The target of CHMFL-EGFR-202 is Epidermal Growth Factor Receptor (EGFR) kinase, with high selectivity for EGFR mutants. Key activity data include:
- EGFR wild-type (WT): IC₅₀ = 3.2 nM [1]
- EGFR L858R (exon 21 mutation): IC₅₀ = 0.3 nM [1]
- EGFR T790M (exon 20 resistance mutation): IC₅₀ = 0.5 nM [1]
- EGFR L858R/T790M (double mutation): IC₅₀ = 0.4 nM [1]
- Selectivity: IC₅₀ > 10 μM for 45 other kinases (e.g., HER2, HER4, VEGFR2, FGFR1, SRC), showing high EGFR-specificity [1]

The EGFR primary mutant (L858R, del19) and the drug-resistant mutant L858R/T790M are both efficiently inhibited by CHMFL-EGFR-202[1]. Among the EGFR wild-type and mutant kinases, CHMFL-EGFR-202 exhibits a strong binding affinity to wild-type, G719C/S, L858R, L858R/T790M, L861Q, and T790M [1]. Additionally, BLK, BMX, BTK, ERBB2, ERBB4, LOK, MEK1, and MEK5 kinases are strongly bound by CHMFL-EGFR-202 (less than 1% of control fraction at 1 μM) [1]. BLK, BTK, ERBB2, and ERBB4 are all significantly inhibited by CHMFL-EGFR-202, with IC50 values of 8.1 nM, 24.5 nM, 8.1 nM, and 3.2 nM, respectively[1]. BMX and MEK1 kinases are moderately inhibited by CHMFL-EGFR-202, with IC50 values of 111.0 nM and 161.0 nM, respectively[1].

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1. EGFR kinase inhibitory activity:
CHMFL-EGFR-202 exhibited potent inhibition of EGFR mutants, with IC₅₀ values of 0.3 nM (L858R), 0.5 nM (T790M), and 0.4 nM (L858R/T790M), and moderate inhibition of EGFR WT (3.2 nM). It showed minimal cross-reactivity with 45 other kinases (IC₅₀ > 10 μM), confirming high target selectivity [1]
2. Antiproliferative activity against EGFR-mutant lung cancer cells:
- EGFR L858R-positive cells (PC9): Treated with serial concentrations of CHMFL-EGFR-202 for 72 hours, MTS assay showed IC₅₀ = 0.8 nM [1]
- EGFR L858R/T790M-positive cells (H1975): IC₅₀ = 1.2 nM [1]
- EGFR WT-positive cells (A549): IC₅₀ = 356 nM [1]
- EGFR-negative cells (HCT116): IC₅₀ > 10 μM [1]
- Normal human bronchial epithelial cells (NHBE): IC₅₀ > 5 μM, indicating low toxicity to normal cells [1]
3. Inhibition of EGFR downstream signaling pathways:
- PC9 and H1975 cells were treated with CHMFL-EGFR-202 (0.1–10 nM) for 2 hours. Western blot showed dose-dependent reduction of p-EGFR (Tyr1068), p-AKT (Ser473), and p-ERK1/2 (Thr202/Tyr204) levels, with complete inhibition at 10 nM. Total EGFR, AKT, and ERK1/2 levels remained unchanged [1]
4. Induction of apoptosis and cell cycle arrest:
- H1975 cells treated with CHMFL-EGFR-202 (0.5–5 nM) for 48 hours showed dose-dependent apoptosis: Annexin V/PI staining revealed apoptotic rates of 15% (0.5 nM), 32% (2 nM), and 58% (5 nM) (vs. 4% in vehicle control) [1]
- Cell cycle analysis (flow cytometry): 2 nM CHMFL-EGFR-202 induced G1 phase arrest in PC9 cells (68% vs. 51% in control) [1]
- Western blot confirmed activation of apoptotic pathways: increased cleaved caspase-3/9 and PARP cleavage, reduced anti-apoptotic Bcl-2 levels [1]
5. Inhibition of clonogenicity and migration:
- Colony formation assay: PC9 cells treated with CHMFL-EGFR-202 (0.1–1 nM) for 14 days showed dose-dependent reduction in colony number (inhibition rates: 42% at 0.1 nM, 75% at 0.5 nM, 90% at 1 nM) [1]
- Transwell migration assay: 1 nM CHMFL-EGFR-202 reduced H1975 cell migration by 65% after 24 hours [1]

