Reversine 656820-32-5_Adenosine Receptor_GPCR & G Protein_Signaling Pathways_Products

Reversine is a cell-permeable, selective and ATP-competitive inhibitor of human A3 adenosine receptor and a pan-aurora A/B/C kinase inhibitor as well with potential anticancer activity. It inhibits pan-aurora A/B/C kinase with IC50s of 12 nM/13 nM/20 nM and the human A3 adenosine receptor with a Ki of 0.66 μM, respectively. It exhibits strong antiproliferative properties in vitro and strong antitumor efficaciousness in vivo.

Physicochemical Properties

Molecular Formula

C21H27N7O

Molecular Weight

393.23

Exact Mass

393.227

Elemental Analysis

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

CAS #

656820-32-5

Related CAS #

656820-32-5

PubChem CID

210332

Appearance

White solid powder

Density

1.343±0.06 g/cm3

Boiling Point

736.4±70.0 °C at 760 mmHg

Melting Point

305 ºC (decomp)

Flash Point

399.2±35.7 °C

Vapour Pressure

0.0±2.4 mmHg at 25°C

Index of Refraction

1.712

LogP

1.23

Hydrogen Bond Donor Count

Hydrogen Bond Acceptor Count

Rotatable Bond Count

Heavy Atom Count

Complexity

503

Defined Atom Stereocenter Count

SMILES

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]

InChi Key

ZFLJHSQHILSNCM-UHFFFAOYSA-N

InChi Code

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)

Chemical Name

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

Synonyms

Reversine

HS Tariff Code

2934.99.9001

Storage

Powder-20°C 3 years

4°C 2 years

In solvent -80°C 6 months

-20°C 1 month

Shipping Condition

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Biological Activity

Targets

Aurora A ( IC50 = 400 nM ); Aurora B ( IC50 = 500 nM ); Aurora C ( IC50 = 400 nM )

ln Vitro

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]

ln Vivo

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]

Enzyme Assay

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.

Cell Assay

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.

Animal Protocol

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).

References

[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.

Additional Infomation

Reversine is a member of the class of purines that is 9H-purine in which the hydrogens at positions 2 and 6 are replaced by a [4-(morpholin-4-yl)phenyl]nitrilo group and a cyclohexylamino group, respectively. It has a role as an antineoplastic agent, a cell dedifferentiation agent, an adenosine A3 receptor antagonist and an Aurora kinase inhibitor. It is a member of purines, a member of morpholines, a secondary amino compound and a tertiary amino compound. It is functionally related to a 9H-purine-2,6-diamine.

Solubility Data

Solubility (In Vitro)

DMSO: 5~20 mg/mL (12.7~50.8 mM)

Water: <1 mg/mL

Ethanol: <1 mg/mL

Solubility (In Vivo)

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.

Injection Formulations
(e.g. IP/IV/IM/SC) 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).
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)

Oral Formulations 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).
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

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.

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

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	2.5430 mL	12.7152 mL	25.4304 mL
	5 mM	0.5086 mL	2.5430 mL	5.0861 mL
	10 mM	0.2543 mL	1.2715 mL	2.5430 mL

*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.

PeptideDB

Reversine 656820-32-5

CAS No.: 656820-32-5

Physicochemical Properties

Biological Activity

Solubility Data