1. PI3K/Akt/mTOR
  2. PI3K
  3. AMG319

AMG319 is a potent and selective PI3Kδ kinase inhibitor with IC50 of 18 nM.

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AMG319 Chemical Structure

AMG319 Chemical Structure

CAS No. : 1608125-21-8

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Customer Review

Based on 1 publication(s) in Google Scholar

Top Publications Citing Use of Products
  • Biological Activity

  • Protocol

  • Purity & Documentation

  • References

  • Customer Review

Description

AMG319 is a potent and selective PI3Kδ kinase inhibitor with IC50 of 18 nM.

IC50 & Target[1]

PI3Kδ

18 nM (IC50)

PI3Kγ

850 nM (IC50)

PI3Kβ

2.7 μM (IC50)

PI3Kα

33 μM (IC50)

Cellular Effect
Cell Line Type Value Description References
B cell IC50
8.6 nM
Compound: 3; AMG 319
Inhibition of cell proliferation of human B cells pre-incubated for 30 mins before anti-IgM/CD40L stimulation and measured after 72 hrs by [3H]thymidine incorporation assay
Inhibition of cell proliferation of human B cells pre-incubated for 30 mins before anti-IgM/CD40L stimulation and measured after 72 hrs by [3H]thymidine incorporation assay
[PMID: 27411843]
Sf9 IC50
18 nM
Compound: 3; AMG 319
Inhibition of polyHis tagged full length recombinant PI3Kdelta (unknown origin) expressed in Sf9 cells co-expressing p85 incubated for 20 mins by alpha screen assay
Inhibition of polyHis tagged full length recombinant PI3Kdelta (unknown origin) expressed in Sf9 cells co-expressing p85 incubated for 20 mins by alpha screen assay
[PMID: 27411843]
Sf9 IC50
2700 nM
Compound: 3; AMG 319
Inhibition of polyHis tagged full length recombinant PI3Kbeta (unknown origin) expressed in Sf9 cells co-expressing p85 incubated for 20 mins by alpha screen assay
Inhibition of polyHis tagged full length recombinant PI3Kbeta (unknown origin) expressed in Sf9 cells co-expressing p85 incubated for 20 mins by alpha screen assay
[PMID: 27411843]
Sf9 IC50
33000 nM
Compound: 3; AMG 319
Inhibition of polyHis tagged full length recombinant PI3Kalpha (unknown origin) expressed in Sf9 cells co-expressing p85 incubated for 20 mins by alpha screen assay
Inhibition of polyHis tagged full length recombinant PI3Kalpha (unknown origin) expressed in Sf9 cells co-expressing p85 incubated for 20 mins by alpha screen assay
[PMID: 27411843]
In Vitro

AMG319 inhibits PI3Kδ, PI3Kγ, PI3Kβ and PI3Kα with IC50s of 18 nM, 850 nM, 2.7 μM and 33 μM, respectively. AMG319, a compound with an IC50 of 16 nM in a human whole blood assay (HWB), excellent selectivity over a large panel of protein kinases, and a high level of in vivo efficacy as measured by two rodent disease models of inflammation. AMG319 has minimal CYP3A4/2D6 inhibition and does not inhibit CYPs (1A2, 2C8, 2C9, 2C19, 2E1, all >20 μM). There is no time dependent inhibition (TDI) against CYPs 3A4, 2D6, 1A2, and 2C9 nor CYP induction (3A4, 2D6, 1A2, 2B6) as measured in hepatocytes. AMG319 is clean in a hERG binding assay (>25 μM), and an Ames micronucleus test proved negative. AMG319 has minimal effects in a BSEP assay up to >200 μM[1].

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.

In Vivo

The study is performed to determine the correlation between biochemical coverage (i.e., pAKT) with functional activity in vivo. AMG319 achieves this coverage at the 3 mg/kg level, which also coveres the human whole blood assay (HWB) (CD-69) IC90 at trough for a full 24 h period. The lower doses 0.1, 0.3, and 1 mg/kg cover trough concentrations between the HWB IC50 and IC90 and evince partial efficacy. Similarly, the plasma concentration of AMG319 covers the IC90 at the 1 mg/kg dose of the mouse anti-IgM pAKT in vitro assay[1].

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.

Clinical Trial
Molecular Weight

385.40

Formula

C21H16FN7

CAS No.
Appearance

Solid

Color

Light yellow to yellow

SMILES

C[C@H](NC1=C2N=CNC2=NC=N1)C3=CC4=CC=C(F)C=C4N=C3C5=NC=CC=C5

Shipping

Room temperature in continental US; may vary elsewhere.

Storage
Powder -20°C 3 years
4°C 2 years
In solvent -80°C 2 years
-20°C 1 year
Solvent & Solubility
In Vitro: 

DMSO : 50 mg/mL (129.74 mM; Need ultrasonic; Hygroscopic DMSO has a significant impact on the solubility of product, please use newly opened DMSO)

Preparing
Stock Solutions
Concentration Solvent Mass 1 mg 5 mg 10 mg
1 mM 2.5947 mL 12.9735 mL 25.9471 mL
5 mM 0.5189 mL 2.5947 mL 5.1894 mL
View the Complete Stock Solution Preparation Table

* Please refer to the solubility information to select the appropriate solvent. Once prepared, please aliquot and store the solution to prevent product inactivation from repeated freeze-thaw cycles.
Storage method and period of stock solution: -80°C, 2 years; -20°C, 1 year. When stored at -80°C, please use it within 2 years. When stored at -20°C, please use it within 1 year.

