CN119306700A - Dnmt1-hdac双靶点抑制剂及其制备方法和应用 - Google Patents
Dnmt1-hdac双靶点抑制剂及其制备方法和应用 Download PDFInfo
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Abstract
本发明涉及一种如式(I)所示DNMT1‑HDAC双靶点抑制剂及其制备方法和应用，本发明化合物不仅表现出良好的DNMT1和HDAC酶抑制活性，而且对实体瘤和血液瘤均有显著抗肿瘤活性。该类化合物作为首次报道的基于DNMT1和HDACs的双靶点抑制剂，在癌症治疗方面具有很大前景；
Description
技术领域
本发明涉及一种DNMT1-HDAC双靶点抑制剂及其制备方法和应用，属于医药技术领域。
背景技术
癌症是一种以基因异常为特征的疾病，其特点是对治疗有抗药性，死亡率高。然而，表观遗传学已经逐渐被认为是导致癌症行为的一个因素，它不能仅仅用基因改变来解释。通常，肿瘤细胞在生长潜力、侵袭性、恶性转化能力和对治疗干预的抗性方面的异质性可归因于表观遗传调控的干扰，与正常细胞相比，癌细胞在表观基因组中引起广泛而深刻的改变，包括DNA甲基化、组蛋白乙酰化、组蛋白甲基化和染色质重塑。其中，组蛋白尾部赖氨酸乙酰化异常和肿瘤抑制基因启动子区CpG岛超甲基化是肿瘤进展和预后不良的驱动因素和生物标志物。
DNA甲基化是由DNA甲基转移酶(DNMT)调控的。人类DNMT家族包括5个成员，可进一步分为两组，包括典型成员(DNMT1、DNMT3A和DNMT3B)，它们可以将s-腺苷-l-蛋氨酸(SAM)衍生的甲基添加到CpG岛胞嘧啶的C-5位置，以及非典型成员(DNMT2和DNMT3L)，该家族成员不具备催化活性。与DNMT3A和DNMT3B从头开始建立甲基化模式不同，DNMT1负责维持DNA甲基化并将甲基化模式从模式传递到子链。此外，与其他DNMT相比，DNMT1的水平在正常组织和转移期的癌症组织中都占主导地位。目前，DNMT抑制剂分为共价核苷抑制剂和非核苷的可逆性抑制剂。前者以阿扎胞苷(AZA)、地西他滨(DAC)、西布拉林为代表，后者以RG108、SGI-1027为代表。
组蛋白去乙酰化酶(HDACs)可以清除组蛋白尾部或非组蛋白底物中赖氨酸的乙酰基团。人类已经发现了18种HDAC，并根据催化机制和细胞内定位将其分为四类，包括锌依赖的I类(HDAC1、2、3、8)，IIa类(HDAC4、5、7、9)，IIb类(HDAC 6、10)，IV类(HDAC11)和NAD+依赖的III类(Sirtuin1-7)。迄今为止，HDAC抑制剂通常用于治疗血液系统恶性肿瘤，部分抑制剂已获得FDA批准，包括伏立诺他(SAHA)、贝利司他(PDX101)、帕比司他(LBH589)、西达苯胺(CS055)和罗米地辛(FK 228)。
DNMT抑制剂和HDAC抑制剂联合应用于多种癌症治疗，如急性髓细胞白血病(AML)，多发性骨髓瘤(MM)，弥漫性大B细胞淋巴瘤(DLBCL)，T细胞淋巴瘤(TCL)，和骨髓增生异常综合征等。例如，AML患者接受阿扎胞苷和帕比司的联合治疗所观察到的的临床疗效优于帕比司他单药治疗。虽然DNMT和HDAC抑制剂联合使用在治疗AML和肿瘤免疫治疗中具有一定的优势，但不利的是药物-药物相互作用限制了协同作用的实现，如复杂的给药方案和对药代动力学的负面影响。相比之下，设计DNMT/HDAC双靶抑制剂可规避这些潜在的缺点。然而，目前报道的DNMT/HDAC双靶抑制剂对DNMT或HDAC的抑制效果不佳，且缺乏对DNMT1的亚型选择性，导致与单药治疗相比，没有显示出明显的治疗优势。
因此，研发一种有效的DNMT1/HDAC双重抑制剂具有重要的临床意义。
发明内容
针对现有技术的不足，本发明提供一种DNMT1-HDAC双靶点抑制剂及其制备方法和应用。
本发明的技术方案如下：
DNMT1-HDAC双靶点抑制剂，所述的抑制剂的结构式如式(I)所示或式(I)在药学上可接受的盐：
其中，X为芳香杂环或以下连接基团之一：
A为以下结构之一：
R1为羟基或2-氨基苯基；
R2为以下结构之一
R3为氢原子为以下结构之一：
DNMT1-HDAC双靶点抑制剂选自以下化合物其中之一：
N1-(1-(6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)-N4-羟基丁二酰胺(11a)；
N1-(1-(6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)-N5-羟基戊二酰胺(11b)；
N1-(1-(6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)-N6-羟基己二酰胺(11c)；
N1-(1-(6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)-N7-羟基庚二酰胺(11d)；
N1-(1-(6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)-N8-羟基辛二酰胺(11e)；
N1-(1-(6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)-N9-羟基壬二酰胺(11f)；
N1-(1-(6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)-N4-羟基对甲苯二酰胺(11g)；
N1-(1-(6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)-4-((4-(羟基氨甲酰基)苄基)氧基)苯甲酰胺(11h)；
N1-(1-(6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)-N4-(4-(羟基氨甲酰基)苯基)丁二酰胺(11i)；
N1-(1-(6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)-N5-(4-(羟基氨甲酰基)苯基)戊二酰胺(11j)；
N1-(1-(6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)-N6-(4-(羟基氨甲酰基)苯基)己二酰胺(11k)；
N1-(1-(6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)-N4-(4-(羟基氨甲酰基)苯基)丁二酰胺(11l)；
N1-(1-(6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)-N5-(4-(羟基氨甲酰基)苯基)戊二酰胺(11m)；
N1-(1-(6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)-N4-(4-(3-(羟基氨基)-3-氧代丙基-1-烯-1-基)苯基)丁二酰胺(11n)；
4-(((1-(6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)氨基)甲基)-N-羟基苯甲酰胺(11o)；
(E)-3-4-(((1-(6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)氨基)甲基)苯基)-N-羟基丙烯酰胺(11p)；
1-(6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)-N-(4-((4-(羟基氨甲酰基)苯基)氨甲酰基)苯基)哌啶4-铵盐(11q)；
(E)-1-(6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)-N-(4-((4-(3-羟基氨基)-3-氧代丙基-1-烯-1-基)苄基)氨甲酰基)苄基)哌啶-4-铵盐(11r)；
(E)-1-(6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)-N-(4-((4-(3-羟基氨基)-3-氧代丙基-1-烯-1-基)苄基)氧代)苄基)哌啶-4-铵盐(11s)；
1-(6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)-N-(4-((7-(羟氨基)-7-氧代庚基)氧代)苄基)哌啶-4-铵盐(11t)；
2-((1-(6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)氨基)-N-羟基嘧啶-5-甲酰胺(11u)；
2-((1-(6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)(甲基)氨基)-N-羟基嘧啶-5-甲酰胺(11v)；
2-(4-((6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)(甲基)氨基)哌啶-1-基)-N-羟基嘧啶-5-甲酰胺(15a)
2-(4-((6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)(甲基)(甲基)氨基)哌啶-1-基)-N-羟基嘧啶-5-甲酰胺(15b)；
(E)-3-(4-((4-(6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)哌嗪-1-基)甲基)苯基)-N-羟基丙烯酰胺(19a)；
2-(4-((6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)哌嗪-1-基)-N-羟基嘧啶-5-甲酰胺(19b)；
(E)-3-(4-((((1-(6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)甲基)氨基)甲基)苯基)-N-羟基丙烯酰胺(23a)；
(R，E)-3-(4-((((1-(6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)甲基)氨基)甲基)苯基)-N-羟基丙烯酰胺((R)-23a)；
(S，E)-3-(4-((((1-(6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)甲基)氨基)甲基)苯基)-N-羟基丙烯酰胺((S)-23a)；
2-(((1-(6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)甲基)氨基)-N-羟基嘧啶-5-甲酰胺(23b)；
(E)-3-(4-((4-(6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)哌嗪-1-基)甲基)苯基)-N-羟基丙烯酰胺(27)；
N1-(1-(6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)-N5-(2-氨基苯基)戊二酰胺(30a)；
N1-(1-(6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)-N4-(2-氨基苯基)对苯二甲酰胺(30b)；
4-(2-((1-(6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)氨基)-2-氧代乙基)-N-(2-氨基苯基)苯甲酰胺(30c)；
4-(((1-(6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)氨基)甲基)-N-(2-氨基苯基)苯甲酰胺(30d)；
2-((1-(6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)(甲基)氨基)-N-(2-氨基苯基)嘧啶-5-甲酰胺(30e)；
N1-(2-((3,5-二氰基-4-乙基-6-((4-(N-甲基甲基磺酰氨基)苯基)硫代)吡啶-2-基)(甲基)氨基)乙基)-N4-羟基丁二酰胺(40a)；
N1-(2-((3,5-二氰基-4-乙基-6-((4-(N-甲基甲基磺酰氨基)苯基)硫代)吡啶-2-基)(甲基)氨基)乙基)-N5-羟基戊二酰胺(40b)；
N1-(3-((3,5-二氰基-4-乙基-6-((4-(N-甲基甲基磺酰氨基)苯基)硫代)吡啶-2-基)(甲基)氨基)丙基)-N4-羟基丁二酰胺(40c)；
2-((2-((3,5-二氰基-4-乙基-6-((4-(N-甲基甲基磺酰氨基)苯基)硫代)吡啶-2-基)(甲基)氨基)乙基)(甲基)氨基)-N-羟基嘧啶-5-甲酰胺(40d)；
N1-(1-(3,5-二氰基-4-乙基-6-((4-(N-甲基甲基磺酰氨基)苯基)硫代)吡啶-2-基)哌啶-4-基)-N5-羟基戊二酰胺(44a)；
(E)-(1-(3,5-二氰基-4-乙基-6-((4-(N-甲基甲基磺酰氨基)苯基)硫代)吡啶-2-基)哌啶-4-基)-N5-羟基戊二酰胺(44b)；
(E)-3-(4-(((1-(3，5-二氰基-4-乙基-6-((4-(N-甲基甲基磺酰氨基)苯基)硫代)吡啶-2-基)哌啶-4-基)氨基)甲基)苯基)-N-羟基丙烯酰胺(44c)；
2-(4-(3，5-二氰基-4-乙基-6-((4-(N-甲基甲基磺酰氨基)苯基)硫代)吡啶-2-基)哌嗪-1-基)-N-羟基嘧啶-5-甲酰胺(48)。
DNMT1-HDAC双靶点抑制剂选自以下结构的一种：
本发明的第二个目的是提供DNMT1-HDAC双靶点抑制剂的制备方法，所述制备方法包括以下任一方法：
化合物11a-11V的制备方法如下：
试剂和条件：(a)对甲苯磺酰氯，N，N-二异丙基乙胺，4-二甲氨基吡啶，乙腈，80℃，6小时；(b)正丙醛，N-甲基吗啉，无水乙醇，室温，30小时；(c)N，N-二异丙基乙胺，N，N-二甲基甲酰胺，85℃，5小时；(d)亚硝酸异戊酯，氯化铜，乙腈，80℃，2.5小时；(e)氨基哌啶衍生物，碳酸钾，二氯甲烷，4小时；(f)4M盐酸/乙酸乙酯，室温，12小时；(g)1)9a-n,氯甲酸异丁酯，N-甲基吗啉，二氯甲烷/四氢呋喃(1：1)，室温，6小时；2)9o-t，三乙酰氧基硼氢化钠，醋酸，室温，12小时；3)9u，碳酸钾，乙腈，80℃，6小时；(h)4M盐酸/乙酸乙酯，室温，12小时；
化合物15a、15b、19a、19b的制备方法如下：
试剂和条件：(a)氨基哌啶衍生物，碳酸钾，二氯甲烷，4小时；(b)4M盐酸/乙酸乙酯，室温，12小时；(c)9u，碳酸钾，乙腈，80℃，6小时；(d)4M盐酸/乙酸乙酯，室温，12小时；(e)哌嗪-1-叔丁基羧酸酯，碳酸钾，二氯甲烷，4小时；(f)4M盐酸/乙酸乙酯，室温，12小时；(g)1)9p，三乙酰氧基硼氢化钠，醋酸，室温，12小时；2)9u，碳酸钾，乙腈，80℃，6小时；(h)4M盐酸/乙酸乙酯，室温，12小时；
化合物23a、(R)-23a、(S)-23a、23b、27、30a-e的制备方法如下：
试剂和条件：(a)哌啶-4-氨甲基叔丁基甲酸酯，碳酸钾，二氯甲烷，4小时；(b)4M盐酸/乙酸乙酯，室温，12小时；(c)1)9p，三乙酰氧基硼氢化钠，醋酸，室温，12小时；2)9u，碳酸钾，乙腈，80℃，6小时；(d)4M盐酸/乙酸乙酯，室温，12小时；(e)叔丁基(2-(甲氨基)乙基)氨基甲酸酯，碳酸钾，二氯甲烷，4小时；(f)4M盐酸/乙酸乙酯，室温，12小时；(g)9p，三乙酰氧基硼氢化钠，醋酸，室温，12小时；(h)4M盐酸/乙酸乙酯，室温，12小时；(i)1)28a-c,氯甲酸异丁酯，N-甲基吗啉，二氯甲烷/四氢呋喃(1：1)，室温，6小时；2)28d，三乙酰氧基硼氢化钠，醋酸，室温，12小时；3)28e，碳酸钾，乙腈，80℃，6小时；(j)4M盐酸/乙酸乙酯，室温，12小时；
化合物40a-d的制备方法如下：
试剂和条件：(a)甲磺酰氯，三乙胺，二氯甲烷，室温，4小时；(b)二异丁基氢化铝，四氢呋喃，-20℃，6小时；(c)对甲苯磺酰氯，三乙胺，DMAP，乙腈，80℃，4小时；(d)4，三乙胺，DMF，85℃，5小时；(e)亚硝酸异戊酯，氯化铜，乙腈，80℃，2.5小时；(f)2-甲氨基衍生物，碳酸钾，二氯甲烷，4小时；(g)4M盐酸/乙酸乙酯，室温，12小时；(h)1)9a or 9b,氯甲酸异丁酯，N-甲基吗啉，二氯甲烷/四氢呋喃(1：1)，室温，6小时；2)9u，碳酸钾，乙腈，80℃，6小时；(i)4M盐酸/乙酸乙酯，室温，12小时；
化合物44a-c、48的制备方法如下：
试剂和条件：(a)4-氨基甲酸叔丁酯，碳酸钾，二氯甲烷，4小时；(b)4M盐酸/乙酸乙酯，室温，12小时；(c)1)9b,氯甲酸异丁酯，N-甲基吗啉，二氯甲烷/四氢呋喃(1：1)，室温，6小时；2)9p，三乙酰氧基硼氢化钠，醋酸，室温，12小时；3)9u，碳酸钾，乙腈，80℃，6小时；(d)4M盐酸/乙酸乙酯，室温，12小时；(e)哌嗪-1-叔丁基羧酸酯，碳酸钾，二氯甲烷，4小时；(f)4M盐酸/乙酸乙酯，室温，12小时；(g)9u，碳酸钾，乙腈，80℃，6小时；(h)4M盐酸/乙酸乙酯，室温，12小时。
一种药物组合物，含有治疗有效量的所述的DNMT1和HDAC双靶点抑制剂及药学上可接受的辅料。
所述的DNMT1-HDAC双靶点抑制剂在制备治疗与靶点有关的肿瘤疾病的药物中的应用。
所述肿瘤为肺癌、肝癌、非小细胞肺癌、结肠癌、皮肤癌、胰腺癌、卵巢癌、乳腺癌、膀胱癌、淋巴瘤、食管癌、胃肠道癌、鼻咽癌、白血病或骨髓瘤。
本发明的技术特点及优点:
1.本发明化合物不仅表现出良好的DNMT1和HDAC酶抑制活性，在细胞内能够抑制DNMT1和HDAC活性，而且具有较强的体内外抗肿瘤活性，能够显著的延缓肿瘤生长。
2.本发明的化合物作为首次报道的基于DNMT1和HDAC的双靶点抗肿瘤药物，具有进一步的开发和研究价值。
附图说明
图1是化合物(R)-23a、联合用药组(GSK5032和SAHA)对人急性单核细胞白血病细胞MV4-11体内抗肿瘤活性结果图，其中，A：荷瘤小鼠体重变化；B：荷瘤小鼠肿瘤体积生长曲线。
图2是化合物(R)-23a、联合用药组(GSK5032和SAHA)对小鼠结肠癌细胞MC38体内抗肿瘤活性结果图，其中，A：荷瘤小鼠体重变化；B：荷瘤小鼠肿瘤体积生长曲线。
具体实施方式
下面通过实例进一步描述本发明的特征，但本发明并不局限于下述实例。
实施例中试剂、原料或中间体均市售可得或可按文献方法制备。下列实施例中未注明具体条件的实验方法，通常按照常规条件，或按照制造厂商所建议的条件。
实施例1：中间体6的制备
2-氨基-2-氧代-1-苯基乙基-4-甲基苯磺酸酯(2)
将2-羟基-2-苯基乙酰胺(10g,66.15mmol)、N,N-二异丙基乙胺(DMAP,25.65g,198.46mmol)和4-二甲氨基吡啶(1.21g,9.92mmol)在乙腈(100mL)中加入4-甲基苯磺酰氯(18.92g,99.23mmol)，在室温下搅拌6h，然后旋转蒸发除去溶剂。所得残渣用100mL水洗涤，混合物用乙酸乙酯(30mL×3)萃取，分离出的有机相用无水硫酸钠干燥后浓缩得到粗产物，粗产物经硅胶柱层析纯化得到化合物2，化合物(R)-2、(S)-2和34的合成方法同该操作步骤。
白色固体,产率77.2％,1H NMR(400MHz,DMSO-d6)δ7.77(s,2H),7.48(d,J＝7.9Hz,1H),7.43(d,J＝8.1Hz,4H),7.34(s,4H),5.68(s,1H),2.40(s,3H).
(R)-2-氨基-2-氧代-1-苯基乙基-4-甲基苯磺酸酯((R)-2)
白色固体,产率69.1％,1H NMR(400MHz,DMSO)δ7.79–7.73(m,3H),7.45–7.38(m,3H),7.38–7.28(m,5H),5.68(s,1H),2.40(s,3H).
(S)-2-氨基-2-氧代-1-苯基乙基-4-甲基苯磺酸酯((S)-2)
白色固体,产率71.5％,1H NMR(400MHz,DMSO)δ7.80–7.72(m,3H),7.47–7.38(m,3H),7.37–7.28(m,5H),5.68(s,1H),2.40(s,3H).
4-甲基吗啉-4-铵盐-6-氨基-3,5-二氰-4-乙基吡啶-2-硫代酸酯(4)
将正丙醛(870mg,14.98mmol)加入到2-氰基乙酰胺(3g,29.96mmol)和N-甲基啉(3.03g,29.96mmol)的EtOH(40mL)溶液中，在室温下搅拌30小时，过滤反应液，收集滤饼并用乙醇充分洗涤滤饼，干燥过夜，得到化合物4。
棕色固体,产率30.4％,1H NMR(400MHz,DMSO-d6)δ7.58(s,2H),3.67(s,2H),2.72(s,2H),2.62(q,J＝7.5Hz,2H),2.51(s,3H),1.19(t,J＝7.6Hz,3H).
2-((6-氨基-3，5-二氰基-4-乙基吡啶-2-基)硫代)-2-苯基乙酰胺(5)
将化合物4(661mg,1.96mmol)和N,N-二异丙基乙胺(760mg,5.88mmol)溶于DMF(5mL)再向其中加入化合物2(598mg,1.96mmol)，升温至85℃反应5小时，然后旋转蒸发除去溶剂。所得残渣用50mL水洗涤，混合物用乙酸乙酯(15mL×3)萃取，分离出的有机相用无水硫酸钠干燥后浓缩得到粗产物，粗产物经硅胶柱层析纯化得到化合物5，化合物(R)-5、(S)-5和35的合成方法同操作步骤。淡黄色固体,产率68.1％,1H NMR(400MHz,DMSO-d6)δ7.90(s,2H),7.73(s,1H),7.59(d,J＝7.3Hz,2H),7.41–7.27(m,4H),5.56(s,1H),2.69(q,J＝7.5Hz,2H),1.18(t,J＝7.6Hz,3H).
(R)-2-((6-氨基-3，5-二氰基-4-乙基吡啶-2-基)硫代)-2-苯基乙酰胺((R)-5)
黄色固体,产率40.7％。1H NMR(400MHz,DMSO)δ7.90(s,2H),7.73(s,1H),7.63–7.55(m,2H),7.37–7.28(m,4H),5.57(s,1H),2.68(t,J＝7.6Hz,2H),1.18(t,J＝7.6Hz,3H).
(S)-2-((6-氨基-3，5-二氰基-4-乙基吡啶-2-基)硫代)-2-苯基乙酰胺((S)-5)
黄色固体,产率45.1％。1H NMR(400MHz,DMSO)δ7.90(s,2H),7.73(s,1H),7.62–7.55(m,2H),7.41–7.26(m,4H),5.56(s,1H),2.68(t,J＝7.6Hz,2H),1.18(t,J＝7.6Hz,3H).
2-((6-氯-3，5-二氰基-4-乙基吡啶-2-基)硫代)-2-苯基乙酰胺(6)
将化合物5a(402mg,1.19mmol)和氯化铜(797mg,5.93mmol)溶于乙腈(20mL)再向其中加入亚硝酸异戊酯(278mg,2.37mmol)，升温至80℃反应2.5小时，然后旋转蒸发除去溶剂。所得残渣用100mL水洗涤，混合物用乙酸乙酯(30mL×3)萃取，分离出的有机相用无水硫酸钠干燥后浓缩得到粗产物，粗产物经硅胶柱层析纯化得到化合物6，化合物(R)-6、(S)-6和36的合成方法同该操作步骤。淡黄色固体,产率31.3％。1H NMR(400MHz,Chloroform-d)δ7.61–7.47(m,3H),7.40(d,J＝6.6Hz,4H),5.63(s,1H),2.99(q,J＝7.5Hz,2H),1.36(t,J＝7.7Hz,3H).
