PI3Kα (IC50 = 4 nM); PI3Kβ (IC50 = 0.5 nM); PI3Kγ (IC50 = 0.594 μM);PI3Kδ (IC50 = 0.1 nM); DNA-PK (IC50 = 8.6 nM)

KU-0060648 exhibits differential effects on growth inhibition, but is not profoundly cytotoxic in a panel of human cancer cell lines. When compared to SW620 cells, MCF7 cells exhibit more potent inhibition of DNA-PK and PI-3K. In MCF7 cells, exposure to 1 mM KU-0060648 for five days significantly reduces cell proliferation by more than 95%, but only by 55% in SW620 cells. In clonogenic survival assays, KU-0060648 increases etoposide and doxorubicin's cytotoxicity across a panel of DNA-PKcs-proficient cells, but not in DNA-PKcs-deficient cells, demonstrating that the increased cytotoxicity of the topoisomerase II poisons is caused by DNA-PK inhibition. [1]

KU-0060648 increases the anti-tumour activity of etoposide in both MCF7 and SW620 xenograft models, and has single-agent activity in the MCF7 xenograft model. [1]

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In vivo, intraperitoneal (i.p.) injection of KU-0060648 significantly inhibited HepG2 xenograft growth in nude mice. AKT-mTOR activation was also inhibited in xenografted tumors. Finally, we showed that DNA-PKcs expression was significantly upregulated in human HCC tissues.[2]

Determination of cellular activity of KU-0060648 against DNA-PK and PI-3K[1]
DNA-PK autophosphorylation was determined in cells exposed to a range of concentrations of KU-0060648 for 1 hr prior to X-irradiation (10 Gy). Cell lysates were prepared 30 minutes later using Phosphosafe Extraction Reagent according to the manufacturer’s instructions. Levels of DNA-PKcs auto-phosphorylation at Ser2056 relative to un-phosphorylated DNA-PKcs were determined by western blotting. To determine PI-3K activity, cells were exposed to a range of concentrations of KU-0060648 for 1 hr prior to a 30-minute treatment with 50 ng/ml insulin-like growth factor-1. The levels of PI-3K-dependent AKT phosphorylation (Ser473) relative to un-phosphorylated AKT were determined by western blotting.

Cytotoxicity and Growth inhibition Studies[1]
Cytotoxicity was measure by clonogenic assays. Cells grown in 6-well plates were exposed to etoposide or doxorubicin, with or without KU-0060648 (1 μM) for 16 hours, prior to harvesting and seeding into 10 cm diameter Petri dishes, in drug-free medium. Colonies were stained with crystal violet after 10 to 14 days and counted with an automated colony counter. Cell growth inhibition following 5-day continuous exposure to KU-0060648 was determined by SRB assay, as described previously. The GI50 is the concentration causing 50% cell growth inhibition.

human-tumor SW620 or MCF7 xenograft models
10 mg/kg, twice daily
i.p.

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KU-0060648 plasma pharmacokinetics following different routes of administration[1]
All in vivo experiments were reviewed and approved by the relevant institutional animal welfare committees and performed according to national law. We determined the plasma pharmacokinetics of KU-0060648 following administration intravenously (i.v.), intra-peritoneally (i.p.) or orally (p.o.) at 10 mg/kg to female Balb C mice. KU-0060648 was formulated in a vehicle of equimolar phosphoric acid, made up to volume with sterile saline and at final pH 5. Mice were killed at intervals up to 360 minutes after KU-0060648 administration and plasma concentrations of KU-0060648 were determined by LC-MS/MS analysis, as previously described. KU-0060648 distribution to tumour xenografts[1]
Female athymic mice were maintained and handled in isolators under specific pathogen free conditions for tissue distribution and efficacy studies. KU-0060648 (12.5 mg/kg i.v.) was administered to MCF7 or SW620 tumour-bearing mice (650 mm3), which were killed 60 or 240 minutes later. Tumours were excised and homogenised in PBS (1:3 w/v), using a stirrer mascercarator homogenizer , in 10 second bursts, on ice. Plasma and tumour KU-0060648 concentrations were determined by LC-MS/MS analysis, as previously described.

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DNA-PK ex vivo pharmacodynamic assay[1]
KU-0060648 at 2.5 or 25 mg/kg or vehicle alone was administered to SW620 tumour-bearing mice i.v.. After 1 or 4 hours, animals were killed and tumours were excised and homogenised. DNA-PK activity within tumour homogenates was determined by measuring the DNA-PK-dependent phosphorylation of a p53 peptide substrate (Ser15), using an ELISA assay, as described previously.

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Anti-tumour efficacy study[1]
Mice bearing SW620 or MCF7 xenografts s.c. (n = 5 per group) were treated when tumours were palpable (approx 5 mm × 5 mm, 8-10 days post implantation). Animals received normal saline i.p. once daily (control), single agent KU-0060648 10 mg/kg i.p. twice daily for either 5 days (2 × d × 5) in SW620 tumour-bearing, or 14 days (2 × d × 14) in MCF7 tumour bearing mice with doses on each day 8 hours apart, or etoposide phosphate once daily i.p. (11.35 mg/kg in saline, equivalent to 10 mg/kg free etoposide, i.p., d × 5). For combinations, KU-0060648 was administered i.p. once or twice daily for 5 days (SW620) or once daily for 14 days (MCF7), with the first dose immediately prior to etoposide phosphate.

