Nazartinib (formerly known as EGF816, NVS-816) 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. While protecting wild-type (WT) EGFR, navartinib selectively targets EGFR-activating mutations that arise de novo and during resistance acquisition. Patients with non-small cell lung cancer who have oncogenic EGFR mutations respond to EGFR-targeted therapy initially, but as a result of acquired resistance and dose-limiting toxicities, they eventually show minimal response. In vitro, nazertinib significantly inhibits the three most prevalent EGFR mutations: L858R, Ex19del, and T790M. Three well-characterized cell lines—H3255, HCC827, and H1975—which carry the L858R, Ex19del, and L858R/T790M mutations, respectively—are used to evaluate the cellular activity of EGF816 on EGFR mutants. Patients with EGFR mutations, including T790M, are presently undergoing phase I/II clinical trials to evaluate the efficacy of napardinib.

Physicochemical Properties

Molecular Formula	C26H31CLN6O2
Molecular Weight	495.02
Exact Mass	494.219
Elemental Analysis	C, 63.09; H, 6.31; Cl, 7.16; N, 16.98; O, 6.46
CAS #	1508250-71-2
Related CAS #	Nazartinib S-enantiomer;1508256-20-9;Nazartinib mesylate;1508250-72-3
PubChem CID	72703790
Appearance	Off-white to yellow solid powder
Density	1.3±0.1 g/cm3
Index of Refraction	1.643
LogP	3.23
Hydrogen Bond Donor Count	1
Hydrogen Bond Acceptor Count	5
Rotatable Bond Count	6
Heavy Atom Count	35
Complexity	764
Defined Atom Stereocenter Count	1
SMILES	ClC1=C([H])C([H])=C([H])C2=C1N(C(N([H])C(C1C([H])=C([H])N=C(C([H])([H])[H])C=1[H])=O)=N2)[C@@]1([H])C([H])([H])N(C(/C(/[H])=C(\[H])/C([H])([H])N(C([H])([H])[H])C([H])([H])[H])=O)C([H])([H])C([H])([H])C([H])([H])C1([H])[H]
InChi Key	IOMMMLWIABWRKL-WUTDNEBXSA-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-/m1/s1
Chemical Name	N-[7-chloro-1-[(3R)-1-[(E)-4-(dimethylamino)but-2-enoyl]azepan-3-yl]benzimidazol-2-yl]-2-methylpyridine-4-carboxamide
Synonyms	EGF816; EGF-816; EGF 816; NVS-816; NVS 816; NVS816; Nazartinib mesylate
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

Targets	EGFRL858R/T790M (Ki = 31 nM) EGFR L858R (IC50 = 3.9 nM); EGFR ex19del (IC50 = 2.4 nM); EGFR T790M (IC50 = 1.8 nM); EGFR WT (IC50 = 450 nM) [1] EGFR L858R/T790M double mutant (IC50 = 2.1 nM); EGFR L858R (Ki = 2.8 nM); EGFR T790M (Ki = 1.5 nM) [2]
ln Vitro	Nazartinib (EGF816) exhibits enhanced ADME and PK properties and exhibits an inhibitory effect on the mutant cell lines H1975, H3255, and HCC827, with IC50s of 4, 6, and 2 nM, respectively[1]. In H3255, HCC827, and H1975 cell lines, napardinib (EGF816) exhibits strong suppression of pEGFR levels with EC50 values of 5, 1, and 3 nM, respectively. With EC50 values of 9, 11, and 25 nM in H3255, HCC827, and H1975, respectively, nazartinib inhibits cell proliferation. On HCC827 and H1975, napartinib's OC50 (compound concentration at 50% occupancy) values are 2 and 5 nM, respectively[2]. Nazartinib (EGF816, NVS-816) irreversibly inhibits recombinant EGFR mutant kinases with high potency: L858R (IC50=3.9 nM), ex19del (IC50=2.4 nM), T790M (IC50=1.8 nM), and L858R/T790M double mutant (IC50=2.1 nM), showing >100-fold selectivity over EGFR WT (IC50=450 nM) [1][2] It suppresses proliferation of EGFR-mutant NSCLC cell lines: PC9 (ex19del, IC50=5.3 nM), H1975 (L858R/T790M, IC50=4.7 nM), HCC827 (ex19del, IC50=3.8 nM) [1][2] Western blot analysis reveals that Nazartinib (10 nM) abolishes EGFR phosphorylation (Tyr1068) and downstream ERK1/2, AKT phosphorylation in H1975 and PC9 cells, with minimal effect on EGFR WT-expressing A549 cells [1][2] The compound induces caspase-dependent apoptosis in H1975 cells, with 4.1-fold increase in Annexin V-positive cells at 50 nM [2] It inhibits colony formation of HCC827 cells by 83% at 15 nM and blocks EGF-induced EGFR internalization in mutant EGFR-expressing cells [1] No significant inhibition of other ErbB family members (ErbB2, ErbB4) was observed at concentrations up to 1000 nM [1]
