EGF816

Novel covalent inhibitor of mutant-selective EGFR CAS# 1508250-71-2

EGF816

Catalog No. BCC6428----Order now to get a substantial discount!

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Chemical structure

EGF816

3D structure

Chemical Properties of EGF816

Cas No. 1508250-71-2 SDF Download SDF
PubChem ID 72703790 Appearance Powder
Formula C26H31ClN6O2 M.Wt 495.02
Type of Compound N/A Storage Desiccate at -20°C
Synonyms Nazartinib
Solubility DMSO : ≥ 242 mg/mL (488.87 mM)
*"≥" means soluble, but saturation unknown.
Chemical Name N-[7-chloro-1-[(3R)-1-[(E)-4-(dimethylamino)but-2-enoyl]azepan-3-yl]benzimidazol-2-yl]-2-methylpyridine-4-carboxamide
SMILES CC1=NC=CC(=C1)C(=O)NC2=NC3=C(N2C4CCCCN(C4)C(=O)C=CCN(C)C)C(=CC=C3)Cl
Standard InChIKey IOMMMLWIABWRKL-WUTDNEBXSA-N
Standard InChI 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-/m1/s1
General tips For obtaining a higher solubility , please warm the tube at 37 ℃ and shake it in the ultrasonic bath for a while.Stock solution can be stored below -20℃ for several months.
We recommend that you prepare and use the solution on the same day. However, if the test schedule requires, the stock solutions can be prepared in advance, and the stock solution must be sealed and stored below -20℃. In general, the stock solution can be kept for several months.
Before use, we recommend that you leave the vial at room temperature for at least an hour before opening it.
About Packaging 1. The packaging of the product may be reversed during transportation, cause the high purity compounds to adhere to the neck or cap of the vial.Take the vail out of its packaging and shake gently until the compounds fall to the bottom of the vial.
2. For liquid products, please centrifuge at 500xg to gather the liquid to the bottom of the vial.
3. Try to avoid loss or contamination during the experiment.
Shipping Condition Packaging according to customer requirements(5mg, 10mg, 20mg and more). Ship via FedEx, DHL, UPS, EMS or other couriers with RT, or blue ice upon request.

Biological Activity of EGF816

DescriptionEGF816 is a novel, covalent mutant-selective EGFR inhibitor, with Ki and Kinact of 31 nM and 0.222 min−1 on EGFR(L858R/790M) mutant, respectively.In Vitro: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[1]. 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[2].In 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)[1]. 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[2].

References:
[1]. Lelais G, et al. Discovery of (R,E)-N-(7-Chloro-1-(1-[4-(dimethylamino)but-2-enoyl]azepan-3-yl)-1H-benzo[d]imidazol-2-yl)-2-methylisonicotinamide (EGF816), a Novel, Potent, and WT Sparing Covalent Inhibitor of Oncogenic (L858R, ex19del) and Resistant (T79 [2]. Jia Y, et al. EGF816 Exerts Anticancer Effects in Non-Small Cell Lung Cancer by Irreversibly and Selectively Targeting Primary and Acquired Activating Mutations in the EGF Receptor. Cancer Res. 2016 Mar 15;76(6):1591-602.

Protocol

Kinase Assay [1]
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 [2]
Cells are seeded 500 cells/well in solid white 384-well plates in maintenance media. Serial diluted compounds are transferred to cells. After 3 days, cell viability is measured by CellTiter-Glo. BaF3 cell viability is measured 2 days after compound treatment using Bright-Glo Luciferase Assay System. Luminescent readout is normalized to 0.1% DMSO-treated cells and empty wells. Five EGFR TKI-resistant cell lines are generated at Massachusetts General Hospital. MGH134, MGH121, MGH141, and MGH157 are derived from patients who developed resistance to erlotinib with acquired T790M mutation. MGH119-R is generated in vitro by treating MGH119 with gefitinib for a prolonged period. The sensitivity of EGF816 on these lines is tested.

Animal Administration [1]
Foxn1 nude mice bearing the H1975 tumors are randomized and used for efficacy studies. Compounds are formulated in 0.5% MC, 0.5% Tween 80 suspension formulation and administered by oral gavage at a dosing volume of 10 μL/g of the animal body weight. Animals in each group receive one oral dose of either vehicle (n=6) or the different test compounds (n=6 in each dose group). Plasma samples are collected for PK measurements at 30 min, 3 h, 7 h, and 24 h after last dose on day 5. Body weight is monitored daily, and the % change in body weight is calculated. Data are presented as percent body weight change from the day of treatment initiation. Tumor sizes are assessed three times during the efficacy study for 5 days. Tumor sizes are determined by using caliper measurements. Tumor volumes are calculated.

References:
[1]. Lelais G, et al. Discovery of (R,E)-N-(7-Chloro-1-(1-[4-(dimethylamino)but-2-enoyl]azepan-3-yl)-1H-benzo[d]imidazol-2-yl)-2-methylisonicotinamide (EGF816), a Novel, Potent, and WT Sparing Covalent Inhibitor of Oncogenic (L858R, ex19del) and Resistant (T79 [2]. Jia Y, et al. EGF816 Exerts Anticancer Effects in Non-Small Cell Lung Cancer by Irreversibly and Selectively Targeting Primary and Acquired Activating Mutations in the EGF Receptor. Cancer Res. 2016 Mar 15;76(6):1591-602.

