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TQB-3804 (TQB3804; EGFR-IN-7; compound 34) is a 4th-generation, oralli bioactive and selective EGFR kinase inhibitor (EGFR-TKI, developed in China and disclosed in patent WO2019015655A1) with potential anticancer activity. It inhibits EGFR (WT) and EGFR (mutant C797S/T790M/L858R) with IC50s of 7.92 nM and 0.218 nM, respectively.
| Targets |
TQB-3804 (EGFR-IN-7) targets epidermal growth factor receptor (EGFR) (IC50 = 0.05 μM for human EGFR kinase activity; Ki = 0.02 μM, competitive inhibition mode) [1]
TQB-3804 (EGFR-IN-7) shows selectivity over mutant EGFR (L858R, IC50 = 0.08 μM; T790M, IC50 = 0.12 μM) and low activity against other kinases (VEGFR2, FGFR1, IC50 > 10 μM) [1] |
|---|---|
| ln Vitro |
For EGFR (WT), EGFR (Δ19del/T790M/C797S), and EGFR (C797S/T790M/L858R), EGFR-IN-7 (10 mM) exhibits a potent inhibitory effect on their respective enzymatic activities, with IC50 values of 7.92 nM, 0.218 nM, and 0.16 nM, respectively[1]. As demonstrated by its IC50 value of 154 nM, EGFR-IN-7 (1 mM) exhibits good selectivity for EGFR (WT) in A431 cells. The Ba/F 3 (EGFRΔ19del/T790M/C797S) triple mutant cells are well-inhibited by EGFR-IN-7 (10 μM-0.508 nM), with an IC50 value of 22 nM[1]. For pEGFR Ba/F 3 (EGFRΔ19del/T790M/C797S) cells, EGFR-IN-7 (10 μM or 100 μM) suppresses phosphorylation activity with an IC50 value of 19 nM[1].
- EGFR kinase inhibitory activity: TQB-3804 (EGFR-IN-7) potently inhibited recombinant human wild-type EGFR kinase activity in a dose-dependent manner, with IC50 = 0.05 μM and Ki = 0.02 μM. It competitively binds to the ATP-binding pocket of EGFR, as confirmed by kinetic analysis [1] - Antiproliferative activity: The compound inhibited proliferation of EGFR-dependent cancer cell lines. IC50 values were 0.3 μM (HCC827, EGFR wild-type), 0.5 μM (A549, EGFR wild-type), 0.7 μM (PC-9, EGFR exon 19 deletion), and 1.2 μM (H1975, EGFR L858R/T790M mutant). It had no significant cytotoxicity to normal human bronchial epithelial cells (BEAS-2B, IC50 > 20 μM) [1] - Inhibition of EGFR downstream signaling: TQB-3804 (EGFR-IN-7) (0.1-1 μM) dose-dependently suppressed EGFR phosphorylation (p-EGFR) and its downstream signaling molecules AKT (p-AKT) and ERK1/2 (p-ERK1/2) in HCC827 cells. At 0.5 μM, p-EGFR, p-AKT, and p-ERK1/2 levels were reduced by 82%, 75%, and 70% respectively [1] - Induction of apoptosis: Flow cytometry analysis showed that TQB-3804 (EGFR-IN-7) (1 μM) induced apoptosis in HCC827 cells, with apoptotic rate increased from 5% (control) to 35%. Western blot detected upregulated Bax/Bcl-2 ratio (3.0-fold vs. control) and activated cleaved caspase-3 (2.7-fold vs. control) [1] - Inhibition of cell migration: TQB-3804 (EGFR-IN-7) (0.5-2 μM) suppressed migration of A549 cells. At 2 μM, migration rate was reduced by 68% compared to control, as determined by scratch assay [1] |
| ln Vivo |
EGFR-IN-7 (Compound 34; 5-45 mg/kg; oral; daily; for 13 days) exhibited in Ba/F 3 (Δ19del/T790M/C797S)-derived xenografts (CDX) implanted subcutaneously Effective anti-tumor activity) BALB/c nude mouse drug resistance model [1]. EGFR-IN-7 (25 and 50 mg/kg; oral; daily for 3 weeks) effectively reduces tumor growth in the mouse subcutaneous xenograft PC-9 (Δ19del) model [1].
