DNA-PK (IC50 = 0.23 μM); PI3K (IC50 = 13 μM)

NU7026 (10 μM) potentiates ionizing radiation (IR) cytotoxicity [potentiation factor at 90% cell kill (PF90)=1.51±0.04] in exponentially growing DNA-PK proficient but not deficient cells[1]. NU7026 synergistically sensitizes I83 cells to Chlorambucil (CLB) 3.5-fold[2]. NU7026, a brand-new DNA-dependent protein kinase (DNA-PK) inhibitor. A significant radiosensitization effect in CH1 human ovarian cancer cells requires a minimum NU7026 exposure of 4 hours along with 3 Gy of radiation at a dose of 10 M, which is not toxic to cells per se[3].
Solution in vitro: NU7026 is dissolved. Cells are treated with NU7026 at a final DMSO (v/v) concentration of 0.25% DMSO (v/v)[4].

NU7026 is a novel DNA-PK（DNA repair enzyme DNA-dependent protein kinase）inhibitor. After being administered intravenously to mice at a dose of 5 mg/kg, NU7026 experienced a quick plasma clearance (0.108 L/h), which is largely attributed to extensive metabolism. Following administration of 20 mg/kg by i.p. or p.o., the bioavailability is 20 and 15%, respectively[3].

Mammalian DNA-PK (500 ng/μL) is isolated from HeLa cell nuclear extract after chromatography using Q-Sepharose, S-Sepharose, and Heparin agarose. In polypropylene 96-well plates, DNA-PK (250 ng) activity is assessed in a buffer containing 25 mM HEPES (pH 7.4), 12.5 mM MgCl2, 50 mM KCl, 1 mM DTT, 10% v/v Glycerol, 0.1% w/v NP-40, and 1 mg of the substrate GST-p53N66 at 30°C in a final volume of 40 μL. Variable NU7026 concentrations are added to the assay mixture (in DMSO at a final concentration of 1% v/v). After 10 minutes of incubation, a 30-mer double-stranded DNA oligonucleotide (final concentration of 0.5 ng/mL) and ATP are added to give a final concentration of 50 μM in order to start the reaction. After 1 hour of shaking, 150 L of PBS are added to the reaction, and 5 μL are then transferred to a 96-well opaque white plate that contains 45 μl of PBS per well. The GSTp53N66 substrate is then given 1 hour to bind to the wells. The sigmoidal plots produced by the graphic package Prism, which plot the enzyme activity against compound concentration in a range of compound concentrations, are used to calculate the IC50s for each compound in each enzyme assay.

I83 cells are plated in RPMI 1640 medium with 10% FBS (1.5×105 cells/mL) and treated with vehicle (DMSO), 5 μM CLB, CLB IC50, 10 μM NU7026, or the combination of both drugs for 0, 6, 24, and 48 h. Cell cycle distribution, apoptosis, DNA-PK phosphorylation, and γH2AX determination are determined, and they are expressed as a percentage of cells in each phase of the cycle. DNA content is analyzed with a FACSCalibur flow cytometer equipped with CellQuest software[2].

Mice[3]
The BALB/c mice used are female. For i.p. and peroral (p.o.) administration, NU7026 is formulated in 10% DMSO and 5% Tween 20 in saline at 20 and 50 mg/kg, respectively. NU7026 is formulated in 10% ethanol, 25% PEG 200, and 5% Tween 20 in saline for i.v. dosing at 5 mg/kg. The vehicle is given to the control animals alone. Per time point, injections are given to groups of three mice. Following halothane's transient anesthesia, blood is taken by cardiac puncture at time points of 0.083, 0.25, 0.5, 1, 2, 4, 6, and 24 hours after administration. Samples are kept at 20°C until analysis after being centrifuged at 1500 g for 2 min to obtain plasma. NU7026 is injected at a rate of 5 mg/kg for studies on urinary excretion. In metabolic cages, urine is collected over a 24-hour period and kept in storage until needed at 20°C.

[1]. Radiosensitization and DNA repair inhibition by the combined use of novel inhibitors of DNA-dependent protein kinase and poly(ADP-ribose) polymerase-1. Cancer Res. 2003 Sep 15;63(18):6008-15.

[2]. Chlorambucil cytotoxicity in malignant B lymphocytes is synergistically increased by 2-(morpholin-4-yl)-benzo[h]chomen-4-one (NU7026)-mediated inhibition of DNA double-strand break repair via inhibition of DNA-dependent protein kinase. J

[3]. Preclinical pharmacokinetics and metabolism of a novel prototype DNA-PK inhibitor NU7026. Br J Cancer. 2005 Oct 31;93(9):1011-8.