(R)-2-((6-氯-3，5-二氰基-4-乙基吡啶-2-基)硫代)-2-苯基乙酰胺((R)-6)
淡黄色固体,产率31.7％。1H NMR(400MHz,DMSO)δ7.99(s,1H),7.61–7.51(m,2H),7.43(s,1H),7.38(dd,J＝8.2,6.3Hz,2H),7.37–7.28(m,1H),5.69(s,1H),2.86(q,J＝7.6Hz,2H),1.23(t,J＝7.7Hz,3H).
(S)-2-((6-氯-3，5-二氰基-4-乙基吡啶-2-基)硫代)-2-苯基乙酰胺((S)-6)
淡黄色固体,产率30.1％,。1H NMR(400MHz,DMSO)δ7.99(s,1H),7.61–7.54(m,2H),7.45–7.28(m,4H),5.69(s,1H),2.86(q,J＝7.6Hz,2H),1.23(t,J＝7.7Hz,3H).
实施例2：中间体7a的制备
将化合物6(200mg,0.56mmol)和碳酸钾(232mg,1.68mmol)的CH2Cl2(10mL)溶液中加入哌啶-4-氨基甲酸叔丁酯(224mg,1.12mmol)，在室温下搅拌4小时，然后用100mL水洗涤。然后将有机相分离，用无水硫酸钠干燥，浓缩得到粗产品。后者经硅胶柱层析纯化得到7a，化合物7b、12a、12b、16、20、(R)-20、(S)-20、24、37a-c、41和45的合成方法同该操作步骤。
叔丁基(1-6((2-氨基-2-氧代-1-苯乙基)硫代)-3，5-二氰基-4-乙基吡啶2-基)哌啶4-基)氨基甲酸酯(7a)
白色固体，产率68.5％。1H NMR(400MHz,DMSO)δ7.91(s,1H),7.52(d,J＝7.4Hz,2H),7.41–7.33(m,4H),6.94(d,J＝7.7Hz,1H),5.52(s,1H),4.45(d,J＝13.3Hz,2H),3.61(s,2H),2.76(q,J＝7.5Hz,2H),1.88(d,J＝12.9Hz,2H),1.41(s,9H),1.21(t,J＝7.5Hz,3H).
叔丁基(1-6((2-氨基-2-氧代-1-苯乙基)硫代)-3，5-二氰基-4-乙基吡啶2-基)哌啶4-基)(甲基)氨基甲酸酯(7b)
白色固体，产率69.5％，1H NMR(400MHz,DMSO)δ7.91(s,1H),7.52(d,J＝7.4Hz,2H),7.42–7.34(m,4H),6.94(d,J＝7.7Hz,1H),5.52(s,1H),4.45(d,J＝13.3Hz,2H),3.61(s,2H),2.76(q,J＝7.5Hz,2H),1.88(d,J＝12.9Hz,2H),1.41(s,9H),1.21(t,J＝7.5Hz,3H).
实施例3：中间体8a的制备
1-(6-((2-氨基-2-氧代-1-苯乙基)硫代)-3，5-二氰基-4-乙基吡啶-2-基)哌啶-4-铵盐(8a)
将化合物7a(292mg,0.56mmol)加入饱和氯化氢乙酸乙酯(10mL)中，在室温下搅拌12小时，过滤后用乙酸乙酯(20mL)重结晶纯化滤饼得到8a，化合物8b、13a、13b、17、21、(R)-21、(S)-21、25、38a-c、42和46的合成方法符合该操作步骤。白色固体，产率85.8％。1H NMR(400MHz,DMSO)δ8.22(s,3H),8.01(s,1H),7.54(d,J＝7.4Hz,2H),7.41–7.32(m,4H),5.58(s,1H),4.59(d,J＝13.7Hz,2H),3.32–3.20(m,2H),2.77(q,J＝7.6Hz,2H),2.09(d,J＝12.6Hz,2H),1.65–1.57(m,2H),1.22(t,J＝7.6Hz,3H).
1-(6-((2-氨基-2-氧代-1-苯乙基)硫代)-3，5-二氰基-4-乙基吡啶-2-基)-N-甲基哌啶-4-铵盐(8b)
白色固体，产率90.4％。1H NMR(400MHz,DMSO)δ9.03(s,2H),8.01(s,1H),7.53(d,J＝7.4Hz,2H),7.39(dd,J＝14.7,7.3Hz,4H),5.57(s,1H),4.63(d,J＝13.6Hz,2H),3.20(t,J＝12.2Hz,3H),2.77(q,J＝7.6Hz,2H),2.57(t,J＝5.3Hz,3H),2.17(d,J＝12.6Hz,2H),1.60(t,J＝12.7Hz,2H),1.21(t,J＝7.6Hz,3H).
实施例4：中间体9a-f的制备
将O-(四氢-2H-吡喃-2-基)羟胺(1g,8.54mmol)、相应的环状酸酐(8.54mmol)和三乙胺(2.6g,25.61mmol)加入15mL CH2Cl2中，在室温下搅拌反应3小时。然后用饱和NaCl溶液(30ml)洗涤有机相，分出有机相用无水Na2SO4干燥，浓缩得到粗产物，粗产物经硅胶柱层析纯化得到化合物9a-f。
4-氧代-4-(((四氢-2H-吡喃-2-基)氧代)氨基)丁酸(9a)
白色固体，产率53％。1H NMR(400MHz,DMSO)δ4.79(d,J＝3.3Hz,1H),3.95–3.87(m,1H),3.50(dd,J＝10.2,5.7Hz,1H),2.37(t,J＝7.0Hz,2H),2.20(t,J＝7.1Hz,2H),1.72–1.57(m,3H),1.57–1.44(m,3H).
5-氧代-5-(((四氢-2H-吡喃-2-基)氧代)氨基)戊酸(9b)
白色固体，产率45.7％。1H NMR(400MHz,DMSO)δ4.81(d,J＝3.7Hz,1H),3.95–3.89(m,1H),3.50(dd,J＝10.1,5.5Hz,1H),2.19(t,J＝7.4Hz,2H),2.02(t,J＝7.4Hz,2H),1.74–1.60(m,5H),1.52(d,J＝11.6Hz,3H).
6-氧代-6-(((四氢-2H-吡喃-2-基)氧代)氨基)己酸(9c)
白色固体，产率48.3％。1H NMR(400MHz,DMSO)δ10.92(s,1H),4.81(d,J＝3.2Hz,1H),3.96–3.88(m,1H),3.50(dd,J＝10.3,5.3Hz,1H),2.22–2.15(m,2H),2.03–1.95(m,2H),1.71–1.58(m,3H),1.56–1.43(m,7H).
7-氧代-7-(((四氢-2H-吡喃-2-基)氧代)氨基)庚酸(9d)
无色油状液体，产率48.1％。1H NMR(400MHz,DMSO)δ12.01(s,1H),10.90(s,1H),4.79(d,J＝3.3Hz,1H),3.95–3.87(m,1H),3.50(dd,J＝10.1,5.5Hz,1H),2.18(t,J＝7.4Hz,2H),1.97(t,J＝7.1Hz,2H),1.71–1.59(m,3H),1.53–1.39(m,7H),1.24–1.17(m,2H).
8-氧代-8-(((四氢-2H-吡喃-2-基)氧代)氨基)辛酸(9e)
白色固体，产率46％。1H NMR(400MHz,DMSO)δ10.90(s,1H),4.80(d,J＝3.2Hz,1H),3.96–3.88(m,1H),3.53–3.45(m,1H),2.28(t,J＝7.4Hz,2H),1.96(t,J＝7.3Hz,2H),1.68–1.60(m,3H),1.54–1.39(m,7H),1.24(h,J＝3.9Hz,4H).
9-氧代-9-(((四氢-2H-吡喃-2-基)氧代)氨基)癸酸(9f)
白色固体，产率64.3％。1H NMR(400MHz,DMSO)δ11.97(s,1H),10.90(s,1H),4.80(d,J＝3.3Hz,1H),3.95–3.89(m,1H),3.49(s,1H),2.19(t,J＝7.4Hz,2H),1.96(d,J＝7.4Hz,2H),1.69–1.60(m,3H),1.52–1.45(m,7H),1.24(d,J＝4.0Hz,6H).
实施例5：中间体9g-9u的制备
4-((四氢-2H-吡喃-2-基)氨甲酰基)苯甲酸(9g)
将相应的羧酸(5.31mmol)，EDCI(1.23g,6.4mmol)，DMAP(843mg,6.9mmol)，和吡喃羟胺(622mg,5.31mmol)加入到15mL CH2Cl2中，室温下反应8小时。反应结束后，反应液依次用1M盐酸水溶液(100mL)、饱和氯化钠水溶液(100mL)洗涤。然后用无水Na2SO4干燥有机相并浓缩。经硅胶柱层析纯化得到9g，化合物9h-u的合成方法同该操作步骤。无色油状液体，产率70.8％。1H NMR(400MHz,DMSO)δ11.81(s,1H),8.02(d,J＝8.1Hz,2H),7.87(d,J＝8.2Hz,2H),5.03(d,J＝3.1Hz,1H),4.06(t,J＝9.5Hz,1H),3.56–3.51(m,1H),1.73(s,3H),1.59–1.51(m,3H).
4-((4-(((四氢-2H-吡喃-2-基)氧代)氨甲酰基)苄基)氧代)苯甲酸(9h)
白色固体，产率90.2％。1H NMR(400MHz,DMSO)δ12.67(s,1H),11.67(s,1H),7.93–7.87(m,2H),7.79(d,J＝8.0Hz,2H),7.55(d,J＝8.1Hz,2H),7.14–7.07(m,2H),5.26(s,2H),5.00(d,J＝3.0Hz,1H),4.11–4.01(m,1H),3.57–3.48(m,1H),1.72(s,3H),1.57–1.51(m,3H).
4-氧代-4-((4-(((四氢-2H-吡喃-2-基)氧代)氨甲酰基)苯基)氨基)丁酸(9i)
白色固体，产率44.5％。1H NMR(400MHz,DMSO)δ11.52(s,1H),10.25(s,1H),7.87–7.53(m,4H),4.98(d,J＝3.1Hz,1H),4.08–4.02(m,1H),3.51(d,J＝11.4Hz,1H),2.58(t,J＝6.9Hz,2H),2.52(s,2H),1.82–1.63(m,3H),1.57–1.49(m,3H).
5-氧代-5-((4-(((四氢-2H-吡喃-2-基)氧代)氨甲酰基)苯基)氨基)戊酸(9j)
白色固体，产率27.9％。1H NMR(400MHz,DMSO)δ11.54(s,1H),10.17(s,1H),7.95–7.53(m,4H),4.98(d,J＝3.1Hz,1H),4.09–4.01(m,1H),3.52(dd,J＝9.8,5.4Hz,1H),2.38(t,J＝7.4Hz,2H),2.28–2.22(m,2H),1.85–1.78(m,2H),1.74–1.67(m,3H),1.59–1.51(m,3H).
6-氧代-6-((4-(((四氢-2H-吡喃-2-基)氧代)氨甲酰基)苯基)氨基)己酸(9k)
淡黄色固体，产率54％。1H NMR(400MHz,DMSO)δ11.50(s,1H),10.15(s,1H),7.87–7.57(m,4H),4.98(d,J＝3.2Hz,1H),4.12–4.00(m,1H),3.57–3.44(m,1H),2.34(t,J＝7.1Hz,2H),2.22(t,J＝7.3Hz,2H),1.71(s,3H),1.60–1.48(m,7H).
4-氧代-4-((4-(((四氢-2H-吡喃-2-基)氨甲酰基)苄基)氨基)丁酸(9l)
白色固体，产率54.2％。1H NMR(400MHz,DMSO)δ11.52(s,1H),10.22(s,1H),7.72(d,J＝8.7Hz,2H),7.66(d,J＝8.8Hz,2H),4.97(d,J＝3.2Hz,1H),4.06(s,1H),3.70(t,J＝6.5Hz,2H),3.51(d,J＝11.4Hz,1H),2.53(s,2H),2.07–2.01(m,2H),1.71(s,3H),1.54(d,J＝5.7Hz,3H).
5-氧代-5-((4-(((四氢-2H-吡喃-2-基)氨甲酰基)苄基)氨基)戊酸(9m)
白色固体，产率48.1％。1H NMR(400MHz,DMSO)δ11.63(s,1H),8.45(t,J＝6.0Hz,1H),7.72(d,J＝8.1Hz,2H),7.32(d,J＝8.0Hz,2H),4.99(d,J＝3.1Hz,1H),4.30(d,J＝5.9Hz,2H),4.07(d,J＝8.6Hz,1H),3.59(s,1H),2.24–2.17(m,4H),1.77–1.69(m,5H),1.54(d,J＝5.7Hz,3H).
4-氧代-4-((4-(3-氧代-3-(((四氢-2H-吡喃-2-基)氧代)氨基)丙基-1-烯-1-基)苯基)氨基)丁酸(9n)
白色固体，产率38.0％。1H NMR(400MHz,DMSO)δ12.15(s,1H),11.18(s,1H),10.15(s,1H),7.63(d,J＝8.3Hz,2H),7.51(d,J＝8.3Hz,2H),7.42(d,J＝15.6Hz,1H),6.39(d,J＝15.9Hz,1H),4.90(s,1H),3.96(s,1H),3.54(dd,J＝11.1,4.8Hz,1H),2.58(t,J＝6.6Hz,2H),2.53(d,J＝5.8Hz,2H),1.69(s,3H),1.54(s,3H).
4-甲酰基-N-((四氢-2H-吡喃-2-基)氧代)苯甲酰胺(9o)
白色固体，产率57.2％。1H NMR(400MHz,DMSO)δ11.88(s,1H),10.09(s,1H),8.04–7.94(m,4H),5.04(d,J＝2.8Hz,1H),4.10–4.03(m,1H),3.54(dd,J＝11.3,4.6Hz,1H),1.79–1.71(m,3H),1.61–1.53(m,3H).
(E)-3-(4-甲酰基苯基)-N-((四氢-2H-吡喃-2-基)氧代)丙烯酰胺(9p)
白色固体，产率39.1％。1H NMR(400MHz,DMSO)δ11.37(s,1H),10.02(s,1H),7.95(d,J＝8.0Hz,2H),7.81(d,J＝8.0Hz,2H),7.58(d,J＝15.9Hz,1H),6.67(d,J＝15.9Hz,1H),4.93(s,1H),3.97(s,1H),3.55(dd,J＝11.1,5.5Hz,1H),1.70(s,3H),1.55(s,3H).
4-甲酰基-N-(4-(((四氢-2H-吡喃-2-基)氧代)氨甲酰基)苄基)苯甲酰胺(9q)
白色固体，产率31.6％。1H NMR(400MHz,DMSO)δ11.61(s,1H),10.09(s,1H),9.33(t,J＝5.9Hz,1H),8.08(d,J＝8.2Hz,2H),8.02(d,J＝8.2Hz,1H),7.73(d,J＝8.2Hz,2H),7.42(d,J＝8.0Hz,2H),4.98(s,1H),4.55(d,J＝6.0Hz,2H),4.06(s,1H),1.72(s,2H),1.55(s,2H).
4-甲酰基-N-(4-(3-氧代-3-(((四氢-2H-吡喃-2-基)氧代)氨基)丙基-1-烯-1基)苄基)苯甲酰胺(9r)
白色固体，产率47.7％。1H NMR(400MHz,DMSO)δ11.24(s,1H),10.09(s,1H),9.24(t,J＝6.0Hz,1H),8.09(d,J＝8.2Hz,1H),8.02(d,J＝8.1Hz,1H),7.95(d,J＝8.3Hz,1H),7.74(d,J＝8.3Hz,1H),7.55(d,J＝7.7Hz,2H),7.48(d,J＝15.5Hz,1H),7.37(dd,J＝8.0,5.1Hz,2H),6.48(d,J＝15.8Hz,1H),4.91(s,1H),4.52(t,J＝6.8Hz,2H),3.96(s,1H),3.55(s,1H),1.73–1.66(m,3H),1.54(s,3H).
(E)-3-(4-((4-甲酰基苯氧基)甲基)苯基)-N-((四氢-2H-吡喃-2-基)氧代)丙烯酰胺(9s)
白色固体，产率52.8％。1H NMR(400MHz,DMSO)δ11.27(s,1H),9.87(s,1H),7.92–7.84(m,2H),7.62(d,J＝7.9Hz,2H),7.55–7.46(m,3H),7.26–7.18(m,2H),6.53(d,J＝15.8Hz,1H),5.27(s,2H),4.92(s,1H),3.98(d,J＝9.7Hz,1H),3.54(d,J＝11.5Hz,1H),1.75–1.67(m,3H),1.54(s,3H).
7-(4-甲酰基苯氧基)-N-((四氢-2H-吡喃-2-基)氧代)庚酰胺(9t)
白色固体，产率62.9％。1H NMR(400MHz,DMSO)δ10.92(s,1H),9.86(s,1H),7.91–7.81(m,2H),7.15–7.07(m,2H),4.81(d,J＝3.2Hz,1H),4.07(t,J＝6.4Hz,2H),3.95–3.88(m,1H),3.52–3.45(m,1H),2.01–1.97(m,2H),1.73(p,J＝6.7Hz,3H),1.68–1.60(m,3H),1.51(q,J＝7.6Hz,6H),1.43–1.37(m,2H),1.31(q,J＝4.5Hz,2H).
2-氯-N-((四氢-2H-吡喃-2-基)氧代)嘧啶-5-甲酰胺(9u)
白色固体，产率29.4％。1H NMR(400MHz,DMSO)δ11.46(s,1H),8.66(s,2H),4.95(d,J＝3.3Hz,1H),4.08–3.97(m,1H),1.75–1.65(m,3H),1.57–1.50(m,3H).
实施例6：中间体10a-10n的制备
N1-(1-(6-((2-氨基-2氧代-1-苯乙基)硫代)-3，5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)-N4-((四氢-2H-吡喃-2-基)氧代)丁二酰胺(10a)
在THF(10mL)中加入9a(190mg,0.875mmol)和NMM(110mg,1.09mmol)，加入氯碳酸异丁酯(120mg,0.875mmol)，0℃下搅拌0.5h。将所得滤液在0℃下滴加到含有8a(200mg,0.437mmol)和N-甲基吗啉(44mg,0.437mmol)的CH2Cl2(10mL)中。将混合物在室温下搅拌6小时，然后旋转蒸发除去溶剂，得到的残渣用100mL水洗涤，用乙酸乙酯(30mL×3)萃取。依次用1M盐酸水溶液(100mL)、饱和碳酸氢钠水溶液(100mL)、饱和氯化钠水溶液(100mL)洗涤有机相。然后用无水Na2SO4干燥有机相并浓缩。经硅胶柱层析纯化得到10a，化合物10b-n、29a-c、39a-c和43a的合成方法同该操作步骤。白色固体，产率44.1％。1H NMR(400MHz,DMSO)δ10.97(s,1H),7.91(d,J＝8.4Hz,2H),7.55–7.48(m,2H),7.44–7.30(m,4H),5.52(s,1H),4.79(s,1H),4.42(s,2H),3.91(s,2H),3.49(d,J＝11.2Hz,3H),2.76(q,J＝7.4Hz,2H),2.32(t,J＝7.7Hz,2H),2.24(d,J＝7.2Hz,2H),1.88(d,J＝12.7Hz,2H),1.64(s,3H),1.50(s,3H),1.43(t,J＝10.3Hz,2H),1.20(t,J＝7.6Hz,3H).
N1-(1-(6-((2-氨基-2氧代-1-苯乙基)硫代)-3，5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)-N5-((四氢-2H-吡喃-2-基)氧代)戊二酰胺(10b)
白色固体，产率50.7％。1H NMR(400MHz,DMSO)δ10.98(s,1H),8.00(s,1H),7.90(d,J＝7.6Hz,1H),7.56–7.51(m,2H),7.41–7.30(m,4H),5.56(s,1H),4.82(s,1H),4.48–4.37(m,2H),3.91(d,J＝11.5Hz,2H),3.55–3.43(m,3H),2.76(q,J＝7.6Hz,2H),2.08(t,J＝7.5Hz,2H),2.00(t,J＝7.5Hz,2H),1.89(d,J＝13.5Hz,2H),1.78–1.74(m,2H),1.69–1.61(m,3H),1.54–1.40(m,5H),1.20(t,J＝7.5Hz,3H).
N1-(1-(6-((2-氨基-2氧代-1-苯乙基)硫代)-3，5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)-N6-((四氢-2H-吡喃-2-基)氧代)己二酰胺(10c)
白色固体，产率60.4％。11H NMR(400MHz,DMSO)δ10.91(s,1H),7.92(s,1H),7.83(d,J＝7.6Hz,1H),7.57–7.49(m,2H),7.40–7.31(m,4H),5.53(s,1H),4.80(s,1H),4.43(s,2H),3.91(s,2H),3.49(d,J＝11.4Hz,1H),3.09(d,J＝7.2Hz,2H),2.76(q,J＝7.6Hz,2H),2.07(s,2H),1.99(d,J＝6.5Hz,2H),1.89(d,J＝12.7Hz,2H),1.63(s,3H),1.53–1.40(m,7H),1.21(dd,J＝10.9,7.4Hz,5H).
N1-(1-(6-((2-氨基-2氧代-1-苯乙基)硫代)-3，5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)-N7-((四氢-2H-吡喃-2-基)氧代)庚二酰胺(10d)
白色固体，产率66.2％。1H NMR(400MHz,DMSO)δ10.91(s,1H),7.94(s,1H),7.83(d,J＝7.6Hz,1H),7.55–7.49(m,2H),7.42–7.30(m,4H),5.53(s,1H),4.79(t,J＝3.0Hz,1H),4.49–4.37(m,2H),3.97–3.84(m,2H),3.49(d,J＝11.4Hz,1H),3.37(s,2H),2.75(q,J＝7.5Hz,2H),2.05(t,J＝7.4Hz,2H),1.97(d,J＝7.3Hz,2H),1.88(d,J＝12.7Hz,2H),1.68–1.58(m,3H),1.53–1.39(m,9H),1.24–1.17(m,5H).
