CN112574217B - 非环核苷先导化合物及其制备方法和在制药中的应用 - Google Patents

非环核苷先导化合物及其制备方法和在制药中的应用 Download PDF

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CN112574217B
CN112574217B CN202011489709.4A CN202011489709A CN112574217B CN 112574217 B CN112574217 B CN 112574217B CN 202011489709 A CN202011489709 A CN 202011489709A CN 112574217 B CN112574217 B CN 112574217B
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郭海明
王洋
郝二军
李恭欣
梁玉茹
谢明胜
王东超
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Abstract

本发明公开了如下通式结构具有显著抗肿瘤活性的新型非环核苷化合物、合成方法及其在抗肿瘤药物研发中的应用，属于药物化学技术领域。本发明还包括所述化合物、其药学盐及其复方药物在制备预防或治疗与肿瘤相关疾病的药物中的应用。本发明通过氮杂迈克尔加成反应、烯丙基胺化反应等反应合成了系列嘌呤2/6/8位及嘌呤侧链1’位带有不同取代基的非环核苷化合物，包括消旋体、R‑构型和S‑构型，通式结构为
Figure DDA0002833956430000011
(*为手性中心)。本发明提供的化合物或其在药学上可接受的盐在体外、体内有效抑制肿瘤细胞的生长，可应用于制备预防或治疗与肿瘤相关疾病的药物。所述与肿瘤相关疾病包括良、恶性肿瘤以及肿瘤引起的其他疾病。

Description

非环核苷先导化合物及其制备方法和在制药中的应用
技术领域
本发明涉及非环核苷类化合物筛选，具体涉及显著抗肿瘤活性的非环核苷类化合物、合成方法、体内外抗肿瘤活性以及该类化合物及其可接受的药学盐或以其为成分之一的复方药物在制备预防和治疗肿瘤相关疾病的药物中的应用，属于药物化学技术领域。
背景技术
恶性肿瘤严重威胁人类的生命和健康，是人类死亡的主要病因之一。抗肿瘤药物的研发一直是药物开发中的重要方向之一。人工合成的核苷类化合物与天然核苷化合物在结构上相似，在进入人体后可以抑制病毒或肿瘤细胞复制，实现抗病毒或抗肿瘤的目的。
人们对于核苷类抗肿瘤和抗病毒药物的研发越来越重视。非环核苷类化合物是核苷类化合物中独特的一类化合物，具有独特的理化性质，已成为核苷药物研发的重点。
目前非环核苷化合物是已上市的抗病毒药物中重要的一类，但是关于非环核苷类化合物的抗肿瘤活性研究却比较少。越来越多的证据显示病毒性疾病与肿瘤的发生有密切关系，对非环核苷抗肿瘤药物的创新研究不仅可以打破国际对核苷药物的垄断，而且具有重要的科学意义和潜在的应用前景。
发明内容
为了提供更多功能结构的核苷类先导化合物，本发明公开了具有抗结肠癌活性非环核苷先导化合物。合成方法上：通过氮杂迈克尔加成反应、烯丙基胺化反应等反应合成了嘌呤2/6/8位及嘌呤侧链1’位带有不同取代基的系列非环核苷化合物，该类结构中同时包括了消旋体、R-构型和S-构型三种结构类型。
本发明所述一种非环核苷化合物，结构通式如下：
Figure GDA0002949731700000021
其中，*代表手性中心，每个通式结构各自包括消旋体、R-构型和S-构型三种结构。
R1、R3、R4各自独立选自氢、卤素、C1-C4烷氧基、氨基、C1-C4烷胺基、双C1-C4烷胺基。R2选自氢、C1-C15烷基、-CH2-OR，其中R为氢、苄基、苯甲酰基、乙酰基、C1-C4烷基硅基等。卤素为氟、氯、溴或碘。
进一步优选，R1为氢、氟、氯；R3为氢、氯、甲氧基、氨基、甲胺基、二甲胺基；R4为氢、溴；R2为氢、C1-C12直链烷烃、BnOCH2-、BzOCH2-、HOCH2-、TBDMSOCH2-。
进一步优选，R1为氢；R3为氯；R4为氢；R2为氢、C1-C12直链烷烃、BnOCH2-、BzOCH2-、HOCH2-、TBDMSOCH2-。
进一步优选，R3为氯；R4为氢；R2为C1-C12直链烷烃。更进一步优选，R1为氢；R3为氯；R4为氢；R2为n-C8H17或n-C9H19；
更进一步优选，通式结构为R-构型结构化合物。R-构型通常都比S-构型和消旋体取得较好的抗肿瘤活性。
本发明第二个目的，提供了上述非环核苷化合物及其在药学上可接受的盐在制备预防和治疗与肿瘤相关疾病的药物中的应用，上述非环核苷先导化合物在抗肿瘤活性药物中的应用。
进一步地，在上述技术方案中，相关疾病包括间质肉瘤、绒癌、恶性葡萄胎、甲状腺癌、头颈部鳞状细胞癌、宫颈癌、前列腺癌、肾癌、膀胱癌、卵巢癌、乳腺癌、结直肠癌、胰腺癌、食管癌、骨肉瘤、胃癌、肺癌、肝癌、黑色素瘤、淋巴瘤、脑胶质瘤、鼻咽癌、神经内分泌癌、未分化癌、恶性畸胎瘤以及良性肿瘤。
进一步地，所述药学上可接受的盐，包括非环核苷化合物与有机酸或无机酸形成的盐。有机酸选自苹果酸、乳酸、樟脑磺酸、枸橼酸、富马酸或草酸中的一种或多种，无机酸选自磷酸、氢卤酸、硫酸或硝酸中一种或多种。
所述药学上可接受的盐，具体地可列举为与苹果酸、乳酸、樟脑磺酸、枸橼酸、富马酸、草酸等有机酸，以及磷酸、氢卤酸、硫酸、硝酸等无机酸形成的盐。
最优选条件下，用于预防和治疗与肿瘤相关疾病是指抗结肠癌。尤其是结肠癌疾病中，在抗HCT-116或SW480活性方面。
进一步地，在上述技术方案中，上述非环核苷化合物为：R1为氢；R3为氯；R4为氢；R2为n-C8H17或n-C9H19；构型为R-构型。
本发明还提供了一种用于预防和治疗与肿瘤相关疾病的复方药物。
进一步地，在上述技术方案中，所述与肿瘤相关疾病是甲状腺癌、肺癌、肝癌、黑色素瘤、淋巴瘤、前列腺癌、头颈部鳞状细胞癌、宫颈癌、卵巢癌、乳腺癌、结直肠癌、胰腺癌、食管癌、骨肉瘤、肾癌、未分化癌、间质肉瘤、绒癌、胃癌、膀胱癌、恶性胶质母细胞瘤、鼻咽癌、神经内分泌癌、恶性葡萄胎、恶性畸胎瘤以及良性肿瘤。
最优选条件下，用于预防和治疗与肿瘤相关疾病是指抗结肠癌。尤其是结肠癌疾病中，在抗HCT-116或SW480活性方面。
进一步地，在上述技术方案中，上述非环核苷化合物为：R1为氢；R3为氯；R4为氢；R2为n-C8H17或n-C9H19；构型为R-构型。
本发明第四个目的，提供了上述结构通式非环核苷类化合物的合成方法，包括如下步骤：
A：对于消旋体而言，采用反应方程式为：
Figure GDA0002949731700000041
B：对于R-构型或S-构型而言，采用反应方程式为：
Figure GDA0002949731700000042
其中，R1、R3、R4各自独立选自氢、卤素、C1-C4烷氧基、氨基、C1-C4烷胺基、双C1-C4烷胺基。R2选自氢、C1-C15烷基、-CH2-OR，其中R为氢、苄基、苯甲酰基、乙酰基、三C1-C4烷基硅基等。卤素为氟、氯、溴或碘。
进一步地，在上述技术方案中，所述催化剂D-和L-Proline催化下均生成消旋体产物。
进一步地，在上述技术方案中，反应条件为Proline、PhCO2H与嘌呤化合物摩尔比为0.05-0.20:0.05-0.20:1。在最优选条件下Proline和PhCO2H采用等摩尔比。
进一步地，在上述技术方案中，反应条件为NaBH4与嘌呤化合物摩尔比为2-5:1。
为了研究非环核苷类化合物的抗肿瘤活性，通过氮杂迈克尔加成反应、烯丙基胺化反应等反应合成了嘌呤2位/6位/8位以及嘌呤侧链的1’位带有不同取代基的系列非环核苷化合物。
实验结果表明：在消旋体中，化合物9b在体外对结肠癌细胞系的两种细胞(HCT-116和SW480)表现出较强的抑制增殖的能力，IC50分别为0.89μM和1.15μM，对照药物5-氟尿嘧啶(5-FU)对两种细胞的IC50分别为10.81μM和9.70μM。在手性结构中，R-构型手性非环核苷类化合物比S-构型表现出更强的抗肿瘤活性。其中，(R)-8b在体外对结肠癌细胞系(SW480)表现出较强抑制增殖的能力，IC50为0.32μM；(R)-9b在体外对结肠癌细胞系(HCT-116)表现出较强抑制增殖的能力，IC50为0.45μM。上述结果为具有抗结肠癌活性药物的设计提供了非环核苷先导化合物基础结构单位，在此基础上可以进行进一步结构细微修改和改造。
通过对非环核苷的合成及体内外抗肿瘤活性评价研究，获得活性较好的抗肿瘤先导化合物或候选药物分子，为手性非环核苷类药物提供合理的合成策略和方法，为开发具有自主知识产权的手性抗肿瘤非环核苷药物奠定了良好基础。
附图说明
图1为实施例4中化合物9b诱导SW480细胞凋亡的细胞流式实验图；
图2为实施例4中化合物9b对凋亡相关蛋白调控Western Blot实验图；
图3为实施例4中线粒体膜电位(MMP)测定实验图；
图4为实施例4中化合物9b对HCT-116细胞和SW480细胞集落形成影响实验图；
图5为实施例4中裸鼠移植瘤实验图。
具体实施方式
实施例1：
Figure GDA0002949731700000061
代表性试验操作：在反应瓶中，依次加入6-氯嘌呤(30.9mg,0.2mmol)、L-脯氨酸(2.3mg,0.02mmol)和苯甲酸(2.4mg,0.02mmol)，接着加入1.5mL甲醇，最后向反应管中加入反-2-十二烯醛(64.4μL,0.3mmol)，室温下反应24h。TLC(石油醚/乙酸乙酯＝1.5/1)检测反应，反应结束后，移至冰水浴中，待温度降下后缓慢加入硼氢化钠(22.7mg,0.6mmol)，反应约10min。TLC(石油醚/乙酸乙酯＝1.5/1)检测反应。反应结束后加饱和氯化铵溶液淬灭反应，减压蒸馏后加水，乙酸乙酯萃取，有机相用无水硫酸钠干燥，石油醚/乙酸乙酯(3:1)柱层析，得到白色固体9b；Mp:101-102℃.1H NMR(400MHz,CDCl3)δ8.69(s,1H),8.17(s,1H),4.86-4.77(m,1H),3.65-3.57(m,1H),3.26-3.17(m,1H),2.93(s,1H),2.23-2.10(m,3H),2.01-1.90(m,1H),1.25-1.20(m,4H),1.20-1.14(m,9H),1.11-1.02(m,1H),0.82(t,J＝6.8Hz,3H)；13C NMR(101MHz,CDCl3)δ152.1,151.7,151.2,144.7,131.8,58.3,54.2,37.5,34.3,31.9,29.44,29.36,29.2,29.1,26.3,22.7,14.2；ESI-HRMS(m/z)calcd C17H28ClN4O(M+H+),339.1952；found,339.1945.
