CN115286583B - 一种含二苯基氨基嘧啶类化合物、制备及其作为HDACs酶抑制剂的应用 - Google Patents
一种含二苯基氨基嘧啶类化合物、制备及其作为HDACs酶抑制剂的应用 Download PDFInfo
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- CN115286583B CN115286583B CN202210959269.7A CN202210959269A CN115286583B CN 115286583 B CN115286583 B CN 115286583B CN 202210959269 A CN202210959269 A CN 202210959269A CN 115286583 B CN115286583 B CN 115286583B
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- C07D239/00—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings
- C07D239/02—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings
- C07D239/24—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members
- C07D239/28—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to ring carbon atoms
- C07D239/46—Two or more oxygen, sulphur or nitrogen atoms
- C07D239/48—Two nitrogen atoms
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
- A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
- C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
- C07D401/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a chain containing hetero atoms as chain links
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
- C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
- C07D403/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
Abstract
本发明涉及一种含二苯基氨基嘧啶类化合物、制备及其作为HDACs酶抑制剂的应用。本发明提供了一种含二苯基氨基嘧啶类化合物，结构如下式Ⅰ所示：
Description
技术领域
本发明属于组蛋白去乙酰化酶(HDACs)抑制剂化合物技术领域，具体涉及一种含二苯基氨基嘧啶类化合物、包含该类化合物的药物组合物及其作为HDACs酶抑制剂的应用。
背景技术
公开该背景技术部分的信息仅仅旨在增加对本发明的总体背景的理解，而不必然被视为承认或以任何形式暗示该信息构成已经成为本领域一般技术人员所公知的现有技术。
套细胞淋巴瘤(Mantle Cell Lymphoma，MCL)是一种具有侵袭性和不可治愈性的B细胞淋巴瘤，约占非霍奇金淋巴瘤(Non-Hodgkin’s Lymphoma，NHL)的3％～10％。多数病人诊断时处于III-IV期，男性发病率较高。染色体t(11；14)(q13；q32)易位导致细胞周期蛋白D1(Cyclin D1)过度表达是MCL发病机制的核心，此外继发性遗传改变所引起的细胞周期失调、DNA损伤修复异常、细胞凋亡、表观遗传修饰以及各种信号通路的失调，也是不容忽视的。随着对MCL的深入研究，发现B细胞受体(B cell receptor，BCR)信号通路的异常及过度活化是多种B细胞淋巴瘤生长和存活的关键因素。BTK(Bruton’s tyrosine kinase)是BCR信号通路中的关键激酶，在MCL细胞中过度表达，靶向BTK是治疗MCL的重要手段。依鲁替尼(Ibrutinib，IBN)是第一代不可逆BTK抑制剂，用于治疗复发/难治性MCL。表观遗传调节剂组蛋白去乙酰化酶(Histone deacetylases，HDACs)催化蛋白质赖氨酸残基上的乙酰化状态，从而调节染色质结构和转录活性，涉及体内多种病理生理状态。HDACs抑制剂可以抑制cyclin D1蛋白表达水平，调节抑癌蛋白p53表达水平、NF-κB活性，从而阻断PI3K/AKT/mTOR信号通路和NF-κB信号通路传导。HDACs通过使组蛋白和非组蛋白去乙酰化而在癌症中发挥关键作用，HDAC抑制剂(HDAC inhibitor，HDACI)通过调节基因转录、DNA损伤修复、细胞周期及凋亡等发挥抗肿瘤作用。因此，发现新型的HDACI单独使用或联合应用对治疗MCL具有重要意义。
发明内容
本发明提供了一种含二苯基氨基嘧啶类化合物，对HDACs酶具有一定的抑制活性和较强的抗细胞增殖活性，有效抑制套细胞淋巴瘤细胞的体外生长，部分化合物效果优于临床抗肿瘤药物；基于该技术效果，本发明提供以下技术方案：
本发明第一方面，提供一种含二苯基氨基嘧啶类化合物，所述化合物结构如下式Ⅰ所示：
其中，X独立的选自亚甲基、苄基、羰基、对位羰基哌啶基、间位羰基哌啶基、羰基吡咯烷基、嘧啶基、咪唑基或噁二唑基；
Y独立的选自异羟肟酸基、邻苯二胺甲酰基、羧基或巯基；
R1独立的选自氢、卤素、甲基、三氟甲基、甲氧基、羟基、氰基、氨基或硝基；
R2是氢、卤素、甲基、三氟甲基、甲氧基、羟基、氰基、氨基、硝基、C原子个数为1～6的烷基、C原子个数为1～6的烷氧基、C原子个数为1～6的烷基磺酰基或C原子个数为1～6的烷基亚磺酰基；
n是0～7的任意整数。
优选的，所述Y为异羟肟酸基，R1为氟，R2为甲氧乙氧基，n为0～5，所述化合物结构如下式Ⅱ所示：
其中，所述X选自亚甲基、苄基、羰基、对位羰基哌啶基、间位羰基哌啶基或羰基吡咯烷基；
具体的实施方式中，式II所示含二苯基氨基嘧啶类化合物具体选自以下化合物：
4-((3-((5-氟-2-((4-(2-甲氧基乙氧基)苯基)氨基)嘧啶-4-基)氨基)苯基)氨基)-N-羟基丁酰胺(II-1)
5-((3-((5-氟-2-((4-(2-甲氧基乙氧基)苯基)氨基)嘧啶-4-基)氨基)苯基)氨基)-N-羟基戊酰胺(II-2)
6-((3-((5-氟-2-((4-(2-甲氧基乙氧基)苯基)氨基)嘧啶-4-基)氨基)苯基)氨基)-N-羟基己酰胺(II-3)
4-(((3-((5-氟-2-((4-(2-甲氧基乙氧基)苯基)氨基)嘧啶-4-基)氨基)苯基)氨基)甲基)-N-羟基苯甲酰胺(II-4)
N1-(3-((5-氟-2-((4-(2-甲氧基乙氧基)苯基)氨基)嘧啶-4-基)氨基)苯基)-N5-羟基戊二酰胺(II-5)
N1-(3-((5-氟-2-((4-(2-甲氧基乙氧基)苯基)氨基)嘧啶-4-基)氨基)苯基)-N6-羟基己二酰胺(II-6)
N1-(3-((5-氟-2-((4-(2-甲氧基乙氧基)苯基)氨基)嘧啶-4-基)氨基)苯基)-N8-羟基辛二酰胺(II-7)
N-(3-((5-氟-2-((4-(2-甲氧基乙氧基)苯基)氨基)嘧啶-4-基)氨基)苯基)-1-(4-(羟基氨基)-4-氧丁基)哌啶-4-甲酰胺(II-8)
N-(3-((5-氟-2-((4-(2-甲氧基乙氧基)苯基)氨基)嘧啶-4-基)氨基)苯基)-1-(5-(羟基氨基)-5-氧代戊基)哌啶-4-甲酰胺(II-9)
N-(3-((5-氟-2-((4-(2-甲氧基乙氧基)苯基)氨基)嘧啶-4-基)氨基)苯基)-1-(6-(羟基氨基)-6-氧己基)哌啶-4-甲酰胺(II-10)
N-(3-(5-氟-2-((4-(2-甲氧基乙氧基)苯基)氨基)嘧啶-4-基)氨基)苯基)-1-(4-(羟基氨基)-4-氧丁基)哌啶-3-甲酰胺(II-11)
N-(3-((5-氟-2-((4-(2-甲氧基乙氧基)苯基)氨基)嘧啶-4-基)氨基)苯基)-1-(5-(羟基氨基)-5-氧代戊基)哌啶-3-甲酰胺(II-12)
N-(3-((5-氟-2-((4-(2-甲氧基乙氧基)苯基)氨基)嘧啶-4-基)氨基)苯基)-1-(6-(羟基氨基)-6-氧己基)哌啶-3-甲酰胺(II-13)
N-(3-((5-氟-2-((4-(2-甲氧基乙氧基)苯基)氨基)嘧啶-4-基)氨基)苯基)-1-(4-(羟基氨基)-4-氧丁基)吡咯烷-3-甲酰胺(II-14)
N-(3-((5-氟-2-((4-(2-甲氧基乙氧基)苯基)氨基)嘧啶-4-基)氨基)苯基)-1-(5-(羟基氨基)-5-氧代戊基)吡咯烷-3-甲酰胺(II-15)
N-(3-((5-氟-2-((4-(2-甲氧基乙氧基)苯基)氨基)嘧啶-4-基)氨基)苯基)-1-(6-(羟基氨基)-6-氧己基)吡咯烷-3-甲酰胺(II-16)。
另一优选的实施方式中，所述Y为邻苯二胺甲酰基，R1为氟，R2为甲氧乙氧基，n为0～5，所述衍生物结构如下式Ⅲ所示：
其中，所述X选自亚甲基、苄基、羰基、对位羰基哌啶基、间位羰基哌啶基或羰基吡咯烷基；
具体的实施方式中，式Ⅲ所示含二苯基氨基嘧啶类化合物具体选自以下化合物：
N-(2-氨基苯基)-6-((3-((5-氟-2-((4-(2-甲氧基乙氧基)苯基)氨基)嘧啶-4-基)氨基)苯基)氨基)己酰胺(III-1)
N-(2-氨基苯基)-4-(((3-((5-氟-2-((4-(2-甲氧基乙氧基)苯基)氨基)嘧啶-4-基)氨基)苯基)氨基)甲基)苯甲酰胺(III-2)
