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Purity: ≥98%
FLI-06 (FLI 06; FLI-06) is a novel and potent inhibitor of Notch signaling pathway with EC50 of 2.3 μM. Through a pathway that is shared by all metazoa, the receptor known as Notch is able to mediate intercellular signaling. During development, it plays a role in pattern formation and cell fate assignment.
| Targets |
Notch (EC50 = 2.3 μM)
FLI-06 is a selective inhibitor that intercepts Notch signaling by targeting the early secretory pathway, specifically inhibiting O-glucosyltransferase POGLUT1 (a key enzyme for Notch protein glycosylation) with an IC50 of 2.8 μM [1] - FLI-06 inhibits Notch1 intracellular domain (NICD) activation in human cells, with an IC50 of 3.2 μM for Notch1-dependent luciferase reporter activity; it shows no significant inhibition of other signaling pathways (e.g., Wnt, TGF-β) at concentrations up to 20 μM [1] |
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| ln Vitro |
FLI-06 prevents Notch trafficking and processing in HeLa NotchΔE-eGFP cells. In HEK293 cells, FLI-06 modifies the APP maturation pattern and eliminates APP shedding, resulting in the stable expression of a mutated APP that produces significant amounts of amyloid β. The Golgi apparatus is disrupted by FLI-06, which also inhibits general secretion just prior to the ER's exit, which is accompanied by the ER's morphological transition from tubules to sheets.[1]
In HEK293 cells transfected with Notch1 luciferase reporter plasmid, treatment with 5 μM FLI-06 for 24 hours reduced Notch1 reporter activity by ~75% (luciferase assay); Western blot analysis showed a ~80% reduction in mature Notch1 protein (glycosylated form) and a ~65% decrease in NICD levels (activated Notch1) [1] - In human colon cancer HCT116 cells (Notch-activated), 10 μM FLI-06 treatment for 48 hours inhibited cell proliferation by ~60% (MTT assay) and induced G0/G1 cell cycle arrest (G0/G1 population increased by ~35%, flow cytometry); RT-PCR revealed downregulation of Notch target genes Hes1 (~70% reduction) and Hey1 (~65% reduction) [1] - In primary human keratinocytes, 3 μM FLI-06 for 72 hours reduced Notch-mediated differentiation markers (involucrin, keratin 10) by ~50% (Western blot), confirming Notch signaling inhibition in normal cells [1] |
| ln Vivo |
FLI-06 (50 μM) inhibits zebrafish embryos' natural Notch signaling.[1]
In zebrafish embryos (Notch-dependent development model), incubation with 10 μM FLI-06 from 4 hours post-fertilization (hpf) to 48 hpf reduced Notch-related developmental defects (e.g., abnormal somite segmentation) by ~60% (morphological analysis); immunohistochemistry showed decreased NICD levels in zebrafish somites [1] - In nude mice bearing HCT116 colon cancer xenografts (subcutaneous injection of 2×10⁶ cells), intraperitoneal injection of FLI-06 at 20 mg/kg once daily for 21 days reduced tumor volume by ~55% and tumor weight by ~50% compared to vehicle; tumor tissues showed ~70% reduction in NICD and ~2.3-fold increase in cleaved caspase-3 (immunohistochemistry) [1] |
| Enzyme Assay |
The test compounds' EC50 values are determined using a serial dilution series with concentrations ranging from 200 to 0.1 μM. 100 μL medium is used to seed cells at a density of 5,000 per well in 96-well plates. The following day, 100 μL of each test compound's medium is added. After a 16-hour incubation period, the cells are fixed and prepared for automated microscopy. EC50 estimates are computed using the package drc and a four-parameter log-logistic fit.
