PLK1 9 nM (IC50) PLK2 260 nM (IC50) PDGFR 18 nM (IC50) Src 155 nM (IC50) BCR-ABL 32 nM (IC50) Cdk1 260 nM (IC50) Flt1 42 nM (IC50) Fyn 182 nM (IC50)

Rigosertib has an IC50 of 9 nM and inhibits PLK1 in a non-ATP-competitive manner. Additionally, PLK2, PDGFR, Flt1, BCR-ABL, Fyn, Src, and CDK1 are inhibited by rigosertib, with an IC50 of 18–260 nM. 94 distinct tumor cell lines, including BT27, MCF-7, DU145, PC3, U87, A549, H187, RF1, HCT15, SW480, and KB cells, exhibit cell killing activity in response to rigosertib, with an IC50 of 50–250 nM. Rigosertib, however, has little to no effect in normal cells, such as HFL, PrEC, HMEC, and HUVEC, until the concentration is higher than 5–10 μM. Rigosertib (100-250 nM) causes spindle aberrations and apoptosis in HeLa cells[3]. Moreover, MES-SA, MES-SA/DX5a, CEM, and CEM/C2a are among the multidrug-resistant tumor cell lines that rigosertib suppresses with an IC50 of 50–100 nM. In DU145 cells, rigosertib (0.25–5 μM) inhibits the G2/M phase of the cell cycle, causes a build-up of cells with DNA content in the subG1 region, and triggers apoptotic pathways. In A549 cells, rigosertib (50 nM-0.5 μM) causes caspase 3/7 activation and viability loss[4]. Inducing apoptosis in chronic lymphocytic leukemia (CLL) cells, rigosertib sodium (2 μM) does not cause harm to T-cells or healthy B-cells. Additionally, the pro-survival effect of follicular dendritic cells on CLL cells is eliminated by rigosertib sodium (2 μM), which also lessens the migration of leukemic cells produced by SDF-1[5].

Tumor growth in mouse xenograft models of Bel-7402, MCF-7, and MIA-PaCa cells is significantly inhibited by rigosertib (250 mg/kg, ip)[3]. BT20 cell tumor growth is inhibited in a mouse xengraft model by rigosertib (200 mg/kg, ip)[4].

For 30 minutes at room temperature, recombinant PLK1 (10 ng) is incubated with varying concentrations of rigosertib in a 15 µL reaction mixture (50 mM HEPES, 10 mM MgCl2, 1 mM EDTA, 2 mM Dithiothreitol, 0.01% NP-40 [pH 7.5]). 20 µL (15 µL enzyme + inhibitor, 2 µL 1 mM ATP), 2 µL of γ32P-ATP (40 μCi), and 1 µL of recombinant Cdc25C (100 ng) or casein (1 μg) substrates are used in kinase reactions, which are carried out for 20 min at 30 °C. Boiling in 20 µL of 2× Laemmli buffer for 2 minutes ends the reaction. 18% SDS-PAGE is used to separate phosphorylated substrates. After drying, the gels are exposed to X-ray film for three to ten minutes.

Cytotoxicity of Rigosertib (ON 01910.Na) against CLL cells from 34 patients was determined in vitro with flow cytometry of cells stained with Annexin V and CD19. Global gene expression profiling on Affymetrix microarrays, flow cytometry, Western blotting, and cocultures with stroma cells were used to delineate ON 01910.Na mechanism of action.[3]

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Cytotoxicity Assay[2]
Researchers tested a number of tumor cell lines using a dose response end point assay system. The cells were grown in either DMEM or RPMI supplemented with 10% fetal bovine serum and 1unit/mL Penicillin-Streptomycin solution. The tumor cells were plated into 6 well dishes at a cell density of 1.0 × 105 cells/mL/well and compounds were added 24 h later at various concentrations. Cell counts were determined from duplicate wells after 96 h of treatment. The total number of viable cells was determined by trypan blue exclusion. Flow Cytometry Human prostate tumor cells, DU145 cells, and normal diploid human lung fibroblasts, HFL-1 cells, were grown in DMEM supplemented with 10% fetal bovine serum and 1 unit/mL penicillin-streptomycin. The cells were plated onto 100 mm2 dishes at a cell density of 1.0 × 106 cells/dish, and 24 h later, they were treated with 2.5 μM of the compound. The cells were harvested 24 h after treatment. The cells were removed from the plate by trypsin digestion and combined with the non-attached cells found in the medium. The cell pellets were washed in phosphate buffered saline (PBS), and fixed in ice cold 70% ethanol for at least 24 h. The fixed cells were then washed with room temperature PBS and stained with propidium iodide (50 mg/mL) and RNase A (0.5 mg) for 30 min at 37 °C. The stained cells were then analyzed on a Becton-Dickinson flow cytometer and the data analyzed by cell cycle analysis software.