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Mass (g) = Concentration (mol/L) × Volume (L) × Molecular Weight (g/mol)

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Concentration (start) × Volume (start) = Concentration (final) × Volume (final)

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In Vivo:

Select the appropriate dissolution method based on your experimental animal and administration route.

For the following dissolution methods, please ensure to first prepare a clear stock solution using an In Vitro approach and then sequentially add co-solvents:
To ensure reliable experimental results, the clarified stock solution can be appropriately stored based on storage conditions. As for the working solution for in vivo experiments, it is recommended to prepare freshly and use it on the same day.
The percentages shown for the solvents indicate their volumetric ratio in the final prepared solution. If precipitation or phase separation occurs during preparation, heat and/or sonication can be used to aid dissolution.

  • Protocol 1

    Add each solvent one by one:  10% DMSO    40% PEG300    5% Tween-80    45% Saline

    Solubility: ≥ 2.5 mg/mL (6.49 mM); Clear solution

    This protocol yields a clear solution of ≥ 2.5 mg/mL (saturation unknown).

    Taking 1 mL working solution as an example, add 100 μL DMSO stock solution (25.0 mg/mL) to 400 μL PEG300, and mix evenly; then add 50 μL Tween-80 and mix evenly; then add 450 μL Saline to adjust the volume to 1 mL.

    Preparation of Saline: Dissolve 0.9 g sodium chloride in ddH₂O and dilute to 100 mL to obtain a clear Saline solution.
  • Protocol 2

    Add each solvent one by one:  10% DMSO    90% (20% SBE-β-CD in Saline)

    Solubility: ≥ 2.5 mg/mL (6.49 mM); Clear solution

    This protocol yields a clear solution of ≥ 2.5 mg/mL (saturation unknown).

    Taking 1 mL working solution as an example, add 100 μL DMSO stock solution (25.0 mg/mL) to 900 μL 20% SBE-β-CD in Saline, and mix evenly.

    Preparation of 20% SBE-β-CD in Saline (4°C, storage for one week): 2 g SBE-β-CD powder is dissolved in 10 mL Saline, completely dissolve until clear.
In Vivo Dissolution Calculator
Please enter the basic information of animal experiments:

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Recommended: Prepare an additional quantity of animals to account for potential losses during experiments.
Please enter your animal formula composition:
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Recommended: Keep the proportion of DMSO in working solution below 2% if your animal is weak.
The co-solvents required include: DMSO, . All of co-solvents are available by MedChemExpress (MCE). , Tween 80. All of co-solvents are available by MedChemExpress (MCE).
Calculation results:
Working solution concentration: mg/mL
Method for preparing stock solution: mg drug dissolved in μL  DMSO (Stock solution concentration: mg/mL).
The concentration of the stock solution you require exceeds the measured solubility. The following solution is for reference only. If necessary, please contact MedChemExpress (MCE).
Method for preparing in vivo working solution for animal experiments: Take μL DMSO stock solution, add μL . μL , mix evenly, next add μL Tween 80, mix evenly, then add μL Saline.
 If the continuous dosing period exceeds half a month, please choose this protocol carefully.
Please ensure that the stock solution in the first step is dissolved to a clear state, and add co-solvents in sequence. You can use ultrasonic heating (ultrasonic cleaner, recommended frequency 20-40 kHz), vortexing, etc. to assist dissolution.
Purity & Documentation
References
Kinase Assay
[1]

A PI3K Alphascreen assay is used to measure the activity of a panel of four phosphoinositide 3-kinases: PI3Kα, PI3Kβ, PI3Kγ, and PI3Kδ. Enzyme reaction buffer is prepared using sterile water and 50 mM Tris-HCl, pH 7, 14 mM MgCl2, 2 mM sodium cholate, and 100 mM NaCl. 2 mM DTT is added fresh on the day of the experiment. The Alphascreen buffer is made using sterile water and 10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.10% Tween 20, and 30 mM EDTA. Then 1 mM DTT is added fresh on the day of the experiment. Compound source plates used for this assay are 384-well Greiner clear polypropylene plates containing test compounds at 5 mM and diluted 1:2 over 22 concentrations. Columns 23 and 24 contained only DMSO, as these wells comprised the positive and negative controls, respectively. Source plates are replicated by transferring 0.5 μL per well into 384-well Optiplates. Each PI3K isoform is diluted in enzyme reaction buffer to 2× working stocks. PI3Kα is diluted to 1.6 nM, PI3Kβ is diluted to 0.8 nM, PI3Kγ is diluted to 15 nM, and PI3Kδ is diluted to 1.6 nM. PI(4,5)P2 is diluted to 10 μM, and ATP is diluted to 20 μM. This 2× stock is used in the assays for PI3Kα and PI3Kβ. For assay of PI3Kγ and PI3Kδ, PI(4,5)P2 is diluted to 10 μM and ATP is diluted to 8 μM to prepare a similar 2× working stock. Alphascreen reaction solutions are made using beads from the anti-GST Alphascreen kit. Two 4× working stocks of the Alphascreen reagents are made in Alphascreen reaction buffer. In one stock, biotinylated-IP4 is diluted to 40 nM and streptavadin-donor beads are diluted to 80 μg/mL. In the second stock, PIP3-binding protein is diluted to 40 nM and anti-GST-acceptor beads are diluted to 80 μg/mL. The plates are incubated at room temperature for 30 min. Still in the dark, 10 μL/well of the acceptor bead solution is added to columns 1-24 of the plates. The plates are then incubated in the dark for 1.5 h. The plates are read on an Envision multimode plate reader using a 680 nm excitation filter and a 520-620 nm emission filter[1].

