Home Angiogenesis FLT3 inhibitor - Aurora Kinase inhibitor - Bcr-Abl inhibitor KW-2449

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KW-2449 FLT3 inhibitor

Cat.No.S2158

KW-2449 is a multiple-targeted inhibitor, mostly for Flt3 with IC50 of 6.6 nM, modestly potent to FGFR1, Bcr-Abl and Aurora A; little effect on PDGFRβ, IGF-1R, EGFR. Phase 1.
KW-2449 FLT3 inhibitor Chemical Structure

Chemical Structure

Molecular Weight: 332.4

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Chemical Information, Storage & Stability

Molecular Weight 332.4 Formula

C20H20N4O

 Storage (From the date of receipt)
CAS No. 1000669-72-6 Download SDF Storage of Stock Solutions

Synonyms N/A Smiles C1CN(CCN1)C(=O)C2=CC=C(C=C2)C=CC3=NNC4=CC=CC=C43

Solubility

In vitro
Batch:

DMSO : 67 mg/mL (201.56 mM)
(Moisture-contaminated DMSO may reduce solubility. Use fresh, anhydrous DMSO.)

Ethanol : 67 mg/mL

Water : Insoluble

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Mass Concentration Volume Molecular Weight

In vivo
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Mechanism of Action

Features
Investigated as a FLT3 inhibitor in clinical trials, with others in early development.
Targets/IC50/Ki
FLT3 (D835Y) [1]
(Cell-free assay)
1 nM
Abl (T315I) [1]
(Cell-free assay)
4 nM
FLT3 [1]
(Cell-free assay)
6.6 nM
Abl [1]
(Cell-free assay)
14 nM
FGFR1 [1]
(Cell-free assay)
36 nM
Aurora A [1]
(Cell-free assay)
48 nM
JAK2 [1]
(Cell-free assay)
150 nM
Kit [1]
(Cell-free assay)
300 nM
Src [1]
(Cell-free assay)
400 nM
In vitro
KW-2449, a multikinase inhibitor of FLT3, ABL, ABL-T315I, and Aurora kinase, is under investigation to treat leukemia patients. This compound shows the potent growth inhibitory effects on leukemia cells with FLT3 mutations by inhibition of the FLT3 kinase, resulting in the down-regulation of phosphorylated-FLT3/STAT5, G1 arrest, and apoptosis. Oral administration of this chemical shows dose-dependent and significant tumor growth inhibition in FLT3-mutated xenograft model with minimum bone marrow suppression. In FLT3 wild-type human leukemia, it induces the reduction of phosphorylated histone H3, G2/M arrest, and apoptosis. In Imatinib-resistant leukemia, this agent contributes to release of the resistance by the simultaneous down-regulation of BCR/ABL and Aurora kinases. The inhibitory activity of this compound is not affected by the presence of human plasma protein, such as α1-acid glycoprotein. It has potent growth inhibitory activity against various types of leukemia by several mechanisms of action. This inhibitor has significant activity and warrants clinical study in leukemia patients with FLT3 mutations as well as Imatinib-resistant mutations. Phosphorylation levels of FLT3 and STAT5 are decreased by this chemical in a dose-dependent manner. In addition, it potently inhibits ABL-T315I, which is associated with IM resistance, with IC50 of 4 nM. On the other hand, this compound has little effect on PDGFRβ, IGF-1R, EGFR, and various serine/threonine kinases even at a concentration of 1 μM. It has the potent growth inhibitory activities against not only FLT3/ITD-expressing leukemia cells but also FLT3/KDM-activated and wild-type FLT3-overexpressing leukemia cells. In accordance with growth inhibitory effect, this agent suppresses the phosphorylations of FLT3 (P-FLT3) and its downstream molecule phospho-STAT5 (P-STAT5) in MOLM-13 cells in a dose-dependent manner. Furthermore, it increases the percentage of cells in the G1 phase of the cell cycle and reciprocally reduces the percentage of cells in the S phase, resulting in the increase of apoptotic cell population. This compound