5-氮杂-5'-脱氧胞苷的合成工艺优化及其对SKOV3作用的研究
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摘要
多数肿瘤细胞的DNA甲基转移酶(DNA methyltransferase, DNMT)水平表达明显高于正常细胞,并与抑癌基因启动子高甲基化状态相关,DNMT抑制剂类药物是一类通过抑制DNMT的活性、逆转基因的异常甲基化状态,从而达到抗癌效果的药物。
胞苷类似物作为DNA甲基化酶抑制剂,能够逆转基因的甲基化状态,从而恢复基因的表达水平,纠正肿瘤细胞的异常生物学特征,在肿瘤研究中受到极大的关注。本课题前期以双氰胺为原料合成、分离得到5-氮杂-5'-脱氧胞(5-Aza-5'- deoxycytidine, 5A5DC),实验证实了β-异构体有着较强的生物活性。但在合成方面,存在催化剂价格过高、反应时间长、纯化方法复杂等缺点。因此本文旨在优化其合成工艺、降低成本、提高产率,并探讨5A5DC对SKOV3的增殖及细胞周期的影响。
具体研究工作分为以下两个部分:
1. 5-氮杂-5'-脱氧胞苷(5A5DC)的合成及工艺优化
通过分析各种核苷类化合物的合成方法,确定了本文的合成路线: 5-氮杂胞嘧啶经硅烷化保护,在路易斯酸作用下与1,2,3-三乙酰基-5-脱氧-D-核糖进行偶联得2',3'-二乙酰氧基-5'-脱氧-5-氮杂胞苷,最后经碱性脱保护、乙酸乙酯重结晶得5A5DC。通过正交设计,比较在Hilbert-Johnson偶联反应中不同Lewis酸、溶剂和反应时间对合成5A5DC的影响。以无水SnCl4为Lewis酸,1,2-二氯乙烷为催化剂、反应时间为8 h时,5A5DC的收率最高。以双氰胺计,总收率为31 %。
2. 5A5DC对卵巢癌细胞株SKOV3的影响
2.1. 5A5DC对SKOV3细胞生长曲线的影响
SKOV3对数期生长期细胞,加入不同浓度的5A5DC,台盼蓝染色后光镜下进行细胞计数,连续计数7 d,绘制生长曲线。5A5DC对SKOV3细胞增殖具有不同程度的抑制作用,呈现剂量依赖关系。剂量愈大,生长曲线愈低平。其中剂量大于100μmol/L抑制作用较为明显。
2.2. MTT法检测5A5DC对SKOV3细胞增殖的影响
SKOV3对数期生长期细胞,加入不同浓度的5A5DC,MTT法检测5A5DC对SKOV3细胞增殖抑制的影响。5A5DC作用细胞72 h后,对SKOV3具有不同程度的抑制作用,呈现剂量依赖关系,其中以200μmol/L抑制效应最为强。
2.3.流式细胞术检测对SKOV3细胞周期的影响
流式细胞仪检测5A5DC作用SKOV3细胞72 h后,SKOV3细胞周期变化情况,当5A5DC的浓度达到100μmol/L以上,细胞周期的G0/G1期明显受到阻滞。
The expression level of DNA methyltransferase (DNA methyltransferase, DNMT) of most tumor cells was higher than normal cells, and relevant with the hypermethylation status of tumor suppressor gene promoter, DNMT inhibitors are a class of drugs by inhibiting the activity of DNMT to reverse the abnormal gene methylation status, to achieve the anti-cancer effect.
Cytidine analogs as DNA methyltransferase inhibitors, could reverse the methylation status of genes, thereby restoring expression of the gene, correct the abnormal biological characteristics of tumor cells, ware concerned in cancer research.5 - aza -5 '- deoxy-cell (5-Aza-5'-deoxycytidine 5A5DC) was synthesised and isolated,and the strong biological activity of theβ-isomer has been confirmed. There was high price of the catalyst,time of the reaction, complexity of purification and other shortcomings in the synthesis. The aim of this article was to optimize the synthesis process, reduce costs and improve yield; study the effect of 5A5DC on the inhibition of proliferation of SKOV3 and its cell cycle .