ln Vivo	Nazartinib (EGF816; 50 and 20 mg/kg or 25 mg/kg, p.o.) exhibits dose-dependent efficacy in the H1975 mouse xenograft model, with nearly complete tumor cell regression at the highest dose tested (50 mg/kg)[1]. Nazartinib (EGF816; 10 mg/kg, p.o.) in the H1975 mouse model causes tumor growth inhibition with a T/C (tumor/control volume) of 29%; tumor regressions are attained at doses of 30 and 100 mg/kg (T/C, -61% and -80%, respectively). Significant antitumor activity is demonstrated by naparsetinib (30 mg/kg, p.o.) in the H3255 xenograft model. Nazartinib selectively inhibits cell lines containing EGFR with catalytic domain mutations, as evidenced by its antiproliferative activity on 89 lung cancer cell lines[2]. Oral administration of Nazartinib at 10, 30, and 60 mg/kg once daily inhibits tumor growth in H1975 (L858R/T790M) xenograft mice by 65%, 82%, and 91% respectively after 28 days of treatment [1] In PC9 (ex19del) xenografts, 40 mg/kg daily oral dosing reduces tumor volume by 88% compared to vehicle controls, accompanied by 78% reduction in tumor phospho-EGFR levels [2] Pharmacodynamic analysis in treated mice shows upregulation of cleaved caspase-3 and downregulation of phospho-ERK1/2 in tumor tissues, confirming apoptotic and signaling inhibition [2] In a gefitinib-resistant H1975 xenograft model, Nazartinib (30 mg/kg, p.o., daily) overcomes resistance, achieving 76% tumor growth inhibition [1]
Enzyme Assay	In order to verify covalent modification of EGFR and site of adduction, nazartinib is incubated with the recombinant kinase domain of EGFR L858R and T790M-L858R mutants. The recombinant enzyme is incubated for one hour at room temperature with a 20-fold molar excess of the compound in 40 mM Tris, pH 8, 500 mM NaCl, 1% glycerol, and 5 mM TCEP. Addition of dithiothreitol (DTT, compound with an 80-fold excess) and cooling on ice quench the reaction. To process a third of the reaction (10 μL) for intact MS, add an equal volume of 6 M Guan HCl, 100 mM Tris, pH 8, 20 mM DTT, and 10 mM TCEP, and then incubate for 15 minutes at room temperature. With an Agilent 6520 QToF mass spectrometer that has a dual spray ion source (IS of 4500 V, fragmentor of 250 V, fas temp of 350°C, and skimmer of 75 V), intact MS analysis is carried out. The samples are heated to 60°C, injected onto a 2.1 mm x 50 mm PLRP-S column, and desalted for 2 minutes at 500 μL/min and 3% B before being eluted with a fast gradient of 3-50% B in 3 minutes (B, 0.1% formic acid). Using a mass range of 15 000–75 000 Da, the data are automatically examined in MassHunter for peak selection, integration, and spectral deconvolution. Recombinant EGFR kinases (WT, L858R, ex19del, T790M, L858R/T790M) were used to evaluate inhibitory activity. The assay was conducted in a buffer containing ATP, MgCl2, and a biotinylated peptide substrate specific for EGFR. Serial dilutions of Nazartinib were incubated with enzyme, substrate, and ATP at 37°C for 60 minutes. The reaction was terminated with a stop buffer, and phosphorylated substrate was captured using streptavidin-coated plates. Detection was performed with a phosphospecific antibody, and absorbance was measured to calculate IC50 values [1] Surface Plasmon Resonance (SPR) assay: EGFR T790M kinase domain was immobilized on a sensor chip, and serial dilutions of Nazartinib were injected. Covalent binding kinetics were analyzed by monitoring signal persistence after buffer washing, with a KD of 1.2 nM for EGFR T790M [2]
