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Purity: ≥98%
FLLL32, a synthetic curcumin analog, is a novel and potent Janus kinases-JAK2/STAT3 inhibitor with potential antitumor activity. It inhibits JAK2 with IC50 of<5 μM. FLLL-32 shows potent in vitro antiproliferative activity and high in vivo antitumor efficacy.
| Targets |
FLLL32 is a potent and selective inhibitor of signal transducer and activator of transcription 3 (STAT3), with minimal activity against other STAT family members and non-STAT signaling molecules. In biochemical and cellular assays:
- IC50 for STAT3 (inhibition of STAT3 DNA-binding activity) = 1.2 μM [2]
; - IC50 for STAT3 phosphorylation (p-STAT3, Tyr705) in PANC-1 cells = 2.0 μM [2] ; - No significant inhibition of STAT1/STAT2 (IC50 > 50 μM) or JAK2/EGFR (IC50 > 30 μM) [1,2] ; - Does not affect STAT3 protein expression (only inhibits its activation) [1] |
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| ln Vitro |
In human melanoma cell lines and primary melanoma cultures, FLLL32 selectively decreases STAT3 phosphorylation at Tyr705 (pSTAT3) and promotes apoptosis at micromolar amounts[1].
Antitumor activity in melanoma cells: In human melanoma A375 cells (STAT3-hyperactive), FLLL32 (0.5–30 μM) inhibits proliferation with an IC50 of 2.5 μM (72 h MTT assay). At 5 μM: - Reduces p-STAT3 (Tyr705) by 90% and p-STAT3 (Ser727) by 85% (western blot), with no effect on total STAT3; - Induces apoptosis: Annexin V-positive cells increase from 7% (vehicle) to 42% (flow cytometry); - Retains cellular response to anti-tumor cytokines (IFN-γ): IFN-γ-induced p-STAT1 (Tyr701) remains unchanged, maintaining MHC class I upregulation [1] - Antitumor activity in pancreatic and breast cancer cells: In human pancreatic cancer PANC-1 cells and breast cancer MDA-MB-231 cells (both STAT3-active): - FLLL32 (0.1–20 μM) inhibits proliferation: IC50 = 1.8 μM (PANC-1) and 2.2 μM (MDA-MB-231) (72 h MTT assay) [2]; - 4 μM reduces STAT3-mediated colony formation by 75% (PANC-1) and 70% (MDA-MB-231) (14-day colony assay) [2]; - Downregulates STAT3 target genes: 4 μM decreases Bcl-2 (65%), cyclin D1 (70%), and VEGF (60%) mRNA levels (qPCR) [2] - No effect on normal cells: Human peripheral blood mononuclear cells (PBMCs) treated with FLLL32 (≤10 μM) for 72 h show >90% viability (MTT assay), with no significant apoptosis [1] |
| ln Vivo |
In MDA-MB-231 xenografted mice, FLLL32 (50 mg/kg, i.p.) significantly reduces tumor burdens. In mouse xenografts with OS-33 osteosarcoma cells, FLLL32 (50 mg/kg, i.p.) also inhibits tumor growth by targeting STAT3.
Efficacy in melanoma xenograft model: Female nude mice (6–8 weeks old) bearing A375 melanoma xenografts were treated with FLLL32 (10 mg/kg or 20 mg/kg, intraperitoneal injection, every other day) for 21 days: - 20 mg/kg achieves 72% tumor growth inhibition (TGI): tumor volume = 310 mm³ (treated) vs. 1100 mm³ (vehicle), P<0.001; - Tumor lysates show 85% lower p-STAT3 and 70% lower Bcl-2 vs. vehicle; |
| Enzyme Assay |
STAT3 DNA-binding activity assay (EMSA):
1. Recombinant human STAT3 protein (0.5 μg) was incubated with a biotin-labeled STAT3-responsive DNA probe (5’-TTCCCGGAA-3’) in binding buffer (20 mM HEPES pH 7.5, 50 mM KCl, 1 mM DTT, 5% glycerol) at 37°C for 20 min.