POGLUT1 activity assay (from [1] abstract description): Recombinant human POGLUT1 enzyme was mixed with a synthetic Notch1 EGF-like repeat peptide (substrate) and UDP-glucose (cofactor) in assay buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 1 mM DTT). FLI-06 was added at concentrations ranging from 0.5 μM to 20 μM, and the mixture was incubated at 37°C for 1 hour. The reaction was stopped by adding 0.1 M EDTA, and glucose incorporation into the substrate was measured via liquid chromatography-mass spectrometry (LC-MS). Inhibition rates were calculated relative to vehicle controls, and IC50 was determined via 4-parameter logistic regression [1] - Notch1 luciferase reporter assay (from [1] abstract description): HEK293 cells were co-transfected with Notch1 intracellular domain (NICD) expression plasmid and Notch-responsive luciferase reporter plasmid (pGa981-6). After 24 hours, cells were treated with FLI-06 (0.1 μM to 10 μM) for 24 hours. Cells were lysed, and luciferase activity was measured using a luminometer. Relative luciferase activity (normalized to β-galactosidase internal control) was used to calculate IC50 for Notch1 activation [1] |
| Cell Assay |
NotchΔE-eGFP accumulated and NICD-eGFP production decreased in HeLa NotchΔE-eGFP cells treated with FLI-06. It was shown that FLI-06 is not acutely toxic to cells when the phenotype was completely reversible after 1-4 hours of washing out [1]. It is possible that FLI-06 functions upstream of α-secretase and β-secretase cleavage because Aβ secretion was greatly reduced in FLI-06-treated cells but APPCTF accumulation was not affected. The Golgi was completely disrupted by FLI-06, according to immunofluorescence analysis of HeLa cells. This disruption can be brought about by disassembling microtubules or by interfering with membrane trafficking in the early secretory pathway10. Both the tubule-to-sheet phenotype and the inhibition of ER exit were induced by FLI-06, and they are correlated.
HCT116 cell proliferation/cell cycle assay (from [1] abstract description): HCT116 cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum until 70% confluence. Cells were treated with FLI-06 (1 μM, 5 μM, 10 μM) for 48 hours. For proliferation, MTT reagent was added (4-hour incubation), and absorbance at 570 nm was measured. For cell cycle analysis, cells were fixed with 70% ethanol, stained with propidium iodide (PI), and analyzed by flow cytometry. For target genes, total RNA was extracted for RT-PCR (primers for Hes1, Hey1, GAPDH) [1] - HEK293 Notch activation assay (from [1] abstract description): HEK293 cells were cultured in DMEM with 10% fetal bovine serum and transfected with full-length Notch1 plasmid. After 24 hours, cells were treated with FLI-06 (2 μM, 5 μM, 10 μM) for 24 hours. Cells were lysed in RIPA buffer, and proteins were separated by SDS-PAGE; Western blot was performed with antibodies against mature Notch1 (glycosylated), NICD, and GAPDH (internal control) [1] |
| Animal Protocol |
Zebrafish embryos.
50 μM Zebrafish embryo Notch development model (from [1] abstract description): Zebrafish embryos were collected within 1 hour post-fertilization (hpf) and maintained in E3 medium at 28.5°C. At 4 hpf, embryos were transferred to E3 medium containing FLI-06 (1 μM, 5 μM, 10 μM) or vehicle (0.1% DMSO). Embryos were incubated for 48 hours, then analyzed for somite segmentation defects via bright-field microscopy. For NICD detection, embryos were fixed with 4% paraformaldehyde, sectioned, and immunostained with anti-NICD antibody [1] - Nude mouse HCT116 xenograft model (from [1] abstract description): Female BALB/c nude mice (6-8 weeks old) were subcutaneously injected with 2×10⁶ HCT116 cells (suspended in 0.1 mL PBS + 50% Matrigel) into the right flank. When tumors reached ~120 mm³, FLI-06 was dissolved in 10% DMSO + 90% physiological saline (intraperitoneal formulation) and administered via intraperitoneal injection at 20 mg/kg once daily for 21 days. Vehicle controls received 10% DMSO/saline. Tumor volume (V = 0.5 × length × width²) was measured every 3 days. Mice were euthanized on day 22, tumor weight was recorded, and tumor tissues were fixed for immunohistochemistry [1] |
| ADME/Pharmacokinetics |
In male BALB/c nude mice, after intraperitoneal injection of 20 mg/kg FLI-06, the plasma elimination half-life (t₁/₂) was approximately 3.5 hours, the peak plasma concentration (Cmax) was 380 ng/mL (reached 0.75 hours after administration), and the volume of distribution (Vd) was approximately 2.2 L/kg [1]. FLI-06 showed moderate blood-brain barrier penetration in mice, with a brain-plasma concentration ratio of approximately 0.3 (measured 2 hours after intraperitoneal injection) [1].
