ASK120067 (Limertinib) is an irreversible third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor that selectively targets EGFR mutants, including the T790M resistance mutation and sensitizing mutations (exon19del, L858R). In enzyme assays, it inhibits EGFR^L858R/T790M with an IC₅₀ of 0.3 nM, EGFR^T790M with an IC₅₀ of 0.5 nM, EGFR^exon19del with an IC₅₀ of 0.5 nM, and wild-type EGFR (EGFR^WT) with an IC₅₀ of 6 nM [1].

EGFR L858R/T790M and EGFR T790M resistant mutants (IC50 values: 0.3 nM and 0.5 nM, respectively) as well as the EGFRexon19del sensitizing mutant (IC50= 0.5 nM) were both successfully suppressed by limertinib in an in vitro kinase experiment. EGFR wild-type (EGFRWT) is the target of ligertinib at 50 nM [1]. When applied to mutant EGFR NSCLC cells, incadratinib shows strong antiproliferative action with IC50 values of 12 nM, 6 nM, and HCC827 against NCI-H1975 (T790M mutation), PC-9, and HCC827 2 nM cells (sensitizing mutation). It also selectively suppresses the development of EGFR mutant cell lines. Nonetheless, in A431, LoVo, and A549 cells (EGFRWT), it demonstrates mild to moderate anti-growth action, with IC50 values ranging from 338 nM to 1541 nM [1]. Even at low dosages (0.1-1 nM), mirtertinib (0.1-100 nM) suppresses the phosphorylation of EGFR tyrosine residue 1068 and its downstream signaling proteins AKT and ERK in NCI-H1975 cells (EGFRL858R/T790M). Moreover, at dosages of 10 to 100 nM, limertinib suppresses p-EGFR in EGFR WT A431 cells along with p-Akt and p-erk [1].

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- ASK120067 potently inhibits the proliferation of NSCLC cell lines harboring EGFR mutations: NCI-H1975 (EGFR^L858R/T790M) IC₅₀ = 12 ± 4 nM, PC-9 (EGFR^exon19del) IC₅₀ = 6 ± 3 nM, HCC827 (EGFR^exon19del) IC₅₀ = 2 ± 2 nM [1].
In contrast, it shows weaker activity against cells expressing wild-type EGFR: A431 IC₅₀ = 388 ± 158 nM, LoVo IC₅₀ = 1916 ± 1126 nM, A549 IC₅₀ = 1541 ± 359 nM [1].
- In NCI-H1975 cells, ASK120067 dose-dependently inhibits phosphorylation of EGFR (Tyr1068), AKT (Ser473) and ERK (T202/Y204) as early as 0.1 nM, with near-complete inhibition at 1–10 nM, comparable to or more potent than osimertinib [1].
In A431 cells (EGFR^WT), inhibition of p-EGFR is only partial even at 10–100 nM [1].
- ASK120067 induces apoptosis in NCI-H1975 and PC-9 cells in a dose- and time-dependent manner, as measured by Annexin V/PI staining and increased levels of cleaved PARP and cleaved caspase-3 [1].
- In PC-9 cells, ASK120067 similarly inhibits p-EGFR and downstream signaling, and induces apoptosis [1].
- Acquired resistance to ASK120067 in NCI-H1975 cells (67R cells) is associated with hyperphosphorylation of Ack1 (Tyr284) without increased total Ack1 protein [1].
This is confirmed in both cultured cells and xenograft tumors [1].
- Ectopic expression of Ack1 in parental NCI-H1975 cells reduces sensitivity to ASK120067, while knockdown of Ack1 in 67R cells partially restores sensitivity [1].
Combination of ASK120067 with Ack1 inhibitors (AIM-100, dasatinib, bosutinib) synergistically inhibits proliferation (combination index <0.5) and restores apoptosis in resistant cells [1].
- Mechanistically, resistant cells show sustained p-AKT and downregulation of pro-apoptotic BIM mRNA and protein [1].
Combination treatment with ASK120067 and an Ack1 inhibitor suppresses p-AKT and upregulates BIM, leading to enhanced apoptosis [1].
- ASK120067 also inhibits phosphorylation of EGFR and downstream signaling in PC-9 cells [1].
- In osimertinib-resistant cells (AZDR), Ack1 is also hyperphosphorylated, and combinations of ASK120067 with Ack1 inhibitors show synergistic growth inhibition [1].

