Physicochemical Properties
| Molecular Formula | C20H19N3O3 |
| Molecular Weight | 349.38 |
| Exact Mass | 349.142 |
| Elemental Analysis | C, 68.75; H, 5.48; N, 12.03; O, 13.74 |
| CAS # | 2407782-01-6 |
| PubChem CID | 138319675 |
| Appearance | White to light yellow solid |
| Density | 1.3±0.1 g/cm3 |
| Boiling Point | 596.3±50.0 °C at 760 mmHg |
| Flash Point | 314.4±30.1 °C |
| Vapour Pressure | 0.0±1.7 mmHg at 25°C |
| Index of Refraction | 1.647 |
| LogP | 2.25 |
| Hydrogen Bond Donor Count | 1 |
| Hydrogen Bond Acceptor Count | 4 |
| Rotatable Bond Count | 3 |
| Heavy Atom Count | 26 |
| Complexity | 503 |
| Defined Atom Stereocenter Count | 0 |
| SMILES | O1C2C=C(C=CC=2CN2C(=CC(C3C=CC=CC=3OC)=N2)C1)NC(C)=O |
| InChi Key | IFOIINXSFCQLOV-UHFFFAOYSA-N |
| InChi Code | InChI=1S/C20H19N3O3/c1-13(24)21-15-8-7-14-11-23-16(12-26-20(14)9-15)10-18(22-23)17-5-3-4-6-19(17)25-2/h3-10H,11-12H2,1-2H3,(H,21,24) |
| Chemical Name | N-(2-(2-Methoxyphenyl)-4H,10H-benzo[f]pyrazolo[5,1-c][1,4]oxazepin-7-yl)acetamide |
| HS Tariff Code | 2934.99.9001 |
| 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) |
Biological Activity
| Targets | Gasdermin D[1]. |
| ln Vitro | LDC7559 (1 and 5 μM) dramatically reduces the lethal effect of human and mouse GSDMD NT domains transfected into HEK293T cells and decreases the release of IL-1β upon inflammasome activation [3]. Rather than interfering with the activation and cleavage mechanism, LDC7559 acts directly by preventing the GSDMD NT domain from functioning [3]. |
| Cell Assay |
Transfection of GSDMD constructs in HEK293T cells [2] HEK293T cells were seeded the day before transfection at a density of 5 × 105 cells/ml. Transfection was done in Opti-MEM using 2 μg of DNA and 7 μl of Lipofectamine 2000 for a six-well plate or respective scaling for other surface areas. LDC7559 or caspase inhibitors were added 2 hours after transfection, and lactate dehydrogenase (LDH) release of cells was measured 16 hours after transfection. Incubation of HEK293T lysates with recombinant proteases[2] HEK293T cell lysate was harvested 16 hours after transfection by changing the medium of a six well to 200 μl of Opti-MEM containing 0.05% (v/v) Triton X-100 and scraping cells off the bottom of the well. After 10 min of centrifugation at full speed and 4°C in a tabletop centrifuge, supernatants were used for cleavage assays. Fifty microliters of lysate were incubated with 10 mU of purified human NE, 2 U of recombinant caspase-4, 4 μg of purified CG (RP-77525), or 6.25 μg of recombinant PR3 (C628) for the indicated time points at 25°C on a shaking incubator. Reactions were stopped by directly adding 50 μl of 2× SDS loading buffer (at 98°C) containing 200 mM DTT and by immediately boiling samples before loading on SDS–polyacrylamide gel electrophoresis gels. |
| Animal Protocol |
Live imaging of neutrophils [2] Cells were resuspended in an assay medium supplemented with 500 nM SYTOX Green and 2.5 μM DRAQ5 and seeded at a density of 5 × 105 cells per well into μ-Slide 8 Well ibiTreat dishes (ibidi). After LDC7559 treatment and NET induction, images were collected at 2-min intervals on a Leica TCS SP8 confocal microscope equipped with a climate chamber at 37°C and with a Leica HC PL APO 20×/0.75 IMM CORR CS2 objective using glycerol immersion. The recording of DRAQ5 (cell-permeable DNA dye) was used to track individual nuclei over time and to determine nuclear area. The recording of SYTOX Green (cell-impermeable dye) was used to determine permeability of cells. Bright-field recording was added for a video representation of the data. Automated algorithms were used to determine the time point of permeabilization and to categorize nuclei into “non-expanded,” “fully expanded” (filling the entire cell), or “NET” (extending beyond the maximal area of a cell). The results were then mapped back to a video representation of the data and manually inspected for accuracy before they were used for further analysis. The complete image analysis pipeline consisting of ImageJ and R scripts will be described elsewhere in detail. |
| References |
[1]. Gasdermin D opens the way for NETs. Nat Rev Rheumatol. 2018 Dec;14(12):690-692. [2]. Gasdermin D plays a vital role in the generation of neutrophil extracellular traps. Sci Immunol. 2018 Aug 24;3(26). pii: eaar6689. [3]. Gasdermin D (GSDMD) as a New Target for the Treatment of Infection. Medchemcomm. 2019 Apr 4;10(5):660-667. |
Solubility Data
| Solubility (In Vitro) | DMSO : 100 mg/mL (286.22 mM) |
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (5.95 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 20.8 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: ≥ 2.08 mg/mL (5.95 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 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 3: ≥ 2.08 mg/mL (5.95 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 | 2.8622 mL | 14.3111 mL | 28.6221 mL | |
| 5 mM | 0.5724 mL | 2.8622 mL | 5.7244 mL | |
| 10 mM | 0.2862 mL | 1.4311 mL | 2.8622 mL |