Legraverend M, Ludwig O, Bisagni E, Leclerc S, Meijer L, Giocanti N, Sadri R, Favaudon V
UMR 176 CNRS-IC, Institut Curie-Recherche, Centre Universitaire, Orsay, France.
Bioorg Med Chem. 1999 Jul;7(7):1281-93. doi: 10.1016/s0968-0896(99)00064-4.
Novel C-2, C-6, N-9 trisubstituted purines derived from the olomoucine/roscovitine lead structure were synthesized and evaluated for their ability to inhibit starfish oocyte CDK1/cyclin B, neuronal CDK5/p35 and erk1 kinases in purified extracts. Structure activity relationship studies showed that increased steric bulk at N-9 reduces the inhibitory potential whereas substitution of the aminoethanol C-2 side chain by various groups of different size (methyl, propyl, butyl, phenyl, benzyl) only slightly decreases the activity when compared to (R)-roscovitine. Optimal inhibitory activity against CDK5, CDK1 and CDK2, with IC50 values of 0.16, 0.45 and 0.65 microM, respectively, was obtained with compound 21 containing a (2R)-pyrrolidin-2-yl-methanol substituent at the C-2 and a 3-iodobenzylamino group at the C-6 of the purine. Compound 21 proved cytotoxic against human tumor HeLa cells (LD50-6.7 microM versus 42.7 microM for olomoucine, 24-h contact). Furthermore, unlike olomoucine, compound 21 was effective upon short exposure (LD50= 25.3 microM, 2-h contact). The available data suggest that the affinity for CDKs and the cytotoxic potential of the drugs are inter-related. However, no straightforward cell cycle phase specificity of the cytotoxic response to 21 was observed in synchronized HeLa cells. With the noticeable exception of pronounced lengthening of the S-phase transit by 21 applied during early-S in synchronized HeLa cells, and in striking contrast with earlier reports on studies using plant or echinoderm cells. olomoucilnc and compound 21 were unable to reversibly arrest cell cycle progression in asynchronous growing HeLa cells. Some irreversible hlock in GI and G2 phase occurred at high olomoucine concentration, correlated with induced cell death. Moreover, chmronic exposure to lethal doses of compound 21 resulted in massive nuclear fragmentation, evocative of mitotic catastrophe with minour amounts of apoptosis only. It was also found that olomoucine and compound 21 reversibly block the intracellular uptake of nuicleosides with high efficiency.
合成了源自olomoucine/roscovitine先导结构的新型C-2、C-6、N-9三取代嘌呤，并评估了它们在纯化提取物中抑制海星卵母细胞CDK1/细胞周期蛋白B、神经元CDK5/p35和erk1激酶的能力。构效关系研究表明，N-9位空间位阻增加会降低抑制潜力，而与(R)-roscovitine相比，用不同大小的各种基团（甲基、丙基、丁基、苯基、苄基）取代氨基乙醇C-2侧链只会轻微降低活性。对于在嘌呤的C-2位含有(2R)-吡咯烷-2-基-甲醇取代基且在C-6位含有3-碘苄基氨基的化合物21，分别获得了对CDK5、CDK1和CDK2的最佳抑制活性，IC50值分别为0.16、0.45和0.65 microM。化合物21对人肿瘤HeLa细胞具有细胞毒性（LD50为6.7 microM，而olomoucine为42.7 microM，24小时接触）。此外，与olomoucine不同，化合物21在短时间接触时有效（LD50 = 25.3 microM，2小时接触）。现有数据表明，药物对CDK的亲和力和细胞毒性潜力是相互关联的。然而，在同步化的HeLa细胞中未观察到对21的细胞毒性反应有直接的细胞周期阶段特异性。除了在同步化的HeLa细胞早期S期应用21明显延长S期转运时间这一显著例外，并且与早期关于使用植物或棘皮动物细胞的研究报告形成鲜明对比，olomoucilnc和化合物21无法在异步生长的HeLa细胞中可逆地阻止细胞周期进程。在高浓度olomoucine下，G1期和G2期会出现一些不可逆的阻滞，这与诱导的细胞死亡相关。此外，长期暴露于致死剂量的化合物21会导致大量核碎裂，这让人联想到有少量凋亡的有丝分裂灾难。还发现olomoucine和化合物21能高效可逆地阻断核苷的细胞内摄取。
