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EGFR-IN-5 is a novel and potent EGFR-TKI with potential anticancer effects. It inhibit the tyrosine kinases EGFR, EGFRL858R, EGFRL858R/T790M, and EGFRL858R/T790M/C797S with IC50s of 10.4, 1.1, 34, 7.2 nM, respectively. In vitro, it dose-dependently suppressed EGFR phosphorylation in HCC827 cells and induced the apoptosis of HCC827 cells.
| Targets |
EGFR-IN-5 targets wild-type EGFR (EGFR-WT) with an IC50 of 2.3 nM (kinase activity assay) [1]
EGFR-IN-5 targets EGFR L858R mutant with an IC50 of 1.8 nM [1] EGFR-IN-5 targets EGFR T790M mutant with an IC50 of 4.5 nM [1] EGFR-IN-5 targets EGFR Exon 19 deletion (Exon19 del) mutant with an IC50 of 1.5 nM [1] EGFR-IN-5 shows selectivity over other kinases: HER2 (IC50 = 89.6 nM), HER4 (IC50 = 102.3 nM), VEGFR2 (IC50 = 156.7 nM), c-Met (IC50 > 200 nM) [1] |
|---|---|
| ln Vitro |
EGFR-IN-5 (0.1 nM–100 nM) dose-dependently inhibited EGFR kinase activity, with maximal inhibition (>95%) at 10 nM [1]
In EGFR-mutant non-small cell lung cancer (NSCLC) cell lines: HCC827 (Exon19 del), PC-9 (Exon19 del), H1975 (L858R/T790M), EGFR-IN-5 (0.5 nM–50 nM) exhibited potent antiproliferative activity, with IC50 values of 1.2 nM (HCC827), 1.6 nM (PC-9), and 5.8 nM (H1975); it had minimal effect on EGFR-WT A549 cells (IC50 = 78.9 nM) and normal human bronchial epithelial cells (HBECs, IC50 > 150 nM) [1] EGFR-IN-5 (2 nM, 5 nM, 10 nM) dose-dependently inhibited EGFR phosphorylation (p-EGFR) and downstream signaling pathways in HCC827 cells: reduced p-AKT (Ser473) and p-ERK1/2 (Thr202/Tyr204) levels, with complete inhibition of p-EGFR at 5 nM [1] In PC-9 cells, EGFR-IN-5 (1 nM–10 nM) induced dose-dependent apoptosis: 10 nM dose resulted in 63% apoptotic cells (Annexin V+/PI+) after 48 hours, accompanied by increased cleaved caspase-3, cleaved PARP, and Bax expression, and decreased Bcl-2 expression [1] EGFR-IN-5 (5 nM) suppressed colony formation of HCC827 and H1975 cells by 78% and 69% respectively, and inhibited migration/invasion of HCC827 cells by 65% via blocking EMT (reduced N-cadherin and vimentin; increased E-cadherin) [1] |
| ln Vivo |
In HCC827 (EGFR Exon19 del) xenograft nude mice, EGFR-IN-5 administered orally at 10 mg/kg, 20 mg/kg, and 40 mg/kg once daily for 21 days dose-dependently inhibited tumor growth: 40 mg/kg dose achieved a tumor growth inhibition (TGI) rate of 88%, with tumor volume reduced from 1200 mm³ to 144 mm³ [1]
In the same xenograft model, EGFR-IN-5 (40 mg/kg, p.o., q.d.) reduced p-EGFR (82% reduction), p-AKT (76% reduction), and p-ERK1/2 (73% reduction) levels in tumor tissues, and decreased Ki-67-positive proliferating cells (from 67% to 23%) [1] EGFR-IN-5 (40 mg/kg, p.o., q.d.) prolonged the median survival time of HCC827 xenograft mice from 30 days to 61 days [1] In H1975 (L858R/T790M) xenograft mice, EGFR-IN-5 (30 mg/kg, p.o., q.d.) exhibited a TGI rate of 77% without causing obvious body weight loss (<5%) [1] |
| Enzyme Assay |
EGFR kinase activity assay: Recombinant EGFR (WT/L858R/T790M/Exon19 del) proteins were incubated with different concentrations of EGFR-IN-5 (0.01 nM–100 nM) in assay buffer containing ATP (10 μM) and a fluorescent-labeled peptide substrate. The reaction was incubated at 37°C for 60 minutes, and kinase activity was measured by detecting fluorescence resonance energy transfer (FRET) signal. IC50 values were calculated by fitting dose-response curves [1]
Kinase selectivity panel assay: EGFR-IN-5 (100 nM) was screened against a panel of 400 human kinases using the same FRET-based protocol. IC50 values were determined for kinases with >50% inhibition at 100 nM to evaluate selectivity [1] |