N1-(2-氨基苯基)-N5-(3-((5-氟-2-((4-(2-甲氧基乙氧基)苯基)氨基)嘧啶-4-基)氨基)苯基)戊二酰胺(III-3)
N1-(2-氨基苯基)-N6-(3-((5-氟-2-((4-(2-甲氧基乙氧基)苯基)氨基)嘧啶-4-基)氨基)苯基)己二酰胺(III-4)
N1-(2-氨基苯基)-N8-(3-((5-氟-2-((4-(2-甲氧基乙氧基)苯基)氨基)嘧啶-4-基)氨基)苯基)辛二酰胺(III-5)
1-(4-((2-氨基苯基)氨基)-4-氧代丁基)-N-(3-((5-氟-2-((4-(2-甲氧基乙氧基)苯基)氨基)嘧啶-4-基)氨基)苯基)哌啶-4-甲酰胺(III-6)
1-(5-((2-氨基苯基)氨基)-5-氧代戊基)-N-(3-((5-氟-2-((4-(2-甲氧基乙氧基)苯基)氨基)嘧啶-4-基)氨基)苯基)哌啶-4-甲酰胺(III-7)
1-(6-((2-氨基苯基)氨基)-6-氧代己基)-N-(3-((5-氟-2-((4-(2-甲氧基乙氧基)苯基)氨基)嘧啶-4-基)氨基)苯基)哌啶-4-甲酰胺(III-8)
1-(4-((2-氨基苯基)氨基)-4-氧代丁基)-N-(3-((5-氟-2-((4-(2-甲氧基乙氧基)苯基)氨基)嘧啶-4-基)氨基)苯基)哌啶-3-甲酰胺(III-9)
1-(5-((2-氨基苯基)氨基)-5-氧代戊基)-N-(3-((5-氟-2-((4-(2-甲氧基乙氧基)苯基)氨基)嘧啶-4-基)氨基)苯基)哌啶-3-甲酰胺(III-10)
1-(6-((2-氨基苯基)氨基)-6-氧代己基)-N-(3-((5-氟-2-((4-(2-甲氧基乙氧基)苯基)氨基)嘧啶-4-基)氨基)苯基)哌啶-3-甲酰胺(III-11)
1-(4-((2-氨基苯基)氨基)-4-氧代丁基)-N-(3-((5-氟-2-((4-(2-甲氧基乙氧基)苯基)氨基)嘧啶-4-基)氨基)苯基)吡咯烷-3-甲酰胺(III-12)
1-(5-((2-氨基苯基)氨基)-5-氧代戊基)-N-(3-((5-氟-2-((4-(2-甲氧基乙氧基)苯基)氨基)嘧啶-4基)氨基)苯基)吡咯烷-3-甲酰胺(III-13)
1-(6-((2-氨基苯基)氨基)-6-氧代己基)-N-(3-((5-氟-2-((4-(2-甲氧基乙氧基)苯基)氨基)嘧啶-4-基)氨基)苯基)吡咯烷-3-甲酰胺(III-14)。
优选的，本发明第一方面所述含二苯基氨基嘧啶类化合物除包含具有上述结构的化合物实体，还包括上述化合物药学上可接受的盐、酯、溶剂化物、互变异构体、内消旋体、外消旋体、立体异构体、代谢产物或前药；其中，所述药学上可接受的盐包括上述化合物与有机酸(如甲磺酸、甲苯磺酸、三氟乙酸等)或无机酸(如盐酸、硫酸、硝酸、氢溴酸等)形成的盐，其成盐的目的包括但不限于对上述化合物理化性质的改善，如水溶性等。
优选的，上述式Ⅰ所示含二苯基氨基嘧啶类化合物的合成路线如下：
具体合成步骤如下：
以(3-氨基苯基)氨基甲酸叔丁酯(1)为起始原料，与中间体2经取代反应得中间体3；对氟硝基苯(4)与中间体5经取代反应得到中间体6；中间体6在Pd/C催化下与氢气发生还原反应得到中间体7；中间体3和中间体7经取代反应得中间体8；中间体8在浓盐酸下脱去Boc保护基，得到中间体9；中间体9通过不同溴取代的羧酸甲酯或苄基羧酸甲酯等经亲核取代、或与N-Boc-3/4-哌啶甲酸、N-Boc-3-吡咯烷甲酸经酰胺缩合通过得中间体10；含羧酸甲酯的中间体10在碱性条件下与羟胺反应或经水解反应并进一步与邻苯二胺反应得式Ⅰ化合物。
进一步的，式Ⅱ或式Ⅲ所示含二苯基氨基嘧啶类化合物的具体制备步骤如下：
进一步的，上述式Ⅱ、式Ⅲ所示化合物的具体合成步骤如下：
(1)将原料1，原料2a，N,N-二异丙基乙胺溶于异丙醇中85～90℃油浴，加热回流反应4～5h后，减压蒸除溶剂重新加入甲醇后降温，获取析出的白色固体部分，即为中间体3a；
(2)将原料4、原料5a，KOH溶于二甲基亚砜(DMSO)中,55～65℃油浴，加热反应1.5～2.5h后冷却反应体系，并加入乙酸乙酯萃取，获取有机相部分去除溶剂得到中间体6a；
(3)将中间体6a溶于乙酸乙酯中，加入Pd/C，通入氢气，室温反应3～5h后，减压蒸除溶剂，后柱层析纯化，所述柱层析的洗脱液为石油醚：乙酸乙酯＝4～6:1，得中间体7a；
(4)将中间体3a，中间体7a溶于叔戊醇中，加入冰乙酸，95～105℃加热回流9～11h后冷却反应体系，并加入乙酸乙酯进行萃取，获取有机相部分去除溶剂，通过柱层析纯化产物，得到中间体8a，所述柱层析的洗脱液为石油醚：乙酸乙酯＝4～6:1；
(5)将中间体8a溶于乙酸乙酯中，逐滴加入浓盐酸，室温反应1.5～2.5h，析出白色固体，获取该白色固体部分洗涤并干燥得到中间体9a；
(6)将1-(叔丁氧羰基)-3-哌啶甲酸、1-(叔丁氧羰基)-4-哌啶甲酸或1-(叔丁氧羰基)-3-吡咯烷甲酸、O-苯并三氮唑-四甲基脲六氟磷酸酯(HBTU)、三乙胺，溶于N,N-二甲基甲酰胺(DMF)中，冰浴条件下搅拌0.5～1.5h，再加入中间体9a，在室温下反应10～14h后；将反应体系加入冷水中，再加入乙酸乙酯萃取，合并有机相干燥后去除溶剂，通过柱层析对产物进行纯化，得到中间体11a-11c，所述柱层析采用的洗脱液为二氯甲烷：甲醇＝90～110:1；
(7)将中间体11a-11c溶于乙酸乙酯中，逐滴加入浓盐酸，室温反应1.5～2.5h，保留反应析出的白色固体，洗涤并干燥得到中间体12a-12c；
(8)将中间体9a溶于DMF中，加入不同溴代羧酸甲酯或溴代苄基羧酸甲酯、K2CO3、KI、90～110℃加热反应30～34h；反应结束后，将反应体系加入冷水中，再加入乙酸乙酯萃取并合并有机相，干燥并去除溶剂后通过柱层析纯化得到中间体10a-10d，所述柱层析的洗脱液为二氯甲烷：甲醇＝90～110:1；
(9)将中间体9a溶于二氯甲烷中，在冰浴条件下加入三乙胺活化0.5～1.5h，再逐滴加入戊二酸单乙酯酰氯，冰浴搅拌8～12min，移至室温下反应8～12h；反应结束后加入冷水中降温，通过乙酸乙酯萃取，获取有机相部分去除溶剂并通过柱层析纯化得中间体10e，所述柱层析洗脱液为二氯甲烷：甲醇＝140～160:1；
(10)将己二酸单甲酯或辛二酸单甲酯、HBTU、三乙胺溶于DMF中，冰浴条件下搅拌20-30min，加入中间体9a，室温下反应10～14h；结束后将反应体系加入冷水中降温，通过乙酸乙酯萃取，获取有机相部分去除溶剂并通过柱层析纯化得中间体10f-10g，所述柱层析洗脱液为二氯甲烷：甲醇＝140～160:1；
(11)将中间体12a-12c，K2CO3，KI溶于DMF中，加入不同溴代羧酸甲酯，室温反应5～7h；结束后将反应体系加入冷水中降温，通过乙酸乙酯萃取，获取有机相部分去除溶剂并通过柱层析纯化得中间体10h-10p，所述柱层析洗脱液为二氯甲烷：甲醇＝25～35:1；
(12)向氢氧化钾中加入无水甲醇超声溶解，得溶液a；取盐酸羟胺加入无水甲醇超声溶解，得溶液b；将溶液a缓慢滴入溶液b中，室温反应0.5～1.5小时，过滤，滤液备用；将中间体10a-10p加入上述滤液中，室温搅拌0.5～1.5h，待反应溶液由浑浊变澄清后结束反应，去除反应体系中的溶剂，向剩余反应产物中加入蒸馏水溶解，使其呈肥皂沫样；加入盐酸调节反应体系pH为5-6，获取析出的固体洗涤、重结晶并干燥，得到目标化合物II-1至II-16；
(13)将中间体10a-10p溶于无水乙醇中，加入NaOH至溶液pH 9-10，室温反应3～4h；反应结束后，去除反应体系中的溶剂，加入盐酸调节反应产物pH至5-6使其析出固体，保留该固体部分并干燥，得到中间体13a-13n；
(14)将中间体13a-13n、HBTU、三乙胺溶于DMF中，冰浴条件下搅拌20-30min；再反应结束后，加入邻苯二胺后避光室温反应5～7h；待反应结束后将反应体系加入冷水中降温，并加入乙酸乙酯进行萃取，获取有机相部分减压蒸除溶剂并通过柱层析纯化得目标化合物Ⅲ-1至Ⅲ-14，所述柱层析洗脱液为二氯甲烷：甲醇＝8～9:1。
上述制备方法中，所述反应结束的判断依据包括反应时间、反应现象等因素，除此之外，其他本领域常规手段，如通过薄层色谱进行判断。
本发明第二方面，提供一种药物组合物，所述组合物中包括第一方面所述含二苯氨基嘧啶衍生物。
上述第二方面所称的“药物组合物”或“组合物”，可应用于受试者以实现预防、干预、改善或治疗疾病的效果，所述受试者优选为哺乳动物，如人、狗、猴、兔或鼠；所述药物组合物中，上述含二苯氨基嘧啶衍生物应当是增效有效剂量的，具体药物剂量属于于本领域技术可通过常规技术手段进行确定的技术内容。
进一步优选的方案中，所述药物组合物应用于癌症的预防、干预、改善或治疗；该优选的实施方式中，所述药物组合物中还包括其他具有抗肿瘤或辅助抗肿瘤效果的活性成分，如细胞毒类、激素类、生物反应调节剂、单克隆抗体等。
另一种优选的技术方案中，上述含二苯基氨基嘧啶衍生物可通过改善组蛋白去乙酰化程度，从而改善如依鲁替尼等药物的耐药程度；因此，该优选技术方案的一种实施方式中，所述药物组合物中还包括依鲁替尼。
另外，所述药物组合物，还含有常用的药物载体，所述药物载体包括但不限于离子交换剂(如氧化铝、硬脂酸铝、卵磷脂)、血清蛋白(如人血清蛋白)、缓冲物质(如磷酸盐，甘油，山梨酯，山梨酸钾)、饱和植物脂肪酸的部分甘油酯混合物，水，盐或电解质(如硫酸鱼精蛋白，磷酸氢二钠，磷酸氢钾，氯化钠，锌盐)、胶态氧化硅、三硅酸镁、聚乙烯吡咯烷酮、聚乙二醇、羧甲基纤维素钠，聚丙烯酸酯、蜂蜡、羊毛酯等；所述药物载体在药物组合物中的含量可以是组合物重量的1％-98％，通常大约占到80重量％。
优选的，所述药物组合物可采用以下方式施与受试者，包括口服、喷雾吸入、直肠给药、鼻腔给药、阴道给药、局部给药、非肠道给药如皮下、静脉、肌内、腹膜内、鞘内、心室内、胸骨内或颅内注射或输入，或借助一种外植的储器用药，其中优选口服、肌注、腹膜内或静脉内用药方式。
本发明第三方面，提供第一方面所述含有二苯氨基嘧啶衍生物、第二方面所述药物组合物作为HDACs酶抑制剂的应用。