Figure GDA0002949731700000071
化合物13a合成：氮气保护下，在反应瓶内加入2-氟-6-氯嘌呤(1.55g,10mmol)、碳酸钾(1.69g,12mmol)和25.0mLDMF，维持0℃下搅拌1h，缓慢加入3-溴丙酸甲酯(1.3mL,12mmol)，室温下搅拌48h。石油醚/乙酸乙酯＝4:1检测反应，加入水，乙酸乙酯萃取，有机相旋干，石油醚/乙酸乙酯＝8:1柱层析，得到白色或淡黄色固体。
氮气保护下，将上述中间体(25.8mg,0.1mmol)加入1.0mL二氯甲烷中，冷却至-78℃，缓慢滴加1.0M DIBAL-H/甲苯溶液(0.3mL,0.3mmol，滴加完毕继续搅拌20min。10％NaOH溶液淬灭反应，无气泡生成时室温搅拌10min，石油醚/乙酸乙酯＝1:1检测反应。二氯甲烷和水萃取，无水硫酸钠干燥有机相，石油醚/乙酸乙酯＝3:1柱层析，得到白色固体13a。
化合物16b合成：向15mL反应管中依次加入15b(35.7mg,0.1mmol)、四丁基氟化铵(52.4mg,0.2mmol)和1.0mL四氢呋喃，室温下搅拌2h。二氯甲烷/甲醇＝30:1检测反应，反应完成后减压蒸馏溶剂，二氯甲烷/甲醇＝50:1过柱，得到白色化合物16b。
Figure GDA0002949731700000081
化合物4j-6j合成(以4j为例)：向10mL圆底烧瓶中，依次加入4b(26.9mg,0.1mmol)，钯碳(10wt％)和1.0mL甲醇，氮气置换后，充入氢气，室温下反应过夜。二氯甲烷/甲醇＝30:1检测反应，反应完成后使用硅藻土过滤，真空蒸发溶剂，二氯甲烷/甲醇＝50:1柱层析，得到无色油状化合物4j。
化合物5k-10k合成(以5k为例)：氮气保护下，将6-氯嘌呤(927.4mg,6mmol)、K2CO3(829.3mg,6mmol)和DABCO(22.6mg,0.2mmol)加入到圆底烧瓶中，随后加入MBH碳酸酯(484.3mg,2mmol)/15.0mL二氯甲烷溶液，室温搅拌24h。石油醚/乙酸乙酯＝3:1检测反应，反应完全后，水和二氯甲烷萃取，无水硫酸钠干燥，石油醚/乙酸乙酯＝5:1柱层析，得到无色油状物。
氮气保护下，加入上述无色油状物(32.3mg,0.1mmol)和1.0mL二氯甲烷，溶液冷却至-78℃，缓慢滴加1.0M DIBAL-H(0.3mL,0.3mmol)，-78℃继续搅拌20min。石油醚/乙酸乙酯＝1.5/1检测反应。反应完全后，10％氢氧化钠溶液淬灭反应，无气泡生成时室温搅拌10min，二氯甲烷和水萃取，无水硫酸钠干燥，石油醚/乙酸乙酯＝3/1过柱，得到无色油状物5k。
Figure GDA0002949731700000091
化合物9ba合成：在0℃下，将化合物9b(169.1mg,0.5mmol)，Et3N(173.0μL,1.25mmol)，加入10.0mL二氯甲烷中，向混合溶液中缓慢滴加甲磺酰氯(58.0μL,0.75mmol)，室温下反应搅拌4h。石油醚/乙酸乙酯＝1.5:1检测反应，反应完全后，加饱和NaHCO3溶液淬灭反应，萃取，无水硫酸钠干燥有机相，真空浓缩，得到残余物。将获得的残余物以石油醚/乙酸乙酯＝3:1柱层析，得到白色固体9ba。
化合物9bb合成：在0℃下，向9ba(124.8mg,0.3mmol)/5.0mL DMF冷溶液中加入NaN3(97.5mg,1.5mmol)，然后将反应在25℃下反应6h。石油醚/乙酸乙酯＝2:1检测反应，反应完全后，将10.0mL水加到混合物中淬灭反应。将得到的混合物用乙酸乙酯萃取，合并有机相，并使用饱和盐水洗涤有机相，无水硫酸钠干燥有机相，石油醚/乙酸乙酯＝4:1柱层析，得到淡黄色油状物9bb。
化合物9bc的合成：向1.0mL乙腈中加入化合物9bb(36.3mg,0.1mmol)、苯乙炔(16.5μL,0.15mmol)和Cu(OAc)2(0.9mg,0.005mmol)。搅拌混合物直到Cu(OAc)2完全溶解。移液枪添加DIPEA(1.7μL,0.01mmol)，0℃下搅拌6h。石油醚/乙酸乙酯＝2:1检测反应，反应结束后加入2.0mL水淬灭反应，乙酸乙酯萃取，合并有机相，饱和盐水洗涤有机相，无水硫酸钠干燥有机相，石油醚/乙酸乙酯＝4:1柱层析，得到白色固体9bc。
化合物9bd合成：将化合物9b(169.1mg,0.5mmol)/0.5mL二氯甲烷溶液冷却至-20℃，并加入吡啶(44.5μL,1.55mmol)，-20℃搅拌5min，滴加磺酰氯(44.5μL,1.55mmol)/0.5mL二氯甲烷溶液，-20℃搅拌15min，0℃继续搅拌15min。石油醚/乙酸乙酯＝3:1检测反应，反应结束后蒸发二氯甲烷。将残余物悬浮在乙醚中，并将沉淀物过滤，真空去除有机相，石油醚/乙酸乙酯＝6:1柱层析，得到无色油状物9bd。
化合物9be合成：氮气保护下，在-15℃向搅拌加有NaH(12.0mg,0.5mmol)/0.5mL四氢呋喃溶液中缓慢加入含有9b(33.8mg,0.1mmol)/0.5mL四氢呋喃溶液，搅拌5min，缓慢滴加磷酸酯2.19(90.0mg,0.3mmol)/1.0mL四氢呋喃溶液，升温至0℃搅拌7h。甲醇/二氯甲烷＝30:1检测反应，反应完全，加水淬灭反应，二氯甲烷萃取，无水硫酸钠干燥，真空蒸发有机相，甲醇/二氯甲烷＝50:1柱层析，得到无色油状物9be。
代表性化合物表征数据如下：
3-(6-Chloro-2-fluoro-9H-purin-9-yl)butan-1-ol(1a).White solid.Whitesolid.mp:109-110℃.1H NMR(400MHz,CDCl3)δ8.15(s,1H),4.98-4.87(m,1H),3.72-3.63(m,1H),3.44-3.35(m,1H),2.30-2.25(m,1H),2.25-2.11(m,2H),1.71(d,J＝6.8Hz,3H)；13CNMR(100MHz,CDCl3)δ158.1,155.9,153.6,153.5,152.9,152.7,145.1,145.0,130.7,130.6,58.6,50.1,38.3,20.5；ESI-HRMS(m/z)calcdC9H11ClFN4O(M+H+),245.0600；found,245.0601.
3-(6-Chloro-2-fluoro-9H-purin-9-yl)pentan-1-ol(2a).White solid.mp:98-100℃.1H NMR(400MHz,CDCl3)δ8.11(s,1H),4.69-4.59(m,1H),3.69-3.61(m,1H),3.37-3.28(m,1H),2.45(s,1H),2.28-2.17(m,2H),2.17-2.09(m,1H),2.08-1.96(m,1H),0.83(t,J＝7.2Hz,3H).13C NMR(100MHz,CDCl3)δ158.1,155.9,153.8,153.7,152.8,152.6,145.73,145.7,130.6,130.5,58.5,56.6,36.5,27.4,10.9；ESI-HRMS(m/z)calcd C10H13ClFN4O(M+H+)259.0756；found,259.0762.
3-(6-Chloro-2-fluoro-9H-purin-9-yl)decan-1-ol(7a).White solid.mp:104-107℃.1H NMR(400 MHz,CDCl3)δ8.13(s,1H),4.77-4.67(m,1H),3.67-3.59(m,1H),3.35-3.26(m,1H),2.91(s,1H),2.26-2.15(m,2H),2.15-2.05(m,1H),1.99-1.87(m,1H),1.25-1.13(m,9H),1.10-0.98(m,1H),0.80(t,J＝7.2 Hz,3H)；13C NMR(100 MHz,CDCl3)δ158.1,155.9,153.8,153.7,152.9,152.7,145.64,145.61,130.61,130.56,58.5,54.9,36.9,34.2,31.7,29.1,26.3,22.6,14.1；ESI-HRMS(m/z)calcd C15H23ClFN4O(M+H+),329.1539；found,329.1540.
