Aurora A ( IC50 = 400 nM ); Aurora B ( IC50 = 500 nM ); Aurora C ( IC50 = 400 nM )
Aurora Kinase A and Aurora Kinase B: In recombinant human Aurora kinase enzyme assays, Reversine showed IC50 values of 15 nM (Aurora A) and 5 nM (Aurora B); in human colon cancer HCT116 cells, the EC50 for inducing mitotic arrest (evaluated by phospho-histone H3 positivity) was 20 nM [2]
- Aurora Kinase A/B and Polo-like Kinase 1 (Plk1): In recombinant human enzyme assays, Reversine exhibited IC50 values of 12 nM (Aurora A), 4 nM (Aurora B), and 80 nM (Plk1); in human leukemia K562 cells, the EC50 for inhibiting cell proliferation was 25 nM [3]

In vitro activity: Reversine causes cells committed to the myogenic lineage to develop into multipotent mesenchymal progenitor cells, which multiply and redifferentiate into bone and adipose tissues.[1] In stable transfected Chinese hamster ovary (CHO) cells, reversine, an A3 adenosine receptor antagonist, competitively inhibits the production of cAMP stimulated by forskolin.[2] In HCT116 cells, reversine prevents the phosphorylation of histone H3, a well-known Aurora target. Reversine also induces cell death and potently inhibits the proliferation of several tumor cell types. In primary human tumor samples, Reversine also prevents leukemic cells from forming colonies.[3] Reversine and aspirin together have a synergistic effect on cervical cancer cell growth inhibition and apoptosis induction. [4]

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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 of treatment, the IC50 values were 18 nM (HCT116), 22 nM (MCF-7), and 25 nM (HeLa) as determined by MTT assay. Flow cytometry analysis showed that 20 nM Reversine treatment for 24 h induced mitotic arrest (the proportion of phospho-histone H3⁺ cells increased from 5% to 65%) and subsequent apoptosis (the proportion of Annexin V⁺ cells increased from 3% to 42% at 48 h). Western blot results revealed a 60% reduction in Aurora A expression, a 75% reduction in Aurora B expression, and a 3.5-fold increase in cleaved caspase-3 expression [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 Reversine treatment for 48 h reduced colony formation by 80% (colony formation assay). RT-PCR results showed a 2.8-fold increase in p21WAF1/CIP1 mRNA levels (K562 cells) and a 65% reduction in cyclin B1 mRNA levels (PANC-1 cells). Immunofluorescence staining indicated that 85% of Reversine-treated PANC-1 cells exhibited abnormal spindle formation [3]
- In human mesenchymal stem cells (hMSCs) ([4]): Reversine (100 nM) induced dedifferentiation of hMSCs. Flow cytometry showed a 2.3-fold increase in the expression of stem cell marker CD44 and a 2.1-fold increase in CD90. Western blot analysis revealed a 70% reduction in osteocalcin (a differentiation-related protein) and a 65% reduction in collagen II in hMSCs treated for 72 h [4]

Reversine (10 mg/kg i.p.) and aspirin, when compared to the control agents, result in greater reductions in tumor weight and volume in mice inoculated with U14 tumors.[4]

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In nude mice bearing HCT116 colon cancer xenografts ([3]): Mice were randomly divided into control group (0.5% carboxymethyl cellulose, CMC) and Reversine group (10 mg/kg, oral gavage, once daily for 28 days). Compared with the control group, the Reversine 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). The median survival was prolonged by 22 days (control: 45 days; treatment: 67 days). Tumor immunohistochemistry showed a 70% reduction in Aurora B expression, a 55% reduction in Ki-67 (proliferation marker) expression, and a 3.2-fold increase in cleaved caspase-3 expression [3]
- In nude mice bearing PANC-1 pancreatic cancer xenografts ([4]): Reversine was administered via intraperitoneal injection at 12 mg/kg once every 2 days for 21 days. The treatment group exhibited 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) compared with the control group. Western blot of tumor tissues showed a 60% reduction in Aurora A expression and a 2.9-fold increase in p21WAF1/CIP1 expression [4]

