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Bioconjug Chem. 2021 Apr 21;32(4):794-800. doi: 10.1021/acs.bioconjchem.1c00075. Epub 2021 Apr 6.
β-Elimination of drugs tethered to macromolecular carbamates provides a platform for drug half-life extension. However, the macromolecular Michael acceptor products formed upon drug release can potentially react with biological amines and thiols and may raise concerns about safety. We desired to mitigate this possibility by developing linkers that have predictable rates of β-elimination but suppressed rates of nucleophilic addition to their Michael acceptor products. We prepared Michael acceptor products of β-eliminative linkers that contained a methyl group at the Cβ carbon or a -dimethyl group at the Cγ carbon and studied the kinetics of their reactions with the most prevalent biological nucleophiles-amine and thiol groups. Aza-Michael reactions with glycine are slowed about 20-fold by methylation of the β-carbon and 175-fold with a -dimethyl group at the γ-carbon. Likewise, addition of the glutathione thiol to γ--dimethyl Michael acceptors was retarded 7-24-fold compared to parent unsubstituted linkers. It was estimated that in an in vivo environment of ∼0.5 mM macromolecular thiols or ∼20 mM macromolecular amines-as in plasma-the reaction half-life of a typical Michael acceptor with a γ--dimethyl linker could exceed 3 years for thiols or 25 years for amines. We also prepared a large series of γ--dimethyl β-eliminative linkers and showed excellent structure-activity relationships of elimination rates with corresponding unsubstituted parent linkers. Finally, we compared the first-generation unsubstituted and new -dimethyl β-eliminative linkers in a once-monthly drug delivery system of a 39 amino acid peptide. Both linkers provided the desired half-life extension of the peptide, but the Michael acceptor formed from the -dimethyl linker was much less reactive. We conclude that the γ--dimethyl β-eliminative linkers provide high flexibility and greatly reduce potential reactions of Michael acceptor products with biologically important nucleophiles.
β-消除与大分子碳酸酯连接的药物为延长药物半衰期提供了一个平台。然而，药物释放后形成的大分子迈克尔受体产物可能与生物胺和硫醇发生反应，并可能引发安全性担忧。我们希望通过开发具有可预测β-消除速率但抑制其迈克尔受体产物亲核加成速率的连接物来减轻这种可能性。我们制备了含有β-碳原子上甲基或γ-碳原子上二甲基的β-消除连接物的迈克尔受体产物，并研究了它们与最常见的生物亲核试剂-胺和硫醇基团的反应动力学。β-碳原子的甲基化使氮杂迈克尔反应的反应速率减慢约 20 倍，而γ-碳原子上的二甲基化则减慢 175 倍。同样，与未取代的连接物相比，谷胱甘肽巯基与γ--二甲基迈克尔受体的加成反应延迟了 7-24 倍。据估计，在体内环境中（约 0.5 mM 大分子硫醇或约 20 mM 大分子胺，如在血浆中），具有γ--二甲基连接物的典型迈克尔受体的反应半衰期对于硫醇而言可能超过 3 年，对于胺而言可能超过 25 年。我们还制备了一系列γ--二甲基β-消除连接物，并显示出与相应未取代的母体连接物的消除速率的优异结构-活性关系。最后，我们在一个 39 个氨基酸肽的每月一次药物递送系统中比较了第一代未取代和新型的γ--二甲基β-消除连接物。两种连接物都为肽提供了所需的半衰期延长，但来自γ--二甲基连接物的迈克尔受体产物的反应性要低得多。我们得出结论，γ--二甲基β-消除连接物提供了高度的灵活性，并大大降低了迈克尔受体产物与生物重要亲核试剂潜在反应的可能性。
