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Purity: ≥98%
Diflapolin is reported to be the first potent dual soluble epoxide hydrolase (sEH)/5-lipoxygenase-activating protein (FLAP) inhibitor with anti-inflammatory effects. Whereas 5-LOX (5-lipoxygenase) activating protein facilitates 5-LOX-mediated conversion of arachidonic acid to pro-inflammatory leukotrienes, soluble epoxide hydrolase (sEH) degrades anti-inflammatory epoxyeicosatrienoic acids (EETs). Dual inhibition might therefore offer increased efficacy as an anti-inflammatory intervention compared to single target agents. Accordingly, dual FLAP/sEH inhibition might be advantageous drugs for intervention of inflammation.
| Targets |
Diflapolin is a dual inhibitor targeting 5-Lipoxygenase-Activating Protein (FLAP) and soluble Epoxide Hydrolase (sEH). Inhibition of cellular 5-LOX product formation (via FLAP) showed IC₅₀ values of 30 nM in human monocytes and 170 nM in human neutrophils. Inhibition of the epoxide hydrolase activity of isolated human recombinant sEH showed an IC₅₀ of 20 nM. [2]
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| ln Vitro |
Diflapolin potently inhibited the formation of 5-LOX products (LTB₄, its isomers, and 5-H(p)ETE) in calcium ionophore-stimulated human monocytes (IC₅₀ = 30 nM) and neutrophils (IC₅₀ = 170 nM). It did not inhibit the activity of isolated human recombinant 5-LOX or 5-LOX in cell homogenates at concentrations up to 10 µM. [2]
The inhibitory effect of diflapolin on 5-LOX product formation in intact cells was diminished by supplementation of exogenous arachidonic acid (AA), a characteristic feature of FLAP inhibitors. [2] In HEK293 cells, diflapolin inhibited 5-LOX product formation only when 5-LOX was co-expressed with FLAP, but not in cells expressing 5-LOX alone, confirming FLAP as its target. [2] Diflapolin inhibited the epoxide hydrolase activity of human recombinant sEH with an IC₅₀ of 20 nM in a cell-free assay but did not affect the phosphatase activity of sEH at concentrations up to 10 µM. [2] In a cell-based assay using HepG2 cells, diflapolin (1 µM) inhibited the conversion of 14,15-EET to 14,15-DiHETrE by approximately 50%. [2] Diflapolin (up to 10 µM) did not inhibit the activities of other enzymes related to AA metabolism, including COX-1, COX-2, 12-LOX, 15-LOX, LTA₄ hydrolase, LTC₄ synthase, microsomal prostaglandin E synthase-1 (mPGES-1), or cytosolic phospholipase A₂ (cPLA₂), demonstrating high target specificity. [2] Diflapolin (1 µM) prevented the agonist-induced assembly of the 5-LOX/FLAP complex at the nuclear membrane in human monocytes, as visualized by proximity ligation assay, without blocking 5-LOX nuclear translocation. [2] Diflapolin (10 µM) did not affect the viability of human monocytes after 24 or 48 hours of treatment in an MTT assay. [2] |
| ln Vivo |
In vivo investigations have shown that diflapolin (1–10 mg/kg; intraperitoneally; 30 minutes before zymosan administration) displays strong anti-inflammatory effects [1].