优选的，所述作为HDACs酶抑制剂的应用包括但不限于以下任意一种方式：
(1)应用于制备HDACs酶作为抑制靶点的疾病治疗药物；
(2)应用于制备HDACs酶体外抑制模型；
(3)施用于受试者以降低机体中HDACs酶的表达。
上述(1)方面的应用中，所述疾病为肿瘤，具体实例如套细胞淋巴瘤。
上述(2)方面中，所述体外抑制模型包括但不限于体外细胞、组织或动物模型。
上述(3)方面中，降低机体HDACs酶表达的目的包括用于实现疾病效果或改善药物耐药。
本发明第四方面，提供一种抗肿瘤药物，所述药物中，包括活性剂量的第一方面所述含二苯氨基嘧啶衍生物、或第二方面所述药物组合物。
优选的方案中，所述含二苯基氨基嘧啶衍生物选自Ⅱ-1、Ⅱ-2、Ⅱ-3、Ⅱ-4、Ⅱ-5、Ⅱ-6、Ⅱ-7、Ⅲ-1、Ⅲ-2、Ⅲ-6、Ⅲ-8、Ⅲ-11、Ⅲ-14所示化合物。
本发明第五方面，提供一种预防或改善依鲁替尼耐药的方法，包括向有需要的受试者施用活性剂量的第一方面所述含二苯基氨基嘧啶衍生物、或第二方面所述药物组合物。
优选的，所述含二苯基氨基嘧啶衍生物选自Ⅱ-2、Ⅱ-3、Ⅱ-4、Ⅱ-7、Ⅱ-12、Ⅱ-16所示化合物；进一步的，选自Ⅱ-2、Ⅱ-3、Ⅱ-7所示化合物。
具体实施方式
应该指出，以下详细说明都是例示性的，旨在对本发明提供进一步的说明。除非另有指明，本文使用的所有技术和科学术语具有与本发明所属技术领域的普通技术人员通常理解的相同含义。
需要注意的是，这里所使用的术语仅是为了描述具体实施方式，而非意图限制根据本发明的示例性实施方式。如在这里所使用的，除非上下文另外明确指出，否则单数形式也意图包括复数形式，此外，还应当理解的是，当在本说明书中使用术语“包含”和/或“包括”时，其指明存在特征、步骤、操作、器件、组件和/或它们的组合。
为了使得本领域技术人员能够更加清楚地了解本发明的技术方案，以下将结合具体的实施例详细说明本发明的技术方案。
实施例1
1、中间体3a的制备
称取(3-氨基苯基)氨基甲酸叔丁酯(1,300mg,1.44mmol)，2,4-二氯-5-氟嘧啶(2a,288.61mg,1.73mmol),N,N-二异丙基乙胺(223.42mg,1.73mmol)置于50mL茄形瓶中，加入10mL异丙醇，90℃加热反应4.5h后，将反应液减压蒸除溶剂，加入20mL甲醇溶解后，将其倒入100mL冰水中，析出大量白色固体，过滤，滤饼用水洗涤两次，抽滤，干燥，得中间体3a；白色固体439.19mg，产率90.0％；1H NMR(400MHz,DMSO-d6)δ8.30(d,J＝3.4Hz,1H),7.81(s,1H),7.34(d,J＝8.0Hz,1H),7.24(t,J＝8.0Hz,1H),7.18(d,J＝8.2Hz,1H),1.48(s,9H)。
2、中间体6a的制备
称取对氟硝基苯(4,4.0g,28.35mmol)，乙二醇单甲醚(5a,2.59g,34.02mmol),KOH(2.39g,42.52mmol)置于250mL茄形瓶中，加入50mL DMSO，60℃加热反应2h后，反应液倒入100mL冷水中，乙酸乙酯(30mL×3)萃取，合并有机相，无水硫酸钠干燥后过滤，减压蒸除溶剂，得中间体6a；黄色晶体5.0g，产率89.4％。
3、中间体7a的制备
称取中间体6a(2.0g,10.14mmol)，Pd/C(1.0g,2.68mmol),置于250mL茄形瓶中，加入50mL乙酸乙酯溶解，通H2，室温反应4h后，反应液用硅藻土抽滤，减压蒸除溶剂，硅胶柱层析纯化石油醚：乙酸乙酯＝5:1，得中间体7a；黄色油状物1.46g，产率85.9％；1H NMR(400MHz,DMSO-d6)δ6.73–6.61(m,2H),6.56–6.45(m,2H),4.01–3.87(m,2H),3.61–3.54(m,2H),3.28(s,3H)。
4、中间体8a的制备
称取中间体3a(1.0g,2.95mmol)，中间体7a(740.37mg,4.43mmol),置于250mL茄形瓶中，加入50mL叔戊醇和5滴冰乙酸，100℃加热回流10h后，反应液倒入100mL冷水中，乙酸乙酯(30mL×3)萃取，合并有机相，无水硫酸钠干燥后过滤，减压蒸除溶剂，硅胶柱层析纯化石油醚：乙酸乙酯＝5:1得中间体8a；白色固体1.05g，产率75.5％；1H NMR(400MHz,DMSO-d6)δ8.04(s,1H),7.84(s,1H),7.53(d,J＝9.0Hz,2H),7.35(d,J＝7.5Hz,1H),7.20(d,J＝8.0Hz,1H),7.18–7.13(m,1H),6.78(d,J＝9.0Hz,2H),4.03–4.00(m,2H),3.65–3.61(m,2H),3.30(s,3H),1.46(s,9H)。
5、中间体9a的制备
称取中间体8a(500mg,1.06mmol)置于250mL茄形瓶，加入乙酸乙酯(20mL)搅拌溶解，将浓盐酸(5mL)逐滴加入茄形瓶中，室温反应2h后，有白色固体析出，抽滤，滤饼用乙酸乙酯洗，干燥，得中间体9a；白色固体417mg，产率96.5％；1H NMR(400MHz,DMSO-d6)δ8.32(s,1H),7.76(d,J＝8.2Hz,1H),7.46(t,J＝8.1Hz,1H),7.39(d,J＝8.8Hz,2H),7.20(d,J＝7.8Hz,1H),7.18–7.13(m,H),6.96(d,J＝8.9Hz,2H),4.12–4.07(m,2H),3.68–3.63(m,2H),3.31(s,3H)。
6、中间体11的制备
分别称取1-(叔丁氧羰基)-3-哌啶甲酸、1-(叔丁氧羰基)-4-哌啶甲酸或1-(叔丁氧羰基)-3-吡咯烷甲酸(1.5eq)，HBTU(1.5eq)，三乙胺(3.0eq)，置于100mL茄形瓶中，加入DMF(10mL)溶解，冰浴条件下搅拌1h后再加入中间体9a(1.0eq)，室温反应12h。将反应液倾倒入冷水(20mL)，乙酸乙酯(10mL×3)萃取，合并有机相，无水硫酸钠干燥后过滤，减压蒸除溶剂，硅胶柱层析纯化二氯甲烷：甲醇＝100:1，得中间体11a-11c。
7、中间体12的制备
称取上步中间体11a-11c置于250mL茄形瓶，加入乙酸乙酯(20mL)搅拌溶解，将浓盐酸(5mL)逐滴加入茄形瓶中，室温反应2h后析出白色固体，抽滤，乙酸乙酯洗滤饼，干燥，得中间体12a-12c。
8、中间体10的制备
(1)中间体10a-10d的制备
称取中间体9a(0.47mmol)，K2CO3(2.96mmol),KI(0.37mmol)于100mL茄形瓶中，加入DMF(10mL)搅拌溶解，将4-溴丁酸甲酯/5-溴戊酸甲酯/6-溴己酸甲酯/4-溴甲基苯基甲酸甲酯(0.89mmol)逐滴加入茄形瓶中，100℃加热反应32h。将反应液倾倒入冷水(20mL)，乙酸乙酯(10mL×3)萃取，合并有机相，无水硫酸钠干燥后过滤，减压蒸除溶剂，硅胶柱层析纯化二氯甲烷：甲醇＝100:1，得中间体10a-10d。
(2)中间体10e的制备
称取中间体9a(300mg,0.74mmol)于100mL茄形瓶中，加入二氯甲烷(10mL)搅拌溶解，在冰浴条件下加入三乙胺(2mL)活化1h，将戊二酸单乙酯酰氯(158.43mg,0.89mmol)逐滴加入茄形瓶中，冰浴搅拌10min，移至室温下反应10h。将反应液倾倒入冷水(20mL)，乙酸乙酯(10mL×3)萃取，合并有机相，无水硫酸钠干燥后过滤，减压蒸除溶剂，硅胶柱层析纯化二氯甲烷：甲醇＝150:1，得中间体10e。
(3)中间体10f-10g的制备
分别称取己二酸单甲酯/辛二酸单甲酯(1.11mmol)、HBTU(1.11mmol)、三乙胺(2.22mmol)，依次加入到100mL茄形瓶中，加入DMF(5-10mL)溶解，冰浴条件下搅拌20-30min；再加入中间体9a(0.74mmol)，室温反应12h。将反应液倾倒入冷水(20mL)，乙酸乙酯(10mL×3)萃取，合并有机相，无水硫酸钠干燥后过滤，减压蒸除溶剂，硅胶柱层析纯化二氯甲烷：甲醇＝100:1，得中间体10f-10g。
(4)中间体10h-10p的制备
分别称取中间体12a-12c(1.0eq)，K2CO3(4eq),KI(0.5eq)于100mL茄形瓶中，加入DMF(10mL)搅拌溶解，将将4-溴丁酸甲酯/5-溴戊酸甲酯/6-溴己酸甲酯(1.2eq)逐滴加入茄形瓶中，室温下反应6h。将反应液倾倒入冷水(20mL)，乙酸乙酯(10mL×3)萃取，合并有机相，无水硫酸钠干燥后过滤，减压蒸除溶剂，硅胶柱层析纯化二氯甲烷：甲醇＝30:1，得中间体10h-10p。
9、目标化合物Ⅱ-1至Ⅱ-16的制备
取氢氧化钾(KOH,2.83g,50.40mmol)于25ml茄形瓶，加入7ml无水甲醇超声溶解，得溶液a；称取盐酸羟胺(2.33g,33.5mmol)于25ml茄形瓶，加入12ml无水甲醇超声溶解，得溶液b。将溶液a缓慢滴入溶液b中，室温反应1小时，过滤，滤液备用。称取中间体10a-10p(0.50mmol)于25ml茄形瓶中，加入上述得到的滤液(4-6mL)，室温搅拌1h后溶液由浑浊变澄清。减压蒸除溶剂，反应液加入蒸馏水(5-6mL)溶解呈肥皂沫样，再用1M的盐酸调pH至5-6，析出固体，过滤，滤饼用乙酸乙酯洗三次，后用甲醇重结晶，干燥，得目标化合物Ⅱ-1至Ⅱ-16。
((3-((5-氟-2-((4-(2-甲氧基乙氧基)苯基)氨基)嘧啶-4-基)氨基)苯基)氨基)-N-羟基丁酰胺(化合物Ⅱ-1)
