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, which has an IC50 of 9 nM, is a non-ATP-competitive inhibitor of PLK1. Moreover, rigosertib shows inhibition with an IC50 of 18-260 nM against PLK2, PDGFR, Flt1, BCR-ABL, Fyn, Src, and CDK1. 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, unless the concentration is higher than 5–10 μM. Rigosertib (100-250 nM) causes spindle abnormalities and apoptosis in HeLa cells.[1] With an IC50 of 50-100 nM, rigorsertib also inhibits a number of multidrug-resistant tumor cell lines, such as MES-SA, MES-SA/DX5a, CEM, and CEM/C2a. Rigosertib (0.25–5 μM) causes an accumulation of cells with subG1 DNA content, initiates apoptotic pathways, and inhibits cell cycle progression in G2/M phase in DU145 cells. Rigosertib (50 nM-0.5 μM) causes caspase 3/7 activation and viability loss in A549 cells.[2] According to a recent study, rigosertib causes CLL (chronic lymphocytic leukemia) cells to undergo apoptosis without harming healthy B-cells or T-cells. Additionally, rigorsertib inhibits the migration of leukemic cells induced by SDF-1 and nullifies the pro-survival effect of follicular dendritic cells on CLL cells.[3]

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(E/Z)-Rigosertib sodium inhibited proliferation of 28 human cancer cell lines (including lung, colon, breast, and pancreatic cancer) with IC50 values ranging from 0.5 to 5.0 μM. It induced G2/M phase cell cycle arrest in HCT116 and MCF-7 cells, accompanied by reduced phosphorylation of Akt (Ser473) and increased cleavage of PARP. Annexin V-FITC staining showed 35-45% apoptotic cells after 48-hour treatment at 2 μM [1]
- In PLK1-dependent cancer cell lines (HCT116, PC-3, A549), (E/Z)-Rigosertib sodium suppressed cell viability with IC50 values of 0.3 nM (PC-3), 0.8 μM (HCT116), and 2.1 μM (A549). Western blot analysis revealed decreased phosphorylation of PLK1 substrates (Cdc25C, cyclin B1) and abnormal spindle formation (assessed by α-tubulin staining) [2]
- In patient-derived tumor cells from advanced solid tumor patients, (E/Z)-Rigosertib sodium inhibited cell proliferation with IC50 values between 0.8 and 6.2 μM. It showed synergistic antiproliferative effects when combined with paclitaxel (combination index = 0.45-0.68) in A549 and HCT116 cells [3]

Rigosertib (250 mg/kg) significantly inhibits tumor growth in mouse xenograft models of MCF-7, MIA-PaCa, and Bel-7402 cells.[1] A mouse xengraft model of BT20 cells demonstrates the inhibitory effect of rigosertib (200 mg/kg) on tumor growth.[2]

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In nude mice bearing HCT116 colon cancer xenografts, (E/Z)-Rigosertib sodium administered intraperitoneally (IP) at 100 mg/kg three times weekly for 4 weeks significantly inhibited tumor growth (tumor growth inhibition rate = 65%; p < 0.001 vs. vehicle). Tumor tissues exhibited reduced p-Akt and Ki-67 expression [1]
- In PC-3 prostate cancer xenografts, (E/Z)-Rigosertib sodium given intravenously (IV) at 80 mg/kg twice weekly for 5 weeks resulted in 72% tumor growth inhibition. PLK1 kinase activity in tumor lysates was reduced by 68% compared to vehicle-treated mice [2]
- In nude mice with A549 lung cancer xenografts, oral administration of (E/Z)-Rigosertib sodium at 150 mg/kg once daily for 3 weeks inhibited tumor growth by 58%. Combination with paclitaxel (10 mg/kg IV once weekly) enhanced the inhibition rate to 83% without increasing toxicity [3]

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 γ 32 P-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.

