Novel compounds and pharmaceutical compositions thereof for the treatment of fibrosis
A technology for compounds and solvates, applied in the field of preparing the compounds of the present invention, can solve the problems of interfering with vascular endothelial barrier and the like
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Embodiment 1
[0851] Example 1. Synthesis of intermediates for exemplary compounds of the present invention
[0852] 1.1. Intermediate 1: 4-methyl-2H-phthalazin-1-one
[0853]
[0854] Hydrazine hydrate 78% (41 mL, 635 mmol, 1.3 equiv) was added to a solution of 2-acetylbenzoic acid (80 g, 488 mmol, 1 equiv) in iPrOH (488 mL). The mixture was stirred at 85°C for 1 hour. A precipitate formed and was filtered off. The filtrate was concentrated to give a precipitate which was filtered off. The two precipitates were combined and the resulting solid washed with H 2 O (3×3L) was washed thoroughly. To remove remaining water, the solid was dissolved in THF and the solvent was removed under reduced pressure (2 x 1 L) to give the desired product.
[0855] 1.2. General method A: Alkylation of 4-methylphthalazinone derivatives
[0856]
[0857] 4-Methyl-2H-phthalazin-1-one derivative (1 equivalent), Cs 2 CO 3 (2 equiv) and the 2-bromoester derivative (1.1 equiv) in DMF was stirred at room...
Embodiment 2
[1611] Example 2. In vitro analysis
[1612] 2.1. Ca 2+ analyze
[1613] Administration of sphingosine-1-phosphate triggers S1PR2 leading to intracellular Ca 2+ Instantaneous increase. Ca 2+ Flux analysis by using Ca 2+ Intracellular measurement of Ca with sensitive fluorescent dyes 2+ release. The assay was first performed in agonist mode (incubating compound alone) to ensure that the measured Ca 2+ The release was not caused by agonistic test compounds. Next, the analysis was continued in the antagonist mode (sphingosine-1-phosphate added to the incubation medium containing the test compound).
[1614] 2.1.1. S1PR2 agonist analysis
[1615] CHO cells stably overexpressing the human GPCR sphingosine 1-phosphate receptor 2 (CHO-S1PR2Perkin Elmer; ES-594-A) were seeded from frozen stock into sterile 384-well microplates (50 μL; 7,500 cells / well) and at 37°C and 5% CO 2 Incubate overnight. The next day, cells were washed twice with starvation medium (F-12 Ham's medi...
Embodiment 3
[1784] Example 3 hADME
[1785] 3.1. Water solubility
[1786] Serial dilutions of compounds were prepared in DMSO starting from 10 mM stock solutions in DMSO. The dilution series were transferred to F-bottom 96 NUNC Maxisorb plates and either 0.1 M phosphate buffer pH 7.4 or 0.1 M citrate buffer pH 3.0 were added at room temperature.
[1787] Final concentrations ranged from 18.75 to 300 μM in 5 identical dilution steps. The final DMSO concentration does not exceed 3%.
[1788] 200 μΜ pyrene was added to the corner points of each 96-well plate and used as a reference point on the microscope to correct the Z-axis.
[1789] Assay plates were sealed and incubated for 1 hour at 37°C with shaking at 230 rpm. The plates were then scanned under a white light microscope, producing individual images for each concentration of precipitate. The first concentration at which the compound appears to dissolve completely is the reported concentration; however, the actual concentration is...
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