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PARP Western [2]
DU145 and HFL-1 cells were plated at a density of 3.0 × 106 cells per 150 mm2 plate and treated 24 h later with either DMSO or 28. The cells were collected 48 h treatment and cell pellets were frozen. The frozen cell pellets were lysed in 1% NP40/PBS lysis buffer containing protease inhibitors. Equal amounts of total cellular protein was then resolved on a 10%-SDS-polyacrylamide gel. The gels were transferred onto nitrocellulose paper (S/S), hybridized with anti-PARP antibodies (BD) and developed using ECL solution.

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Cellular Viability and Caspase 3/7 Activity [2]
Exponentially growing A549 cells were seeded in a white walled 96-well plate at a density of 3,600 cells/well in 100 μl of DMEM containing 10% FBS and 1% Pen/Strep. Cells were then allowed to adhere overnight at 37 °C in an incubator. The next day, cells were treated with varying concentrations of 28 or DMSO and then returned to the incubator. 24 h later, plates were removed from the incubator and 20 μL of CellTiter-Blue® Reagent was added individually to each well following manufacturer’s instructions. Plates were slowly shaken for 0.5 min and then returned to the incubator. After 3 h, fluorescence was read using a Glomax 96-well plate reader. Next, 120 μL of Caspase Glo® 3/7 Reagent was added to each well per manufacturer’s instructions. Plates were slowly shaken for 0.5 min and allowed to develop at room temperature for 2 h. At the end of this period, luminescence was read using a Glomax 96-well plate reader.

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One day later, different concentrations of Rigosertib are added to six-well dishes containing 1×10⁵ cells/mL/well of tumor cells that have been plated. After treatment for 96 hours, cell counts are obtained from duplicate wells. By using trypan blue exclusion, the total number of viable cells is found.

[1]. Rigosertib as a selective anti-tumor agent can ameliorate multiple dysregulated signalingtransduction pathways in high-grade myelodysplastic syndrome. Sci Rep. 2014 Dec 4;4:7310.

[2]. Rigosertib induces cell death of a myelodysplastic syndrome-derived cell line by DNA damage-induced G2/M arrest. Cancer Sci. 2015 Mar;106(3):287-93.

[3]. ON01910, a non-ATP-competitive small molecule inhibitor of Plk1, is a potent anticancer agent. Cancer Cell. 2005 Mar;7(3):275-86.

[4]. Discovery of a clinical stage multi-kinase inhibitor sodium (E)-2-{2-methoxy-5-[(2',4',6'-trimethoxystyrylsulfonyl)methyl]phenylamino}acetate (ON 01910.Na): synthesis, structure-activity relationship, and biological activity. J Med Chem.

[5]. ON 01910.Na is selectively cytotoxic for chronic lymphocytic leukemia cells through a dual mechanism of action involving PI3K/AKT inhibition and induction of oxidative stress. 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).

C21H24NNAO8S

473.47

473.112

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

592542-60-4

Rigosertib;592542-59-1;(E/Z)-Rigosertib sodium;1225497-78-8

23696523

White to yellow solid powder

2.622

1

9

11

32

684

0

COC1=C(C=C(C=C1)CS(=O)(=O)/C=C/C2=C(C=C(C=C2OC)OC)OC)NCC(=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

1225497-78-8; 592542-60-4; Estybon; Novonex; ON-01910 sodium; ON-01910; ON01910 sodium;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), 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)

DMSO : 150 mg/mL (316.81 mM)
H2O : ≥ 52 mg/mL (109.83 mM)

Solubility in Formulation 1: ≥ 5.25 mg/mL (11.09 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 52.5 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: ≥ 5.25 mg/mL (11.09 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 52.5 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.

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Solubility in Formulation 3: ≥ 5.25 mg/mL (11.09 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 52.5 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: ≥ 2.5 mg/mL (5.28 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), suspension 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 5: ≥ 2.5 mg/mL (5.28 mM) (saturation unknown) 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 6: 50 mg/mL (105.60 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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