Tandutinib (TND) is a novel, oral small molecule designed for treating acute myeloid leukemia (AML) by inhibiting type III receptor tyrosine kinases. This study reports the use of in silico, in vivo, and in vitro methods to investigate the metabolism and possible metabolic bioactivation of TND. First, in silico metabolism of TND was assessed using the WhichP450™ module of the StarDrop® software to determine labile sites of metabolism in the TND chemical structure. Second, the XenoSite reactivity model, a web-based metabolism prediction software, was used to determine probable bioactive centers. Based on the in silico outcomes, a list of predicted metabolites and reactive intermediates were prepared. Third, in vitro and in vivo experiments were performed. In vitro TND metabolites were generated through incubation of TND with rat liver microsomes (RLMs). Another incubation of TND with RLMs was separately performed in the presence of GSH and KCN to check for the generation of reactive intermediates (soft and hard electrophiles). In vitro phase II metabolism was assessed by incubation of TND with isolated perfused rat hepatocytes. In vivo metabolism was investigated by oral gavage of TND (37 mg kg⁻¹) in Sprague Dawley rats. Five in vitro phase I metabolites, one in vitro phase II and five reactive iminium intermediates (cyano adducts), six in vivo phase I, and one in vivo phase II metabolites of TND were characterized. The in vitro and in vivo metabolic pathways involved were O-dealkylation, α-hydroxylation, α-carbonyl formation, reduction, glucuronide, and sulfate conjugation. No GSH conjugate or its catabolic products were detected either in vitro or in vivo. Two cyclic tertiary rings of TND (piperazine and piperidine) were metabolically bioactivated to generate reactive iminium intermediates forming cyano adducts with KCN. The formed reactive intermediates may be the reason behind TND toxicity. In silico toxicological studies were performed for TND and its related (in vitro and in vivo) metabolites were evaluated using the DEREK software tool.

中文翻译：

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使用液相色谱离子阱质谱法鉴定和研究坦度替尼的体外，体内和反应性代谢产物

Tandutinib（TND）是一种新颖的口服小分子药物，旨在通过抑制III型受体酪氨酸激酶来治疗急性髓细胞性白血病（AML）。这项研究报告了使用计算机模拟，体内和体外方法研究TND的代谢和可能的代谢生物活化作用。首先，使用StarDrop®软件的WhereP450™模块评估TND的计算机代谢，以确定TND化学结构中不稳定的代谢位点。其次，使用基于网络的新陈代谢预测软件XenoSite反应性模型来确定可能的生物活性中心。基于计算机模拟结果，准备了一系列预测的代谢物和反应性中间体。第三，进行了体外和体内实验。通过将TND与大鼠肝微粒体（RLM）孵育，可以生成体外TND代谢产物。在GSH和KCN的存在下分别进行TND与RLM的另一次孵育，以检查反应性中间体（软和硬亲电试剂）的生成。通过将TND与分离的灌注大鼠肝细胞孵育来评估体外II期代谢。通过口服灌胃TND（37 mg kg ^-1）在Sprague Dawley大鼠中研究体内代谢。五种体外I期代谢产物，一种体外II期代谢产物和五种反应性亚胺中间体（氰基加合物），六种表征了TND的体内I期和II种体内II期代谢产物。涉及的体外和体内代谢途径是O-脱烷基化，α-羟基化，α-羰基形成，还原，葡糖醛酸苷和硫酸盐结合。在体外或体内均未检测到GSH结合物或其分解产物。TND的两个环状叔环（哌嗪和哌啶）被代谢生物活化以生成反应性亚胺中间体，与KCN形成氰基加合物。形成的反应性中间体可能是TND毒性的原因。在计算机上对TND及其相关的毒理学研究（在体外和在体内）的代谢物使用德里克软件工具进行评价。