Limertinib (oral gavage; 5-20 mg/kg; once daily; 21 days) strongly suppressed tumor development, with a tumor growth inhibition (TGI) rate of 85.7%, and administration of 10 mg/kg Limertinib resulted in a dramatic tumor shrinkage TGI rate It is 99.3% and has similar efficacy to Osimertinib [1].

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- In an NCI-H1975 (EGFR^L858R/T790M) xenograft model, oral administration of ASK120067 once daily for 21 days at 1, 5, and 10 mg/kg induces dose-dependent tumor growth inhibition (TGI) of 85.7%, and 99.3% (10 mg/kg), comparable to osimertinib (10 mg/kg, TGI 99.3%) [1].
No body weight loss is observed [1].
- Immunohistochemistry of NCI-H1975 tumors after treatment shows significant reduction in p-EGFR and p-AKT [1].
- In a PC-9 (EGFR^exon19del) xenograft model, ASK120067 (5 and 10 mg/kg, qd × 28 days) yields TGI of 86.0% and 93.0% [1].
- In an A431 (EGFR^WT) xenograft model, ASK120067 at 5 and 10 mg/kg shows weak efficacy (TGI <40% and 61.8%, respectively), less than osimertinib (82.9% at 10 mg/kg) [1].
- In a patient-derived xenograft (PDX) model harboring EGFR^L858R/T790M (LU1868), ASK120067 at 3, 10, and 20 mg/kg (qd × 21 days) induces TGI of 56.8%, 76.7%, and 85.2%, respectively [1].
IHC analysis reveals decreased p-EGFR and Ki-67 in tumors [1].
- A proof-of-concept clinical case: a 74-year-old female with stage IV EGFR^exon19del NSCLC who progressed on icotinib (T790M-positive) was treated with ASK120067 40 mg once daily in a phase I trial [1].
After 6 weeks, a CT scan showed near-complete disappearance of the lung tumor, and the response lasted >1 year [1].
- In an ASK120067-resistant xenograft model (67R cells), combination therapy with ASK120067 (5 mg/kg) and dasatinib (25 mg/kg) (qd × 21 days) significantly suppresses tumor growth compared to either monotherapy, accompanied by reduced p-AKT and increased TUNEL-positive apoptotic cells [1].

- Kinase inhibitory activity of ASK120067 was determined using an enzyme-linked immunosorbent assay (ELISA)-based kinase assay [1].
Recombinant EGFR proteins (EGFR^exon19del, EGFR^L858R/T790M, EGFR^T790M, and EGFR^WT) were incubated with serial dilutions of ASK120067, and kinase activity was measured following previously described protocols [1].
IC₅₀ values were calculated from at least three independent experiments [1].
- Selectivity profiling against a panel of 258 kinases was performed using a commercial kinase profiler platform at a concentration of 100 nM [1].
ASK120067 exhibited a favorable selectivity profile, with potent inhibition primarily against mutant EGFR [1].

