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Purity: ≥98%
RO-3306 (RO3306; RO 3306) is a novel, potent, ATP-competitive, and selective CDK1 inhibitor with potential antineoplastic activity. It has a Ki of 20 nM for CDK1 inhibition and >15-fold selectivity against several closely related human kinases. RO-3306 treatment of growing AML cells resulted in dose- and time-dependent G2/M-phase cell cycle arrest and apoptosis. RO-3306 inhibited p53-mediated induction of p21 and MDM2, and downregulated the expression of the antiapoptotic proteins Bcl-2 and survivin. In order to encourage apoptosis, RO-3306 actively amplifies downstream p53 signaling.
| Targets |
CDK1 (Ki = 20 nM); CDK1/cyclinB1 (Ki = 35 nM); CDK1/cyclin A (Ki = 110 nM); CDK2/cyclinE (Ki = 340 nM); PKCδ (Ki = 318 nM); SGK (Ki = 497 nM); ERK (Ki = 1980 nM)
Cyclin-dependent kinase 1 (CDK1)/cyclin B (IC50 = 35 nM, human) [1] - Cyclin-dependent kinase 2 (CDK2)/cyclin E (IC50 = 2.3 μM, human; >65-fold lower potency than CDK1) [1] - Cyclin-dependent kinase 4 (CDK4)/cyclin D1 (IC50 = 10 μM, human; >285-fold lower potency than CDK1) [1] - Cyclin-dependent kinase 6 (CDK6)/cyclin D3 (IC50 = 12 μM, human; >340-fold lower potency than CDK1) [1] - No significant affinity for CDK5/p25 (IC50 > 20 μM) [1] |
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| ln Vitro |
RO-3306 is an ATP-competitive inhibitor, and inhibits CDK1/cyclin A complexes with Ki of 110 nM. In the G2/M phase of human cancer cells, RO-3306 inhibits the cell cycle. RO-3306 (4 μM) causes cancer cells to undergo apoptosis. AML cells undergo time-dependent apoptosis and G2/M-phase cell cycle arrest when exposed to RO-3306 (5 μM). Without changing the cell cycle distribution, RO-3306 treatment dramatically raises the proportion of Annexin V-positive cells in G1-phase cells. RO-3306 enhances p53-mediated apoptosis. In order to activate Bax and trigger mitochondrial apoptosis, RO-3306 collaborates with Nutlin-3. In AML, the antiapoptotic p21, Bcl-2, and survivin protein expression are downregulated by RO-3306 (5 μM). P53-induced p21 synthesis is inhibited by RO-3306. According to reference [2], RO-3306 does not prevent RNA polymerase II CTD phosphorylation. Oocyte maturation is successfully stopped by RO-3306 (10 μM). In oocytes, RO-3306 inhibits the formation of blastocysts[3].
Ro-3306 is a potent, selective inhibitor of cyclin-dependent kinase 1 (CDK1), with minimal activity against other CDK subtypes [1][2][3] - In human cancer cell lines (HeLa, U2OS), Ro-3306 (0.1-5 μM) dose-dependently inhibited CDK1-mediated mitotic entry, inducing reversible G2/M phase cell cycle arrest (G2/M population increased by 70-80% at 1 μM) and preventing chromosome condensation [1] - In human acute myeloid leukemia (AML) cells (HL-60, U937) with wild-type p53, Ro-3306 (0.5-5 μM) induced caspase-dependent apoptosis (apoptosis rate up to 60% at 2 μM), enhanced p53-mediated Bax activation (Bax mitochondrial translocation increased by 2.5-fold), and reduced anti-apoptotic Bcl-2 expression by 50% [2] - In porcine oocytes during in vitro maturation, Ro-3306 (1-10 μM) reversibly arrested meiosis at the metaphase I stage (arrest rate >90% at 5 μM), without affecting oocyte viability or subsequent fertilization potential after drug withdrawal [3] - It specifically blocked CDK1/cyclin B phosphorylation of histone H1 (inhibition rate 95% at 0.5 μM), while having no significant effect on CDK2-mediated Rb phosphorylation [1] |
| ln Vivo |
RO-3306 is an ATP-competitive, and selective CDK1 inhibitor.
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| Enzyme Assay |
Recombinant human CDK/cyclin complexes—CDK1/cyclin B1, CDK1/cyclin A, CDK2/cyclin E, and CDK4/cyclin D—expressed and separated from Hi5 insect cells are used in the CDK assays. The assay makes use of GST-cyclin B1, CDK1, GST-cyclin-E, CDK2, GST-CDK4, and cyclin D. Together with their partners, the GST-tagged proteins are coexpressed and purified in complex. A His-6-tagged portion of pRB (amino acids 385–928) is used as the substrate in all assays. From a construct, the protein is expressed. The protein is expressed in M15 Escherichia coli cells, bound to an agarose column that has been Ni-chalated, pretreated with 1 mM imidazole, and then eluted with 500 mM imidazole. Following an aliquoting process and dialyzation against 20 mM Hepes at pH 7/6.25 mM MgCl2/1.5 mM DTT, the eluted protein is kept at -80°C.
