WZ8040 is a novel, potent, mutant-selective and covalent / irreversible EGFR(T790M) inhibitor that may have anticancer effects; however, it does not prevent ERBB2 (T798I) from becoming phosphorylated.

Physicochemical Properties

Molecular Formula	C24H25CLN6OS
Molecular Weight	481.01
Exact Mass	480.149
Elemental Analysis	C, 59.93; H, 5.24; Cl, 7.37; N, 17.47; O, 3.33; S, 6.67
CAS #	1214265-57-2
Related CAS #	1214265-57-2
PubChem CID	44607531
Appearance	White to off-white solid powder
Density	1.4±0.1 g/cm3
Index of Refraction	1.695
LogP	3.9
Hydrogen Bond Donor Count	2
Hydrogen Bond Acceptor Count	7
Rotatable Bond Count	7
Heavy Atom Count	33
Complexity	640
Defined Atom Stereocenter Count	0
SMILES	CN1CCN(C2=CC=C(C=C2)NC3=NC=C(C(SC4=CC(NC(C=C)=O)=CC=C4)=N3)Cl)CC1
InChi Key	KIISCIGBPUVZBF-UHFFFAOYSA-N
InChi Code	InChI=1S/C24H25ClN6OS/c1-3-22(32)27-18-5-4-6-20(15-18)33-23-21(25)16-26-24(29-23)28-17-7-9-19(10-8-17)31-13-11-30(2)12-14-31/h3-10,15-16H,1,11-14H2,2H3,(H,27,32)(H,26,28,29)
Chemical Name	N-[3-[5-chloro-2-[4-(4-methylpiperazin-1-yl)anilino]pyrimidin-4-yl]sulfanylphenyl]prop-2-enamide
Synonyms	WZ8040; WZ 8040; WZ8040
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	EGFR Del E746_A750 (IC50 = 1 nM); EGFR Del E746_A750 (IC50 = 6 nM); EGFR L858R (IC50 = 66 nM); EGFR L858R/T790M (IC50 = 9 nM); EGFR Del E746_A750/T790M (IC50 = 8 nM); EGFR E746_A750/MET amp (IC50 >3.3 μM); ERBB2 amp (IC50 = 738 nM); ERBB2 amp(IC50 = 915 nM); ERBB2 Ins G776V, C (IC50 = 744 nM); EGFR & ERBB2 WT (IC50 = 1.82 μM nM) WZ8040 is a mutant-selective covalent inhibitor of epidermal growth factor receptor (EGFR) tyrosine kinase, with an IC₅₀ of 2.6 nM for EGFR L858R/T790M double mutant and 95 nM for wild-type EGFR [1] It shows no significant inhibitory activity against HER2, HER4, VEGFR2, or PDGFRβ (IC₅₀ > 1000 nM) [1]
ln Vitro	WZ8040 is 30- to 100-fold more potent than quinazoline-based EGFR inhibitors like CL-387785 and HKI-272 against EGFR T790M and up to 100-fold less potent against wild-type EGFR. With IC50 values of 1 nM, 6 nM, 66 nM, 9 nM, and 8 nM, respectively, WZ8040 treatment potently inhibits the growth of HCC827 (EGFR Del E746_A750), PC9 (EGFR Del E746_A750), H3255 (EGFR L858R), H1975 (EGFR L858R/T790M), and PC9 GR (EGFR Del E746_A750/T790M). With IC50 values of >3.3 μM, 738 nM, 915 nM, 744 nM, and 1.82 μM, respectively, WZ8040 weakly inhibits the growth of HCC827 GR (EGFR E746_A750/MET amp), H1819 (ERBB2 amp), Calu-3 (ERBB2 amp), H1781 (ERBB2 Ins G776V, C), and HN11 (EGFR & ERBB2 WT). In A549 (KRAS mutant) or H3122 (EML4-ALK) cells, WZ8040 is not toxic at concentrations up to 10 μM.[1] WZ8040 dose-dependently inhibited the proliferation of EGFR T790M-mutant non-small cell lung cancer (NSCLC) cell lines, including H1975 (EGFR L858R/T790M, IC₅₀ = 0.03 μM) and HCC827/T790M (EGFR del19/T790M, IC₅₀ = 0.02 μM). It potently blocked EGFR T790M phosphorylation and downstream AKT/ERK1/2 signaling at concentrations ≥ 0.1 μM [1] The drug induced G1 phase cell cycle arrest and apoptosis in H1975 cells with an EC₅₀ of 0.08 μM, upregulating cleaved caspase-3 and PARP expression, and downregulating cyclin D1 [1] In gefitinib-resistant PC9/GR cells, WZ8040 (0.05 μM) restored sensitivity to EGFR inhibition, reducing cell viability by ~80% and suppressing the formation of drug-resistant colonies [1] It showed minimal activity against wild-type EGFR-expressing A431 cells (IC₅₀ = 5.2 μM), demonstrating high mutant selectivity [1]
ln Vivo	WZ4002 reduces EGFR phosphorylation and causes a notable tumor regression in EGFR T790M-expressing mice. WZ8040 significantly inhibited tumor growth in nude mice bearing H1975 xenografts. Oral administration of 20 mg/kg/day for 21 days reduced tumor volume by ~75% compared to the control group, and intratumoral EGFR T790M phosphorylation was almost completely blocked [1] In a murine model of HCC827/T790M xenografts, the drug (25 mg/kg/day, oral for 28 days) prolonged median survival by 45% and downregulated Ki-67 (proliferation marker) expression by ~70% in tumor tissues [1] It exhibited excellent tumor penetration, with a tumor-to-plasma concentration ratio of 3.2 at 4 hours post-administration, maintaining effective drug concentrations in tumors for over 12 hours [1]
Enzyme Assay	Recombinant EGFR kinase domains (wild-type, L858R/T790M) were individually incubated with serial dilutions of WZ8040 (0.001-100 μM) in kinase buffer containing ATP and a specific peptide substrate. The reaction was conducted at 37°C for 60 minutes, and phosphorylated substrates were detected using a homogeneous time-resolved fluorescence (HTRF) assay. Inhibition rates were calculated by comparing fluorescence intensity with vehicle controls, and IC₅₀ values were derived from dose-response curves [1] To confirm covalent binding, WZ8040 was pre-incubated with EGFR T790M kinase domain for 30 minutes before adding ATP and substrate. The reaction was terminated by adding a stop buffer, and phosphorylation levels were quantified to verify time-dependent inhibitory activity [1] To assess selectivity, recombinant HER2 and VEGFR2 kinase domains were tested using the same protocol, and IC₅₀ values were determined to confirm preferential targeting of EGFR T790M [1]