N1-(1-(6-((2-氨基-2氧代-1-苯乙基)硫代)-3，5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)-N8-((四氢-2H-吡喃-2-基)氧代)辛二酰胺(10e)
白色固体，产率31.1％。1H NMR(400MHz,DMSO)δ10.92(s,1H),7.94(s,1H),7.90–7.76(m,1H),7.55–7.49(m,2H),7.48–7.17(m,4H),5.53(s,1H),4.84(s,1H),4.57–4.41(m,2H),4.00–3.87(m,2H),3.55–3.51(m,3H),2.76(d,J＝7.8Hz,2H),2.34–2.26(m,4H),2.05(t,J＝7.4Hz,2H),1.86(s,2H),1.53–1.40(m,10H),1.25(m,7H).
N1-(1-(6-((2-氨基-2氧代-1-苯乙基)硫代)-3，5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)-N9-((四氢-2H-吡喃-2-基)氧代)辛二酰胺(10f)
白色固体，产率83％。1H NMR(400MHz,DMSO)δ10.91(s,1H),7.94(s,1H),7.83(d,J＝7.6Hz,1H),7.60–7.49(m,2H),7.46–7.29(m,4H),5.54(s,1H),4.79(s,1H),4.44(dd,J＝13.1,8.9Hz,2H),3.96–3.86(m,2H),3.49(d,J＝11.0Hz,1H),2.76(q,J＝7.6Hz,2H),2.06(t,J＝7.4Hz,2H),1.97(d,J＝7.4Hz,2H),1.88(d,J＝12.6Hz,2H),1.69–1.58(m,3H),1.56–1.38(m,9H),1.22–1.17(m,9H).
N1-(1-(6-((2-氨基-2氧代-1-苯乙基)硫代)-3，5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)-N4-((四氢-2H-吡喃-2-基)氧代)对苯二甲酰胺(10g)
白色固体，产率48.2％。1H NMR(400MHz,DMSO)δ11.79(s,1H),8.51(d,J＝7.8Hz,1H),7.95(d,J＝7.8Hz,2H),7.85(d,J＝8.2Hz,2H),7.52(d,J＝7.5Hz,2H),7.44–7.30(m,4H),5.55(s,1H),5.02(d,J＝3.3Hz,1H),4.58(t,J＝14.1Hz,2H),4.27–4.13(m,1H),4.08(d,J＝7.7Hz,1H),3.54(q,J＝6.6Hz,3H),2.77(q,J＝7.6Hz,2H),2.05–1.97(m,2H),1.77–1.53(m,8H),1.19(t,J＝7.5Hz,3H).
N1-(1-(6-((2-氨基-2氧代-1-苯乙基)硫代)-3，5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)-4-((4-(((四氢-2H-吡喃-2-基)氧代)氨甲酰基)苯基)氧代)苯甲酰胺(10h)
白色固体，产率41.4％。1H NMR(400MHz,DMSO)δ11.67(s,1H),8.21(d,J＝7.8Hz,1H),7.94(s,1H),7.82(dd,J＝21.7,8.3Hz,4H),7.53(t,J＝7.8Hz,4H),7.47–7.25(m,4H),7.09(d,J＝8.6Hz,2H),5.54(s,1H),5.25(s,2H),5.00(s,1H),4.57(t,J＝13.7Hz,2H),4.17(s,2H),2.77(t,J＝7.6Hz,2H),1.99(s,2H),1.69–1.49(m,8H),1.21(t,J＝7.5Hz,3H).
N1-(1-(6-((2-氨基-2氧代-1-苯乙基)硫代)-3，5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)-N4-(4-(((四氢-2H-吡喃-2-基)氧代)氨甲酰基)苯基)丁二酰胺(10i)
白色固体，产率38.6％。1H NMR(400MHz,DMSO)δ11.56(s,1H),10.36(s,1H),8.04(s,1H),7.71(q,J＝8.7Hz,4H),7.53(d,J＝7.4Hz,2H),7.40–7.31(m,5H),5.57(s,1H),4.98(s,1H),4.43(d,J＝13.0Hz,2H),3.91(s,2H),3.51(d,J＝11.8Hz,2H),2.75(d,J＝7.7Hz,2H),2.61(t,J＝6.9Hz,2H),2.43(t,J＝7.2Hz,2H),1.89(d,J＝12.5Hz,2H),1.71(s,2H),1.54(s,3H),1.45(t,J＝12.6Hz,3H),1.22(t,J＝7.6Hz,3H).
N1-(1-(6-((2-氨基-2氧代-1-苯乙基)硫代)-3，5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)-N5-(4-(((四氢-2H-吡喃-2-基)氧代)氨甲酰基)苯基)戊二酰胺(10j)
白色固体，产率39.2％。1H NMR(400MHz,DMSO)δ11.54(s,1H),10.32(s,1H),8.01(s,2H),7.71(q,J＝8.7Hz,4H),7.52(d,J＝7.4Hz,2H),7.42–7.30(m,5H),5.57(s,1H),4.98(s,1H),4.41(d,J＝13.0Hz,2H),4.05(d,J＝7.6Hz,1H),3.91(s,1H),3.51(d,J＝11.7Hz,2H),2.75(q,J＝7.7Hz,2H),2.61(t,J＝6.9Hz,2H),2.41(t,J＝7.0Hz,2H),1.87(d,J＝12.6Hz,2H),1.71(s,2H),1.66–1.30(m,8H),1.20(t,J＝7.6Hz,3H).
N1-(1-(6-((2-氨基-2氧代-1-苯乙基)硫代)-3，5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)-N6-(4-(((四氢-2H-吡喃-2-基)氧代)氨甲酰基)苯基)己二酰胺(10k)
白色固体，产率41.4％。1H NMR(400MHz,DMSO)δ11.60(s,1H),10.52(s,1H),8.13(s,1H),8.06(d,J＝7.6Hz,1H),7.73(s,4H),7.58–7.52(m,2H),7.40–7.29(m,4H),5.61(s,1H),4.98(s,1H),4.42(d,J＝13.3Hz,2H),4.10–4.01(m,1H),3.91(s,1H),3.51(d,J＝11.2Hz,3H),2.75(q,J＝7.6Hz,2H),2.40(t,J＝6.8Hz,2H),2.18–2.10(m,2H),1.87(d,J＝12.8Hz,2H),1.71(s,3H),1.55(s,9H),1.20(t,J＝7.6Hz,3H).
N1-(1-(6-((2-氨基-2氧代-1-苯乙基)硫代)-3，5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)-N4-(4-(((四氢-2H-吡喃-2-基)氧代)氨甲酰基)苄基)丁二酰胺(10l)
白色固体，产率42.5％。1H NMR(400MHz,DMSO)δ11.60(s,1H),8.46(t,J＝6.0Hz,1H),8.00(s,1H),7.94(d,J＝7.5Hz,1H),7.72(d,J＝7.9Hz,2H),7.52(d,J＝7.5Hz,2H),7.40–7.31(m,6H),5.56(s,1H),4.98(s,1H),4.40(d,J＝10.8Hz,2H),4.30(d,J＝6.0Hz,2H),4.05(d,J＝6.2Hz,1H),3.90(s,1H),3.54–3.43(m,2H),2.75(q,J＝7.6Hz,2H),2.43–2.33(m,4H),1.87(d,J＝12.5Hz,2H),1.71(s,2H),1.62–1.35(m,6H),1.21(d,J＝7.6Hz,3H).
N1-(1-(6-((2-氨基-2氧代-1-苯乙基)硫代)-3，5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)-N5-(4-(((四氢-2H-吡喃-2-基)氧代)氨甲酰基)苄基)戊二酰胺(10m)
白色固体，产率41.3％。1H NMR(400MHz,DMSO)δ11.58(s,1H),8.42(t,J＝6.0Hz,1H),7.98(s,1H),7.92(d,J＝7.5Hz,1H),7.70(d,J＝7.9Hz,2H),7.48(d,J＝7.5Hz,2H),7.42–7.32(m,6H),5.57(s,1H),4.96(s,1H),4.40(d,J＝10.8Hz,2H),4.38(d,J＝6.0Hz,2H),4.08(d,J＝6.2Hz,1H),3.92(s,1H),3.52–3.41(m,2H),2.76(q,J＝7.6Hz,2H),2.41–2.31(m,4H),1.88(d,J＝12.5Hz,2H),1.72(s,2H),1.64–1.31(m,8H),1.21(t,J＝7.6Hz,3H).
(E)-N1-(1-(6-((2-氨基-2氧代-1-苯乙基)硫代)-3，5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)-N4-(4-(3-氧代-3-(((四氢-2H-吡喃-2-基)氧代)氨基)丙基-1-烯-1-基)苯基)丁二酰胺(10n)
白色固体，产率35.9％。1H NMR(400MHz,DMSO)δ11.22(s,1H),10.22(s,1H),7.99(d,J＝8.2Hz,2H),7.65(d,J＝8.3Hz,2H),7.59–7.45(m,4H),7.45–7.24(m,5H),6.40(d,J＝15.8Hz,1H),5.55(s,1H),4.90(s,1H),4.42(s,2H),3.93(s,2H),3.53(d,J＝11.8Hz,2H),2.76(q,J＝7.0Hz,2H),2.59(t,J＝7.2Hz,2H),2.43(t,J＝7.2Hz,2H),1.93–1.85(m,2H),1.69(s,3H),1.58–1.39(m,5H),1.21(t,J＝7.6Hz,3H).
实施例7：中间体10o-10t的制备
4-(((1-(6-((2-氨基-2氧代-1-苯乙基)硫代)-3，5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)氨基)甲基)-N-((四氢-2H-吡喃-2-基)氧代)苯甲酰胺(10o)
在MeOH/CH2Cl2(1:1,10mL)的混合溶剂中加入8a(350mg,0.766mmol)和9o(287mg,1.15mmol)，室温下反应9小时后加入三乙酰氧基硼氢化钠(649mg,3.06mmol)，室温下继续搅拌3小时。旋蒸除去溶剂后，所得残渣用50mL水洗涤，混合物用乙酸乙酯(30mL×3)萃取。分离出的有机相依次用1M盐酸水溶液(100mL)、饱和碳酸氢钠水溶液(100mL)、饱和氯化钠水溶液(100mL)洗涤有机相。然后用无水Na2SO4干燥有机相并浓缩。经硅胶柱层析纯化得到10o，化合物10p-t、18a、22、(R)-22、(S)-22、26、29d和43b的合成方法同该操作步骤。白色固体，产率41.2％。1H NMR(400MHz,DMSO)δ11.87(s,1H),9.58(d,J＝6.5Hz,2H),8.04(s,1H),8.00(d,J＝8.0Hz,4H),7.95(d,J＝8.0Hz,2H),7.51(d,J＝7.5Hz,2H),7.36(m,4H),5.61(s,1H),5.03(d,J＝2.8Hz,1H),4.65(d,J＝13.4Hz,2H),4.26(t,J＝6.0Hz,2H),4.08(m,1H),3.53–3.42(m,2H),3.52–3.39(m,1H),3.22(d,J＝13.0Hz,2H),2.77(q,J＝7.6Hz,2H),2.29(d,J＝12.5Hz,2H),1.85–1.69(m,5H),1.58–1.53(m,3H),1.21(t,J＝7.7Hz,3H).
(E)-3-(4-(((1-(6-((2-氨基-2氧代-1-苯乙基)硫代)-3，5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)氨基)甲基)苯基)-N-((四氢-2H-吡喃-2-基)氧代)丙烯酰胺(10p)
白色固体，产率39.6％。1H NMR(400MHz,DMSO)δ11.23(s,1H),7.93(d,J＝2.1Hz,1H),7.56–7.49(m,4H),7.47–7.39(m,3H),7.39–7.28(m,4H),6.48(d,J＝15.7Hz,1H),5.53(s,1H),4.91(s,1H),4.39(d,J＝13.1Hz,2H),3.96(s,1H),3.54(dd,J＝10.0,5.5Hz,2H),2.75(q,J＝7.3Hz,3H),1.94(d,J＝12.5Hz,2H),1.70(s,3H),1.54(d,J＝4.8Hz,3H),1.44–1.32(m,2H),1.20(t,J＝7.6Hz,3H).
4-(((1-(6-((2-氨基-2氧代-1-苯乙基)硫代)-3，5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)氨基)甲基)-N-(4-(((四氢-2H-吡喃-2-基)氧代)氨甲酰基)苄基)苯甲酰胺(10q)
白色固体，产率38.2％。1H NMR(400MHz,DMSO)δ11.20(s,1H),9.21(t,J＝6.1Hz,1H),7.99(s,1H),7.98(d,J＝8.0Hz,2H),7.80–7.70(m,4H),7.54–7.52(m,2H),7.41–7.27(m,6H),5.61(s,1H),4.88(s,1H),4.66(d,J＝13.4Hz,2H),4.53(d,J＝5.9Hz,2H),4.28(t,J＝6.1Hz,2H),3.91(s,1H),3.36(m,1H),3.20(t,J＝12.8Hz,2H),2.77(q,J＝7.5Hz,2H),2.30(d,J＝12.4Hz,2H),1.77(dt,J＝15.2,7.2Hz,2H),1.65(s,3H),1.54(s,3H),1.21(t,J＝7.8Hz,3H)
(E)-4-(((1-(6-((2-氨基-2氧代-1-苯乙基)硫代)-3，5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)氨基)甲基)-N-(4-(((四氢-2H-吡喃-2-基)氧代)氨甲酰基)丙基-1-烯-1-基)苄基)苯甲酰胺(10r)
白色固体，产率35.1％。1H NMR(400MHz,DMSO)δ11.25(s,1H),9.07(t,J＝6.1Hz,1H),7.95(s,1H),7.89(d,J＝8.0Hz,2H),7.62–7.43(m,7H),7.43–7.27(m,6H),6.48(d,J＝15.9Hz,1H),5.54(s,1H),4.91(s,1H),4.47(dd,J＝27.3,9.5Hz,4H),3.92(d,J＝29.0Hz,4H),2.75(q,J＝7.5Hz,2H),2.03–1.95(m,2H),1.70(s,3H),1.53(d,J＝5.0Hz,3H),1.42(s,2H),1.20(t,J＝7.6Hz,3H).
(E)-3-(4-((4-(((1-(6-((2-氨基-2氧代-1-苯乙基)硫代)-3，5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)氨基)甲基)苯氧基)甲基)苯基)-N-(4-(((四氢-2H-吡喃-2-基)氧代)丙烯酰胺(10s)
白色固体，产率33.8％。1H NMR(400MHz,DMSO)δ11.29(s,1H),7.98(s,1H),7.60(d,J＝7.9Hz,2H),7.51(dd,J＝15.5,7.7Hz,5H),7.39(dd,J＝8.2,6.2Hz,4H),7.37–7.30(m,2H),7.03(d,J＝8.1Hz,2H),6.53(d,J＝15.9Hz,1H),5.56(s,1H),5.15(s,2H),4.92(s,1H),4.51(d,J＝13.1Hz,2H),3.93(d,J＝24.3Hz,2H),3.57–3.51(m,1H),3.23(s,2H),2.76(d,J＝7.6Hz,2H),2.08(s,2H),1.70(s,2H),1.54(s,5H),1.21(t,J＝7.6Hz,3H).
7-(4-(((1-(6-((2-氨基-2氧代-1-苯乙基)硫代)-3，5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)氨基)甲基)苯氧基)-N-(4-(((四氢-2H-吡喃-2-基)氧代)苯甲酰胺(10t)
白色固体，产率32.1％。1H NMR(400MHz,DMSO)δ10.90(s,1H),7.93(s,1H),7.52(d,J＝7.4Hz,2H),7.45–7.22(m,6H),6.92(s,2H),5.54(s,1H),4.80(s,1H),4.47(s,2H),3.95(t,J＝6.5Hz,2H),3.48(s,2H),2.76(q,J＝7.5Hz,2H),1.99(t,J＝7.3Hz,2H),1.74–1.59(m,5H),1.55–1.34(m,8H),1.31(d,J＝7.4Hz,2H),1.21(t,J＝7.6Hz,3H).
实施例8：中间体10u的制备
在乙腈(10mL)中加入8a(356mg,0.78mmol)，9u(242mg,0.94mmol)和K2CO3(162mg,1.17mmol)，80℃下反应6小时。反应液用50mL水稀释后用乙酸乙酯(20mL×3)萃取。分离出的有机相依次用无水Na2SO4干燥有机相并浓缩得到粗产物。后者经硅胶柱层析纯化得到10u，化合物10v、14a、14b、18b、22b、29e、39d、43c和47的合成方法同该操作步骤。
2-((1-(6-((2-氨基-2氧代-1-苯乙基)硫代)-3，5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)氨基)-N-(4-(((四氢-2H-吡喃-2-基)氧代)嘧啶-5-甲酰胺(10u)
白色固体，产率29.6％。1H NMR(400MHz,DMSO)δ11.51(s,1H),8.66(s,2H),7.97–7.89(m,2H),7.51(d,J＝7.5Hz,2H),7.44–7.30(m,4H),5.53(s,1H),4.95(s,1H),4.55(s,2H),4.18(s,1H),4.03(s,1H),2.76(q,J＝7.3Hz,2H),2.03(d,J＝13.3Hz,2H),1.71(s,3H),1.55(s,5H),1.21(t,J＝7.6Hz,3H).
2-((1-(6-((2-氨基-2氧代-1-苯乙基)硫代)-3，5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)(甲基)氨基)-N-(4-(((四氢-2H-吡喃-2-基)氧代)嘧啶-5-甲酰胺(10u)
白色固体，产率31.1％。1H NMR(400MHz,DMSO)δ11.54(s,1H),8.71(s,2H),7.92(s,1H),7.52(d,J＝7.3Hz,2H),7.44–7.31(m,4H),5.55(s,1H),4.98(d,J＝16.4Hz,2H),4.74(d,J＝13.5Hz,2H),4.04(s,1H),3.51(s,1H),3.27(d,J＝12.0Hz,2H),3.02(s,3H),2.77(q,J＝7.3Hz,3H),1.75(d,J＝27.5Hz,7H),1.55(s,3H),1.22(t,J＝7.5Hz,3H).
实施例9：目标化合物的制备
将实施例6制得的化合物10a(234mg,0.378mmol)溶于饱和氯化氢乙酸乙酯(10mL)中，在室温下搅拌12h，过滤后用EA或MeOH(20mL)重结晶纯化滤饼得到11a，化合物11b-v、15a、15b、19a、19b、23a、(R)-23a、(S)-23a、23b、27、30a-e、40a-d、44a-c和48的合成方法同该操作步骤。
N1-(1-(6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)-N4-羟基丁二酰胺(11a)
白色固体，产率44.1％，mp170-171℃。1H NMR(400MHz,DMSO)δ10.30(s,1H),8.02–7.93(m,2H),7.53(d,J＝7.4Hz,2H),7.42–7.30(m,4H),5.56(s,1H),4.48–4.37(m,2H),3.90(s,1H),3.41–3.30(m,2H),2.76(q,J＝7.5Hz,2H),2.33(t,J＝7.4Hz,2H),2.20(t,J＝7.4Hz,2H),1.92–1.83(m,2H),1.48–1.39(m,2H),1.18(t,J＝7.5Hz,3H).13C NMR(101MHz,DMSO)δ174.32,174.06,170.81,170.04,169.99,164.87,164.78,164.40,158.46,136.66,129.12,128.67,128.63,116.43,115.15,93.78,87.59,53.64,47.47,46.93,45.68,32.04,30.55,29.62,29.24,27.58,14.01.HRMS-ESI(m/z)calc.for C26H28N7O4S[M–H]-534.1929,found 534.1932.HPLC tR＝6.19min(99.89％purity).
N1-(1-(6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)-N5-羟基戊二酰胺(11b)
白色固体，产率55.8％，mp171-173℃。1H NMR(400MHz,DMSO)δ10.31(s,1H),8.01(s,1H),7.91(d,J＝7.6Hz,1H),7.57–7.50(m,2H),7.43–7.30(m,4H),5.56(s,1H),4.45–4.41(m,2H),3.91(d,J＝8.6Hz,1H),3.40–3.30(m,2H),2.76(q,J＝7.5Hz,2H),2.08(t,J＝7.4Hz,2H),2.01–1.94(m,2H),1.91–1.85(m,2H),1.76–1.85(m,2H),1.51–1.40(m,2H),1.21(t,J＝7.5Hz,3H).13C NMR(101MHz,DMSO)δ174.66,171.46,170.00,164.79,164.40,158.45,136.69,129.11,128.66,128.61,116.43,115.15,93.77,87.56,53.63,46.96,45.79,45.59,34.97,33.48,33.22,32.06,32.00,27.58,21.19,14.01,8.90.HRMS-ESI(m/z)calc.for C27H30N7O4S[M-H]+548.2085,found 548.2088.HPLC tR＝6.65min(97.33％purity).
N1-(1-(6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)-N6-羟基己二酰胺(11c)
白色固体，产率34.5％，mp173-175℃。1H NMR(400MHz,DMSO)δ8.05(s,1H),7.94(d,J＝7.6Hz,1H),7.54(d,J＝7.4Hz,2H),7.43–7.30(m,4H),5.58(s,1H),4.43(d,J＝14.0Hz,2H),3.91(s,1H),3.23–3.15(m,2H),2.76(q,J＝7.6Hz,2H),2.07(d,J＝7.6Hz,2H),1.98(d,J＝9.2Hz,2H),1.88(d,J＝12.7Hz,2H),1.48(s,6H),1.18(t,J＝7.5Hz,3H).13C NMR(101MHz,DMSO)δ171.98,170.04,169.60,164.81,164.37,158.46,136.83,129.05,128.69,128.56,116.44,115.17,93.77,87.54,53.59,47.04,46.95,45.54,35.87,32.71,32.06,28.84,27.57,25.70,25.52,14.00.HRMS-ESI(m/z)calc.for C28H32N7O4S[M-H]+562.2242,found 562.2246.HPLC tR＝6.49min(98.83％purity).
