Lck (IC50 = 4 nM); Fyn (IC50 = 5 nM)[1]
Src family kinases: Lck (IC₅₀ ≈ 4 nM), Fyn (IC₅₀ ≈ 5 nM), Src (IC₅₀ ≈ 6 nM); non-Src kinases: EGFR (IC₅₀ > 1000 nM), Abl (IC₅₀ > 1000 nM), PKC (IC₅₀ > 1000 nM) (showing high selectivity for Src family kinases) [1]
- Src kinase (pancreatic adenocarcinoma cells): PP2 (AG1879) inhibited Src phosphorylation (Tyr416) at concentrations of 5 μM–10 μM, with no effect on total Src protein levels [2]

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].

---

In human peripheral blood T cells: PP2 (AG1879) (1 μM–10 μM) concentration-dependently inhibited Lck/FynT-dependent T cell activation. At 10 μM: (1) Anti-CD3/CD28-induced IL-2 secretion decreased by ~80% (ELISA); (2) CD69 (early activation marker) expression on T cell surface reduced by ~75% (flow cytometry); (3) Western blot showed reduced phosphorylation of Lck (Tyr394), Fyn (Tyr417), and downstream ZAP-70 (Tyr493) [1]
- In human pancreatic adenocarcinoma cell lines (AsPC-1, BxPC-3) and gemcitabine-resistant sublines (AsPC-1/GemR, BxPC-3/GemR): PP2 (AG1879) (5 μM–10 μM) reversed gemcitabine resistance: (1) In AsPC-1/GemR, gemcitabine IC₅₀ decreased from ~150 nM (control) to ~30 nM (10 μM PP2); (2) Cell proliferation (MTT assay) was inhibited by ~50% (10 μM PP2, 72 h) in resistant sublines; (3) Apoptosis (Annexin V/PI staining) increased from ~5% (control) to ~35% (10 μM PP2, 48 h); (4) Western blot confirmed reduced p-Src (Tyr416), p-FAK (Tyr397), and p-ERK1/2 (Thr202/Tyr204) levels [2]

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/

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].

---

Recombinant Src family kinase activity assay: Recombinant human Lck, Fyn, Src, EGFR, or Abl kinase domains were incubated with reaction buffer (50 mM Tris-HCl pH 7.4, 10 mM MgCl₂, 1 mM DTT) containing 10 μM ATP (including [γ-³²P]ATP) and a Src-specific peptide substrate (sequence: KKEEEEYMMMM). PP2 (AG1879) was added at concentrations of 0.1 nM–1000 nM (solvent as control). The reaction mixture (25 μL) was incubated at 37°C for 30 minutes, then terminated by spotting 20 μL onto phosphocellulose filter paper. Filters were washed 3 times with 0.75% phosphoric acid to remove unincorporated ATP, and radioactivity was measured via liquid scintillation counting. Inhibition rates were calculated, and IC₅₀ values were determined by fitting data to a dose-response curve [1]

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].

---

T cell activation assay: Human peripheral blood T cells were isolated via density gradient centrifugation and resuspended in RPMI 1640 medium with 10% FBS. Cells (1×10⁶ cells/mL) were pretreated with PP2 (AG1879) (0 μM, 1 μM, 5 μM, 10 μM) for 1 hour, then stimulated with anti-CD3 (5 μg/mL) + anti-CD28 (2 μg/mL) for 24 hours. (1) IL-2 secretion: Culture supernatant was collected, and IL-2 concentration was measured via sandwich ELISA; (2) CD69 expression: Cells were stained with anti-CD69-PE antibody for 30 minutes, then analyzed via flow cytometry; (3) Signaling analysis: Cells were lysed, and Western blot was performed to detect p-Lck (Tyr394), p-Fyn (Tyr417), p-ZAP-70 (Tyr493), and total Lck/Fyn/ZAP-70 [1]
- Pancreatic adenocarcinoma cell proliferation and apoptosis assay: (1) Proliferation: AsPC-1/GemR/BxPC-3/GemR cells were seeded in 96-well plates (5×10³ cells/well), treated with PP2 (AG1879) (0 μM–10 μM) alone or combined with gemcitabine (0 nM–200 nM) for 72 hours. MTT reagent was added, and absorbance at 570 nm was measured to calculate cell viability and IC₅₀; (2) Apoptosis: Cells were treated with 10 μM PP2 (AG1879) for 48 hours, stained with Annexin V-FITC/PI, and analyzed via flow cytometry; (3) Signaling analysis: Cells were lysed after 2 hours of PP2 (AG1879) treatment, and Western blot was used to detect p-Src (Tyr416), total Src, p-FAK (Tyr397), p-ERK1/2 (Thr202/Tyr204), and β-actin [2]

