5-carboxamido substitued thiazole derivatives that interact with ion channels, in particular with ion channels from the kv family
A technology of metabolites and alkyl groups, which is applied in the field of preparing this pharmaceutical composition and preparing the compound, and can solve problems such as non-drugs
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Embodiment 1
[0237] Example 1: Preparation of compounds according to the invention
[0238] The practice of the present invention will employ, unless otherwise indicated, conventional techniques in the fields of synthetic organic chemistry, biological testing and the like, which are within the skill of the art. Such techniques are explained fully in the literature. Unless otherwise indicated, the purity of the compounds was confirmed by liquid chromatography / mass spectrometry (LC / MS) according to Method A or Method B as follows:
[0239] Method A (5 minute gradient):
[0240] HPLC: Waters Alliance 2690 with Photo=- / Diode Array Detector Waters 996. Mass spectrometer: Micromass Platform ZMD LC. Ionization: Electrospray (polarity: negative and positive).
[0241] method:
[0242] Phase: Tosohaas TSK-gel super ODS ( 2 μ m), column: 4.6 * 50mm; Solvent A: water and formic acid (26.5mM); Solvent B: acetonitrile and formic acid (17mM); Flow rate: 2.75ml / min; Gradient 5 minutes: from 100% A...
Embodiment 2
[0474] Example 2: Biological Assay Using C. elegans Screening
[0475] A C. elegans-based high-throughput screen for Kv4.3 modulators has been used to establish the in vivo SAR (structure-activity relationship: effect of chemical structure on biological activity) of compounds according to the invention for Kv4.3.
[0476] The test employed a stable transgenic C. elegans strain that functionally expresses human Kv4.3 in the pharynx and a visual selection GFP marker in the body wall muscle.
[0477] Methods describing the construction of transgenic C. elegans strains expressing human Kv4.3 are described in WO03 / 097682. Briefly, 5 ng / μl pGV8 plasmid (human Kv4.3), 20 ng / μl pDW2821 (GFP-tagged), and 40 ng / μl genomic C. elegans DNA were microinjected into the gonads of wild-type strain N2 to generate practical The strain UG1755. Transgenic animals were isolated and extrachromosomal arrays were integrated into the C. elegans genome. 50% of the cell lines delivering functionally e...
Embodiment 3
[0490] Example 3: Patch clamp analysis
[0491] Cell culture:
[0492] For this assay, a recombinant CHO-K1 cell line stably expressing the human Kv4.3 / KChIP2.2 potassium channel was used. The cells used in this experiment were grown under standard conditions (37°C, supplemented with 7% CO 2air) maintained in continuous culture. CHO-K1 Kv4.3 / KChIP2.2 cells were maintained in Iscove's modified DMEM (Dulbecco's modified Eagle's medium) medium (IMEM), in which the medium was supplemented with 100 U / ml penicillin, 100 μg / ml streptomycin, 7% Fetal calf serum (FCS), 2.5 μg / ml amphotericin, 400 μg / ml G418 and 400 μg / ml Zeocin TM . Cells were passaged every 3-4 days after detachment with trypsin solution. Ensure the quality of cultured cells with viability and contamination testing. Cell culture was performed as described in Protocol D hereinafter.
[0493] Protocol D: Cells in 5% CO 2 and cultured in 94mm Petri dishes at 37°C. Subcultures were performed every 3-4 days by r...
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