ETP-46464 1345675-02-6_ATM(ATR)_DNA Damage_Signaling Pathways_Products

ETP-46464 (ETP46464) is a novel, potent and selective inhibitor of ATR (Ataxia-telangiectasia mutated) and mTOR (mammalian target of rapamycin) with potential anticancer activity. It has an IC50 of 25 nM for ATR inhibition and 0.6 nM for mTOR inhibition. At IC50s of 170 and 545 nM, respectively, it also inhibits ATM and PI3Kα. Stalled replication forks break chromosomally when ATR, a DNA damage response kinase, is inhibited by ETP-46464. This is known to cause replicative stress.

Physicochemical Properties

Molecular Formula

C30H22N4O2

Molecular Weight

470.52

Exact Mass

470.174

Elemental Analysis

C, 76.58; H, 4.71; N, 11.91; O, 6.80

CAS #

1345675-02-6

Related CAS #

1345675-02-6

PubChem CID

72199292

Appearance

Light yellow to yellow solid powder

Density

1.3±0.1 g/cm3

Boiling Point

709.3±60.0 °C at 760 mmHg

Flash Point

382.7±32.9 °C

Vapour Pressure

0.0±2.3 mmHg at 25°C

Index of Refraction

1.697

LogP

3.58

Hydrogen Bond Donor Count

Hydrogen Bond Acceptor Count

Rotatable Bond Count

Heavy Atom Count

Complexity

858

Defined Atom Stereocenter Count

SMILES

O=C1N(C2=CC=C(C(C)(C#N)C)C=C2)C3=C4C(C=CC(C5=CN=C(C=CC=C6)C6=C5)=C4)=NC=C3CO1

InChi Key

DPLMXAYKJZOTKO-UHFFFAOYSA-N

InChi Code

InChI=1S/C30H22N4O2/c1-30(2,18-31)23-8-10-24(11-9-23)34-28-22(17-36-29(34)35)16-33-27-12-7-19(14-25(27)28)21-13-20-5-3-4-6-26(20)32-15-21/h3-16H,17H2,1-2H3

Chemical Name

2-methyl-2-[4-(2-oxo-9-quinolin-3-yl-4H-[1,3]oxazino[5,4-c]quinolin-1-yl)phenyl]propanenitrile

Synonyms

ETP46464;ETP 46464;ETP-46464; ATM Inhibitor III; ATR Inhibitor III

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

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

mTOR ( IC50 = 0.6 nM ); ATR ( IC50 = 14 nM ); ATM ( IC50 = 545 nM ); DNA-PK ( IC50 = 36 nM ); PI3Kα ( IC50 = 170 nM )

ln Vitro

ETP-46464 (ATRi) also inhibits DNA-PK, PI3Kα and ATM with IC50s of 36 nM, 170 nM and 545 nM, respectively[1]. Cell lines with platinum-sensitive and -resistant ovarian, endometrial, and cervical cancer are treated for 72 hours with different dosages of cisplatin (0-50 µM), with or without KU55933 (10.0 µM) and/or ETP-46464 (5.0 µM). A subset of cell lines' single-agent dose response analyses for ETP-46464 and KU55933 show broad LD50 ranges of 10.0±8.7 and 38.3±7.6 µM, respectively. These investigations, along with previously documented evidence of phospho-Chk1 (Ser345) and phospho-ATM (Ser1981) inhibition after ionizing radiation exposure and dose response treatments with ETP-46464 and KU55933, are used to determine co-treatment doses. In all tested cell lines, treatment with ETP-46464 dramatically boosts the response of Cisplatin, resulting in 52–89% increase in activity and synergistic effects. When ATR and ATM are inhibited simultaneously, the response of Cisplatin is increased to a degree comparable to when ETP-46464 is used alone. These effects are seen in all tested gynecologic (GYN) cancer cells and are not dependent on the p53 status. These GYN cancer cell lines respond better to carboplatin when treated with ETP-46464 rather than KU55933, which also makes the cell lines more sensitive to cisplatin[2].

ln Vivo

N/A

Enzyme Assay

Compounds (e.g., ETP-46464) and master suppressors are introduced straight into the cell medium (100 μL in each well) using a multi-well pipette, with an ultimate concentration of 10 μM. By meticulously vortexing plates at 500 rpm, media content is homogenized. Before adding 4-hydroxy-tamoxifen (4-OHT), compounds (like ETP-46464) are incubated for 15 minutes at 37ºC. All wells are then treated with 4-OHT and incubated for 60 minutes at 37ºC to trigger ATR activity. Cells are then prepared for IF after being fixed with paraformaldehyde. Each compound (such as ETP-46464) undergoes at least three separate experiments for analysis[1].

Cell Assay

The cells are plated in 96-well plates at 5000 cells per well for KLE, HEC1B, and HELA, and 10,000 cells per well for OVCAR3, A2780, A2780-CP20, and SIHA. The cells are trypsinized with 0.25% Trypsin-EDTA, and they are counted with 0.4% Trypan Blue using an automated cell counter. Following 24-48 hours after seeding, the media is removed and replaced with new media containing either 0.78, 1.56, 3.13, 6.25, 12.5, 25, 50, or 100 µM of Cisplatin or 0.56, 3.13, 6.25, 12.5, 25, 50, or 100 µM of Carboplatin in 0.15% DMSO, 5 µM ETP-46464, 10 µM KU55933, or a combination of 5 µM ETP-46464 and 10 µM KU55933 and incubated for 72 hours. The final ETP-46464 and KU55933 concentrations used are determined by previous data showing inhibition of ATR and ATM signaling, respectively. In a subset of cell lines, single-agent dose response analyses of ETP-46464 and KU55933 showed a broad LD50 range (10.0±8.7 and 38.3±7.6 µM, respectively). In a similar manner, new media containing ciprofloxacin (0, 0.78, 1.56, 3.13, 6.25, 12.5, 25 or 50 µM) in 0.08% DMSO and 5 µM VE-821 are applied to the cells. The MTS CellTiter 96 Aqueous One Solution Cell Proliferation Assay is used to determine the viability of cells. A microplate spectrophotometer is used to measure absorbance at 490 nm following a 2-hour incubation period at 37°C. For every cell line, three biological replicates are carried out, and each experiment's inhibitor(s) and/or ciplatin concentration is measured in triplicate[2].

Animal Protocol

N/A

References

[1]. A cell-based screen identifies ATR inhibitors with synthetic lethal properties for cancer-associated mutations. Nat Struct Mol Biol. 2011 Jun;18(6):721-7.

[2]. Pharmacologic inhibition of ATR and ATM offers clinically important distinctions to enhancing platinum or radiation response in ovarian, endometrial, and cervical cancer cells. Gynecol Oncol. 2015 Mar;136(3):554-61.

Solubility Data

Solubility (In Vitro)

DMSO: 5~6 mg/mL (10.6~12.8 mM)
Water: <1 mg/mL
Ethanol: <1 mg/mL

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 0.5 mg/mL (1.06 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 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 5.0 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.

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

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	2.1253 mL	10.6265 mL	21.2531 mL
	5 mM	0.4251 mL	2.1253 mL	4.2506 mL
	10 mM	0.2125 mL	1.0627 mL	2.1253 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.

PeptideDB

ETP-46464 1345675-02-6

CAS No.: 1345675-02-6

Physicochemical Properties

Biological Activity

Solubility Data

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PeptideDB

ETP-46464 1345675-02-6

CAS No.: 1345675-02-6

Physicochemical Properties

Biological Activity

Solubility Data

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Latest release

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