PIM-1/MNKs激酶双重抑制剂的设计合成与抗肿瘤活性研究

A series of novel 4,6-bis-substituted pyridine-triazines and 4,6-bis-substituted pyridine-pyrimidines will be designed and synthesized by analyzing the characteristics of PIM-1 and MNKs kinases active pockets, which is based on compounds PTZ510 and PPM103 with moderate inhibitory activities against PIM-1 and MNK2. The inhibitory effects of the compounds towards PIM-1 and MNKs will be determined, the anti-proliferation, cell cycle arrest and apoptosis induction effects against target-high-expressing tumor cell lines will be investigated, and the effects upon PIM-1/MNKs signaling will be studied through evaluating the levels of p-BAD、p-eIF4E、p-4E-BP1. Dual PIM-1/MNKs inhibitors will be further studied with their in vivo antitumor activities. We aim to develop potent and selective dual PIM-1/MNKs inhibitors as novel antitumor agents.

以前期研究得到的具有PIM-1和MNK2双重抑制活性的PTZ510和PPM103为先导化合物，基于PIM-1与MNK激酶活性口袋的结构特征，设计合成4,6-双取代吡啶并三嗪类化合物和4,6-双取代吡啶并嘧啶类化合物；进行PIM-1/MNKs激酶活性筛选，选取PIM-1/MNKs高表达的肿瘤细胞株考察抑制剂的生长抑制、周期阻滞及诱导凋亡作用；选择活性化合物，考察其对PIM-1/MNKs通路中p-BAD、p-eIF4E、p-4E-BP1等蛋白表达的影响，确定该类化合物的作用机制；探讨PIM-1/MNKs双靶点抑制剂的体内抗肿瘤效果，寻找活性强选择性好的PIM-1/MNKs激酶双重抑制剂。

在前期研究的基础上，综合考虑MNK和PIM两种激酶ATP结合位点氨基酸残基的特点，采用基于片段的药物设计方法，设计并合成104个4,6-双取代吡啶并[3,2-d]嘧啶类化合物，开展活性筛选，总结构效关系，得到活性显著化合物84和99。化合物84（(4-(4-((4-氟-2-异丙氧基苯基)氨基)吡啶并[3,2-d]嘧啶-6-基)苯基)(哌嗪-1-基)甲酮）和化合物99（(S)-(4-(4-((4-氟苯基)氨基)吡啶并[3,2-d]嘧啶-6-基)苯基)(3-甲基哌嗪-1-基)甲酮）对MNK均表现出较强的抑制效果，IC50值在个位数纳摩尔水平（84：MNK1 1.0 nM，MNK2 7.0 nM；99：MNK1 0.4 nM, MNK2 9 nM）。化合物84对PIM也表现出较好的抑制活性（PIM1 43 nM, PIM2 232 nM, PIM3 774 nM）。化合物84在Molm-13和K562细胞中能够剂量依赖性下调MNK和PIM的下游底物eIF4E、BAD和4E-BP1的磷酸化水平，也能下调与肿瘤细胞存活相关蛋白c-myc、cyclin-D1和Mcl-1的表达，且使细胞周期阻滞在G0/G1期，并促进细胞凋亡。化合物84对小鼠Molm-13异种移植模型表现出较好的体内抗肿瘤效果，腹腔注射给药5和10 mg/kg时，抑瘤率分别为41.22%和57.65%。细胞水平和体内实验结果表明化合物84为全新结构的PIM/MNK双靶点抑制剂，具有进一步研究的价值。