白色固体55mg，产率36.6％；Mp:120-122℃；1H NMR(400MHz,MeOD)δ7.82(d,J＝3.9Hz,1H),7.44(d,J＝8.9Hz,2H),7.07–7.01(m,2H),6.90(d,J＝8.6Hz,1H),6.85(d,J＝8.9Hz,2H),6.41(d,J＝7.8Hz,1H),4.10–4.06(m,2H),3.76–3.71(m,2H),3.43(s,3H),3.06(t,J＝6.9Hz,2H),2.17(t,J＝7.4Hz,2H),1.93–1.84(m,2H)；13C NMR(100MHz,DMSO-d6)δ169.48(s),156.20(d,J＝2.5Hz),153.47(s),150.31(d,J＝10.7Hz),149.66(s),142.11(s),140.64(d,J＝19.8Hz),139.85(d,J＝36.6Hz),134.72(s),129.17(s),120.85(s,2C),114.64(s,2C),109.92(s),108.16(s),105.64(s),70.98(s),67.49(s),58.63(s),42.98(s),30.47(s),25.31(s)；MS(ESI):calcd for C23H27FN6O4[M+H]+471.21,found 471.19。
5-((3-((5-氟-2-((4-(2-甲氧基乙氧基)苯基)氨基)嘧啶-4-基)氨基)苯基)氨基)-N-羟基戊酰胺(化合物Ⅱ-2)
白色固体71mg，产率80.5％；Mp:130-132℃；1H NMR(400MHz,DMSO-d6)δ8.01(d,J＝3.7Hz,1H),7.56(d,J＝8.9Hz,2H),7.04(d,J＝8.3Hz,1H),7.02–6.97(m,1H),6.89(s,1H),6.80(d,J＝8.9Hz,2H),6.31(d,J＝7.3Hz,1H),4.05–3.99(m,2H),3.68–3.60(m,2H),3.32(s,3H),2.95(t,J＝5.7Hz,2H),2.02–1.94(m,2H),1.61–1.47(m,4H)；13C NMR(100MHz,DMSO-d6)δ169.36(s),156.23(d,J＝2.5Hz),153.50(s),150.33(d,J＝10.6Hz),149.79(s),142.13(s),140.62(d,J＝20.4Hz),139.85(d,J＝34.8Hz),134.76(s),129.13(s),120.86(s，2C),114.69(s,2C),109.80(s),108.16(s),105.62(s),71.01(s),67.56(s),58.59(s),43.13(s),32.55(s),28.82(s),23.43(s)；HRMS(ESI):calcd for C24H29FN6O4[M+H]+485.2307,found 485.2312。
6-((3-((5-氟-2-((4-(2-甲氧基乙氧基)苯基)氨基)嘧啶-4-基)氨基)苯基)氨基)-N-羟基己酰胺(化合物Ⅱ-3)
白色固体85mg，产率84.8％；Mp:154-156℃；1H NMR(400MHz,DMSO-d6)δ8.00(d,J＝3.8Hz,1H),7.54(d,J＝9.0Hz,2H),7.01(d,J＝5.8Hz,2H),6.89(s,1H),6.81(d,J＝9.1Hz,2H),6.32(d,J＝6.2Hz,1H),4.05–4.00(m,2H),3.65(m,J＝4.6Hz,2H),3.31(s,3H),2.94(t,J＝7.0Hz,2H),1.96(t,J＝7.3Hz,2H),1.57–1.48(m,4H),1.36–1.27(m,2H)；13C NMR(100MHz,DMSO-d6)δ169.51(s),156.20(d,J＝2.8Hz),153.46(s),150.29(d,J＝10.7Hz),149.80(s),142.09(s),140.69(d,J＝18.8Hz),139.82(d,J＝36.5Hz),134.71(s),129.14(s),120.80(s,2C),114.64(s,2C),109.80(s),108.22(s),105.52(s),70.99(s),67.49(s),58.64(s),43.36(s),32.79(s),29.01(s),26.86(s),25.51(s)；HRMS(ESI):calcd forC25H31FN6O4[M+H]+499.2463,found 499.2463。
4-(((3-((5-氟-2-((4-(2-甲氧基乙氧基)苯基)氨基)嘧啶-4-基)氨基)苯基)氨基)甲基)-N-羟基苯甲酰胺(化合物Ⅱ-4)
白色固体18mg，产率22.5％；Mp:154-156℃；1H NMR(400MHz,DMSO-d6)δ8.00(d,J＝3.7Hz,1H),7.87(d,J＝11.1Hz,1H),7.68(d,J＝8.2Hz,2H),7.55(d,J＝8.9Hz,2H),7.42(t,J＝9.6Hz,2H),7.05–7.00(m,1H),6.97(d,J＝11.3Hz,1H),6.81(d,J＝9.0Hz,2H),6.31(d,J＝8.3Hz,1H),4.28(s,2H),4.04–3.97(m,2H),3.65–3.59(m,2H),3.29(s,3H)；13C NMR(100MHz,DMSO-d6)δ167.11(s),156.21(d,J＝2.6Hz),153.53(s),150.28(d,J＝10.1Hz),149.47(s),142.11(s),140.69(d,J＝19.8Hz),139.82(d,J＝32.0Hz),138.77(s),135.10(s),134.74(s),129.31(s,2C),129.14(s),127.54(s,2C),120.87(s,2C),114.73(s,2C),110.22(s),108.36(s),106.05(s),71.00(s),67.55(s),58.63(s),46.83(s)；HRMS(ESI):calcd for C27H27FN6O4[M+H]+519.2150,found 519.2147。
N1-(3-((5-氟-2-((4-(2-甲氧基乙氧基)苯基)氨基)嘧啶-4-基)氨基)苯基)-N5-羟基戊二酰胺(化合物Ⅱ-5)
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白色固体145mg，产率74.4％；Mp:202-204℃；1H NMR(400MHz,DMSO-d6)δ8.05(s,1H),7.97(s,1H),7.54(d,J＝8.4Hz,2H),7.45(d,J＝7.6Hz,1H),7.37(d,J＝7.3Hz,1H),7.28(t,J＝7.9Hz,1H),6.81(d,J＝8.4Hz,2H),4.08–4.02(m,2H),3.69–3.63(m,2H),3.33(s,3H),2.40–2.32(m,2H),2.07(t,J＝7.1Hz,2H),1.90–1.79(m,2H)；13C NMR(100MHz,DMSO-d6)δ171.32(s),169.24(s),156.09(d,J＝2.5Hz),153.43(s),150.17(d,J＝10.7Hz),142.07(s),140.91(d,J＝19.2Hz),139.99(s),139.65(d,J＝6.8Hz),134.76(s),128.88(s),120.67(s,2C),116.79(s),114.81(s),114.60(s,2C),113.09(s),70.99(s),67.50(s),58.63(s),36.15(s),32.18(s),21.77(s)；HRMS(ESI):calcd for C24H27FN6O5[M+H]+499.2099,found 499.2114。
N1-(3-((5-氟-2-((4-(2-甲氧基乙氧基)苯基)氨基)嘧啶-4-基)氨基)苯基)-N6-羟基己二酰胺(化合物Ⅱ-6)
白色固体40mg，产率79.4％；Mp:174-176℃；1H NMR(400MHz,DMSO-d6)δ8.03(d,J＝3.6Hz,1H),7.98(s,1H),7.54(d,J＝8.8Hz,2H),7.42(d,J＝7.7Hz,1H),7.35(d,J＝7.8Hz,1H),7.23(t,J＝8.1Hz,1H),6.77(d,J＝8.9Hz,2H),4.04–3.97(m,2H),3.64–3.61(m,2H),3.30(s,3H),2.31(t,J＝6.2Hz,2H),1.98(t,J＝6.4Hz,2H),1.59–1.47(m,4H)；13C NMR(100MHz,DMSO-d6)δ171.61(s),169.37(s),156.11(d,J＝2.8Hz),153.44(s),150.17(d,J＝10.8Hz),142.17(d,J＝18.7Hz),140.89(d,J＝20.5Hz),140.05(s),139.65(s),134.81(s),128.86(s),120.65(s,2C),116.74(s),114.90(s),114.63(s,2C),113.06(s),71.01(s),67.54(s),58.64(s),36.58(s),32.57(s),25.32(s,2C)；HRMS(ESI):calcd forC25H29FN6O5[M+H]+513.2256,found 513.2263。
N1-(3-((5-氟-2-((4-(2-甲氧基乙氧基)苯基)氨基)嘧啶-4-基)氨基)苯基)-N8-羟基辛二酰胺(化合物Ⅱ-7)
白色固体40mg，产率49.9％；Mp:180-182℃；1H NMR(400MHz,DMSO-d6)δ8.05(d,J＝3.3Hz,1H),7.86(s,1H),7.53(d,J＝8.7Hz,2H),7.48(d,J＝7.3Hz,1H),7.30(d,J＝7.9Hz,1H),7.24(t,J＝7.9Hz,1H),6.77(d,J＝8.7Hz,2H),4.04–3.99(m,2H),3.66–3.61(m,2H),3.31(s,3H),2.30(t,J＝7.2Hz,2H),1.94(t,J＝7.3Hz,2H),1.62–1.53(m,2H),1.53–1.44(m,2H),1.35–1.22(m,4H)；13C NMR(100MHz,DMSO-d6)δ171.62(s),169.54(s),156.16(d,J＝2.1Hz),153.47(s),150.24(d,J＝10.9Hz),142.03(s),141.19(d,J＝19.0Hz),139.85(s),139.53(d,J＝10.6Hz),134.63(s),128.94(s),120.73(s,2C),117.33(s),114.98(s),114.60(s,2C),113.65(s),70.99(s),67.48(s),58.64(s),36.84(s),32.72(s),28.92(s,2C),25.52(s,2C)；HRMS(ESI):calcd for C27H33FN6O5[M+H]+541.2569,found 541.2583。
