Zipalertinib (TAS6417; CLN-081; TAS-6417) is a novel, potent, orally bioavailable and selective covalent/irreversible EGFR inhibitor with anticancer activity. It acts by binding covalently to cysteine residue at 797 in the ATP-binding site of the EGFR hinge region. TAS6417 is an efficacious drug candidate for patients with NSCLC, with IC50 values ranging from 1.1-8.0 nM. TAS6417 inhibited EGFR with various exon 20 insertion mutations more potently than it inhibited the WT. TAS6417 inhibited EGFR phosphorylation and downstream molecules in NSCLC cell lines expressing EGFR exon 20 insertions, resulting in caspase activation. These characteristics led to marked tumor regression in vivo in both a genetically engineered model and in a patient-derived xenograft model. Furthermore, TAS6417 provided a survival benefit with good tolerability in a lung orthotopic implantation mouse model.

Physicochemical Properties

Molecular Formula	C23H20N6O
Molecular Weight	396.4445
Exact Mass	396.17
Elemental Analysis	C, 69.68; H, 5.09; N, 21.20; O, 4.04
CAS #	1661854-97-2
PubChem CID	117918742
Appearance	White to yellow solid powder
LogP	2.3
Hydrogen Bond Donor Count	2
Hydrogen Bond Acceptor Count	5
Rotatable Bond Count	3
Heavy Atom Count	30
Complexity	710
Defined Atom Stereocenter Count	1
SMILES	CC1=C[C@@H](CN2C1=C(C3=C(N=CN=C32)N)C4=CC5=CC=CC=C5N=C4)NC(=O)C=C
InChi Key	MKCYPWYURWOKST-INIZCTEOSA-N
InChi Code	InChI=1S/C23H20N6O/c1-3-18(30)28-16-8-13(2)21-19(15-9-14-6-4-5-7-17(14)25-10-15)20-22(24)26-12-27-23(20)29(21)11-16/h3-10,12,16H,1,11H2,2H3,(H,28,30)(H2,24,26,27)/t16-/m0/s1
Chemical Name	(S)-N-(4-amino-6-methyl-5-(quinolin-3-yl)-8,9-dihydropyrimido[5,4-b]indolizin-8-yl)acrylamide
Synonyms	Zipalertinib; TAS-6417; CLN-081; TAS6417; CLN081; TAS 6417;
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 Note: (1). This product requires protection from light (avoid light exposure) during transportation and storage.(2). Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture.
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 NSCLC cell lines containing EGFR exon 20 insertions, zipalertinib (TAS6417) suppresses EGFR phosphorylation and downstream molecules, resulting in caspase activation [1]. Targeting the most prevalent EGFR (exon 19 deletion and L858R), zipalertinib (TAS6417) works best against cells with the EGFR-T790M (1/2 generation TKI mutations) mutation [2]. Recombinant EGFR cysteine residue 797 is covalently modified by zipalertinib (TAS6417). Cell growth is inhibited by zipalertinib (TAS6417), which blocks the transmission of the EGFR signal. In intermittently driven NSCLC cells, EGFR exon 20 insertion causes cell abnormalities as well [1]. In EGFR-tagged NSCLC cell lines, zipalertinib (TAS6417) (0–10 μM) suppresses both EGFR signaling and cell growth, regardless of whether the prevalent mutation T790M is present or not [2].
ln Vivo	In an EGFR exon 20 insertion-driven tumor model, continuous tumor regression is produced in vivo by zipalertinib (TAS6417) (10–200 mg/kg). In NCI-H23 or NCI-H460 cells, TAS6417 inhibits bright EGFR in tumors but not WT EGFR in skin tissue [1]. Zipalertinib (TAS6417) has no effect on EGFR non-proliferative proliferation. At 20 mg/kg, zipalertinib (TAS6417) can significantly lower pEGFR and totally inhibit tumor growth. as a result, pAKT and pERK decreased at one hour, inhibition of inhibition was observed at six hours, and EGFR, ATK, and ERK phosphorylation was restored at the 24-hour mark [1]. TAS6417 (zipalertinib) 100 and 200 mg/kg/day
Cell Assay	Apoptosis analysis [2] Cell Types: PC 1]. -9, H1975, BID007, BID019, BEAS-2B cells. Tested Concentrations: 0-10μM. Incubation Duration: 24-48 hrs (hours). Experimental Results: Cell apoptosis was induced by inhibiting mutant EGFR.
Animal Protocol	Animal/Disease Models: Mice implanted with NCI-H1975 EGFR D770_N771insSVD xenografts [1]. Doses: 50 and 100 mg/kg. Route of Administration: po (po (oral gavage)) one time/day for 14 days. Experimental Results: Demonstrated significant tumor growth inhibition with treatment/control (T/C) ratios of 51% and 19%, respectively.
References	[1]. TAS6417, A Novel EGFR Inhibitor Targeting Exon 20 Insertion Mutations. Mol Cancer Ther. 2018 Aug;17(8):1648-1658. [2]. TAS6417/CLN-081 Is a Pan-Mutation-Selective EGFR Tyrosine Kinase Inhibitor with a Broad Spectrum of Preclinical Activity against Clinically Relevant EGFR Mutations. Mol Cancer Res. 2019 Nov;17(11):2233-2243.
Additional Infomation	Zipalertinib is an orally available selective inhibitor of a broad spectrum of epidermal growth factor receptor (EGFR) mutations, including EGFR exon 20 insertion mutations (EGFR Ex20ins; Ex20ins mutations), with potential antineoplastic activity. CLN-081 is also active against other EGFR mutations including exon 19 deletions (exon19del), L858R, and T790M, as well as the less common G719X, L861Q and S768I mutations. Upon administration, zipalertinib specifically and covalently binds to and inhibits a variety of EGFR mutations, with particularly high selectivity against EGFR Ex20ins, which prevents EGFR mutant-mediated signaling and leads to cell death in EGFR mutant-expressing tumor cells. Compared to some other EGFR inhibitors, CLN-081 may have therapeutic benefits in tumors with EGFR Ex20ins, as most EGFR mutant-selective inhibitors are not active against EGFR Ex20ins. This agent shows minimal activity against wild-type EGFR (wt EGFR), and does not cause dose-limiting toxicities that occur during the use of non-selective EGFR inhibitors, which also inhibit wt EGFR. EGFR, a receptor tyrosine kinase mutated in many tumor cell types, plays a key role in tumor cell proliferation and tumor vascularization.

Solubility Data

Solubility (In Vitro)

DMSO : ~22.73 mg/mL (~57.34 mM)

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.08 mg/mL (5.25 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 20.8 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: ≥ 2.08 mg/mL (5.25 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly. Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution. Solubility in Formulation 3: ≥ 2.08 mg/mL (5.25 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 20.8 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	2.5224 mL	12.6122 mL	25.2245 mL
	5 mM	0.5045 mL	2.5224 mL	5.0449 mL
	10 mM	0.2522 mL	1.2612 mL	2.5224 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.