Compounds useful as inhibitors of atr kinase and combination therapies thereof

Inactive Publication Date: 2014-02-13
VERTEX PHARMA INC
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  • Abstract
  • Description
  • Claims
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Benefits of technology

[0008]The present invention relates to compounds useful as inhibitors of ATR protein kinase. The invention also relates to pharmaceutically acceptable compositions comprising the compounds of this invention; methods of treating of various diseases, disorders, and conditions using the compounds of this invention; processes for preparing the compounds of

Problems solved by technology

In addition, many cancer cells express activated oncogenes or lack key tumour suppressors, and this can

Method used

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  • Compounds useful as inhibitors of atr kinase and combination therapies thereof
  • Compounds useful as inhibitors of atr kinase and combination therapies thereof
  • Compounds useful as inhibitors of atr kinase and combination therapies thereof

Examples

Experimental program
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Effect test

example 2

Cellular ATR Inhibition Assay

[0236]Compounds can be screened for their ability to inhibit intracellular ATR using an immunofluorescence microscopy assay to detect phosphorylation of the ATR substrate histone H2AX in hydroxyurea treated cells. HT29 cells are plated at 14,000 cells per well in 96-well black imaging plates (BD 353219) in McCoy's 5A media (Sigma M8403) supplemented with 10% foetal bovine serum (JRH Biosciences 12003), Penicillin / Streptomycin solution diluted 1:100 (Sigma P7539), and 2 mM L-glutamine (Sigma G7513), and allowed to adhere overnight at 37° C. in 5% CO2. Compounds are then added to the cell media from a final concentration of 25 μM in 3-fold serial dilutions and the cells are incubated at 37° C. in 5% CO2. After 15 min, hydroxyurea (Sigma H8627) is added to a final concentration of 2 mM.

[0237]After 45 min of treatment with hydroxyurea, the cells are washed in PBS, fixed for 10 min in 4% formaldehyde diluted in PBS (Polysciences Inc 18814), washed in 0.2% Twe...

example 3

ATR Inhibition Assay

[0240]Compounds can be screened for their ability to inhibit ATR kinase using a radioactive-phosphate incorporation assay. Assays are carried out in a mixture of 50 mM Tris / HCl (pH 7.5), 10 mM MgCl2 and 1 mM DTT. Final substrate concentrations are 10 μM [γ-33P]ATP (3 mCi 33P ATP / mmol ATP, Perkin Elmer) and 800 μM target peptide (ASELPASQPQPFSAKKK).

[0241]Assays are carried out at 25° C. in the presence of 5 nM full-length ATR. An assay stock buffer solution is prepared containing all of the reagents listed above, with the exception of ATP and the test compound of interest. 13.5 μL of the stock solution is placed in a 96 well plate followed by addition of 2 μL of DMSO stock containing serial dilutions of the test compound (typically starting from a final concentration of 15 μM with 3-fold serial dilutions) in duplicate (final DMSO concentration 7%). The plate is pre-incubated for 10 minutes at 25° C. and the reaction initiated by addition of 15 μL [γ-33P]ATP (final...

example 4

Cisplatin Sensitization Assay

[0244]Compounds can be screened for their ability to sensitize HCT116 colorectal cancer cells to Cisplatin using a 96 h cell viability (MTS) assay. HCT116 cells, which possess a defect in ATM signaling to Cisplatin (see, Kim et al.; Oncogene 21:3864 (2002); see also, Takemura et al.; JBC 281:30814 (2006)) are plated at 470 cells per well in 96-well polystyrene plates (Costar 3596) in 150 μl of McCoy's 5A media (Sigma M8403) supplemented with 10% foetal bovine serum (JRH Biosciences 12003), Penicillin / Streptomycin solution diluted 1:100 (Sigma P7539), and 2 mM L-glutamine (Sigma G7513), and allowed to adhere overnight at 37° C. in 5% CO2. Compounds and Cisplatin are then both added simultaneously to the cell media in 2-fold serial dilutions from a top final concentration of 10 μM as a full matrix of concentrations in a final cell volume of 200 μl, and the cells are then incubated at 37° C. in 5% CO2. After 96 h, 40 μl of MTS reagent (Promega G358a) is add...

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Abstract

The present invention relates to compounds useful as inhibitors of ATR protein kinase and combination therapies thereof. The invention also relates to pharmaceutically acceptable compositions comprising the compounds of this invention; methods of treating of various diseases, disorders, and conditions using the compounds of this invention; processes for preparing the compounds of this invention; intermediates for the preparation of the compounds of this invention; and methods of using the compounds in in vitro applications, such as the study of kinases in biological and pathological phenomena; the study of intracellular signal transduction pathways mediated by such kinases; and the comparative evaluation of new kinase inhibitors.
The compounds of this invention have formula I:
wherein the variables are as defined herein.

Description

CROSS REFERENCE TO RELATED APPLICATIONS[0001]This present invention claims the benefit, under 35 U.S.C. §119, of U.S. Provisional Application No. 61 / 620,717, filed Apr. 5, 2012, the entire contents of which are incorporated herein by reference.BACKGROUND OF THE INVENTION[0002]ATR (“ATM and Rad3 related”) kinase is a protein kinase involved in cellular responses to DNA damage. ATR kinase acts with ATM (“ataxia telangiectasia mutated”) kinase and many other proteins to regulate a cell's response to DNA damage, commonly referred to as the DNA Damage Response (“DDR”). The DDR stimulates DNA repair, promotes survival and stalls cell cycle progression by activating cell cycle checkpoints, which provide time for repair. Without the DDR, cells are much more sensitive to DNA damage and readily die from DNA lesions induced by endogenous cellular processes such as DNA replication or exogenous DNA damaging agents commonly used in cancer therapy.[0003]Healthy cells can rely on a host of differen...

Claims

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Application Information

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IPC IPC(8): A61K31/497C07D241/28C07D413/04A61K33/24A61K45/06A61K31/4965A61K31/55A61K33/243
CPCA61K31/497A61K31/4965C07D241/28C07D413/04A61K33/24A61K45/06A61K31/55A61P17/02A61P17/06A61P35/00A61P43/00A61K33/243A61K31/496
Inventor POLLARD, JOHN ROBERTREAPER, PHILIP MICHAELASMAL, MOHAMMED
Owner VERTEX PHARMA INC
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