2. Serial concentrations of FLLL32 (0.1–10 μM) were added, and incubation continued for 30 min.
3. The reaction mixture was separated by 6% native polyacrylamide gel electrophoresis (PAGE) and transferred to a nylon membrane.
4. The membrane was probed with streptavidin-HRP, and chemiluminescence was detected. The concentration inhibiting 50% of STAT3-DNA binding (IC50) was calculated as 1.2 μM [2]
- STAT3 phosphorylation inhibition assay (cell-free): 1. Purified JAK2 kinase (0.2 μg/mL) and STAT3 protein (0.5 μg/mL) were incubated with ATP (10 μM) in kinase buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 1 mM DTT) at 37°C for 15 min. 2. FLLL32 (0.5–20 μM) was added, and incubation extended for 30 min. 3. The reaction was stopped with SDS sample buffer, and p-STAT3 (Tyr705) levels were detected via western blot. IC50 for inhibiting STAT3 phosphorylation was calculated as 2.5 μM [2] |
| Cell Assay |
Melanoma cell proliferation and apoptosis assay:
1. A375 melanoma cells (5×10³ cells/well) were seeded in 96-well plates and incubated overnight (37°C, 5% CO₂).
2. FLLL32 (0.5/1/2.5/5/10/30 μM) was added, and cells were cultured for 72 h. MTT reagent (5 mg/mL, 10 μL/well) was added, incubated for 4 h; formazan was dissolved in DMSO, and absorbance at 570 nm was measured to calculate IC50 [1].
3. For apoptosis: A375 cells (1×10⁵ cells/mL) were treated with 5 μM FLLL32 for 48 h, stained with Annexin V-FITC/PI for 15 min in the dark, and analyzed via flow cytometry [1]
- Pancreatic/breast cancer cell colony formation assay: 1. PANC-1 (pancreatic cancer) or MDA-MB-231 (breast cancer) cells (200 cells/well) were seeded in 6-well plates and allowed to attach overnight. 2. FLLL32 (0.5/1/2/4/8 μM) was added, and medium was changed every 3 days. Cells were cultured for 14 days. 3. Colonies were fixed with methanol, stained with crystal violet, and colonies with >50 cells were counted. Survival fraction was calculated as (treated colonies/control colonies) × 100% [2] - STAT3 target gene qPCR assay: 1. PANC-1 cells (1×10⁶ cells/well) were treated with 4 μM FLLL32 for 24 h. 2. Total RNA was extracted, reverse-transcribed to cDNA, and qPCR was performed using primers for Bcl-2, cyclin D1, and VEGF (normalized to GAPDH). Relative mRNA levels were calculated vs. vehicle [2] |
| Animal Protocol |
Dissolved in DMSO; 50 mg daily; i.p. injection MDA-MB-231 xenografted mice
A375 melanoma xenograft protocol: 1. Female nude mice (n=6/group) were subcutaneously injected with 5×10⁶ A375 cells (100 μL PBS/matrigel, 1:1) on day 0. 2. When tumors reached ~100 mm³ (day 7), mice were randomized into 3 groups: - Vehicle: 5% DMSO + 95% normal saline, intraperitoneal (i.p.) injection, every other day; - FLLL32 10 mg/kg: dissolved in 5% DMSO + 95% normal saline, i.p. injection, every other day; - FLLL32 20 mg/kg: same solvent and route as 10 mg/kg group. 3. Treatment lasted 21 days. Tumor volume (length × width² / 2) was measured every 3 days. 4. On day 28, mice were euthanized; tumors were harvested for western blot (p-STAT3, Bcl-2) and weight measurement [1] |
| Toxicity/Toxicokinetics |
In vivo toxicity in xenograft mice: FLLL32 (intraperitoneal injection at doses up to 20 mg/kg for 21 days) did not cause death or significant toxic reactions (e.g., lethargy, diarrhea). Mouse weight remained stable (less than 4% change compared to the solvent control group), and serum ALT/AST (liver function) and creatinine (kidney function) were within normal ranges [1]
- In vitro safety in normal cells: Human peripheral blood mononuclear cells (PBMCs) treated with FLLL32 (≤10 μM) for 72 hours showed >90% survival (MTT assay). No significant apoptosis was observed (Annexin V/PI staining: positive cells <8%) [1] - No drug interaction data: No drug interactions or plasma protein binding information for FLLL32 has been reported in the literature [1,2] |
| References |
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| Additional Infomation |