Dissolved in 1% DMSO; 5 mg/kg; i.p. injection
SCID mice inoculated HT29 cells in the spleen Intracisternal administration of drugs was performed as previously described (Ueda et al., 1979). Mice were briefly anesthetized with isoflurane, and GRP (1 nmol, n = 9) or NMDA (1 nmol, n = 4; 5 nmol, n = 4; 10 nmol, n = 6; 20 nmol, n = 8; 40 nmol, n = 4) was injected i.cist. at a volume of 10 μL. Then, the number of scratching bouts, made with a hind paw, was counted after recording mice on a video for 30 min. To examine the involvement of trigeminal NMDA receptors in scratching behaviors, we injected D-AP5 (10.1 nmol, n = 8), CP101,606 (30.5 nmol, n = 8) or PP2 (1 nmol, n = 7) i.cist. 30 min before the injection of chloroquine into the cheek. For the GRPR antagonist experiment, RC-3095 (5 nmol) was i.cist. injected simultaneously with GRP (1 nmol, n = 5) or NMDA (20 nmol, n = 6) at a volume of 10 μL, and the number of scratching bouts over a 30-min period was counted. For chemically induced pain, capsaicin (98.2 nmol, n = 5) was injected intradermally into the left cheek at a volume of 10 μL, and the number of wipings with a fore limb was counted.[4]

---

Sprague-Dawley rats were exposed to transient (90 min) middle cerebral artery occlusion (MCAO) and evaluated after 1 day of survival. PP2 (1.5 mg/kg i.p.) or vehicle was given 30 min after MCAO. The lesions were examined with magnetic resonance imaging (MRI), tri-phenyl tetrazolium chloride (TTC) staining and the functional outcome was determined using neurological scoring according to Bederson et al. Acta Neurol Scand. 2004 Sep;110(3):175-9. https://pubmed.ncbi.nlm.nih.gov/15285775/

[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.

PP2 belongs to the pyrazolopyrimidine class of compounds. Its structure is pyrazolo[3,4-d]pyrimidine-4-amine, with tert-butyl and 4-chlorophenyl substituents at positions 1 and 3, respectively. It is a potent ATP-competitive Src family protein tyrosine kinase inhibitor. It has multiple functions, including as an EC 2.7.10.2 (non-specific protein tyrosine kinase) inhibitor, a β-adrenergic antagonist, and an anti-aging agent. PP2 is a pyrazolopyrimidine class of compounds, belonging to the aromatic amine class and also to the monochlorobenzene class of compounds. PP2 (AG1879) is a small molecule inhibitor with high selectivity for Src family kinases (Lck, Fyn, Src) and very low activity against non-Src kinases. It is widely used as a tool compound for studying Src family-mediated signaling pathways, especially in T cell activation (where Lck/Fyn are key regulators) [1].
In gemcitabine-resistant pancreatic adenocarcinoma cells, PP2 (AG1879) restores gemcitabine sensitivity by inhibiting Src kinase activation and downstream FAK/ERK signaling pathways, suggesting its potential as an adjuvant to overcome chemotherapy resistance in pancreatic cancer. However, it has not been developed for clinical use due to a lack of in vivo efficacy and toxicity data [2].
References [3] (focusing on AP23846, a different Src inhibitor) and [4] (focusing on phosphorylation of NMDA receptor GluN2B in pruritus) do not contain information related to PP2 (AG1879) [3][4].

C15H16CLN5

431.53

301.109

C, 59.70; H, 5.34; Cl, 11.75; N, 23.21

172889-27-9

4878

White to off-white solid powder

1.4±0.1 g/cm3

493.5±40.0 °C at 760 mmHg

214-216ºC

252.3±27.3 °C

0.0±1.3 mmHg at 25°C

1.676

3.22

1

4

2

21

364

0

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

PBBRWFOVCUAONR-UHFFFAOYSA-N

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)

1-(tert-butyl)-3-(4-chlorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine

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.

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

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.)