EGFR (IC50 = 1.9 μM); EGFRL858R (IC50 = 0.019 μM); EGFRT790M (IC50 = 0.19 μM); EGFRL858R/T790M (IC50 = 0.002 μM)

EAI045, but not HaCaT cells, potently suppresses EGFR Y1173 phosphorylation in H1975 cells (EC50=2 nM). At 1 mM ATP, EAI045 is a 1000-fold selective inhibitor of the L858R/T790M mutant compared to wild-type EGFR. EAI045 exhibits remarkable selectivity when compared to a panel of 250 protein kinases; at 1 μM, no other kinase is inhibited by more than 20%[1]. High potency and selectivity are seen for the L858R/T790M mutation in EAI045. EAI045 reduces EGFR autophosphorylation in L858R/T790M-mutant NSCLC cell line H1975 cells, although it does not entirely eliminate it. EAI045 exhibits the same activity in stably transfected NIH-3T3 cells expressing the L858R/T790M EGFR mutation. EAI045 has moderate activity in H3255 cells carrying the L858R mutation. EAI045 does not exhibit any action of suppressing EGFR phosphorylation in the HaCaT cells, a keratinocyte cell line with wild-type EGFR. It validates EAI045's specificity for EGFR mutants[2].

L858R/T790M-mutant mice treated with a combination of EAI045 and cetuximab showed remarkable tumor regression in a genetically engineered mouse model of L858R/T790Mmutant-driven lung cancer. In mice treated with EAI045 alone, there is no noticeable reaction. Both mice bearing the L858R/T790M/C797S tumor xenografts and Ba/F3 cells engineered with these mutations exhibit the same effect. These tests unequivocally demonstrate that acquired T790M and C797S mutation resistance can be overcome by EAI045[2].

Phospho-EGFR (Y1173) Target Modulation Assay[1]
HaCaT cells were stimulated with 10 ng/mL EGF for 5 minutes at room temperature. Constitutively activated EGFR mutant cell lines (H1975 and H3255) were not stimulated with EGF. The media was reduced to 20 μL using a Bio-Tek ELx 405 SelectTM plate washer. Cells were lysed with 20 μL of 2X Lysis buffer containing protease and phosphatase inhibitors (2% Triton X-100, 40 mM Tris, pH 7.5, 2 mM EDTA, 2 mM EGTA, 300 mM NaCl, 2X complete cocktail inhibitor, 2X Phosphatase Inhibitor Cocktail Set II and Set III)). The plates were shaken for 20 minutes. An aliquot of 25 μL from each well was transferred to prepared ELISA plates for analysis.[1]
For the experiment studying the effect of EGF pre-treatment on EAI045 target modulation, H1975 cells were harvested and plated in 0.5% FBS/RPMI Pen/Strep. On the following day, cells were pre-treated with 0.5% FBS/RPMI media with or without 10 ng EGF/mL for 5 minutes. Compound was added and assay was carried out as described above. The experiment was performed twice with duplicate samples in each experiment.

H1975, H3255 and HaCaT Proliferation Assays[1]
H1975, H3255 and HaCaT cell lines were plated in solid white 384-well plates at 500 cells per well in 10% FBS RPMI P/S media. Using a Pin Tool, 50 nL of serial diluted compounds were transferred to the cells. After 3 days, cell viability was measured by CellTiter-Glo according to manufacturer's instructions. Luminescent readout was normalized to 0.1% DMSO-treated cells and empty wells. Data was analyzed by non-linear regression curve fitting and EC50 values were reported.

---

Ba/F3 cell proliferation models[1]
The EGFR mutant L858R, L858R/T790M, DelE746_A750/T790M, L858R/T790M/C797S and Del/T790M/C797S Ba/F3 cells have been previously described15. The EGFR I941R mutation was introduced via site directed mutagenesis using the Quick Change Site-Directed Mutagenesis kit according to the manufacturer's instructions. All constructs were confirmed by DNA sequencing. The constructs were shuttled into the retroviral vector JP1540 using the BD Creator™ System. Ba/F3 cells were infected with retrovirus and according to standard protocols, as described previously30. Stable clones were obtained by selection in puromycin (2 μg/ml).[1]
Growth and inhibition of growth was assessed by MTS assay and was performed according to previously established methods15. Ba/F3 cells of different EGFR genotypes were exposed to treatment for 72 hours and the number of cells used per experiment determined empirically and has been previously established. All experimental points were set up in six wells and all experiments were repeated at least three times. The data was graphically displayed using GraphPad Prism version 5.0 for Windows, (GraphPad Software; www.graphpad.com). The curves were fitted using a non-linear regression model with a sigmoidal dose response.

---

NIH-3T3 cell studies[1]
NIH-3T3 cells were infected with retroviral constructs expressing EGFR mutants according to standard protocols, as described previously15,19. Stable clones were obtained by selection in puromycin (2 μg/ml).