3-(6-Chloro-2-fluoro-9H-purin-9-yl)dodecan-1-ol(9a).White solid.mp:113-115℃.1H NMR(400 MHz,CDCl3)δ8.10(s,1H),4.76-4.67(m,1H),3.68-3.60(m,1H),3.35-3.26(m,1H),2.50(s,1H),2.26-2.15(m,2H),2.14-2.06(m,1H),1.98-1.88(m,1H),1.27-1.21(m,4H),1.21-1.14(m,9H),1.10-1.00(m,1H),0.82(t,J＝6.8 Hz,3H)；13C NMR(100 MHz,CDCl3)δ158.1,155.9,153.8,153.6,152.8,152.6,145.72,145.68,130.6,130.5,58.5,55.0,36.8,34.2,31.9,29.5,29.4,29.3,29.1,26.3,22.7,14.1；ESI-HRMS(m/z)calcd C17H27ClFN4O(M+H+),357.1852；found,357.1850.
3-(6-Chloro-2-fluoro-9H-purin-9-yl)propan-1-ol(13a).White solid,mp:111-112℃.1H NMR(400 MHz,CDCl3)δ8.15(s,1H),4.43(t,J＝6.8 Hz,2H),3.65(t,J＝5.4Hz,2H),2.24(s,1H),2.18-2.08(m,2H)；13C NMR(100 MHz,CDCl3)δ158.5,156.3,154.0,153.8,153.0,152.8,146.6,146.5,130.5,130.4,58.5,41.4,31.8；ESI-HRMS(m/z)calcdC8H9ClFN4O(M+H+),231.0443；found,231.0442.
3-(6-Chloro-9H-purin-9-yl)butan-1-ol(1b).White solid.mp:104-106℃.1HNMR(400 MHz,CDCl3)δ8.73(s,1H),8.19(s,1H),5.08-4.98(m,1H),3.71-3.59(m,1H),3.33-3.21(m,1H),2.80(s,1H),2.26-2.04(m,2H),1.75(d,J＝6.8 Hz,3H)；13C NMR(150MHz,CDCl3)δ151.9,151.7,151.4,144.0,131.9,58.4,49.3,39.2,20.7；ESI-HRMS(m/z)calcd C9H12ClN4O(M+H+),227.0694；found,227.0694.
3-(6-Chloro-9H-purin-9-yl)pentan-1-ol(2b).White solid.mp:100-103℃.1HNMR(400MHz,CDCl3)δ8.70(s,1H),8.14(s,1H),4.79-4.70(m,1H),3.67-3.60(m,1H),3.28-3.19(m,1H),2.69(s,1H),2.28-2.12(m,3H),2.12-1.99(m,1H),0.85(t,J＝7.2 Hz,3H)；13CNMR(150MHz,CDCl3)δ152.2,151.8,151.3,144.6,131.9,58.4,55.8,37.2,27.6,11.0；ESI-HRMS(m/z)calcd C10H14ClN4O(M+H+),241.0851；found,241.0855.
3-(6-Chloro-9H-purin-9-yl)heptan-1-ol(4b).White solid.mp:87-89℃.1HNMR(400 MHz,CDCl3)δ8.73(s,1H),8.14(s,1H),4.88-4.78(m,1H),3.68-3.58(m,1H),3.25-3.15(m,1H),2.49(s,1H),2.28-2.07(m,3H),2.05-1.94(m,1H),1.39-1.19(m,3H),1.15-1.02(m,1H),0.83(t,J＝7.6 Hz,3H)；13C NMR(150 MHz,CDCl3)δ152.2,151.8,151.4,144.5,131.9,58.4,54.1,37.7,34.1,28.5,22.3,13.9；ESI-HRMS(m/z),calcd C12H18ClN4O(M+H+)269.1164；found,269.1166.
3-(6-Chloro-9H-purin-9-yl)dodecan-1-ol(9b).White solid.mp:101-102℃.1H NMR(400MHz,CDCl3)δ8.69(s,1H),8.17(s,1H),4.86-4.77(m,1H),3.65-3.57(m,1H),3.26-3.17(m,1H),2.93(s,1H),2.23-2.10(m,3H),2.01-1.90(m,1H),1.25-1.20(m,4H),1.20-1.14(m,9H),1.11-1.02(m,1H),0.82(t,J＝6.8 Hz,3H)；13C NMR(100 MHz,CDCl3)δ152.1,151.7,151.2,144.7,131.8,58.3,54.2,37.5,34.3,31.9,29.44,29.36,29.2,29.1,26.3,22.7,14.2；ESI-HRMS(m/z)calcd C17H28ClN4O(M+H+),339.1946；found,339.1945.
4-(Tert-butyldimethylsilyl)-3-(6-chloro-9H-purin-9-yl)butan-1-ol(15b).White solid.mp:105-107℃.1H NMR(400 MHz,CDCl3)δ8.66(s,1H),8.32(s,1H),5.01-4.93(m,1H),4.08(dd,J＝10.8,5.6 Hz,1H),3.95(dd,J＝10.8,3.2 Hz,1H),3.72-3.63(m,1H),3.40-3.30(m,1H),3.02(s,1H),2.33-2.23(m,1H),2.21-2.12(m,1H),0.79(s,9H),-0.07(d,J＝7.6 Hz,6H)；13C NMR(100 MHz,CDCl3)δ152.0,151.6,151.1,145.6,131.5,64.4,58.2,54.8,33.5,25.7,18.1,-5.6,-5.7；ESI-HRMS(m/z)calcd C15H26ClN4O2Si(M+H+),357.1508；found,357.1511.
2-(6-Chloro-9H-purin-9-yl)butane-1,4-diol(16b).White solid.mp:143-144℃.1H NMR(600 MHz,CD3OD)δ8.71(s,1H),8.62(s,1H),4.98-4.92(m,1H),4.12(dd,J＝12.0,7.8 Hz,1H),3.92(dd,J＝12.0,4.2 Hz,1H),3.62-3.56(m,1H),3.44-3.38(m,1H),2.42-2.35(m,1H),2.24-2.16(m,1H)；13C NMR(150 MHz,CD3OD)δ153.5,152.6,151.1,148.3,132.5,63.8,59.0,58.0,33.6；ESI-HRMS(m/z)calcd C9H12ClN4O2(M+H+),243.0643；found,243.0644.
3-(2,6-Dichloro-9H-purin-9-yl)heptan-1-ol(4c).White solid.mp:143-144℃.1H NMR(400MHz,CDCl3)δ8.11(s,1H),4.80-4.71(m,1H),3.69-3.61(m,1H),3.35-3.26(m,1H),2.29-2.16(m,3H),2.16-2.07(m,1H),2.02-1.91(m,1H),1.33-1.23(m,3H),1.12-0.99(m,1H),0.83(t,J＝7.2 Hz,3H)；13C NMR(100 MHz,CDCl3)δ153.4,152.8,151.9,145.5,131.1,58.6,54.8,37.1,34.0,28.4,22.2,13.9；ESI-HRMS(m/z)calcd C12H17Cl2N4O(M+H+),303.0774；found,303.0775.
3-(2,6-Dichloro-9H-purin-9-yl)dodecan-1-ol(9c).White solid.mp:94-96℃.1H NMR(600MHz,CDCl3)δ8.15(s,1H),4.78-4.71(m,1H),3.66-3.60(m,1H),3.34-3.28(m,1H),2.74(s,1H),2.25-2.15(m,2H),2.15-2.06(m,1H),1.98-1.89(m,1H),1.25-1.19(m,5H),1.19-1.11(m,8H),1.08-1.00(m,1H),0.82(t,J＝7.2 Hz,3H)；13C NMR(100 MHz,CDCl3)δ153.4,152.8,151.9,145.5,131.0,58.6,54.8,37.0,34.3,31.9,29.5,29.4,29.3,29.1,26.3,22.7,14.2；ESI-HRMS(m/z)calcd C17H27Cl2N4O(M+H+),373.1556；found,373.1561.
3-(6-Methoxy-9H-purin-9-yl)heptan-1-ol(4d).White solid.mp:68-71℃.1HNMR(600MHz,CDCl3)δ8.51(s,1H),7.94(s,1H),4.83-4.76(m,1H),4.19(s,3H),3.61-3.55(m,1H),3.27(s,1H),3.12(td,J＝10.8,3.6 Hz,1H),2.23-2.16(m,1H),2.16-2.09(m,1H),2.01-1.91(m,2H),1.35-1.25(m,3H),1.18-1.10(m,1H),0.84(t,J＝6.6 Hz,3H)；13C NMR(150 MHz,CDCl3)δ161.4,152.5,152.1,140.8,121.5,58.2,54.5,52.7,38.7,34.2,28.6,22.3,14.0；ESI-HRMS(m/z)calcd C13H21N4O2(M+H+)265.1659；found,265.1660.
3-(6-Methoxy-9H-purin-9-yl)tridecan-1-ol(10d).White solid.mp:49-51℃.1H NMR(400MHz,CDCl3)δ8.50(s,1H),7.93(s,1H),4.83-4.73(m,1H),4.18(s,3H),3.62-3.53(m,1H),3.39(s,1H),3.13(td,J＝11.2,3.6 Hz,1H),2.24-2.07(m,2H),2.01-1.90(m,2H),1.28-1.22(m,5H),1.22-1.16(m,11H),0.85(t,J＝6.8 Hz,3H)；13C NMR(150 MHz,CDCl3)δ161.3,152.5,152.0,140.8,121.5,58.1,54.4,52.8,38.6,34.5,32.0,29.6,29.5,29.42,29.36,29.2,26.4,22.8,14.2；ESI-HRMS(m/z)calcd C19H33N4O2(M+H+),349.2598；found,349.2606.
