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Purity: ≥98%
ZSTK474 is a novel, potent and ATP-competitive pan-PI3K inhibitor (class I) with potential anticancer activity. IThe activity of all class I PI3K isoforms, including PI3Kα, PI3-Kβ, PI3-Kγ, PI3-Kδ, which have IC50 values of 16 nM, 44 nM, 49 nM, and 4.6 nM, respectively, is directly inhibited. This effectively slows the growth of tumor cells. In comparison to other PI3K and protein kinase classes, ZSTK474 exhibits high selectivity. Additionally, ZSTK474 prevented mature osteoclasts from resorbing bone. Studies revealed that 0.1μM ZSTK474 completely stopped osteoclasts from forming pits.
| Targets |
PI3Kδ (IC50 = 4.6 nM); PI3Kα (IC50 = 16 nM); PI3Kβ (IC50 = 44 nM); PI3Kγ (IC50 = 49 nM); Autophagy
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| ln Vitro |
ZSTK474 inhibits all four PI3K isoforms in an ATP-competitive manner, according to analysis of Lineweaver-Burk plots. The four PI3K isoforms' Ki values revealed that ZSTK474 inhibited the PI3K isoform most potently with a Ki of 1.8 nM, while the other isoforms were inhibited with Ki values that were 4–10 times higher. ZSTK474 should be thought of as a pan-PI3K inhibitor as a result. Using ZSTK474 and LY294002, we also calculated the IC50 values for inhibiting the four PI3K isoforms. The IC50 values of ZSTK474 (16, 44, 4.6 and 49 nM for PI3Kα, PI3Kβ, PI3Kδ and PI3Kγ, respectively) are shown to be consistent with the Ki values (6.7, 10.4, 1.8 and 11.7 nM for PI3Kα, PI3Kβ, PI3Kδ and PI3Kγ, respectively), which further supported the idea that ZSTK474 inhibits PI3Kδ most potently. ZSTK474 only moderately inhibits mTOR activity even at a concentration of 100 µM[1].
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| ln Vivo |
Treatment with ZSTK474 is examined in mice that have undergone MCAO at doses of 50, 100, 200, and 300 mg/kg. Since the 200 mg/kg dose produces significant improvement and no obvious toxic effects (P<0.01), mice are treated with ZSTK474 at a dose of 200 mg/kg/day daily for three post-MCAO days during the remaining experiments of this study. Neurological function is examined in mice suffered from MCAO followed by 24, 48, and 72 h of reperfusion. With the exception of the corner test, ZSTK474 group scores on neurological function are significantly higher than those of the control group[2].
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| Enzyme Assay |
The linear phase of each kinetic reaction is defined at the respective enzyme amount (0.05, 0.1, 0.12 and 1 µg/mL for PI3Kα, PI3β, PI3δ and PI3γ, respectively) and reaction time (20 min). PI3K activity is assayed at various concentrations of ATP (5, 10, 25, 50, 100 µM) in the presence of increasing concentrations of ZSTK474. A Lineweaver-Burk plot is developed by plotting 1/v (the inverse of v, where v is obtained by subtracting the HTRF signal of the kinase test sample from the HTRF signal of the minus-enzyme control) versus 1/[ATP] (the inverse of the ATP concentration). PIP2 is incubated with ATP without kinase for the minus-enzyme control. To determine the Ki value (inhibition constant) of ZSTK474 for each PI3K isoform, the slope of the respective Lineweaver-Burk plot is replotted against the ZSTK474 concentration. GraphPad Prism 4 is used to analyze and calculate the Ki values[1].
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| Cell Assay |
ZSTK474 is applied to cells for 48 hours at progressively higher concentrations. By measuring changes in total cellular protein using a sulforhodamine B assay, the inhibition of cell proliferation is evaluated. Apoptosis is assessed by chromatin condensation or by flow cytometry. For chromatin condensation assay, cells are stained with Hoechst 33342 and examined by fluorescence microscopy. Morphologic changes induced by ZSTK474, such as the condensation of chromatin, are indicative of apoptosis. For flow cytometry analysis, cells are harvested, washed with ice-cold PBS, and fixed in 70% ethanol. Cells are then washed twice with ice-cold PBS again, treated with RNase A (500 μg/mL) at 37 °C for 1 hour, and stained with propidium iodide (25 μg/mL). The DNA content of the cells is analyzed with a flow cytometer.
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| Animal Protocol |
Mice: Mice are randomly assigned to receive different doses of ZSTK474 (50, 100, 200, and 300 mg/kg) to determine the optimum dose; in our experiment, the optimum dose is 200 mg/kg. Then, mice are randomized into one of three groups: the ZSTK474-treated group (MCAO+ZSTK474), the control group (MCAO+PBS), or the group that received phosphate-buffered saline (PBS) as a sham operation. The mice in the ZSTK474-treated group receive the drug at the recommended dosage of 200 mg/kg. Mice are given an identical amount of PBS in the control and sham groups. For a total of three days, beginning six hours after the onset of focal ischemia and continuing daily for two additional days, all mice receive the same dose of medication via oral gavage.