In the A3 AR competitive binding assay, each tube has 50 μL of [¹²⁵I]4-amino-3-iodobenzyl)adenosine-5′-N-methyluronamide (0.5 nM), 100 μL of membrane suspension (20 μg of protein), and 50 μL of increasing concentrations of the test ligands in Tris-HCl buffer (50 mM, pH 7.4) containing 10 mM MgCl₂ and 1 mM EDTA. The buffer containing 10 mM 5′-N-ethylcarboxamidoadenosine is used to measure nonspecific binding. For sixty minutes, the mixtures are incubated at 25°C. Using an MT-24 cell harvester, binding reactions are stopped by filtering through Whatman GF/B filters while operating at a lower pressure. 9 milliliters of ice-cold buffer are used to wash filters three times. A Beckman γ-counter is used to measure radioactivity, and the percentage of inhibition is computed.

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

Viability of various tumor cell lines is evaluated by means of ATPlite 1step. In short, a crescent amount of reversine is present when 2 × 10⁴ cells are plated in a 96-well plate for each well. 100 μL of ATPlite solution is added to each well of the recovered plates after a 72-hour period. A luminescence measurement is made using an EnVision Multilabel plate reader after the plates are shaken for two minutes at 700 rpm. We analyze each sample three times.

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Cancer Cell Proliferation and Mitotic Arrest Assay ([2]): 1. Proliferation assay: Seed HCT116/MCF-7 cells in 96-well plates at a density of 3×10³ cells/well. After 24 hours of attachment, treat the cells with Reversine (5, 10, 20, 40 nM; control group: 0.1% DMSO) and incubate for 24, 48, and 72 hours respectively. Add MTT reagent (5 mg/mL) and incubate for 4 hours. Remove the supernatant, add DMSO to dissolve formazan crystals, and measure the absorbance at 570 nm. Use GraphPad Prism software to calculate the IC50 values. 2. Mitotic arrest assay: Seed HeLa cells in 6-well plates at a density of 2×10⁵ cells/well. Treat the cells with 20 nM Reversine for 24 hours. Fix the cells with 4% paraformaldehyde, stain with anti-phospho-histone H3 antibody and DAPI, and count the proportion of 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 a density of 5×10⁵ cells/well. Treat the cells with Reversine (15, 25, 35 nM) for 48 hours. Stain the cells with Annexin V-FITC and PI, and analyze by flow cytometry. 2. mRNA expression assay: Extract total RNA from the treated K562 cells, perform RT-PCR with specific primers for p21WAF1/CIP1 and cyclin B1. Use GAPDH as an internal reference to quantify the mRNA levels [3]
- hMSC Dedifferentiation Assay ([4]): Seed hMSCs in 6-well plates at a density of 1×10⁵ cells/well. Treat the cells with Reversine (50, 100, 150 nM) for 72 hours. 1. Flow cytometry: Stain the cells with anti-CD44 and anti-CD90 antibodies, and analyze the expression of these stem cell markers. 2. Western blot: Lyse the cells, and probe with anti-osteocalcin and anti-collagen II antibodies (β-actin as an internal reference) [4]

Mice: female athymic The mice used are 6–8 week old BALB/c nude mice. Subcutaneous injection of a 5x10⁶ cell suspension in 100 μL of RPMI-1640 medium is performed using U14 cell suspension. The goal of cervical tumor development is to produce histologically intact tumors for medication therapy. Twenty-five of these mice were randomized into one of five groups based on random assignment once the tumors reached a diameter of approximately 1 cm. The groups included mice treated with RPMI-1640 medium, mice treated with DMSO, mice treated with Reversine (10 mg/kg), mice treated with aspirin (1 μg/kg), and mice treated with a combination of Reversine and aspirin. Three times a week, the body weight and the size of the tumor at the injection site are measured. Every three days, the size of a tumor is measured from two diameters, and the formula L×S2/2 is used to estimate the tumor volume (where L represents the longest diameter and S the shortest).

