Nazartinib S-enantiomer (EGF816) 1508256-20-9_EGFR_JAK / STAT_Signaling Pathways_Products

Nazartinib S-enantiomer is the S-isomer of Nazartinib which is a novel, covalent/irreversible, mutant-selective EGFR inhibitor with Ki and Kinact of 31 nM and 0.222 min−1 on EGFR(L858R/790M) mutant, respectively. Nazartinib specifically targets EGFR-activating mutations arising de novo and upon resistance acquisition, while sparing wild-type (WT) EGFR. Non-small cell lung cancer patients carrying oncogenic EGFR mutations initially respond to EGFR-targeted therapy, but later elicit minimal response due to dose-limiting toxicities and acquired resistance.Nazartinib potently inhibits the most common EGFR mutations L858R, Ex19del, and T790M in vitro. The cellular activity of EGF816 on EGFR mutants are assessed using three well-characterized cell lines, H3255, HCC827, and H1975, which harbor the L858R, Ex19del, and L858R/T790M mutations, respectively. Nazartinib is currently under clinical evaluation (phase I/II clinical trials) in patients harboring EGFR mutations, including T790M.

Physicochemical Properties

Molecular Formula

C₂₆H₃₁CLN₆O₂

Molecular Weight

495.02

Exact Mass

494.219

CAS #

1508256-20-9

Related CAS #

Nazartinib;1508250-71-2;Nazartinib mesylate;1508250-72-3

PubChem CID

118223934

Appearance

White to off-white solid powder

Density

1.3±0.1 g/cm3

Index of Refraction

1.643

LogP

3.23

Hydrogen Bond Donor Count

Hydrogen Bond Acceptor Count

Rotatable Bond Count

Heavy Atom Count

Complexity

764

Defined Atom Stereocenter Count

SMILES

O=C(C1=CC(C)=NC=C1)NC2=NC3=CC=CC(Cl)=C3N2[C@@H]4CN(C(/C=C/CN(C)C)=O)CCCC4

InChi Key

IOMMMLWIABWRKL-OEMHODTASA-N

InChi Code

InChI=1S/C26H31ClN6O2/c1-18-16-19(12-13-28-18)25(35)30-26-29-22-10-6-9-21(27)24(22)33(26)20-8-4-5-15-32(17-20)23(34)11-7-14-31(2)3/h6-7,9-13,16,20H,4-5,8,14-15,17H2,1-3H3,(H,29,30,35)/b11-7+/t20-/m0/s1

Chemical Name

N-[7-chloro-1-[(3S)-1-[(E)-4-(dimethylamino)but-2-enoyl]azepan-3-yl]benzimidazol-2-yl]-2-methylpyridine-4-carboxamide

Synonyms

EGF816 S-enantiomer; EGF-816 S-enantiomer; EGF 816 S-enantiomer; NVS-816 S-enantiomer; NVS 816; NVS816; Nazartinib S-enantiomer

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

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

ln Vitro

In vitro activity: EGF816 has inhibitory effect on the mutant cell lines with IC50s of 4, 6, 2 nM in H1975, H3255, and HCC827, respectively, and demonstrates improved ADME and PK properties. EGF816 shows potent inhibition of pEGFR levels in H3255, HCC827, and H1975 cell lines with EC50 values of 5, 1, and 3 nM, respectively. EGF816 inhibits cell proliferation, with EC50 values of 9, 11, and 25 nM in H3255, HCC827, and H1975, respectively. EGF816 has an OC50 (compound concentration at 50% occupancy) value of 2 and 5 nM on HCC827 and H1975, respectively.

Kinase Assay: Recombinant kinase domain of EGFR L858R and T790M-L858R mutants are incubated with EGF816 to confirm covalent modification of EGFR and site of adduction. Recombinant enzyme is incubated at room temperature with a 20-fold molar excess of compound in 40 mM Tris, pH 8, 500 mM NaCl, 1% glycerol, 5 mM TCEP for 1 h. The reaction is quenched by addition of dithiothreitol (DTT, 80-fold excess to compound) and transfer to ice. A third of the reaction (10 μL) is processed for intact MS by adding an equal volume of 6 M Guan HCl, 100 mM Tris, pH 8, 20 mM DTT, 10 mM TCEP and incubating at room temperature for 15 min. Intact MS analysis is performed on an Agilent 6520 QToF mass spectrometer equipped with a dual spray ion source (IS of 4500 V, fragmentor of 250 V, fas temp of 350°C, and skimmer of 75 V). The samples are injected onto a PLRP-S column (2.1 mm × 50 mm), heated to 60°C, and desalted for 2 min at 500 μL/min and 3% B prior to elution with a fast gradient of 3-50% B in 3 min (B, 0.1% formic acid). The data are analyzed in MassHunter for automatic peak selection, integration, and spectral deconvolution with a mass range of 15 000-75 000 Da.

Cell Assay: H1975, H3255, HCC827, A431, and HaCaT cells are maintained in RPMI media supplemented with antibiotics and 10% FBS, maintained in a 37°C, 5% CO2 humidified incubator. After an overnight incubation in 384-well plates, serial diluted compounds are transferred to cells and incubated for 3 hours. HaCaT cells are stimulated with 10 ng/mL EGF (50 ng/mL EGF for A431) for 5 minutes. Cells are lysed in 1% Triton X-100 buffer containing protease and phosphatase inhibitors. Lysates are analyzed by sandwich ELISA utilizing goat anti-EGFR capture antibody, anti-phospho-EGFR(Y1173), and anti-rabbit HRP. Signal is measured by chemiluminescent detection.

ln Vivo

In H1975 mouse xenograft model, EGF816 (50 and 20 mg/kg or 25 mg/kg, p.o.) demonstrates dose-dependent efficacy with near complete tumor cells regression at the highest dose tested (50 mg/kg). In H1975 mouse model, EGF816 (10 mg/kg, p.o.) induces tumor growth inhibition with a T/C (tumor/control volume) of 29%, and when doses are 30 and 100 mg/kg, tumor regressions are achieved (T/C, −61% and −80%, respectively). In the H3255 xenograft model, EGF816 (30 mg/kg, p.o.) shows significant antitumor activity. Antiproliferative activity of EGF816 on 89 lung cancer cell lines indicates that EGF816 selectively inhibits cell lines containing EGFR with catalytic domain mutations.

Animal Protocol

Dissolved in 5% ethanol, 25% PEG300, and 70% D5W (5% dextrose in water); 50 mg/kg; Oral administration.

balb/c mice and male Wistar rats

References

Cancer Res.2016 Mar 15;76(6):1591-602;J Med Chem.2016 Jul 28;59(14):6671-89.

Solubility Data

Solubility (In Vitro)

DMSO: 99 mg/mL (200mM)

Water:<1 mg/mL

Ethanol:99 mg/mL (200mM)

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.75 mg/mL (5.56 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (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 27.5 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

Solubility in Formulation 2: ≥ 2.75 mg/mL (5.56 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 27.5 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

(Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	2.0201 mL	10.1006 mL	20.2012 mL
	5 mM	0.4040 mL	2.0201 mL	4.0402 mL
	10 mM	0.2020 mL	1.0101 mL	2.0201 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.

PeptideDB

Nazartinib S-enantiomer (EGF816) 1508256-20-9

CAS No.: 1508256-20-9

Physicochemical Properties

Biological Activity

Solubility Data