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| References |
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| Additional Infomation |
ZSTK-474 is a triamino-1,3,5-triazine that is 1,3,5-triazine in which two of the hydrogens have been replaced by morpholin-4-yl groups while the third hydrogen has been replaced by a 2-(difluoromethyl)benzimidazol-1-yl group. It is an inhibitor of phosphatidylinositol 3-kinase. It has a role as an EC 2.7.1.137 (phosphatidylinositol 3-kinase) inhibitor and an antineoplastic agent. It is a member of morpholines, a member of benzimidazoles, a triamino-1,3,5-triazine and an organofluorine compound.
ZSTK474 has been used in trials studying the treatment of Neoplasms. PI3K Inhibitor ZSTK474 is an orally available, s-triazine derivative, ATP-competitive phosphatidylinositol 3-kinase (PI3K) inhibitor with potential antineoplastic activity. PI3K inhibitor ZSTK474 inhibits all four PI3K isoforms. Inhibiting the activation of the PI3K/AKT kinase (or protein kinase B) signaling pathway results in inhibition of tumor cell growth and survival in susceptible tumor cell populations. Dysregulated PI3K signaling may contribute to tumor resistance to a variety of antineoplastic agents. This agent does not induce apoptosis but rather induces strong G(0)/G(1) arrest, which might contribute to its favorable efficacy in tumor cells. |
| Molecular Formula |
C19H21F2N7O2
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|---|---|
| Molecular Weight |
417.4125
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| Exact Mass |
417.172
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| Elemental Analysis |
C, 54.67; H, 5.07; F, 9.10; N, 23.49; O, 7.67
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| CAS # |
475110-96-4
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| Related CAS # |
19545-26-7
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| PubChem CID |
11647372
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| Appearance |
White to off-white solid powder
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| Density |
1.6±0.1 g/cm3
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| Boiling Point |
640.3±65.0 °C at 760 mmHg
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| Flash Point |
341.0±34.3 °C
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| Vapour Pressure |
0.0±1.9 mmHg at 25°C
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| Index of Refraction |
1.711
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| LogP |
-0.21
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| Hydrogen Bond Donor Count |
0
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| Hydrogen Bond Acceptor Count |
10
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| Rotatable Bond Count |
4
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| Heavy Atom Count |
30
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| Complexity |
539
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| Defined Atom Stereocenter Count |
0
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| SMILES |
FC([H])(C1=NC2=C([H])C([H])=C([H])C([H])=C2N1C1=NC(=NC(=N1)N1C([H])([H])C([H])([H])OC([H])([H])C1([H])[H])N1C([H])([H])C([H])([H])OC([H])([H])C1([H])[H])F
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| InChi Key |
HGVNLRPZOWWDKD-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C19H21F2N7O2/c20-15(21)16-22-13-3-1-2-4-14(13)28(16)19-24-17(26-5-9-29-10-6-26)23-18(25-19)27-7-11-30-12-8-27/h1-4,15H,5-12H2
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| Chemical Name |
4-[4-[2-(difluoromethyl)benzimidazol-1-yl]-6-morpholin-4-yl-1,3,5-triazin-2-yl]morpholine
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| Synonyms |
ZSTK-474; ZSTK474; ZSTK 474
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO: ~21 mg/mL (~50.3 mM)
Water: <1 mg/mL (slightly soluble or insoluble) Ethanol: <1 mg/mL |
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| Solubility (In Vivo) |
Formulation 1: 0.5% hydroxyethyl cellulose: 30mg/mL
Formulation 2: suspended in 5% hydroxypropyl cellulose (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.3957 mL | 11.9786 mL | 23.9573 mL | |
| 5 mM | 0.4791 mL | 2.3957 mL | 4.7915 mL | |
| 10 mM | 0.2396 mL | 1.1979 mL | 2.3957 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
| NCT Number | Status | Interventions | Conditions | Sponsor/Collaborators | Start Date | Phases |
| NCT01280487 | Completed | Drug: ZSTK474 | Neoplasms | Zenyaku Kogyo Co., Ltd. | January 2011 | Phase 1 |
| NCT01682473 | Completed | Drug: ZSTK474 | Neoplasms | Zenyaku Kogyo Co., Ltd. | September 20, 2012 | Phase 1 |
ZSTK474 alleviates neurological deficits and reduces infarct volume in a model of ischemic reperfusion.J Neuroinflammation. 2016; 13(1): 192. |
ZSTK474 inhibits the expression of Iba-1 and GFAP at 72h after cerebral reperfusion injury.J Neuroinflammation. 2016; 13(1): 192. |
Double immunofluorescent staining for the microglial/macrophage marker Iba-1 and detrimental or restorative state markers.J Neuroinflammation. 2016; 13(1): 192. |
ZSTK474 inhibits pro-inflammatory cytokines and promotes anti-inflammatory cytokines.J Neuroinflammation. 2016; 13(1): 192. |
ZSTK474 reduces P-AKT and P-p70S6k protein levels. ZSTK474 suppresses phosphorylation of AKT and other downstream compounds.J Neuroinflammation. 2016; 13(1): 192. |
Summary diagram illustrates a hypothesis for how ZSTK474 exerts its effects on ischemic reperfusion injury.J Neuroinflammation. 2016; 13(1): 192. |
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