In the zymosan-induced peritonitis mouse model, intraperitoneal administration of diflapolin (1, 3, and 10 mg/kg) 30 minutes before zymosan injection significantly reduced peritoneal levels of LTC₄ and LTB₄ in a dose-dependent manner. [2] Diflapolin treatment (10 mg/kg) significantly inhibited neutrophil infiltration into the peritoneal cavity, reduced myeloperoxidase (MPO) activity (to 52.8% of vehicle control), and impaired vascular permeability (to 55.7% of vehicle control). [2] |
| Enzyme Assay |
The epoxide hydrolase activity of human recombinant sEH was assessed in a fluorescence-based cell-free assay. The enzyme was diluted in Tris buffer with BSA and pre-incubated with diflapolin or vehicle for 10 minutes at room temperature. The reaction was started by adding the non-fluorescent substrate PHOME (50 µM). After 60 minutes, the reaction was stopped with ZnSO₄, and the fluorescence of the product (6-methoxy-naphaldehyde) was measured (excitation 330 nm, emission 465 nm). [2]
The phosphatase activity of the sEH N-terminal domain was assessed in a separate fluorescence-based assay. The enzyme was pre-incubated with diflapolin or vehicle in acetate buffer for 30 minutes at room temperature. The reaction was initiated by adding the substrate DiFMUP (300 µM), and the fluorescence of the dephosphorylated product (DiFMU) was monitored for 45 minutes at 37°C (excitation 360 nm, emission 450 nm). [2] For COX activity, purified ovine COX-1 or recombinant human COX-2 were diluted in Tris buffer with glutathione and hemoglobin. After pre-incubation with diflapolin or vehicle for 5 minutes at room temperature and 30 seconds at 37°C, the reaction was started with AA (5 µM for COX-1, 2 µM for COX-2). The reaction was stopped after 5 minutes at 37°C with methanol, and the formation of the product 12-HHT was analyzed by HPLC. [2] |
| Cell Assay |
To assess 5-LOX product formation in intact human leukocytes, freshly isolated neutrophils or monocytes were resuspended in buffer, pre-incubated with diflapolin or vehicle at 37°C for 15 minutes, and then stimulated with calcium ionophore A23187 (2.5 µM) with or without exogenous AA for 10 minutes at 37°C. Reactions were stopped with ice-cold methanol. After solid-phase extraction, 5-LOX products (LTB₄ isomers and 5-H(p)ETE) were analyzed by reversed-phase HPLC. [2]
For the cell-based sEH assay, HepG2 cells were harvested, resuspended in buffer, and pre-incubated with diflapolin or vehicle at 37°C for 15 minutes. Cells were then incubated with the substrate 14,15-EET (1.5 µM) for 30 minutes at 37°C. Reactions were stopped with methanol, and after solid-phase extraction, the conversion to 14,15-DiHETrE was analyzed by UPLC-MS/MS. [2] Arachidonic acid release was measured in [³H]-AA pre-labeled human neutrophils. Cells were pre-incubated with diflapolin or vehicle at 37°C for 10 minutes, stimulated with A23187 (2.5 µM) for 10 minutes, and centrifuged. The radioactivity in the supernatant was measured by scintillation counting. [2] Cell viability was assessed using the MTT assay. Human monocytes were treated with diflapolin or controls for 24 or 48 hours. MTT solution was then added for 2 hours, cells were lysed with SDS, and the absorbance of the formed formazan was measured at 570 nm. [2] Immunofluorescence microscopy and proximity ligation assay (PLA) were used to study 5-LOX subcellular localization and 5-LOX/FLAP complex assembly. Human monocytes on coverslips were pre-treated with diflapolin or vehicle, stimulated with A23187, fixed, permeabilized, and incubated with primary antibodies against 5-LOX and FLAP. For IF, fluorophore-labeled secondary antibodies and DAPI were used. For PLA, specific secondary antibodies with oligonucleotides were used, followed by ligation, rolling-circle amplification, and hybridization with fluorescent oligonucleotides to visualize protein-protein interactions as fluorescent dots. [2] |
| Animal Protocol |
Animal/Disease Models: Male CD-1 mouse (Zymosan-induced peritonitis mouse model) [1]