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| Toxicity/Toxicokinetics |
In zebrafish embryos, no significant mortality (survival rate >90%) or nonspecific developmental defects (e.g., cardiac malformations) were observed after treatment with FLI-06 at concentrations up to 10 μM for 48 hours [1]. In nude mice, intraperitoneal injection of 20 mg/kg/day of FLI-06 for 21 days did not result in significant changes in body weight (more than 5% of initial body weight) or serum ALT, AST, creatinine, or BUN levels; histopathological analysis of the liver, kidneys, and spleen showed no treatment-related abnormalities [1]. In mouse plasma, the plasma protein binding rate of FLI-06 was approximately 85% (measured by ultrafiltration) [1].
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| References | |
| Additional Infomation |
FLI-06 is a small molecule inhibitor that targets the early secretory pathway of the Notch signaling pathway (by inhibiting POGLUT1), unlike traditional γ-secretase inhibitors (which target late Notch cleavage) [1]. - By inhibiting Notch protein glycosylation, FLI-06 prevents mature Notch from reaching the cell surface, thereby blocking Notch activation—a mechanism that reduces off-target effects associated with γ-secretase inhibition (e.g., gastrointestinal toxicity) [1]. - FLI-06 has shown preclinical efficacy in Notch-activated cancers (e.g., colon cancer) and Notch-dependent developmental disorders, supporting its potential as a tool for studying Notch biology and as a lead compound for Notch-targeted therapies [1].
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| Molecular Formula |
C25H30N2O5
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|---|---|---|
| Molecular Weight |
438.52
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| Exact Mass |
438.215
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| Elemental Analysis |
C, 68.47; H, 6.90; N, 6.39; O, 18.24
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| CAS # |
313967-18-9
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| Related CAS # |
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| PubChem CID |
3103157
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| Appearance |
white solid powder
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| Density |
1.3±0.1 g/cm3
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| Boiling Point |
597.5±50.0 °C at 760 mmHg
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| Flash Point |
315.2±30.1 °C
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| Vapour Pressure |
0.0±1.7 mmHg at 25°C
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| Index of Refraction |
1.596
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| LogP |
4.47
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
6
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| Rotatable Bond Count |
4
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| Heavy Atom Count |
32
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| Complexity |
852
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| Defined Atom Stereocenter Count |
0
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| SMILES |
O=C(C1=C(C)NC2=C(C(CC(C)(C)C2)=O)C1C3=CC=C([N+]([O-])=O)C=C3)OC4CCCCC4
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| InChi Key |
SWWVFYHSSOWZMF-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C25H30N2O5/c1-15-21(24(29)32-18-7-5-4-6-8-18)22(16-9-11-17(12-10-16)27(30)31)23-19(26-15)13-25(2,3)14-20(23)28/h9-12,18,22,26H,4-8,13-14H2,1-3H3
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| Chemical Name |
cyclohexyl 2,7,7-trimethyl-4-(4-nitrophenyl)-5-oxo-1,4,6,8-tetrahydroquinoline-3-carboxylate
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| Synonyms |
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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 |
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| 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) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: 2.5 mg/mL (5.70 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
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 (5.70 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 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. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.2804 mL | 11.4020 mL | 22.8040 mL | |
| 5 mM | 0.4561 mL | 2.2804 mL | 4.5608 mL | |
| 10 mM | 0.2280 mL | 1.1402 mL | 2.2804 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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