The conjugate N-adducts of thio-1,3,4-diazole and 2-thiazoline with levoglucosenone were synthesized via a stereoselective, base-catalyzed conjugate N-Michael addition to levoglucosenone at C-4. Structural assignments were established using 1H and 13C NMR analysis, and X-ray single-crystal analysis for one of the compounds. The biological properties of the novel compounds were tested on a cell model. Cytotoxicity was analyzed via colorimetric assay. Two distinct types of cell death, apoptosis and necrosis, were analyzed by determining the phosphatidylserine levels from the outer leaflet of the plasma membrane, caspase activation, and lactate dehydrogenase release. We also evaluated DNA damage using an alkaline comet assay. The level of oxidative stress was measured with a modified comet assay and an H2DCFDA probe. The thio-1,3,4-diazole adduct (FCP23) and the 2-thiazoline adduct (FCP26) exhibit similar cytotoxicity values for cancer cells (ovarian (A2780), breast (MCF-7), cervix (HeLa), colon (LoVo), and brain (MO59J and MO59K)), but their mechanism of action is drastically different. While FCP23 induces oxidative stress, DNA damage, and necrosis, FCP26 induces apoptosis through caspase activation.
通过立体选择性碱催化共轭N-迈克尔加成反应，在左旋葡烯糖酮C-4位合成了硫代-1,3,4-二唑与2-噻唑啉的共轭N-加合物。利用氢谱、碳谱核磁共振分析及其中一种化合物的X射线单晶衍射确定了结构。通过细胞模型测试了新型化合物的生物学特性：采用比色法分析细胞毒性；通过测定质膜外叶磷脂酰丝氨酸水平、半胱天冬酶活化及乳酸脱氢酶释放量，分析了细胞凋亡与坏死两种死亡模式；采用碱性彗星实验评估DNA损伤；通过改良彗星实验与H2DCFDA探针检测氧化应激水平。硫代-1,3,4-二唑加合物（FCP23）与2-噻唑啉加合物（FCP26）对卵巢癌（A2780）、乳腺癌（MCF-7）、宫颈癌（HeLa）、结肠癌（LoVo）及脑胶质瘤（MO59J和MO59K）细胞系表现出相近的细胞毒性，但作用机制截然不同：FCP23通过诱发氧化应激、DNA损伤导致细胞坏死，而FCP26通过激活半胱天冬酶途径诱导细胞凋亡。
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