【作者】 郭刚；

【导师】 赵临襄；

【作者基本信息】 沈阳药科大学 ， 药物化学， 2008， 博士

【摘要】 氮杂胞苷和地西他宾是DNA甲基转移酶抑制剂,临床用于治疗骨髓增生异常综合征。这两种药物在高剂量下具有细胞毒作用,在低剂量下,通过抑制DNA甲基转移酶而达到抑制肿瘤生长的作用。一些肿瘤抑癌基因由于DNA高甲基化引起基因的静息,去甲基化后,能恢复基因的表达,实验结果证实氮杂胞苷和地西他宾具有恢复抑癌基因表达的作用。氮杂胞苷和地西他宾具有呋喃糖结构,因此它们能结合到DNA或RNA中从而导致细胞毒作用。在碱性及中性条件下,4-氨基-1,3,5-三嗪-2（1H）-酮片段开环形成无活性的N-（甲酰氨基-亚氨甲基）-N’-[（2-去氧-）-β-D-呋喃核糖基]脲,因此在三嗪6位引入甲基、乙基、苯基,用硫原子代替氧原子形成硫酮,并用吡喃单糖/双糖代替呋喃糖,设计合成了52个化合物,其中48个为新化合物,包括4-氨基-6-烷基-1-吡喃糖基/呋喃糖基-1,3,5-三嗪-2（1H）-酮（N₁-苷）,6-氨基-4-烷基/芳香基-1-吡喃糖基-1,3,5-三嗪-2（1H）-酮（N₃-苷）和4-氨基-6-烷基-1,3,5-三嗪-2-基-1-硫代-吡喃糖/呋喃糖苷（S-苷）等三类化合物,以期获得毒副作用低、活性好的化合物。对目标化合物进行肿瘤细胞生长抑制、诱导分化以及与ATRA的协同作用等活性筛选,初步证明了设计思想,DNA甲基转移酶抑制作用及选择性抑制作用研究正在进行中。目标化合物结构经¹H-NMR、IR、MS图谱数据确认。苷的构型可以从J_1’2’的偶合常数确定,其符合Baker反式规则,甘露糖苷、鼠李糖苷为α构型;葡萄糖苷、木糖苷、乳糖苷、麦芽糖苷为β构型。采用三种方法合成目标化合物,三甲基硅烷化法适用于6-甲基-N₁-苷、N₃-苷及S-苷,异氰酸酯法适用于N₁-硫酮苷的合成,N-乙酰基吡喃糖基-N’-[（甲酰氨基）-亚胺亚甲基)]脲环合法可用于合成6位具有较大取代基的N₁-苷的合成。我们对三甲基硅烷化法的反应条件对反应区域选择性的影响规律进行了初步探讨,在Vorbrüggen耦联反应中Lewis酸的酸性和空间位阻效应决定Vorbrüggen耦联反应的区域选择性。N-三甲基硅基-4-三甲基硅氧基-1,3,5-三嗪-2-胺与酰化吡喃糖成苷时,只得到N₁-苷。4-甲基-N-三甲基硅基-6-三甲基硅硫基-1,3,5-三嗪-2-胺与酰化吡喃糖成苷时,以SnCl₄为催化剂,产物以N₃-苷为主;当降低Lewis酸的酸性,以TMSOTf为催化剂时,产物以N₁-苷为主。当碱基为4-乙基/苯基-N-三甲基硅基-6-三甲基硅氧基-1,3,5-三嗪-2-胺时,空间位阻效应成主要影响因素,以SnCl₄或TMSOTf做催化剂,产物都以N₃-苷为主。当碱基为（4-甲基/乙基-）N-三甲基硅基-6-三甲基硅硫基-1,3,5-三嗪-2-胺时,产物以S-苷为主。由于反异头碳效应的作用,在合成4-氨基-6-烷基-1-甘露糖基/鼠李糖基-1,3,5-三嗪-2（1H）-酮和6-氨基-4-烷基/芳香基-1-甘露糖基/鼠李糖基-1,3,5-三嗪-2（1H）-酮的过程中,乙酰化甘露糖的C1构象和乙酰化鼠李糖的1C构象发生翻转,生成了相应的1C构象的甘露糖苷和C1构象的鼠李糖苷。由于异头碳效应,4-氨基-6-烷基-1,3,5-三嗪-2-基-1-硫代-甘露糖/鼠李糖苷的构象和相应的乙酰化甘露糖和鼠李糖构象保持一致。以DNA甲基转移酶抑制剂氮杂胞苷和RG108为阳性对照药,测试了目标化合物对HL-60和MCF-7细胞的生长抑制作用,GG-07、GG-30和GG-50对HL-60细胞的GI₅₀分别为18.5μM、1.7μM和1.48μM,细胞毒活性略低于氮杂胞苷,其余目标化合物在50μM对HL-60的生长抑制率均低于50%。结果表明对氮杂胞苷进行结构改造,向其6位引入甲基、乙基、苯基,吡喃糖代替呋喃糖及形成电子等排体吡喃硫苷,对HL-60和MCF-7两种细胞系具有微弱的生长抑制作用,与DNA甲基转移酶选择性抑制剂RG108相当;与先导化合物氮杂胞苷相比,细胞毒作用显著降低。化合物GG-16、GG-37、GG-39、GG-48在50μM可使0.1μM ATRA对MCF-7的生长抑制率由38.7%提高到50.0%以上,表现出微弱的协同作用;化合物GG-02、GG-04、GG-38与ATRA对HL-60细胞和NB-4细胞具有微弱的分化协同作用。更多还原

【Abstract】 5-Azacytidine（5-aza-CR） and 5-aza-2’-deoxycytidine（5-aza-CdR,Decitabine） are known DNA methyltransferase inhibitors and have been approved for the treatment of myelodysplastic syndrome（MDS）.Although both agents are characterized by significant cytotoxicity at high concentrations,they have been thought to mediate their therapeutic effects in MDS at low concentrations through the inhibition of DNA methyltransferase.Aberrant DNA methylation in the promoter region can silence the expression of many genes.Some tumor suppressor genes have been found to be silenced due to DNA hypermethylation and they