Abstract: Mixed Lineage Kinase domain-Like pseudokinase (MLKL) is implicated in a broad range of diseases due to its role as the ultimate effector of necroptosis and has therefore emerged as an attractive drug target. Here, we describe the development of PROteolysis TArgeting Chimeras (PROTACs) as a novel approach to knock down MLKL through chem. means. A series of candidate degraders were synthesized from a high-affinity pyrazole carboxamide-based MLKL ligand leading to the identification of a PROTAC mol. that effectively degraded MLKL and completely abrogated cell death in a TSZ model of necroptosis. By leveraging the innate ability of these PROTACs to degrade MLKL in a dose-dependent manner, the quant. relationship between MLKL levels and necroptosis was interrogated. This work demonstrates the feasibility of targeting MLKL using a PROTAC approach and provides a powerful tool to further our understanding of the role of MLKL within the necroptotic pathway.
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With N-ethyl-N,N-diisopropylamine; N-[(dimethylamino)-3-oxo-1H-1,2,3-triazolo[4,5-b]pyridin-1-yl-methylene]-N-methylmethanaminium hexafluorophosphate; In N,N-dimethyl-formamide; at 20℃; for 1h;
Methyl 3-(2-aminoacetamido)-4-fluorobenzoate (I-58)HATU (8.2 mmol) was added to a mixture of N-Boc glycine (6.8 mmol), <strong>[369-26-6]methyl 3-amino-4-fluorobenzoate</strong> (6.2 mmol), diisopropylethylamine (3.6 mL, 20.5 mmol) and DMF (15 mL). The reaction was stirred at room temperature for 1 hr. LCMS indicated that the reaction was complete. The reaction was diluted with water and extracted with ethyl acetate. The organic extracts were dried over MgSC and concentrated. No further purification was necessary. The crude amide (I-57) (9.2 mmol) was dissolved in dichloromethane (15 mL) and treated with trifluoroacetic acid (8 mL). The reaction stirred at room temperature for 1 hr. LCMS indicated that the reaction was complete. The reaction was concentrated to dryness and the crude material was purified by preparative HPLC to give I-58.
To a 40 mL reaction vessel was added reagents in the following order: N-(tert- butoxycarbonyl)glycine (1.0 mmol), THF (4.0 mL), N-methyl morpholine (c.a. 0.2 mL, 2.0 mmol). This was stirred until complete dissolution. Next was added 2-chloro-4,6-dimethoxy-1,3,5- triazine (1.0 mmol) and this solution was stirred for 20 mins at 50 °C until a white precipitate fully formed. The precipitate was physically agitated to ensure all solids were well stirred. Next was added <strong>[369-26-6]methyl 3-amino-4-fluorobenzoate</strong> (1.3 mmol) and the reaction was stirred for 30 mins at 50 °C and then at room temperature for 2 hrs, then diluted with 5 mL of MeOH and then purified by being poured into water (rapidly stirred, 25 mL). A white solid was collected and treated with dichloromethane (5 mL) and TFA (5 mL), and heated to 34 °C for about 1 hr until removal of the Boc group was complete (by LCMS monitoring). The material was concentrated to dryness and subjected to reverse-phase chromatography (10 to 40 percent water/ACN) to give I-69. The desired fractions were then passed through polymer bound ion- exchange free base cartridges to removal any residual TFA (using product Stratospheres, SPE PL HC03 MP SPE 0.9 mmol nominal load x 2 units). Mobilizing eluent was MeOH (10 mL total flush volume).
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