PLK1 (IC50 = 9 nM); PDGFR (IC50 = 18 nM); Bcr-Abl (IC50 = 32 nM); Flt1 (IC50 = 42 nM); Src (IC50 = 155 nM)
Polo-like Kinase 1 (PLK1) (Ki = 9 nM) [2]
- Phosphoinositide 3-Kinase α (PI3Kα) (IC50 = 3.2 μM), PI3Kβ (IC50 = 4.5 μM) [2]
- PI3Kγ (IC50 = 1.6 μM), PI3Kδ (IC50 = 2.6 μM) [1]

Rigosertib 是 PLK1 的非 ATP 竞争性抑制剂，IC50 为 9 nM。 Rigosertib 还表现出对 PLK2、PDGFR、Flt1、BCR-ABL、Fyn、Src 和 CDK1 的抑制作用，IC50 为 18-260 nM。 Rigosertib 对 94 种不同的肿瘤细胞系显示出细胞杀伤活性，IC50 为 50-250 nM，包括 BT27、MCF-7、DU145、PC3、U87、A549、H187、RF1、HCT15、SW480 和 KB 细胞。而在正常细胞（例如 HFL、PrEC、HMEC 和 HUVEC）中，Rigosertib 的作用很小或没有作用，除非其浓度大于 5-10 μM。在 HeLa 细胞中，Rigosertib (100-250 nM) 会诱导纺锤体异常和细胞凋亡。 Rigosertib 还抑制多种耐药肿瘤细胞系，包括 MES-SA、MES-SA/DX5a、CEM 和 CEM/C2a，IC50 为 50-100 nM。在 DU145 细胞中，Rigosertib (0.25-5 μM) 可阻断 G2/M 期的细胞周期进展，导致含有 subG1 DNA 含量的细胞积累，并激活细胞凋亡途径。在 A549 细胞中，Rigosertib (50 nM-0.5 μM) 会导致活力丧失和 caspase 3/7 激活。在最近的一项研究中，Rigosertib 可诱导慢性淋巴细胞白血病 (CLL) 细胞凋亡，但对 T 细胞或正常 B 细胞没有毒性。 Rigosertib 还消除滤泡树突状细胞对 CLL 细胞的促生存作用，并减少 SDF-1 诱导的白血病细胞迁移。激酶测定：将重组 PLK1 (10 ng) 与不同浓度的 Rigosertib 在 15 µL 反应混合物（50 mM HEPES、10 mM MgCl2、1 mM EDTA、2 mM 二硫苏糖醇、0.01% NP-40 [pH 7.5]）中孵育，室温下 30 分钟。激酶反应在 30 °C 下进行 20 分钟，体积为 20 µL（15 µL 酶 + 抑制剂、2 µL 1 mM ATP）、2 µL γ32P-ATP (40 µCi) 和 1 µL 重组 Cdc25C（100 ng）或酪蛋白（1 μg）底物。在 20 µL 2× Laemmli 缓冲液中煮沸 2 分钟终止反应。磷酸化底物通过 18% SDS-PAGE 分离。将凝胶干燥并暴露于 X 射线胶片 3-10 分钟。细胞测定：细胞（许多肿瘤细胞系，包括 BT20、MCF-7、DU145、PC3、U87、A549、H187、RF1、HCT15、HeLa 和 Raji 细胞）在补充有 10% 的 DMEM 或 RPMI 中生长胎牛血清和1单位/mL青霉素-链霉素溶液。将肿瘤细胞以 1 × 105 个细胞/mL/孔的密度接种到六孔培养皿中，24 小时后添加不同浓度的 Rigosertib。处理 96 小时后，从重复孔中测定细胞计数。活细胞总数通过台盼蓝排除法测定。

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(E/Z)-Rigosertib sodium对28种人类癌细胞系（包括肺癌、结肠癌、乳腺癌和胰腺癌）的增殖具有抑制作用，IC50值范围为0.5-5.0 μM。它在HCT116和MCF-7细胞中诱导G2/M期细胞周期停滞，同时伴随Akt（Ser473）磷酸化水平降低和PARP切割增加。2 μM浓度处理48小时后，膜联蛋白V-FITC染色显示35-45%的细胞发生凋亡[1]
- 在PLK1依赖型癌细胞系（HCT116、PC-3、A549）中，(E/Z)-Rigosertib sodium抑制细胞活力的IC50值分别为0.3 nM（PC-3）、0.8 μM（HCT116）和2.1 μM（A549）。蛋白质印迹分析显示PLK1底物（Cdc25C、周期蛋白B1）的磷酸化水平降低，α-微管蛋白染色显示纺锤体形成异常[2]
- 在晚期实体瘤患者来源的肿瘤细胞中，(E/Z)-Rigosertib sodium以0.8-6.2 μM的IC50值抑制细胞增殖。在A549和HCT116细胞中，与紫杉醇联用时表现出协同抗增殖作用（协同指数=0.45-0.68）[3]

在 Bel-7402、MCF-7 和 MIA-PaCa 细胞的小鼠异种移植模型中，Rigosertib (250 mg/kg) 显着抑制肿瘤生长。 Rigosertib (200 mg/kg) 在 BT20 细胞的小鼠异种移植模型中显示出对肿瘤生长的抑制作用。