can dephosphorylate constitutively active WT-FLT3 kinase but not inhibit the proliferation of leukemia cells if they are not mainly addicted to FLT3 the kinase. [1] It is rapidly absorbed and converted to a major metabolite M1.Preclinical studies reveal that this chemical is converted by monoamine oxidase-B (MAO-B) and aldehyde oxidase into its major metabolite M1.This agent mediates cytotoxicity thru inhibition of FLT3/ITD.It is a direct inhibitor of FLT3 and induces inhibition of its downstream target STAT5. [2] This compound interacts synergistically with HDACIs to induce apoptosis in Ph+ CML cells in a time- and concentration-dependent manner. It synergistically enhances the lethality of vorinostat/SNDX275 in CML cells.This inhibitor regimens are active against additional IM-resistant Bcr/Abl+ leukemia cells. It moderately reduces phosphorylation of histone H3, an indicator of Aurora B activity, in nocodozole-treated K562 cells. [3]
Kinase Assay
FLT3 phosphorylation
Leukemia cells are washed in phosphate-buffered saline (PBS), then lysed by resuspending the cells in lysis buffer (20 mM Tris pH 7.4, 100 mM NaCl, 1% Igepal, 1 mM EDTA, 2 mM NaVO4, plus Complete protease inhibitor KW-2449 for 30 minutes while rocking. The extract is clarified by centrifugation at 1.6 × 104 g and the supernatant is assayed for protein (Bio-Rad). A 50-μg aliquot is removed as a whole-cell lysate for analysis of STAT5, and the remainder is used for immunoprecipitation with anti-FLT3. Anti-FLT3 antibody is added to the extract for overnight incubation, then protein A sepharose is added for 2 additional hours. Separate sodium dodecyl sulfate–polyacrylamide electrophoresis (SDS-PAGE) gels for whole-cell lysate and immunoprecipates are run in parallel. After transfer to Immobilon membranes, immunoblotting is performed with antiphosphotyrosine antibody (4G10) to detect phosphorylated FLT3 or, for the whole-cell lysate gels, with a rat monoclonal antibody against phosphorylated STAT5 (residue Y694) then stripped and reprobed with anti-FLT3 antibody to measure total FLT3. Proteins are visualized using chemiluminescence, exposed on Kodak BioMax XAR film, developed, and scanned using a Bio-Rad GS800 densitometer. The concentration of this compound for which the phosphorylation of FLT3 or STAT5 is inhibited to 50% of its baseline (IC50) is determined using linear regression analysis of the dose response curves. For direct analysis of FLT3 and STAT5 in circulating blasts, peripheral blood is collected in heparinized tubes and promptly chilled on ice. Samples are centrifuged for 10 minutes at 900 g, at 4 °C. The plasma is removed and stored frozen at −80 °C. The buffy coat is carefully transferred to ice-cold PBS, layered onto chilled Ficoll-Hypaque, and centrifuged for 5 minutes at 600 g, at 4 °C. All subsequent steps are carried out at 4 °C. Mononuclear cells are collected and washed rapidly once in red blood cell lysis buffer (0.155 M NH4Cl, 0.01 M KHCO3, 0.1 mM EDTA), then washed once in PBS. Cells are then lysed as described for FLT3 and STAT5 analysis.
In vivo
In the MOLM-13 tumor xenograft model, oral administration of KW-2449 for 14 days shows a potent and significant antitumor effect in a dose-dependent manner. [1]
References

Clinical Trial Information

(data from https://clinicaltrials.gov, updated on 2024-05-22)

NCT Number Recruitment Conditions Sponsor/Collaborators Start Date Phases
NCT00346632 Terminated
Acute Myelogenous Leukemia|Acute Lymphoblastic Leukemia|Myelodysplastic Syndromes|Chronic Myelogenous Leukemia
Kyowa Kirin Inc.
 June 2006 Phase 1

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