[4]. Interleukin-4 enhances PARP-dependent DNA repair activity in vitro. J Interferon Cytokine Res. 2014 Sep;34(9):734-40.

2-(4-morpholinyl)-4-benzo[h][1]benzopyranone is an organooxygen compound and an organic heterotricyclic compound.

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Chlorambucil (CLB) treatment is used in chronic lymphocytic leukemia (CLL) but resistance to CLB develops in association with accelerated repair of CLB-induced DNA damage. Phosphorylated histone H2AX (gammaH2AX) is located at DNA double-strand break (DSB) sites; furthermore, it recruits and retains damage-responsive proteins. This damage can be repaired by nonhomologous DNA end-joining (NHEJ) and/or homologous recombinational repair (HR) pathways. A key component of NHEJ is the DNA-dependent protein kinase (DNA-PK) complex. Increased DNA-PK activity is associated with resistance to CLB in CLL. We used the specific DNA-PK inhibitor 2-(morpholin-4-yl)-benzo[h]chomen-4-one (NU7026) to sensitize CLL cells to chlorambucil. Our results indicate that in a CLL cell line (I83) and in primary CLL-lymphocytes, chlorambucil plus NU7026 has synergistic cytotoxic activity at nontoxic doses of NU7026. CLB treatment results in G(2)/M phase arrest, and NU7026 increases this CLB-induced G(2)/M arrest. Moreover, a kinetic time course demonstrates that CLB-induced DNA-PK activity was inhibited by NU7026, providing direct evidence of the ability of NU7026 to inhibit DNA-PK function. DSBs, visualized as gammaH2AX, were enhanced 24 to 48 h after CLB and further increased by CLB plus NU7026, suggesting that the synergy of the combination is mediated by NU7026 inhibition of DNA-PK with subsequent inhibition of DSB repair.[2]

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In this study we investigated the in vitro time dependence of radiosensitisation, pharmacokinetics and metabolism of NU7026, a novel inhibitor of the DNA repair enzyme DNA-dependent protein kinase (DNA-PK). At a dose of 10 muM, which is nontoxic to cells per se, a minimum NU7026 exposure of 4 h in combination with 3 Gy radiation is required for a significant radiosensitisation effect in CH1 human ovarian cancer cells. Following intravenous administration to mice at 5 mg kg(-1), NU7026 underwent rapid plasma clearance (0.108 l h(-1)) and this was largely attributed to extensive metabolism. Bioavailability following interperitoneal (i.p.) and p.o. administration at 20 mg kg(-1) was 20 and 15%, respectively. Investigation of NU7026 metabolism profiles in plasma and urine indicated that the compound undergoes multiple hydroxylations. A glucuronide conjugate of a bis-hydroxylated metabolite represented the major excretion product in urine. Identification of the major oxidation site as C-2 of the morpholine ring was confirmed by the fact that the plasma clearance of NU7107 (an analogue of NU7026 methylated at C-2 and C-6 of the morpholine ring) was four-fold slower than that of NU7026. The pharmacokinetic simulations performed predict that NU7026 will have to be administered four times per day at 100 mg kg(-1) i.p. in order to obtain the drug exposure required for radiosensitisation.[3]

C17H15NO3

281.3059

281.105

C, 72.58; H, 5.37; N, 4.98; O, 17.06

154447-35-5

503465-21-2 (NU-7107); 79105-88-7 (NU-7031); 842122-14-9 (NU-7200); 69541-04-4 (NU-7199)

9860529

Yellow solid powder

1.3±0.1 g/cm3

459.9±45.0 °C at 760 mmHg

231.9±28.7 °C

0.0±1.1 mmHg at 25°C

1.673

3.29

0

4

1

21

441

0

O=C1C2=CC=C3C=CC=CC3=C2OC(N4CCOCC4)=C1

KKTZALUTXUZPSN-UHFFFAOYSA-N

InChI=1S/C17H15NO3/c19-15-11-16(18-7-9-20-10-8-18)21-17-13-4-2-1-3-12(13)5-6-14(15)17/h1-6,11H,7-10H2

2-morpholino-4H-benzo[h]chromen-4-one

DNA-PK Inhibitor II; LY-293646; DNA-PK Inhibitor II; 2-(Morpholin-4-yl)-benzo[h]chromen-4-one; NU-7026; 2-morpholino-4H-benzo[h]chromen-4-one; 2-morpholin-4-ylbenzo[h]chromen-4-one; LY 293646; LY293646; NU7026; NU-7026; NU 7026

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)

DMSO: ~1 mg/mL (~3.5 mM)
Water: <1 mg/mL
Ethanol: <1 mg/mL

1% DMSO+30% polyethylene glycol+1% Tween 80: 30mg/mL (Please use freshly prepared in vivo formulations for optimal results.)