3-(6-Methoxy-9H-purin-9-yl)tetradecan-1-ol(11d).White solid.mp:57-58℃.1H NMR(400MHz,CDCl3)δ8.50(s,1H),7.93(s,1H),4.83-4.73(m,1H),4.18(s,3H),3.62-3.53(m,1H),3.37(s,1H),3.12(td,J＝11.2,3.6 Hz,1H),2.24-2.06(m,2H),2.01-1.90(m,2H),1.29-1.23(m,5H),1.23-1.16(m,13H),0.85(t,J＝7.2 Hz,3H)；13C NMR(100 MHz,CDCl3)δ161.3,152.5,152.0,140.8,121.5,58.1,54.4,52.8,38.6,34.5,32.0,29.66,29.65,29.5,29.43,29.41,29.2,26.4,22.8,14.2；ESI-HRMS(m/z)calcd C20H35N4O2(M+H+),363.2755；found,363.2759.
3-(6-(Methylamino)-9H-purin-9-yl)octan-1-ol(5e).White solid.mp:109-111℃.1H NMR(400 MHz,CDCl3)δ8.33(s,1H),7.72(s,1H),6.48-6.41(m,1H),4.75-4.65(m,1H),4.34(s,1H),3.57-3.50(m,1H),3.23-3.08(m,4H),2.19-2.09(m,1H),2.09-2.00(m,1H),1.95-1.83(m,2H),1.25-1.10(m,6H),0.79(t,J＝6.8 Hz,3H)；13C NMR(100 MHz,CDCl3)δ155.7,153.1,149.2,138.0,119.6,57.9,52.3,38.8,34.5,31.3,27.7,26.1,22.5,14.0；ESI-HRMS(m/z)calcd C14H24N5O(M+H+),278.1975；found,278.1985.
3-(6-(Methylamino)-9H-purin-9-yl)dodecan-1-ol(9e).White solid.mp:92-94℃.1H NMR(400 MHz,CDCl3)δ8.38(s,1H),7.75(s,1H),5.93(s,1H),4.81-4.66(m,1H),4.01(s,1H),3.61-3.51(m,1H),3.22(s,3H),3.09(t,J＝11.6 Hz,1H),2.25-2.13(m,1H),2.13-2.04(m,1H),2.00-1.89(m,1H),1.81(t,J＝12.8 Hz,1H),1.29-1.18(m,14H),0.86(t,J＝7.2 Hz,3H).13C NMR(100 MHz,CDCl3)δ155.7,153.1,149.4,137.9,119.6,57.9,52.3,38.9,34.5,31.9,29.5,29.4,29.3,29.2,27.7,26.4,22.7,14.2；ESI-HRMS(m/z)calcd C18H32N5O(M+H+),334.2601；found,334.2600.
3-(6-(methylamino)-9H-purin-9-yl)tetradecan-1-ol(11e).White solid.mp:90-91℃.1H NMR(400 MHz,CDCl3)δ8.37(s,1H),7.75(s,1H),6.03(s,1H),4.79-4.68(m,1H),4.04(s,1H),3.60-3.52(m,1H),3.21(s,3H),3.13-3.05(m,1H),2.23-2.12(m,1H),2.12-2.04(m,1H),1.99-1.90(m,1H),1.85-1.77(m,1H),1.30-1.23(m,8H),1.23-1.18(m,10H),0.86(t,J＝6.8 Hz,3H)；13C NMR(150 MHz,CDCl3)δ155.7,153.2,149.3,137.8,119.7,58.0,52.0,39.3,34.5,32.0,29.8,29.6,29.6,29.5,29.4,29.3,27.7,26.5,22.8,14.2；ESI-HRMS(m/z)calcd C20H36N5O(M+H+),362.2914；found,362.2919.
3-(6-(Dimethylamino)-9H-purin-9-yl)octan-1-ol(5f).Colorless oil.1HNMR(400 MHz,CDCl3)δ8.24(s,1H),7.71(s,1H),4.74-4.64(m,1H),3.96(s,1H),3.72-3.30(m,7H),3.05(td,J＝11.2,3.2 Hz,1H),2.18-1.96(m,2H),1.94-1.83(m,1H),1.82-1.70(m,1H),1.30-1.13(m,6H),0.78(t,J＝6.8 Hz,3H)；13C NMR(100 MHz,CDCl3)δ155.1,152.3,151.0,136.2,119.8,57.8,51.5,39.3,38.7,34.4,31.4,26.1,22.5,14.0；ESI-HRMS(m/z)calcd C15H26N5O(M+H+),292.2132；found,292.2131.
3-(6-(Dimethylamino)-9H-purin-9-yl)decan-1-ol(7f).Colorless oil.1HNMR(400 MHz,CDCl3)δ8.24(s,1H),7.70(s,1H),4.74-4.64(m,1H),4.09(s,1H),3.75-3.23(m,7H),3.05(td,J＝11.2,3.2 Hz,1H),2.16-1.96(m,2H),1.94-1.82(m,1H),1.82-1.71(m,1H),1.26-1.08(m,10H),0.78(t,J＝6.8 Hz,3H)；13C NMR(100 MHz,CDCl3)δ155.0,152.3,151.0,136.3,119.8,57.8,51.6,39.3,38.7,34.5,31.7,29.14,29.06,26.4,22.6,14.1；ESI-HRMS(m/z)calcd C17H30N5O(M+H+),320.2445；found,320.2447.
3-(6-(Dimethylamino)-9H-purin-9-yl)tridecan-1-ol(10f).Colorlessoil.1H NMR(400 MHz,CDCl3)δ8.29(s,1H),7.73(s,1H),4.78-4.68(m,1H),4.27(s,1H),3.75-3.33(m,7H),3.09-2.99(m,1H),2.22-2.10(m,1H),2.10-2.01(m,1H),1.99-1.87(m,2H),1.79-1.67(m,1H),1.28-1.23(m,6H),1.22-1.18(m,10H),0.85(t,J＝6.8 Hz,3H)；13CNMR(150 MHz,CDCl3)δ155.1,152.4,151.1,136.1,119.9,57.9,51.4,39.5,38.7,34.5,32.0,29.63,29.57,29.5,29.4,29.3,26.5,22.8,14.2；ESI-HRMS(m/z)calcd C20H36N5O(M+H+),362.2914；found,362.2917.
3-(6-Amino-9H-purin-9-yl)heptan-1-ol(4g).White solid.mp:118-121℃.1HNMR(400MHz,CDCl3)δ8.30(s,1H),7.81(s,1H),6.27(s,2H),4.79-4.67(m,1H),4.19(s,1H),3.61-3.53(m,1H),3.20-3.09(m,1H),2.23-2.03(m,2H),2.00-1.86(m,2H),1.34-1.22(m,3H),1.21-1.09(m,1H),0.83(t,J＝6.8 Hz,3H)；13C NMR(150 MHz,CDCl3)δ155.9,152.9,150.5,138.9,119.5,58.0,52.4,38.8,34.2,28.6,22.3,14.0；ESI-HRMS(m/z)calcdC12H20N5O(M+H+)250.1662；found,250.1664.
3-(6-amino-9H-purin-9-yl)tridecan-1-ol(10g).White solid.mp:105-106℃.1H NMR(400MHz,CDCl3)δ8.31(s,1H),7.81(s,1H),6.11(s,2H),4.79-4.69(m,1H),4.06(s,1H),3.61-3.54(m,1H),3.14(td,J＝11.2,2.8 Hz,1H),2.23-2.13(m,1H),2.13-2.04(m,1H),2.00-1.83(m,2H),1.28-1.23(m,5H),1.23-1.18(m,11H),0.85(t,J＝6.8 Hz,3H)；13CNMR(100 MHz,CDCl3)δ155.8,153,0,150.5,138.9,119.4,58.0,52.4,38.9,34.5,32.0,29.63,29.58,29.5,29.4,29.2,26.5,22.8,14.2；ESI-HRMS(m/z)calcd C18H32N5O(M+H+),334.2601；found,334.2605.
3-(6-amino-9H-purin-9-yl)tetradecan-1-ol(11g).White solid.mp:93-94℃.1H NMR(400MHz,CDCl3)δ8.31(s,1H),7.81(s,1H),6.12(s,2H),4.78-4.69(m,1H),4.05(s,1H),3.61-3.54(m,1H),3.14(td,J＝11.2,3.2 Hz,1H),2.24-2.12(m,1H),2.12-2.04(m,1H),2.01-1.83(m,2H),1.28-1.24(m,5H),1.24-1.18(m,13H),0.86(t,J＝6.8 Hz,3H)；13CNMR(100 MHz,CDCl3)δ155.8,153.0,150.5,138.9,119.4,58.0,52.4,38.9,34.5,32.0,29.7,29.6,29.5,29.4,29.2,26.5,22.8,14.2；ESI-HRMS(m/z)calcd C19H34N5O(M+H+),348.2758；found,348.2768.
3-(6-Amino-2-chloro-9H-purin-9-yl)octan-1-ol(5h).White solid.mp:154-156℃.1H NMR(400 MHz,CDCl3)δ7.78(s,1H),6.09(s,2H),4.73-4.61(m,1H),3.65-3.55(m,1H),3.29-3.18(m,1H),2.98-2.86(m,1H),2.24-2.12(m,1H),2.12-2.04(m,1H),2.03-1.88(m,2H),1.28-1.25(m,5H),1.20-1.14(m,1H),0.84(t,J＝6.8 Hz,3H)；13C NMR(150 MHz,CDCl3)δ156.3,154.1,151.6,139.5,118.3,58.4,53.1,38.4,34.5,31.4,26.1,22.5,14.0；ESI-HRMS(m/z)calcd C13H21ClN5O(M+H+),298.1429；found,298.1433.
3-(6-Amino-2-chloro-9H-purin-9-yl)nonan-1-ol(6h).White solid.mp:120-123℃.1H NMR(600 MHz,CDCl3)δ7.79(s,1H),6.79(s,2H),4.69-4.62(m,1H),3.66-3.56(m,2H),3.31-3.25(m,1H),2.20-2.11(m,1H),2.08-1.99(m,2H),1.95-1.86(m,1H),1.28-1.15(m,7H),1.14-1.07(m,1H),0.82(t,J＝7.2 Hz,3H)；13C NMR(150 MHz,CDCl3)δ156.5,154.0,151.4,139.5,118.3,58.3,53.2,38.2,34.6,31.6,28.9,26.3,22.6,14.1；ESI-HRMS(m/z)calcd C14H23ClN5O(M+H+),312.1586；found,312.1594.