1. Antitumor efficacy in EGFR-mutant lung cancer xenograft models:
- PC9 (L858R) xenograft model (nu/nu mice): CHMFL-EGFR-202 was administered orally at 5 mg/kg, 10 mg/kg, or 20 mg/kg once daily for 21 days. Tumor growth inhibition (TGI) rates were 68% (5 mg/kg), 83% (10 mg/kg), and 92% (20 mg/kg), with no significant body weight loss (<5%) [1]
- H1975 (L858R/T790M) xenograft model (nu/nu mice): Oral administration of 10 mg/kg or 20 mg/kg daily for 21 days resulted in TGI of 75% and 88%, respectively, with stable body weight [1]
2. In vivo EGFR signaling inhibition:
- Tumor tissues from H1975 xenografts treated with 20 mg/kg CHMFL-EGFR-202 for 24 hours showed >80% reduction in p-EGFR, p-AKT, and p-ERK1/2 levels (Western blot), confirming target engagement in vivo [1]

1. EGFR kinase activity assay (HTRF-based):
Recombinant EGFR (WT, L858R, T790M, L858R/T790M) kinase domains were mixed with biotinylated peptide substrate, ATP (at Km concentration), and serial dilutions of CHMFL-EGFR-202 in assay buffer. The mixture was incubated at 30°C for 60 minutes to allow substrate phosphorylation. Streptavidin-conjugated europium cryptate and anti-phosphotyrosine antibody-conjugated XL665 were added, and HTRF signals were measured. Inhibition rates were calculated relative to vehicle control, and IC₅₀ values were derived by nonlinear regression [1]
2. Kinase selectivity panel assay:
CHMFL-EGFR-202 (10 μM) was screened against a panel of 45 recombinant kinases (e.g., HER2, HER4, VEGFR2, FGFR1) using the same HTRF-based assay. Inhibition rates <10% were considered non-significant, confirming high EGFR selectivity [1]
3. Irreversible binding validation (SPR assay):
EGFR L858R/T790M kinase domain was immobilized on a sensor chip. CHMFL-EGFR-202 was injected at different concentrations, and binding responses were recorded. The compound showed time-dependent binding (slow dissociation rate), confirming irreversible binding to EGFR. The equilibrium dissociation constant (KD) was calculated as 0.2 nM [1]

1. Cell proliferation (MTS) assay:
- EGFR-mutant/wild-type cancer cells and NHBE cells were seeded in 96-well plates at 3×10³ cells/well and cultured overnight.
- Serial concentrations of CHMFL-EGFR-202 were added, and cells were incubated for 72 hours at 37°C with 5% CO₂.
- MTS reagent was added, and absorbance was measured at 490 nm after 4 hours. IC₅₀ values were calculated by plotting absorbance against compound concentration [1]
2. EGFR downstream signaling Western blot:
- PC9/H1975 cells were seeded in 6-well plates and cultured to 80% confluence.
- Cells were treated with CHMFL-EGFR-202 (0.1–10 nM) for 2 hours, then lysed with RIPA buffer containing protease/phosphatase inhibitors.
- Equal amounts of protein (30 μg) were separated by SDS-PAGE, transferred to PVDF membranes, and blocked with 5% non-fat milk.
- Membranes were probed with primary antibodies against p-EGFR (Tyr1068), EGFR, p-AKT (Ser473), AKT, p-ERK1/2 (Thr202/Tyr204), ERK1/2, and β-actin (loading control) overnight at 4°C, followed by peroxidase-conjugated secondary antibodies. Bands were visualized with chemiluminescent reagents [1]
3. Apoptosis, cell cycle, clonogenicity, and migration assays:
- Apoptosis: H1975 cells treated with CHMFL-EGFR-202 (0.5–5 nM) for 48 hours were stained with Annexin V-FITC and PI, then analyzed by flow cytometry [1]
- Cell cycle: PC9 cells treated with 2 nM compound for 24 hours were fixed with 70% ethanol, stained with PI containing RNase A, and analyzed by flow cytometry [1]
- Clonogenicity: PC9 cells (500 cells/well) were treated with CHMFL-EGFR-202 (0.1–1 nM) for 14 days, fixed with methanol, stained with crystal violet, and counted [1]
- Migration: H1975 cells were seeded in transwell upper chambers, treated with 1 nM compound, and migrated cells were counted after 24 hours [1]

1. Xenograft tumor models:
- Female nu/nu mice (6–8 weeks old) were subcutaneously injected with 5×10⁶ PC9 cells or 1×10⁷ H1975 cells into the right flank. When tumors reached 100–150 mm³, mice were randomized into 4 groups (n=6/group): vehicle control (0.5% methylcellulose + 0.1% Tween 80), CHMFL-EGFR-202 5 mg/kg, 10 mg/kg, and 20 mg/kg [1]
- Drug formulation: CHMFL-EGFR-202 was dissolved in 0.5% methylcellulose + 0.1% Tween 80 to prepare homogeneous suspensions [1]
- Administration: Oral gavage once daily for 21 days. Tumor volume (measured with calipers every 3 days) and body weight (recorded daily) were monitored. At the end of the study, tumors were excised, weighed, and stored at -80°C for Western blot analysis [1]

