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    • In terms of the ring B

    2022-12-15

    In terms of the ring B C5′ position, while C5′-F (compound ) was tolerated, C5′-Me (compound ) and C5′-Cl (compound ) substitutions resulted in a loss in potency. However, the C5′-OMe substitution (compound ) resulted in a ∼3-fold increase in potency compared to . Modeling suggested a pseudo hydrogen bond interaction between the C5′-OMe group and the carbonyl of Gly269 (, b). A C5′-OEt substitution (compound ) resulted in a decrease in potency, speculated to be due to the bulkier ethoxy group and the ensuing desolvation of Asn257, Arg256 and Gly269. The SAR around the NoD linker moiety which connects rings A and B was also explored (). This effort primarily focused on one-atom-length tethers, due to the concern that an extended linker would simply push ring A into a steric clash with the protein, as suggested by modeling and SAR previously established and highlighted in . Various bioisosteric replacements to the oxygen linker in AG-120 sale were generally tolerated with the observation of less than threefold loss of potency (compounds –). Several variants of the carbon based linker such as carbonyl (compound ) and alcohols (compound , ) showed increased potency over compound . This was rationalized through the model where these linkers formed hydrogen bond interactions with Lys158. Several 5,6-cyclized systems (compounds , ) suffered significant losses of potency, speculated to be due to the inability to adopt the required bioactive conformation possessed by (). Subsequently, we successfully obtained a co-crystal structure of compound , a close analog of , with the ACK1 kinase domain (PDB code: ). The interactions revealed in this co-crystal structure essentially confirmed the binding mode proposed by modeling, including the predicted enlargement of the back pocket to accommodate ring A of the NoD moiety (). While expanding the SAR around the NoD moiety, we conducted a mouse PK study on compound to benchmark its DMPK properties. As summarized in , at an oral dose of 20mg/kg, compound displayed good oral bioavailability (50%) with a moderate exposure as indicated by a plasma of 0.9μM. The intravenous clearance was higher than mouse liver blood flow (∼90mL/min/kg), as was predicted by the high mouse hepatic extraction ratio (mouse ER=0.89) observed upon in vitro incubation in microsomes. Furthermore, two significant M+16 metabolites were detected in both oral (∼65% of parent AUC) and intravenous plasma samples (∼20% of parent AUC). Mass fragmentation analysis suggested that both metabolites were produced from hydroxylation of the cyclobutyl southern domain moiety (SoD) via a phase-I mechanism. Compound was synthesized and confirmed as one of the metabolites based on LC–MS/MS (the other metabolite was speculated to be the -isomer). Compound was subsequently synthesized where the C3 position of cyclobutyl moiety was fully substituted in order to block this primary route of metabolism. To our satisfaction, compound maintained similar potency (biochem IC=0.16μM) to that of compound while showing significantly improved in vitro microsomal stability (mouse ER=0.60). A mouse PK study with compound showed significantly improved oral exposure, decreased intravenous clearance with no significant metabolites detected in plasma. Subsequently, a systematic SAR exploration around the SoD (data not shown) revealed that the methyl cyclobutanol moiety shown in compound was a preferred pharmacophoric element for this region of the molecule. After extensive SAR exploration on key pharmacophoric elements as described above, preferred SoD and NoD groups were merged, with a representative list of compounds highlighted in . Compounds with carbonyl and methylene NoD linkers (–) gave both good biochemical and cellular mechanistic potencies but less optimal DMPK properties as indicated by relatively high extraction ratios (low microsomal stability) in both mouse and human liver microsomes. Compound , with a gem-difluoromethylene linker, showed improved microsomal stability, however, it suffered a moderate potency loss. Compound with the alcohol linker motif and compounds – with an oxygen linker demonstrated both good potency and good microsomal stability. Key compounds were progressed to mouse PK studies and the results are summarized in . All three compounds tested had good oral exposures and were highly bioavailable with moderate rates of clearance. It should be noted that in these studies, compound was evaluated as a racemic mixture. The enantiomers were later separated by chiral supercritical fluid chromatography (SFC) and subjected to mouse PK studies. Both enantiomers showed similar PK profiles to , with no racemization detected in vivo by chiral HPLC analysis of the plasma samples.