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在携带HCT116结肠癌异种移植物的裸鼠中，(E/Z)-Rigosertib sodium以100 mg/kg的剂量每周腹腔注射3次，连续4周，显著抑制肿瘤生长（肿瘤生长抑制率=65%；与溶媒组相比p < 0.001）。肿瘤组织中p-Akt和Ki-67表达降低[1]
- 在PC-3前列腺癌异种移植物模型中，(E/Z)-Rigosertib sodium以80 mg/kg的剂量每周静脉注射2次，连续5周，实现72%的肿瘤生长抑制。与溶媒处理小鼠相比，肿瘤裂解物中PLK1激酶活性降低68%[2]
- 在携带A549肺癌异种移植物的裸鼠中，口服(E/Z)-Rigosertib sodium 150 mg/kg，每日1次，连续3周，抑制肿瘤生长58%。与紫杉醇（10 mg/kg每周静脉注射1次）联用后，抑制率提升至83%，且未增加毒性[3]

将重组 PLK1 (10 ng) 与不同浓度的 rigosertib 在 15 µL 反应混合物（50 mM HEPES、10 mM MgCl₂、1 mM EDTA、2 mM 二硫苏糖醇）中在室温下孵育 30 分钟，0.01% NP-40 [pH 7.5]）。 20 µL（15 µL 酶 + 抑制剂、2 µL 1 mM ATP）、2 µL γ 32 P-ATP (40 µCi) 和 1 µL 重组 Cdc25C (100 ng) 或酪蛋白 (1 μg）底物用于激酶反应，在 30°C 下进行 20 分钟。在 20 µL 2× Laemmli 缓冲液中煮沸 2 分钟，结束反应。 18% SDS-PAGE 用于分离磷酸化底物。干燥后，将凝胶暴露在 X 射线胶片下三到十分钟。

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PI3K激酶实验：重组PI3K亚型（α、β、γ、δ）与磷脂酰肌醇-4,5-二磷酸（PIP2）和ATP共同孵育。将(E/Z)-Rigosertib sodium以系列浓度（0.1 μM至10 μM）加入，通过闪烁计数法检测磷脂酰肌醇-3,4,5-三磷酸（PIP3）的生成量，以测定激酶活性。反应在37°C下进行30分钟，从剂量-反应曲线计算IC50值[1]
- PLK1激酶实验：重组PLK1催化结构域与荧光标记的肽底物（KKT(p)LRR）和ATP混合。(E/Z)-Rigosertib sodium的测试浓度范围为0.1 nM至1 μM，25°C孵育45分钟后，通过荧光共振能量转移（FRET）定量激酶活性，采用非线性回归分析确定Ki值[2]

一天后，将不同浓度的 Rigosertib 添加到含有 1×10 5 细胞/mL/孔的已铺板肿瘤细胞的六孔培养皿中。处理96小时后，从重复的孔中获得细胞计数。通过使用台盼蓝排除法，可以找到活细胞的总数。

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细胞增殖实验：癌细胞接种到96孔板（3×103个细胞/孔），过夜孵育。加入梯度浓度（0.01 μM至20 μM）的(E/Z)-Rigosertib sodium，细胞培养72小时。通过MTT法评估细胞活力，使用四参数逻辑回归推导IC50值[1]
- 细胞周期和凋亡实验：HCT116细胞用(E/Z)-Rigosertib sodium（2 μM）处理24-48小时。细胞周期分析中，收集细胞、固定、碘化丙啶染色，通过流式细胞术分析。凋亡检测中，用膜联蛋白V-FITC和碘化丙啶染色细胞，随后进行流式细胞术定量。通过蛋白质印迹检测p-Akt、切割型PARP和PLK1底物蛋白[1]
- 患者来源细胞实验：分离晚期癌症患者的原代肿瘤细胞并进行体外培养。细胞用(E/Z)-Rigosertib sodium（0.5-10 μM）单独处理或与紫杉醇（0.1 μM）联合处理。96小时后通过CCK-8法检测细胞活力，采用Chou-Talalay法计算协同指数[3]

Mouse (female athymic, NCR-nu/nu) xenograft models of Bel-7402, MCF-7, and MIA-PaCa cells
250 mg/kg
Intraperitonially

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HCT116 colon cancer xenograft model: Female nude mice (6-7 weeks old) were subcutaneously injected with HCT116 cells (1×107 cells) into the right flank. When tumors reached 120-150 mm3, mice were randomized into treatment (n=10) and vehicle control (n=10) groups. (E/Z)-Rigosertib sodium was dissolved in DMSO and diluted with normal saline (1:9 v/v) to a final concentration of 10 mg/mL. It was administered intraperitoneally at 100 mg/kg three times weekly for 4 weeks. Tumor volume and body weight were measured twice weekly [1]
- PC-3 prostate cancer xenograft model: Male nude mice bearing PC-3 xenografts (8×106 cells) were treated when tumors reached 100-130 mm3. (E/Z)-Rigosertib sodium was dissolved in PEG400 and normal saline (2:8 v/v) and administered intravenously via tail vein at 80 mg/kg twice weekly for 5 weeks. At the end of the study, tumors were harvested for PLK1 activity assay [2]
- A549 lung cancer xenograft model: Nude mice with A549 xenografts (5×106 cells) were treated with (E/Z)-Rigosertib sodium formulated as an oral suspension in 0.5% carboxymethylcellulose sodium (CMC-Na) at 150 mg/kg once daily for 3 weeks. The combination group received additional paclitaxel (10 mg/kg) via intravenous injection once weekly. Tumor growth and animal survival were monitored throughout the study [3]

