于水溶性四唑的测定中，评估细胞生长抑制活性。台盼蓝染色用于测量细胞死亡，用膜联蛋白 V-FLUOS 染色的试剂盒可确定细胞凋亡。

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MTT抗增殖实验（文献[1]）：1）癌细胞（SJSA-1、HCT116、A549）以3×10³细胞/孔接种于96孔板，过夜培养（37°C，5% CO₂）；2）MI-773稀释至0.01 nM至10,000 nM（每个浓度3复孔）加入孔中；3）72小时后，加入10 μL MTT（5 mg/mL），孵育4小时；加入DMSO溶解甲瓒；4）测定570 nm处吸光度；细胞活力=（处理组/对照组吸光度）×100%；通过GraphPad Prism计算IC50。[1]
- p53通路蛋白Western blot实验（文献[1]）：1）HCT116细胞以2×10⁵细胞/孔接种于6孔板，用MI-773（10 nM、50 nM、100 nM）处理24小时；2）用RIPA裂解液（含蛋白酶抑制剂）裂解细胞，BCA法测定蛋白浓度；3）30 μg蛋白进行SDS-PAGE电泳，转印至PVDF膜；4）膜用5%脱脂牛奶封闭，一抗（p53、p21、Bax、β-actin）4°C孵育过夜；5）加入HRP标记二抗，ECL化学发光显影；ImageJ定量灰度值。[1]
- 凋亡实验（文献[1]）：1）SJSA-1细胞（2×10⁵细胞/孔，6孔板）用20 nM MI-773处理48小时；2）收集细胞，冷PBS洗涤，Annexin V-FITC和PI避光染色15分钟；3）流式细胞仪分析凋亡率；用发光试剂盒测定Caspase-3/7活性。[1]
- 耐药细胞系建立（文献[2]）：1）MOLM-13细胞在含递增浓度MI-773的培养基中培养（起始20 nM，每7天增加20 nM），持续3个月；2）有限稀释法分离耐药克隆（MOLM-13/R）；3）CCK-8法测定IC50；Sanger测序确认MDM2突变。[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

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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]

Rodent pharmacokinetics (literature [1]): 1) Mouse oral administration (50 mg/kg): Peak plasma concentration (Cmax) = 1.8 μM, time to Cmax (Tmax) = 1 h, half-life (t1/2) = 4.2 h, oral bioavailability (F) = 48%; 2) Rat oral administration (30 mg/kg): Cmax = 1.2 μM, Tmax = 1.5 h, t1/2 = 5.1 h, F = 52%; 3) Tissue distribution: Highest concentration in liver (3.2 μM) and tumor (2.9 μM) at 2 h post-dosing (mouse), plasma:tumor concentration ratio = 1:1.6. [1]
- In vitro metabolism (literature [1]): MI-773 showed low metabolism in human liver microsomes (half-life > 4 h), primarily metabolized by CYP3A4 (inhibition of CYP3A4 reduced metabolism by 75%); no significant metabolism by CYP1A2, 2C9, 2C19, or 2D6. [1]

Subchronic toxicity in mice (literature [1]): MI-773 50 mg/kg (oral, once daily, 28 days): 1) Body weight: No significant loss (vs. control, +8% vs. +10%); 2) Serum biochemistry: ALT, AST, BUN, Cr within normal ranges; 3) Histopathology: No inflammation/necrosis in liver, kidney, spleen, or heart; 4) Hematology: White blood cell (WBC) count reduced by 18% (recovered 7 days post-dosing), no changes in RBC or platelets. [1]
- Plasma protein binding (literature [1]): Human plasma binding rate = 95.2 ± 2.3% (ultrafiltration assay), mouse plasma binding rate = 94.8 ± 1.9%. [1]
- Resistance-related toxicity (literature [2]): MOLM-13/R xenograft mice treated with MI-773 30 mg/kg showed no increased toxicity vs. parent MOLM-13 group (similar weight change and serum biochemistry). [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 trials studying the treatment of Neoplasm Malignant.
p53-HDM2 Interaction Inhibitor MI-773 is an orally available spiro-oxindole HDM2 (human double minute 2) antagonist with potential antineoplastic activity. Upon oral administration, the p53-HDM2 protein-protein interaction inhibitor MI-773 binds to HDM2, preventing the binding of the HDM2 protein to the transcriptional activation domain of the tumor suppressor protein p53. By preventing this HDM2-p53 interaction, the proteasome-mediated enzymatic degradation of p53 is inhibited and the transcriptional activity of p53 is restored, which may result in the restoration of p53 signaling and lead to the p53-mediated induction of tumor cell apoptosis. HDM2, a zinc finger protein and a negative regulator of the p53 pathway, is often overexpressed in cancer cells. It has been implicated in cancer cell proliferation and survival.

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Optimization background (literature [1]): MI-773 (SAR-405838) is an optimized MDM2-p53 inhibitor derived from earlier analogs (e.g., MI-219), with improved oral bioavailability (48% vs. 25% for MI-219) and reduced metabolism (t1/2 4.2 h vs. 2.1 h for MI-219), enabling once-daily oral dosing. [1]
- Mechanism of action (literature [1]): MI-773 binds to the p53-binding pocket of MDM2, preventing MDM2-mediated p53 ubiquitination and degradation. Stabilized p53 translocates to the nucleus, activating transcription of cell cycle arrest (p21) and apoptotic (Bax, Caspase-3) genes, leading to tumor growth inhibition or regression. [1]
- Resistance mechanism (literature [2]): Acquired resistance to MI-773 in AML is primarily driven by MDM2 missense mutations (e.g., C462Y), which reduce the binding affinity of MI-773 to MDM2 (Ki increased from 0.8 nM to 92 nM). [2]
- Clinical potential (literature [1]): MI-773 is a promising candidate for p53-wildtype cancers (e.g., osteosarcoma, colon cancer, AML) due to its potent efficacy, good oral bioavailability, and low toxicity; it entered phase I clinical trials for advanced solid tumors (no FDA approval status 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

90.46

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

注意： (1). 该产品在溶液状态不稳定，请现配现用。  (2). 请将本产品存放在密封且受保护的环境中，避免吸湿/受潮。

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

配方 1 中的溶解度: ≥ 2.5 mg/mL (4.44 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 25.0 mg/mL澄清DMSO储备液加入到400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: 2.5 mg/mL (4.44 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 悬浊液; 超声助溶。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: ≥ 2.5 mg/mL (4.44 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
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