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

Cell Assay
[1]

B cells are purified from human peripheral blood mononuclear cells (PBMCs) by negative selection. Approximately 3×104 purified B cells per well are seeded into a 96-well plate. Compounds (e.g., AMG319) are dissolved in DMSO at a concentration of 10 mM, and a 10-point, 3-fold serial dilution of the compound is carried out in DMSO. Then 0.5 μL of compound is added to each well in duplicates so that the final DMSO concentration is 0.25% and the highest compound concentration is 10 μM. After preincubating for 30 min, B cells are treated with 2 μg/mL of anti-human IgM antibody plus 300 ng/mL human CD40L or 5 ng/mL human IL-4 plus 200 ng/mL of CD40L as a counterscreen to evaluate the off-target effects. The plates are incubated at 37°C and 5% CO2 for 72 h, then pulsed with 0.5 μCi per well 3H thymidine for 18 h, and B cells are collected to count the incorporation of 3H thymidine[1].

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

Animal Administration
[1]

Mice[1]
IgM membrane only homozygous transgenic mice (6- to 12-week-old female) are orally dosed with AMG319 or vehicle control (n=5 per group). At 15 min after treatment, mice are tail iv injected with 50 μg of Endotoxin-free FITC-labeled μ chain specific anti-IgM or PBS only control. Blood and spleen tissue are collected after 30 min of stimulation for drug concentration and B cell pAKT analysis via flow cytometry. Briefly, blood and dispersed splenocytes are fixed with BD Phosflow lyse/fix buffer, pelleted, and permeabilized with cold 90% MeOH. Cells are then stained with pAKT and Alexa-647 secondary and B220-Pacific Blue for FACS analysis. Stimulated B220+/anti-IgM FITC+ B cells are analyzed for pAKT levels with B220+/FITC-B cells from anti-IgM untreated mice serving as a control. Mice are maintained and experiments are performed.
Rats[1]
Female Lewis rats (N=8/dose group) are dosed po with AMG319 or vehicle (2% HPMC, 1% Pluronic F68, 10% Captisol, pH 2.0) once a day for 10 days at various doses. Two hours after the first dosing, 200 μL of PBS containing 60 μg of KLH is administered to each rat intravenously. Ten days after the KLH priming, rats are euthanized and blood is taken by cardiac puncture for the measurement of KLH specific IgG and IgM by ELISA.

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

References

Complete Stock Solution Preparation Table

* Please refer to the solubility information to select the appropriate solvent. Once prepared, please aliquot and store the solution to prevent product inactivation from repeated freeze-thaw cycles.
Storage method and period of stock solution: -80°C, 2 years; -20°C, 1 year. When stored at -80°C, please use it within 2 years. When stored at -20°C, please use it within 1 year.

Optional Solvent Concentration Solvent Mass 1 mg 5 mg 10 mg 25 mg
DMSO 1 mM 2.5947 mL 12.9735 mL 25.9471 mL 64.8677 mL
5 mM 0.5189 mL 2.5947 mL 5.1894 mL 12.9735 mL
10 mM 0.2595 mL 1.2974 mL 2.5947 mL 6.4868 mL
15 mM 0.1730 mL 0.8649 mL 1.7298 mL 4.3245 mL
20 mM 0.1297 mL 0.6487 mL 1.2974 mL 3.2434 mL
25 mM 0.1038 mL 0.5189 mL 1.0379 mL 2.5947 mL
30 mM 0.0865 mL 0.4325 mL 0.8649 mL 2.1623 mL
40 mM 0.0649 mL 0.3243 mL 0.6487 mL 1.6217 mL
50 mM 0.0519 mL 0.2595 mL 0.5189 mL 1.2974 mL
60 mM 0.0432 mL 0.2162 mL 0.4325 mL 1.0811 mL
80 mM 0.0324 mL 0.1622 mL 0.3243 mL 0.8108 mL
100 mM 0.0259 mL 0.1297 mL 0.2595 mL 0.6487 mL
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    Species cross-reactivity must be investigated individually for each product. Many human cytokines will produce a nice response in mouse cell lines, and many mouse proteins will show activity on human cells. Other proteins may have a lower specific activity when used in the opposite species.

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