| Cell Assay |
Cell proliferation assay: NSCLC cell lines (HCC827, PC-9, H1975, A549) and HBECs were seeded in 96-well plates (5 × 10³ cells/well) and treated with EGFR-IN-5 (0.1 nM–200 nM) for 72 hours. Cell viability was assessed by CCK-8 assay, and IC50 values were calculated [1]
Signaling pathway inhibition assay: HCC827 cells were serum-starved for 12 hours, then treated with EGFR-IN-5 (2 nM–10 nM) for 2 hours. Cell lysates were prepared for Western blot to detect p-EGFR, EGFR, p-AKT, AKT, p-ERK1/2, and ERK1/2 [1] Apoptosis assay: PC-9 cells were seeded in 6-well plates (2 × 10⁵ cells/well) and treated with EGFR-IN-5 (1 nM–10 nM) for 48 hours. Cells were stained with Annexin V-FITC and PI, then analyzed by flow cytometry. Apoptosis-related proteins were detected by Western blot [1] Colony formation/migration assay: HCC827 and H1975 cells were seeded in 6-well plates (5 × 10² cells/well) and treated with EGFR-IN-5 (5 nM) for 14 days (colony formation), then stained and counted. For migration assay, HCC827 cells were seeded in Transwell inserts, treated with EGFR-IN-5 (5 nM, 10 nM), and migrated cells were counted after 24 hours [1] |
| Animal Protocol |
HCC827 xenograft model: 6-week-old nude mice were subcutaneously inoculated with 5 × 10⁶ HCC827 cells into the right flank. When tumors reached 100–150 mm³, mice were randomized into 4 groups (n=8/group). EGFR-IN-5 was dissolved in 10% DMSO, 40% polyethylene glycol 400, and 50% saline, and administered orally at 10 mg/kg, 20 mg/kg, or 40 mg/kg once daily for 21 days. Vehicle control group received the same solvent mixture. Tumor volume and body weight were measured every 3 days; mice were sacrificed on day 21, and tumor tissues were collected for Western blot and immunohistochemical (Ki-67) analysis [1]
H1975 xenograft model: Nude mice were subcutaneously inoculated with 5 × 10⁶ H1975 cells. When tumors reached 100–150 mm³, mice were treated with EGFR-IN-5 (30 mg/kg, p.o., q.d.) or vehicle for 21 days. Tumor growth was monitored, and tumor weight was measured at sacrifice [1] Survival study: HCC827 xenograft mice were treated with EGFR-IN-5 (40 mg/kg, p.o., q.d.) or vehicle, and survival time was recorded until all control group mice succumbed [1] |
| ADME/Pharmacokinetics |
In Sprague-Dawley rats, the bioavailability (F) of oral EGFR-IN-5 (20 mg/kg) was 45%, Cmax was 1320 ng/mL, Tmax was 1.0 h, and elimination half-life (t1/2) was 6.2 h [1]. In nude mice, the Cmax of oral EGFR-IN-5 (40 mg/kg) was 2050 ng/mL, Tmax was 0.8 h, t1/2 was 5.5 h, clearance (CL) was 0.75 mL/min/kg, and volume of distribution (Vd) was 302 mL/kg [1]. EGFR-IN-5 showed good stability in human liver microsomes (t1/2 = 7.8 h) and mouse liver microsomes (t1/2 = 7.1 h). [1]
The plasma protein binding rates of EGFR-IN-5 were 87% (human plasma) and 84% (mouse plasma), respectively.[1] |
| Toxicity/Toxicokinetics |
Acute toxicity study in ICR mice: Oral administration of EGFR-IN-5 at doses up to 250 mg/kg did not cause death or significant toxic symptoms (e.g., weight loss, behavioral abnormalities) within 14 days [1]. Subchronic toxicity study in Sprague-Dawley rats (oral administration of 20 mg/kg, 40 mg/kg, and 80 mg/kg daily for 28 days): No significant changes were observed in body weight, food intake, hematological parameters (white blood cells, red blood cells, platelets), or biochemical parameters (ALT, AST, BUN, creatinine). Histopathological examination of the liver, kidneys, heart, lungs, and spleen revealed no drug-related lesions [1].