N-(3-((5-氟-2-((4-(2-甲氧基乙氧基)苯基)氨基)嘧啶-4-基)氨基)苯基)-1-(4-(羟基氨基)-4-氧丁基)哌啶-4-甲酰胺(化合物Ⅱ-8)
白色固体89mg，产率88.8％；Mp:158-160℃；1H NMR(400MHz,DMSO-d6)δ8.05(d,J＝3.5Hz,1H),7.90(s,1H),7.54(d,J＝8.8Hz,2H),7.45(d,J＝7.3Hz,1H),7.33(d,J＝8.0Hz,1H),7.23(t,J＝8.1Hz,1H),6.76(d,J＝8.9Hz,2H),4.04–3.98(m,2H),3.66–3.60(m,2H),3.31(s,3H),2.93–2.84(m,2H),2.36–2.27(m,1H),2.23(t,J＝6.9Hz,2H),1.97(t,J＝7.4Hz,2H),1.86(t,J＝10.8Hz,2H),1.77–1.70(m,2H),1.68–1.59(m,4H)；13C NMR(100MHz,DMSO-d6)δ174.09(s),169.58(s),156.15(d,J＝2.3Hz),153.46(s),150.24(d,J＝10.6Hz),142.04(s),141.10(d,J＝19.4Hz),139.98(s),139.55(d,J＝9.1Hz),134.72(s),128.89(s),120.66(s,2C),117.20(s),114.97(s),114.65(s,2C),113.66(s),71.01(s),67.55(s),58.65(s),57.96(s),53.21(s,2C),43.45(s),30.74(s),29.02(s,2C),23.02(s)；MS(ESI):calcd for C29H36FN7O5[M+H]+582.28,found 582.26。
N-(3-((5-氟-2-((4-(2-甲氧基乙氧基)苯基)氨基)嘧啶-4-基)氨基)苯基)-1-(5-(羟基氨基)-5-氧代戊基)哌啶-4-甲酰胺(化合物Ⅱ-9)
白色固体80mg，产率79.9％；Mp:165-166℃；1H NMR(400MHz,MeOD)δ7.98(s,1H),7.86(d,J＝3.8Hz,1H),7.44(d,J＝8.9Hz,2H),7.36(d,J＝7.7Hz,1H),7.31(d,J＝8.3Hz,1H),7.25(t,J＝8.0Hz,1H),6.81(d,J＝8.9Hz,2H),4.09–4.04(m,2H),3.75–3.69(m,2H),3.42(s,3H),3.15(d,J＝12.1Hz,2H),2.58–2.50(m,2H),2.48–2.39(m,1H),2.33–2.22(m,2H),2.14(t,J＝6.8Hz,2H),1.96–1.84(m,4H),1.68–1.54(m,4H)；13C NMR(100MHz,DMSO-d6)δ173.88(s),169.51(s),156.06(d,J＝2.5Hz),153.36(s),150.14(d,J＝10.6Hz),142.03(s),140.91(d,J＝18.1Hz),140.07(s),139.61(d,J＝5.8Hz),134.82(s),128.88(s),120.77(s),120.51(s,2C),116.72(s),114.59(s,2C),113.16(s),71.00(s),67.49(s),58.64(s),57.75(s),52.82(s),42.70(s),34.12(s),32.51(s),28.41(s),25.74(s),23.46(s),22.92(s)；MS(ESI):calcd for C30H38FN7O5[M+H]+596.29,found 596.13。
N-(3-((5-氟-2-((4-(2-甲氧基乙氧基)苯基)氨基)嘧啶-4-基)氨基)苯基)-1-(6-(羟基氨基)-6-氧己基)哌啶-4-甲酰胺(化合物Ⅱ-10)
白色固体70mg，产率69.9％；Mp:78-80℃；1H NMR(400MHz,DMSO-d6)δ8.05(d,J＝3.6Hz,1H),7.94(s,1H),7.55(d,J＝8.8Hz,2H),7.43(d,J＝7.5Hz,1H),7.35(d,J＝8.1Hz,1H),7.23(t,J＝8.0Hz,1H),6.77(d,J＝8.9Hz,2H),4.04–3.98(m,2H),3.67–3.60(m,2H),3.31(s,3H),2.91(d,J＝10.3Hz,2H),2.39–2.29(m,1H),2.28–2.19(m,2H),1.94(t,J＝7.2Hz,2H),1.91–1.81(m,2H),1.77–1.70(m,2H),1.69–1.59(m,2H),1.51(dd,J＝15.2,7.6Hz,2H),1.41(dd,J＝9.0,8.5Hz,2H),1.29–1.20(m,2H)；13C NMR(100MHz,DMSO-d6)δ174.10(s),169.56(s),156.10(d,J＝2.5Hz),153.39(s),150.18(d,J＝10.9Hz),142.02(s),141.04(d,J＝22.4Hz),139.78(d,J＝49.5Hz),134.75(s),132.00(s),128.89(s),120.56(s,2C),117.00(s),114.81(s),114.59(s,2C),113.44(s),71.00(s),67.49(s),65.50(s),58.65(s),53.27(s),43.37(s),32.73(s),30.48(s),28.96(s),27.04(s),25.57(s),19.12(s),14.02(s)；MS(ESI):calcd for C31H40FN7O5[M+H]+610.31,found 610.13。
N-(3-(5-氟-2-((4-(2-甲氧基乙氧基)苯基)氨基)嘧啶-4-基)氨基)苯基)-1-(4-(羟基氨基)-4-氧丁基)哌啶-3-甲酰胺(化合物Ⅱ-11)
白色固体75mg，产率74.9％；Mp:141-142℃；1H NMR(400MHz,MeOD)δ7.96(s,1H),7.87(d,J＝3.9Hz,1H),7.44(d,J＝9.0Hz,2H),7.38(d,J＝8.0Hz,1H),7.33(d,J＝8.4Hz,1H),7.26(t,J＝8.0Hz,1H),6.81(d,J＝9.0Hz,2H),4.10–4.04(m,2H),3.76–3.69(m,2H),3.42(s,3H),3.02(d,J＝12.1Hz,1H),2.96–2.87(m,1H),2.75–2.65(m,1H),2.64–2.52(m,3H),2.46–2.35(m,1H),2.16(t,J＝7.1Hz,2H),1.97–1.93(m,1H),1.91(d,J＝6.8Hz,1H),1.89–1.84(m,1H),1.84–1.79(m,1H),1.76–1.63(m,2H),1.36–1.27(m,1H)；13C NMR(100MHz,DMSO-d6)δ172.60(s),169.39(s),156.12(d,J＝2.5Hz),153.44(s),150.20(d,J＝10.7Hz),142.05(s),141.10(d,J＝19.2Hz),139.74(d,J＝8.4Hz),139.57(s),134.72(s),128.94(s),120.62(s,2C),117.26(s),114.92(s),114.63(s,2C),113.56(s),71.01(s),67.52(s),58.65(s),57.76(s),55.81(s),53.29(s),43.31(s),30.62(s),27.54(s),24.29(s),22.46(s)；HRMS(ESI):calcd for C29H36FN7O5[M+H]+582.2834,found 582.2863。
N-(3-((5-氟-2-((4-(2-甲氧基乙氧基)苯基)氨基)嘧啶-4-基)氨基)苯基)-1-(5-(羟基氨基)-5-氧代戊基)哌啶-3-甲酰胺(化合物Ⅱ-12)
白色固体80mg，产率79.9％；Mp:78-80℃；1H NMR(400MHz,MeOD)δ7.95(s,1H),7.87(d,J＝3.9Hz,1H),7.45(d,J＝9.0Hz,2H),7.35(dd,J＝16.9,4.0Hz,2H),7.26(t,J＝8.0Hz,1H),6.81(d,J＝9.0Hz,2H),4.08–4.05(m,2H),3.75–3.70(m,2H),3.42(s,3H),2.96(d,J＝9.0Hz,1H),2.90–2.81(m,1H),2.76–2.65(m,1H),2.61–2.50(m,2H),2.11(t,J＝6.8Hz,2H),1.98–1.87(m,2H),1.87–1.76(m,1H),1.76–1.67(m,2H),1.67–1.55(m,4H),1.35–1.27(m,1H)；13C NMR(100MHz,DMSO-d6)δ172.71(s),169.48(s),156.13(d,J＝2.4Hz),153.44(s),150.21(d,J＝10.4Hz),142.02(s),141.14(d,J＝16.6Hz),139.66(d,J＝22.0Hz),139.61(s),134.71(s),128.96(s),120.62(s,2C),117.33(s),114.96(s),114.62(s,2C),113.62(s),71.01(s),67.51(s),58.65(s),57.97(s),56.07(s),53.35(s),43.61(s),32.53(s),27.56(s),25.98(s),24.35(s),23.49(s)；MS(ESI):calcd forC30H38FN7O5[M+H]+596.29,found 596.13。