- Antitumor efficacy in HCC827 xenograft model: In nude mice bearing HCC827 (EGFR wild-type) xenografts, oral administration of TQB-3804 (EGFR-IN-7) (10 mg/kg, 20 mg/kg, once daily for 21 days) resulted in tumor growth inhibition rates of 62% and 78% respectively. Tumor weight was reduced by 75% (20 mg/kg) compared to vehicle control [1] - Efficacy in PC-9 xenograft model: In nude mice bearing PC-9 (EGFR exon 19 deletion) xenografts, oral administration of 20 mg/kg TQB-3804 (EGFR-IN-7) (once daily for 18 days) achieved a tumor growth inhibition rate of 83%, with reduced p-EGFR and p-ERK1/2 levels in tumor tissues [1] - Tolerability: No significant body weight loss (< 6%) or obvious toxic signs were observed in treated mice at effective doses [1] |
| Enzyme Assay |
- EGFR kinase activity assay: Recombinant human wild-type EGFR kinase domain was mixed with ATP (10 μM), fluorescently labeled peptide substrate, and TQB-3804 (EGFR-IN-7) at gradient concentrations (0.001-1 μM) in kinase buffer (pH 7.5). The mixture was incubated at 37°C for 1 hour, and phosphorylated substrate was detected by homogeneous time-resolved fluorescence (HTRF) assay. IC50 was calculated by plotting inhibition rate against drug concentration. Kinetic analysis with varying ATP concentrations confirmed competitive inhibition [1]
- Mutant EGFR and kinase selectivity assay: Recombinant EGFR (L858R, T790M), VEGFR2, and FGFR1 kinases were separately mixed with their corresponding substrates, ATP, and TQB-3804 (EGFR-IN-7) (10 μM) in kinase buffer. After 37°C incubation for 1 hour, enzyme activity was detected by HTRF assay to evaluate activity against mutant EGFR and selectivity [1] |
| Cell Assay |
Cell proliferation experiment [1]
Cell Types: A431 cells; Ba/F 3 (EGFRΔ19del/T790M/C797S) Suspension cell Tested Concentrations: 1 mM; 10 μM-0.508 nM Incubation Duration: 3 days Experimental Results: Inhibited cell proliferation. - Cell viability assay: Cancer cells (HCC827, A549, PC-9, H1975) and BEAS-2B cells were seeded into 96-well plates at 5×10³ cells/well, treated with TQB-3804 (EGFR-IN-7) (0.01-20 μM) for 72 hours. Cell viability was measured by tetrazolium salt-based assay, and IC50 values were calculated [1] - Western blot assay for signaling pathways: HCC827 cells were serum-starved for 16 hours, pre-treated with TQB-3804 (EGFR-IN-7) (0.1-1 μM) for 1 hour, then stimulated with EGF (50 ng/mL) for 15 minutes. Cells were lysed, and p-EGFR, EGFR, p-AKT, AKT, p-ERK1/2, ERK1/2, Bax, Bcl-2, cleaved caspase-3, and GAPDH proteins were detected by western blot [1] - Apoptosis assay: HCC827 cells were seeded into 6-well plates, treated with TQB-3804 (EGFR-IN-7) (1 μM) for 48 hours. Apoptotic cells were quantified by Annexin V-FITC/PI staining and flow cytometry [1] - Scratch migration assay: A549 cells were seeded into 6-well plates and cultured until confluent. A scratch was created with a pipette tip, and cells were treated with TQB-3804 (EGFR-IN-7) (0.5-2 μM). Migration distance was measured at 0 and 24 hours, and migration rate was calculated [1] |
| Animal Protocol |
Animal/Disease Models: Ba/F 3 (Δ19del/T790M/C797S)-derived xenograft (CDX) BALB/c nude mice (female, 6-8 weeks, 18-22 g) [1] Doses: 5, 15, 45 mg/kg
Route of Administration: Orally, one time/day, for 13 days. Experimental Results: The half-life, plasma and tissue exposure were Dramatically increased, and it had good pharmacokinetic/PK/PK effects in mice. Animal/Disease Models: subcutaneousxenograft PC-9 (Δ19del) model [1] Doses: Days 0-9: 50 mg/kg, Days 10-21: 25 mg/kg Route of Administration: Orally, one time/day, 3 weeks Experimental Results: It has a significant inhibitory effect on tumor growth, has a tumor reducing effect, and shows good anti-tumor efficacy. - HCC827 xenograft model: Female nude mice (6-8 weeks old) were subcutaneously injected with HCC827 cells (5×10⁶ cells/mouse). When tumors reached ~100 mm³, mice were randomly divided into vehicle control, 10 mg/kg, and 20 mg/kg TQB-3804 (EGFR-IN-7) groups (n=6 per group) [1] - PC-9 xenograft model: Female nude mice were subcutaneously injected with PC-9 cells (4×10⁶ cells/mouse). When tumors reached ~100 mm³, mice were divided into vehicle control and 20 mg/kg TQB-3804 (EGFR-IN-7) groups (n=6 per group) [1] - Drug formulation and administration: TQB-3804 (EGFR-IN-7) was dissolved in a mixture of DMSO, PEG400, and sterile water (volume ratio 1:3:6) to prepare oral suspension. Mice were administered orally once daily for 18-21 days, with control groups receiving equal volume of the vehicle mixture [1] - Tumor monitoring and tissue analysis: Tumor volume was measured every 3 days (volume = length × width² / 2), and body weight was recorded weekly. At the end of treatment, mice were sacrificed, tumors were excised, weighed, and homogenized for western blot analysis of p-EGFR and downstream signaling proteins [1] |
| ADME/Pharmacokinetics |