The study includes two parts as follows:
1. 5 - aza -5 '- deoxycytidine (5A5DC) synthesis and process optimization The synthetic route was determined by analyzing a variety of methods of nucleoside compounds: 5 - aza cytosine coupling with 1,2,3 - triethylene acyl -5-deoxy - D-ribose to 2 ', 3'-acetoxy-5'-deoxy -5-azacytidine with Lewis acid and protection of silylation, then recrystallized by ethyl acetate. Compare he Hilbert-Johnson-coupling reaction of different Lewis acid and catalysts on the synthesis of 5A5DC by orthogonal design. SnCl4 as the catalyst, 1,2 - dichloroethane as the Lewis acid, reaction time 8 h, The yield of 5A5DC was highest. The total yield was 31% to dicyandiamide.
2. The effect of 5A5DC on the proliferation of ovarian cancer cell SKOV3
2.1 The effect of 5A5DC on SKOV3 cell growth curve
Logarithmic growth phase of SKOV3 cells with different concentrations of 5A5DC, cells were stained by trypan blue and counted under light microscope, continuous counting 7 d, drawn the growth curve. 5A5DC with different degrees of inhibition of SKOV3 cells with different degrees of inhibition in a dose dependent manner. The growth curve was lower when the dose was larger. The inhibition was obvious when the dose of more than 100μmol/L.
2.2. Assay 5A5DC inhibition of SKOV3 cell proliferation by MTT
Logarithmic growth phase of SKOV3 cells with different concentrations of 5A5DC, Assay 5A5DC inhibition of SKOV3 cell proliferation by MTT. After 72 h of 5A5DC treatment, varying degrees of inhibition of SKOV3 with a dose dependent manner, of which 200μmol / L inhibitory effect is most significant.
2.3. Detect the cell cycle of SKOV3 by flow cytometry
The cycle changes of SKOV3 cells after 72 h of 5A5DC treatment was detected by flow cytometry, when the concentration of 5A5DC was 100μmol / L or more, the G0/G1 cell cycle was obvious blocked.
胞苷类似物作为DNA甲基化酶抑制剂,能够逆转基因的甲基化状态,从而恢复基因的表达水平,纠正肿瘤细胞的异常生物学特征,在肿瘤研究中受到极大的关注。本课题前期以双氰胺为原料合成、分离得到5-氮杂-5'-脱氧胞(5-Aza-5'- deoxycytidine, 5A5DC),实验证实了β-异构体有着较强的生物活性。但在合成方面,存在催化剂价格过高、反应时间长、纯化方法复杂等缺点。因此本文旨在优化其合成工艺、降低成本、提高产率,并探讨5A5DC对SKOV3的增殖及细胞周期的影响。
具体研究工作分为以下两个部分:
1. 5-氮杂-5'-脱氧胞苷(5A5DC)的合成及工艺优化
通过分析各种核苷类化合物的合成方法,确定了本文的合成路线: 5-氮杂胞嘧啶经硅烷化保护,在路易斯酸作用下与1,2,3-三乙酰基-5-脱氧-D-核糖进行偶联得2',3'-二乙酰氧基-5'-脱氧-5-氮杂胞苷,最后经碱性脱保护、乙酸乙酯重结晶得5A5DC。通过正交设计,比较在Hilbert-Johnson偶联反应中不同Lewis酸、溶剂和反应时间对合成5A5DC的影响。以无水SnCl4为Lewis酸,1,2-二氯乙烷为催化剂、反应时间为8 h时,5A5DC的收率最高。以双氰胺计,总收率为31 %。
2. 5A5DC对卵巢癌细胞株SKOV3的影响
2.1. 5A5DC对SKOV3细胞生长曲线的影响
SKOV3对数期生长期细胞,加入不同浓度的5A5DC,台盼蓝染色后光镜下进行细胞计数,连续计数7 d,绘制生长曲线。5A5DC对SKOV3细胞增殖具有不同程度的抑制作用,呈现剂量依赖关系。剂量愈大,生长曲线愈低平。其中剂量大于100μmol/L抑制作用较为明显。
2.2. MTT法检测5A5DC对SKOV3细胞增殖的影响
SKOV3对数期生长期细胞,加入不同浓度的5A5DC,MTT法检测5A5DC对SKOV3细胞增殖抑制的影响。5A5DC作用细胞72 h后,对SKOV3具有不同程度的抑制作用,呈现剂量依赖关系,其中以200μmol/L抑制效应最为强。
2.3.流式细胞术检测对SKOV3细胞周期的影响
流式细胞仪检测5A5DC作用SKOV3细胞72 h后,SKOV3细胞周期变化情况,当5A5DC的浓度达到100μmol/L以上,细胞周期的G0/G1期明显受到阻滞。
