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| Targets |
E2F family transcription factors (mainly E2F4/DP2 heterodimer); in vivo IC₅₀ for inhibiting E2F4 DNA-binding activity in A375 cells: 29.8 ± 7.6 μM[1]
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| ln Vitro |
At 10 and 20 µM, HLM006474 has little effect on E2F4 DNA binding in A375 cells. At 40 µM, it significantly inhibits E2F4 DNA binding, and at 60 and 80 µM, it gradually reduces this effect. By inhibiting its DNA-binding activity and downregulating its expression, HLM006474 (40 µM) suppresses E2F4 activity. In A375 and 231 cell lines, HLM006474 (40 µM) also markedly induced apoptosis over the course of 24 hours. HLM006474 is a strong inducer of PARP cleavage and significantly lowers the expression of the cyclin D3 protein [1]. HLM006474, whose IC50 ranges from 15 to 75 µM, decreases the viability of SCLC and NSCLC cell lines. In H292 and H1299 cell lines, HLM006474 (60 µM) temporarily increases the expression of several known E2F-regulated genes. In H1299 cells, HLM006474 (20 µM) exhibits weak synergistic effects with paclitaxel, but antagonistic effects with cisplatin and gemcitabine [2]. HLM006474 causes the levels of MAD2 mRNA to drop. Furthermore, in human lung cancer A549 cells, HLM006474 significantly reduced the rise in Mad2 protein and pRb-S780 signal, but not the level of Skp2 protein [3].
HLM006474 inhibits DNA‑binding activity of E2F4 in A375 cells in a concentration‑dependent manner, with obvious inhibition at 40 μM and enhanced effect at 60–80 μM[1] HLM006474 (40 μM) suppresses E2F4 DNA‑binding activity starting at 9 h and reduces total E2F4 protein level at 24 h in A375 cells[1] HLM006474 (40 μM, 24 h) significantly induces apoptosis in A375 and MDA‑MB‑231 cells, with no obvious apoptosis in MCF‑7 and HFF cells[1] HLM006474 triggers apoptosis in A375 cells starting at ~9 h, with marked PARP cleavage observed at 12 h[1] HLM006474 induces apoptosis via a p53‑independent pathway, downregulates cyclin D3 and slightly reduces Mcl‑1 expression, distinct from cisplatin, doxorubicin and VP16[1] HLM006474 shows synergistic effect with cisplatin, doxorubicin or VP16 in eliminating E2F4 expression in A375 cells[1] E2F4‑null MEFs are less sensitive to HLM006474‑induced apoptosis than wild‑type MEFs, confirming partial E2F4 dependence[1] HLM006474 inhibits proliferation and invasion of A375 melanoma cells in 3D skin reconstruction model, reduces E2F4 level and Ki‑67 positive rate[1] |
| Enzyme Assay |
Electrophoretic mobility shift assay (EMSA) was used to detect E2F4 DNA‑binding activity: 20 μg of whole cell extract was incubated with ³²P‑labeled E2F‑specific oligonucleotide probe; the reaction mixture was separated by non‑denaturing polyacrylamide gel electrophoresis; gel signals were captured with a phosphoimager and band intensity was quantified with ImageQuant software; quantitative assays were performed in triplicate[1]
Supershift EMSA was performed using specific antibodies against E2F4, Rb, p107, p130, E2F1, E2F2, E2F3 to verify the composition of E2F complexes[1] |
| Cell Assay |
Cell viability assay: Cells were seeded in culture plates, treated with gradient concentrations of HLM006474, and incubated for a set period; cell viability was measured by MTS assay to evaluate antiproliferative effect[1]
Apoptosis detection by TUNEL assay: Cells were treated with HLM006474, collected and fixed; apoptotic cells were labeled using APO‑BRDU kit and analyzed by flow cytometry[1] Cell cycle and sub‑G1 analysis: Cells were digested, washed, fixed with 70% ethanol, treated with RNase A and propidium iodide; DNA content was detected by flow cytometry to assess cell cycle distribution and sub‑G1 apoptotic cell ratio[1] Western blot assay: 50 μg of whole cell extract was loaded per lane; proteins were separated by SDS‑PAGE, transferred to membrane, and incubated with primary antibodies against E2F4, E2F1, PARP, cyclin D3, p53, Mcl‑1, β‑actin; signals were visualized by HRP‑conjugated secondary antibody and chemiluminescence[1] 3D cell culture assay: A375 cells and normal human epidermal keratinocytes were mixed at 1:10 ratio and seeded on fibroblast‑contracted collagen gels; after differentiation in serum‑free medium for ~7 days, cultures were treated with 0, 40 or 80 μM HLM006474 and harvested at set time points; tissues were fixed, embedded, sectioned and stained with H&E or processed for IHC of S‑100, E2F4, activated caspase‑3 and Ki‑67[1] |