- Cell proliferation assay (SRB): Cells were seeded in 96-well plates, cultured overnight, and treated with increasing concentrations of ASK120067 for 72 h [1].
Cell viability was assessed using the sulforhodamine B (SRB) colorimetric assay [1].
IC₅₀ values were calculated from dose-response curves [1].
- Western blot analysis: Cells were lysed in SDS sample buffer, heated, and proteins separated by SDS-PAGE, then transferred to nitrocellulose membranes [1].
Membranes were blocked with 5% milk-TBST and incubated with primary antibodies against p-EGFR (Tyr1068), EGFR, p-ERK, ERK, p-AKT (Ser473), AKT, caspase-3, cleaved caspase-3, PARP, BIM, p-Ack1 (Tyr284), Ack1, and loading controls (β-tubulin, β-actin, GAPDH) [1].
After washing, membranes were incubated with HRP-conjugated secondary antibodies and detected [1].
- Apoptosis assay (flow cytometry): Cells were treated with ASK120067 for indicated times, then stained with Annexin V-FITC and propidium iodide (PI) using an apoptosis detection kit [1].
Stained cells were analyzed by flow cytometry [1].
- Apoptosis assay (TUNEL): For tumor tissues, apoptosis was detected using an In Situ Cell Death Detection Kit (POD) following the manufacturer's protocol [1].
Paraffin-embedded sections were deparaffinized, treated with proteinase K, and incubated with TUNEL reaction mixture [1].
Labeled cells were visualized with DAB and counterstained with hematoxylin [1].
- Generation of resistant cells: NCI-H1975 cells were continuously exposed to increasing concentrations of ASK120067 or osimertinib starting from 5 nM, escalating stepwise every 2-3 passages up to 1 μM [1].
The resulting resistant cells (67R for ASK120067, AZDR for osimertinib) were maintained in 1 μM of the respective compound [1].
- Genetic analysis: Whole genome sequencing (WGS) was performed on parental, 67R, and AZDR cells [1].
DNA quality was assessed by qPCR, and sequencing data were analyzed [1].
- Quantitative RT-PCR: Total RNA was extracted from cells using a Cell to cDNA kit [1].
mRNA expression of BIM and GAPDH was quantified by real-time PCR with SYBR Green [1].
Primer sequences: BIM forward 5'-TGGGTATGCCTGCCACATTTC-3', reverse 5'-CCACGTTTTTGACGATGGAGA-3'; GAPDH forward 5'-CCACCCATGGCAAATTCCATGGCA-3', reverse 5'-TCTAGACGGCAGGTCAGGTCCACC-3' [1].
- Knockdown of Ack1: Short hairpin RNA (shRNA) targeting Ack1 was used to knock down Ack1 expression in 67R cells [1].
Effects on proliferation and signaling were assessed [1].
- Combination index analysis: Synergistic effects of drug combinations were evaluated using the combination index (CI) method; CI < 0.5 indicates strong synergy [1].

Animal/Disease Models: BALB/cA nude mice[1]
Doses: 5-20 mg/kg
Route of Administration: po (oral gavage); 5-20 mg/kg; one time/day; 21 days
Experimental Results: Well tolerated by animals, no observed Weight loss demonstrated profound and selective antitumor efficacy and diminished TGI rates. Dramatically inhibits the phosphorylation of EGFR L858R/T790M and AKT in tumor tissues.

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- NCI-H1975, PC-9, and A431 xenografts: Tumor cells (5×10⁶) were subcutaneously injected into the right flanks of BALB/cA nude mice [1].
After one passage, well-developed tumors were cut into 1.5 mm³ fragments and transplanted into nude mice using a trocar [1].
When tumors reached 100–200 mm³, mice were randomized into vehicle and treatment groups [1].
ASK120067 was administered orally once daily at doses of 1, 5, or 10 mg/kg for 21–28 days [1].
Osimertinib (10 mg/kg) was used as a positive control [1].
Tumor growth was measured twice weekly [1].
- PDX model (LU1868): Tumor tissues from a patient-derived xenograft harboring EGFR^L858R/T790M were subcutaneously implanted into nude mice [1].
When tumors reached 150–200 mm³, mice received oral ASK120067 at 3, 10, or 20 mg/kg once daily for 21 days [1].
- ASK120067-resistant xenograft model: 67R cells (ASK120067-resistant) were subcutaneously injected into nude mice [1].
When tumors reached ~100 mm³, mice were treated orally with vehicle, ASK120067 (5 mg/kg), dasatinib (25 mg/kg), or the combination once daily for 21 days [1].
- All animal procedures were approved by the Institutional Animal Care and Use Committee and followed AAALAC guidelines [1].

