Src family tyrosine kinases (Src, Lck, Lyn, Hck, Fyn, Fgr). Specifically, A-419259 exhibits potent inhibitory activity against Lck (IC50 < 0.003 μM) and Lyn (IC50 < 0.003 μM), with high selectivity over c-Abl (IC50 = 3.0 μM) and PKC (IC50 > 33 μM) in in vitro kinase assays. [1]

In contrast to other cytoplasmic tyrosine kinases, A 419259 TriHClide is a second-generation pyrrolopyrimidine that is intended to improve selectivity for the Src family. K-562 cells are inhibited by A-419259 with an IC50 of 0.1–0.3 μM, while Meg-01 proliferation is inhibited with an IC50 of about 0.1 μM. Moreover, A-419259 potently triggered apoptosis in K-562 cells, with dose-dependent increases beginning at 0.1 μM. In two Ph+ cell lines (K-562 and Meg-01), PP2 inhibits Src kinase autophosphorylation with an IC50 ranging from 3 to 10 μM, while A-419259 prevents kinase activation with an IC50 ranging from 0.1 to 0.3 μM. With an IC50 ranging from 0.1 to 0.3 μM, A-419259 significantly suppresses the proliferation of DAGM/Bcr-Abl cells in the absence of IL-3 [1]. A-419259 is a broad-spectrum pyrrolopyrimidine inhibitor that causes CML cell lines to undergo apoptosis and stops growth. In K562 and other CML cell lines, A-419259 suppresses total SFK activity with IC50 values ranging from 0.1 to 0.3 μM [2].

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A-419259 dose-dependently inhibits the proliferation of Philadelphia chromosome-positive (Ph+) chronic myelogenous leukemia (CML) cell lines K-562 (IC50 between 0.1 and 0.3 μM) and Meg-01 (IC50 ~0.1 μM) over a 4-day period, as measured by a fluorescent microplate viability assay. [1]
A-419259 (0.1-1 μM) induces apoptosis in K-562 cells in a dose-dependent manner after 72 hours of treatment, as assessed by annexin-V binding and caspase-3 activation assays. [1]
A-419259 (0.1-0.3 μM) inhibits Src family kinase autophosphorylation (activation) in Ph+ cell lines (K-562, Meg-01) and in Bcr-Abl-transformed DAGM myeloid cells, correlating with its anti-proliferative and pro-apoptotic effects. [1]
At concentrations that inhibit cell growth and Src kinases (e.g., 0.3 μM), A-419259 does not markedly reduce the phosphotyrosine content of Bcr-Abl or the phosphorylation status of its direct substrate CrkL in K-562 and Meg-01 cells, indicating its primary target is not Bcr-Abl itself. [1]
A-419259 (0.1-0.3 μM) blocks the constitutive activation of downstream signaling pathways in K-562 cells, including Stat5 (inhibiting its DNA-binding activity and tyrosine phosphorylation) and the Ras/Erk pathway (reducing Erk phosphorylation). [1]
A-419259 downregulates the expression of the anti-apoptotic Stat5 target gene Bcl-XL in K-562 cells at concentrations that inhibit Stat5 activation. [1]
Transformation of IL-3-dependent DAGM myeloid cells with Bcr-Abl sensitizes them to the growth-inhibitory effects of A-419259 (IC50 0.1-0.3 μM in the absence of IL-3), while the untransformed parental cells (DAGM/Neo) are unaffected. IL-3 can partially rescue the growth inhibition in Bcr-Abl-transformed cells. [1]
A-419259 has little to no effect on the proliferation or induction of apoptosis in Philadelphia chromosome-negative (Ph-) myeloid leukemia cell lines TF-1 and HEL at concentrations up to 1 μM. [1]

In vitro kinase inhibitory activities (IC50) of A-419259 against various kinases were determined using ELISA-based assays for Src, Lck, Lyn, and c-Abl, and a radioactive assay for PKC. For the ELISA-based assays, 96-well plates were coated with a Poly(Glu,Tyr) 4:1 substrate. After washing, serial dilutions of the inhibitor were added to the wells along with the respective kinase enzyme in an assay buffer containing ATP. Following incubation, plates were washed again, and plate-bound phosphotyrosine was detected using an anti-phosphotyrosine-HRP conjugate followed by colorimetric development. For the PKC assay, the enzyme was incubated with a substrate peptide, ATP (including [γ-³³P]ATP), and the inhibitor in a kinase buffer. Reactions were stopped, spotted onto phosphocellulose filters, washed, and radiolabel incorporation was quantified by scintillation counting. The amounts of each kinase per reaction were optimized for signal-to-noise. [1]

