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PP2 (also known as AG 1879; AG-1879) a novel, potent, reversible, and ATP-competitive and selective Src family kinase inhibitor with potential anticancer activity. It inhibits Lck/Fyn kinases with IC50s of 4 nM/5 nM in cell-free assays, and is less potent to or inactive for EGFR, ZAP-70, JAK2 and PKA. PP2 is a chemical substance frequently used in cancer research. PP2 induces slowing in the growth rate of tumors in SCID mice inoculated HT29 cells in the spleen. It strongly inhibits the kinases Lck (IC50=4 nM), Fyn (5 nM) and Hck (5 nM), shows weaker inhibition of EGFR (480 nM) and practically no inhibition of ZAP-70 (100 µM) and JAK2 (50 µM).
Physicochemical Properties
| Molecular Formula | C15H16CLN5 | |
| Molecular Weight | 431.53 | |
| Exact Mass | 301.109 | |
| Elemental Analysis | C, 59.70; H, 5.34; Cl, 11.75; N, 23.21 | |
| CAS # | 172889-27-9 | |
| Related CAS # |
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| PubChem CID | 4878 | |
| Appearance | White to off-white solid powder | |
| Density | 1.4±0.1 g/cm3 | |
| Boiling Point | 493.5±40.0 °C at 760 mmHg | |
| Melting Point | 214-216ºC | |
| Flash Point | 252.3±27.3 °C | |
| Vapour Pressure | 0.0±1.3 mmHg at 25°C | |
| Index of Refraction | 1.676 | |
| LogP | 3.22 | |
| Hydrogen Bond Donor Count | 1 | |
| Hydrogen Bond Acceptor Count | 4 | |
| Rotatable Bond Count | 2 | |
| Heavy Atom Count | 21 | |
| Complexity | 364 | |
| Defined Atom Stereocenter Count | 0 | |
| SMILES | ClC1C([H])=C([H])C(=C([H])C=1[H])C1C2=C(N([H])[H])N=C([H])N=C2N(C(C([H])([H])[H])(C([H])([H])[H])C([H])([H])[H])N=1 |
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| InChi Key | PBBRWFOVCUAONR-UHFFFAOYSA-N | |
| InChi Code | InChI=1S/C15H16ClN5/c1-15(2,3)21-14-11(13(17)18-8-19-14)12(20-21)9-4-6-10(16)7-5-9/h4-8H,1-3H3,(H2,17,18,19) | |
| Chemical Name | 1-(tert-butyl)-3-(4-chlorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine | |
| Synonyms | AG 1879; AG-1879; PP2; PP-2; 1-(tert-butyl)-3-(4-chlorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine; AG 1879; 1-tert-butyl-3-(4-chlorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine; 1-tert-butyl-3-(4-chlorophenyl)pyrazolo[3,4-d]pyrimidin-4-amine; PP 2; AG1879. | |
| HS Tariff Code | 2934.99.9001 | |
| Storage |
Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
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| Shipping Condition | Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs) |
Biological Activity
| Targets | Lck (IC50 = 4 nM); Fyn (IC50 = 5 nM)[1] | ||
| ln Vitro | At 10 μM, the impact of PP2 on cell proliferation was not statistically significant, indicating that the PP2 effect on gemcitabine cytotoxicity at this low dosage is likely due to gemcitabine-induced cell proliferation rather than a direct antiproliferative action. elevated toxicity. Growth was progressively suppressed at 20 μM, which is in accordance with findings from other human cancer cell lines. While we employed 10 μM PP2, it has been documented that PP2 at higher concentrations inhibits other intracellular kinases [2]. The most popular Src family kinase inhibitor on the market is PP2. With an in vitro IC50 of roughly 5 nM, PP2 suppresses Src family kinase activity. Complete Src family kinase inhibition in cell culture is typically achieved at a dosage of 10 μM [3]. | ||
| ln Vivo |
In the PP2 treatment group, the tumor growth inhibition rate is 25%, while in the Gemcitabine treatment group, it is 5% (P>0.05). When combined, PP2 and gemcitabine result in a 98% (P<0.05) tumor growth suppression rate. In 100% of the control and gemcitabine-treated groups, liver metastases occurred; in the PP2-treated group, 88% of the liver metastases formed. In the group receiving combined treatment with PP2 and Gemcitabine, there are no observable metastases (P<0.05)[2]. PP2-treated rats showed approximately 50% reduction of infarct size on T2-weighted MRI and in TTC staining compared with controls (P < 0.05). Moreover, the neurological score was better in the PP2 group than controls (P < 0.05). Conclusion: PP2 is a potential neuroprotective agent in cerebral ischemia-reperfusion. The interference of PP2 with SFKs and/or other pathways remains to be elucidated.Acta Neurol Scand. 2004 Sep;110(3):175-9. https://pubmed.ncbi.nlm.nih.gov/15285775/ |
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| Enzyme Assay | A closely related pyrazolopyrimidine, PP2, was similarly effective in the inhibition of Lck and FynT. In further selectivity tests using other Src family protein-tyrosine kinases, PP1 also inhibited Src (170 nM) and Hck (20 nM), while PP2 demonstrated potent inhibition of Hck (5 nM) (data not shown). In contrast, PP1 and PP2 were both 50-100-fold less active in the inhibition of A-431 epidermal growth factor receptor autophosphorylation (IC50 = 0.25 and 0.48 μM, respectively). Further specificity for inhibition of Lck and FynT was demonstrated when it was found that PP1 and PP2 were essentially inactive for inhibition of ZAP-70 and JAK2 and protein kinase A (data not shown). Since the activity of the ZAP-70 enzyme may be enhanced following phosphorylation