NU6027 (NU-6027; NU 6027) is a novel, potent and ATP competitive ATR/CDK (ataxia telangiectasia and Rad3-related/Cyclin-dependent kinases) inhibitor with potential antitumor activity. At 2.5 μM/1.3 μM, 0.4 μM, and 2.2 μM, it inhibits CDK1/2, ATR, and DNA-PK.

Physicochemical Properties

Molecular Formula	C11H17N5O2
Molecular Weight	251.28
Exact Mass	251.138
Elemental Analysis	C, 52.58; H, 6.82; N, 27.87; O, 12.73
CAS #	220036-08-8
Related CAS #	220036-08-8
PubChem CID	398148
Appearance	Pale purple to purple solid powder
Density	1.5±0.1 g/cm3
Boiling Point	549.2±60.0 °C at 760 mmHg
Melting Point	252.5-253.7 °C(lit.)
Flash Point	286.0±32.9 °C
Vapour Pressure	0.0±1.5 mmHg at 25°C
Index of Refraction	1.699
LogP	3.71
Hydrogen Bond Donor Count	2
Hydrogen Bond Acceptor Count	7
Rotatable Bond Count	3
Heavy Atom Count	18
Complexity	272
Defined Atom Stereocenter Count	0
SMILES	C1CCC(CC1)COC2=NC(=N)NC(=C2N=O)N
InChi Key	DGWXOLHKVGDQLN-UHFFFAOYSA-N
InChi Code	InChI=1S/C11H17N5O2/c12-9-8(16-17)10(15-11(13)14-9)18-6-7-4-2-1-3-5-7/h7H,1-6H2,(H4,12,13,14,15)
Chemical Name	6-(cyclohexylmethoxy)-5-nitrosopyrimidine-2,4-diamine
Synonyms	NU-6027; NU 6027; NU6027
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 Note: This product requires protection from light (avoid light exposure) during transportation and storage.
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	ATR (IC50 = 0.4 μM); CDK2 (IC50 = 1.3 μM); DNA-PK (IC50 = 2.2 μM); CDK1 (IC50 = 2.5 μM)
ln Vitro	Nu6027 is soaked into monomeric CDK2 crystals, and 1.85 Å resolution is achieved in the structure refinement process. The growth of human tumor cells with a mean GI50 of 10 μM is inhibited by NU6027 (100μM). In MCF7 cells, NU6027 reduces the number of cells in S-phase but not in G1 or G2/M.[1] With an IC50 of 6.7 μM in MCF7 cells and 2.8 μM in GM847KD cells, NU6027 is a strong inhibitor of cellular ATR activity. It also increases hydroxyurea and cisplatin cytotoxicity in an ATR-dependent way. At 10 μM, NU6027 inhibits pRbT821 mediated by CDK2 by 42% and pCHK1S345 by 70%. The sensitivity of doxorubicin (1.3-fold at 4 μM and 2.5-fold at 10 μM), camptothecin (1.4-fold at 4 μM and 2-fold at 10 μM), hydroxyurea (1.8-fold at 4 μM), and cisplatin (1.4-fold at 4 μM and 8.7-fold at 10 μM) against MCF7 cells is significantly increased by NU6027. Additionally, at concentrations above and below their LC50, NU6027 increases the cytotoxicity of camptothecin and temozolomide (a DNA methylating agent) and potentiates 2Gy IR in a concentration-dependent manner. In MCF7 cells, NU6027 (10 μM) increases the cytotoxicity of the major classes of DNA-damaging anticancer cytotoxic therapy, but not of the antimitotic drug paclitaxel. It also inhibits the formation of RAD51 foci and attenuates G2/M arrest following DNA damage. When XRCC1 defects in MCF7 cells or inhibition of poly(ADP-ribose) polymerase (PARP) compromise DNA single-strand break repair, NU6027 (4 μM) becomes synthetically lethal.[3] After treating EM-C11 cells for 48 hours, NU6027 (4 μM) raises the percentage of cells in early apoptosis to 7.5% from 1.73% in untreated cells. Comparing XRCC1 deficient OVCAR-4 cells to proficient cells, NU6027 (10 μM) treatment decreases cell survival. When applied to OVCAR-3 cells lacking XRCC1, NU6027 increases the cytotoxicity of cisplatin in comparison to cells with XRCC1. When DSB accumulation is induced by Cisplatin in OVCAR-3 cells lacking XRCC1, NU6027 increases it.[4]
ln Vivo
Cell Assay	Using an enzyme made from starfish oocytes, the inhibition of cyclin B1/CDK1 is measured. The assay buffer consists of 50 mM Tris-HCl pH 7.5 containing 5 mM MgCl2, and a similar procedure is used to determine the inhibition of cyclinA3/CDK2. As for NU6027, the IC50 concentration is the amount needed to inhibit enzyme activity by 50% in the assay conditions employed, and the final ATP concentration in both CDK tests is 12.5 μM. In order to ascertain the Ki values for NU6027 and the Km for ATP for cyclin B1/CDK1 and cyclin A3/CDK2, assays are conducted both with and without NU6027, at two fixed concentrations of NU6027 (5 μM and 10 μM), and with ATP concentrations ranging from 6.25 μM to 800 μM. Regression using unweighted nonlinear least squares is used to fit the data to the Michaelis-Menten equation.
Animal Protocol	The normal 48-hour exposure assay and NU6027 concentrations ranging from 10-9 to 10-4 M are used to assess the growth inhibitory activity of NU6027 in the NCl in vitro cell line panel. The COMPARE algorithm is utilized to examine correlations between the cell growth inhibition profile generated by NU6027 and that of conventional anticancer agents, as well as the well-established CDK inhibitors flavopiridol and olomoucine.
References	[1]. J Med Chem . 2000 Jul 27;43(15):2797-804. [2]. J Med Chem . 2011 Apr 14;54(7):2320-30. [3]. Br J Cancer . 2011 Jul 26;105(3):372-81. [4]. PLoS One . 2013;8(2):e57098.

Solubility Data

Solubility (In Vitro)

DMSO: ~50 mg/mL (~199 mM) Water: <1 mg/mL Ethanol: ~3 mg/mL (~11.9 mM)

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 1.25 mg/mL (4.97 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 12.5 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: ≥ 1.25 mg/mL (4.97 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 12.5 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.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	3.9796 mL	19.8981 mL	39.7962 mL
	5 mM	0.7959 mL	3.9796 mL	7.9592 mL
	10 mM	0.3980 mL	1.9898 mL	3.9796 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.