3-(6-Amino-2-chloro-9H-purin-9-yl)tridecan-1-ol(10h).White solid.mp:131-132℃.1H NMR(400 MHz,CDCl3)δ7.78(s,1H),6.81(s,2H),4.71-4.62(m,1H),3.66-3.56(m,2H),3.33-3.23(m,1H),2.21-2.11(m,1H),2.11-1.99(m,2H),1.95-1.85(m,1H),1.27-1.17(m,15H),1.15-1.07(m,1H),0.84(t,J＝6.8 Hz,3H)；13C NMR(100 MHz,CDCl3)δ156.4,154.1,151.6,139.5,118.4,58.4,53.1,38.4,34.6,29.64,29.58,29.5,29.4,29.2,26.4,22.8,14.2；ESI-HRMS(m/z)calcd C18H31ClN5O(M+H+),368.2212；found,368.2214.
3-(8-Bromo-6-chloro-9H-purin-9-yl)octan-1-ol(5i).White solid.mp:74-75℃.1H NMR(400 MHz,CDCl3)δ8.65(s,1H),4.88(s,1H),3.69-3.61(m,1H),3.39-3.29(m,1H),2.66(s,1H),2.50-2.37(m,1H),2.22-2.11(m,1H),1.95-1.85(m,1H),1.84-1.74(m,1H),1.28-1.17(m,5H),1.04-0.93(m,1H),0.80(t,J＝7.2 Hz,3H)；13C NMR(150 MHz,CDCl3)δ152.6,151.4,149.6,136.3,132.2,59.0,58.2,35.3,33.0,31.3,26.0,22.5,14.0；ESI-HRMS(m/z)calcd C13H19BrClN4O(M+H+),361.0425；found,361.0430.
3-(8-Bromo-6-chloro-9H-purin-9-yl)dodecan-1-ol(9i).White solid.mp:70-71℃.1H NMR(600 MHz,CDCl3)δ8.64(s,1H),4.85(s,1H),3.67-3.61(m,1H),3.38-3.30(m,1H),2.67(s,1H),2.48-2.39(m,1H),2.20-2.11(m,1H),1.95-1.80(m,2H),1.26-1.20(m,4H),1.20-1.14(m,9H),1.02-0.93(m,1H),0.83(t,J＝7.2 Hz,3H)；13C NMR(150 MHz,CDCl3)δ152.5,151.4,149.6,136.2,132.2,58.9,58.2,35.3,33.0,31.9,29.5,29.4,29.3,29.1,26.3,22.7,14.2；ESI-HRMS(m/z)calcd C17H27BrClN4O(M+H+),417.1051；found,417.1058.
3-(9H-Purin-9-yl)heptan-1-ol(4j).Colorless oil.1H NMR(400 MHz,CDCl3)δ9.13(s,1H),8.93(s,1H),8.12(s,1H),4.89-4.79(m,1H),3.64-3.57(m,1H),3.24-3.08(m,2H),2.26-2.07(m,3H),2.05-1.92(m,1H),1.33-1.20(m,3H),1.15-1.02(m,1H),0.82(t,J＝7.2 Hz,3H)；13C NMR(150 MHz,CDCl3)δ152.5,151.8,149.0,144.4,134.2,58.4,53.1,38.0,34.2,28.6,22.3,13.9；ESI-HRMS(m/z),calcd C12H19N4O(M+H+),235.1553；found,235.1561.
3-(9H-Purin-9-yl)nonan-1-ol(6j).Colorless oil.1H NMR(400 MHz,CDCl3)δ9.18(s,1H),8.97(s,1H),8.13(s,1H),4.92-4.81(m,1H),3.66-3.57(m,1H),3.22-3.13(m,1H),2.60(s,1H),2.28-2.13(m,2H),2.10-1.96(m,2H),1.28-1.24(m,6H),1.22-1.20(m,2H),0.84(t,J＝6.8 Hz,3H)；13C NMR(100 MHz,CDCl3)δ152.5,151.9,149.1,144.2,134.3,58.4,52.9,38.2,34.5,31.6,28.9,26.4,22.6,14.1；ESI-HRMS(m/z)calcd C14H23N4O(M+H+),263.1866；found,263.1865.
3-(6-Chloro-9H-purin-9-yl)-2-methyleneoctan-1-ol(5k).Colorless oil.1HNMR(600 MHz,CDCl3)δ8.75(s,1H),8.18(s,1H),5.36(s,1H),5.36(t,J＝7.2 Hz,1H),5.25(s,1H),4.13-4.01(m,2H),2.28-2.18(m,3H),1.27-1.23(m,5H),1.21-1.16(m,1H),0.84(t,J＝7.2 Hz,3H)；13C NMR(150 MHz,CDCl3)δ152.0,151.4,148.8,146.3,144.1,136.1,115.0,64.5,56.4,32.5,31.3,26.1,22.5,14.0；ESI-HRMS(m/z)calcd C14H20ClN4O(M+H+),295.1320；found,295.1326.
3-(6-Chloro-9H-purin-9-yl)-2-methylenedodecan-1-ol(9k).Colorlessoil.1H NMR(400MHz,CDCl3)δ8.72(s,1H),8.18(s,1H),5.36(s,1H),5.32(t,J＝8.0 Hz,1H),5.23(s,1H),4.14-4.00(m,2H),2.72(s,1H),2.26-2.15(m,2H),1.29-1.09(m,14H),0.85(t,J＝6.8 Hz,3H)；13C NMR(100 MHz,CDCl3)δ152.01,151.97,151.3,146.2,144.2,131.7,114.9,64.4,56.5,32.5,31.9,29.5,29.4,29.3,29.2,26.4,22.8,14.2；ESI-HRMS(m/z)calcd C18H28ClN4O(M+H+),351.1946；found,351.1945.
3-(6-Chloro-9H-purin-9-yl)dodecyl methanesulfonate(9ba).1H NMR(400MHz,CDCl3)δ8.71(s,1H),8.12(s,1H),4.75-4.65(m,1H),4.25-4.18(m,1H),3.98-3.90(m,1H),2.91(s,3H),2.71-2.60(m,1H),2.47-2.35(m,1H),2.24-2.14(m,1H),2.00-1.88(m,1H),1.26-1.13(m,13H),1.07-0.95(m,1H),0.85(t,J＝7.2Hz,3H)；13C NMR(100MHz,CDCl3)δ151.9,151.8,151.5,145.0,132.3,65.8,54.8,37.6,34.2,33.9,31.9,29.5,29.4,29.3,29.0,26.2,22.7,14.2；ESI-HRMS(m/z)calcdC18H30ClN4O3S(M+H+),417.1722found,417.1719.
9-(1-Azidododecan-3-yl)-6-chloro-9H-purine(9bb).1H NMR(400MHz,CDCl3)δ8.73(s,1H),8.09(s,1H),4.71-4.61(m,1H),3.38-3.29(m,1H),3.07-2.99(m,1H),2.44-2.34(m,1H),2.23-2.09(m,2H),1.99-1.88(m,1H),1.27-1.15(m,13H),1.09-0.98(m,1H),0.85(t,J＝6.8Hz,3H)；13CNMR(100MHz,CDCl3)δ152.0,151.9,151.4,144.6,132.2,55.3,48.1,34.3,33.7,31.9,29.5,29.4,29.3,29.0,26.2,22.8,14.2；ESI-HRMS(m/z)calcdC17H27ClN7(M+H+),364.2011；found,364.2014.
6-Chloro-9-(1-(4-phenyl-1H-1,2,3-triazol-1-yl)dodecan-3-yl)-9H-purine(9bc).1H NMR(400MHz,CDCl3)δ8.73(s,1H),8.16(s,1H),7.80-7.74(m,2H),7.67(s,1H),7.42(t,J＝7.2Hz,2H),7.37-7.31(m,1H),4.61-4.50(m,1H),4.37-4.28(m,1H),4.20-4.09(m,1H),2.99-2.86(m,1H),2.66-2.55(m,1H),2.26-2.13(m,1H),1.96-1.85(m,1H),1.25-1.12(m,13H),1.01-0.90(m,1H),0.84(t,J＝6.8Hz,3H)；13C NMR(100MHz,CDCl3)δ151.9,151.8,151.6,148.1,145.2,132.3,130.3,129.1,128.5,125.8,120.0,55.6,47.0,34.7,34.3,31.9,29.4,29.31,29.25,29.0,26.2,22.7,14.2；ESI-HRMS(m/z)calcdC25H33ClN7(M+H+),466.2480；found,466.2484.
6-Chloro-9-(1-chlorododecan-3-yl)-9H-purine(9bd).1H NMR(400MHz,CDCl3)δ8.70(s,1H),8.09(s,1H),4.81-4.70(m,1H),3.55-3.46(m,1H),3.18-3.08(m,1H),2.75-2.64(m,1H),2.39-2.28(m,1H),2.26-2.14(m,1H),1.98-1.86(m,1H),1.26-1.11(m,13H),1.06-0.94(m,1H),0.82(t,J＝6.8Hz,3H)；13C NMR(100MHz,CDCl3)δ151.8,151.7,151.4,145.1,132.3,55.6,41.0,36.4,33.8,31.9,29.4,29.3,29.2,29.0,26.2,22.7,14.2；ESI-HRMS(m/z)calcdC17H27Cl2N4(M+H+),357.1607；found,357.1608.
Diethyl(((3-(6-chloro-9H-purin-9-yl)dodecyl)oxy)methyl)phosphonate(9be).Colorless oil.1H NMR(600MHz,CDCl3)δ8.70(s,1H),8.14(s,1H),4.76-4.67(m,1H),4.20-4.06(m,4H),3.69-3.58(m,2H),3.58-3.52(m,1H),3.24-3.12(m,1H),2.44-2.34(m,1H),2.28-2.16(m,2H),1.99-1.87(m,1H),1.37-1.29(m,6H),1.26-1.23(m,3H),1.23-1.16(m,10H),1.06-0.97(m,1H),0.85(t,J＝7.2Hz,3H)；13C NMR(150MHz,CDCl3)δ151.9,151.7,151.2,145.6,132.3,69.6,69.6,65.9,64.8,62.54,62.50,55.3,34.2,34.1,31.9,29.5,29.4,29.3,29.1,26.4,22.8,16.7,16.6,14.2；ESI-HRMS(m/z)calcdC22H39ClN4O4P(M+H+),489.2392；found,489.2396.
