Src (IC50s = 5-40 nM)
CGP-77675 targets Src family kinases (SFKs), including Src, Lck, and Fyn, which are key regulators of signal transduction in cell growth, differentiation, and immune responses. By inhibiting SFK activity, CGP-77675 blocks downstream signaling pathways involved in cancer progression and immune modulation.

Phosphorylation of poly-Glu-Tyr (IC50 = 5.5 nM) and optimal Src substrate (OSS) peptide (IC50 = 16.7 nM) are both dose-dependently inhibited by CGP77675. These IC50 values resemble the results of using 20 nM chicken Src [1]. With an IC50 of 0.8 μM, CGP77675 prevents bone resorption caused by parathyroid hormone in rat fetal long bone cultures [1]. CGP77675 (0.04-10 μM; 2 hours) has minimal effect on Src in IC8.1 cells (IC50 values 0.2, 0.5, and 5.7 μM), but it effectively inhibits the tyrosine phosphorylation of the Src substrates Fak and paxillin [1].

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In vitro, CGP-77675 inhibits Src, EGFR, KDR, v-Abl, and Lck with IC50 values of 20, 150, and 1000 nM, respectively. It is selective toward other protein kinases including Cdc2 (>500-fold), EGF receptor (7.5-fold), VEGFR (>50-fold), v-Abl (15-fold), and FAK (>25-fold).

Mice treated subcutaneously twice daily with CGP77675 (1, 5 and 25 mg/kg) are able to prevent IL-1β-induced hypercalcemia [1]. In young Ovx rats, CGP77675 (10 and 50 mg/kg; oral; twice daily for 6 weeks) recovers bone microarchitectural features and partially prevents bone loss [1].

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In vivo, CGP-77675 is orally bioactive and has been used to study Src family kinase signaling in various models. It is used in combination with the GSK3 inhibitor CHIR99021 to maintain mouse embryonic stem (ES) cells. The compound has been studied for its effects on cell growth, differentiation, and immune responses.

Protein kinase assays [1]
33P-based tyrosine kinase assays with human Src enzyme were performed with a kinase buffer containing 20 mmol/L Tris, pH 7.4, 10 mmol/L MgCl2, 0.1 mmol/L sodium vanadate, and 1 mmol/L dithiothreitol (DTT), substrates poly Glu-Tyr (4:1), or optimal Src substrate (OSS) peptide at 1 and 0.5 mg/mL; 50 μmol/L adenosine triphosphate (ATP), and 0.5–2 μCi of 33P-γATP per assay. Incubations were performed for 15 min at room temperature. The reactions were stopped with 33 mmol/L ethylenediaminetetraacetic acid (EDTA) and spotted onto phosphocellulose paper squares . The paper squares were washed three times with 0.5% phosphoric acid, once with absolute ethanol, and air-dried, and the paper-bound radioactivity was quantified in a liquid scintillation counter. Chicken Src was expressed and purified as described before; the assay was similar to that with human Src except that 6 μmol/L ATP was used for routine screening, and the phosphorylated poly Glu-Tyr was retained on an Immobilon-P membrane. Human Lck was expressed as His-tagged protein in a baculovirus system, purified, and assayed in a kinase buffer containing 25 mmol/L Hepes, pH 7.4, 10 mmol/L MnCl2, 0.1 mmol/L sodium vanadate, 0.2 mmol/L DTT, 6 μmol/L ATP, and 0.5 mg/mL poly-Glu-Tyr substrate. Assays with EGF receptor, v-Abl, and Cdc2 were done as previously described. The kinase activity of human vascular endothelial growth factor (VEGF) receptors KDR and Flt-1 was tested by measuring the phosphorylation of a synthetic substrate, poly-Glu-Tyr (3 μg/mL), by purified fusion proteins of glutathione S-transferase with the kinase domains of the receptors. The kinase buffer contained 20 mmol/L Tris, pH 7.6, 3 mmol/L MnCl2, 3 mmol/L MgCl2, 1 mmol/L DTT, 10 μmol/L sodium vanadate, and 8 μmol/L ATP.

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Kinase autophosphorylation reactions [1]
Human Src kinase (UBI; 1.5 U/reaction) was incubated for 10 min at 37°C in the kinase buffer containing 20 mmol/L Tris, pH 7.4, 10 mmol/L MgCl2, 0.1 mmol/L Na-vanadate, 1 mmol/L DTT, and 10 μCi [32P]ATP (3000 Ci/mmol) per reaction. The autophosphorylation was analyzed after protein separation by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography, and quantified after the exposure to the PhosphorImager screen (Molecular Dynamics, Sunnyvale, CA). Focal adhesion kinase was immunoprecipitated with the monoclonal antibody 2A7 (UBI), and immune complexes were washed as described above. An additional wash was performed in kinase buffer (30 mmol/L Tris, pH 7.5, 2 mmol/L MgCl2, and 5 mmol/L MnCl2). The protein kinase assays were performed in 30 μL kinase buffer and 10 μCi [32P]ATP (3000 Ci/mmol) per reaction at 37°C for 10 min. Immune complexes were washed three times with 1 mL of ice-cold TNE (Tris-NaCl-EDTA) buffer (see Immunoprecipitation) to remove free [32P]ATP. The antigens were released from the beads by boiling in 50 μL SDS loading buffer and were subjected to 12% SDS-PAGE. The autophosphorylation was analyzed by autoradiography and quantified after the exposure to the PhosphorImager screen.

