sEH

Inflammatory responses are orchestrated by a plethora of lipid mediators, and perturbations of their biosynthesis or degradation hinder resolution and lead to uncontrolled inflammation, which contributes to diverse pathologies. Small molecules that induce a switch from pro-inflammatory to anti-inflammatory lipid mediators are considered valuable for the treatment of chronic inflammatory diseases. Commonly used non-steroidal anti-inflammatory drugs (NSAIDs) are afflicted with side effects caused by the inhibition of beneficial prostanoid formation and redirection of arachidonic acid (AA) into alternative pathways. Multi-target inhibitors like diflapolin, the first dual inhibitor of soluble epoxide hydrolase (sEH) and 5-lipoxygenase-activating protein (FLAP), promise improved efficacy and safety but are confronted by poor solubility and bioavailability. Four series of derivatives bearing isomeric thiazolopyridines as bioisosteric replacement of the benzothiazole core and two series additionally containing mono- or diaza-isosteres of the phenylene spacer were designed and synthesized to improve solubility. The combination of thiazolo[5,4-b]pyridine, a pyridinylen spacer and a 3,5-Cl2-substituted terminal phenyl ring (46a) enhances solubility and FLAP antagonism, while preserving sEH inhibition. Moreover, the thiazolo[4,5-c]pyridine derivative 41b, although being a less potent sEH/FLAP inhibitor, additionally decreases thromboxane production in activated human peripheral blood mononuclear cells. We conclude that the introduction of nitrogen, depending on the position, not only enhances solubility and FLAP antagonism (46a), but also represents a valid strategy to expand the scope of application towards inhibition of thromboxane biosynthesis.[1]

nhibitory activity on isolated sEH[1]
Human recombinant sEH was expressed and purified and epoxide hydrolase activity was determined as previously reported. Briefly, isolated sEH was diluted in 25 mM Tris buffer (pH 7) containing 0.1 mg/mL BSA and pre-incubated for 10 min with vehicle (0.1% DMSO, v/v), compounds or the control inhibitor AUDA (1 µM) at RT. The reaction was initiated by addition of 3-phenyl-cyano-(6-methoxy-2-naphthalenyl)methylester-2-oxiraneacetic acid (PHOME) (50 µM), stopped after 60 min with 200 mM ZnSO4, and the resulting fluorescence was detected (λem = 465 nm and λex = 330 nm). In case of fluorescent test compounds, the read-out was corrected by subtracting their internal fluorescence.[1]

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Isolated 5-LOX assay[1]
Human recombinant 5-LOX (30 U) was pre-incubated with vehicle (DMSO, 0.1%) or test compounds plus Triton X-100 for 10 min on ice (1 mL PBS pH 7.4 plus 1 mM EDTA and 1 mM ATP) before CaCl2 (2 mM) and AA (20 µM) were added. The enzymatic reaction was stopped after 10 min at 37 °C with ice-cold methanol (1 mL), and PGB1 (2 ng) was added as internal standard. Samples were acidified (530 µL PBS pH 7.4 plus 60 mM HCl), centrifuged (750 × g, 10 min, 4 °C), and loaded onto Clean-Up C18 Endcapped SPE cartridges (100 mg, 10 mL), which were conditioned with MeOH (1 mL, twice) and equilibrated with water (1 mL). After washing with water (1 mL) and MeOH/water (25:75, 1 mL), oxylipins were eluted with MeOH (300 µL) and diluted by addition of water (120 µL). Samples were centrifuged (21,100 × g, 10 min, 4 °C) and subjected to UPLC-PDA analysis. 5-LOX products, including all trans-isomers of LTB4 and 5-HETE, were separated on a Kinetex C-18 LC column (100 Å, 1.3 µm, 2.1 × 50 mm) using a Nexera X2 UHPLC system at a flow rate of 0.45 mL/min and 40 °C oven temperature. A step gradient of mobile phase A (water/methanol, 50/50, 0.05% trifluoroacetic acid) and mobile phase B (methanol, 0.05% trifluoroacetic acid) was employed for the chromatographic separation: 14% B for 2 min, followed by 46% B for 2 min and 90% B for another 2 min. LTB4 isomers and 5-HETE were detected with a photodiode array detector at 280 nm and 235 nm, respectively. Lipid quantities refer to an internal calibration using PGB1 as reference standard.

Inhibitory activity on 5-LOX product formation in PMNLs[1]
The inhibitory activity on 5-LOX product formation was evaluated in a cell-based assay using freshly isolated human neutrophils (5 × 106 cells per mL) obtained from leukocyte concentrates of healthy and fasted adult donors, provided by the Institute for Transfusion Medicine of the University Hospital Jena, as previously described. Briefly, leukocyte concentrates were prepared from freshly withdrawn blood by centrifugation (4,000 × g, 20 min, 20 °C). PMNLs were subsequently isolated by dextran sedimentation, purified via density gradient centrifugation and hypotonic lysis of erythrocytes. Cells were resuspended in PGC buffer (PBS pH 7.4, glucose 0.1%, CaCl2 1 mM), preincubated with vehicle (0.1% DMSO, v/v), compounds or MK-886 (0.03 µM) for 15 min (37 °C) and treated with the Ca2+-ionophore A23187 (2.5 µM) for 10 min (37 °C). The reaction was terminated with an equal volume of ice-cold methanol, the samples were acidified, PGB1 (200 ng) was added as internal standard, and formed lipid mediators were purified by solid phase extraction using C18 RP-columns (100 mg). 5-LOX products were chromatographically separated on a C-18 Radial-PAK column and quantified at 235 or 280 nm, as described before

[1]. Novel thiazolopyridine derivatives of diflapolin as dual sEH/FLAP inhibitors with improved solubility. Bioorg Chem. 2023 Oct;139:106685.

C19H13CL2N5O2S

446.31

Typically exists as solid at room temperature

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)