N-Acylphosphatidylethanolamine phospholipase D (NAPE-PLD) is regarded as the main enzyme responsible for the biosynthesis of N-acylethanolamines (NAEs), a family of bioactive lipid mediators. Previously, we reported N-(cyclopropylmethyl)-6-((S)-3-hydroxypyrrolidin-1-yl)-2-((S)-3-phenylpiperidin-1-yl)pyrimidine-4-carboxamide (1, LEI-401) as the first potent and selective NAPE-PLD inhibitor that decreased NAEs in the brains of freely moving mice and modulated emotional behavior [Mock Nat Chem. Biol., 2020, 16, 667−675]. Here, we describe the structure–activity relationship (SAR) of a library of pyrimidine-4-carboxamides as inhibitors of NAPE-PLD that led to the identification of LEI-401. A high-throughput screening hit was modified at three different substituents to optimize its potency and lipophilicity. Conformational restriction of an N-methylphenethylamine group by replacement with an (S)-3-phenylpiperidine increased the inhibitory potency 3-fold. Exchange of a morpholine substituent for an (S)-3-hydroxypyrrolidine reduced the lipophilicity and further increased activity by 10-fold, affording LEI-401 as a nanomolar potent inhibitor with drug-like properties. LEI-401 is a suitable pharmacological tool compound to investigate NAPE-PLD function in vitro and in vivo.

中文翻译：

---

嘧啶-4-羧酰胺作为N-酰基磷脂酰乙醇胺磷脂酶D抑制剂的构效关系研究

N-酰基磷脂酰乙醇胺磷脂酶D（NAPE-PLD）被认为是负责生物活性脂质介体家族N-酰基乙醇胺（NAE）生物合成的主要酶。以前，我们报道了N-（环丙基甲基）-6-（（S）-3-羟基吡咯烷-1-基）-2-（（S）-3-苯基哌啶-1-基）嘧啶-4-羧酰胺（1，LEI -401）是第一种有效且选择性的NAPE-PLD抑制剂，可降低自由移动小鼠大脑中的NAE并调节情绪行为[嘲笑 纳特化学 生物学 ， 2020， 16，667-675]。在这里，我们描述作为NAPE-PLD抑制剂的嘧啶-4-羧酰胺库的结构-活性关系（SAR），这导致了LEI-401的鉴定。在三个不同的取代基上修饰了高通量筛选命中物，以优化其效力和亲脂性。通过用（ S）-3-苯基哌啶取代来构象限制N-甲基苯乙胺基团将抑制能力提高了3倍。（ S）-3-羟基吡咯烷的吗啉取代基交换降低了亲脂性，并进一步将活性提高了10倍，提供了LEI-401作为具有药物样特性的纳摩尔强效抑制剂。LEI-401是一种合适的药理学工具化合物，可用于研究NAPE-PLD在体外和体内的功能。