The expression level of DNA methyltransferase (DNA methyltransferase, DNMT) of most tumor cells was higher than normal cells, and relevant with the hypermethylation status of tumor suppressor gene promoter, DNMT inhibitors are a class of drugs by inhibiting the activity of DNMT to reverse the abnormal gene methylation status, to achieve the anti-cancer effect.
Cytidine analogs as DNA methyltransferase inhibitors, could reverse the methylation status of genes, thereby restoring expression of the gene, correct the abnormal biological characteristics of tumor cells, ware concerned in cancer research.5 - aza -5 '- deoxy-cell (5-Aza-5'-deoxycytidine 5A5DC) was synthesised and isolated,and the strong biological activity of theβ-isomer has been confirmed. There was high price of the catalyst,time of the reaction, complexity of purification and other shortcomings in the synthesis. The aim of this article was to optimize the synthesis process, reduce costs and improve yield; study the effect of 5A5DC on the inhibition of proliferation of SKOV3 and its cell cycle .
The study includes two parts as follows:
1. 5 - aza -5 '- deoxycytidine (5A5DC) synthesis and process optimization The synthetic route was determined by analyzing a variety of methods of nucleoside compounds: 5 - aza cytosine coupling with 1,2,3 - triethylene acyl -5-deoxy - D-ribose to 2 ', 3'-acetoxy-5'-deoxy -5-azacytidine with Lewis acid and protection of silylation, then recrystallized by ethyl acetate. Compare he Hilbert-Johnson-coupling reaction of different Lewis acid and catalysts on the synthesis of 5A5DC by orthogonal design. SnCl4 as the catalyst, 1,2 - dichloroethane as the Lewis acid, reaction time 8 h, The yield of 5A5DC was highest. The total yield was 31% to dicyandiamide.
2. The effect of 5A5DC on the proliferation of ovarian cancer cell SKOV3
2.1 The effect of 5A5DC on SKOV3 cell growth curve
Logarithmic growth phase of SKOV3 cells with different concentrations of 5A5DC, cells were stained by trypan blue and counted under light microscope, continuous counting 7 d, drawn the growth curve. 5A5DC with different degrees of inhibition of SKOV3 cells with different degrees of inhibition in a dose dependent manner. The growth curve was lower when the dose was larger. The inhibition was obvious when the dose of more than 100μmol/L.
2.2. Assay 5A5DC inhibition of SKOV3 cell proliferation by MTT
Logarithmic growth phase of SKOV3 cells with different concentrations of 5A5DC, Assay 5A5DC inhibition of SKOV3 cell proliferation by MTT. After 72 h of 5A5DC treatment, varying degrees of inhibition of SKOV3 with a dose dependent manner, of which 200μmol / L inhibitory effect is most significant.
2.3. Detect the cell cycle of SKOV3 by flow cytometry
The cycle changes of SKOV3 cells after 72 h of 5A5DC treatment was detected by flow cytometry, when the concentration of 5A5DC was 100μmol / L or more, the G0/G1 cell cycle was obvious blocked.
引文
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