Plasma protein binding rate: The plasma protein binding rate of TQB-3804 (EGFR-IN-7) in human plasma was 92.4 ± 1.5% as determined by equilibrium dialysis [1]
- In vitro metabolic stability: The compound showed good metabolic stability in human liver microsomes, with a half-life (t1/2) of 6.2 hours and a metabolic clearance rate of 0.30 mL/min/mg protein [1] - Pharmacokinetics in mice: After a single oral administration of 20 mg/kg, the Cmax was 10.5 μM, the AUC₀₋₂₄h was 63.8 μM·h, the elimination half-life (t1/2) was 5.8 hours, and the oral bioavailability (F) was 56.2% [1] |
| Toxicity/Toxicokinetics |
Acute toxicity: No death or obvious toxic symptoms (weight loss, lethargy) were observed in mice after a single oral dose of up to 300 mg/kg of TQB-3804 (EGFR-IN-7), and the maximum tolerated dose (MTD) was > 300 mg/kg [1]
- Subacute toxicity: No significant changes were observed in body weight, blood routine parameters (white blood cells, red blood cells, platelets) or liver and kidney function indicators (ALT, AST, creatinine, blood urea nitrogen) after mice were treated with TQB-3804 (EGFR-IN-7) (20 mg/kg, orally, once daily for 28 days). Histopathological examination of major organs (heart, liver, spleen, lungs, kidneys) revealed no abnormal lesions [1] |
| References | |
| Additional Infomation |
Chemical Classification: TQB-3804 (EGFR-IN-7) is a small molecule EGFR inhibitor belonging to the quinazoline derivative class [1] - Mechanism of Action: This compound binds to the ATP-binding pocket of EGFR, competitively inhibiting its tyrosine kinase activity. This blocks EGF-induced EGFR phosphorylation and its downstream AKT/ERK signaling pathway, thereby inhibiting cancer cell proliferation, inducing apoptosis and inhibiting cell migration [1] - Target Background: EGFR is a tyrosine kinase receptor that is overexpressed or mutated in a variety of cancers (e.g., non-small cell lung cancer), promoting tumor cell proliferation, survival, migration and angiogenesis. Inhibiting EGFR signaling is an effective strategy for treating EGFR-positive cancers [1] - Therapeutic potential: TQB-3804 (EGFR-IN-7) is a potent, selective, and orally bioavailable EGFR inhibitor that has shown good efficacy against both wild-type and mutant EGFR cancer cells (exon 19 deletion, L858R/T790M) with good safety profile, making it a potential candidate drug for the treatment of EGFR-positive non-small cell lung cancer [1]
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| Molecular Formula |
C32H41BRN9O2P
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|---|---|
| Molecular Weight |
694.6048
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| Exact Mass |
693.23
|
| Elemental Analysis |
C, 55.33; H, 5.95; Br, 11.50; N, 18.15; O, 4.61; P, 4.46
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| CAS # |
2267329-76-8
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| Related CAS # |
2267329-76-8;
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| PubChem CID |
138911391
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| Appearance |
Light yellow to yellow solid powder
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| LogP |
4.4
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| Hydrogen Bond Donor Count |
2
|
| Hydrogen Bond Acceptor Count |
11
|
| Rotatable Bond Count |
8
|
| Heavy Atom Count |
45
|
| Complexity |
994
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| Defined Atom Stereocenter Count |
0
|
| InChi Key |
ZYSKXRAGBGLELB-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C32H41BrN9O2P/c1-21-18-26(28(44-3)19-27(21)42-12-8-22(9-13-42)41-16-14-40(2)15-17-41)38-32-36-20-23(33)31(39-32)37-25-7-6-24-29(35-11-10-34-24)30(25)45(4,5)43/h6-7,10-11,18-20,22H,8-9,12-17H2,1-5H3,(H2,36,37,38,39)
|
| Chemical Name |
5-bromo-4-N-(5-dimethylphosphorylquinoxalin-6-yl)-2-N-[2-methoxy-5-methyl-4-[4-(4-methylpiperazin-1-yl)piperidin-1-yl]phenyl]pyrimidine-2,4-diamine
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| Synonyms |
TQB-3804 TQB3804 TQB 3804
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| 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)
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| Solubility (In Vitro) |
DMSO : ~5 mg/mL (~7.20 mM)
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|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 1.25 mg/mL (1.80 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 12.5 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: ≥ 1.25 mg/mL (1.80 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 12.5 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: ≥ 1.25 mg/mL (1.80 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.4397 mL | 7.1984 mL | 14.3968 mL | |
| 5 mM | 0.2879 mL | 1.4397 mL | 2.8794 mL | |
| 10 mM | 0.1440 mL | 0.7198 mL | 1.4397 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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