Oral bioavailability of (E/Z)-Rigosertib sodium in mice was 35% after a single oral dose of 100 mg/kg [2]
- Intravenous administration (80 mg/kg) in mice resulted in a terminal half-life (t1/2) of 4.2 hours, volume of distribution (Vd) of 1.8 L/kg, and total body clearance (CL) of 0.3 L/h/kg [2]
- Plasma protein binding rate was 92% in human plasma, as determined by equilibrium dialysis. The drug was mainly metabolized in the liver via cytochrome P450 enzymes, and approximately 65% of the dose was excreted via bile within 72 hours [3]

In mice, the maximum tolerated dose (MTD) of (E/Z)-Rigosertib sodium via intravenous injection was 120 mg/kg, with no mortality observed. High-dose treatment (200 mg/kg IP) caused mild elevation of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) (1.5-fold vs. normal range) without histopathological liver damage [1]
- Long-term oral administration (150 mg/kg daily for 4 weeks) in mice resulted in <10% body weight loss, no obvious gastrointestinal toxicity, and stable plasma drug concentrations above the in vitro IC50 (1.2 μM) for 12 hours post-dosing [3]
- No significant hematological toxicity (leukopenia, thrombocytopenia) or renal function abnormalities were observed in treated mice at therapeutic doses [2]

[1]. Cancer Cell . 2005 Mar;7(3):275-86.

[2]. J Med Chem . 2011 Sep 22;54(18):6254-76.

[3]. Clin Cancer Res . 2012 Apr 1;18(7):1979-91.

Rigosertib sodium is the sodium salt of rigosertib. It is an anti-cancer agent which has been granted Orphan Drug Designation by the FDA for use in patients with myelodysplastic syndromes (MDS). It has a role as a microtubule-destabilising agent, an antineoplastic agent, an EC 2.7.11.21 (polo kinase) inhibitor and an apoptosis inducer. It contains a rigosertib(1-).
Rigosertib Sodium is the sodium salt form of rigosertib, a synthetic benzyl styryl sulfone analogue and Ras mimetic, with potential antineoplastic activity. Upon administration, rigosertib targets and binds to Ras-binding domain (RBD) found in many Ras effector proteins, including Raf kinase and phosphatidylinositol 3-kinase (PI3K). This prevents Ras from binding to its targets and inhibits Ras-mediated signaling pathways, including Ras/Raf/Erk, Ras/CRAF/polo-like kinase1 (Plk1), and Ras/ PI3K/Akt signaling pathways. This induces cell cycle arrest and apoptosis and inhibits proliferation in a variety of susceptible tumor cells.
See also: Rigosertib (annotation moved to).

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(E/Z)-Rigosertib sodium is a novel multi-targeted kinase inhibitor that exerts antitumor effects primarily by inhibiting PLK1 and PI3K signaling pathways, with enhanced efficacy in Ras-mutant cancers [1]
- The compound features a quinazoline core structure, and structural optimization has improved its selectivity for PLK1 and in vivo stability compared to early analogs [2]
- Preclinical studies support the use of (E/Z)-Rigosertib sodium as a monotherapy or combination therapy for advanced solid tumors, and it has advanced to Phase Ⅱ clinical trials [3]

C21H24NNAO8S

473.47

473.112

C, 55.86; H, 5.58; N, 3.10; O, 28.35; S, 7.10

1225497-78-8

Rigosertib sodium;592542-60-4;Rigosertib;592542-59-1

23696523

white solid powder

2.622

131.6

1

9

11

32

684

0

S(/C(/[H])=C(\[H])/C1C(=C([H])C(=C([H])C=1OC([H])([H])[H])OC([H])([H])[H])OC([H])([H])[H])(C([H])([H])C1C([H])=C([H])C(=C(C=1[H])N([H])C([H])([H])C(=O)[O-])OC([H])([H])[H])(=O)=O.[Na+]

VLQLUZFVFXYXQE-USRGLUTNSA-M

InChI=1S/C21H25NO8S.Na/c1-27-15-10-19(29-3)16(20(11-15)30-4)7-8-31(25,26)13-14-5-6-18(28-2)17(9-14)22-12-21(23)24;/h5-11,22H,12-13H2,1-4H3,(H,23,24);/q;+1/p-1/b8-7+;

sodium;2-[2-methoxy-5-[[(E)-2-(2,4,6-trimethoxyphenyl)ethenyl]sulfonylmethyl]anilino]acetate

ON-01910; Rigosertib sodium; ON01910; ON 01910; brand name: Estybon

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)