CDK1/cyclin B kinase activity assay: Recombinant human CDK1/cyclin B complex was incubated with [γ-³²P]-ATP, histone H1 substrate, and Ro-3306 (0.001-20 μM) at 30°C for 45 minutes. Phosphorylated histone H1 was separated by SDS-PAGE and quantified by autoradiography to calculate IC50 values [1] - CDK subtype selectivity assay: Recombinant human CDK2/cyclin E, CDK4/cyclin D1, CDK6/cyclin D3, CDK5/p25 complexes were individually incubated with their specific peptide substrates, [γ-³²P]-ATP, and Ro-3306 (0.01-50 μM) under the same conditions as CDK1 assay. Phosphorylated substrates were quantified to assess off-target inhibition [1] |
| Cell Assay |
In 96-well plates, log phase cells (25,000) are seeded and CO2 is added to an incubator set at 37°C. In order to ascertain the drug concentrations necessary to attain a 50% growth inhibition (IC50), various doses of RO-3306 are given after a 24-hour period. The cells are incubated for 4 hours after MTT (20 μL, 5 mg/mL stock solution in saline) is added to each well. After the removal of supernatants, 200 μL of anhydrous DMSO is used to dissolve the formazan crystals from viable cells. 565 nm is the wavelength at which the absorbance is measured using a microplate reader model 550.
Cancer cell cycle arrest assay: HeLa/U2OS cells were seeded in 24-well plates, treated with Ro-3306 (0.1-5 μM) for 24 hours, stained with propidium iodide, and cell cycle distribution was analyzed by flow cytometry. Chromosome condensation was visualized by DAPI staining [1] - AML cell apoptosis assay: HL-60/U937 cells were seeded in 24-well plates, treated with Ro-3306 (0.5-5 μM) for 48 hours. Apoptosis rate was analyzed by flow cytometry (annexin V-FITC/PI staining). Bax mitochondrial translocation and Bcl-2 expression were detected by Western blot and immunofluorescence [2] - Porcine oocyte meiosis assay: Porcine cumulus-oocyte complexes (COCs) were isolated and cultured in maturation medium containing Ro-3306 (1-10 μM) for 44 hours. Oocyte maturation status was evaluated by Hoechst 33342 staining to observe chromosome alignment and meiotic stage [3] |
| Animal Protocol |
Female BALB/c mice
1.5 mg/kg i.n. |
| References |
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| Additional Infomation |
Ro-3306 is a potent and selective CDK1 inhibitor widely used as a research tool to elucidate the specific function of CDK1 in mitosis and meiosis [1][3]
- Its core mechanism involves the specific inhibition of the CDK1/cyclin B complex, blocking the initiation of mitosis (in somatic cells) or the progression of meiosis (in oocytes), and enhancing p53-mediated mitochondrial apoptosis in AML cells [1][2][3] - Research applications include studying CDK1-dependent cell cycle regulation, the mechanism of AML cell apoptosis, and reversible meiotic arrest in oocytes for assisted reproductive research [1][2][3] - Compared with other CDKs, Ro-3306 has extremely high selectivity for CDK1 (≥65-fold), enabling it to precisely target the mitosis/meiosis pathway without off-target effects on other cell cycle regulators [1] |
| Molecular Formula |
C18H13N3OS2
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| Molecular Weight |
351.45
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| Exact Mass |
351.049
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| Elemental Analysis |
C, 61.52; H, 3.73; N, 11.96; O, 4.55; S, 18.24
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| CAS # |
872573-93-8
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| Related CAS # |
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| PubChem CID |
135400873
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| Appearance |
Yellow to brown solid powder
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| Density |
1.4±0.1 g/cm3
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| Boiling Point |
569.1±60.0 °C at 760 mmHg
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| Flash Point |
298.0±32.9 °C
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| Vapour Pressure |
0.0±1.6 mmHg at 25°C
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| Index of Refraction |
1.746
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| LogP |
3.24
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
5
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| Rotatable Bond Count |
3
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| Heavy Atom Count |
24
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| Complexity |
548
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| Defined Atom Stereocenter Count |
0
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| SMILES |
C(=C1/C(=O)N=C(NCC2SC=CC=2)S/1)\C1C=CC2N=CC=CC=2C=1
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| InChi Key |
XOLMRFUGOINFDQ-YBEGLDIGSA-N
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| InChi Code |
InChI=1S/C18H13N3OS2/c22-17-16(24-18(21-17)20-11-14-4-2-8-23-14)10-12-5-6-15-13(9-12)3-1-7-19-15/h1-10H,11H2,(H,20,21,22)/b16-10-
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| Chemical Name |
(5Z)-5-(quinolin-6-ylmethylidene)-2-(thiophen-2-ylmethylimino)-1,3-thiazolidin-4-one
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| Synonyms |
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
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| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 1.67 mg/mL (4.75 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 16.7 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. Solubility in Formulation 2: ≥ 1.67 mg/mL (4.75 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), suspension solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 16.7 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. View More
Solubility in Formulation 3: 5 mg/mL (14.23 mM) in 0.5% CMC-Na/saline water (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.8454 mL | 14.2268 mL | 28.4535 mL | |
| 5 mM | 0.5691 mL | 2.8454 mL | 5.6907 mL | |
| 10 mM | 0.2845 mL | 1.4227 mL | 2.8454 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.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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