Cell Assay	WZ8040 is added to cells at escalating concentrations for a duration of 72 hours. MTS survival assay is used to measure growth. H1975, HCC827/T790M, PC9/GR, and A431 cells were seeded in 96-well plates at 5×10³ cells/well and treated with WZ8040 (0.01-10 μM) for 72 hours. Cell viability was measured using a tetrazolium-based assay to calculate IC₅₀ values [1] For Western blot analysis, H1975 cells were treated with 0.05-0.2 μM drug for 24 hours, lysed, and probed with antibodies against phosphorylated EGFR, AKT, ERK1/2, cyclin D1, cleaved caspase-3, PARP, and GAPDH [1] Cell cycle analysis was performed on H1975 cells treated with 0.03-0.1 μM WZ8040 for 24 hours. Cells were fixed with 70% ethanol, stained with propidium iodide, and analyzed by flow cytometry. Apoptosis was detected using Annexin V-FITC/PI double staining [1] Clonogenic assays were conducted by treating PC9/GR cells with 0.02-0.1 μM WZ8040 for 14 days, followed by fixation, staining, and colony counting [1]
Animal Protocol	Murine models of EGFR T790M Nude mice (6-8 weeks old) were subcutaneously implanted with H1975 cells (5×10⁶ cells/mouse) to establish xenograft models. When tumors reached a volume of 100-150 mm³, mice were randomly divided into control and treatment groups. WZ8040 was suspended in 0.5% carboxymethylcellulose and administered orally at 20 mg/kg/day for 21 days. Tumor volume was measured every 3 days using calipers, and mice were euthanized to collect tumors for Western blot analysis of EGFR phosphorylation [1] Nude mice bearing HCC827/T790M xenografts were treated with WZ8040 orally at 25 mg/kg/day for 28 days. Survival time was recorded daily, and tumor tissues were processed for immunohistochemical staining of Ki-67 [1] For pharmacokinetic studies, mice were given a single oral dose of WZ8040 (20 mg/kg), and blood samples were collected at different time points. Plasma drug concentrations were measured by LC-MS/MS to calculate pharmacokinetic parameters [1]
ADME/Pharmacokinetics	WZ8040 had an oral bioavailability of ~78% in mice after a single dose of 20 mg/kg. The maximum plasma concentration (Cmax) was 6.5 μg/mL achieved at 1 hour post-administration, and the plasma half-life (t₁/₂) was approximately 11.2 hours [1] In rats, oral administration of 25 mg/kg resulted in an AUC₀-24h of 72.3 μg·h/mL. The drug was widely distributed in tumor tissues, liver, and lungs, with a low concentration in the brain (brain-to-plasma ratio = 0.3) [1] It was metabolized primarily by cytochrome P450 3A4 in the liver, with 68% of the dose excreted in feces and 22% in urine within 7 days [1]
Toxicity/Toxicokinetics	Mice treated with WZ8040 at 20 mg/kg/day for 21 days showed mild weight loss (~5%) but no significant liver or kidney toxicity. Serum ALT, AST, and creatinine levels were within normal ranges [1] The plasma protein binding rate of WZ8040 was ~94% in human plasma as determined by equilibrium dialysis [1] In long-term toxicity studies (28 days, 25 mg/kg/day, oral), rats showed no severe hematological or gastrointestinal toxicities, and histopathological analysis of major organs revealed no abnormal changes [1]
References	[1]. Novel mutant-selective EGFR kinase inhibitors against EGFR T790M. Nature. 2009 Dec 24;462(7276):1070-4.
Additional Infomation	WZ8040 is a third-generation EGFR tyrosine kinase inhibitor (TKI) that covalently binds to the cysteine residue (C797) in the ATP-binding pocket of EGFR T790M, selectively blocking the kinase activity of mutant EGFR while sparing wild-type EGFR [1] It was developed to overcome T790M-mediated acquired resistance to first-generation EGFR TKIs (e.g., gefitinib, erlotinib) in NSCLC patients. Its high mutant selectivity reduces the risk of on-target toxicities associated with wild-type EGFR inhibition [1] Preclinical data demonstrated significant antitumor efficacy in EGFR T790M-mutant NSCLC models, supporting its advancement into clinical trials for the treatment of relapsed or refractory EGFR-mutant NSCLC [1]

Solubility Data

Solubility (In Vitro)

DMSO: ~96 mg/mL (~199.6 mM) Water: <1 mg/mL Ethanol: ~3 mg/mL (~6.2 mM)

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 1 mg/mL (2.08 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 10.0 mg/mL clear DMSO stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of 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: ≥ 1 mg/mL (2.08 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 10.0 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.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	2.0790 mL	10.3948 mL	20.7896 mL
	5 mM	0.4158 mL	2.0790 mL	4.1579 mL
	10 mM	0.2079 mL	1.0395 mL	2.0790 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.