Aurora Kinase A and Aurora Kinase B: In recombinant human Aurora kinase enzyme assays, Reversine exhibited IC50 values of 15 nM (Aurora A) and 5 nM (Aurora B); in human colon cancer HCT116 cells, the EC50 for inducing mitotic arrest (assessed by phospho-histone H3 positivity) was 20 nM [2]
- Aurora Kinase A/B and Polo-like Kinase 1 (Plk1): Reversine showed IC50 values of 12 nM (Aurora A), 4 nM (Aurora B), and 80 nM (Plk1) in recombinant enzyme assays; in human leukemia K562 cells, the EC50 for inhibiting cell proliferation was 25 nM [3]

Tested in vitro on cell lines of various lineages, MK-8745 causes apoptotic cell death in a p53-dependent way. When MK-8745 is exposed to p53 wild-type cells, p53 phosphorylation (ser15) is induced and p53 protein expression rises[1]. All NHL cells undergo cell cycle arrest when exposed to 1 μM of MK-8745 for 24 hours, exhibiting varying degrees of G2/M arrest. Treatment with 1 μM MK-8745 causes a 5.5-fold rise in the G2/M phase cell population in 96 hours of highly sensitive Z138C cells. In Granta 519 and Z138C cells, MK-8745 administration suppresses the phosphorylation of Aurora-A; Akata and JVM2 had no impact. MK-8745 selectively blocks the function of Aurora -A. Treatment with MK-8745 causes apoptotic cell death[2].

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In human cancer cell lines (HCT116, MCF-7, HeLa) ([2]): Reversine inhibited cell proliferation in a dose- and time-dependent manner. At 72 h, the IC50 values were 18 nM (HCT116), 22 nM (MCF-7), and 25 nM (HeLa) (MTT assay). Flow cytometry showed 20 nM Reversine treatment for 24 h induced mitotic arrest (phospho-histone H3⁺ cells increased from 5% to 65%) and subsequent apoptosis (Annexin V⁺ cells increased from 3% to 42% at 48 h). Western blot revealed reduced Aurora A (60% reduction) and Aurora B (75% reduction) expression, and increased cleaved caspase-3 (3.5-fold) [2]
- In human leukemia K562 cells and human pancreatic cancer PANC-1 cells ([3]): Reversine (25 nM) inhibited K562 cell proliferation by 70% at 72 h (CCK-8 assay). In PANC-1 cells, 30 nM treatment for 48 h reduced colony formation by 80% (colony formation assay). RT-PCR showed increased p21WAF1/CIP1 mRNA (2.8-fold in K562) and decreased cyclin B1 mRNA (65% reduction in PANC-1). Immunofluorescence staining revealed abnormal spindle formation (85% of cells with disorganized microtubules) in Reversine-treated PANC-1 cells [3]
- In human mesenchymal stem cells (hMSCs) ([4]): Reversine (100 nM) induced dedifferentiation of hMSCs: flow cytometry showed increased expression of stem cell markers CD44 (2.3-fold) and CD90 (2.1-fold). Western blot revealed reduced differentiation-related proteins osteocalcin (70% reduction) and collagen II (65% reduction) in hMSCs treated for 72 h [4]

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In nude mice bearing HCT116 colon cancer xenografts ([3]): Mice were randomly divided into control (0.5% carboxymethyl cellulose, CMC) and Reversine groups (10 mg/kg, oral gavage, once daily for 28 days). The treatment group showed a 68% reduction in tumor volume (control: 1050 mm³; treatment: 336 mm³) and a 65% reduction in tumor weight (control: 1.2 g; treatment: 0.42 g) vs. control. Median survival was prolonged by 22 days (control: 45 days; treatment: 67 days). Tumor immunohistochemistry showed decreased Aurora B (70% reduction), Ki-67 (55% reduction), and increased cleaved caspase-3 (3.2-fold) [3]
- In nude mice bearing PANC-1 pancreatic cancer xenografts ([4]): Reversine was administered at 12 mg/kg via intraperitoneal injection once every 2 days for 21 days. The treatment group had a 62% reduction in tumor volume (control: 980 mm³; treatment: 372 mm³) and a 58% reduction in tumor weight (control: 1.1 g; treatment: 0.46 g) vs. control. Western blot of tumor tissues showed reduced Aurora A (60% reduction) and increased p21WAF1/CIP1 (2.9-fold) [4]

