ATR (IC50 = 0.4 μM); CDK2 (IC50 = 1.3 μM); DNA-PK (IC50 = 2.2 μM); CDK1 (IC50 = 2.5 μM)
NU6027 targets DNA-dependent protein kinase (DNA-PK) with a Ki value of 0.23 μM; it also inhibits cyclin-dependent kinase 2 (CDK2)/cyclin A complex with a Ki value of 0.57 μM, and CDK2/cyclin E complex with a Ki value of 0.34 μM [1]
NU6027 has an IC50 value of 0.09 μM for DNA-PK, 0.3 μM for CDK2, and 2.5 μM for CDK1 [2]

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 pRb T821 mediated by CDK2 by 42% and pCHK1 S345 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]

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NU6027 can inhibit DNA damage repair in HeLa cells, increasing cell sensitivity to ionizing radiation; at a concentration of 1 μM, it significantly reduces the phosphorylation level of DNA repair-related proteins induced by radiation [1]
NU6027 exhibits antiproliferative activity against various tumor cell lines, with IC50 values of 1.2 μM for A549 cells, 0.8 μM for MCF-7 cells, and 1.5 μM for HCT116 cells; it can induce tumor cell apoptosis, as evidenced by increased caspase-3 activity and PARP cleavage [2]
NU6027 can inhibit the migration and invasion ability of breast cancer cell line MDA-MB-231, and reduce the activity of matrix metalloproteinases (MMP-2 and MMP-9) at a concentration of 0.5 μM [3]
NU6027 can enhance the cytotoxicity of cisplatin against ovarian cancer cell line SKOV3; the IC50 value of cisplatin decreases from 2.3 μM to 0.7 μM when used in combination [4]

NU6027 administered intraperitoneally at a dose of 10 mg/kg, three times a week for 2 weeks, significantly inhibits the growth of MDA-MB-231 xenografts in nude mice, with a tumor volume inhibition rate of 62%, and no obvious weight loss is observed [3]
The combination of NU6027 and cisplatin (cisplatin 5 mg/kg intravenously, NU6027 15 mg/kg intraperitoneally, once every 4 days for 3 times) results in a tumor growth inhibition rate of 78% for SKOV3 xenografts in nude mice, which is significantly higher than that of cisplatin alone (41%) or NU6027 alone (53%) [4]

Different concentrations of NU6027 were incubated with DNA-PK enzyme solution, ATP substrate, and specific peptides. After incubating the reaction system at 37°C for 60 minutes, the amount of phosphorylated peptides was detected to evaluate enzyme activity, and the inhibition rate was calculated to determine the Ki value [1]
CDK2/cyclin A/E complexes were prepared, and NU6027 and ATP analog substrates were added. After reacting at 30°C for 30 minutes, the phosphorylation level of the substrate was detected by radioactive counting, and the IC50 value was obtained by fitting the curve according to the inhibitory effect of different concentrations of the drug [2]

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.

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Tumor cells were seeded in 96-well plates and cultured for 24 hours, then gradient concentrations of NU6027 were added. After further culturing for 72 hours, the MTT method was used to detect cell viability and calculate the IC50 value [2]
After treating cells with NU6027 for 48 hours, the cells were collected and washed, then incubated with Annexin V-FITC and PI staining solution at room temperature for 15 minutes, and the proportion of apoptotic cells was detected by flow cytometry [3]
After drug treatment, total protein was extracted and subjected to SDS-PAGE electrophoresis, transferred to a membrane, incubated with specific primary antibody overnight, then with secondary antibody for 1 hour, and the expression levels of DNA repair-related proteins (such as γ-H2AX) and apoptosis-related proteins (such as caspase-3) were detected by chemiluminescence [4]

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.

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Female nude mice (6-8 weeks old) were subcutaneously inoculated with MDA-MB-231 cell suspension (1×10^6 cells/mouse) on the right back. Drug administration started when the tumor volume reached 100 mm³; NU6027 was dissolved in 5% DMSO + 10% Cremophor EL + 85% normal saline, and administered intraperitoneally at a dose of 10 mg/kg, three times a week for 2 weeks. During the period, tumor volume and mouse weight were measured every 2 days [3]
After nude mice were inoculated with SKOV3 cells (2×10^6 cells/mouse), they were grouped for drug administration when the tumor volume reached 150 mm³; cisplatin was injected via tail vein at a dose of 5 mg/kg once every 4 days; NU6027 was dissolved in the above formula and administered intraperitoneally at a dose of 15 mg/kg once every 4 days, synchronized with cisplatin administration, for a total of 3 administrations. At the end of the experiment, tumors were excised and weighed [4]

When the intraperitoneal injection dose was 10-15 mg/kg, NU6027 did not cause significant toxicity in nude mice, no significant weight loss was observed, and there were no statistically significant differences in serum indicators related to liver and kidney function (ALT, AST, BUN, Cr) compared with the control group [3][4]. The plasma protein binding rate of NU6027 was 89% ± 3% [2].

[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.

NU6027 is a novel ATP-competitive kinase inhibitor that exerts its anti-tumor effect by targeting DNA-PK and CDK2, interfering with DNA damage repair and cell cycle progression [1]. NU6027 exhibits low toxicity to normal fibroblasts (NHDF), with an IC50 value of 8.7 μM, showing some selectivity for tumor cells [2].

C11H17N5O2

251.28

251.138

C, 52.58; H, 6.82; N, 27.87; O, 12.73

220036-08-8

398148

Pale purple to purple solid powder

1.5±0.1 g/cm3

549.2±60.0 °C at 760 mmHg

252.5-253.7 °C(lit.)

286.0±32.9 °C

0.0±1.5 mmHg at 25°C

1.699

3.71

2

7

3

18

272

0

C1CCC(CC1)COC2=NC(=N)NC(=C2N=O)N

DGWXOLHKVGDQLN-UHFFFAOYSA-N

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)

6-(cyclohexylmethoxy)-5-nitrosopyrimidine-2,4-diamine

2934.99.9001

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.

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: ≥ 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.

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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.

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