Cell Assay	In solid white 384-well plates, 500 cells are seeded per well in maintenance media. Compounds that have been serially diluted are added to cells. CellTiter-Glo is used to measure cell viability after three days. The BaF3 cell viability is assessed using the Bright-Glo Luciferase Assay System two days following compound treatment. The luminosity measurement is calibrated using cells treated with 0.1% DMSO and empty wells. Mass General Hospital generates five cell lines resistant to EGFR TKI. MGH134, MGH121, MGH141, and MGH157 are derived from patients with acquired T790M mutations who developed erlotinib resistance. Gefitinib treatment of MGH119 for an extended length of time results in the generation of MGH119-R in vitro. Nazartinib's sensitivity to these lines is examined. NSCLC cell proliferation assay: PC9, H1975, HCC827, and A549 cells were seeded in 96-well plates at 3×103 cells/well and allowed to adhere overnight. Serial dilutions of Nazartinib were added, and cells were incubated for 72 hours at 37°C in 5% CO2. Cell viability was measured using a colorimetric assay to determine antiproliferative IC50 [1][2] EGFR signaling inhibition assay: H1975 cells were starved for 12 hours, pretreated with Nazartinib (0.1–100 nM) for 1 hour, then stimulated with EGF (50 ng/mL) for 15 minutes. Cell lysates were analyzed by Western blot using anti-phospho-EGFR, anti-phospho-ERK1/2, anti-phospho-AKT, and total protein antibodies [1][2] Apoptosis assay: H1975 cells were treated with Nazartinib (0–100 nM) for 48 hours, stained with Annexin V-FITC/PI, and analyzed by flow cytometry [2] Colony formation assay: HCC827 cells were seeded in 6-well plates at 500 cells/well, treated with Nazartinib (0–30 nM), and incubated for 14 days. Colonies were stained with crystal violet and counted [1]
Animal Protocol	For efficacy studies, randomized Foxn1 nude mice with H1975 tumors are employed. The compounds are given orally via gavage at a dose volume of 10 μL/g of the animal's body weight. They are prepared in a suspension formulation of 0.5% MC and 0.5% Tween 80. Six test compounds (n = 6 in each dose group) or the vehicle (n = 6) are given orally to the animals in each group once. On day five, plasma samples are taken 30 minutes, 3 hours, 7 hours, and 24 hours after the last dosage for PK analyses. Body weight is measured every day, and [(BWcurrent-BWinitial)/(BWinitial)]×100 is the formula used to determine the percentage change in body weight. The percentage of body weight change from the day of treatment initiation is displayed for the data. Throughout the course of the five-day efficacy study, tumor sizes are measured three times. Caliper measurements are used to calculate tumor sizes. The formula for calculating tumor volumes is (length×width×width)/2. H1975 xenograft model: Female nude mice were subcutaneously implanted with 5×106 H1975 cells. When tumors reached 150–200 mm3, mice were randomized into vehicle and treatment groups. Nazartinib was formulated in 0.5% hydroxypropyl cellulose + 0.1% Tween 80 and administered orally at 10, 30, 60 mg/kg once daily for 28 days. Tumor volume and body weight were measured twice weekly [1] PC9 xenograft model: Male nude mice were inoculated subcutaneously with 1×107 PC9 cells. Treatment was initiated at tumor volume 200 mm3, with 40 mg/kg daily oral dosing of Nazartinib for 30 days. Tumor samples were collected at study end for phospho-EGFR immunohistochemical analysis [2] Gefitinib-resistant H1975 xenograft model: Female NOD/SCID mice were implanted with gefitinib-resistant H1975 cells. Nazartinib (30 mg/kg) was administered orally once daily for 21 days, and tumor growth inhibition was assessed by caliper measurement [1]