EGF816 Dilution Calculator

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EGF816 Molarity Calculator

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Preparing Stock Solutions of EGF816

1 mg 5 mg 10 mg 20 mg 25 mg
1 mM 2.0201 mL 10.1006 mL 20.2012 mL 40.4024 mL 50.503 mL
5 mM 0.404 mL 2.0201 mL 4.0402 mL 8.0805 mL 10.1006 mL
10 mM 0.202 mL 1.0101 mL 2.0201 mL 4.0402 mL 5.0503 mL
50 mM 0.0404 mL 0.202 mL 0.404 mL 0.808 mL 1.0101 mL
100 mM 0.0202 mL 0.101 mL 0.202 mL 0.404 mL 0.505 mL
* Note: If you are in the process of experiment, it's necessary to make the dilution ratios of the samples. The dilution data above is only for reference. Normally, it's can get a better solubility within lower of Concentrations.

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Background on EGF816

Non-small cell lung cancer (NSCLC) patients with activating epidermal growth factor receptor (EGFR) mutations respond to EGFR tyrosine kinase inhibitors (TKI) but ultimately develop resistance to these therapies. The most common resistance mechanism is a second site gate-keeper mutation within exon 20 of EGFR (T790M). EGF816 is identified as a novel covalent inhibitor of mutant-selective epidermal growth factor receptor.

In vitro: EGF816 showed sustained inhibition of pEGFR, which is consistent with the irreversible binding mechanism of EGF816. EGF816 also performs exceptionally well in long term dosing studies providing durable responses in the preclinical models [1].

In vivo: EGF816 demonstrated strong in vivo tumor regressions in several EGFR activating and resistant tumor models. In all of the models EGF816 inhibited tumor growth dose-dependently and achieved regressions of established tumors at well tolerated doses [1].

Clinical trial: A phase I/II study of EGFRmut-TKI EGF816 is being conducted to investigate the efficacy in adult patients with EGFRmut solid malignancies.

Reference:
[1] Shailaja Kasibhatla, Jie Li, Celin Tompkins, Mei-Ting Vaillancourt, Jennifer Anderson, AnneMarie Culazzo Pferdekamper, Chun Li, Oliver Long, Mathew McNeill, Robert Epple, Debbie Liao, Eric Murphy, Steve Bender, Yong Jia, Gerald Lelais.  EGF816, a novel covalent inhibitor of mutant-selective epidermal growth factor receptor, overcomes T790M-mediated resistance in NSCLC. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1733. doi:10.1158/1538-7445.AM2014-1733

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References on EGF816

Discovery of (R,E)-N-(7-Chloro-1-(1-[4-(dimethylamino)but-2-enoyl]azepan-3-yl)-1H-benzo[d]imid azol-2-yl)-2-methylisonicotinamide (EGF816), a Novel, Potent, and WT Sparing Covalent Inhibitor of Oncogenic (L858R, ex19del) and Resistant (T790M) EGFR Mutants for the Treatment of EGFR Mutant Non-Small-Cell Lung Cancers.[Pubmed:27433829]

J Med Chem. 2016 Jul 28;59(14):6671-89.

Over the past decade, first and second generation EGFR inhibitors have significantly improved outcomes for lung cancer patients with activating mutations in EGFR. However, both resistance through a secondary T790M mutation at the gatekeeper residue and dose-limiting toxicities from wild-type (WT) EGFR inhibition ultimately limit the full potential of these therapies to control mutant EGFR-driven tumors and new therapies are urgently needed. Herein, we describe our approach toward the discovery of 47 (EGF816, nazartinib), a novel, covalent mutant-selective EGFR inhibitor with equipotent activity on both oncogenic and T790M-resistant EGFR mutations. Through molecular docking studies we converted a mutant-selective high-throughput screening hit (7) into a number of targeted covalent EGFR inhibitors with equipotent activity across mutants EGFR and good WT-EGFR selectivity. We used an abbreviated in vivo efficacy study for prioritizing compounds with good tolerability and efficacy that ultimately led to the selection of 47 as the clinical candidate.

EGF816 Exerts Anticancer Effects in Non-Small Cell Lung Cancer by Irreversibly and Selectively Targeting Primary and Acquired Activating Mutations in the EGF Receptor.[Pubmed:26825170]

Cancer Res. 2016 Mar 15;76(6):1591-602.

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. EGF816 is a novel, irreversible mutant-selective EGFR inhibitor that specifically targets EGFR-activating mutations arising de novo and upon resistance acquisition, while sparing wild-type (WT) EGFR. EGF816 potently inhibited the most common EGFR mutations L858R, Ex19del, and T790M in vitro, which translated into strong tumor regressions in vivo in several patient-derived xenograft models. Notably, EGF816 also demonstrated antitumor activity in an exon 20 insertion mutant model. At levels above efficacious doses, EGF816 treatment led to minimal inhibition of WT EGFR and was well tolerated. In single-dose studies, EGF816 provided sustained inhibition of EGFR phosphorylation, consistent with its ability for irreversible binding. Furthermore, combined treatment with EGF816 and INC280, a cMET inhibitor, resulted in durable antitumor efficacy in a xenograft model that initially developed resistance to first-generation EGFR inhibitors via cMET activation. Thus, we report the first preclinical characterization of EGF816 and provide the groundwork for its current evaluation in phase I/II clinical trials in patients harboring EGFR mutations, including T790M.

Description

Nazartinib (EGF816) is a novel, covalent mutant-selective EGFR inhibitor, with Ki and Kinact of 31 nM and 0.222 min−1 on EGFR(L858R/790M) mutant, respectively.

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