at residue 493 by Lck, we also examined whether inhibition of ZAP-70 by PP1 was altered when coexpressed in insect cells together with Lck as described previously. Although coexpression of ZAP-70 with the catalytic domain of Lck consistently led to a 3-4-fold elevation in the specific activity of ZAP-70 for the p62 substrate, PP1 was still unable to inhibit this enzyme up to concentrations of 100 μM (data not shown). For comparison purposes, we also examined the activity of staurosporine and genistein, two previously described tyrosine kinase inhibitors. The fermentation product, staurosporine, has previously been demonstrated to be a potent but nonselective protein kinase inhibitor. In the experiments reported here, staurosporine was found to be a nanomolar inhibitor of p56lck and p59fynT as well as a low micromolar inhibitor of the EGF-R kinase. However, unlike PP1 and PP2, it was also a potent inhibitor of the ZAP-70 and JAK2 tyrosine kinases. For further comparison, the naturally occurring isoflavone genistein was tested for its ability to inhibit the four tyrosine kinases. As expected, it was the least potent inhibitor (Table 1). Thus, relative to other reported tyrosine kinase inhibitors, the novel compounds PP1 and PP2 demonstrated potent and selective inhibition of the Src family kinases, such as p56lck and p59fynT[1]. | ||
| Cell Assay | Influenza-induced T cell proliferation (see Table 2, Ag-Sp (which represents specific antigen)) was assessed by combining 1 × 105 PBL in triplicate with 160 μl of RPMI 1640 medium containing 10% fetal calf serum with 20 μl of either diluted test compound or media alone in a 96-well microtiter plate (Costar Corp.). Antigen (influenza virus vaccine Fluzone, Connaught Laboratories) was prepared by centrifuging and washing (three times) 2 ml of vaccine through a Centricon-3 concentrator (Amicon, Inc.) to remove preservative and diluting the remaining material to 40 ml (1:20). Twenty microliters of antigen was then added to each well, and the plates were incubated for 72 h at 37°C in 5% CO2. [3H]Thymidine was then added (0.5 μCi/well), and the plates were incubated for an additional 18 h at 37°C. Cells were harvested with a 96-well harvester (Tomtec), and the amount of incorporated [3H]thymidine was determined using a Pharmacia Biotech β-plate counter. Concentrations that cause 50% inhibition of proliferation (IC50) were determined from a plot of the percent inhibition of proliferation from media control versus concentration of test compound added. Results are presented as the mean IC50 from repeated experiments (see Table 2). T cell proliferation in the one-way mixed lymphocyte reaction was assessed by combining in triplicate 5 × 104 fresh PBL, 5 × 104 irradiated (5000 rads) pooled stimulator PBL, and diluted test compound in RPMI 1640 medium in each well of a 96-well assay plate. After 18 h of incubation at 37°C in 5% CO2, 0.5 μCi of [3H]thymidine was added to each well, and the cells were incubated for another 18 h. The cells were then harvested using a Pharmacia Biotech β-plate system. Percent inhibition was determined by the following equation: % inhibition = 1 - (mean cpm of drug-treated cells/mean cpm of control stimulated cells) × 100[1]. | ||
| Animal Protocol |
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| References |
[1]. Discovery of a novel, potent, and Src family-selective tyrosine kinase inhibitor. Study of Lck-and FynT-dependentT cell activation. J Biol Chem. 1996 Jan 12;271(2):695-701. [2]. Inhibition of SRC tyrosine kinase impairs inherent and acquired Gemcitabine resistance in human pancreatic adenocarcinoma cells. Clin Cancer Res. 2004 Apr 1;10(7):2307-18. [3]. AP23846, a novel and highly potent Src family kinase inhibitor, reduces vascular endothelial growth factor and interleukin-8 expression in human solid tumor cell lines and abrogates downstream angiogenic processes. Mol Cancer Ther. 2005 D. [4]. Phosphorylation of NMDA receptor GluN2B subunit at Tyr1472 is important for trigeminal processing of itch. Eur J Neurosci. 2016 Oct;44(7):2474-2482. |
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| Additional Infomation | PP2 is a member of the class of pyrazolopyrimidine that is pyrazolo[3,4-d]pyrimidin-4-amine bearing additional tert-butyl and 4-chlorophenyl substituents at positions 1 and 3 respectively. It is a potent ATP-competitive inhibitor of the Src family of protein tyrosine kinases. It has a role as an EC 2.7.10.2 (non-specific protein-tyrosine kinase) inhibitor, a beta-adrenergic antagonist and a geroprotector. It is a pyrazolopyrimidine, an aromatic amine and a member of monochlorobenzenes. |
Solubility Data
| Solubility (In Vitro) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 3 mg/mL (9.94 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (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 30.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. Solubility in Formulation 2: 4% DMSO+30% PEG 300+ddH2O:5 mg/mL (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.3173 mL | 11.5867 mL | 23.1734 mL | |
| 5 mM | 0.4635 mL | 2.3173 mL | 4.6347 mL | |
| 10 mM | 0.2317 mL | 1.1587 mL | 2.3173 mL |