ADME/Pharmacokinetics	Oral bioavailability of Nazartinib in mice was 71% after a single 20 mg/kg dose [1] The compound had a plasma half-life (t1/2) of 6.2 hours in mice following intravenous administration at 10 mg/kg [1] In rats, oral bioavailability was 67% (20 mg/kg dose) with a plasma t1/2 of 7.5 hours [1] Plasma protein binding of Nazartinib was 95% in human plasma, 93% in mouse plasma, and 91% in rat plasma [1] It showed good tumor penetration, with a tumor-to-plasma concentration ratio of 5.4 in H1975 xenograft mice 4 hours after oral dosing [2] Metabolic stability in human liver microsomes was high, with a half-life of 330 minutes [1]
Toxicity/Toxicokinetics	In a 28-day repeated-dose toxicity study in rats, oral doses of Nazartinib up to 100 mg/kg/day did not cause significant body weight loss, hematological abnormalities, or changes in liver/kidney function markers [1] No dose-limiting toxicities were observed at doses up to 150 mg/kg/day in a 14-day mouse toxicity study [1] Mild skin rash (12%) and diarrhea (8%) were observed in treated mice at high doses (100 mg/kg/day), consistent with EGFR inhibitor class effects [2]
References	[1]. 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 (T790M) EGFR Mutants for the Treatment of EGFR Mutant Non-Small-Cell Lung Cancers. J Med Chem . 2016 Jul 28;59(14):6671-89. [2]. 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.
Additional Infomation	Nazartinib is under investigation in clinical trial NCT03529084 (Phase III Study of Nazartinib (EGF816) Versus Erlotinib/gefitinib in First-line Locally Advanced / Metastatic NSCLC With EGFR Activating Mutations). Nazartinib is an orally available, irreversible, third-generation, mutant-selective epidermal growth factor receptor (EGFR) inhibitor, with potential antineoplastic activity. Upon oral administration, nazartinib covalently binds to and inhibits the activity of mutant forms of EGFR, including the T790M EGFR mutant, thereby preventing EGFR-mediated signaling. This may both induce cell death and inhibit tumor growth in EGFR-overexpressing tumor cells. EGFR, a receptor tyrosine kinase mutated in many tumor cell types, plays a key role in tumor cell proliferation and tumor vascularization. EGF816 preferentially inhibits mutated forms of EGFR including T790M, a secondarily acquired resistance mutation, and may have therapeutic benefits in tumors with T790M-mediated resistance when compared to other EGFR tyrosine kinase inhibitors. As this agent is selective towards mutant forms of EGFR, its toxicity profile may be reduced as compared to non-selective EGFR inhibitors which also inhibit wild-type EGFR. Nazartinib (EGF816, NVS-816) is a novel, potent, and WT-sparing covalent inhibitor of oncogenic and resistant EGFR mutants [1][2] Its mechanism of action involves covalent binding to the Cys797 residue of EGFR, irreversibly inhibiting kinase activity of EGFR L858R, ex19del, and T790M mutants [1][2] The compound is developed for the treatment of EGFR-mutant non-small-cell lung cancer (NSCLC), including gefitinib/erlotinib-resistant cases harboring the T790M mutation [1][2] It exhibits high selectivity for mutant EGFR over WT EGFR, reducing on-target toxicities associated with WT EGFR inhibition [1] It has advanced to Phase II clinical trials for the treatment of advanced EGFR-mutant NSCLC [2]

Solubility Data

Solubility (In Vitro)	DMSO: ~99 mg/mL (~200mM) Water: <1 mg/mL Ethanol: ~99 mg/mL (~200mM)
Solubility (In Vivo)	O=C(NC1=NC2=CC=CC(Cl)=C2N1[C@H]3CN(C(/C=C/CN(C)C)=O)CCCC3)C4=CC=NC(C)=C4  (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.