The EAI045 compound was dissolved in 10% NMP (10% 1-methyl-2-pyrrolidinone: 90% PEG-300), and was dosed at 60 mg/kg daily by oral gavage. Cetuximab was administrated at 1 mg/mouse every other day by intraperitoneal injection. The TL, TD and TLCS mice were monitored by MRI to quantify lung tumor burden before being assigned to various study treatment cohorts, which were non-blinded and not formally randomized. All treated mice had an equal initial tumor burden. MRI evaluation was repeated every 2 weeks during treatment. The animals were imaged with a rapid acquisition with relaxation enhancement sequence (TR = 2000 ms, TE effect = 25 ms) in the coronal and axial planes with a 1-mm slice thickness gating with respiratory rates. The detailed procedure for MRI scanning has been previously described27. The tumor burden volumes were quantified using 3-dimensional Slicer software. Source data for tumor volume measurements are provided in Supplementary Figure 2.[1]

---

Formulated in 10% NMP (10% 1-methyl-2-pyrrolidinone:90% PEG-300); 60 mg/kg; Oral gavage

EGFR(TL) (bearing L858R/T790M point mutations) and EGFR(TD) (bearing exon19del/T790M point mutations) mice

[1]. Overcoming EGFR(T790M) and EGFR(C797S) resistance with mutant-selective allosteric inhibitors. Nature. 2016 Jun 2;534(7605):129-32.

[2]. EAI045: The fourth-generation EGFR inhibitor overcoming T790M and C797S resistance. Cancer Lett. 2017 Jan 28;385:51-54.

The epidermal growth factor receptor (EGFR)-directed tyrosine kinase inhibitors (TKIs) gefitinib, erlotinib and afatinib are approved treatments for non-small cell lung cancers harbouring activating mutations in the EGFR kinase, but resistance arises rapidly, most frequently owing to the secondary T790M mutation within the ATP site of the receptor. Recently developed mutant-selective irreversible inhibitors are highly active against the T790M mutant, but their efficacy can be compromised by acquired mutation of C797, the cysteine residue with which they form a key covalent bond. All current EGFR TKIs target the ATP-site of the kinase, highlighting the need for therapeutic agents with alternative mechanisms of action. Here we describe the rational discovery of EAI045, an allosteric inhibitor that targets selected drug-resistant EGFR mutants but spares the wild-type receptor. The crystal structure shows that the compound binds an allosteric site created by the displacement of the regulatory C-helix in an inactive conformation of the kinase. The compound inhibits L858R/T790M-mutant EGFR with low-nanomolar potency in biochemical assays. However, as a single agent it is not effective in blocking EGFR-driven proliferation in cells owing to differential potency on the two subunits of the dimeric receptor, which interact in an asymmetric manner in the active state. We observe marked synergy of EAI045 with cetuximab, an antibody therapeutic that blocks EGFR dimerization, rendering the kinase uniformly susceptible to the allosteric agent. EAI045 in combination with cetuximab is effective in mouse models of lung cancer driven by EGFR(L858R/T790M) and by EGFR(L858R/T790M/C797S), a mutant that is resistant to all currently available EGFR TKIs. More generally, our findings illustrate the utility of purposefully targeting allosteric sites to obtain mutant-selective inhibitors.[1]

---

The third-generation tyrosine kinase inhibitors (TKI), AZD9291 (osimertinib) and CO-1686 (rociletinib) of epidermal growth factor receptor (EGFR) are highly active against T790M positive non-small cell lung cancer (NSCLC). However, resistance develops rapidly. EGFR C797S mutation was reported to be a leading mechanism of resistance to the third-generation inhibitors. The C797S mutation appears to be an ideal target for overcoming the acquired resistance to the third-generation inhibitors. This review summarizes the latest development on the discovery of a fourth-generation EGFR TKI, EAI045.3.[2]

C19H14FN3O3S

383.40

383.074

C, 59.52; H, 3.68; F, 4.96; N, 10.96; O, 12.52; S, 8.36

1942114-09-1

1942114-09-1;

121231412

White to khaki solid powder

1.5±0.1 g/cm3

1.729

2.47

2

6

4

27

580

0

0

YTUFHOKUFOQRDF-UHFFFAOYSA-N

InChI=1S/C19H14FN3O3S/c20-12-5-6-15(24)14(9-12)16(17(25)22-19-21-7-8-27-19)23-10-11-3-1-2-4-13(11)18(23)26/h1-9,16,24H,10H2,(H,21,22,25)

2-(5-fluoro-2-hydroxyphenyl)-2-(3-oxo-1H-isoindol-2-yl)-N-(1,3-thiazol-2-yl)acetamide

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.52 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.

---

Solubility in Formulation 2: 2.5 mg/mL (6.52 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
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 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.

---

View More

Solubility in Formulation 3: ≥ 2.5 mg/mL (6.52 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 25.0 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.)