N1-(1-(6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)-N7-羟基庚二酰胺(11d)
白色固体，产率59.6％，mp 173-175℃。1H NMR(400MHz,DMSO)δ10.47(s,1H),8.70(s,1H),8.14(s,1H),7.97(d,J＝7.5Hz,1H),7.55(d,J＝7.2Hz,2H),7.41–7.30(m,4H),5.62(s,1H),4.44(d,J＝14.3,2H),3.90(s,1H),3.33(d,J＝13.4Hz,2H),2.75(q,J＝7.5Hz,2H),2.07(t,J＝7.3Hz,2H),1.95(t,J＝7.3Hz,2H),1.87(d,J＝12.6Hz,2H),1.60–1.36(m,6H),1.21(q,J＝7.1Hz,5H).13C NMR(101MHz,DMSO)δ171.94,170.05,169.57,164.81,164.38,158.45,136.78,129.07,128.69,128.58,116.44,115.16,93.74,87.52,53.59,47.02,46.94,45.54,35.78,32.62,32.06,28.68,27.57,25.53,25.39,14.01.HRMS-ESI(m/z)calc.for C29H35N7O4S[M+Na]+600.2375,found 600.2379.HPLC tR＝12.41min(98.19％purity)。
N1-(1-(6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)-N8-羟基辛二酰胺(11e)
淡黄色固体，产率35.2％，mp 177-179℃。1H NMR(400MHz,DMSO)δ10.46(s,1H),8.70(s,1H),8.15(s,1H),7.97(d,J＝7.5Hz,1H),7.55(d,J＝7.3Hz,2H),7.35(dq,J＝13.2,7.4Hz,4H),5.62(s,1H),4.51–4.38(m,2H),3.90(s,1H),3.34(s,2H),2.75(q,J＝7.5Hz,2H),2.06(q,J＝6.9Hz,2H),1.94(t,J＝7.2Hz,2H),1.87(d,J＝12.6Hz,2H),1.471.61–1.35(m,6H),1.23(dd,J＝12.8,8.4Hz,7H).13C NMR(101MHz,DMSO)δ171.98,170.04,169.60,164.81,164.37,158.46,136.83,129.05,128.69,128.56,116.44,115.17,93.77,87.54,53.59,47.04,46.95,45.54,35.87,32.71,32.06,28.84,27.57,25.70,25.52,14.00.HRMS-ESI(m/z)calc.for C30H37N7O4S[M+H]+592.2706,found 592.2708.HPLC tR＝6.89min(99.98％purity)。
N1-(1-(6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)-N9-羟基壬二酰胺(11f)
白色固体，产率48.2％，mp 177-179℃。1H NMR(400MHz,DMSO)δ10.37(s,1H),7.98(s,1H),7.86(d,J＝7.6Hz,1H),7.56–7.49(m,2H),7.43–7.29(m,4H),5.55(s,1H),4.47–4.42(m,2H),3.34(t,J＝12.2Hz,2H),2.76(q,J＝7.6Hz,2H),2.06(t,J＝7.4Hz,2H),1.98–1.83(m,4H),1.55–1.35(m,6H),1.21(dd,J＝14.7,7.1Hz,9H).13C NMR(101MHz,DMSO)δ171.97,169.99,169.57,164.78,164.40,158.48,136.67,129.12,128.67,116.43,115.14,93.79,87.60,53.64,47.00,45.56,35.91,32.70,32.04,29.00,28.97,28.93,27.57,25.74,25.56,14.01.HRMS-ESI(m/z)calc.for C31H39N7O4S[M+Na]+628.2682,found628.2681.HPLC tR＝11.64min(99.9％purity).
N1-(1-(6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)-N4-羟基对甲苯二酰胺(11g)
白色固体，产率67.6％，mp 214-216℃。1H NMR(400MHz,DMSO)δ11.37(s,1H),8.50(d,J＝7.7Hz,1H),7.98(s,1H),7.93(d,J＝8.3Hz,2H),7.84(d,J＝8.2Hz,2H),7.56–7.49(m,2H),7.44–7.29(m,4H),5.56(s,1H),4.65–4.52(m,2H),4.29–4.17(m,1H),3.34(q,J＝4.3Hz,2H),2.77(q,J＝7.3Hz,2H),1.98(d,J＝6.2Hz,2H),1.67(q,J＝12.3Hz,2H),1.21(t,J＝7.5Hz,3H)).13C NMR(101MHz,DMSO)δ169.98,165.47,164.87,164.44,163.90,158.51,137.20,136.62,135.48,129.15,128.68,128.65,127.86,127.29,116.45,115.14,93.83,87.64,53.68,47.33,47.23,46.73,31.96,31.86,27.60,14.03.HRMS-ESI(m/z)calc.for C30H29N7O4S[M+Na]+606.1905,found 606.1908.HPLC tR＝6.73min(97.35％purity).
N1-(1-(6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)-4-((4-(羟基氨甲酰基)苄基)氧基)苯甲酰胺(11h)
白色固体，产率53.0％，mp 202-204℃。1H NMR(400MHz,DMSO)δ11.34(s,1H),8.33(d,J＝7.6Hz,1H),8.19(s,1H),7.89(d,J＝8.5Hz,2H),7.80(d,J＝7.9Hz,2H),7.54(dd,J＝16.1,7.7Hz,4H),7.43–7.30(m,4H),7.07(d,J＝8.5Hz,2H),5.65(s,1H),5.24(s,2H),4.64–4.52(m,2H),4.25–4.13(m,1H),3.30(d,J＝13.0Hz,2H),2.76(q,J＝7.5Hz,2H),1.98–1.92(m,2H),1.66(dd,J＝26.5,12.7Hz,2H),1.21(t,J＝7.6Hz,3H).13C NMR(101MHz,DMSO)δ170.05,165.53,164.89,164.39,160.84,158.50,140.34,136.80,132.73,129.71,129.06,128.70,128.57,127.87,127.57,127.49,116.46,115.18,114.72,93.78,87.59,69.20,60.24,53.62,47.49,47.32,46.58,32.04,27.59,21.25,14.56,14.03.HRMS-ESI(m/z)calc.for C37H34N7O5S[M-H]-688.2348,found 688.2348.HPLC tR＝4.86min(99.92％purity).
N1-(1-(6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)-N4-(4-(羟基氨甲酰基)苯基)丁二酰胺(11i)
白色固体，产率28.2％，mp 225-227℃。1H NMR(400MHz,DMSO)δ11.12(s,1H),10.33(s,1H),8.93(s,1H),8.08–7.97(m,2H),7.68(q,J＝8.7Hz,3H),7.53(d,J＝7.4Hz,2H),7.43–7.27(m,4H),5.57(s,1H),4.43(d,J＝13.1Hz,2H),3.91(s,1H),2.75(q,J＝7.5Hz,2H),2.61(t,J＝7.1Hz,2H),2.43(t,J＝7.2Hz,2H),1.88(d,J＝12.7Hz,2H),1.54–1.37(m,2H),1.20(t,J＝7.6Hz,3H).13C NMR(101MHz,DMSO)δ170.03,164.76,164.36,163.09,158.45,138.23,136.73,135.03,130.31,129.10,128.68,128.60,127.95,119.84,116.46,115.16,93.68,87.58,53.64,48.02,30.29,27.56,14.00.HRMS-ESI(m/z)calc.forC33H33N8O5S[M-H]+653.2300,found653.2302.HPLC tR＝8.39min(99.06％purity).
N1-(1-(6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)-N5-(4-(羟基氨甲酰基)苯基)戊二酰胺(11j)
白色固体，产率49.3％，mp214-216℃。1H NMR(400MHz,DMSO)δ10.25(s,1H),8.02(s,1H),7.94(d,J＝7.6Hz,1H),7.70(t,J＝6.1Hz,4H),7.53(d,J＝7.5Hz,2H),7.41–7.29(m,4H),5.56(s,1H),4.43(t,J＝10.4Hz,2H),3.92(d,J＝10.4Hz,1H),3.40–3.28(m,2H),2.75(q,J＝7.5Hz,2H),2.37(t,J＝7.4Hz,2H),2.15(t,J＝7.4Hz,2H),1.92–1.79(m,4H),1.51–1.37(m,2H),1.20(s,3H).13C NMR(101MHz,DMSO)δ171.79,171.50,169.99,164.79,164.39,158.46,142.35,136.68,129.11,128.67,128.10,127.38,118.72,116.43,115.15,93.77,87.57,53.63,46.97,45.60,36.20,35.12,32.08,27.58,21.64,14.01.HRMS-ESI(m/z)calc.for C34H36N8O5S[M+H]+669.2612,found 669.2615.HPLC tR＝10.52min(97.52％purity).
N1-(1-(6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)-N6-(4-(羟基氨甲酰基)苯基)己二酰胺(11k)
白色固体，产率33.1％，mp 211-213℃。1H NMR(400MHz,DMSO)δ11.10(s,1H),10.19(s,1H),7.99–7.95(m,1H),7.90(d,J＝7.6Hz,1H),7.74–7.64(m,4H),7.52(d,J＝7.4Hz,2H),7.40–7.31(m,4H),5.54(s,1H),4.44(t,J＝10.4Hz,2H),3.97–3.89(m,1H),3.32(d,J＝12.1Hz,2H),2.75(q,J＝7.5Hz,2H),2.35(t,J＝6.8Hz,2H),2.11(t,J＝6.8Hz,2H),1.97–1.81(m,2H),1.62–1.53(m,4H),1.47–1.37(m,2H),1.20(t,J＝7.6Hz,3H).13CNMR(101MHz,DMSO)δ172.01,171.79,169.98,164.78,164.40,158.46,142.33,136.65,129.12,128.66,128.63,128.11,127.40,118.72,116.43,115.14,93.79,87.59,53.66,46.97,45.60,36.73,35.75,32.08,27.58,25.48,25.18,14.01.HRMS-ESI(m/z)calc.forC35H37N8O5S[M-H]-681.2613,found681.2617.HPLC tR＝6.4min(97.15％purity).
N1-(1-(6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)-N4-(4-(羟基氨甲酰基)苯基)丁二酰胺(11l)
白色固体，产率28.7％，mp 207-209℃。1H NMR(400MHz,DMSO)δ11.18(s,1H),8.41(t,J＝6.0Hz,1H),7.97–7.87(m,2H),7.70(d,J＝7.9Hz,2H),7.51(d,J＝7.4Hz,2H),7.42–7.29(m,6H),5.53(s,1H),4.47–4.35(m,2H),4.30(d,J＝5.9Hz,2H),3.95–3.87(m,1H),2.76(q,J＝7.5Hz,2H),2.46–2.31(m,4H),1.88(d,J＝12.7Hz,2H),1.51–1.36(m,2H),1.21(t,J＝7.6Hz,3H).13C NMR(101MHz,DMSO)δ171.97,171.14,169.98,164.78,164.56,164.41,158.49,143.45,136.62,131.65,129.15,128.66,127.43,127.33,116.43,115.14,93.81,87.63,53.67,46.93,45.64,42.22,32.07,31.98,31.24,31.15,27.58,14.01.HRMS-ESI(m/z)calc.for C34H35N8O5S[M-H]-667.2466,found 667.2467.HPLC tR＝11.36min(99.30％purity).
N1-(1-(6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)-N5-(4-(羟基氨甲酰基)苯基)戊二酰胺(11m)
白色固体，产率43.2％，mp 193-195℃。1H NMR(400MHz,DMSO)δ11.19(s,1H),8.40(t,J＝6.0Hz,1H),7.96(s,1H),7.88(d,J＝7.6Hz,1H),7.70(d,J＝8.1Hz,2H),7.52(d,J＝7.1Hz,2H),7.42–7.26(m,6H),5.54(s,1H),4.48–4.37(m,2H),4.29(d,J＝5.9Hz,2H),3.96–3.89(m,1H),3.37–3.31(m,2H),2.76(q,J＝7.7Hz,2H),2.17(t,J＝7.5Hz,2H),2.10(t,J＝7.5Hz,2H),1.94–1.83(m,2H),1.80–1.72(m,2H),1.48–1.40(m,2H),1.20(t,J＝7.6Hz,3H).13CNMR(101MHz,DMSO)δ172.34,171.56,169.99,164.79,164.54,164.40,158.47,143.47,136.65,131.68,129.85,129.13,128.66,127.47,127.37,116.44,115.14,93.78,87.59,53.65,46.94,45.59,42.20,35.36,35.19,32.09,32.02,27.58,22.05,14.02.HRMS-ESI(m/z)calc.for C35H37N8O5S[M-H]-681.2613,found 681.2618.HPLC tR＝6.21min(99.91％purity).
(E)-N1-(1-(6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)-N4-(4-(3-(羟基氨基)-3-氧代丙基-1-烯-1-基)苯基)丁二酰胺(11n)
白色固体,产率30.8％,mp 212-214℃。1H NMR(400MHz,DMSO)δ10.71(s,1H),10.14(s,1H),8.01–7.90(m,2H),7.64(d,J＝8.3Hz,2H),7.51(t,J＝8.9Hz,4H),7.36(dt,J＝22.5,7.6Hz,5H),6.36(d,J＝15.8Hz,1H),5.54(s,1H),4.47–4.36(m,2H),3.95–3.88(m,1H),3.34(d,J＝12.0Hz,2H),2.76(q,J＝7.5Hz,2H),2.60(t,J＝7.1Hz,2H),2.43(t,J＝7.1Hz,2H),1.94–1.82(m,2H),1.54–1.39(m,2H),1.21(t,J＝7.6Hz,3H).13C NMR(101MHz,DMSO)δ171.18,171.03,170.00,164.77,164.40,163.52,158.46,140.94,138.48,136.62,129.82,129.14,128.66,119.41,117.65,116.43,115.14,93.79,87.60,53.66,46.92,45.71,32.13,32.08,32.01,30.71,27.58,14.02.HRMS-ESI(m/z)calc.for C35H35N8O5S[M-H]+679.2457,found 679.2462.HPLC tR＝6.44min(97.25％purity).
4-(((1-(6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)氨基)甲基)-N-羟基苯甲酰胺(11o)
白色固体,产率26％,mp 201-203℃。1H NMR(400MHz,DMSO)δ10.76(s,1H),9.03(s,1H),7.95(s,1H),7.53(dd,J＝7.8,5.8Hz,4H),7.45(dd,J＝11.7,4.0Hz,3H),7.41–7.28(m,4H),6.46(d,J＝15.8Hz,1H),5.55(s,1H),4.43(d,J＝13.3Hz,2H),3.86(s,2H),3.27(s,2H),2.86(s,1H),2.75(q,J＝7.6Hz,2H),1.44(s,2H),1.20(t,J＝7.6Hz,3H).13CNMR(101MHz,DMSO)δ170.05,164.94,164.40,164.05,158.62,136.67,135.58,133.57,130.72,130.55,129.94,129.15,128.70,128.64,127.56,116.32,115.00,94.43,88.17,54.16,53.64,47.05,46.39,46.27,45.90,28.41,27.59,13.98,8.93.HRMS-ESI(m/z)calc.for C30H32N7O3S[M+H]+570.2287,found 570.2297.HPLC tR＝9.58min(97.78％purity).
(E)-3-4-(((1-(6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)氨基)甲基)苯基)-N-羟基丙烯酰胺(11p)
白色固体,产率29.3％,mp 199-201℃。1H NMR(400MHz,CDCl3)δ9.63(s,2H),8.11(d,J＝3.6Hz,1H),7.75–7.66(m,4H),7.59(d,J＝7.2Hz,2H),7.53(d,J＝15.8Hz,1H),7.47–7.36(m,4H),6.59(dd,J＝15.9,1.7Hz,1H),5.65(s,1H),4.70(d,J＝13.4Hz,2H),4.28(t,J＝6.0Hz,2H),3.47(d,J＝6.0Hz,1H),3.24(t,J＝12.9Hz,2H),2.81(q,J＝7.6Hz,2H),2.39–2.30(m,2H),1.88–1.73(m,2H),1.26(t,J＝7.7Hz,3H).13C NMR(101MHz,DMSO)δ170.05,164.92,164.41,162.92,158.59,137.97,136.60,135.86,133.65,131.13,129.18,128.70,128.13,120.50,116.34,115.01,94.36,88.13,60.24,54.01,53.62,47.10,46.36,46.25,28.45,27.60,21.25,14.56,14.01.HRMS-ESI(m/z)calc.for C32H33N7O3S[M+H]+596.2438,found 596.2440.HPLC tR＝8.11min(98.52％purity).
1-(6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)-N-(4-((4-(羟基氨甲酰基)苄基)氨甲酰基)苄基)哌啶4-铵盐(11q)
白色固体,产率28％,mp 197-199℃。1H NMR(400MHz,DMSO)δ11.20(s,1H),9.66(s,2H),9.24(t,J＝6.0Hz,1H),8.06(s,1H),7.98(d,J＝8.0Hz,2H),7.73(dd,J＝10.7,8.1Hz,4H),7.58–7.52(m,2H),7.44–7.31(m,6H),5.61(s,1H),4.66(d,J＝13.4Hz,2H),4.53(d,J＝5.9Hz,2H),4.28(t,J＝6.1Hz,2H),3.45(m,1H),3.20(t,J＝12.8Hz,2H),2.77(q,J＝7.5Hz,2H),2.30(d,J＝12.4Hz,2H),1.85–1.75(m,2H),1.21(t,J＝7.8Hz,3H).13CNMR(101MHz,DMSO)δ170.05,166.17,164.92,164.51,164.41,158.59,143.32,136.62,135.76,134.92,131.78,130.53,129.89,129.18,128.70,128.01,127.68,127.55,127.42,116.34,115.02,94.36,88.14,54.09,53.62,46.99,46.37,46.26,42.89,28.46,27.61,14.02.HRMS-ESI(m/z)calc.for C38H39N8O4S[M+H]+703.2816,found 703.2821.HPLC tR＝4.04min(96.86％purity).
(E)-1-(6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)-N-(4-((4-(3-羟基氨基)-3-氧代丙基-1-烯-1-基)苄基)氨甲酰基)苄基)哌啶-4-铵盐(11r)
白色固体,产率68.4％,mp 207-208℃。1H NMR(400MHz,DMSO)δ10.86(s,1H),9.77(s,2H),9.26(t,J＝5.9Hz,1H),8.13(s,1H),7.99(d,J＝7.9Hz,2H),7.77(d,J＝8.0Hz,2H),7.54(dd,J＝13.3,7.6Hz,4H),7.44–7.24(m,7H),6.48(d,J＝15.8Hz,1H),5.63(s,1H),4.66(d,J＝13.2Hz,2H),4.51(d,J＝5.9Hz,2H),4.28(t,J＝6.0Hz,2H),3.25–3.16(m,2H),3.11–3.16(m,1H),2.77(q,J＝7.7Hz,2H),2.36–2.27(m,2H),1.83–1.70(m,2H),1.21(t,J＝7.6Hz,3H).13CNMR(101MHz,DMSO)δ170.07,166.15,164.92,164.40,163.20,158.57,141.58,138.45,136.65,135.74,134.96,133.90,130.54,129.16,128.70,128.66,128.25,127.98,127.96,119.19,116.34,115.03,94.34,88.11,54.07,53.60,46.97,46.38,46.26,42.92,28.43,27.61,14.01.HRMS-ESI(m/z)calc.for C40H41N8O4S[M+H]+729.2975,found729.2979.HPLC tR＝4.02min(97.99％purity).
(E)-1-(6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)-N-(4-((4-(3-羟基氨基)-3-氧代丙基-1-烯-1-基)苄基)氧代)苄基)哌啶-4-铵盐(11s)
白色固体,产率58.4％,mp 187-189℃。1H NMR(400MHz,DMSO)δ10.83(s,1H),9.35(s,2H),8.05(s,1H),7.58(d,J＝7.9Hz,2H),7.54(d,J＝7.7Hz,4H),7.50–7.46(m,2H),7.46–7.30(m,5H),7.08(d,J＝8.5Hz,2H),6.49(d,J＝15.8Hz,1H),5.60(s,1H),5.18(s,2H),4.65(d,J＝13.3Hz,2H),4.14(t,J＝5.9Hz,2H),3.39(s,2H),3.19(t,J＝13.0Hz,2H),2.77(q,J＝7.6Hz,2H),2.25(s,2H),1.72(t,J＝13.7Hz,2H),1.21(t,J＝7.6Hz,3H).13CNMR(101MHz,DMSO)δ170.04,164.92,164.41,159.07,158.59,138.69,138.26,136.60,134.91,132.14,129.18,128.69,128.55,128.06,124.67,119.80,116.33,115.44,115.00,94.37,88.13,69.27,53.77,53.63,46.98,46.35,46.26,28.46,27.60,14.01.HRMS-ESI(m/z)calc.for C40H41N8O4S[M+Na]+724.2682,found 724.2676.HPLC tR＝6.45min(97.29％purity).
1-(6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)-N-(4-((7-(羟氨基)-7-氧代庚基)氧代)苄基)哌啶-4-铵盐(11t)
白色固体,产率45.2％,mp 191-193℃。1H NMR(400MHz,DMSO)δ10.38(s,1H),9.36–9.31(m,2H),8.05(s,1H),7.53(t,J＝8.4Hz,4H),7.46–7.33(m,4H),7.00(d,J＝8.2Hz,2H),5.60(s,1H),4.65(d,J＝13.3Hz,2H),4.14(t,J＝6.0Hz,2H),3.98(t,J＝6.5Hz,2H),3.20(t,J＝12.7Hz,2H),2.77(q,J＝7.5Hz,2H),2.27(d,J＝10.7Hz,2H),1.95(t,J＝7.3Hz,2H),1.78–1.66(m,5H),1.54–1.48(m,2H),1.43–1.38(m,2H),1.34–1.27(m,2H),1.20(dt,J＝14.5,7.3Hz,5H).13C NMR(101MHz,DMSO)δ170.07,169.58,164.92,164.41,159.57,158.58,136.60,132.12,129.17,128.69,124.16,116.33,115.02,114.98,94.34,88.10,67.94,53.71,53.62,47.00,46.37,46.26,32.66,28.95,28.78,28.45,27.60,25.69,25.52,14.01.HRMS-ESI(m/z)calc.for C36H43N7O4S[M+H]+670.3170,found670.3177.HPLC tR＝7.85min(98.24％purity)
2-((1-(6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)氨基)-N-羟基嘧啶-5-甲酰胺(11u)
白色固体,产率38.3％,mp 185-187℃。1H NMR(400MHz,DMSO)δ8.66(s,2H),7.95(s,1H),7.90(d,J＝7.8Hz,1H),7.52(d,J＝7.4Hz,2H),7.41–7.31(m,4H),5.55(s,1H),4.58–4.49(m,2H),4.18(t,J＝9.2Hz,1H),3.37(dd,J＝11.2,6.5Hz,2H),2.77(q,J＝7.6Hz,2H),2.03(d,J＝12.8Hz,2H),1.65–1.55(m,2H),1.22(t,J＝7.6Hz,3H).13C NMR(101MHz,DMSO)δ170.04,164.80,164.48,162.35,158.42,136.51,129.17,128.68,116.45,115.39,115.15,93.77,87.55,53.63,47.76,46.99,31.77,27.58,14.00.HRMS-ESI(m/z)calc.for C28H29N9O3S[M+Na]+580.1811,found 580.1810.HPLC tR＝5.74min(97.25％purity).