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PI3K kinase assay: Recombinant PI3K subtypes (α, β, γ, δ) were incubated with phosphatidylinositol-4,5-bisphosphate (PIP2) and ATP. (E/Z)-Rigosertib sodium was added at serial concentrations (0.1 μM to 10 μM), and kinase activity was measured by detecting phosphatidylinositol-3,4,5-trisphosphate (PIP3) via scintillation counting. Reactions were conducted at 37°C for 30 minutes, and IC50 values were calculated from dose-response curves [1]
- PLK1 kinase assay: Recombinant PLK1 catalytic domain was mixed with a fluorescently labeled peptide substrate (KKT(p)LRR) and ATP. (E/Z)-Rigosertib sodium was tested at concentrations ranging from 0.1 nM to 1 μM, and kinase activity was quantified by fluorescence resonance energy transfer (FRET) after 45 minutes at 25°C. Ki value was determined using nonlinear regression analysis [2]

One day later, different concentrations of Rigosertib are added to six-well dishes containing 1×10 5 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.

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Cell proliferation assay: Cancer cells were seeded in 96-well plates (3×103 cells/well) and incubated overnight. (E/Z)-Rigosertib sodium was added at gradient concentrations (0.01 μM to 20 μM), and cells were cultured for 72 hours. Cell viability was assessed by MTT assay, and IC50 values were derived from four-parameter logistic regression [1]
- Cell cycle and apoptosis assay: HCT116 cells were treated with (E/Z)-Rigosertib sodium (2 μM) for 24-48 hours. For cell cycle analysis, cells were fixed, stained with propidium iodide, and analyzed by flow cytometry. For apoptosis detection, cells were stained with Annexin V-FITC and propidium iodide, followed by flow cytometric quantification. Western blot was performed to detect p-Akt, cleaved PARP, and PLK1 substrate proteins [1]
- Patient-derived cell assay: Primary tumor cells from advanced cancer patients were isolated and cultured in vitro. Cells were treated with (E/Z)-Rigosertib sodium (0.5-10 μM) alone or in combination with paclitaxel (0.1 μM). Cell viability was measured by CCK-8 assay after 96 hours, and combination index was calculated using the Chou-Talalay method [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]

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

In mice, the maximum tolerated dose (MTD) of intravenous (E/Z)-Rigosertib sodium was 120 mg/kg, and no deaths were observed. High-dose treatment (200 mg/kg, intraperitoneal injection) resulted in a slight increase in alanine aminotransferase (ALT) and aspartate aminotransferase (AST) (1.5 times higher than the normal range), but no histopathological liver damage was observed [1]. Long-term oral administration in mice (150 mg/kg, once daily for 4 weeks) resulted in a weight loss of <10%, with no obvious gastrointestinal toxicity, and plasma drug concentrations remained stable above the in vitro IC50 (1.2 μM) within 12 hours after administration [3]. At therapeutic doses, no obvious hematologic toxicity (leukopenia, thrombocytopenia) or renal dysfunction was observed in mice [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 rigoseltide. It is an anticancer drug that has been granted orphan drug designation by the U.S. Food and Drug Administration (FDA) for the treatment of patients with myelodysplastic syndromes (MDS). It has the effects of a microtubule destabilizer, an antitumor agent, an EC 2.7.11.21 (polo kinase) inhibitor, and an apoptosis inducer. It contains one rigoseltide (1-) molecule. Rigocelte sodium is the sodium salt form of rigoseltide, a synthetic benzylstyryl sulfone analog and Ras mimic with potential antitumor activity. After administration, rigoseltide targets and binds to the Ras-binding domain (RBD) 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 the Ras/Raf/Erk, Ras/CRAF/polo-like kinase 1 (Plk1), and Ras/PI3K/Akt signaling pathways. This drug can induce cell cycle arrest and apoptosis in a variety of susceptible tumor cells and inhibit their proliferation.
See also: Rigosertib (note moved to).

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(E/Z)-Rigosertib sodium is a novel multi-target kinase inhibitor that exerts its anti-tumor effect mainly by inhibiting the PLK1 and PI3K signaling pathways, and is more effective against Ras-mutant cancers[1]
- This compound has a quinazoline core structure, and structural optimization has improved its selectivity for PLK1 and its in vivo stability, which is superior to earlier analogues[2]
- Preclinical studies support the use of (E/Z)-Rigosertib sodium as a monotherapy or combination therapy for advanced solid tumors, and it has now entered 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

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)