364042-47-7
364042-47-7 structure
- Name: A 419259
- Chemical Name: 7-[trans-4-(4-Methyl-1-piperazinyl)cyclohexyl]-5-(4-phenoxyphenyl )-7H-pyrrolo[2,3-d]pyrimidin-4-amine trihydrochloride
- CAS Number: 364042-47-7
- Molecular Formula: C29H34N6O
- Molecular Weight: 482.62
- Catalog: Signaling Pathways Protein Tyrosine Kinase/RTK Src
- Create Date: 2016-02-07 10:13:58
- Modify Date: 2024-01-12 00:27:33
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A 419259 is a broad-spectrum pyrrolo-pyrimidine inhibitor, designed to enhance selectivity towards the Src family with IC50 of 9 nM, <3 nM and <3 nM for Src, Lck and Lyn, respectively.
| Name | 7-[trans-4-(4-Methyl-1-piperazinyl)cyclohexyl]-5-(4-phenoxyphenyl )-7H-pyrrolo[2,3-d]pyrimidin-4-amine trihydrochloride |
|---|---|
| Synonyms |
7H-Pyrrolo[2,3-d]pyrimidin-4-amine, 7-[trans-4-(4-methyl-1-piperazinyl)cyclohexyl]-5-(4-phenoxyphenyl)-, hydrochloride (1:3)
A-419259 trihydrochloride A 419259 |
| Description | A 419259 is a broad-spectrum pyrrolo-pyrimidine inhibitor, designed to enhance selectivity towards the Src family with IC50 of 9 nM, <3 nM and <3 nM for Src, Lck and Lyn, respectively. |
|---|---|
| Related Catalog | |
| Target |
IC50: 9 nM (Src), <3 nM (Lck), <3 nM (Lyn), 3 μM (Abl)[1] |
| In Vitro | A 419259 is a second-generation pyrrolo-pyrimidine designed to enhance selectivity towards the Src family relative to other cytoplasmic tyrosine kinases. A-419259 inhibits K-562 cells with an IC50 between 0.1 and 0.3 μM, and Meg-01 proliferation with an IC50 of approximately 0.1 μM. A-419259 also potently induces apoptosis in K-562 cells beginning at 0.1 μM and increasing in a dose-dependent manner. PP2 inhibits Src kinase autophosphorylation in both Ph+ cell lines (K-562 and Meg-01) with an IC50 between 3 and 10 μM, while A-419259 blocks kinase activation between 0.1 and 0.3 μM. A-419259 strongly inhibits DAGM/Bcr-Abl cell proliferation in the absence of IL-3 with an IC50 between 0.1 and 0.3 μM[1]. A-419259 is a broad-spectrum pyrrolo-pyrimidine inhibitor, and blocks proliferation and induces apoptosis in CML cell lines. A-419259 inhibits overall SFK activity in K562 and other CML cell lines with an IC50 value of 0.1-0.3 μM[2]. |
| Kinase Assay | In vitro kinase assays are performed on His(6)-tagged Lck (residues 62-509) and full-length c-Abl purified from Sf-9 cells, and commercial sources of Lyn, Src and PKC. Lck, Lyn, Src and Abl activities are measured with an ELISA-based assay. Flat bottom 96-well ELISA plates are incubated with a 200 μg/mL solution of Poly(Glu,Tyr) 4 : 1 substrate in phosphate buffered saline (PBS) for 1 h at 37°C and then washed with PBS containing 0.1% Tween-20 (PBS-T). Inhibitor dilutions are added to the washed plates already containing the appropriate enzyme in kinase assay buffer (250 mM Mopso, pH 6.75, 10 mM MgCl2, 2 mM MnCl2, 2.5 mM DTT, 0.02% BSA, 2 mM Na3VO4, 5% DMSO, 5 μM ATP). After incubation at room temperature for 20 min, plates are washed three times with PBS-T and plate-bound phosphotyrosine is detected with an anti-phosphotyrosine-HRP antibody conjugate and subsequent color development with K-Blue reagents. All assays are optimized to use the least amount of enzyme necessary for a reproducible signal-to-noise ratio[1]. |
| Cell Assay | K-562 cells are grown in RPMI 1640 supplemented with 10% fetal calf serum (FCS), and 50 g/mL Gentamycin. Meg-01 cells are cultured in Vitacell modified RPMI 1640 (ATCC), supplemented with 10% FCS and 50 μg/mL Gentamycin. The human GM-CSF-dependent myeloid leukemia cell line TF-1 is grown in RPMI 1640 supplemented with 10% FCS, 50 μg/mL Gentamycin, and 1 ng/mL of recombinant human GM-CSF. DAGM murine myeloid leukemia cells are cultured in RPMI 1640 supplemented with 10% FCS, 50 μg/mL Gentamycin, and 0.5 ng/mL recombinant IL-3. Concentrated stock solutions of PP2 (5 mM) and A-419259 (2 mM) are prepared in DMSO and stored at -20°C. Cellular proliferation is measured using the Live/Dead growth assay. This assay employs calcein-AM, a fluorogenic esterase substrate that is taken up by viable cells and hydrolyzed intracellularly, releasing a green fluorescent product. Briefly, 104 cells are plated per well in 96-well plates for each day of a 4-day time course. Various concentrations of PP2, A-419259 or vehicle control are added to the wells (five wells per concentration per day) and the plates are incubated at 37°C. At each time point, one plate is centrifuged at 1500 g for 10 min to pellet the cells. Cells are washed with phosphate buffered saline (PBS), and calcein-AM is added to each well to a final concentration of 1 μM. Plates are incubated in the dark at room temperature for 1 h. The plates are then read at 485/530 nm (excitation/emission) using a SpectraMax Gemini XS fluorescent plate reader and data are analysed with SoftMax Pro software[1]. |
| References |
| Molecular Formula | C29H34N6O |
|---|---|
| Molecular Weight | 482.62 |
| PSA | 72.44000 |
| LogP | 8.06690 |
| Storage condition | 2-8℃ |