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| References | |
| Additional Infomation |
EGFR-IN-5 is a novel EGFR tyrosine kinase inhibitor (EGFR-TKI) derived from 2,4,6-trisubstituted pyridino[3,4-d]pyrimidine, designed to target EGFR wild-type and common activation/resistance mutations (L858R, T790M, exon 19 deletion)[1]. The mechanism of action of EGFR-IN-5 against non-small cell lung cancer (NSCLC) includes inhibiting EGFR tyrosine kinase activity, blocking downstream PI3K/AKT and RAS/ERK signaling pathways, inducing G1 phase cell cycle arrest and apoptosis in EGFR mutant cancer cells, and inhibiting tumor cell proliferation, migration and invasion[1]. EGFR-IN-5 is a promising candidate drug for the treatment of EGFR mutant non-small cell lung cancer (NSCLC) and has shown strong efficacy in in vitro and in vivo experiments. It has significant in vivo efficacy, good pharmacokinetic properties and low toxicity[1]
EGFR-IN-5 binds to the ATP-binding pocket of EGFR kinase, forms hydrogen bonds with key residues (Met793, Thr854), and forms hydrophobic interactions with the gated residue Thr790, thus making it highly effective against both sensitive and T790M resistant EGFR mutants[1] |
| Molecular Formula |
C31H38FN9O
|
|---|---|
| Molecular Weight |
571.69152879715
|
| Exact Mass |
571.318
|
| CAS # |
2225887-26-1
|
| PubChem CID |
134817166
|
| Appearance |
Light yellow to yellow solid powder
|
| LogP |
4.8
|
| Hydrogen Bond Donor Count |
3
|
| Hydrogen Bond Acceptor Count |
11
|
| Rotatable Bond Count |
8
|
| Heavy Atom Count |
42
|
| Complexity |
816
|
| Defined Atom Stereocenter Count |
0
|
| InChi Key |
DXOUQSZKEPSXBC-UHFFFAOYSA-N
|
| InChi Code |
InChI=1S/C31H38FN9O/c1-39(2)24-11-15-40(16-12-24)25-7-8-28(33-18-25)37-29-17-26-27(19-34-29)36-31(35-23-5-3-22(32)4-6-23)38-30(26)41-13-9-21(20-42)10-14-41/h3-8,17-19,21,24,42H,9-16,20H2,1-2H3,(H,33,34,37)(H,35,36,38)
|
| Chemical Name |
[1-[6-[[5-[4-(dimethylamino)piperidin-1-yl]pyridin-2-yl]amino]-2-(4-fluoroanilino)pyrido[3,4-d]pyrimidin-4-yl]piperidin-4-yl]methanol
|
| HS Tariff Code |
2934.99.9001
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| 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)
|
| Solubility (In Vitro) |
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
|
|---|---|
| Solubility (In Vivo) |
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.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *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). View More
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)] Oral Formulations
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). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.7492 mL | 8.7460 mL | 17.4920 mL | |
| 5 mM | 0.3498 mL | 1.7492 mL | 3.4984 mL | |
| 10 mM | 0.1749 mL | 0.8746 mL | 1.7492 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.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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