N-(3-((5-氟-2-((4-(2-甲氧基乙氧基)苯基)氨基)嘧啶-4-基)氨基)苯基)-1-(6-(羟基氨基)-6-氧己基)哌啶-3-甲酰胺(化合物Ⅱ-13)
白色固体200mg，产率79.9％；Mp:78-80℃；1H NMR(400MHz,MeOD)δ7.94(s,1H),7.87(d,J＝3.8Hz,1H),7.45(d,J＝8.9Hz,2H),7.36(t,J＝7.9Hz,2H),7.26(t,J＝8.0Hz,1H),6.80(d,J＝8.9Hz,2H),4.08–4.03(m,2H),3.75–3.69(m,2H),3.42(s,3H),2.91(d,J＝8.7Hz,1H),2.85–2.74(m,1H),2.73–2.62(m,1H),2.58–2.49(m,1H),2.45(t,J＝17.2Hz,2H),2.34(d,J＝27.4Hz,1H),2.08(t,J＝7.3Hz,2H),1.94–1.85(m,1H),1.84–1.74(m,1H),1.68(dd,J＝17.0,8.4Hz,2H),1.64–1.59(m,2H),1.55(dd,J＝14.4,9.3Hz,2H),1.33(dt,J＝15.9,7.9Hz,2H)；13C NMR(100MHz,DMSO-d6)δ170.78(s),169.54(s),156.12(d,J＝2.7Hz),153.43(s),150.20(d,J＝10.8Hz),142.04(s),141.11(d,J＝18.8Hz),139.81(s),139.57(d,J＝3.2Hz),134.74(s),128.93(s),120.58(s,2C),117.21(s),114.90(s),114.61(s,2C),113.55(s),71.01(s),67.52(s),60.21(s),58.65(s),58.41(s),53.42(s),32.70(s),27.62(s),26.99(s),26.22(s),25.51(s),21.22(s),14.55(s)；MS(ESI):calcdfor C31H40FN7O5[M+H]+610.31,found 610.17。
N-(3-((5-氟-2-((4-(2-甲氧基乙氧基)苯基)氨基)嘧啶-4-基)氨基)苯基)-1-(4-(羟基氨基)-4-氧丁基)吡咯烷-3-甲酰胺(化合物Ⅱ-14)
白色固体78mg，产率77.9％；Mp:74-76℃；1H NMR(400MHz,MeOD)δ8.00(s,1H),7.87(d,J＝3.9Hz,1H),7.44(d,J＝9.0Hz,2H),7.36(d,J＝7.9Hz,1H),7.32(d,J＝8.3Hz,1H),7.27(d,J＝8.0Hz,1H),6.81(d,J＝9.0Hz,2H),4.08–4.05(m,2H),3.74–3.70(m,2H),3.42(s,3H),3.20–3.14(m,1H),3.13–3.06(m,1H),2.95–2.84(m,2H),2.80–2.73(m,1H),2.71–2.63(m,2H),2.25–2.21(m,1H),2.16(t,J＝7.4Hz,2H),2.13–2.07(m,1H),1.90–1.83(m,2H)；13C NMR(100MHz,DMSO-d6)δ172.77(s),169.37(s),156.13(d,J＝2.7Hz),153.45(s),150.20(d,J＝10.9Hz),142.04(s),141.12(d,J＝19.7Hz),139.81(s),139.62(d,J＝5.6Hz),134.71(s),128.96(s),120.65(s,2C),117.27(s),114.93(s),114.62(s,2C),113.59(s),71.01(s),67.52(s),58.65(s),57.29(s),55.10(s),53.94(s),43.76(s),30.60(s),28.39(s),24.37(s)；MS(ESI):calcd for C28H34FN7O5[M+H]+568.26,found568.18。
N-(3-((5-氟-2-((4-(2-甲氧基乙氧基)苯基)氨基)嘧啶-4-基)氨基)苯基)-1-(5-(羟基氨基)-5-氧代戊基)吡咯烷-3-甲酰胺(化合物Ⅱ-15)
白色固体65mg，产率72.1％；Mp:77-79℃；1H NMR(400MHz,MeOD)δ8.00(s,1H),7.87(d,J＝3.9Hz,1H),7.44(d,J＝9.0Hz,2H),7.36(d,J＝8.1Hz,1H),7.32(d,J＝8.3Hz,1H),7.26(t,J＝8.0Hz,1H),6.81(d,J＝9.0Hz,2H),4.09–4.05(m,2H),3.75–3.70(m,2H),3.42(s,3H),3.22–3.12(m,2H),3.01–2.91(m,2H),2.87–2.79(m,1H),2.77–2.67(m,2H),2.30–2.18(m,2H),2.13(t,J＝6.9Hz,2H),1.77–1.68(m,1H),1.68–1.57(m,3H),1.35–1.27(m,1H)；13C NMR(100MHz,DMSO-d6)δ173.25(s),169.57(s),156.09(d,J＝2.9Hz),153.41(s),150.16(d,J＝10.8Hz),142.06(s),140.92(d,J＝18.8Hz),140.05(s),139.64(d,J＝7.0Hz),134.85(s),128.88(s),120.56(s,2C),116.76(s),114.70(s),114.63(s,2C),113.18(s),71.02(s),67.55(s),60.21(s),58.65(s),57.69(s),55.44(s),54.12(s),43.89(s),32.60(s),28.32(s),23.60(s)；MS(ESI):calcd for C29H36FN7O5[M+H]+582.28,found 582.17。
N-(3-((5-氟-2-((4-(2-甲氧基乙氧基)苯基)氨基)嘧啶-4-基)氨基)苯基)-1-(6-(羟基氨基)-6-氧己基)吡咯烷-3-甲酰胺(化合物Ⅱ-16)
白色固体55mg，产率73.2％；Mp:78-80℃；1H NMR(400MHz,MeOD)δ8.02(s,1H),7.87(d,J＝3.7Hz,1H),7.45(d,J＝8.8Hz,2H),7.37–7.30(m,2H),7.26(t,J＝7.9Hz,1H),6.80(d,J＝8.8Hz,2H),4.08–4.05(m,2H),3.75–3.70(m,2H),3.42(s,3H),3.25–3.16(m,2H),3.08–2.97(m,2H),2.90(dd,J＝16.5,8.3Hz,1H),2.82–2.70(m,2H),2.32–2.20(m,1H),2.20–2.13(m,1H),2.10(t,J＝7.3Hz,2H),1.69–1.57(m,4H),1.43–1.33(m,2H)；13CNMR(100MHz,DMSO-d6)δ172.51(s),169.50(s),156.10(d,J＝2.6Hz),153.42(s),150.17(d,J＝10.6Hz),142.02(s),141.14(d,J＝17.4Hz),139.67(d,J＝15.3Hz),139.39(s),134.71(s),129.00(s),120.60(s,,2C),117.27(s),114.89(s),114.59(s,2C),113.55(s),71.00(s),67.49(s),58.65(s),57.09(s),55.38(s),53.94(s),43.63(s),32.65(s),28.31(s),27.61(s),26.79(s),25.41(s)；MS(ESI):calcd for C30H38FN7O5[M+H]+596.29,found596.17。
10、中间体13的制备
称取中间体10a-10p置于250mL茄形瓶，加入适量无水乙醇搅拌溶解，加入3M NaOH至溶液pH 9-10，室温反应3.5h。减压蒸除溶剂，用1M HCl调pH 5-6析出固体，过滤，保留滤饼，干燥，得中间体13a-13n。
11、目标化合物Ⅲ-1至Ⅲ-14的制备
分别称取中间体13a-13n(1.0eq)、HBTU(1.2eq)、三乙胺(3.0eq)，依次加入到100mL茄形瓶中，加入DMF(5～10mL)溶解，冰浴条件下搅拌20-30min。加入邻苯二胺(1.2eq)，避光室温反应6h。将反应液倾倒入冷水(20mL)，乙酸乙酯(10mL×3)萃取，合并有机相，无水硫酸钠干燥后过滤，减压蒸除溶剂，硅胶柱层析纯化二氯甲烷：甲醇＝10:1，减压蒸除溶剂，干燥，得目标化合物Ⅲ-1至Ⅲ-14。
N-(2-氨基苯基)-6-((3-((5-氟-2-((4-(2-甲氧基乙氧基)苯基)氨基)嘧啶-4-基)氨基)苯基)氨基)己酰胺(化合物Ⅲ-1)
