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Purity: ≥98%
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.
| 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] |
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| 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 |
In mice, the bioavailability of a single oral dose of 20 mg/kg nazatinib was 71%[1]. Following an intravenous injection of 10 mg/kg, the plasma half-life (t1/2) of the compound in mice was 6.2 hours[1]. In rats, the oral bioavailability was 67% (at a 20 mg/kg dose), and the plasma t1/2 was 7.5 hours[1]. Nazatinib was 95% bound to human plasma, 93% in mouse plasma, and 91% in rat plasma[1]. The drug showed good tumor penetration, with a tumor/plasma concentration ratio of 5.4 four hours after oral administration in H1975 xenograft mice[2]. The drug exhibited high metabolic stability in human liver microsomes, with a half-life of 330 minutes[1].
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| Toxicity/Toxicokinetics |
In a 28-day repeated-dose toxicity study in rats, oral doses up to 100 mg/kg/day of nazatinib did not cause significant weight loss, hematological abnormalities, or changes in liver and kidney function parameters [1]. In a 14-day mouse toxicity study, no dose-limiting toxicities were observed at doses up to 150 mg/kg/day [1]. Mild rash (12%) and diarrhea (8%) were observed in mice treated with high doses (100 mg/kg/day), consistent with the toxic effects of EGFR inhibitors [2].
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| References |
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| Additional Infomation |
Nazartinib is being investigated in the clinical trial NCT03529084 (a phase III study of nalzatinib (EGF816) versus erlotinib/gefitinib as first-line treatment for EGFR-activating mutations in locally advanced/metastatic non-small cell lung cancer). Nazartinib is an oral, irreversible, third-generation mutation-selective epidermal growth factor receptor (EGFR) inhibitor with potential antitumor activity. After oral administration, nalzatinib covalently binds to and inhibits the activity of EGFR mutants (including the T790M EGFR mutant), thereby blocking EGFR-mediated signaling. This may induce EGFR-overexpressing tumor cell death and inhibit tumor growth. EGFR is a receptor tyrosine kinase that is mutated in various tumor cell types and plays a crucial role in tumor cell proliferation and tumor angiogenesis. EGF816 preferentially inhibits mutated forms of EGFR, including the secondary acquired resistance mutation T790M, and may offer therapeutic benefits to tumors with T790M-mediated resistance compared to other EGFR tyrosine kinase inhibitors. Due to its selectivity for EGFR mutants, its toxicity may be reduced compared to non-selective EGFR inhibitors that also inhibit wild-type EGFR. Nazartinib (EGF816, NVS-816) is a novel, potent, and non-affective covalent inhibitor of wild-type EGFR that inhibits oncogenic and resistant EGFR mutants [1][2]. Its mechanism of action involves covalent binding to the Cys797 residue of EGFR, irreversibly inhibiting the kinase activity of EGFR L858R, ex19del, and T790M mutants [1][2].
This compound was developed for the treatment of EGFR-mutant non-small cell lung cancer (NSCLC), including gefitinib/erlotinib resistant cases with the T790M mutation [1][2]. Its selectivity for mutant EGFR is much higher than that for wild-type EGFR. EGFR inhibitors can reduce the targeting toxicity associated with wild-type EGFR inhibition [1]. This drug has entered a phase II clinical trial for the treatment of advanced EGFR-mutant non-small cell lung cancer [2]. |
| Molecular Formula |
C26H31CLN6O2
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| Molecular Weight |
495.02
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| Exact Mass |
494.219
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| Elemental Analysis |
C, 63.09; H, 6.31; Cl, 7.16; N, 16.98; O, 6.46
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| CAS # |
1508250-71-2
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| Related CAS # |
Nazartinib S-enantiomer;1508256-20-9;Nazartinib mesylate;1508250-72-3
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| PubChem CID |
72703790
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| Appearance |
Off-white to yellow solid powder
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| Density |
1.3±0.1 g/cm3
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| Index of Refraction |
1.643
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| LogP |
3.23
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
5
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| Rotatable Bond Count |
6
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| Heavy Atom Count |
35
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| Complexity |
764
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| Defined Atom Stereocenter Count |
1
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| 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]
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| InChi Key |
IOMMMLWIABWRKL-WUTDNEBXSA-N
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| 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
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| Chemical Name |
N-[7-chloro-1-[(3R)-1-[(E)-4-(dimethylamino)but-2-enoyl]azepan-3-yl]benzimidazol-2-yl]-2-methylpyridine-4-carboxamide
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| Synonyms |
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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 |
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| 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) |
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| Solubility (In Vivo) |
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| 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.
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 | Recruitment | interventions | Conditions | Sponsor/Collaborators | Start Date | Phases |
| NCT03040973 | Recruiting | Drug: Capmatinib Drug: Nazartinib |
Advanced Solid Tumors Which Are cMET-dependent |
Novartis Pharmaceuticals | July 4, 2017 | Phase 2 |
| NCT03114319 | Recruiting | Drug: TNO155 in combination with EGF816 (nazartinib) Drug: TNO155 |
Advanced EGFRmutant Non Small Sell Lung Cancer (NSCLC), KRAS G12-mutant NSCLC, Esophageal SquamousCellCancer (SCC), Head/Neck SCC, Melanoma |
Novartis Pharmaceuticals | May 26, 2017 | Phase 1 |
| NCT02335944 | Terminated | Drug: Capmatinib Drug: Nazartinib |
Non Small Cell Lung Cancer | Novartis Pharmaceuticals | January 13, 2015 | Phase 1 Phase 2 |
Cellular activity of EGF816 in mutant and WT EGFR cell lines.Cancer Res.2016 Mar 15;76(6):1591-602. |
In vivo efficacy of EGF816 in EGFR-mutant–containing cell line xenograft models following 14 days (A, B, D) or 21 days (C, E, F) of once daily oral dosing.Cancer Res.2016 Mar 15;76(6):1591-602. |
EGF816 inhibits EGFR phosphorylation and downstream signaling pathways in vivo in H1975 (EGFR L858R/T790M) mouse xenograft model.Cancer Res.2016 Mar 15;76(6):1591-602. |
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