2-((1-(6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)(甲基)氨基)-N-羟基嘧啶-5-甲酰胺(11v)
白色固体,产率36.1％,mp 185-187℃。1H NMR(400MHz,DMSO)δ11.20(s,1H),9.03(s,1H),8.73(s,2H),8.07(s,1H),7.55(d,J＝7.4Hz,2H),7.39–7.30(m,4H),5.61(s,1H),5.00(s,1H),4.76(d,J＝13.2Hz,2H),3.01(s,3H),2.77(q,J＝8.0Hz,2H),1.81(d,J＝22.1Hz,4H),1.22(t,J＝7.6Hz,3H).13C NMR(101MHz,DMSO)δ170.02,164.80,164.40,161.99,158.37,157.48,136.73,129.11,128.66,116.51,115.17,114.92,93.84,87.63,53.64,52.34,47.62,29.49,29.06,27.59,14.01.HRMS-ESI(m/z)calc.for C28H29N9O3S[M+Na]+597.1965,found 594.1967.HPLC tR＝4.52min(95.29％purity)
叔丁基-4-((6-((2-氨基-2-氧代-苯乙基)硫代)-3，5-二氰基-4-乙基吡啶-2-基)(甲基)氨基)哌啶-1-氨基甲酸酯(12a)
白色固体,产率38.3％。1H NMR(400MHz,DMSO)δ7.89(s,1H),7.53–7.46(m,2H),7.44–7.28(m,4H),5.48(s,1H),4.69(s,1H),4.05(s,2H),3.13(s,3H),2.78(q,J＝7.7Hz,2H),1.67(d,J＝9.4Hz,4H),1.41(s,9H),1.22(t,J＝7.6Hz,3H).
叔丁基-4-((6-((2-氨基-2-氧代-苯乙基)硫代)-3，5-二氰基-4-乙基吡啶-2-基)甲基)(甲基)氨基)哌啶-1-氨基甲酸酯(12b)
白色固体,产率42.8％。1H NMR(400MHz,DMSO)δ7.89(s,1H),7.53–7.46(m,2H),7.44–7.28(m,4H),5.48(s,1H),4.69(s,1H),4.05(s,2H),3.13(s,3H),2.78(q,J＝7.7Hz,2H),1.67(d,J＝9.4Hz,4H),1.41(s,9H),1.22(t,J＝7.6Hz,3H).
4-((6-((2-氨基-2-氧代-苯乙基)硫代)-3，5-二氰基-4-乙基吡啶-2-基)(甲基)氨基)哌啶-1-铵盐(13a)
白色固体,产率56.2％。1H NMR(400MHz,DMSO)δ8.82(d,J＝37.0Hz,2H),8.08(s,1H),7.53(d,J＝7.5Hz,2H),7.46–7.34(m,4H),5.53(s,1H),4.89(s,1H),3.17(s,3H),3.15–3.05(m,2H),2.80(t,J＝7.6Hz,2H),2.09(dd,J＝19.2,8.3Hz,2H),1.80(t,J＝14.8Hz,2H),1.23(t,J＝7.6Hz,3H).
4-((6-((2-氨基-2-氧代-苯乙基)硫代)-3，5-二氰基-4-乙基吡啶-2-基)甲基)(甲基)氨基)哌啶-1-铵盐(13b)
白色固体,产率41.7％。1H NMR(400MHz,DMSO)δ8.74(s,2H),8.04(s,1H),7.54(d,J＝7.1Hz,2H),7.45–7.34(m,4H),5.48(s,1H),3.89(dd,J＝14.0,8.3Hz,1H),3.63–3.56(m,1H),3.27(d,J＝12.3Hz,2H),2.83–2.72(m,3H),2.03(d,J＝9.4Hz,1H),1.75–1.66(m,2H),1.56–1.42(m,2H),1.22(t,J＝7.7Hz,3H).
2-(4-((6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)(甲基)氨基)哌啶-1-基)-N-((四氢-2H-吡喃-2-基)氧代)嘧啶-5-甲酰胺(14a)
白色固体,产率37.2％。1H NMR(400MHz,DMSO)δ11.51(s,1H),8.70(s,2H),7.90(s,1H),7.51(d,J＝7.0Hz,2H),7.43–7.32(m,4H),5.52(s,1H),4.96(s,1H),4.87(d,J＝12.6Hz,3H),4.04(s,1H),3.53(d,J＝11.4Hz,1H),3.12(s,4H),2.80(t,J＝7.6Hz,2H),1.80–1.74(m,3H),1.73–1.69(m,2H),1.55(s,3H),1.23(t,J＝7.6Hz,3H).
2-(4-((6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)甲基)(甲基)氨基)哌啶-1-基)-N-((四氢-2H-吡喃-2-基)氧代)嘧啶-5-甲酰胺(14b)
白色固体,产率45.3％。1H NMR(400MHz,DMSO)δ11.51(s,1H),8.69(s,2H),7.92(s,1H),7.46(d,J＝7.5Hz,2H),7.41–7.32(m,4H),7.21(dd,J＝8.4,6.3Hz,1H),5.52(d,J＝4.4Hz,1H),4.96(d,J＝3.4Hz,1H),4.76(t,J＝15.8Hz,2H),4.03(s,1H),3.92(dd,J＝14.1,7.8Hz,1H),3.55(dd,J＝14.3,6.9Hz,2H),2.95–2.83(m,2H),2.77(d,J＝7.7Hz,2H),2.10(s,1H),1.75–1.49(m,8H),1.21(t,J＝7.6Hz,3H).
2-(4-((6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)(甲基)氨基)哌啶-1-基)-N-羟基嘧啶-5-甲酰胺(15a)
白色固体,产率37.2％,mp 172-173℃。1H NMR(400MHz,DMSO)δ11.28(s,1H),9.06(s,1H),8.73(s,2H),8.24(s,1H),7.60–7.50(m,2H),7.45–7.30(m,4H),5.66(s,1H),4.87(d,J＝25.6Hz,3H),3.10(s,5H),2.77(d,J＝7.6Hz,2H),1.72(d,J＝34.2Hz,4H),1.27–1.18(m,3H).13C NMR(101MHz,DMSO)δ170.13,164.41,162.37,161.71,158.61,157.72,136.58,129.09,128.65,128.56,116.75,115.30,114.85,93.61,87.03,56.28,53.48,43.05,42.96,33.60,29.00,28.75,27.62,14.00.HRMS-ESI(m/z)calc.for C28H29N9O3S[M+Na]+594.2007,found594.2002.HPLC tR＝4.67min(96.98％purity).
2-(4-((6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)(甲基)(甲基)氨基)哌啶-1-基)-N-羟基嘧啶-5-甲酰胺(15b)
白色固体,产率32.0％,mp 173-175℃。1H NMR(400MHz,DMSO)δ8.71(s,2H),8.15(s,1H),7.50(d,J＝7.5Hz,2H),7.36(s,1H),7.28(t,J＝7.5Hz,2H),7.18(t,J＝7.3Hz,1H),5.61(s,1H),4.74(t,J＝16.8Hz,2H),3.93(dd,J＝14.2,7.8Hz,1H),3.54(dd,J＝17.9,8.3Hz,2H),3.38(s,2H),2.87(q,J＝13.4Hz,3H),2.76(q,J＝7.4Hz,2H),2.08(s,1H),1.68–1.56(m,2H),1.20(m,5H).13C NMR(101MHz,DMSO)δ170.13,164.50,164.34,161.67,157.91,157.66,144.73,136.87,134.34,129.01,128.52,128.39,116.90,115.33,114.62,93.22,86.12,56.76,53.48,43.79,41.14,35.49,29.15,29.01,27.63,13.97.HRMS-ESI(m/z)calc.for C28H29N9O3S[M+Na]+608.2160,found 608.2155.HPLC tR＝4.55min(95.28％purity).
叔丁基-4-((6-((2-氨基-2-氧代-苯乙基)硫代)-3，5-二氰基-4-乙基吡啶-2-基)哌嗪-1-氨基甲酸酯(16)
白色固体,产率81.2％。1H NMR(400MHz,DMSO)δ7.90(s,1H),7.54–7.49(m,2H),7.45–7.29(m,5H),5.53(s,1H),3.89(t,J＝5.3Hz,4H),3.25–3.18(m,4H),2.77(q,J＝7.6Hz,3H),1.39(s,9H),1.21(t,J＝7.5Hz,3H).
4-((6-((2-氨基-2-氧代-苯乙基)硫代)-3，5-二氰基-4-乙基吡啶-2-基)哌嗪-1-铵盐(17)
白色固体,产率90.2％。1H NMR(400MHz,DMSO)δ11.23(s,1H),7.87(s,1H),7.57(d,J＝7.7Hz,2H),7.54–7.46(m,3H),7.43–7.27(m,6H),6.50(d,J＝15.9Hz,1H),5.51(s,1H),4.92(s,1H),3.97(s,1H),3.90(s,4H),3.57(s,2H),3.53(s,1H),2.76(q,J＝7.6Hz,2H),1.70(s,3H),1.55(s,3H),1.20(t,J＝7.6Hz,3H).
(E)-3-(4-((4-(6-((2-氨基-2氧代-1-苯乙基)硫代)-3，5-二氰基-4-乙基吡啶-2-基)哌嗪-1-基)甲基)苯基)-N-((四氢-2H-吡喃-2-基)氧代)丙烯酰胺(18a)
白色固体,产率48.8％。1H NMR(400MHz,DMSO)δ11.23(s,1H),7.87(s,1H),7.57(d,J＝7.7Hz,2H),7.54–7.46(m,3H),7.43–7.27(m,6H),6.50(d,J＝15.9Hz,1H),5.51(s,1H),4.92(s,1H),3.97(s,1H),3.90(s,4H),3.57(s,2H),3.53(s,1H),2.76(q,J＝7.6Hz,2H),1.70(s,3H),1.55(s,3H),1.20(t,J＝7.6Hz,3H).
2-(4-((6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)哌嗪-1-基)-N-((四氢-2H-吡喃-2-基)氧代)嘧啶-5-甲酰胺(18b)
白色固体,产率50.2％。1H NMR(400MHz,DMSO)δ11.63(s,1H),8.77(s,2H),7.98(s,1H),7.54(d,J＝7.5Hz,2H),7.40(dd,J＝15.2,7.7Hz,4H),5.59(s,1H),4.98(s,1H),4.03(s,6H),4.00(s,4H),2.79(q,J＝7.6Hz,2H),1.72(s,3H),1.55(s,3H),1.23(t,J＝7.6Hz,3H).
(E)-3-(4-((4-(6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)哌嗪-1-基)甲基)苯基)-N-羟基丙烯酰胺(19a)
白色固体,产率30.3％,mp 187-189℃。1H NMR(400MHz,DMSO)δ10.81(s,1H),9.04(s,1H),7.93(s,1H),7.55(d,J＝7.7Hz,2H),7.50(d,J＝7.3Hz,2H),7.46(d,J＝15.5Hz,1H),7.40–7.29(m,7H),6.48(d,J＝15.8Hz,1H),5.54(s,1H),3.90(s,4H),3.56(s,2H),2.75(q,J＝7.4Hz,2H),1.20(t,J＝7.6Hz,3H).13C NMR(101MHz,DMSO)δ169.96,164.79,164.44,158.51,136.55,129.97,129.13,128.64,127.91,119.34,116.37,115.07,94.07,87.85,61.74,53.63,52.86,47.93,27.55,13.98.HRMS-ESI(m/z)calc.for C31H31N7O3S[M+Na]+604.2058,found 604.2059.HPLC tR＝5.16min(95.27％purity).
2-(4-((6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)哌嗪-1-基)-N-羟基嘧啶-5-甲酰胺(19b)
白色固体,产率31.2％,mp 211-213℃。1H NMR(400MHz,DMSO)δ11.16(s,1H),8.75(s,2H),7.97(s,1H),7.54(d,J＝7.1Hz,2H),7.45–7.34(m,4H),5.58(s,1H),4.04(dd,J＝7.3,3.6Hz,4H),3.98(dd,J＝7.3,3.6Hz,4H),2.79(q,J＝7.5Hz,2H),1.23(t,J＝7.6Hz,3H).13C NMR(101MHz,DMSO)δ170.02,164.91,164.39,162.19,161.68,160.15,158.62,157.62,136.42,129.21,128.73,128.70,116.37,115.52,115.06,94.14,87.93,53.71,47.31,43.54,27.57,14.01.HRMS-ESI(m/z)calc.for C26H25N9O3S[M+Na]+566.1659,found566.1655.HPLC tR＝4.51min(96.89％purity).
叔丁基-4-((6-((2-氨基-2-氧代-苯乙基)硫代)-3，5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)甲基)-1-氨基甲酸酯(20)
白色固体,产率77.8％。1H NMR(400MHz,DMSO)δ7.92(s,1H),7.55–7.48(m,2H),7.41–7.30(m,4H),6.93(t,J＝5.9Hz,1H),5.53(s,1H),4.55(d,J＝13.3Hz,2H),3.11(t,J＝12.3Hz,2H),2.86(t,J＝6.1Hz,2H),2.75(q,J＝7.5Hz,2H),1.76(d,J＝12.8Hz,3H),1.39(s,9H),1.25–1.16(m,5H).
叔丁基-(R)-((1-(6-((2-氨基-2-氧代-苯乙基)硫代)-3，5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)甲基)-1-氨基甲酸酯((R)-20)
白色固体,产率63.5％。1H NMR(400MHz,DMSO)δ7.92(s,1H),7.52(dt,J＝6.0,1.4Hz,2H),7.42–7.28(m,4H),6.94(q,J＝5.7Hz,1H),5.54(s,1H),4.55(d,J＝13.3Hz,2H),3.10(d,J＝10.5Hz,2H),2.85(q,J＝6.2Hz,2H),2.75(q,J＝7.5Hz,2H),1.80–1.68(m,3H),1.38(d,J＝5.7Hz,9H),1.29–1.15(m,5H).
叔丁基-(S)-((1-(6-((2-氨基-2-氧代-苯乙基)硫代)-3，5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)甲基)-1-氨基甲酸酯((S)-20)
白色固体,产率66.2％。1H NMR(400MHz,DMSO)δ7.92(s,1H),7.55–7.46(m,2H),7.42–7.28(m,4H),6.93(t,J＝5.9Hz,1H),5.54(s,1H),4.55(d,J＝13.2Hz,2H),3.11(t,J＝12.6Hz,2H),2.85(q,J＝6.3Hz,2H),2.75(q,J＝7.6Hz,2H),1.76–1.70(m,2H),1.38(d,J＝5.6Hz,9H),1.29–1.15(m,5H).
(1-(6-((2-氨基-2-氧代-苯乙基)硫代)-3，5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)甲铵盐(21)
白色固体,产率88.2％。1H NMR(400MHz,DMSO)δ7.94(s,1H),7.81(s,3H),7.52(d,J＝7.5Hz,2H),7.43–7.31(m,4H),5.54(s,1H),4.58(d,J＝13.2Hz,2H),3.17(d,J＝5.0Hz,2H),3.10(s,2H),2.76(d,J＝8.2Hz,2H),1.88(t,J＝13.2Hz,3H),1.35–1.17(m,5H).
(R)-(1-(6-((2-氨基-2-氧代-苯乙基)硫代)-3，5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)甲铵盐((R)-21)
白色固体,产率67.6％。1H NMR(400MHz,DMSO)δ8.08(s,4H),7.54(d,J＝7.5Hz,2H),7.43–7.29(m,4H),5.57(s,1H),4.57(d,J＝13.4Hz,2H),3.14(q,J＝13.7Hz,2H),2.76(q,J＝6.0Hz,4H),1.88(d,J＝12.5Hz,3H),1.43–1.28(m,2H),1.20(t,J＝7.6Hz,3H).
(S)-(1-(6-((2-氨基-2-氧代-苯乙基)硫代)-3，5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)甲铵盐((S)-21)
白色固体,产率58.5％。1H NMR(400MHz,DMSO)δ8.11(s,4H),7.57–7.52(m,2H),7.41–7.32(m,4H),5.58(s,1H),4.57(d,J＝13.5Hz,2H),3.14(q,J＝13.7Hz,2H),2.79–2.73(m,4H),1.91–1.86(m,3H),1.40–1.30(m,2H),1.20(t,J＝7.6Hz,3H).
(E)-3-(4-((((1-(6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)甲基)氨基)甲基)苯基)-N-(四氢-2H-吡喃-2-基)氧代)丙烯酰胺(22a)
白色固体,产率53.2％。1H NMR(400MHz,DMSO)δ11.22(s,1H),7.92(s,1H),7.51(d,J＝8.3Hz,4H),7.45–7.34(m,5H),6.48(d,J＝15.6Hz,1H),5.52(s,1H),4.91(s,1H),4.57(d,J＝13.5Hz,2H),3.96(s,1H),3.76(s,1H),3.53(d,J＝11.4Hz,1H),3.17–3.03(m,2H),2.72(d,J＝7.6Hz,2H),1.85(s,2H),1.69(s,3H),1.54(s,3H),1.20(t,J＝7.5Hz,3H).
(E)-3-(4-((((1-(6-(((R)-2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)甲基)氨基)甲基)苯基)-N-(四氢-2H-吡喃-2-基)氧代)丙烯酰胺((R)-22a)
白色固体,产率33.0％。White solid,yield 33.0％,1H NMR(400MHz,DMSO)δ11.22(s,1H),7.92(s,1H),7.58–7.48(m,5H),7.48–7.28(m,8H),6.50(d,J＝15.8Hz,1H),5.53(s,1H),4.91(s,1H),4.56(d,J＝13.3Hz,2H),3.96(s,1H),3.81(s,2H),3.61–3.47(m,2H),3.17–3.04(m,2H),2.75(q,J＝7.5Hz,2H),1.87(d,J＝12.2Hz,2H),1.70(s,3H),1.54(s,3H),1.19(t,J＝7.7Hz,3H).
(E)-3-(4-((((1-(6-(((S)-2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)甲基)氨基)甲基)苯基)-N-(四氢-2H-吡喃-2-基)氧代)丙烯酰胺((S)-22a)
白色固体,产率35.2％。1H NMR(400MHz,DMSO)δ11.26(s,1H),7.95(s,1H),7.57(d,J＝7.9Hz,2H),7.52(d,J＝7.3Hz,3H),7.47(d,J＝7.3Hz,2H),7.41–7.32(m,4H),6.51(d,J＝15.8Hz,1H),5.55(s,1H),4.92(s,1H),4.46(d,J＝13.1Hz,2H),3.90(s,4H),3.05(s,2H),2.76(q,J＝7.5Hz,2H),2.02(s,2H),1.72(d,J＝10.7Hz,3H),1.55(s,3H),1.47(s,2H),1.20(t,J＝7.4Hz,3H).
2-(((1-(6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)甲基)氨基)-N-((四氢-2H-吡喃-2-基)氧代)嘧啶-5-甲酰胺(22b)
白色固体,产率48.3％。1H NMR(400MHz,DMSO)δ11.49(s,1H),8.63(d,J＝11.4Hz,2H),7.98(t,J＝6.2Hz,1H),7.92(s,1H),7.51(d,J＝7.6Hz,2H),7.41–7.29(m,4H),5.53(s,1H),4.95(s,1H),4.57(d,J＝13.4Hz,2H),4.03(s,1H),3.28(s,2H),2.75(q,J＝7.4Hz,3H),1.95(s,1H),1.83(d,J＝12.8Hz,2H),1.71(s,3H),1.54(s,3H),1.35–1.22(m,5H).
(E)-1-(1-(6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)-N-(4-(3-(羟氨基)3-氧代丙烷-1-烯-1-基)苄基)甲铵盐(23a)
白色固体,产率33.2％,mp 203-205℃。1H NMR(400MHz,DMSO)δ10.85(s,1H),9.36(s,2H),8.02(s,1H),7.63(s,4H),7.57–7.45(m,3H),7.43–7.28(m,4H),6.53(d,J＝15.8Hz,1H),5.56(s,1H),4.56(d,J＝13.3Hz,2H),4.17(t,J＝5.6Hz,2H),3.14(q,J＝13.0Hz,2H),2.84(d,J＝6.6Hz,2H),2.75(q,J＝7.6Hz,2H),2.15(s,1H),1.92(d,J＝12.8Hz,2H),1.37–1.24(m,2H),1.20(t,J＝7.6Hz,3H).13C NMR(101MHz,DMSO)δ170.03,164.81,164.34,162.93,158.51,137.98,136.69,135.86,133.39,131.31,129.11,128.68,128.61,128.05,120.48,116.43,115.14,93.77,87.73,53.64,51.57,50.46,49.06,47.86,47.72,32.84,30.00,27.56,14.01.HRMS-ESI(m/z)calc.for C33H35N7O3S[M+H]+610.2570,found 610.2552.HPLC tR＝5.76min(97.46％purity).