实施例2：
手性核苷化合物7b-9b(R-andS-)的合成：通用操作如下：在反应管中，依次加入α,β-不饱和醛(0.3mmol)、催化剂(0.02mmol)、苯甲酸(0.02mmol)和无水甲苯1.0mL，室温下搅拌2h，降温到-30℃，在搅拌条件下向反应管中加入6-氯嘌呤(0.2mmol)，控温-30℃反应48h。石油醚/乙酸乙酯＝1.5/1检测反应。反应结束后，保温向混合溶液中加入0.5mL甲醇，再缓慢加入硼氢化钠(0.6mmol)。缓慢升温至0℃反应30min，石油醚/乙酸乙酯＝1.5:1检测反应。反应结束，饱和氯化铵淬灭，减压蒸馏后加水，乙酸乙酯萃取，无水硫酸钠干燥，石油醚/乙酸乙酯＝3:1柱层析，得目标化合物。
Figure GDA0002949731700000171
代表性化合物：
3-(6-Chloro-9H-purin-9-yl)decan-1-ol(7b).White solid.mp:101-102℃.R:[α]D 20-8.37(c 0.14,CHCl3),91％ee[CHIRALCEL ID,n-hexane/2-propanol＝95/5,flowrate＝0.8mL/min,temperature＝25℃,λ＝250nm,retention time:29.500min(major),34.580min(minor)].S:[α]D 208.37(c 0.14,CHCl3),91％ee[CHIRALCEL ID,n-hexane/2-propanol＝95/5,flow rate＝0.8mL/min,temperature＝25℃,λ＝250nm,retentiontime:30.142min(minor),32.460min(major)].1HNMR(400MHz,CDCl3)δ8.69(s,1H),8.13(s,1H),4.86-4.76(m,1H),3.65-3.57(m,1H),3.26-3.16(m,1H),2.84(s,1H),2.25-2.09(m,3H),2.02-1.91(m,1H),1.27-1.12(m,9H),1.10-1.01(m,1H),0.81(t,J＝7.2Hz,3H)；13CNMR(101MHz,CDCl3)δ152.1,151.7,151.2,144.6,131.8,58.3,54.2,37.5,34.3,31.7,29.04,29.03,26.3,22.6,14.1；ESI-HRMS(m/z)calcd C15H23ClN4O(M+H+),311.1639；found,311.1633.
3-(6-Chloro-9H-purin-9-yl)undecan-1-ol(8b).White solid.mp:106-108℃.R:[α]D 20-2.86(c 0.14,CHCl3),92％ee[CHIRALCEL ID,n-hexane/2-propanol＝95/5,flow rate＝0.8mL/min,temperature＝25℃,λ＝250nm,retention time:28.079min(major),32.026min(minor)].S:[α]D 203.00(c 0.20,CHCl3),91％ee[CHIRALCEL ID,n-hexane/2-propanol＝95/5,flow rate＝0.8mL/min,temperature＝25℃,λ＝250 nm,retention time:28.368 min(minor),30.165 min(major)].1H NMR(400 MHz,CDCl3)δ8.69(s,1H),8.13(s,1H),4.86-4.76(m,1H),3.66-3.57(m,1H),3.27-3.15(m,1H),2.80(s,1H),2.25-2.10(m,3H),2.02-1.91(m,1H),1.25-1.20(m,4H),1.20-1.14(m,7H),1.12-1.02(m,1H),0.82(t,J＝7.2 Hz,3H)；13C NMR(101 MHz,CDCl3)δ152.1,151.8,151.3,144.6,131.8,58.3,54.2,37.5,34.4,31.8,29.3,29.2,29.1,26.3,22.7,14.1；ESI-HRMS(m/z)calcdC16H25ClN4O(M+H+),325.1795；found,325.1795.
3-(6-Chloro-9H-purin-9-yl)dodecan-1-ol(9b).White solid.mp:101-102℃.R:[α]D 20-5.00(c 0.12,CHCl3),95％ee[CHIRALCEL ID,n-hexane/2-propanol＝95/5,flow rate＝0.8 mL/min,temperature＝25℃,λ＝250 nm,retention time:25.799 min(major),29.523 min(minor)].R:[α]D 205.00(c 0.12,CHCl3),95％ee[CHIRALCEL ID,n-hexane/2-propanol＝95/5,flow rate＝0.8 mL/min,temperature＝25℃,λ＝250 nm,retention time:26.768 min(minor),28.771 min(major)].1H NMR(400 MHz,CDCl3)δ8.69(s,1H),8.17(s,1H),4.86-4.77(m,1H),3.65-3.57(m,1H),3.26-3.17(m,1H),2.93(s,1H),2.23-2.10(m,3H),2.01-1.90(m,1H),1.25-1.20(m,4H),1.20-1.14(m,9H),1.11-1.02(m,1H),0.82(t,J＝6.8 Hz,3H)；13C NMR(101 MHz,CDCl3)δ152.1,151.7,151.2,144.7,131.8,58.3,54.2,37.5,34.3,31.9,29.44,29.36,29.2,29.1,26.3,22.7,14.2；ESI-HRMS(m/z)calcd C17H27ClN4O(M+H+),339.1952；found,339.1945.
3-(6-Chloro-9H-purin-9-yl)dodecyl methanesulfonate(9ba).Compound R-9ba was synthesised by R-9b.White solid.mp:73-74℃.R:[α]D 20-2.70(c 0.30,CHCl3),94％ee[CHIRALCEL IA,n-hexane/2-propanol＝95/5,flow rate＝0.8 mL/min,temperature＝25℃,λ＝250 nm,retention time:47.048 min(minor),51.301 min(major)].1H NMR(400 MHz,CDCl3)δ8.71(s,1H),8.12(s,1H),4.75-4.65(m,1H),4.25-4.18(m,1H),3.98-3.90(m,1H),2.91(s,3H),2.71-2.60(m,1H),2.47-2.35(m,1H),2.24-2.14(m,1H),2.00-1.88(m,1H),1.26-1.13(m,13H),1.07-0.95(m,1H),0.85(t,J＝7.2Hz,3H)；13C NMR(101 MHz,CDCl3)δ151.9,151.8,151.5,145.0,132.3,65.8,54.8,37.6,34.2,33.9,31.9,29.5,29.4,29.3,29.0,26.2,22.7,14.2；ESI-HRMS(m/z)calcd C18H29ClN4O3S(M+H+),417.1727；found,417.1719.
9-(1-Azidododecan-3-yl)-6-chloro-9H-purine(9bb).Compound R-9bb wassynthesised by R-9ba.Colorless oil.R:[α]D 20-22.5(c 0.08,CHCl3),94％ee[CHIRALCEL IA,n-hexane/2-propanol＝98/2,flow rate＝0.6 mL/min,temperature＝25℃,λ＝250 nm,retention time:34.051 min(minor),39.148 min(major)].Colorlessoil.1H NMR(400 MHz,CDCl3)δ8.73(s,1H),8.09(s,1H),4.71-4.61(m,1H),3.38-3.29(m,1H),3.07-2.99(m,1H),2.44-2.34(m,1H),2.23-2.09(m,2H),1.99-1.88(m,1H),1.27-1.15(m,13H),1.09-0.98(m,1H),0.85(t,J＝6.8 Hz,3H)；13C NMR(101 MHz,CDCl3)δ152.0,151.9,151.4,144.6,132.2,55.3,48.1,34.3,33.7,31.9,29.5,29.4,29.3,29.0,26.2,22.8,14.2；ESI-HRMS(m/z)calcd C17H26ClN7(M+H+),364.2016；found,364.2014.
6-Chloro-9-(1-(4-phenyl-1H-1,2,3-triazol-1-yl)dodecan-3-yl)-9H-purine(9bc).CompoundR-9bc was synthesised by R-9bb.White solid.mp:98-100℃.R:[α]D 20-48.37(c 0.22,CHCl3),94％ee[CHIRALCEL IA,n-hexane/2-propanol＝70/30,flow rate＝0.8 mL/min,temperature＝25℃,λ＝250 nm,retention time:21.317 min(major),25.346 min(minor)].White solid.1H NMR(400 MHz,CDCl3)δ8.73(s,1H),8.16(s,1H),7.80-7.74(m,2H),7.67(s,1H),7.42(t,J＝7.2 Hz,2H),7.37-7.31(m,1H),4.61-4.50(m,1H),4.37-4.28(m,1H),4.20-4.09(m,1H),2.99-2.86(m,1H),2.66-2.55(m,1H),2.26-2.13(m,1H),1.96-1.85(m,1H),1.25-1.12(m,13H),1.01-0.90(m,1H),0.84(t,J＝6.8 Hz,3H)；13C NMR(101 MHz,CDCl3)δ151.9,151.8,151.6,148.1,145.2,132.3,130.3,129.1,128.5,125.8,120.0,55.6,47.0,34.7,34.3,31.9,29.4,29.31,29.25,29.0,26.2,22.7,14.2；ESI-HRMS(m/z)calcd C25H32ClN7(M+H+),466.2486；found,466.2484.
6-Chloro-9-(1-chlorododecan-3-yl)-9H-purine(9bd).Compound R-9bd wassynthesised by R-9b.Colorless oil.R:[α]D 20-21.78(c 0.15,CHCl3),93％ee[CHIRALCEL IA,n-hexane/2-propanol＝95/5,flow rate＝0.8 mL/min,temperature＝25℃,λ＝250 nm,retention time:9.979 min(minor),11.190 min(major)].Colorlessoil.1H NMR(400 MHz,CDCl3)δ8.70(s,1H),8.09(s,1H),4.81-4.70(m,1H),3.55-3.46(m,1H),3.18-3.08(m,1H),2.75-2.64(m,1H),2.39-2.28(m,1H),2.26-2.14(m,1H),1.98-1.86(m,1H),1.26-1.11(m,13H),1.06-0.94(m,1H),0.82(t,J＝6.8 Hz,3H)；13C NMR(101 MHz,CDCl3)δ151.8,151.7,151.4,145.1,132.3,55.6,41.0,36.4,33.8,31.9,29.4,29.3,29.2,29.0,26.2,22.7,14.2；ESI-HRMS(m/z)calcd C17H26Cl2N4(M+H+),357.1613；found,357.1608.