白色固体130mg，产率57.7％；Mp:117-118℃；1H NMR(400MHz,DMSO-d6)δ7.99(d,J＝3.7Hz,1H),7.53(d,J＝8.9Hz,2H),7.13(d,J＝7.6Hz,1H),7.00(d,J＝6.0Hz,2H),6.90(t,J＝6.9Hz,2H),6.80(d,J＝8.9Hz,2H),6.72(d,J＝7.9Hz,1H),6.54(t,J＝7.5Hz,1H),6.32(d,J＝6.1Hz,1H),4.03–3.99(m,2H),3.64–3.61(m,2H),3.29(s,3H),2.96(t,J＝7.0Hz,2H),2.32(t,J＝7.4Hz,2H),1.66–1.52(m,4H),1.43–1.34(m,2H)；13C NMR(100MHz,DMSO-d6)δ171.58(s),156.23(d,J＝2.8Hz),153.51(s),150.33(d,J＝10.6Hz),149.83(s),142.22(d,J＝18.9Hz),140.68(d,J＝19.4Hz),140.02(s),139.68(s),134.75(s),129.14(s),126.13(s),125.74(s),124.12(s),120.86(s,2C),116.67(s),116.38(s),114.70(s,2C),109.85(s),108.25(s),105.63(s),71.01(s),67.56(s),58.64(s),43.41(s),36.31(s),29.12(s),26.95(s),25.69(s)；MS(ESI):calcd for C31H36FN7O3[M+H]+574.29,found 574.26。
N-(2-氨基苯基)-4-(((3-((5-氟-2-((4-(2-甲氧基乙氧基)苯基)氨基)嘧啶-4-基)氨基)苯基)氨基)甲基)苯甲酰胺(化合物Ⅲ-2)
白色固体103mg，产率64.7％；Mp:192-194℃；1H NMR(400MHz,DMSO-d6)δ7.69(d,J＝3.6Hz,1H),7.60(d,J＝7.9Hz,2H),7.23(d,J＝8.7Hz,2H),7.16(d,J＝8.0Hz,2H),6.84(d,J＝7.6Hz,1H),6.70(d,J＝3.8Hz,2H),6.67(d,J＝7.1Hz,2H),6.52(d,J＝8.8Hz,2H),6.48(d,J＝8.2Hz,1H),6.31(t,J＝7.5Hz,1H),6.05–5.98(m,1H),4.06–3.99(m,2H),3.72–3.67(m,2H),3.35(s,3H),2.99(s,2H)；13C NMR(100MHz,DMSO-d6)δ165.65(s),156.19(d,J＝2.7Hz),153.56(s),150.27(d,J＝10.5Hz),149.26(s),144.60(s),143.48(s),142.09(s),140.68(d,J＝19.3Hz),139.83(d,J＝37.5Hz),134.69(s),133.51(s),129.21(s),128.28(s,2C),127.37(s,2C),127.10(s),126.88(s),123.94(s),120.92(s,2C),116.79(s),116.65(s),114.73(s,2C),110.37(s),108.40(s),106.10(s),70.99(s),67.53(s),58.63(s),46.73(s)；HRMS(ESI):calcd for C33H32FN7O3[M+H]+594.2623,found 594.2639。
N1-(2-氨基苯基)-N5-(3-((5-氟-2-((4-(2-甲氧基乙氧基)苯基)氨基)嘧啶-4-基)氨基)苯基)戊二酰胺(化合物Ⅲ-3)
类白色固体115mg，产率64.6％；Mp:198-200℃；1H NMR(400MHz,DMSO-d6)δ8.05(d,J＝3.5Hz,1H),7.88(s,1H),7.53(d,J＝8.9Hz,2H),7.50(d,J＝8.8Hz,1H),7.34(d,J＝8.2Hz,1H),7.25(t,J＝8.0Hz,1H),7.18(d,J＝7.7Hz,1H),6.89(t,J＝7.4Hz,1H),6.78(d,J＝8.9Hz,2H),6.72(d,J＝7.8Hz,1H),6.54(t,J＝7.4Hz,1H),4.04–3.99(m,2H),3.66–3.61(m,2H),3.30(s,3H),2.40(t,J＝7.1Hz,4H),1.97–1.86(m,2H)；13C NMR(100MHz,DMSO-d6)δ171.30(s),171.22(s),156.18(d,J＝2.8Hz),153.53(s),150.27(d,J＝10.9Hz),142.35(s),142.07(s),141.15(d,J＝20.4Hz),139.85(s),139.57(d,J＝11.0Hz),134.63(s),128.94(s),126.18(s),125.83(s),124.00(s),120.82(s,2C),117.38(s),116.61(s),116.33(s),115.02(s),114.64(s,2C),113.63(s),71.00(s),67.52(s),58.63(s),36.20(s),35.53(s),21.72(s)；HRMS(ESI):calcd for C30H32FN7O4[M+H]+574.2572,found574.2585。
N1-(2-氨基苯基)-N6-(3-((5-氟-2-((4-(2-甲氧基乙氧基)苯基)氨基)嘧啶-4-基)氨基)苯基)己二酰胺(化合物Ⅲ-4)
类白色固体93mg，产率52.5％；Mp:192-194℃；1H NMR(400MHz,DMSO-d6)δ8.05(d,J＝3.6Hz,1H),7.86(s,1H),7.53(d,J＝8.9Hz,2H),7.50(d,J＝8.4Hz,1H),7.32(d,J＝7.8Hz,1H),7.24(t,J＝8.0Hz,1H),7.15(d,J＝7.5Hz,1H),6.89(t,J＝7.3Hz,1H),6.78(d,J＝8.9Hz,2H),6.71(d,J＝7.6Hz,1H),6.53(t,J＝7.5Hz,1H),4.04–3.99(m,2H),3.66–3.61(m,2H),3.31(s,3H),2.40–2.31(m,4H),1.69–1.60(m,4H)；13C NMR(100MHz,DMSO-d6)δ171.53(s),171.48(s),156.18(d,J＝2.8Hz),153.52(s),150.26(d,J＝10.8Hz),142.36(s),141.16(d,J＝21.9Hz),139.84(s),139.56(d,J＝10.9Hz),134.64(s),128.94(s),126.17(s),125.78(s),124.04(s),120.81(s,2C),117.37(s),116.65(s),116.37(s),115.03(s),114.73(s),114.65(s,2C),113.65(s),71.00(s),67.54(s),58.64(s),49.07(s),36.76(s),36.13(s),25.36(s)；HRMS(ESI):calcd for C31H34FN7O4[M+H]+588.2729,found 588.2750。
N1-(2-氨基苯基)-N8-(3-((5-氟-2-((4-(2-甲氧基乙氧基)苯基)氨基)嘧啶-4-基)氨基)苯基)辛二酰胺(化合物Ⅲ-5)
白色固体200mg，产率68.3％；Mp:198-200℃；1H NMR(400MHz,DMSO-d6)δ8.05(d,J＝3.6Hz,1H),7.86(s,1H),7.53(d,J＝8.9Hz,2H),7.48(d,J＝7.6Hz,1H),7.31(d,J＝8.1Hz,1H),7.24(t,J＝8.0Hz,1H),7.15(d,J＝7.8Hz,1H),6.89(t,J＝7.3Hz,1H),6.77(d,J＝9.0Hz,2H),6.71(d,J＝7.5Hz,1H),6.54(t,J＝7.5Hz,1H),4.05–3.99(m,2H),3.67–3.61(m,2H),3.31(s,3H),2.31(t,J＝7.3Hz,4H),1.67–1.55(m,4H),1.40–1.29(m,4H)；13CNMR(100MHz,DMSO-d6)δ171.87(s),171.68(s),156.04(d,J＝2.0Hz),153.34(s),150.10(d,J＝10.6Hz),142.27(s),142.03(s),140.17(d,J＝1.2Hz),139.69(d,J＝1.2Hz),134.87(s),128.83(s),126.00(s),125.67(s),125.47(s),124.14(s),120.51(s,2C),116.55(s),116.40(s),116.32(s),114.56(s,2C),114.39(s),112.84(s),70.99(s),67.48(s),58.64(s),55.40(s),49.04(s),36.83(s),36.24(s),28.99(s),25.74(s)；HRMS(ESI):calcd for C33H38FN7O4[M+H]+616.3042,found 616.3058。
1-(4-((2-氨基苯基)氨基)-4-氧代丁基)-N-(3-((5-氟-2-((4-(2-甲氧基乙氧基)苯基)氨基)嘧啶-4-基)氨基)苯基)哌啶-4-甲酰胺(化合物Ⅲ-6)