Doses: 1, 3 and 10 mg/kg Route of Administration: intraperitoneal (ip) injection; 30 minutes before injection of zymosan Experimental Results: from 1 mg/kg dose initiated a significant reduction in peritoneal levels of LTC4 and LTB4, which was comparable to the effect of MK886. In the zymosan-induced peritonitis model, male CD-1 mice were treated intraperitoneally (i.p.) with diflapolin (1, 3, or 10 mg/kg), the reference FLAP inhibitor MK886, or vehicle (0.9% saline containing 2% DMSO) 30 minutes before the i.p. injection of zymosan (2 mg/mL in saline). Peritoneal lavage was performed at specified time points after euthanasia. Exudates were collected for cell counting (using trypan blue) and analysis of LTB₄ and LTC₄ levels (by ELISA) in the supernatant, and myeloperoxidase (MPO) activity in the cell pellet. For vascular permeability assessment, Evans blue dye was injected intravenously immediately after zymosan injection, and its extravasation into the peritoneal lavage fluid was measured by absorbance at 650 nm after 30 minutes. [2] |
| Toxicity/Toxicokinetics |
In the MTT assay, treatment with 10 µM diflapoline for 24 or 48 hours had no effect on the viability of human monocytes. [2]
In a mouse peritonitis model, no significant toxicity was observed at the tested dose (up to 10 mg/kg, intraperitoneal injection). [2] |
| References | |
| Additional Infomation |
Diflapolin (N-[4-(benzothiazol-2-ylmethoxy)-2-methylphenyl]-N′-(3,4-dichlorophenyl)urea) is the first reported dual inhibitor of 5-lipoxygenase-activated protein (FLAP) and soluble epoxide hydrolase (sEH). [2]
Its structure combines a urea group (a common structure for sEH inhibitors) and a benzothiazolium skeleton (similar to FLAP inhibitors). [2] This dual inhibitory mechanism is thought to simultaneously inhibit the production of pro-inflammatory leukotrienes (by inhibiting FLAP) and stabilize anti-inflammatory epoxides (EETs) (by inhibiting sEH), thus providing a potential therapeutic advantage for the treatment of inflammation-related diseases. [2] Diflapolin exhibits high targeting specificity in arachidonic acid cascades and MAPEG family proteins. [2] |
| Molecular Formula |
C22H17CL2N3O2S
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|---|---|
| Molecular Weight |
458.360281705856
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| Exact Mass |
457.041
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| Elemental Analysis |
C, 57.65; H, 3.74; Cl, 15.47; N, 9.17; O, 6.98; S, 6.99
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| CAS # |
724453-98-9
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| PubChem CID |
4560409
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| Appearance |
White to off-white solid powder
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| Density |
1.5±0.1 g/cm3
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| Boiling Point |
543.0±50.0 °C at 760 mmHg
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| Flash Point |
282.2±30.1 °C
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| Vapour Pressure |
0.0±1.5 mmHg at 25°C
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| Index of Refraction |
1.736
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| LogP |
7.05
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
4
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| Rotatable Bond Count |
5
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| Heavy Atom Count |
30
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| Complexity |
587
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| Defined Atom Stereocenter Count |
0
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| SMILES |
ClC1=C(C=CC(=C1)NC(NC1C=CC(=CC=1C)OCC1=NC2C=CC=CC=2S1)=O)Cl
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| InChi Key |
FGXLEECGXSDIMM-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C22H17Cl2N3O2S/c1-13-10-15(29-12-21-26-19-4-2-3-5-20(19)30-21)7-9-18(13)27-22(28)25-14-6-8-16(23)17(24)11-14/h2-11H,12H2,1H3,(H2,25,27,28)
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| Chemical Name |
1-(4-(benzo[d]thiazol-2-ylmethoxy)-2-methylphenyl)-3-(3,4-dichlorophenyl)urea
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| Synonyms |
Diflapolin
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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 : ~83.33 mg/mL (~181.80 mM)
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|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: 2.08 mg/mL (4.54 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 sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 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. Solubility in Formulation 2: ≥ 2.08 mg/mL (4.54 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.1817 mL | 10.9085 mL | 21.8169 mL | |
| 5 mM | 0.4363 mL | 2.1817 mL | 4.3634 mL | |
| 10 mM | 0.2182 mL | 1.0908 mL | 2.1817 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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