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Compounds useful for inhibiting chk1

A compound and solvate technology, applied in medical preparations containing active ingredients, drug combinations, organic chemistry, etc., can solve the problems of increasing sensitivity and not being able to be used as a treatment option

Inactive Publication Date: 2008-01-30
ELI LILLY & CO
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the dose of caffeine used to achieve cell cycle abrogation exceeds clinically acceptable levels and thus cannot be used as a therapeutic option
In addition, antisense nucleotides of Chk1 kinase have been used to increase sensitivity to the topoisomerase inhibitor BNP1350 (Yin et al., Biochem. Biophys. Res. Commun., 295:435-44, 2002), but it was demonstrated There are issues commonly associated with antisense and gene therapy

Method used

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  • Compounds useful for inhibiting chk1
  • Compounds useful for inhibiting chk1
  • Compounds useful for inhibiting chk1

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0236] Determination of the IC of Chk1 inhibitor 50 value

[0237] Human Chk1 cDNA was identified and cloned as described in the previous International Application Publication No. WO 99 / 11795 (application filed on September 4, 1998). The FLAG(R) tag is inserted in frame according to the reading frame of the amino terminal segment of the full-length Chk1. The 5'primer contains an EcoRI site, Kozak sequence, and also encodes a FLAG(R) tag for affinity purification with M2 antibody (Sigma, Saint Louis, IL). The 3'primer contains a SalI site. The PCR amplified fragment was cloned into pCI-Neo (Invitrogen, Carlsbad, CA) as an EcoRI-SalI fragment, and then subcloned into pFastBacI (Gibco-BRL, Bethesda, MD) as an EcoRI-NotI fragment. Recombinant baculovirus was prepared as described in the Gibco-BRL Bac-to-Bac manual and used to infect Sf-9 cells grown in CCM3 medium (HyClone Laboratories, Logan, UT) to express FLAG(R)-labeled Chk1 protein .

[0238] The FLAG(R)-labeled Chk1 was purified...

Embodiment 2

[0241] Selectivity

[0242] Test the selectivity of the Chk1 inhibitor of the present invention to one or more other protein kinases, namely against DNA-PK, Cdc2, casein kinase I (CKI), Chk2, p38 MAP kinase, ERK kinase, protein kinase A (PKA) And / or calcium-calmodulin kinase II (CaM KII) selectivity. Except for Chk2, the assay procedures for all these kinases have previously been described in the following documents, including U.S. Patent Publication No. 2002-016521 A1 and U.S. Patent Application No. 08 / 184,605 ​​(filed on January 21, 1994), Both of these are cited here for reference.

[0243] The activity of the compound against Chk2 was determined as follows: in 4mM ATP, 1mCi[ 32 P]γ-ATP, 20mM Hepes pH 7.5, 5mM MgCl 2 In the presence of 0.25% NP40, 128ng of purified His-labeled Chk2 was incubated with up to 100mM Chk1 inhibitor for 20 minutes at room temperature. The reaction was terminated with phosphoric acid at a final concentration of 150 mM, and 5 / 8 of the reaction mixtur...

Embodiment 3

[0246] The Chk1 inhibitor of the present invention inhibits the function of Chk1 in cells

[0247] To determine the function of Chk1 inhibitors of the present invention in inhibiting Chk1 in cells, the inhibitors can be tested in cell-based molecular assays. Since it has been proved that mammalian Chk1 phosphorylates Cdc25C in vitro, indicating that it is negatively regulating cyclin B / cdc2 in response to DNA damage, the ability of Chk1 inhibitors to increase the activity of cyclin B / cdc2 can be analyzed. The experiment can be designed as follows: irradiate HeLa cells with 800 rad light and incubate at 37°C for 7 hours. Because these cells are functionally p53 negative, they exclusively stall in the G2 phase. Then, nocodazole at a concentration of 0.5 μg / mL was added, and the cells were incubated at 37° C. for 15 hours. Nocodazole is added to capture any cells arrested to M by G2. Finally, Chk1 inhibitor was added for 8 hours, the cells were harvested, lysed, and the same amount...

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Abstract

The present invention discloses aryl and heteroaryl substituted urea compounds useful in the treatment of diseases and disorders associated with DNA damage or impaired DNA replication. The invention also discloses methods of making the compounds, and the use of the compounds as therapeutic agents for the treatment of, for example, cancer and other diseases characterized by defects in DNA replication, chromosome segregation or cell division.

Description

Invention field [0001] The present invention relates to compounds for inhibiting enzymes that maintain and repair the integrity of genetic material. More specifically, the present invention relates to a series of aryl and heteroaryl substituted urea compounds, to methods of making these compounds, and their use as therapeutic agents in the treatment of, for example, cancer and are characterized by deoxyribonucleic acid (DNA) replication, chromosome Use in other diseases with detachment or cell division defects. Background of the invention [0002] A large number of diseases, disorders and conditions (hereinafter referred to as "indications") are characterized by the involvement of abnormally proliferating cells. As used herein, the term "abnormally proliferating cells" (or "abnormal cell proliferation") means cell proliferation that deviates from the normal, proper or expected course. For example, abnormal cell proliferation includes inappropriate cell proliferation in which DNA ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07D241/20C07D401/04C07D403/04C07D413/04A61K31/4965A61K31/497A61P35/00
CPCC07D241/20C07D413/04C07D403/04C07D401/04A61P17/00A61P17/06A61P29/00A61P35/00A61P35/02A61P43/00A61K31/4965
Inventor F·S·弗鲁兹R·霍尔坎布E·索尔塞特J·J·高迪诺
Owner ELI LILLY & CO
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