(R，E)-1-(1-(6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)-N-(4-(3-(羟氨基)3-氧代丙烷-1-烯-1-基)苄基)甲铵盐((R)-23a)
白色固体,产率50.2％,mp 201-202℃。1H NMR(400MHz,DMSO)δ10.78(s,1H),9.04(s,1H),7.94(s,1H),7.58(d,J＝7.8Hz,2H),7.53(d,J＝1.9Hz,2H),7.51–7.42(m,3H),7.42–7.28(m,4H),6.48(d,J＝15.8Hz,1H),5.54(s,1H),4.56(d,J＝13.2Hz,2H),3.99(s,2H),3.14(d,J＝8.8Hz,2H),2.75(q,J＝7.6Hz,2H),2.67(s,2H),1.98(s,1H),1.89(d,J＝13.2Hz,2H),1.21(q,J＝7.1Hz,5H).13C NMR(101MHz,DMSO)δ170.03,164.83,164.30,162.96,158.50,137.93,136.77,135.84,133.40,131.36,131.31,129.07,128.73,128.68,128.56,127.99,120.53,116.42,115.14,93.77,87.73,56.49,53.64,51.48,50.41,47.90,47.73,45.78,32.81,30.05,27.55,19.03,13.99,8.88.HRMS-ESI(m/z)calc.forC33H35N7O3S[M+H]+610.2570,found 610.2572.HPLC tR＝5.78min(98.02％purity).
(S，E)-1-(1-(6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)-N-(4-(3-(羟氨基)3-氧代丙烷-1-烯-1-基)苄基)甲铵盐((S)-23a)
白色固体,产率47.5％,mp 202-204℃。1H NMR(400MHz,DMSO)δ10.86(s,1H),9.29(s,2H),8.00(s,1H),7.63(s,3H),7.53(dd,J＝7.0,1.9Hz,2H),7.50(s,1H),7.41–7.30(m,4H),6.53(d,J＝15.8Hz,1H),5.56(s,1H),4.56(d,J＝13.3Hz,2H),4.17(t,J＝5.6Hz,2H),3.21–3.07(m,3H),2.85(d,J＝6.0Hz,2H),2.75(q,J＝7.6Hz,2H),2.14(s,1H),1.96–1.88(m,2H),1.31–1.17(m,5H).13C NMR(101MHz,DMSO)δ170.03,164.83,164.31,162.95,158.50,137.95,136.75,135.83,133.40,131.35,131.30,129.08,128.75,128.68,128.57,128.00,120.50,116.43,115.15,93.76,87.73,56.49,53.63,51.49,50.41,47.89,47.73,32.81,30.06,30.03,27.55,14.00.HRMS-ESI(m/z)calc.for C33H35N7O3S[2M+H]+1219.5122,found 1219.5129.HPLC tR＝5.82min(99.24％purity).
2-(((1-(6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)甲基)氨基)-N-羟基嘧啶-5-甲酰胺(23b)
白色固体,产率35.1％,mp 178-180℃。1H NMR(400MHz,DMSO)δ8.65(s,2H),8.07(s,1H),7.96(s,1H),7.52(dd,J＝7.1,1.9Hz,2H),7.40–7.28(m,4H),5.55(s,1H),4.58(d,J＝13.3Hz,2H),3.29(d,J＝5.7Hz,2H),3.12(d,J＝12.6Hz,2H),2.80–2.72(m,2H),2.00–1.92(m,1H),1.89–1.80(m,2H),1.21(m,5H).13C NMR(101MHz,DMSO)δ170.03,164.69,164.37,162.85,158.42,157.61,136.73,129.09,128.67,128.58,116.48,115.20,93.58,87.50,53.61,49.06,48.10,46.36,45.90,35.53,30.25,30.12,27.56,14.00.HRMS-ESI(m/z)calc.for C28H29N9O3S[M+H]+572.2155,found 572.2150.HPLC tR＝11.25min(99.40％purity).
叔丁基(2-((6-((2-氨基-2-氧代-苯乙基)硫代)-3，5-二氰基-4-乙基吡啶-2-基)(甲基)氨基)乙基)氨基甲酸酯(24)
白色固体,产率85.2％。1H NMR(400MHz,DMSO)δ7.87(s,1H),7.57–7.48(m,2H),7.45–7.29(m,4H),6.96(t,J＝6.1Hz,1H),5.59(s,1H),3.87–3.74(m,2H),3.40(s,3H),3.21(t,J＝5.7Hz,2H),2.76(q,J＝7.5Hz,2H),1.34(s,9H),1.23(d,J＝7.5Hz,3H).
2-((6-((2-氨基-2-氧代-苯乙基)硫代)-3，5-二氰基-4-乙基吡啶-2-基)(甲基)氨基)乙基-1-铵盐(25)
白色固体,产率91.2％。1H NMR(400MHz,DMSO)δ8.27(s,4H),7.65–7.57(m,2H),7.47–7.31(m,4H),4.09(s,2H),3.43(d,J＝1.1Hz,3H),3.09(s,2H),2.78(q,J＝7.5Hz,2H),1.21(d,J＝7.5Hz,3H).
(E)-3-(4-(((2-((6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)(甲基)氨基)乙基)氨基)甲基)苯基)-N-(四氢-2H-吡喃-2-基)氧代)丙烯酰胺(26)
白色固体,产率51.5％。1H NMR(400MHz,DMSO)δ11.23(s,1H),7.96(s,1H),7.50(d,J＝7.0Hz,4H),7.44(s,1H),7.42–7.27(m,6H),6.47(d,J＝15.9Hz,1H),5.52(s,1H),4.91(s,1H),3.99–3.91(m,2H),3.85(s,1H),3.72(s,2H),3.53(d,J＝11.3Hz,2H),2.74(q,J＝7.4Hz,2H),1.70(s,3H),1.54(s,3H),1.18(d,J＝7.1Hz,3H).
(E)-2-((6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)(甲基)氨基)-N-(4-(3-(羟氨基)-3-氧代丙烷-1-烯-1-基)苄基)乙基-1-铵盐(27)
白色固体,产率32.7％,mp 197-199℃。1H NMR(400MHz,DMSO)δ10.84(s,1H),9.51(s,2H),9.09(s,1H),8.32(s,1H),7.71–7.58(m,6H),7.55–7.52(m,1H),7.48(d,J＝15.8Hz,1H),7.41–7.32(m,3H),6.52(d,J＝15.9Hz,1H),5.80(s,1H),4.26–4.11(m,4H),3.28–3.20(m,2H),2.79(q,J＝7.6Hz,2H),1.21(t,J＝7.6Hz,3H).13C NMR(101MHz,DMSO)δ170.55,164.48,163.01,158.18,138.04,136.44,135.92,133.44,131.14,129.17,128.79,128.71,128.12,120.48,116.82,115.15,93.97,87.12,52.94,50.58,48.06,44.46,27.62,13.96.HRMS-ESI(m/z)calc.for C28H29N9O3S[M+H]+570.2282,found 570.2284.HPLC tR＝5.18min(96.49％purity).
实施例10：目标化合物30a-e的制备
5-((2-((叔丁氧基氨甲酰基)氨基)苯基)氨基)-5-氧代戊酸(28a)
将相应的羧酸(2.66mmol)，EDCI(615mg,3.2mmol)，DMAP(422mg,3.45mmol)，和叔丁基2-氨基苯基甲酸酯(555mg,2.66mmol)加入到15mL CH2Cl2中，室温下反应8小时。反应结束后，反应液依次用1M盐酸水溶液(100mL)、饱和氯化钠水溶液(100mL)洗涤。然后用无水Na2SO4干燥有机相并浓缩。经硅胶柱层析纯化得到28a，化合物28d-e的合成方法同该操作步骤。白色固体，产率25.8％。1H NMR(400MHz,DMSO)δ12.10(s,1H),9.46(s,1H),8.32(s,1H),7.54(d,J＝8.0Hz,1H),7.41(d,J＝7.8Hz,1H),7.19–7.05(m,2H),2.40(t,J＝7.3Hz,2H),2.29(t,J＝7.4Hz,2H),1.83(p,J＝7.3Hz,2H),1.46(s,9H).
4-((2-((叔丁氧基氨甲酰基)氨基)苯基)氨甲酰基)苯甲酸(28b)
白色固体，产率78.1％。1H NMR(400MHz,DMSO)δ13.31(s,1H),9.98(s,1H),8.74(s,1H),8.11–8.05(m,4H),7.56(t,J＝9.4Hz,2H),7.21–7.14(m,2H).
2-(4-((2-((叔丁氧基氨甲酰基)氨基)苯基)氨甲酰基)苯基)乙酸(28c)
白色固体，产率96.4％。1H NMR(400MHz,DMSO)δ9.83(s,1H),8.69(s,1H),7.90(d,J＝8.2Hz,2H),7.57–7.51(m,2H),7.46–7.40(m,2H),7.22–7.15(m,2H),3.69(s,2H),1.45(s,9H).
叔丁基(2-(4-甲酰基苯甲酰胺基)苯基)氨基甲酸酯(28d)
黄色固体，产率43.9％。1H NMR(400MHz,DMSO)δ10.13(s,1H),10.01(s,1H),8.74(s,1H),8.15(d,J＝8.0Hz,2H),8.07(d,J＝8.3Hz,2H),7.58–7.50(m,2H),7.25–7.11(m,2H),1.44(s,9H).
叔丁基(2-(2-氯嘧啶-5-甲酰胺基)苯基)氨基甲酸酯(28e)
黄色固体，产率72.7％。1H NMR(400MHz,DMSO)δ10.13(s,1H),10.01(s,1H),8.74(s,1H),8.15(d,J＝8.0Hz,2H),8.07(d,J＝8.3Hz,2H),7.59–7.51(m,2H),7.26–7.15(m,2H),1.44(s,9H).
叔丁基(2-(5-((1-(6-((2-氨基-2-氧代-苯乙基)硫代)-3，5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)氨基)-5-氧代戊氨基)苯基)氨基甲酸酯(29a)
白色固体,产率84.3％。1H NMR(400MHz,DMSO)δ9.43(s,1H),8.38(s,1H),7.96–7.87(m,2H),7.58–7.49(m,3H),7.47–7.29(m,5H),7.19–7.08(m,2H),5.53(s,1H),4.52–4.39(m,2H),3.94(s,1H),2.76(q,J＝7.5Hz,2H),2.37(t,J＝7.4Hz,2H),2.16(t,J＝7.5Hz,2H),1.94–1.80(m,4H),1.46(s,9H),1.22(t,J＝6.4Hz,3H).
叔丁基(2-(4-((1-(6-((2-氨基-2-氧代-苯乙基)硫代)-3，5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)氨甲酰基)苯甲酰胺基)苯基)氨基甲酸酯(29b)
白色固体,产率82.2％。1H NMR(400MHz,DMSO)δ10.01(s,1H),8.76(s,1H),8.58(d,J＝7.6Hz,1H),8.05(q,J＝8.6Hz,5H),7.57–7.52(m,4H),7.39(t,J＝7.3Hz,2H),7.34(dd,J＝4.9,2.3Hz,2H),7.24–7.14(m,2H),5.58(s,1H),4.60(t,J＝13.5Hz,2H),4.23(s,1H),2.78(q,J＝7.6Hz,2H),2.03(s,2H),1.68(t,J＝11.9Hz,2H),1.45(s,9H),1.23(t,J＝7.6Hz,3H).
叔丁基(2-(4-(2-((1-(6-((2-氨基-2-氧代-苯乙基)硫代)-3，5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)氨基)-2-氧代乙基)苯甲酰胺基)苯基)氨基甲酸酯(29c)
白色固体,产率75.5％。1H NMR(400MHz,DMSO)δ9.86(s,1H),8.71(s,1H),8.27(d,J＝7.5Hz,1H),7.99–7.95(m,1H),7.91(d,J＝8.0Hz,2H),7.58–7.49(m,4H),7.44(d,J＝8.0Hz,2H),7.41–7.29(m,4H),7.25–7.15(m,2H),5.55(s,1H),4.49–4.39(m,2H),3.95–3.89(m,1H),3.53(s,2H),3.38(d,J＝11.2Hz,2H),2.76(q,J＝7.5Hz,2H),1.91(d,J＝12.8Hz,2H),1.44(s,9H),1.21(t,J＝7.5Hz,3H).
叔丁基(2-(4-(((1-(6-((2-氨基-2-氧代-苯乙基)硫代)-3，5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)氨基)甲基)苯甲酰胺基)苯基)氨基甲酸酯(29d)
白色固体,产率50.2％。1H NMR(400MHz,DMSO)δ9.83(s,1H),8.71(s,1H),7.93(d,J＝6.4Hz,3H),7.61–7.51(m,6H),7.41–7.30(m,4H),7.23–7.15(m,2H),5.54(s,1H),4.43(s,2H),3.88(s,2H),3.30(d,J＝11.4Hz,2H),2.75(q,J＝7.5Hz,3H),2.02–1.90(m,2H),1.45(s,11H),1.20(t,J＝7.6Hz,3H).
叔丁基(2-(2-((1-(6-((2-氨基-2-氧代-苯乙基)硫代)-3，5-二氰基-4-乙基吡啶-2-基)(甲基)氨基)嘧啶-甲酰胺基)苯基)氨基甲酸酯(29e)
白色固体,产率46.0％。1H NMR(400MHz,DMSO)δ9.71(s,1H),8.92(s,2H),8.57(s,1H),7.93(s,1H),7.61(d,J＝7.8Hz,1H),7.54(d,J＝7.1Hz,2H),7.48(dd,J＝7.9,1.6Hz,1H),7.44–7.30(m,4H),7.21–7.15(m,2H),5.57(s,1H),5.09–4.99(m,1H),4.77(d,J＝13.3Hz,2H),3.30(s,2H),3.06(s,3H),2.78(q,J＝7.5Hz,2H),1.93–1.76(m,4H),1.45(s,9H),1.23(t,J＝7.6Hz,3H).
N1-(1-(6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)-N5-(2-氨基苯基)戊二酰胺(30a)
白色固体,产率74.2％,mp 174-176℃。1H NMR(400MHz,DMSO)δ9.09(s,1H),7.89(dd,J＝12.0,4.8Hz,2H),7.58–7.43(m,3H),7.41–7.28(m,4H),7.24–7.05(m,2H),6.95–6.85(m,1H),6.72(d,J＝7.9,1H),6.54(t,J＝7.5,1H),5.53(s,1H),4.46–4.41(m,2H),3.94(s,1H),3.37(d,J＝12.6Hz,2H),2.77(q,J＝7.3Hz,2H),2.34(t,J＝7.4Hz,2H),2.30–2.17(m,2H),2.05–1.80(m,4H),1.45(s,2H),1.21(t,J＝7.5Hz,3H).13C NMR(101MHz,DMSO)δ171.62,171.51,171.27,169.98,164.79,164.41,158.50,155.11,142.37,136.64,129.13,128.66,126.18,125.82,123.98,121.63,116.60,116.42,116.32,115.11,93.85,87.63,53.69,46.96,45.62,35.62,35.32,32.07,28.47,27.57,24.13,22.03,13.99.HRMS-ESI(m/z)calc.for C33H36N8O3S[M+Na]+647.2529,found 647.2529.HPLC tR＝10.24min(99.93％purity).
N1-(1-(6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)-N4-(2-氨基苯基)对苯二甲酰胺(30b)
白色固体,产率76.5％,mp 247-249℃。1H NMR(400MHz,DMSO)δ10.64(s,1H),8.66(d,J＝7.6Hz,1H),8.32–7.99(m,6H),7.67–7.25(m,12H),5.63(s,1H),4.65–4.54(m,2H),4.26–4.18(m,1H),3.34(d,J＝12.1Hz,2H),2.77(q,J＝7.6Hz,2H),2.06–1.98(m,2H),1.80–1.60(m,2H),1.22(t,J＝7.6Hz,3H).13C NMR(101MHz,DMSO)δ170.05,165.45,165.38,164.91,164.40,158.53,137.68,136.79,136.41,129.08,128.71,128.58,128.52,127.82,127.76,126.95,123.70,116.46,115.17,93.82,87.64,53.63,47.43,47.27,46.83,31.92,27.60,14.04.HRMS-ESI(m/z)calc.for C36H34N8O3S[M+Na]+681.2372,found681.2374.HPLC tR＝11.46min(99.31％purity).
4-(2-((1-(6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)氨基)-2-氧代乙基)-N-(2-氨基苯基)苯甲酰胺(30c)
白色固体,产率79.2％,mp 245-247℃。1H NMR(400MHz,DMSO)δ10.41(d,J＝11.4Hz,1H),8.34(t,J＝7.0Hz,1H),8.04(td,J＝9.7,4.1Hz,3H),7.53(d,J＝7.4Hz,3H),7.44(d,J＝8.0Hz,2H),7.40–7.27(m,7H),5.57(d,J＝2.2Hz,1H),4.45(t,J＝10.5Hz,2H),3.92(d,J＝9.9Hz,1H),3.37(dd,J＝13.6,10.5Hz,2H),2.76(q,J＝7.6Hz,2H),1.91(d,J＝12.4Hz,2H),1.48(t,J＝14.4Hz,2H),1.24–1.18(t,J＝7.6Hz,3H).13C NMR(101MHz,DMSO)δ170.00,169.46,166.02,164.80,164.40,158.48,141.21,136.70,132.32,129.39,129.11,128.67,128.48,127.61,127.02,116.45,115.14,93.82,87.61,53.64,46.89,45.89,42.72,31.96,27.59,21.25,14.57,14.02.HRMS-ESI(m/z)calc.for C36H34N8O3S[M–H]-671.2558,found 671.2559.HPLC tR＝5.18min(98.52％purity).
4-(((1-(6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)氨基)甲基)-N-(2-氨基苯基)苯甲酰胺(30d)
白色固体,产率70.2％,mp 221-223℃。1H NMR(400MHz,DMSO)δ10.21(s,1H),9.82(s,2H),8.13(d,J＝8.5Hz,3H),7.81(d,J＝7.9Hz,2H),7.56(d,J＝7.4Hz,2H),7.47–7.27(m,5H),7.14(s,2H),6.98(s,1H),5.63(s,1H),4.67(d,J＝13.1Hz,2H),4.32(d,J＝6.1Hz,2H),3.22(d,J＝13.8Hz,2H),2.77(q,J＝7.6Hz,2H),2.33(d,J＝11.7Hz,2H),1.88–1.75(m,2H),1.22(t,J＝7.5Hz,3H).13C NMR(101MHz,DMSO)δ170.06,165.43,164.93,164.40,158.60,136.66,136.10,134.89,130.46,129.17,128.71,128.64,127.54,127.08,116.34,115.03,94.36,88.14,55.42,54.09,53.61,46.97,46.40,46.28,28.43,27.61,14.02.HRMS-ESI(m/z)calc.for C36H36N8O2S[M+Na]+645.2755,found 645.2756.HPLC tR＝7.35min(97.74％purity).
2-((1-(6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)(甲基)氨基)-N-(2-氨基苯基)嘧啶-5-甲酰胺(30e)
白色固体,产率70.2％,mp 217-219℃。1H NMR(400MHz,DMSO)δ9.52(s,1H),8.93(s,2H),7.95(s,1H),7.57–7.50(m,2H),7.40–7.31(m,4H),7.14(d,J＝7.8Hz,1H),7.01–6.93(m,1H),6.77(d,J＝7.9Hz,1H),6.59(t,J＝7.5Hz,1H),5.57(s,1H),4.95(s,2H),4.76(d,J＝13.3Hz,2H),3.05(s,3H),2.83–2.75(m,2H),1.91–1.77(m,4H),1.22(t,J＝7.6Hz,3H).13C NMR(101MHz,DMSO)δ170.01,164.79,164.43,163.23,162.03,158.42,143.80,136.60,129.16,128.64,127.38,127.06,123.26,116.65,116.57,116.51,116.37,115.16,93.89,87.70,53.71,52.45,47.63,29.58,29.09,29.04,27.60,14.01.HRMS-ESI(m/z)calc.for C34H34N10O2S[M-H]-645.2489,found 645.2494.HPLC tR＝5.21min(96.94％purity).
实施例11：目标化合物40a-d、44a-c、48的制备
甲基4-(N-甲基甲磺酰胺基)苯甲酸酯(32)
在含有31(1g,6.05mmol)和三乙胺(1.22g,12.10mmol)的CH2Cl2(10mL)中于0℃下缓慢加入甲磺酰氯(1.04g,9.08mmol)，室温下反应1.5小时。反应液用50mL CH2Cl2稀释，依次用1M盐酸水溶液(100mL)、饱和碳酸氢钠水溶液(100mL)、饱和氯化钠水溶液(100mL)洗涤有机相。然后用无水Na2SO4干燥有机相并浓缩。经硅胶柱层析纯化得到32。淡黄色固体,产率83.5％。1H NMR(400MHz,DMSO)δ8.02–7.94(m,2H),7.59–7.51(m,2H),3.86(s,3H),3.30(s,3H),3.01(s,3H).
N-(4-(羟甲基)苯基)-N-甲基甲磺酰胺(33)
在32(1g，4.11mmol)的THF溶液中加入DIBAL(8.22mmol)，在-20℃下搅拌6小时，反应结束后用30mL 10％ NaOH溶液淬灭反应。然后用乙酸乙酯萃取混合物(10mL×3)，浓缩后有机相后得粗产物，后者经柱层析纯化后得到33。灰色固体,产率68.9％。1H NMR(400MHz,DMSO)δ7.40–7.30(m,4H),5.22(t,J＝5.7Hz,1H),4.49(d,J＝5.7Hz,2H),3.22(s,3H),2.92(s,3H).
4-(N-甲基甲基磺酰胺基)苄基-4-甲基苯磺酸酯(34)
淡黄色固体,产率45.8％。MS-ESI(m/z)calc.for C16H19N1O5S2[M+H]+370.1.
N-(4-(((6-氨基-3，5-二氰基-4-乙基哌啶-2-基)硫代)甲基)苯基)-N-甲基甲磺酰胺(35)
棕黄色固体,产率71.6％。1H NMR(400MHz,DMSO)δ7.55–7.50(m,2H),7.35–7.30(m,2H),4.46(s,2H),3.21(s,3H),2.93(s,3H),2.69(q,J＝7.8Hz,2H),1.18(t,J＝7.6Hz,3H).