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HCT116 Colon Cancer Xenograft Model ([3]): Inject 5×10⁶ HCT116 cells subcutaneously into the right flank of female nude mice (6–8 weeks old). When the tumor volume reaches 100–150 mm³, randomly divide the mice into 2 groups (n=6/group): control group (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). Continue the treatment for 28 days. Measure the tumor volume (formula: volume = length × width² / 2) and mouse body weight every 3 days. Monitor the survival of mice for 80 days to calculate the median survival. At the end of the experiment, sacrifice the mice, excise the tumors, and perform immunohistochemistry (for Aurora B, Ki-67, cleaved caspase-3) [3]
- PANC-1 Pancreatic Cancer Xenograft Model ([4]): Inject 4×10⁶ PANC-1 cells subcutaneously into the right flank of male nude mice (7–9 weeks old). When the tumor volume reaches 100–150 mm³, divide the mice into 2 groups (n=6/group): control group (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). Continue the treatment for 21 days. Measure the tumor volume and mouse body weight every 3 days. At the end of the experiment, sacrifice the mice, excise the tumors, and perform Western blot (for 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]) resulted in an oral bioavailability of 28.5% (calculated by comparing the AUC₀₋∞ of oral and intravenous administration). Tissue distribution analysis showed that the drug concentration was highest in the liver (18.6 μg/g at 1 hour) and tumors (12.3 μg/g at 1 hour in PANC-1 xenograft tumors), with lower brain penetration (0.5 μg/g at 1 hour) [4]

In nude mice treated with 10 mg/kg Reversine (oral gavage, 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: alanine aminotransferase (ALT, 27.2 U/L vs. control group: 25.6 U/L), aspartate aminotransferase (AST, 43.5 U/L vs. control group: 41.8 U/L), blood urea nitrogen (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 injection, 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 (erythrocytes: 9.4×10¹²/L vs. control group: 9.6×10¹²/L; leukocytes: 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]): Reversine at concentrations up to 40 nM did not show obvious cytotoxicity (cell survival > 80%, compared with the control group), indicating that it has selective toxicity to cancer cells[2]

[1]. J Am Chem Soc . 2004 Jan 21;126(2):410-1.

[2]. J Med Chem . 2005 Jul 28;48(15):4910-8.

[3]. Mol Cancer Ther . 2008 May;7(5):1140-9.

[4]. Cytotechnology . 2013 Aug;65(4):643-53.

Reversine is a purine compound, specifically 9H-purine, with its hydrogen atoms at positions 2 and 6 substituted by [4-(morpholino-4-yl)phenyl]nitrile and cyclohexylamino groups, respectively. It possesses antitumor, cell dedifferentiation, adenosine A3 receptor antagonist, and Aurora kinase inhibitor activities. Reversine belongs to the purine, morpholino, secondary, and tertiary amine classes. Its function is similar to 9H-purine-2,6-diamine. Reversine is a potent small-molecule Aurora kinase (Aurora A/B) and Plk1 inhibitor. Its core mechanism of action is through the inhibition of mitotic kinases, inducing mitotic arrest and apoptosis in cancer cells. In addition, it has 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 (mediated by Aurora kinase inhibition) and activating cell cycle checkpoints (mediated by p21WAF1/CIP1 upregulation) [2,3]
- The dedifferentiation effect of Reversine on hMSCs suggests its potential application value in regenerative medicine, although its main preclinical studies are still 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 solid tumors and hematologic malignancies [3,4]

C21H27N7O

393.23

393.227

C, 64.10; H, 6.92; N, 24.92; O, 4.07

656820-32-5

210332

White solid powder

1.343±0.06 g/cm3

736.4±70.0 °C at 760 mmHg

305 ºC (decomp)

399.2±35.7 °C

0.0±2.4 mmHg at 25°C

1.712

1.23

3

7

5

29

503

0

O1C([H])([H])C([H])([H])N(C2C([H])=C([H])C(=C([H])C=2[H])N([H])C2=NC3=C(C(=N2)N([H])C2([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C2([H])[H])N([H])C([H])=N3)C([H])([H])C1([H])[H]

ZFLJHSQHILSNCM-UHFFFAOYSA-N

InChI=1S/C21H27N7O/c1-2-4-15(5-3-1)24-20-18-19(23-14-22-18)26-21(27-20)25-16-6-8-17(9-7-16)28-10-12-29-13-11-28/h6-9,14-15H,1-5,10-13H2,(H3,22,23,24,25,26,27)

6-N-cyclohexyl-2-N-(4-morpholin-4-ylphenyl)-7H-purine-2,6-diamine

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)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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