can be reactivated by DNA demethylation.Treatments of malignant cells with 5-aza-CR or 5-aza-CdR have been found to be associated with reversal of specific gene suppression.Due to the ribonucleoside structure of 5-aza-CR and 5-aza-CdR,both compounds are incorporated into DNA or RNA and cause substantial cytotoxicity.The structure of 4-Amino-1,3,5-triazin-2（1 H）-one moiety is capable of cleavage to form N-aminoiminomethyl-N’-β-D-（2-deoxy）-ribofuranosyl-urea in alkaline and neutral which is a inactive product.We have synthesized a series of 5-aza-CR analogues with introduction of a methyl,an ethyl or a phenyl group into 6 position of 5-aza-CR and/or with a replacement of O by S or with a replacement of ribofuranosyl moiety by pyranosyl sugars or disaccharides.Fifty-two 5-azacytidine analogues, including 4-amino-6-alkyl-1-pyranosyl/ribofuranosyl-1,3,5-triazin-2（1 H）-ones,6-amino-4-alkyl/ -aryl-1-pyranosyl/ribofuranosyl-1,3,5-triazin-2（1 H）-ones and 4-amino-6-alkyl-1,3,5-triazin-2-y1-1 -thio-pyranosides/ribofuranosides were synthesized.The antiproliferative activities as well as their differentiation effects of these compounds alone and in combination with all-trans retinoic acid （ATRA） were investigated in human leukemia HL-60 and MCF-7 cells.The inhibitory effects of these compounds on DNA methyltransferase is under investigation.The structures of all compounds were confirmed by ¹H-NMR,MS and IR.The configuration of nucleoside was readily deduced from the trans-diaxial coupling between H-1’ and H-2’ of pyranose in ¹H-NMR spectra.The mannopyranosyl/rhamnopyranosyl nucleosides wereα-configuration nucleosides.The residues wereβ-configuration nucleosides.The target compounds were synthesized by three different methods in the process.As a result, it was shown that the trimethylsilylational method was fit for the synthesis of N3-,S-,and 6-methyl-N₁-nucleosides.However,isocyanate procedure was more suitable for S-nucleoside.In addition,it is possible that N-aminoimino -methyl-N’-P-D-pyranosyl-urea cyclization was used for the synthesis of 6-larger substituted groups-N₁-nucleoside.Meanwhile,the regioselectivity of products which were attained by the trimethylsilylational method was discussed simply.Vorbrüggen coupling of N-trimethylsilyl-6-（trimethylsilyl）oxy-1,3,5-triazin-2-amine with acylated sugars and 4-methyl-N-trimethylsilyl-6-（trimethylsilyl）oxy-1,3,5-triazin-2-amine with 1-0 -acetyl-2,3,5-tri-O-benzoyl-β-D-ribose in anhydrous acetonitrile with SnCl₄ or trimethylsilyltriflate （TMSOTf） catalysis gave acylated N₁-nucleosides.However when the coupling procedure was applied to 4-Methyl-N-trimethylsilyl-6-（trimethylsilyl）oxy-1,3,5-triazin-2-amine and acetylated pyranoses in the presence of SnCl₄（2eq ）,acylated N₃-isomers were obtained as the main products. It was observed that N₁-isomers were predominant products while a weaker Lewis acid TMSOTf （2eq ） was used as the catalyst.N3-Nucleosides were predominant products even though TMSOTf was used as catalyst.While ethyl and phenyl were introduced to 6 position the steric hindrance turned to be a major factor determining the regioselectivity.The acylated S-nucleosides which were kinetically controlled products were predominant products during couplings of with acylated sugars in anhydrous acetonitrile with SnCU or TMSOTf catalysis.It was obtained C1 conformation mannopyranoside and 1C conformation rhamnopyranoside formed when vorbriiggen coupling of 4-alkyl/aryl -N-trimethylsilyl-6-（trimethylsilyl）oxy-1,3,5 -triazin-2-amine with the 1C conformation acetylated mannose or C1 conformation acetylated rhamnose because of antianomeric effect.However,the rentention of conformation of 4-amino-6-alkyl-1,3,5-triazin-2-yl-1-thio-mannopyranoside/rhamnopyranoside were obtained when vorbrüggen coupling of 4-alkyl-N-trimethylsilyl-6-（trimethylsilyl）thio-1,3,5-triazin-2-amine with the 1C conformation acetylated mannose or C1 conformation acetylated rhamnose for anomeric effect.Compounds GG-07,GG-30 and GG-50 had a lower GI₅₀ value of 18.5μM,1.7μM and 1.48μM respectively.Other compounds did not inhibit 50.0%cell growth at concentrations less than 50μM.Comparing the antiproliferative effects of 5-aza-CR,it was found that there were lower growth inhibition effects when the replacement of ribofuranosyl moiety with pyranosyl sugars or disaccharides.The replacement of 5-aza-CR into bioisostere ribofuranosyl S-nucleoside and the replacement of H with methyl,ethyl,phenyl group significantly decreased the antiproliferative effect also. All of these compounds alone did not induce NBT differentiation but the treatment of ATRA in combination with compound GG-02,GG-04 or GG-38 induced a significant increase in differentiation in HL-60 and NB-4 cells.The treatment of ATRA in combination with compound GG-16,GG-37,GG-39 or GG-48 induced a moderate growth inhibiton effect and the ratio of growth inhibiton rate was increased from 38.7%into above 50.0%comparing with 0.1μM ATRA alone.更多还原