N-(4-(((6-氯-3，5-二氰基-4-乙基哌啶-2-基)硫代)甲基)苯基)-N-甲基甲磺酰胺(36)
棕黄色固体,产率31.3％。1H NMR(400MHz,DMSO)δ7.51(d,J＝7.2Hz,2H),7.42–7.33(m,2H),4.58(s,2H),3.21(s,3H),2.94(s,3H),2.88(q,J＝8.2Hz,2H),1.25(t,J＝8.6Hz,3H).
叔丁基(2-((3，5-二氰基-4-乙基-6-((4-(N-甲基甲磺酰胺基)苄基)硫代)吡啶2-基)(甲基)氨基)乙基)氨基甲酸酯(37a)
黄色固体,产率79.7％。1H NMR(400MHz,DMSO)δ7.44(d,J＝8.4Hz,2H),7.41–7.34(m,2H),6.94(t,J＝6.0Hz,1H),4.52(s,2H),3.83(t,J＝6.1Hz,2H),3.39(s,3H),3.20(d,J＝8.2Hz,5H),2.92(s,3H),2.77(q,J＝7.6Hz,2H),1.31(s,9H),1.21(t,J＝7.6Hz,3H).
叔丁基(3-((3，5-二氰基-4-乙基-6-((4-(N-甲基甲磺酰胺基)苄基)硫代)吡啶2-基)(甲基)氨基)丙基)氨基甲酸酯(37b)
黄色固体,产率77.2％。1H NMR(400MHz,DMSO)δ7.45(d,J＝8.2Hz,2H),7.37(dd,J＝8.3,1.5Hz,2H),6.85(d,J＝5.6Hz,1H),4.52(s,2H),3.73(t,J＝7.4Hz,2H),3.21(s,3H),2.94(dd,J＝8.3,3.5Hz,5H),2.77(q,J＝7.6Hz,2H),1.79–1.71(m,2H),1.33(s,9H),1.21(t,J＝7.6Hz,3H).
叔丁基(2-((3，5-二氰基-4-乙基-6-((4-(N-甲基甲磺酰胺基)苄基)硫代)吡啶2-基)(甲基)氨基)乙基)(甲基)氨基甲酸酯(37c)
黄色固体,产率74.0％。1H NMR(400MHz,DMSO)δ7.43(d,J＝8.3Hz,1H),7.37(d,J＝8.3Hz,1H),4.53(s,1H),3.91(d,J＝25.3Hz,1H),3.37(d,J＝7.7Hz,3H),2.91(s,2H),1.38(s,2H),1.31(s,2H),1.22(d,J＝6.8Hz,5H).
2-((3，5-二氰基-4-乙基-6-((4-(N-甲基甲磺酰胺基)苄基)硫代)吡啶2-基)(甲基)氨基)乙基-1-铵盐(38a)
淡黄色固体,产率90.1％。1H NMR(400MHz,DMSO)δ8.01(s,3H),7.46(d,J＝8.3Hz,2H),7.38(d,J＝8.5Hz,2H),4.54(s,2H),3.99(t,J＝6.4Hz,2H),3.45(s,3H),3.22(s,3H),3.10(d,J＝7.1Hz,2H),2.94(s,3H),2.80(q,J＝7.6Hz,3H),1.23(t,J＝7.5Hz,3H).
3-((3，5-二氰基-4-乙基-6-((4-(N-甲基甲磺酰胺基)苄基)硫代)吡啶2-基)(甲基)氨基)丙基-1-铵盐(38b)
淡黄色固体,产率84.1％。1H NMR(400MHz,DMSO)δ7.95(s,3H),7.46(d,J＝8.4Hz,2H),7.39(d,J＝8.3Hz,2H),4.54(s,2H),3.99(t,J＝6.4Hz,2H),3.45(s,3H),3.23(s,3H),3.11(t,J＝6.5Hz,2H),2.94(s,3H),2.80(q,J＝7.6Hz,2H),1.23(t,J＝7.6Hz,3H).
2-((3，5-二氰基-4-乙基-6-((4-(N-甲基甲磺酰胺基)苄基)硫代)吡啶2-基)(甲基)氨基)丙基-1-铵盐(38c)
淡黄色固体,产率85.0％。1H NMR(400MHz,DMSO)δ8.82(s,2H),7.50–7.43(m,2H),7.43–7.35(m,2H),4.56(s,2H),4.04(t,J＝6.3Hz,2H),3.46(s,4H),3.22(s,3H),3.17(q,J＝6.8Hz,3H),2.94(s,3H),2.80(q,J＝7.6Hz,2H),2.54(d,J＝5.3Hz,3H),1.23(t,J＝7.5Hz,3H).
N1-2-((3，5-二氰基-4-乙基-6-((4-(N-甲基甲磺酰胺基)苄基)硫代)吡啶2-基)(甲基)氨基)乙基)-N4-((四氢-2H-吡喃-2-基)氧代)丁二酰胺(39a)
淡黄色固体,产率31.8％。1H NMR(400MHz,DMSO)δ10.89(s,1H),7.94(t,J＝5.9Hz,1H),7.45(d,J＝8.5Hz,2H),7.37(d,J＝8.4Hz,2H),4.79(s,1H),4.51(s,2H),3.89(d,J＝11.1Hz,1H),3.82(t,J＝6.2Hz,2H),3.48(d,J＝11.6Hz,1H),3.38(s,3H),3.29(d,J＝6.1Hz,1H),3.21(s,3H),2.92(s,3H),2.78(q,J＝7.5Hz,2H),1.98(dt,J＝20.0,7.5Hz,4H),1.65(p,J＝7.8Hz,5H),1.50(s,3H),1.22(t,J＝7.6Hz,3H).
N1-2-((3，5-二氰基-4-乙基-6-((4-(N-甲基甲磺酰胺基)苄基)硫代)吡啶2-基)(甲基)氨基)乙基)-N5-((四氢-2H-吡喃-2-基)氧代)戊二酰胺(39b)
淡黄色固体,产率44.2％。1H NMR(400MHz,DMSO)δ10.86(s,1H),7.96(t,J＝6.1Hz,1H),7.44(d,J＝8.4Hz,2H),7.37(d,J＝8.3Hz,2H),4.77(s,1H),4.49(s,2H),3.81–3.74(m,2H),3.40(s,3H),3.29(d,J＝6.1Hz,3H),3.20(s,3H),2.91(s,3H),2.76(q,J＝7.5Hz,2H),,1.98(dt,J＝20.0,7.5Hz,4H),1.71–1.64(m,5H),1.50(s,3H),1.22(t,J＝7.6Hz,3H).
N1-2-((3，5-二氰基-4-乙基-6-((4-(N-甲基甲磺酰胺基)苄基)硫代)吡啶2-基)(甲基)氨基)丙基)-N4-((四氢-2H-吡喃-2-基)氧代)丁二酰胺(39c)
淡黄色固体,产率39.5％。1H NMR(400MHz,DMSO)δ10.95(s,1H),7.89(s,1H),7.48–7.41(m,2H),7.41–7.34(m,2H),4.78(s,1H),4.52(s,2H),3.90(s,1H),3.73(s,2H),3.48(s,2H),3.22(s,3H),3.05(d,J＝6.1Hz,2H),2.93(d,J＝1.9Hz,3H),2.77(d,J＝8.0Hz,2H),2.42(d,J＝4.7Hz,2H),2.26(s,2H),2.21(d,J＝6.8Hz,2H),1.75(s,2H),1.62(s,3H),1.49(s,3H),1.21(t,J＝7.7Hz,3H).
2-((2-((3，5-二氰基-4-乙基-6-((4-(N-甲基甲磺酰胺基)苄基)硫代)吡啶2-基)(甲基)氨基)乙基)(甲基)氨基)-N-((四氢-2H-吡喃-2-基)氧代)嘧啶-5-甲酰胺(39d)
淡黄色固体,产率39.2％。1H NMR(400MHz,DMSO)δ11.49(s,1H),8.62(s,2H),7.44–7.32(m,4H),5.75(s,1H),4.93(s,1H),4.47(s,2H),4.08(s,2H),4.02(s,1H),3.91(t,J＝5.8Hz,2H),3.52(d,J＝11.4Hz,1H),3.35(s,3H),3.21(s,3H),3.08(s,3H),2.92(s,3H),2.72(d,J＝7.6Hz,10H),1.71(s,3H),1.54(s,3H),1.17(t,J＝7.6Hz,3H).
N1-(2-((3,5-二氰基-4-乙基-6-((4-(N-甲基甲基磺酰氨基)苯基)硫代)吡啶-2-基)(甲基)氨基)乙基)-N4-羟基丁二酰胺(40a)
淡黄色固体,产率21.2％,mp 152-154℃。1H NMR(400MHz,DMSO)δ10.39(s,1H),8.68(d,J＝1.6Hz,1H),8.04(t,J＝5.9Hz,1H),7.45(d,J＝8.2Hz,2H),7.38(d,J＝8.3Hz,2H),4.52(s,2H),3.81(t,J＝6.2Hz,2H),3.39(s,3H),3.30(d,J＝6.1Hz,3H),3.22(s,3H),2.93(s,3H),2.78(q,J＝7.5Hz,2H),2.25(dd,J＝8.8,6.6Hz,2H),2.15(t,J＝7.4Hz,2H),1.22(t,J＝7.5Hz,3H).13C NMR(101MHz,DMSO)δ171.93,168.79,164.72,164.54,157.63,141.30,135.82,129.88,126.83,116.99,115.36,93.51,86.00,51.69,38.18,36.93,35.47,33.45,31.06,28.17,27.62,14.00.HRMS-ESI(m/z)calc.for C34H34N10O2S[M+Na]+596.1726,found 596.1728.HPLC tR＝3.98min(96.78％purity).
N1-(2-((3,5-二氰基-4-乙基-6-((4-(N-甲基甲基磺酰氨基)苯基)硫代)吡啶-2-基)(甲基)氨基)乙基)-N5-羟基戊二酰胺(40b)
淡黄色固体,产率30％,mp 117-119℃。1H NMR(400MHz,DMSO)δ10.37(s,1H),8.68(s,1H),7.90(t,J＝5.6Hz,1H),7.44(d,J＝8.2Hz,2H),7.37(d,J＝8.1Hz,2H),4.52(s,2H),4.03(td,J＝8.6,5.8Hz,1H),3.73(t,J＝7.5Hz,2H),3.22(s,3H),3.05(q,J＝6.4Hz,3H),2.93(s,3H),2.77(q,J＝7.4Hz,2H),2.30–2.24(m,2H),2.16(t,J＝7.4Hz,2H),1.76(t,J＝7.5Hz,2H),1.22(t,J＝7.9Hz,3H).13C NMR(101MHz,DMSO)δ171.56,168.89,164.80,164.59,157.44,141.32,135.81,129.87,126.82,116.93,115.38,93.41,85.92,50.28,38.18,36.50,35.49,33.43,31.04,28.25,27.64,14.56,13.99.HRMS-ESI(m/z)calc.forC26H33N7O5S2[M+Na]+610.1836,found586.1831.HPLC tR＝4.09min(95.64％purity).
N1-(3-((3,5-二氰基-4-乙基-6-((4-(N-甲基甲基磺酰氨基)苯基)硫代)吡啶-2-基)(甲基)氨基)丙基)-N4-羟基丁二酰胺(40c)
淡黄色固体,产率39.7％,mp 110-112℃。1H NMR(400MHz,DMSO)δ10.41(s,1H),8.70(s,1H),8.04(t,J＝5.9Hz,1H),7.45(d,J＝8.2Hz,2H),7.38(d,J＝8.3Hz,2H),4.52(s,2H),3.82(t,J＝6.2Hz,2H),3.39(s,3H),3.31(q,J＝6.1Hz,3H),3.22(s,3H),2.93(s,3H),2.78(q,J＝7.5Hz,2H),2.01(t,J＝7.6Hz,2H),1.92(t,J＝7.4Hz,2H),1.66(p,J＝7.5Hz,2H),1.22(t,J＝7.5Hz,3H).13C NMR(101MHz,DMSO)δ172.49,169.17,164.75,164.53,157.66,141.31,135.80,129.90,126.81,116.97,115.34,93.52,86.00,51.71,38.19,36.85,35.53,35.19,33.50,32.22,27.61,21.81,13.98.HRMS-ESI(m/z)calc.forC26H33N7O5S2[M-H]-586.1887,found586.1883.HPLC tR＝7.85min(98.24％purity).
2-((2-((3,5-二氰基-4-乙基-6-((4-(N-甲基甲基磺酰氨基)苯基)硫代)吡啶-2-基)(甲基)氨基)乙基)(甲基)氨基)-N-羟基嘧啶-5-甲酰胺(40d)
橘黄色固体,产率27％,mp 155-157℃。1H NMR(400MHz,DMSO)δ11.05(s,1H),9.00(s,1H),8.61(s,2H),7.42(d,J＝8.2Hz,2H),7.36(d,J＝8.2Hz,2H),4.48(s,2H),4.12–4.05(m,2H),3.91(t,J＝5.9Hz,2H),3.35(s,3H),3.32(s,3H),3.07(s,3H),2.93(s,3H),2.72(q,J＝7.7Hz,2H),1.17(t,J＝7.6Hz,3H).13C NMR(101MHz,DMSO)δ172.49,169.17,164.75,164.53,157.66,141.31,135.80,129.90,126.81,116.97,115.34,93.52,86.00,51.71,38.19,36.85,35.53,35.19,33.50,32.22,27.61,21.81,13.98.HRMS-ESI(m/z)calc.for C27H31N9O4S2[M-H]-608.1845,found 608.1640.HPLC tR＝9.90min(98.58％purity).
叔丁基(1-((3，5-二氰基-4-乙基-6-((4-(N-甲基甲磺酰胺基)苄基)硫代)吡啶2-基)哌啶-4-基)氨基甲酸酯(41)
白色固体,产率73.5％。1H NMR(400MHz,DMSO)δ7.44(d,J＝8.3Hz,2H),7.37(d,J＝8.3Hz,2H),6.90(d,J＝7.8Hz,1H),4.50(s,2H),4.39(d,J＝13.5Hz,2H),3.60(s,1H),3.27(s,1H),3.21(s,3H),2.92(s,3H),2.76(q,J＝7.9Hz,2H),1.85(d,J＝12.7Hz,2H),1.44(d,J＝12.6Hz,2H),1.39(s,9H),1.21(t,J＝7.6Hz,3H).
1-((3，5-二氰基-4-乙基-6-((4-(N-甲基甲磺酰胺基)苄基)硫代)吡啶2-基)哌啶-4-铵盐(42)
白色固体,产率80.8％。1H NMR(400MHz,DMSO)δ8.12(s,3H),7.45(d,J＝8.4Hz,2H),7.38(d,J＝8.4Hz,2H),4.53(d,J＝6.8Hz,4H),3.24(d,J＝17.7Hz,5H),2.94(s,3H),2.78(q,J＝7.6Hz,2H),2.06(d,J＝12.6Hz,2H),1.67–1.54(m,2H),1.22(t,J＝7.6Hz,3H).
N1-(1-(3，5-二氰基-4-乙基-6-((4-(N-甲基甲磺酰胺基)苄基)硫代)吡啶2-基)哌啶-4-基)-N5-((四氢-2H-吡喃-2-基)氧代)戊二酰胺(43a)
白色固体,产率37％。1H NMR(400MHz,DMSO)δ10.91(s,1H),7.81(d,J＝7.6Hz,1H),7.44(d,J＝8.3Hz,2H),7.37(d,J＝8.4Hz,2H),4.80(s,1H),4.50(s,2H),4.37(d,J＝13.4Hz,2H),3.91(d,J＝9.6Hz,2H),3.49(d,J＝11.6Hz,1H),3.37(d,J＝12.4Hz,2H),3.21(s,3H),3.17(d,J＝5.2Hz,1H),2.93(s,3H),2.77(q,J＝7.6Hz,2H),2.06(t,J＝7.5Hz,2H),1.99(t,J＝7.4Hz,2H),1.87(d,J＝12.8Hz,2H),1.77–1.60(m,5H),1.54–1.37(m,5H),1.22(t,J＝7.6Hz,3H).
(E)-3-(4-(((1-(3，5-二氰基-4-乙基-6-((N-甲基甲磺酰胺基)苄基)硫代)吡啶2-基)哌啶-4-基)氨基)甲基)苯基)-N-((四氢-2H-吡喃-2-基)氧代)丙烯酰胺(43b)
白色固体,产率45％。1H NMR(400MHz,DMSO)δ11.21(s,1H),7.52(t,J＝7.6Hz,3H),7.44(d,J＝8.6Hz,2H),7.41(d,J＝7.9Hz,2H),7.37(d,J＝8.4Hz,2H),6.48(d,J＝15.8Hz,1H),4.91(s,1H),4.51(s,2H),4.36(d,J＝13.3Hz,2H),3.97(s,1H),3.77(s,2H),3.21(s,3H),2.92(s,3H),2.77(d,J＝7.5Hz,2H),1.95(s,2H),1.70(s,3H),1.55(s,3H),1.37(d,J＝10.5Hz,2H),1.22(t,J＝7.7Hz,3H).
2-((1-(3，5-二氰基-4-乙基-6-((4-(N-甲基甲磺酰胺基)苄基)硫代)吡啶2-基)哌啶-4-基)氨基)-N-((四氢-2H-吡喃-2-基)氧代)嘧啶-5-甲酰胺(43c)
白色固体,产率40.9％。1H NMR(400MHz,DMSO)δ11.51(s,1H),8.64(s,2H),7.91(d,J＝7.9Hz,1H),7.45(d,J＝8.5Hz,2H),7.41–7.33(m,2H),4.95(s,1H),4.48(d,J＝22.6Hz,4H),4.18(s,1H),3.20(s,3H),2.92(s,3H),2.78(q,J＝7.6Hz,2H),1.99(d,J＝12.3Hz,2H),1.71(s,3H),1.56(d,J＝12.7Hz,5H),1.22(t,J＝7.6Hz,3H).
N1-(1-(3,5-二氰基-4-乙基-6-((4-(N-甲基甲基磺酰氨基)苯基)硫代)吡啶-2-基)哌啶-4-基)-N5-羟基戊二酰胺(44a)
白色固体,产率23.5％,mp 149-151℃。1H NMR(400MHz,DMSO)δ10.41(s,1H),8.73–8.66(m,1H),7.91(d,J＝7.7Hz,1H),7.44(d,J＝8.2Hz,2H),7.37(d,J＝8.3Hz,2H),4.50(s,2H),4.38(d,J＝13.4Hz,2H),3.91(s,1H),3.21(s,3H),3.17(d,J＝5.2Hz,1H),2.93(s,3H),2.77(q,J＝7.6Hz,2H),2.06(t,J＝7.5Hz,2H),1.96(dt,J＝14.7,7.4Hz,3H),1.86(d,J＝12.7Hz,2H),1.70(p,J＝7.7Hz,2H),1.44(q,J＝11.4Hz,2H),1.21(t,J＝7.6Hz,3H).13C NMR(101MHz,DMSO)δ171.42,169.22,165.05,164.49,158.59,141.25,136.09,129.79,126.83,116.44,115.18,94.38,87.83,60.23,46.93,45.50,38.18,35.46,35.23,33.53,33.24,32.18,31.92,31.72,27.60,21.92,21.03,14.59,14.03.HPLC tR＝9.90min(98.58％purity).
(E)-(1-(3,5-二氰基-4-乙基-6-((4-(N-甲基甲基磺酰氨基)苯基)硫代)吡啶-2-基)哌啶-4-基)-N5-羟基戊二酰胺(44b)
白色固体,产率27.2％,mp 164-166℃。1H NMR(400MHz,DMSO)δ10.81(s,1H),9.07(s,1H),7.55(d,J＝7.8Hz,2H),7.50–7.41(m,5H),7.38(d,J＝8.1Hz,2H),6.48(d,J＝15.7Hz,1H),4.51(s,2H),4.43(d,J＝13.2Hz,2H),3.90(s,1H),3.29(d,J＝11.9Hz,3H),3.21(s,3H),2.92(s,3H),2.77(q,J＝7.5Hz,2H),2.04(d,J＝12.1Hz,2H),1.49(s,2H),1.21(t,J＝7.6Hz,3H).13C NMR(101MHz,DMSO)δ165.04,164.52,163.17,158.44,141.25,138.45,136.14,129.81,127.91,126.83,116.47,115.19,94.33,87.74,53.23,46.47,38.20,35.48,33.49,27.60,14.02.HRMS-ESI(m/z)calc.for C33H37N7O4S2[M+H]+660.2368,found 660.2371.HPLC tR＝5.47min(96.01％purity).
(E)-3-(4-(((1-(3，5-二氰基-4-乙基-6-((4-(N-甲基甲基磺酰氨基)苯基)硫代)吡啶-2-基)哌啶-4-基)氨基)甲基)苯基)-N-羟基丙烯酰胺(44c)
白色固体,产率29％,mp 137-139℃。1H NMR(400MHz,DMSO)δ11.07(s,1H),9.00(s,1H),8.63(s,2H),7.82(d,J＝7.8Hz,1H),7.45(d,J＝8.1Hz,2H),7.37(d,J＝8.1Hz,2H),4.51(s,2H),4.46(s,2H),4.17(s,1H),3.44(d,J＝7.1Hz,2H),3.20(s,3H),2.92(s,3H),2.78(q,J＝7.7Hz,2H),1.99(d,J＝12.3Hz,2H),1.57(d,J＝11.7Hz,2H),1.22(t,J＝7.5Hz,3H).13C NMR(101MHz,DMSO)δ165.07,164.55,162.54,159.83,158.50,141.22,136.12,129.77,126.85,116.48,115.20,94.37,87.76,64.00,55.39,47.58,46.98,38.17,35.42,33.51,31.71,27.61,14.69,14.03.HRMS-ESI(m/z)calc.for C28H31N9O4S2[M-H]-620.1840,found 620.1838.HPLC tR＝2.27min(99.37％purity).