Diethyl(((3-(6-chloro-9H-purin-9-yl)dodecyl)oxy)methyl)phosphonate(9be).Compound R-9be was synthesised by R-9b.Colorless oil.R:[α]D 20-1.74(c0.23,CHCl3),90％ee[CHIRALCEL ID,n-hexane/2-propanol＝80/20,flow rate＝0.8mL/min,temperature＝25℃,λ＝250 nm,retention time:19.747 min(minor),21.282min(major)].Colorless oil.1H NMR(600 MHz,CDCl3)δ8.70(s,1H),8.14(s,1H),4.76-4.67(m,1H),4.20-4.06(m,4H),3.69-3.58(m,2H),3.58-3.52(m,1H),3.24-3.12(m,1H),2.44-2.34(m,1H),2.28-2.16(m,2H),1.99-1.87(m,1H),1.37-1.29(m,6H),1.26-1.23(m,3H),1.23-1.16(m,10H),1.06-0.97(m,1H),0.85(t,J＝7.2Hz,3H)；13C NMR(151MHz,CD3OD)δ155.5,153.5,152.8,152.6,132.6,71.2,71.1,66.0,64.9,64.04,63.99,56.5,35.3,35.2,33.0,30.4,30.32,30.26,29.9,27.1,23.7,16.8,16.7,14.4；ESI-HRMS(m/z)calcdC22H38ClN4 O4P(M+H+),489.2397；found,489.2396.
Figure GDA0002949731700000201
R-构型手性核苷化合物9ba-9be的合成与实施例1中消旋的核苷化合物9ba-9be的合成方法类似，原料换成R-构型的反应原料就行。
3-(6-Chloro-9H-purin-9-yl)dodecyl methanesulfonate(9ba).Compound R-9ba was synthesised by R-9b.White solid.mp:73-74℃.R:[α]D 20-2.70(c 0.30,CHCl3),94％ee[CHIRALCEL IA,n-hexane/2-propanol＝95/5,flow rate＝0.8mL/min,temperature＝25℃,λ＝250nm,retention time:47.048min(minor),51.301min(major)].1H NMR(400MHz,CDCl3)δ8.71(s,1H),8.12(s,1H),4.75-4.65(m,1H),4.25-4.18(m,1H),3.98-3.90(m,1H),2.91(s,3H),2.71-2.60(m,1H),2.47-2.35(m,1H),2.24-2.14(m,1H),2.00-1.88(m,1H),1.26-1.13(m,13H),1.07-0.95(m,1H),0.85(t,J＝7.2Hz,3H)；13C NMR(101MHz,CDCl3)δ151.9,151.8,151.5,145.0,132.3,65.8,54.8,37.6,34.2,33.9,31.9,29.5,29.4,29.3,29.0,26.2,22.7,14.2；ESI-HRMS(m/z)calcd C18H29ClN4O3S(M+H+),417.1727；found,417.1719.
9-(1-Azidododecan-3-yl)-6-chloro-9H-purine(9bb).Compound R-9bb wassynthesised by R-9ba.Colorless oil.R:[α]D 20-22.5(c 0.08,CHCl3),94％ee[CHIRALCEL IA,n-hexane/2-propanol＝98/2,flow rate＝0.6mL/min,temperature＝25℃,λ＝250nm,retention time:34.051min(minor),39.148min(major)].Colorlessoil.1H NMR(400MHz,CDCl3)δ8.73(s,1H),8.09(s,1H),4.71-4.61(m,1H),3.38-3.29(m,1H),3.07-2.99(m,1H),2.44-2.34(m,1H),2.23-2.09(m,2H),1.99-1.88(m,1H),1.27-1.15(m,13H),1.09-0.98(m,1H),0.85(t,J＝6.8 Hz,3H)；13C NMR(101 MHz,CDCl3)δ152.0,151.9,151.4,144.6,132.2,55.3,48.1,34.3,33.7,31.9,29.5,29.4,29.3,29.0,26.2,22.8,14.2；ESI-HRMS(m/z)calcd C17H26ClN7(M+H+),364.2016；found,364.2014.
6-Chloro-9-(1-(4-phenyl-1H-1,2,3-triazol-1-yl)dodecan-3-yl)-9H-purine(9bc).CompoundR-9bc was synthesised by R-9bb.White solid.mp:98-100℃.R:[α]D 20-48.37(c 0.22,CHCl3),94％ee[CHIRALCEL IA,n-hexane/2-propanol＝70/30,flow rate＝0.8 mL/min,temperature＝25℃,λ＝250 nm,retention time:21.317 min(major),25.346 min(minor)].White solid.1H NMR(400 MHz,CDCl3)δ8.73(s,1H),8.16(s,1H),7.80-7.74(m,2H),7.67(s,1H),7.42(t,J＝7.2 Hz,2H),7.37-7.31(m,1H),4.61-4.50(m,1H),4.37-4.28(m,1H),4.20-4.09(m,1H),2.99-2.86(m,1H),2.66-2.55(m,1H),2.26-2.13(m,1H),1.96-1.85(m,1H),1.25-1.12(m,13H),1.01-0.90(m,1H),0.84(t,J＝6.8 Hz,3H)；13C NMR(101 MHz,CDCl3)δ151.9,151.8,151.6,148.1,145.2,132.3,130.3,129.1,128.5,125.8,120.0,55.6,47.0,34.7,34.3,31.9,29.4,29.31,29.25,29.0,26.2,22.7,14.2；ESI-HRMS(m/z)calcd C25H32ClN7(M+H+),466.2486；found,466.2484.
6-Chloro-9-(1-chlorododecan-3-yl)-9H-purine(9bd).Compound R-9bd wassynthesised by R-9b.Colorless oil.R:[α]D 20-21.78(c 0.15,CHCl3),93％ee[CHIRALCEL IA,n-hexane/2-propanol＝95/5,flow rate＝0.8 mL/min,temperature＝25℃,λ＝250 nm,retention time:9.979 min(minor),11.190 min(major)].Colorlessoil.1H NMR(400 MHz,CDCl3)δ8.70(s,1H),8.09(s,1H),4.81-4.70(m,1H),3.55-3.46(m,1H),3.18-3.08(m,1H),2.75-2.64(m,1H),2.39-2.28(m,1H),2.26-2.14(m,1H),1.98-1.86(m,1H),1.26-1.11(m,13H),1.06-0.94(m,1H),0.82(t,J＝6.8 Hz,3H)；13C NMR(101 MHz,CDCl3)δ151.8,151.7,151.4,145.1,132.3,55.6,41.0,36.4,33.8,31.9,29.4,29.3,29.2,29.0,26.2,22.7,14.2；ESI-HRMS(m/z)calcd C17H26Cl2N4(M+H+),357.1613；found,357.1608.
Diethyl(((3-(6-chloro-9H-purin-9-yl)dodecyl)oxy)methyl)phosphonate(9be).Compound R-9be was synthesised by R-9b.Colorless oil.R:[α]D 20-1.74(c0.23,CHCl3),90％ee[CHIRALCEL ID,n-hexane/2-propanol＝80/20,flow rate＝0.8mL/min,temperature＝25℃,λ＝250 nm,retention time:19.747 min(minor),21.282min(major)].Colorless oil.1H NMR(600 MHz,CDCl3)δ8.70(s,1H),8.14(s,1H),4.76-4.67(m,1H),4.20-4.06(m,4H),3.69-3.58(m,2H),3.58-3.52(m,1H),3.24-3.12(m,1H),2.44-2.34(m,1H),2.28-2.16(m,2H),1.99-1.87(m,1H),1.37-1.29(m,6H),1.26-1.23(m,3H),1.23-1.16(m,10H),1.06-0.97(m,1H),0.85(t,J＝7.2Hz,3H)；13C NMR(151MHz,CD3OD)δ155.5,153.5,152.8,152.6,132.6,71.2,71.1,66.0,64.9,64.04,63.99,56.5,35.3,35.2,33.0,30.4,30.32,30.26,29.9,27.1,23.7,16.8,16.7,14.4；ESI-HRMS(m/z)calcdC22H38ClN4 O4P(M+H+),489.2397；found,489.2396.