For proliferation assays, cells (e.g., K-562, Meg-01, TF-1, HEL, DAGM derivatives) were seeded in 96-well plates. A-419259 or vehicle control (DMSO) was added at indicated concentrations, with five replicate wells per condition. Plates were incubated at 37°C over a 4-day time course. At each time point (days 0, 2, 4), cells were pelleted, washed with PBS, and incubated with 1 μM calcein-AM (a fluorogenic esterase substrate taken up by viable cells) for 1 hour at room temperature in the dark. Fluorescence (excitation/emission 485/530 nm) was measured using a fluorescent plate reader to determine viable cell counts. [1]
For apoptosis assays, cells were treated with A-419259 for 72 hours. Apoptosis was assessed by two methods: 1) Annexin-V binding: Cells were washed, resuspended in binding buffer, and incubated with annexin-V-FITC and propidium iodide, followed by analysis via flow cytometry. 2) Caspase-3 activity: Cells were pelleted and incubated with a cell-permeable, profluorescent caspase-3 substrate (PhiPhiLux). After incubation and washing, intracellular fluorescence generated by caspase-3 cleavage was measured by flow cytometry. [1]
For analysis of protein phosphorylation and expression, cells were treated with A-419259 for 20 hours, lysed in RIPA buffer containing protease and phosphatase inhibitors. Clarified lysates were resolved by SDS-PAGE, transferred to membranes, and immunoblotted with specific antibodies: anti-phospho-Src (Y418) for active Src family kinases; anti-phosphotyrosine (e.g., PY99) and anti-Abl for Bcr-Abl phosphorylation; anti-CrkL to assess its phosphorylation-induced mobility shift; anti-phospho-Stat5 (after immunoprecipitation of Stat5) and anti-Stat5; anti-phospho-Erk and anti-Erk2. Antibody binding was detected using alkaline phosphatase-conjugated secondary antibodies with a colorimetric substrate. [1]
For Stat5 DNA-binding activity (EMSA), nuclear extracts were prepared from treated cells. Extracts were incubated with a ³²P-labeled double-stranded DNA probe containing the γ-activation sequence (GAS) consensus site for Stat5. Binding reactions were performed in the presence or absence of excess unlabeled probe (competitor). Protein-DNA complexes were resolved on non-denaturing polyacrylamide gels, which were then dried and visualized by autoradiography. [1]
For analysis of Bcl-XL mRNA expression (RNase protection assay), total RNA was isolated from treated cells. A ³²P-labeled riboprobe set containing Bcl-2 family gene templates and GAPDH (control) was hybridized to the RNA. After RNase digestion to remove unhybridized probe, the protected RNA duplexes were resolved on denaturing urea gels and visualized by autoradiography/phosphoimaging. Bcl-XL signal intensity was normalized to GAPDH. [1]

[1]. Wilson MB, et al. Selective pyrrolo-pyrimidine inhibitors reveal a necessary role for Src family kinases in Bcr-Abl signal transduction and oncogenesis. Oncogene. 2002 Nov 21;21(53):8075-88.
[2]. Pene-Dumitrescu T, et al. An inhibitor-resistant mutant of Hck protects CML cells against the antiproliferative and apoptotic effects of the broad-spectrum Src family kinase inhibitor A-419259.Oncogene (2008) 27, 7055-7069

A-419259 is a second-generation pyrrolopyrimidine compound designed to enhance selectivity for Src family kinases relative to other cytoplasmic tyrosine kinases. [1] Studies have shown that Src family kinases (e.g., Hck, Lyn) are important intermediates in CML cells that link Bcr-Abl to key downstream signaling pathways (e.g., Stat5 and Ras/Erk). Inhibition of these Src kinases with A-419259 disrupts these pathways, leading to growth arrest and apoptosis in Bcr-Abl-transformed cells. [1] These findings confirm that myeloid-expressed Src kinases are potential therapeutic targets for CML, particularly in cases resistant to the Bcr-Abl inhibitor STI-571 (imatinib). This study suggests that the combined use of Src and Bcr-Abl inhibitors, or the use of bispecific drugs, may be a beneficial therapeutic strategy. [1]

C₂₉H₃₇CL₃N₆O

592.00

590.209

1435934-25-0

A 419259;364042-47-7

76848881

White to yellow solid powder

8.066

4

6

5

39

679

0

ALRMEQIQFCUAMR-UHFFFAOYSA-N

InChI=1S/C29H34N6O.3ClH/c1-33-15-17-34(18-16-33)22-9-11-23(12-10-22)35-19-26(27-28(30)31-20-32-29(27)35)21-7-13-25(14-8-21)36-24-5-3-2-4-6-24;;;/h2-8,13-14,19-20,22-23H,9-12,15-18H2,1H3,(H2,30,31,32);3*1H

7-[4-(4-methylpiperazin-1-yl)cyclohexyl]-5-(4-phenoxyphenyl)pyrrolo[2,3-d]pyrimidin-4-amine;trihydrochloride

RK 20449 trihydrochlorideA419259 RK-20449 A-419259

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O : ~50 mg/mL (~84.46 mM)
DMSO : ~1 mg/mL (~1.69 mM)

Solubility in Formulation 1: 100 mg/mL (168.92 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.

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