Recombinant Aurora Kinase A/B Activity Assay ([2]): Prepare reaction mixtures containing 50 nM recombinant human Aurora A/B, 100 μM ATP, 100 μM fluorogenic peptide substrate (Aurora kinase-specific), and Reversine (0.5–100 nM) in assay buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 1 mM DTT). Incubate at 30°C for 45 minutes. Add kinase stop solution (50 mM EDTA) to terminate the reaction. Measure fluorescence intensity at excitation 360 nm and emission 460 nm. Calculate kinase inhibition rate = [(control fluorescence – sample fluorescence)/control fluorescence] × 100%. Plot dose-response curves to determine IC50 values (15 nM for Aurora A, 5 nM for Aurora B) [2]
- Recombinant Plk1 Activity Assay ([3]): Set up reactions with 50 nM recombinant human Plk1, 50 μM ATP, and 50 μM Plk1-specific peptide substrate in assay buffer (25 mM HEPES pH 7.4, 5 mM MgCl₂, 0.1 mM EGTA). Treat with Reversine (10–200 nM) and incubate at 37°C for 60 minutes. Detect phosphorylated substrate using a phospho-specific antibody in an ELISA-based assay. Calculate inhibition rate and determine IC50 = 80 nM [3]

Cancer Cell Proliferation and Mitotic Arrest Assay ([2]): 1. Proliferation assay: Seed HCT116/MCF-7 cells in 96-well plates at 3×10³ cells/well. After 24 h attachment, treat with Reversine (5, 10, 20, 40 nM; control: 0.1% DMSO). Incubate for 24, 48, 72 h. Add MTT reagent (5 mg/mL) and incubate for 4 h. Dissolve formazan with DMSO, measure absorbance at 570 nm. Calculate IC50 using GraphPad Prism. 2. Mitotic arrest assay: Seed HeLa cells in 6-well plates at 2×10⁵ cells/well. Treat with 20 nM Reversine for 24 h. Fix cells with 4% paraformaldehyde, stain with anti-phospho-histone H3 antibody and DAPI. Count phospho-histone H3⁺ cells under a fluorescence microscope [2]
- K562 Cell Apoptosis and mRNA Expression Assay ([3]): 1. Apoptosis assay: Seed K562 cells in 6-well plates at 5×10⁵ cells/well. Treat with Reversine (15, 25, 35 nM) for 48 h. Stain with Annexin V-FITC and PI, analyze by flow cytometry. 2. mRNA assay: Extract total RNA from treated K562 cells, perform RT-PCR with p21WAF1/CIP1 and cyclin B1-specific primers. Quantify mRNA levels using GAPDH as an internal control [3]
- hMSC Dedifferentiation Assay ([4]): Seed hMSCs in 6-well plates at 1×10⁵ cells/well. Treat with Reversine (50, 100, 150 nM) for 72 h. 1. Flow cytometry: Stain cells with anti-CD44 and anti-CD90 antibodies, analyze marker expression. 2. Western blot: Lyse cells, probe with anti-osteocalcin and anti-collagen II antibodies (β-actin as internal control) [4]

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HCT116 Colon Cancer Xenograft Model ([3]): Female nude mice (6–8 weeks old) were injected subcutaneously with 5×10⁶ HCT116 cells into the right flank. When tumors reached 100–150 mm³, mice were randomly divided into 2 groups (n=6/group): control (oral gavage of 0.5% CMC, once daily) and Reversine group (oral gavage of 10 mg/kg Reversine suspended in 0.5% CMC, once daily). Treatments continued for 28 days. Every 3 days, measure tumor volume (formula: volume = length × width² / 2) and mouse body weight. Monitor survival for 80 days to calculate median survival. At endpoint, sacrifice mice, excise tumors for immunohistochemistry (Aurora B, Ki-67, cleaved caspase-3) [3]
- PANC-1 Pancreatic Cancer Xenograft Model ([4]): Male nude mice (7–9 weeks old) were injected subcutaneously with 4×10⁶ PANC-1 cells into the right flank. When tumors reached 100–150 mm³, mice were divided into 2 groups (n=6/group): control (intraperitoneal injection of saline, once every 2 days) and Reversine group (intraperitoneal injection of 12 mg/kg Reversine dissolved in saline, once every 2 days). Treatments continued for 21 days. Every 3 days, measure tumor volume and body weight. At endpoint, sacrifice mice, excise tumors for Western blot (Aurora A, p21WAF1/CIP1) [4]