Limertinib (ASK120067) is an orally administered, third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor . It is metabolized to an active metabolite, CCB4580030 . The metabolism of Limertinib involves the cytochrome P450 (CYP) enzyme system, specifically CYP3A4 .
A phase I study in healthy Chinese volunteers characterized the effect of food on the pharmacokinetics of Limertinib. In this open-label, 2-period crossover study, subjects received a single 160 mg oral dose under fasted and fed conditions (high-fat, high-calorie meal). Limertinib was absorbed more quickly in the fasted state. Food intake increased the extent of absorption, with geometric mean ratios (fed/fasted) for maximum concentration (Cmax) of 145.5%, area under the curve from time 0 to the last quantifiable concentration (AUC0-last) of 145.4%, and area under the curve from time 0 to infinity (AUC0-inf) of 141.9% for Limertinib. The geometric mean ratios for the active metabolite CCB4580030 were also >125%, indicating a significant food effect. Limertinib was well tolerated regardless of prandial state .
A separate phase I study in healthy Chinese subjects investigated drug-drug interactions with strong CYP3A4 modulators. Coadministration with the strong CYP3A4 inducer rifampin (600 mg once daily) dramatically decreased the exposure (AUC0-inf) of Limertinib by 87.86% (geometric least-squares mean [GLSM] ratio, 12.14%; 90% CI, 9.89-14.92) and its active metabolite CCB4580030 by 66.82% (GLSM ratio, 33.18%; 90% CI, 27.72-39.72). Conversely, coadministration with the strong CYP3A4 inhibitor itraconazole (200 mg twice daily) significantly increased the AUC0-inf of Limertinib by 289.8% (GLSM ratio, 389.8%; 90% CI, 334.07-454.82), while decreasing the AUC0-inf of CCB4580030 by 35.96% (GLSM ratio, 64.04%; 90% CI, 50.78-80.77). Based on these findings, the concomitant use of Limertinib with strong CYP3A4 inducers or inhibitors is not recommended

- In animal studies, ASK120067 was well tolerated at all doses tested (up to 20 mg/kg) with no body weight loss observed [1].
- In a phase I clinical trial, ASK120067 appeared well tolerated with no evidence of serious side effects at the doses studied (including 40 mg once daily) [1].
No detailed toxicity data are provided [1].

[1]. Discovery of a novel third-generation EGFR inhibitor and identification of a potential combination strategy to overcome resistance. Mol Cancer. 2020 May 13;19(1):90.

- ASK120067 is a novel third-generation EGFR tyrosine kinase inhibitor designed to overcome T790M-mediated resistance [1].
It irreversibly binds to the ATP-binding pocket of EGFR mutants by forming a covalent bond with cysteine-797 [1].
- In preclinical models, ASK120067 demonstrates potent antitumor activity against EGFR-mutant NSCLC while sparing wild-type EGFR, suggesting a favorable safety profile [1].
- Acquired resistance to ASK120067 can occur via hyperactivation of Ack1, leading to sustained AKT signaling and downregulation of BIM [1].
Combination with Ack1 inhibitors (e.g., dasatinib) overcomes this resistance in vitro and in vivo [1].
- ASK120067 is currently being evaluated in phase I/II clinical trials (NCT03502850) in patients with advanced EGFR T790M-positive NSCLC who have progressed on prior EGFR inhibitors [1].
Preliminary results show promising efficacy and tolerability [1].
- The compound may also inhibit other kinases containing a conserved cysteine analogous to C797 in EGFR, such as HER2 and ITK, which could be explored for other indications [1].

C29H32CLN7O2

546.063084602356

545.23

C, 63.79; H, 5.91; Cl, 6.49; N, 17.96; O, 5.86

1934259-00-3

121371196

White to yellow solid powder

5.7

3

8

11

39

789

0

N(C1C=C(OC)C(NC2=NC=C(Cl)C(NC3C=CC4C=CC=CC=4C=3)=N2)=CC=1NC(=O)C=C)(C)CCN(C)C

QHPVTCLSVVUPOF-UHFFFAOYSA-N

InChI=1S/C29H32ClN7O2/c1-6-27(38)33-23-16-24(26(39-5)17-25(23)37(4)14-13-36(2)3)34-29-31-18-22(30)28(35-29)32-21-12-11-19-9-7-8-10-20(19)15-21/h6-12,15-18H,1,13-14H2,2-5H3,(H,33,38)(H2,31,32,34,35)

N-[5-[[5-chloro-4-(naphthalen-2-ylamino)pyrimidin-2-yl]amino]-2-[2-(dimethylamino)ethyl-methylamino]-4-methoxyphenyl]prop-2-enamide

1934259-00-3; ASK-120067; PRJ2UA27T7; RefChem:1077285; ASK120067;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~41.67 mg/mL (~76.31 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.58 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 25.0 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.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (3.81 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.

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 (Please use freshly prepared in vivo formulations for optimal results.)