Molecular Weight (MW)

364.44

Formula

C21H24N4O2

CAS No.

663619-89-4

Storage

-20℃ for 3 years in powder form

-80℃ for 2 years in solvent

Solubility (In vitro)

DMSO: 12 mg/mL (32.9 mM)

Water:<1 mg/mL (slightly soluble or insoluble)

Ethanol: <1 mg/mL (slightly soluble or insoluble)

Solubility (In vivo)

1% DMSO+30% polyethylene glycol+1% Tween 80: 30 mg/mL

Chemical Name/Synonyms

9-(1-anilinoethyl)-7-methyl-2-morpholin-4-ylpyrido[1,2-a]pyrimidin-4-one; TGX221; TGX221; TGX 221

In Vitro

Kinase Assay: IC50 values are measured using a standard lipid kinase activity with PI as a substrate. (i)100 μM cold ATP is used instead of 10 μM, (ii) the DMSO concentration is 1%, and (iii) [γ-33P]ATP is used instead of [γ-32P]ATP. The TLC plates are quantified using a phosphorimager screen. The reported IC50 values are determined by non-linear regression analysis on the basis of at least three independent experiments repeated across multiple preparations of recombinant protein.

Cell Assay: For measurement of proliferation, PC3 cells are seeded in triplicate in 96-well culture plates and incubated overnight to allow cell attachment. The cells are incubated with TGX-221 for 24, 48, and 72 hours. At designated time intervals, cells are quantified by a crystal violet staining-based colorimetric assay. Briefly, cells are fixed by addition of 100 μl of 2.5% glutaraldehyde solution and incubated at room temperature for 30 minutes. Plates are washed three times by submersion in PBS solution. Plates are air-dried and stained by addition of 100 μL of 0.1% solution of crystal violet dissolved in deionized water and incubated for 20 minutes at room temperature, excess dye is removed by extensive washing with deionized water, and plates are air-dried prior to bound dye solubilization in 100 μL of 10% acetic acid. The optical density of dye extracts is measured directly in plates using a microplate reader at 570 nm. Drug concentration: 20 μM

The activity of TGX-221 against different isoforms is measured in an in vitro PI3K assay using multiple preparations of recombinant p85/p110. TGX-221 show slow potent to p110δ with IC50 of 211 nM. Furthermore, TGX-221 partially attenuates insulin-induced phosphorylation of Ser473 of PKB in J774.2 macrophage cells. TGX-221 inhibits platelet-ECC interaction, platelet aggregation and platelet-granulocyte binding in an extracorporeal circulation (ECC) model. A recent study shows that after treatment with TGX-221 (0.2, 2, and 20 μM), PC3 cells show inhibition of proliferation with a significant reduction of the activity of the p110β PI3K isoform.

In Vivo

As an anti-thrombotic agent, TGX-221 at doses 1 + 1 (49 %) and 3+3 (88 %) improves integrated blood flow over 30 minutes in a mouse model. In addition, Tail bleeding time (BT) (sec) increases with TGX-221 doses of 3 + 3 (median 1560) and 1 + 1 (1305) and mean renal BT (sec) also increases in all TGX-221 groups.

Animal model

FeCl3-induced arterial thrombosis in mice

Formulation & Dosage

Solubilized in 0% ethanol, 10% cremaphor, 10% N,N-dimethylacetamine, 70% distilled water; 0.1, 1, 3 mg/kg; i.v.

References

[1] Chaussade C, et al. Biochem J. 2007, 404(3), 449-458.; [2] Straub A, et al. Thromb Haemost. 2008, 99(3), 609-615.; [3] Lu XY, et al. Appl Microbiol Biotechnol. 2011, 89(5), 1423-1433.

Biochem J. 2007 Jun 15; 404( Pt 3): 449–458.