叔丁基4-((3，5-二氰基-4-乙基-6-((4-(N-甲基甲磺酰胺基)苄基)硫代)吡啶2-基)哌嗪-1-氨基甲酸酯(45)
白色固体,产率81.1％。1H NMR(400MHz,DMSO)δ7.46(d,J＝8.2Hz,2H),7.38(d,J＝8.2Hz,2H),4.53(s,2H),3.86(t,J＝5.2Hz,4H),3.46(s,3H),3.22(s,3H),2.93(s,3H),2.77(q,J＝7.5Hz,2H),1.42(s,9H),1.22(t,J＝7.5Hz,3H).
4-(3，5-二氰基-4-乙基-6-((4-(N-甲基甲磺酰胺基)苄基)硫代)吡啶2-基)哌嗪-1-铵盐(46)
白色固体,产率86.1％。1H NMR(400MHz,DMSO)δ9.42(s,2H),7.46(d,J＝8.0Hz,2H),7.38(d,J＝8.2Hz,2H),4.54(s,2H),4.05(q,J＝6.5Hz,4H),3.23(s,7H),2.94(s,3H),2.84–2.75(m,2H),1.24(d,J＝7.2Hz,3H).
2-(4-(3，5-二氰基-4-乙基-6-((4-(N-甲基甲基磺酰氨基)苄基)硫代)吡啶-2-基)哌嗪-1-基)-N-((四氢-2-H-吡喃-2-基)氧代)嘧啶-5-甲酰胺(47)
白色固体,产率49.2％。1H NMR(400MHz,DMSO)δ12.05(s,1H),11.56(s,1H),9.05(s,2H),7.47(d,J＝8.3Hz,2H),7.38(d,J＝8.4Hz,2H),4.96(s,1H),4.55(s,2H),4.02–3.99(m,4H),3.96(dd,J＝7.1,3.5Hz,4H),3.55(s,2H),3.21(s,3H),2.92(s,3H),2.80(q,J＝7.8Hz,2H),1.74–1.71(m,3H),1.55(s,3H),1.23(t,J＝7.6Hz,3H).
2-(4-(3，5-二氰基-4-乙基-6-((4-(N-甲基甲基磺酰氨基)苄基)硫代)吡啶-2-基)哌嗪-1-基)-N-羟基嘧啶-5-甲酰胺(48)
白色固体,产率33％,mp 170-171℃。1H NMR(400MHz,DMSO)δ8.73(s,2H),7.47(d,J＝8.4Hz,2H),7.38(d,J＝8.5Hz,2H),4.55(s,2H),4.00(dd,J＝7.2,3.5Hz,4H),3.95(dd,J＝7.1,3.6Hz,4H),3.21(s,3H),2.92(s,3H),2.80(q,J＝7.6Hz,2H),1.23(t,J＝7.6Hz,3H).13CNMR(101MHz,DMSO)δ165.14,164.47,161.72,158.70,157.58,141.27,136.09,129.79,126.81,116.41,115.57,115.12,94.78,88.00,47.19,43.34,38.19,35.54,33.62,27.58,14.01.HRMS-ESI(m/z)calc.for C27H29N9O4S2[M-H]-606.1688,found 606.1683.HPLCtR＝9.16min(99.65％purity).
实验例1：目标化合物对HDACs抑制活性
1.材料：
HDACs酶(HeLa细胞核提取物)；Boc-Lys(Ac)-AMC底物；胰酶；EDTA；LBH-589(10mM，溶于二甲基亚砜)；96孔全黑酶标板；甘油；超纯水
2.方法：
Buffer的配制：
配方为15mM Tris-HCl(pH＝8.0)，250μM EDTA，250mM NaCl，10％甘油。
Trypsin溶液的配制：
配方为10mg/mL胰酶，Buffer，2μMLBH589。
底物溶液的配制：
用DMSO溶解底物配制成30mM的储备液，然后用buffer稀释至300μM，使得
DMSO的含量为1％左右。
酶液的稀释：
用buffer将酶液按1：80的比例稀释。
化合物溶液的配制：
用buffer将化合物(待测化合物和阳性对照药SAHA)稀释成5×终浓度
100％和空白的配制与测定：
50μL buffer与10μL酶液混合，5分钟后加入40μL底物在37℃下反应0.5小时，然后加入100μL Trypsin solution终止上述反应，并在37℃下反应20分钟后在(390nm/460nm)测定荧光强度，即得100％吸收；60μL buffer加入40μL底物后在37℃下反应0.5小时加入100μL Trypsin溶液，并在37℃下反应20分钟后在(390nm/460nm)测定荧光强度，即得空白吸收。
化合物抑制HDACs活性的测定：
50μL含有药物的buffer与10μL酶液混合预先孵育5分钟，加入40μL底物后在37℃下反应0.5小时，然后再加入100μL Trypsin溶液终止上述反应，并在37℃下反应20分钟后，在(390nm/460nm)测定荧光强度，利用GraphPad Prism软件和公式计算抑制率和IC50值。(表1)
表1.目标化合物的HDACs抑制活性
aA，IC50大于5μM；B，IC50在1μM到5μM之间；C，IC50在0.2μM到1μM之间；D，IC50低于0.2μM
b未测试
实验例2：目标化合物对DNMT1抑制活性
1.材料：
MT1hypo-SFRP1-Luc细胞；96孔全黑酶标板；RPMI1640培养基；10％胎牛血清(美国Hyclone公司)；PBS缓冲液；荧光素酶检测试剂，阳性对照药SAHA和GSK5032。
2.方法：
抑制DNMT1可恢复高甲基化的SFRP1基因的表达。因SFRP1插入了荧光素酶基因，所以可通过检测荧光强度来确定其蛋白表达含量。在黑色孔板中种入MT1hypo-SFRP1-Luc细胞(每孔5400个)，置于恒温孵育箱(37℃，5％二氧化碳)中培养16小时。然后加入不同浓度的由相应细胞培养基配制的化合物稀释液，加毕，继续放入恒温孵育箱中培养72小时。最后，每孔加入荧光素酶检测试剂并用酶标仪检测相应的荧光强度。抑制活性用fold change(化合物组的荧光强度/空白对照组的荧光强度)来表示(表2)。
表2.目标化合物的DNMT1抑制活性
aA，fold change小于100；B，fold change在100到200之间；C，fold change在200到300之间；D，fold change大于300
实验例3：目标化合物抗增殖活性实验
1.材料：
MV4-11(人急性骨髓性白血病细胞)，KG-1(人急性骨髓性白血病细胞)，THP-1(人单核细胞白血病细胞)，Jurkat(人T淋巴细胞白血病细胞)，K562(人慢性骨髓性白血病细胞)，MC38(小鼠结肠癌细胞)，10％胎牛血清(美国Hyclone公司)，2.5g·L-1胰蛋白酶(美国Gibco公司)，CCK-8，改良型RPMI1640培养基，DMEM培养基(美国Hyclone公司)，阳性对照药SAHA和GSK5032，96孔板。
2.方法：
常规培养细胞，收集对数生长的细胞进行实验；用含胎牛血清10％的RPMI1640或DMEM培养基将对数生长期实体瘤细胞稀释至4×103个·mL-1(血液瘤细胞稀释至1×104个·mL-1)后接种于96孔板中(每孔加100μL)，不加细胞的作为空白孔，之后放于恒温孵育箱(37℃，5％二氧化碳)中培养8小时；加入用培养基配制的目标化合物溶液和阳性药溶液，不加药的作为100％孔，于恒温孵育箱(37℃,5％二氧化碳)中培养72小时后加入20μL CCK-8，三小时后用酶标仪于450nm波长处测定每孔的吸光度值，计算抑制率和IC50值(表3)。
表3.化合物的抗增殖活性
a A，IC50大于20μM；B，IC50在4μM到20μM之间；C，IC50在0.4μM到4μM之间；D，IC50低于0.4μM
实验例4：化合物对人急性髓细胞白血病的治疗效果
1.材料：
细胞系：人急性髓细胞白血病细胞(MV4-11)。
实验动物：六周龄BALB/c雌性裸鼠，购置于北京维通利华实验动物技术有限公司。
2.方法：
将约2×107个MV4-11细胞重悬于50μL无血清高糖培养基和50μL基质胶中，并皮下植入BALB/c裸鼠右侧腋窝区域。当肿瘤体积达到约100mm3时，开始口服给药，连续28天。治疗组剂量为30mg/kg/d，联合用药组为30mg/kg/d+30mg/kg/d，对照组给予等量生理盐水。在治疗期间每天测量肿瘤大小和小鼠体重。肿瘤体积计算公式：V(mm3)＝长(mm)×宽2(mm)/2；肿瘤生长抑制值(TGI)计算公式：TGI＝(1-治疗组肿瘤平均体积/对照组肿瘤平均体积)×100％。
3.实验结果：
测试结果见图1，目标化合物(R)-23a的肿瘤抑制率(TGI)最好为98％，抑瘤效果明显优于阳性对照药GSK5032与SAHA的联合治疗组。
实验例5：化合物对小鼠结肠癌的治疗效果
1.材料：
细胞系：小鼠结肠癌细胞(MC38)。
实验动物：六周龄BALB/c雌性裸鼠购置于北京维通利华实验动物技术有限公司。
2.方法：
将约1.2×106个MC38细胞重悬于100μL无血清高糖培养基中，并皮下植入BALB/c裸鼠右侧腋窝区域。当肿瘤体积达到约100mm3时，开始口服给药，连续14天。治疗组剂量为30mg/kg/d，联合用药组为30mg/kg/d+30mg/kg/d，对照组给予等量生理盐水。在治疗期间每天测量肿瘤大小和小鼠体重。肿瘤体积计算公式：V(mm3)＝长(mm)×宽2(mm)/2；肿瘤生长抑制值(TGI)计算公式：TGI＝(1-治疗组肿瘤平均体积/对照组肿瘤平均体积)×100％。
3.实验结果：
测试结果见图2，目标化合物(R)-23a在小鼠结肠癌模型中的肿瘤抑制率(TGI)为82％，抑瘤效果优于阳性对照药GSK5032及其与SAHA的联合治疗组。
Claims (10)
1.DNMT1-HDAC双靶点抑制剂，所述的抑制剂的结构式如式(I)所示或式(I)在药学上可接受的盐：
其中，X为芳香杂环或以下连接基团之一：
A为以下结构之一：
R1为羟基或2-氨基苯基；
R2为以下结构之一
R3为氢原子为以下结构之一：
2.根据权利要求1所述的DNMT1-HDAC双靶点抑制剂，其特征在于，抑制剂选自以下化合物其中之一：
N1-(1-(6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)-N4-羟基丁二酰胺(11a)；
N1-(1-(6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)-N5-羟基戊二酰胺(11b)；
N1-(1-(6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)-N6-羟基己二酰胺(11c)；
N1-(1-(6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)-N7-羟基庚二酰胺(11d)；
N1-(1-(6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)-N8-羟基辛二酰胺(11e)；
N1-(1-(6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)-N9-羟基壬二酰胺(11f)；
N1-(1-(6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)-N4-羟基对甲苯二酰胺(11g)；
N1-(1-(6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)-4-((4-(羟基氨甲酰基)苄基)氧基)苯甲酰胺(11h)；
N1-(1-(6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)-N4-(4-(羟基氨甲酰基)苯基)丁二酰胺(11i)；
N1-(1-(6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)-N5-(4-(羟基氨甲酰基)苯基)戊二酰胺(11j)；
N1-(1-(6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)-N6-(4-(羟基氨甲酰基)苯基)己二酰胺(11k)；
N1-(1-(6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)-N4-(4-(羟基氨甲酰基)苯基)丁二酰胺(11l)；
N1-(1-(6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)-N5-(4-(羟基氨甲酰基)苯基)戊二酰胺(11m)；
N1-(1-(6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)-N4-(4-(3-(羟基氨基)-3-氧代丙基-1-烯-1-基)苯基)丁二酰胺(11n)；
4-(((1-(6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)氨基)甲基)-N-羟基苯甲酰胺(11o)；
(E)-3-4-(((1-(6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)氨基)甲基)苯基)-N-羟基丙烯酰胺(11p)；
1-(6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)-N-(4-((4-(羟基氨甲酰基)苯基)氨甲酰基)苯基)哌啶4-铵盐(11q)；
(E)-1-(6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)-N-(4-((4-(3-羟基氨基)-3-氧代丙基-1-烯-1-基)苄基)氨甲酰基)苄基)哌啶-4-铵盐(11r)；
(E)-1-(6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)-N-(4-((4-(3-羟基氨基)-3-氧代丙基-1-烯-1-基)苄基)氧代)苄基)哌啶-4-铵盐(11s)；
1-(6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)-N-(4-((7-(羟氨基)-7-氧代庚基)氧代)苄基)哌啶-4-铵盐(11t)；
2-((1-(6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)氨基)-N-羟基嘧啶-5-甲酰胺(11u)；
2-((1-(6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)(甲基)氨基)-N-羟基嘧啶-5-甲酰胺(11v)；
2-(4-((6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)(甲基)氨基)哌啶-1-基)-N-羟基嘧啶-5-甲酰胺(15a)
2-(4-((6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)(甲基)(甲基)氨基)哌啶-1-基)-N-羟基嘧啶-5-甲酰胺(15b)；
(E)-3-(4-((4-(6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)哌嗪-1-基)甲基)苯基)-N-羟基丙烯酰胺(19a)；
2-(4-((6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)哌嗪-1-基)-N-羟基嘧啶-5-甲酰胺(19b)；
(E)-3-(4-((((1-(6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)甲基)氨基)甲基)苯基)-N-羟基丙烯酰胺(23a)；
(R，E)-3-(4-((((1-(6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)甲基)氨基)甲基)苯基)-N-羟基丙烯酰胺((R)-23a)；
(S，E)-3-(4-((((1-(6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)甲基)氨基)甲基)苯基)-N-羟基丙烯酰胺((S)-23a)；
2-(((1-(6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)甲基)氨基)-N-羟基嘧啶-5-甲酰胺(23b)；
(E)-3-(4-((4-(6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)哌嗪-1-基)甲基)苯基)-N-羟基丙烯酰胺(27)；
N1-(1-(6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)-N5-(2-氨基苯基)戊二酰胺(30a)；
N1-(1-(6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)-N4-(2-氨基苯基)对苯二甲酰胺(30b)；
4-(2-((1-(6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)氨基)-2-氧代乙基)-N-(2-氨基苯基)苯甲酰胺(30c)；
4-(((1-(6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)氨基)甲基)-N-(2-氨基苯基)苯甲酰胺(30d)；
2-((1-(6-((2-氨基-2-氧代-1-苯基乙基)硫代)-3,5-二氰基-4-乙基吡啶-2-基)哌啶-4-基)(甲基)氨基)-N-(2-氨基苯基)嘧啶-5-甲酰胺(30e)；
N1-(2-((3,5-二氰基-4-乙基-6-((4-(N-甲基甲基磺酰氨基)苯基)硫代)吡啶-2-基)(甲基)氨基)乙基)-N4-羟基丁二酰胺(40a)；
N1-(2-((3,5-二氰基-4-乙基-6-((4-(N-甲基甲基磺酰氨基)苯基)硫代)吡啶-2-基)(甲基)氨基)乙基)-N5-羟基戊二酰胺(40b)；
N1-(3-((3,5-二氰基-4-乙基-6-((4-(N-甲基甲基磺酰氨基)苯基)硫代)吡啶-2-基)(甲基)氨基)丙基)-N4-羟基丁二酰胺(40c)；
2-((2-((3,5-二氰基-4-乙基-6-((4-(N-甲基甲基磺酰氨基)苯基)硫代)吡啶-2-基)(甲基)氨基)乙基)(甲基)氨基)-N-羟基嘧啶-5-甲酰胺(40d)；
N1-(1-(3,5-二氰基-4-乙基-6-((4-(N-甲基甲基磺酰氨基)苯基)硫代)吡啶-2-基)哌啶-4-基)-N5-羟基戊二酰胺(44a)；
(E)-(1-(3,5-二氰基-4-乙基-6-((4-(N-甲基甲基磺酰氨基)苯基)硫代)吡啶-2-基)哌啶-4-基)-N5-羟基戊二酰胺(44b)；
(E)-3-(4-(((1-(3，5-二氰基-4-乙基-6-((4-(N-甲基甲基磺酰氨基)苯基)硫代)吡啶-2-基)哌啶-4-基)氨基)甲基)苯基)-N-羟基丙烯酰胺(44c)；
2-(4-(3，5-二氰基-4-乙基-6-((4-(N-甲基甲基磺酰氨基)苯基)硫代)吡啶-2-基)哌嗪-1-基)-N-羟基嘧啶-5-甲酰胺(48)。
3.权利要求2所述的DNMT1-HDAC双靶点抑制剂的制备方法，所述制备方法包括以下任一方法：
化合物11a-11V的制备方法如下：
化合物15a、15b、19a、19b的制备方法如下：
化合物23a、(R)-23a、(S)-23a、23b、27、30a-e的制备方法如下：
化合物40a-d的制备方法如下：
化合物44a-c、48的制备方法如下：
4.权利要求3所述的制备方法，其特征在于，化合物11a-11V的制备方法中，
试剂和条件如下：(a)对甲苯磺酰氯，N，N-二异丙基乙胺，4-二甲氨基吡啶，乙腈，80℃，6小时；(b)正丙醛，N-甲基吗啉，无水乙醇，室温，30小时；(c)N，N-二异丙基乙胺，N，N-二甲基甲酰胺，85℃，5小时；(d)亚硝酸异戊酯，氯化铜，乙腈，80℃，2.5小时；(e)氨基哌啶衍生物，碳酸钾，二氯甲烷，4小时；(f)4M盐酸/乙酸乙酯，室温，12小时；(g)1)9a-n,氯甲酸异丁酯，N-甲基吗啉，二氯甲烷/四氢呋喃(1：1)，室温，6小时；2)9o-t，三乙酰氧基硼氢化钠，醋酸，室温，12小时；3)9u，碳酸钾，乙腈，80℃，6小时；(h)4M盐酸/乙酸乙酯，室温，12小时。
5.权利要求3所述的制备方法，其特征在于，化合物15a、15b、19a、19b的制备方法中，
试剂和条件如下：(a)氨基哌啶衍生物，碳酸钾，二氯甲烷，4小时；(b)4M盐酸/乙酸乙酯，室温，12小时；(c)9u，碳酸钾，乙腈，80℃，6小时；(d)4M盐酸/乙酸乙酯，室温，12小时；(e)哌嗪-1-叔丁基羧酸酯，碳酸钾，二氯甲烷，4小时；(f)4M盐酸/乙酸乙酯，室温，12小时；(g)1)9p，三乙酰氧基硼氢化钠，醋酸，室温，12小时；2)9u，碳酸钾，乙腈，80℃，6小时；(h)4M盐酸/乙酸乙酯，室温，12小时。
6.权利要求3所述的制备方法，其特征在于，化合物23a、(R)-23a、(S)-23a、23b、27、30a-e的制备方法中，
试剂和条件如下：(a)哌啶-4-氨甲基叔丁基甲酸酯，碳酸钾，二氯甲烷，4小时；(b)4M盐酸/乙酸乙酯，室温，12小时；(c)1)9p，三乙酰氧基硼氢化钠，醋酸，室温，12小时；2)9u，碳酸钾，乙腈，80℃，6小时；(d)4M盐酸/乙酸乙酯，室温，12小时；(e)叔丁基(2-(甲氨基)乙基)氨基甲酸酯，碳酸钾，二氯甲烷，4小时；(f)4M盐酸/乙酸乙酯，室温，12小时；(g)9p，三乙酰氧基硼氢化钠，醋酸，室温，12小时；(h)4M盐酸/乙酸乙酯，室温，12小时；(i)1)28a-c,氯甲酸异丁酯，N-甲基吗啉，二氯甲烷/四氢呋喃(1：1)，室温，6小时；2)28d，三乙酰氧基硼氢化钠，醋酸，室温，12小时；3)28e，碳酸钾，乙腈，80℃，6小时；(j)4M盐酸/乙酸乙酯，室温，12小时。
7.权利要求3所述的制备方法，其特征在于，化合物40a-d的制备方法中，
试剂和条件如下：(a)甲磺酰氯，三乙胺，二氯甲烷，室温，4小时；(b)二异丁基氢化铝，四氢呋喃，-20℃，6小时；(c)对甲苯磺酰氯，三乙胺，DMAP，乙腈，80℃，4小时；(d)4，三乙胺，DMF，85℃，5小时；(e)亚硝酸异戊酯，氯化铜，乙腈，80℃，2.5小时；(f)2-甲氨基衍生物，碳酸钾，二氯甲烷，4小时；(g)4M盐酸/乙酸乙酯，室温，12小时；(h)1)9a or 9b,氯甲酸异丁酯，N-甲基吗啉，二氯甲烷/四氢呋喃(1：1)，室温，6小时；2)9u，碳酸钾，乙腈，80℃，6小时；(i)4M盐酸/乙酸乙酯，室温，12小时。
8.权利要求3所述的制备方法，其特征在于，化合物44a-c、48的制备方法中，
试剂和条件如下：(a)4-氨基甲酸叔丁酯，碳酸钾，二氯甲烷，4小时；(b)4M盐酸/乙酸乙酯，室温，12小时；(c)1)9b,氯甲酸异丁酯，N-甲基吗啉，二氯甲烷/四氢呋喃(1：1)，室温，6小时；2)9p，三乙酰氧基硼氢化钠，醋酸，室温，12小时；3)9u，碳酸钾，乙腈，80℃，6小时；(d)4M盐酸/乙酸乙酯，室温，12小时；(e)哌嗪-1-叔丁基羧酸酯，碳酸钾，二氯甲烷，4小时；(f)4M盐酸/乙酸乙酯，室温，12小时；(g)9u，碳酸钾，乙腈，80℃，6小时；(h)4M盐酸/乙酸乙酯，室温，12小时。
9.一种药物组合物，含有治疗有效量的权利要求1所述的DNMT1和HDAC双靶点抑制剂及药学上可接受的辅料。
10.权利要求1所述的DNMT1-HDAC双靶点抑制剂在制备治疗与靶点有关的肿瘤疾病的药物中的应用；
所述肿瘤为肺癌、肝癌、非小细胞肺癌、结肠癌、皮肤癌、胰腺癌、卵巢癌、乳腺癌、膀胱癌、淋巴瘤、食管癌、胃肠道癌、鼻咽癌、白血病或骨髓瘤。
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