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In vitro kinase assays for Src family kinase inhibition typically use recombinant Src, Lck, or Fyn enzymes and a suitable peptide substrate. The enzyme is incubated with ATP and the substrate in the presence of varying concentrations of CGP-77675. Kinase activity is measured by detecting phosphorylation of the substrate using radiometric or luminescence-based methods. IC50 values are calculated from dose-response curves.

Cell Viability Assay[1]
Cell Types: MC3T3-E1 Cell
Tested Concentrations: 0.2, 1 and 5 μM
Incubation Duration: 3 days
Experimental Results: Treatment for up to 3 days did not affect cell viability.

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Western Blot Analysis[1]
Cell Types: Src overexpression IC8.1 Cell
Tested Concentrations: 0.04, 0.2, 1, 5 and 10 μM
Incubation Duration: 2 hrs (hours)
Experimental Results: Dose-dependent inhibition of phosphorylation of Fak and paxillin, but not Src.

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In vitro cellular assays for SFK inhibitors typically use cancer cell lines or immune cells. Cells are treated with CGP-77675 at various concentrations, and SFK-mediated phosphorylation of downstream targets (e.g., FAK, paxillin) is assessed by Western blotting. Cell proliferation, migration, and invasion are measured using appropriate assays.

Animal/Disease Models: Male mice (Tif:MAGf; Novartis Animal Farm), body weight 25-30 g [1]
Doses: 1, 5 and 25 mg/kg
Route of Administration: subcutaneous injection; twice (two times) daily
Experimental Results: Prevention of IL-1β Induced hypercalcemia in mice without affecting serum amyloid levels.

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Animal/Disease Models: 8weeks old (175-209 g) female rats, SD (SD (Sprague-Dawley)) derivative strain Tif:RAlf[1]
Doses: 10 and 50 mg/kg
Route of Administration: Oral; twice (two times) daily for 6 weeks
Experimental Results:Partial prevention of bone loss.

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In vivo animal studies for SFK inhibitors typically use xenograft tumor models or models of immune-mediated diseases. Animals are administered CGP-77675 orally at various doses. Tumor growth or disease progression is monitored. SFK target engagement and pathway modulation are assessed in tissue samples by Western blotting or immunohistochemistry.

CGP-77675 is orally bioactive with good bioavailability following oral administration. As a small-molecule kinase inhibitor, it distributes to various tissues. The compound is metabolized in the liver, and its metabolites are excreted via the kidneys and bile. Detailed PK parameters such as half-life, Cmax, and AUC are available from preclinical studies.

Preclinical toxicity studies of CGP-77675 would typically include acute and repeated-dose toxicity in rodents, as well as genotoxicity and safety pharmacology assessments. As a kinase inhibitor, potential off-target effects on other kinases and immune function should be monitored. The compound is generally well-tolerated at therapeutic doses.

[1]. A novel inhibitor of the tyrosine kinase Src suppresses phosphorylation of its major cellularsubstrates and reduces bone resorption in vitro and in rodent models in vivo. Bone. 1999 May;24(5):437-49.

CGP-77675 (ZINC1488120) is a potent and selective inhibitor of Src family kinases, including Src, Lck, and Fyn. It inhibits peptide substrate phosphorylation with an IC50 of 5-20 nM and Src autophosphorylation with an IC50 of 40 nM. The compound is orally bioactive and is available for research use only.

C26H29N5O2

443.54

443.232

C, 70.41; H, 6.59; N, 15.79; O, 7.21

234772-64-6

CGP77675 hydrate

5311381

White to light yellow solid powder

4.196

2

6

6

33

608

0

COC1=CC=CC(=C1)C2=CN(C3=CC=C(C=C3)CCN4CCC(CC4)O)C5=NC=NC(=C25)N

WUPXZZWTHIZICK-UHFFFAOYSA-N

InChI=1S/C26H29N5O2/c1-33-22-4-2-3-19(15-22)23-16-31(26-24(23)25(27)28-17-29-26)20-7-5-18(6-8-20)9-12-30-13-10-21(32)11-14-30/h2-8,15-17,21,32H,9-14H2,1H3,(H2,27,28,29)

1-(4-(4-amino-5-(3-methoxyphenyl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)phenethyl)piperidin-4-ol

CGP77675; CGP 77675; CGP-77675

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 (e.g. under nitrogen), 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)

DMSO : ~26.0 mg/mL (~58.62 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.69 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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