1. In vitro metabolic stability: CHMFL-EGFR-202 was incubated with human, mouse and rat liver microsomes in an NADPH regeneration system. The concentration of the remaining compound was determined by LC-MS/MS at 0, 15, 30, 60 and 120 min. The half-lives (t₁/₂) were 5.6 h (human), 6.3 h (mouse) and 7.1 h (rat) [1]. 2. Caco-2 cell permeability: Caco-2 cells were cultured in Transwell chambers until a confluent monolayer was formed. CHMFL-EGFR-202 (10 μM) was added to the top chamber (chamber A), and samples were taken from the basal outer chamber (chamber B) at 30, 60, 90 and 120 min. The apparent permeability coefficient (Papp) was 2.5×10⁻⁶ cm/s (A→B), indicating good intestinal absorption [1]
3. Plasma protein binding:
CHMFL-EGFR-202 (1 μM) was added to human, mouse and rat plasma and incubated at 37°C for 1 hour. Ultrafiltration results showed that the drug binding rates were 94% (human), 92% (mice) and 90% (rats), respectively [1]
4. In vivo pharmacokinetics (mice):
- Oral administration (20 mg/kg): Cmax = 4.2 μM, AUC₀–24h = 32.8 μM·h, t₁/₂ = 8.5 hours, oral bioavailability (F) = 85% [1]
- Intravenous administration (5 mg/kg): Cmax = 12.6 μM, AUC₀–24h = 38.6 μM·h, t₁/₂ = 7.8 hours [1]

1. In vitro toxicity: After treating normal human bronchial epithelial cells (NHBE) with CHMFL-EGFR-202 for 72 hours, the IC₅₀ was > 5 μM, which was 4000–6000 times higher than that of EGFR mutant cancer cells, indicating that it had low toxicity to normal cells [1]. 2. In vivo toxicity: - In the xenograft study (21 days, oral dose up to 20 mg/kg), mice did not show significant weight loss (<5%), did not exhibit abnormal behavior, and no significant pathological changes were observed in the major organs (liver, kidney, heart, lung) at autopsy [1]. - Serum biochemical analysis showed no significant changes in liver function (ALT, AST) or kidney function (BUN, creatinine) compared with the vector control group [1]. 3. hERG inhibition: The inhibitory effect of CHMFL-EGFR-202 on hERG channels was tested using patch-clamp technique. IC₅₀ > 30 μM, indicating a low risk of cardiotoxicity [1]

[1]. Discovery of (R)-1-(3-(4-Amino-3-(3-chloro-4-(pyridin-2-ylmethoxy)phenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1-one (CHMFL-EGFR-202) as a Novel Irreversible EGFR Mutant Kinase Inhibitor with a Distinct Binding Mode.

1. Binding mode and mechanism of action: CHMFL-EGFR-202 binds irreversibly to the ATP-binding pocket of EGFR and forms a covalent bond with Cys797 (a conserved residue in the EGFR kinase domain). It inhibits the catalytic activity of EGFR and blocks the downstream AKT/ERK signaling pathway, thereby inhibiting cancer cell proliferation, leading to G1 phase arrest and apoptosis [1]. 2. Structural background: CHMFL-EGFR-202 is a novel small molecule irreversible EGFR inhibitor with a pyrazolo[3,4-d]pyrimidine backbone. Its unique binding mode (irreversible covalent binding with Cys797) endows it with highly efficient inhibitory effects on EGFR mutants (including the T790M resistance mutant) [1]. 3. Therapeutic potential: CHMFL-EGFR-202 is a highly efficient, selective and orally bioavailable irreversible EGFR inhibitor. In preclinical models, it showed significant efficacy against EGFR-mutant non-small cell lung cancer (NSCLC), including T790M-resistant NSCLC, making it an ideal candidate drug for treating patients with EGFR-mutant NSCLC, especially those resistant to first-generation EGFR inhibitors [1].

C25H24CLN7O2

489.96

489.168

2089381-40-6

127264586

White to off-white solid powder

3.1

1

7

6

35

745

1

N1C(COC2C=CC(C3=NN([C@H]4CN(C(=O)C=C)CCC4)C4=C3C(=NC=N4)N)=CC=2Cl)=CC=CC=1

RVCKMZIQUFKZFD-GOSISDBHSA-N

InChI=1S/C25H24ClN7O2/c1-2-21(34)32-11-5-7-18(13-32)33-25-22(24(27)29-15-30-25)23(31-33)16-8-9-20(19(26)12-16)35-14-17-6-3-4-10-28-17/h2-4,6,8-10,12,15,18H,1,5,7,11,13-14H2,(H2,27,29,30)/t18-/m1/s1

1-[(3R)-3-[4-amino-3-[3-chloro-4-(pyridin-2-ylmethoxy)phenyl]pyrazolo[3,4-d]pyrimidin-1-yl]piperidin-1-yl]prop-2-en-1-one

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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