实施例3：
本发明采用上述方法合成了系列消旋、R-构型和S-构型非环核苷化合物，并用MTT法测试了这些化合物对肿瘤细胞的细胞活性。使用3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide(MTT)测定评估合成化合物的细胞活性，将每孔5×103个细胞的密度将细胞接种到96孔板中，将细胞培养在37℃/5％CO2的培养箱中24h。然后用各种梯度稀释浓度的化合物，对照药物5-FU或者稀释剂(DMSO)处理48h。将含化合物，5-FU或者稀释剂(DMSO)的培养基更换成180μL的新鲜培养基以及20μL的MTT溶液(5mg/mLPBS溶液)，放置于37℃，5％CO2的培养箱中孵育4h，然后将含有MTT的培养基更换成DMSO(150μL)，用酶标仪(Multiskan FC，Thermo)在570nm波长处测定每个空的吸光度，每个浓度测试三次。数据通过GraphPadPism 6软件进行分析。
非环核苷化合物的抗肿瘤活性结果如下：
Figure GDA0002949731700000221
Figure GDA0002949731700000231
Figure GDA0002949731700000241
Figure GDA0002949731700000251
Figure GDA0002949731700000252
Figure GDA0002949731700000261
Figure GDA0002949731700000262
申请人选取了抗肿瘤活性较好的3个化合物7b，8b，9b；并合成了三个对应的R和S两种构型化合物，并通过MTT对它们的抗肿瘤活性进行研究。化合物构型对于其抗肿瘤活性略有影响，S-构型化合物抗肿瘤活性没有其对应消旋化合物好，而R-构型化合物抗肿瘤活性比之其对应消旋化合物要好或者相当，并且比对照药物5-氟尿嘧啶的抗肿瘤活性要好。
Figure GDA0002949731700000263
Figure GDA0002949731700000271
申请人又探索了将侧链上羟基进行修饰或者改变时的抗肿瘤活性。非环核苷化合物R-构型抗肿瘤活性比其对应消旋化合物高，同时也合成了侧链上羟基进行修饰或者改变后每个化合物对应R-构型化合物。以9b为底物，将羟基更换为OMs、N3等基团。由图2和表2可知，当羟基更换为N3时，其抗肿瘤活性与9b相当，其它化合物与9b相比，抗肿瘤活性均有所降低。
Figure GDA0002949731700000272
Figure GDA0002949731700000281
本实施例所有表格中，涉及a,b,c角标，标注解释均如下：
a通过MTT测定法测定单个化合物对肿瘤细胞的抗增殖活性。数据是一式三份测定的平均值。b,cHCT-116和SW480均为人结肠癌细胞系。
实施例4
细胞周期测试：将每孔5×105个HCT-116细胞接种到六孔板中，将六孔板放在37℃/5％CO2培养箱中培养24h，用稀释剂(DMSO)或指定浓度化合物9b处理细胞。24h后，使用不加EDTA胰酶溶液消化细胞，离心得到悬浮细胞，PBS洗涤两次，并在-20℃用75％乙醇固定过夜。离心，弃去上清液，PBS清洗两次，然后根据试剂盒说明书在室温下在黑暗条件下用碘化丙啶染料将细胞染色孵育15min，然后进行流式细胞术检测。每次计数10000个细胞。实验结果表明化合物9b可将HCT-116细胞有显著性的阻滞在G0/G1期(p<0.05)。所以，化合物9b对细胞分裂有一定的阻滞作用，可将癌细胞阻滞在G0/G1期。
细胞凋亡测试：将每孔5×105个SW480细胞或HCT-116细胞接种到六孔板中，37℃/5％CO2培养箱中培养24h。用稀释剂(DMSO)或指定浓度化合物9b处理细胞，48h后，使用不加EDTA胰酶溶液消化细胞，离心，弃去上清液，PBS洗涤两次，根据试剂盒说明书在室温下在黑暗条件下将Annexin V-FITC和PI将细胞染色15min，然后1h内进行流式细胞术检测。每次计数10000个细胞。化合物9b在HCT-116细胞中诱导的细胞凋亡，随着化合物浓度的增加，凋亡细胞的比例从12％增加到66％，化合物9b在SW480细胞中诱导的细胞凋亡如图1所示，随着化合物浓度增加，凋亡细胞比例从6％增加到69％，凋亡细胞所占比例均有显著性提高。由此可以得出9b可以诱导HCT-116和SW480的细胞凋亡从而抑制癌细胞增殖。
Western Blot实验：将每孔5×105个SW480细胞接种到六孔板中，37℃/5％CO2培养箱中培养24h。用稀释剂(DMSO)或指定浓度化合物9b处理细胞。48h后，使用胰酶溶液消化细胞，离心，弃去上清液，PBS洗两次，加裂解液裂解细胞，4℃/15000r/min离心15min，弃去上清液，按照试剂盒说明定量蛋白浓度。在SDS-PAGE凝胶上通过电泳分离等量的总细胞蛋白提取物，然后转移至聚偏二氟乙烯(PVDF)膜上，封闭液封闭2h。再将膜与Bax，P53，Bcl-xl和Cleaved-PARP一抗以1：2000稀释度0℃孵育过夜或者室温孵育2h。随后，将膜与适当的荧光二抗0℃孵育过夜或者室温孵育2h，并通过Odyssey CLx红外激光成像系统扫描。由图2可以看出，随着化合物9b浓度的增加，Bax、P53和Cleaved-PARP这3种蛋白表达在逐渐递增，Bcl-xl蛋白表达在逐渐减弱。由此得出化合物9b至少通过促进Bax、P53和Cleaved-PARP表达和抑制Bcl-xl蛋白表达来诱导细胞凋亡。
线粒体膜电位(MMP)测定：将每孔5×105个SW480细胞接种到六孔板中，将六孔板放在37℃/5％CO2的培养箱中培养24h。用稀释剂(DMSO)或指定浓度化合物9b处理细胞。24h后，胰酶溶液消化细胞，离心，加入JC-1缓冲液并混匀细胞，在37℃/5％CO2培养箱中孵育20min，离心，JC-1染色缓冲液清洗3次，并用JC-1染色缓冲液重悬后，使用荧光显微镜观察或者使用流式细胞仪进行分析。由图3中A荧光显微镜获得的图像显示绿色区域(低MMP)随着化合物9b浓度增加而增加。同时，图3中B流式细胞仪结果表明，发射红色荧光的已处理细胞数量明显减少，而发射绿色荧光的已处理细胞数量明显增加，这提示了MMP破坏。结果表明，MMP耗散可能参与化合物9b诱导的细胞凋亡。
细胞集落形成实验：将HCT-116细胞和SW480细胞以每孔1500个细胞的密度接种到六孔板中，将六孔板放在37℃/5％CO2培养箱中培养24h。然后用指定浓度化合物9b处理细胞，48h后更换为常规培养基继续培养，之后每72h更换一次培养基，14天后每孔用PBS清洗两次，加入甲醇固定30min，使用PBS清洗两次，向每孔中加入1％结晶紫染色缓冲液，并孵育30min。最后将每孔用PBS清洗3次，晾干后拍摄照片。由图4可以看出，随着化合物9b的浓度增加，两种细胞的集落的形成受到了明显的抑制作用，并且当化合物9b的浓度为16μM时，集落几乎完全消失。这表明化合物9b可以以剂量依赖性的抑制癌细胞集落的形成，具有很好的抗肿瘤活性。
裸鼠移植瘤实验:所有动物研究和程序均已获得上海复旦大学药学院动物伦理委员会批准。6周龄BALB/C裸鼠购自上海Slac实验动物有限公司(中国上海)，在无特定病原体环境中饲养在上海复旦大学的动物保管单元中。皮下注射SW480细胞(每只小鼠4×106)。注射后7天，将小鼠随机分为4组(n＝8)，并分别用25mg/kg和50mg/kg化合物9b溶于5％乙醇和5％蓖麻油PBS中(每只小鼠0.2mL)50mg/kg 5-FU或媒介物对照(0.2mL稀释剂)进行腹膜内治疗并且用数字卡尺测量肿瘤体积，肿瘤体积计算公式为体积＝最短直径(W)2×最长直径(L)×0.52。每2天进行一次，给药7次。在实验结束时，将小鼠称重并处死，收集肿瘤，用4％多聚甲醛固定并包埋在石蜡中。H&E染色用于进一步分析小鼠的实体瘤。如图5所示，肿瘤质量图5中A，肿瘤体积图5中C均有显著性减小，并且这也可以从裸鼠体内解剖出的肿瘤图5中D可以看出肿瘤有明显减小，而由小鼠体重图5中B无明显变化。和对照药物5-FU相比药效相当且毒性较小。如图5中E所示，对裸鼠中的肿瘤进行H&E染色实验结果表明，给予化合物9b后，小鼠体内肿瘤发生明显坏死。由此可以得出化合物9b可以杀死体内肿瘤细胞并且抑制肿瘤生长，有进一步成为临床药物的潜力。
实施例5
申请人对合成非环核苷类化合物进行体内外抗肿瘤活性评价，选出最优化合物，总结构效关系。针对于结肠癌细胞HCT-116和SW480发现得到化合物9b的抗肿瘤活性最佳。接着，选取了人肝癌细胞(SK-hep-1)、人卵巢癌细胞(SKOV-3)、人宫颈癌细胞(Hela)、人乳腺癌细胞(MCF-7)等测试化合物9b是否对其他癌细胞有抑制作用。如表3所示，化合物9b及其R,S两种构型对这几种癌细胞均有一定的抗肿瘤活性，但是都没有对结肠癌细胞HCT-116和SW480的抗肿瘤活性好，其中在人肝癌细胞(SK-hep-1)，人卵巢癌细胞(SKOV-3)，人宫颈癌细胞(Hela)，人乳腺癌细胞(MCF-7)这几种癌细胞中，对人肝癌细胞(SK-hep-1)的抑制作用最好(表3)。
表3化合物9b对SK-hep-1，SKOV3，Hela，MCF-74种癌细胞的抗肿瘤活性
Figure GDA0002949731700000311
a通过MTT测定法测定单个化合物对肿瘤细胞的抗增殖活性。数据是一式三份测定的平均值。b,c,d,e SK-hep-1，SKOV3，Hela和MCF-7是人癌细胞系。
以上实施例描述了本发明的基本原理、主要特征及优点。本行业的技术人员应该了解，本发明不受上述实施例的限制，上述实施例和说明书的描述的只是说本发明的原理，在不脱离本发明原理的范围下，本发明还会有各种变化和改进，任何非环核苷的结构变化，这些变化和改进均落入本发明保护的范围内。

Claims (5)

1.一种非环核苷化合物，其特征在于，其结构如下：
Figure FDA0003592249930000011
2.非环核苷化合物及其在药学上可接受的盐在制备预防和治疗结肠癌药物中的应用，所述非环核苷类化合物结构为：
Figure FDA0003592249930000012
Figure FDA0003592249930000021
Figure FDA0003592249930000031
Figure FDA0003592249930000041
3.根据权利要求2所述非环核苷化合物及其在药学上可接受的盐在制备预防和治疗结肠癌药物中的应用，其特征在于：所述药学上可接受的盐，包括非环核苷化合物与有机酸或无机酸形成的盐。
4.根据权利要求3所述非环核苷化合物及其在药学上可接受的盐在制备预防和治疗结肠癌药物中的应用，其特征在于：有机酸选自苹果酸、乳酸、樟脑磺酸、枸橼酸、富马酸或草酸中的一种或多种，无 机酸选自磷酸、氢卤酸、硫酸或硝酸中一种或多种。
5.一种用于预防和治疗结肠癌的复方药物，其特征在于，其含有非环核苷类化合物，所述非环核苷类化合物与权利要求2中一致。
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