| References | |
| Additional Infomation |
Cell viability assay: Cells were seeded in culture plates, treated with gradient concentrations of HLM006474, and incubated for a set period; cell viability was measured by MTS assay to evaluate antiproliferative effect[1]
Apoptosis detection by TUNEL assay: Cells were treated with HLM006474, collected and fixed; apoptotic cells were labeled using APO‑BRDU kit and analyzed by flow cytometry[1] Cell cycle and sub‑G1 analysis: Cells were digested, washed, fixed with 70% ethanol, treated with RNase A and propidium iodide; DNA content was detected by flow cytometry to assess cell cycle distribution and sub‑G1 apoptotic cell ratio[1] Western blot assay: 50 μg of whole cell extract was loaded per lane; proteins were separated by SDS‑PAGE, transferred to membrane, and incubated with primary antibodies against E2F4, E2F1, PARP, cyclin D3, p53, Mcl‑1, β‑actin; signals were visualized by HRP‑conjugated secondary antibody and chemiluminescence[1] 3D cell culture assay: A375 cells and normal human epidermal keratinocytes were mixed at 1:10 ratio and seeded on fibroblast‑contracted collagen gels; after differentiation in serum‑free medium for ~7 days, cultures were treated with 0, 40 or 80 μM HLM006474 and harvested at set time points; tissues were fixed, embedded, sectioned and stained with H&E or processed for IHC of S‑100, E2F4, activated caspase‑3 and Ki‑67[1] |
| Molecular Formula |
C25H25N3O2
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| Molecular Weight |
399.484905958176
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| Exact Mass |
399.195
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| CAS # |
353519-63-8
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| PubChem CID |
2895500
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| Appearance |
White to off-white solid powder
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| LogP |
5.625
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
5
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| Rotatable Bond Count |
6
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| Heavy Atom Count |
30
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| Complexity |
533
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| Defined Atom Stereocenter Count |
0
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| SMILES |
OC(C1=NC(C)=CC=C1C=C2)=C2C(NC3=NC=CC=C3)C(C=C4C)=CC=C4OCC
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| InChi Key |
CYNZBLNMIJNBSF-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C25H25N3O2/c1-4-30-21-13-11-19(15-16(21)2)23(28-22-7-5-6-14-26-22)20-12-10-18-9-8-17(3)27-24(18)25(20)29/h5-15,23,29H,4H2,1-3H3,(H,26,28)
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| Chemical Name |
7-[(4-ethoxy-3-methylphenyl)-(pyridin-2-ylamino)methyl]-2-methylquinolin-8-ol
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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 : ~16.67 mg/mL (~41.73 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (6.26 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 (6.26 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 (6.26 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 | 2.5033 mL | 12.5163 mL | 25.0325 mL | |
| 5 mM | 0.5007 mL | 2.5033 mL | 5.0065 mL | |
| 10 mM | 0.2503 mL | 1.2516 mL | 2.5033 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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