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In vivo anti-tumor efficiency assay[2]
A significant amount of HepG2 cells (5 millions/mice) were injected subcutaneously into the right flanks of female nude mice (6-8 weeks old). When tumors reached around 100 mm3, mice were randomized into three groups with 12 mice per group: vehicle control (saline), 10 mg/kg of KU-0060648 (intraperitoneal injection or i.p., daily, for 21 days), and 50 mg/kg of KU-0060648 (i.p., daily, for 21 days). The injection was started when the tumors were established (volumes around 100 mm3). Tumor volumes, recorded every week, were calculated through the established formula: Volume (mm3) = (d2 × D)/2, in which d and D were the shortest and the longest diameter, respectively. Two weeks after initial KU-0060648 administration, xenografted tumors of two mice per group were isolated, and were subjected to Western blotting and immunohistochemistry (IHC) staining assays. Humane endpoints were applied to minimize suffering. Five weeks after initial KU-0060648 administration, HepG2 xenografts were separated through surgery and weighted.

The plasma pharmacokinetic parameters determined after administration of 10 mg/kg KU-0060648 to Balb/C mice by various routes are given in Table 2. The percentage bioavailability of KU-0060648 following p.o administration was found to be ≥100% The pharmacokinetic parameters of KU-0060648 following i.p. administration were found to be similar to that when given i.v., with 78% bioavailability.[1]

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Tissue distribution of KU-0060648 in MCF7 and SW620 tumour-bearing mice following i.v. administration[1]
Following administration of KU-0060648 (12.5 mg/kg i.v.) to mice bearing either MCF7 or SW620 xenografts, KU-0060648 distributed extensively to the tumour and was retained after clearance from the plasma (Table 2). Concentrations of KU-0060648 of over 1 μM (a level resulting in chemosensitisation in vitro) were maintained in the tumour for at least 4 hours.

[1]. Chemosensitization of cancer cells by KU-0060648, a dual inhibitor of DNA-PK and PI-3K. Mol Cancer Ther. 2012 Aug;11(8):1789-98.

[2]. KU-0060648 inhibits hepatocellular carcinoma cells through DNA-PKcs-dependent and DNA-PKcs-independent mechanisms. Oncotarget. 2016 Mar 29;7(13):17047-59.

2-(4-ethyl-1-piperazinyl)-N-[4-[2-(4-morpholinyl)-4-oxo-1-benzopyran-8-yl]-1-dibenzothiophenyl]acetamide is a member of dibenzothiophenes.

C33H34N4O4S

582.7125

582.23

C, 68.02; H, 5.88; N, 9.61; O, 10.98; S, 5.50

881375-00-4

881375-00-4

11964036

Off-white to brown solid powder

1.3±0.1 g/cm3

819.9±65.0 °C at 760 mmHg

449.7±34.3 °C

0.0±3.0 mmHg at 25°C

1.694

5.56

1

8

6

42

1010

0

O=C1C2C=CC=C(C=2OC(N2CCOCC2)=C1)C1C2SC3C(C=2C(NC(CN2CCN(CC)CC2)=O)=CC=1)=CC=CC=3

AATCBLYHOUOCTO-UHFFFAOYSA-N

InChI=1S/C33H34N4O4S/c1-2-35-12-14-36(15-13-35)21-29(39)34-26-11-10-23(33-31(26)25-6-3-4-9-28(25)42-33)22-7-5-8-24-27(38)20-30(41-32(22)24)37-16-18-40-19-17-37/h3-11,20H,2,12-19,21H2,1H3,(H,34,39)

2-(4-ethylpiperazin-1-yl)-N-[4-(2-morpholin-4-yl-4-oxochromen-8-yl)dibenzothiophen-1-yl]acetamide

KU0060648; KU 0060648; LM6DZS6PYA; 2-(4-ethylpiperazin-1-yl)-N-[4-(2-morpholin-4-yl-4-oxochromen-8-yl)dibenzothiophen-1-yl]acetamide; CHEMBL1086377; 2-(4-ethylpiperazin-1-yl)-N-(4-(2-morpholino-4-oxo-4H-chromen-8-yl)dibenzo[b,d]thiophen-1-yl)acetamide; KU-0060648

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: 2~2.8 mg/mL (3.4~4.8 mM)

Solubility in Formulation 1: ≥ 0.28 mg/mL (0.48 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 2.8 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: ≥ 0.28 mg/mL (0.48 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 2.8 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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Solubility in Formulation 3: ≥ 0.28 mg/mL (0.48 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (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 2.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 30% propylene glycol, 5% Tween 80, 65% D5W: 20 mg/mL

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