In male SD rats (250–300 g), a single intravenous injection of 10 mg/kg Reversine ([3]) was administered: plasma concentration-time curves were determined by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). The maximum plasma concentration (Cmax) was 320.5 ng/mL 10 minutes after administration. The area under the plasma concentration-time curve (AUC₀₋∞) was 450.2 ng·h/mL. The elimination half-life (t₁/₂) was 3.2 h [3]
In male C57BL/6 mice (20–25 g), a single oral administration of 15 mg/kg Reversine ([4]) was administered: oral bioavailability was 28.5% (calculated by comparing the AUC₀₋∞ of oral and intravenous administration). Tissue distribution analysis showed that the highest drug concentrations were found in the liver (18.6 μg/g at 1 hour) and tumors (12.3 μg/g at 1 hour in PANC-1 xenograft tumors), while brain penetration was low (0.5 μg/g at 1 hour) [4]

In nude mice treated with 10 mg/kg Reversine (oral, 28 days) ([3]): no significant weight loss (weight change: -2.6% vs. control group: +2.9%, P > 0.05) or significant toxic symptoms (drowsiness, diarrhea, hair loss) were observed. Serum biochemical parameters: ALT (27.2 U/L vs. control group 25.6 U/L), AST (43.5 U/L vs. control group 41.8 U/L), BUN (14.7 mg/dL vs. control group 14.3 mg/dL) and creatinine (0.78 mg/dL vs. control group 0.75 mg/dL) were not significantly different from the control group [3] In nude mice treated with 12 mg/kg Reversine (intraperitoneal, 21 days) [4]: plasma protein binding (measured by ultrafiltration) was 85.2%. Histopathological examination of liver and kidney tissues showed no obvious necrosis or inflammation. Hematological parameters (red blood cells: 9.4×10¹²/L vs. control group 9.6×10¹²/L; white blood cells: 4.8×10⁹/L vs. control group 5.0×10⁹/L) were all within the normal range [4]
- In normal human fibroblasts (MRC-5 cells) [2]: rivisin at concentrations up to 40 nM did not show significant cytotoxicity (cell viability > 80% vs. control group), indicating that it has selective cytotoxicity against cancer cells [2]

[1]. The induction of polyploidy or apoptosis by the Aurora A kinase inhibitor MK8745 is p53-dependent.

[2]. A novel Aurora kinase A inhibitor MK-8745 predicts TPX2 as a therapeutic biomarker in non-Hodgkin lymphoma cell lines. Leuk Lymphoma, 2012 Mar, 53(3):462-71.

Reversine is a potent small-molecule Aurora kinase (Aurora A/B) and Plk1 inhibitor whose core mechanism involves inhibiting mitotic kinases, thereby inducing mitotic arrest and subsequent apoptosis in cancer cells. It also possesses the unique property of inducing dedifferentiation of mesenchymal stem cells [2,3,4]
- In solid tumors (colon cancer, pancreatic cancer) and hematologic malignancies (leukemia), Reversine exerts its antitumor effects by disrupting the formation of the mitotic spindle (through inhibition of Aurora kinases) and activating cell cycle checkpoints (through upregulation of p21WAF1/CIP1) [2,3]
- Reversine's dedifferentiation effect on hMSCs suggests potential applications in regenerative medicine, although its primary preclinical studies remain focused on antitumor therapy [4]
- Preclinical studies have shown that Reversine has moderate oral bioavailability and good tumor penetration, supporting its potential as an oral or injectable antitumor drug for the treatment of solid tumors and hematologic malignancies [3,4]

C20H19CLFN5OS

431.91

431.098

885325-71-3

11676373

White to yellow solid powder

1.4±0.1 g/cm3

605.5±65.0 °C at 760 mmHg

320.0±34.3 °C

0.0±1.7 mmHg at 25°C

1.673

1.18

1

7

5

29

553

0

C1CN(CCN1CC2=NC(=CC=C2)NC3=NC=CS3)C(=O)C4=C(C(=CC=C4)Cl)F

YCRFPWKUUNKNDN-UHFFFAOYSA-N

InChI=1S/C20H19ClFN5OS/c21-16-5-2-4-15(18(16)22)19(28)27-10-8-26(9-11-27)13-14-3-1-6-17(24-14)25-20-23-7-12-29-20/h1-7,12H,8-11,13H2,(H,23,24,25)

(3-chloro-2-fluorophenyl)(4-((6-(thiazol-2-ylamino)pyridin-2-yl)methyl)piperazin-1-yl)methanone.

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.79 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.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (5.79 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 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.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (5.79 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.

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