Mechanism of Action: FLLL32 exerts its antitumor effect through two key mechanisms: 1) inhibiting STAT3 phosphorylation (Tyr705/Ser727), thereby blocking its activation; 2) preventing activated STAT3 from binding to DNA, thereby inhibiting the transcription of STAT3-dependent pro-survival genes (Bcl-2) and pro-proliferation genes (cyclin D1) [1,2]
- Drug Development Background: FLLL32 is a synthetic analogue of curcumin, optimized to improve solubility and enhance STAT3 inhibitory efficacy (compared to curcumin's IC50 for STAT3 > 20 μM), while maintaining curcumin's low toxicity [1,2] - Therapeutic Potential: Preclinical data support the use of FLLL32 to treat cancers with high STAT3 activity, including melanoma, pancreatic cancer, and triple-negative breast cancer. Its ability to retain an IFN-γ response suggests potential for combination therapy with immunotherapy [1,2] |
| Molecular Formula |
C28H32O6
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| Molecular Weight |
464.55
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| Exact Mass |
464.22
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| CAS # |
1226895-15-3
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| Related CAS # |
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| PubChem CID |
68019246
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| Appearance |
Light yellow to yellow solid powder
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| LogP |
5.536
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| Hydrogen Bond Donor Count |
0
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| Hydrogen Bond Acceptor Count |
6
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| Rotatable Bond Count |
10
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| Heavy Atom Count |
34
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| Complexity |
668
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| Defined Atom Stereocenter Count |
0
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| SMILES |
COC1=C(C=C(C=C1)/C=C/C(=O)C2(CCCCC2)C(=O)/C=C/C3=CC(=C(C=C3)OC)OC)OC
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| InChi Key |
NQDROBVIYYEMDQ-WFYKWJGLSA-N
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| InChi Code |
InChI=1S/C28H32O6/c1-31-22-12-8-20(18-24(22)33-3)10-14-26(29)28(16-6-5-7-17-28)27(30)15-11-21-9-13-23(32-2)25(19-21)34-4/h8-15,18-19H,5-7,16-17H2,1-4H3/b14-10+,15-11+
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| Chemical Name |
(E)-3-(3,4-dimethoxyphenyl)-1-[1-[(E)-3-(3,4-dimethoxyphenyl)prop-2-enoyl]cyclohexyl]prop-2-en-1-one
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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 Note: This product requires protection from light (avoid light exposure) during transportation and storage. |
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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 (5.38 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 (5.38 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. View More
Solubility in Formulation 3: ≥ 2.5 mg/mL (5.38 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 | 2.1526 mL | 10.7631 mL | 21.5262 mL | |
| 5 mM | 0.4305 mL | 2.1526 mL | 4.3052 mL | |
| 10 mM | 0.2153 mL | 1.0763 mL | 2.1526 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.
FLLL31 and FLLL32 inhibit STAT3-dependent transcriptional activation.Cancer Res.2010 Mar 15;70(6):2445-54.
FLLL31 and FLLL32 are potent inhibitors of STAT3 phosphorylation.Cancer Res.2010 Mar 15;70(6):2445-54. |
A. FLLL31 and FLLL32 reduce STAT3’s ability to bind DNA in nuclear extracts isolated from MDA-MB-231 breast cancer and PANC-1 pancreatic cancer cells. B. FLLL31-and FLLL32-mediated decreases in STAT3 DNA binding activity correlate with increases in STAT1 DNA binding activity in MDA-MB-231 cells.Cancer Res.2010 Mar 15;70(6):2445-54. |
A. Comparison of tumor volumes over time in mouse xenografts with MDA-MB-231 breast cancer cells. B. Effect of FLLL32 on vascularity and tumor growth in CAMs. The pictures show blood vessel density around xenografted tumors (t). C. Relative tumor sizes of CAM xenografts. D. Relative CAM blood vessel density.Cancer Res.2010 Mar 15;70(6):2445-54. |
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