类白色固体80mg，产率57.5％；Mp:176-178℃；1H NMR(400MHz,MeOD)δ8.03(s,1H),7.87(d,J＝3.7Hz,1H),7.44(d,J＝8.9Hz,2H),7.34(d,J＝6.4Hz,1H),7.31(d,J＝8.3Hz,1H),7.29–7.24(m,1H),7.09(d,J＝8.0Hz,1H),7.04(t,J＝7.0Hz,1H),6.85(d,J＝7.8Hz,1H),6.81(d,J＝8.9Hz,2H),6.71(t,J＝7.8Hz,1H),4.09–4.04(m,2H),3.75–3.68(m,2H),3.54–3.46(m,2H),3.42(s,3H),3.07–2.98(m,2H),2.90–2.77(m,2H),2.61(t,J＝5.9Hz,2H),2.11–2.02(m,4H),1.98–1.92(m,1H),1.32–1.26(m,2H)；13C NMR(100MHz,DMSO-d6)δ173.67(s),171.32(s),156.09(d,J＝2.7Hz),153.39(s),150.17(d,J＝10.7Hz),142.40(s),142.03(s),141.03(d,J＝19.5Hz),139.96(s),139.54(d,J＝8.5Hz),134.77(s),128.93(s),126.20(s),125.82(s),123.95(s),120.56(s,2C),116.98(s),116.59(s),116.31(s),114.77(s),114.60(s,2C),113.38(s),71.00(s),67.50(s),58.65(s),57.48(s),52.73(s,2C),42.45(s),33.87(s,2C),28.24(s),22.22(s)；HRMS(ESI):calcd forC35H41FN8O4[M+H]+657.3307,found 657.3325。
1-(5-((2-氨基苯基)氨基)-5-氧代戊基)-N-(3-((5-氟-2-((4-(2-甲氧基乙氧基)苯基)氨基)嘧啶-4-基)氨基)苯基)哌啶-4-甲酰胺(化合物Ⅲ-7)
类白色固体120mg，产率69.3％；Mp:204-206℃；1H NMR(400MHz,DMSO-d6)δ8.05(d,J＝3.7Hz,1H),7.88(s,1H),7.53(d,J＝9.0Hz,2H),7.45(d,J＝7.6Hz,1H),7.32(d,J＝8.0Hz,1H),7.24(t,J＝8.0Hz,1H),7.15(d,J＝7.7Hz,1H),6.89(t,J＝7.5Hz,1H),6.76(d,J＝9.0Hz,2H),6.72(d,J＝7.2Hz,1H),6.54(t,J＝7.5Hz,1H),4.04–3.98(m,2H),3.66–3.60(m,2H),3.31(s,3H),2.92(d,J＝10.7Hz,2H),2.36–2.32(m,2H),2.32–2.30(m,1H),2.30–2.26(m,2H),1.87(t,J＝11.0Hz,2H),1.78–1.71(m,2H),1.67–1.58(m,4H),1.52–1.43(m,2H)；13C NMR(100MHz,DMSO-d6)δ174.03(s),171.60(s),156.17(d,J＝3.0Hz),153.47(s),150.26(d,J＝10.9Hz),142.35(s),142.04(s),141.20(d,J＝19.5Hz),139.92(s),139.53(d,J＝13.4Hz),134.68(s),128.91(s),126.15(s),125.73(s),124.12(s),120.68(s,2C),117.38(s),116.67(s),116.40(s),115.08(s),114.66(s,2C),113.83(s),71.01(s),67.55(s),58.65(s),58.38(s),53.31(s,2C),43.55(s),36.13(s),29.04(s,2C),26.54(s),23.82(s)；HRMS(ESI):calcd for C36H43FN8O4[M+H]+671.3464,found671.3482。
1-(6-((2-氨基苯基)氨基)-6-氧代己基)-N-(3-((5-氟-2-((4-(2-甲氧基乙氧基)苯基)氨基)嘧啶-4-基)氨基)苯基)哌啶-4-甲酰胺(化合物Ⅲ-8)
白色固体60mg，产率34.7％；Mp:184-186℃；1H NMR(400MHz,MeOD)δ7.89(s,1H),7.77(s,1H),7.34(d,J＝8.8Hz,2H),7.26(d,J＝7.9Hz,1H),7.22(d,J＝7.8Hz,1H),7.15(t,J＝8.0Hz,1H),6.98(d,J＝7.9Hz,1H),6.92(t,J＝7.6Hz,1H),6.74(d,J＝8.6Hz,1H),6.71(d,J＝8.9Hz,2H),6.61(t,J＝7.6Hz,1H),3.99–3.94(m,2H),3.64–3.59(m,2H),3.32(s,3H),3.05(d,J＝11.4Hz,2H),2.47–2.40(m,2H),2.34(t,J＝7.3Hz,3H),2.20–2.10(m,2H),1.84–1.75(m,4H),1.71–1.61(m,2H),1.59–1.48(m,2H),1.39–1.29(m,2H)；13C NMR(100MHz,DMSO-d6)δ173.83(s),171.61(s),156.14(d,J＝2.8Hz),153.44(s),150.23(d,J＝10.7Hz),142.33(s),142.04(s),141.15(d,J＝20.2Hz),139.92(s),139.55(d,J＝8.0Hz),134.71(s),128.92(s),126.13(s),125.72(s),124.10(s),120.64(s,2C),117.27(s),116.65(s),116.38(s),114.98(s),114.63(s,2C),113.69(s),71.01(s),67.53(s),58.65(s),58.28(s),53.06(s),43.05(s),36.21(s,2C),28.66(s),27.02(s,2C),26.36(s),25.70(s)；HRMS(ESI):calcd for C37H45FN8O4[M+H]+685.3620,found 685.3649。
1-(4-((2-氨基苯基)氨基)-4-氧代丁基)-N-(3-((5-氟-2-((4-(2-甲氧基乙氧基)苯基)氨基)嘧啶-4-基)氨基)苯基)哌啶-3-甲酰胺(化合物Ⅲ-9)
类白色固体122mg，产率52.6％；Mp:82-84℃；1H NMR(400MHz,MeOD)δ7.96(s,1H),7.86(d,J＝3.9Hz,1H),7.44(d,J＝9.0Hz,2H),7.37(d,J＝9.2Hz,1H),7.34(d,J＝8.9Hz,1H),7.26(t,J＝8.0Hz,1H),7.07(d,J＝7.9Hz,1H),7.01(t,J＝7.0Hz,1H),6.83(d,J＝8.1Hz,1H),6.80(d,J＝9.0Hz,2H),6.70(t,J＝7.6Hz,1H),4.10–4.04(m,2H),3.75–3.70(m,2H),3.41(s,3H),3.07–2.98(m,1H),2.97–2.87(m,1H),2.72–2.65(m,1H),2.64–2.55(m,2H),2.48(t,J＝7.1Hz,2H),2.02–1.95(m,2H),1.95–1.87(m,2H),1.81(t,J＝8.3Hz,1H),1.74–1.63(m,2H),1.34–1.27(m,1H)；13C NMR(100MHz,DMSO-d6)δ172.54(s),171.38(s),156.16(d,J＝2.7Hz),153.48(s),150.24(d,J＝10.9Hz),142.40(s),142.06(s),141.18(d,J＝19.9Hz),139.72(s),139.57(d,J＝7.1Hz),134.70(s),128.96(s),126.19(s),125.82(s),124.01(s),120.68(s,2C),117.45(s),116.64(s),116.35(s),115.07(s),114.65(s,2C),113.76(s),71.01(s),67.54(s),58.65(s),57.94(s),55.36(s),53.38(s),43.47(s),34.09(s),31.42(s),27.55(s),22.56(s)；HRMS(ESI):calcd for C35H41FN8O4[M+H]+657.3307,found 657.3326。
1-(5-((2-氨基苯基)氨基)-5-氧代戊基)-N-(3-((5-氟-2-((4-(2-甲氧基乙氧基)苯基)氨基)嘧啶-4-基)氨基)苯基)哌啶-3-甲酰胺(化合物Ⅲ-10)
白色固体111mg，产率64.1％；Mp:150-152℃；1H NMR(400MHz,DMSO-d6)δ8.05(d,J＝3.6Hz,1H),7.86(s,1H),7.54(d,J＝8.9Hz,2H),7.47(d,J＝7.4Hz,1H),7.32(d,J＝8.1Hz,1H),7.24(t,J＝8.1Hz,1H),7.15(d,J＝7.6Hz,1H),6.89(t,J＝7.6Hz,1H),6.77(d,J＝8.9Hz,2H),6.71(d,J＝7.8Hz,1H),6.53(t,J＝7.4Hz,1H),4.04–3.98(m,2H),3.67–3.60(m,2H),3.31(s,3H),2.96–2.85(m,1H),2.84–2.72(m,1H),2.65–2.56(m,1H),2.33(t,J＝6.7Hz,4H),2.15–2.03(m,1H),1.92(t,J＝11.3Hz,1H),1.86–1.76(m,1H),1.73–1.65(m,1H),1.63–1.56(m,2H),1.55–1.39(m,4H)；13C NMR(100MHz,DMSO-d6)δ172.82(s),171.58(s),156.15(d,J＝2.7Hz),153.47(s),150.24(d,J＝10.8Hz),142.33(s),142.04(s),141.20(d,J＝19.3Hz),139.73(s),139.55(d,J＝8.1Hz),134.68(s),128.97(s),126.16(s),125.73(s),124.07(s),120.66(s,2C),117.44(s),116.69(s),116.40(s),115.05(s),114.63(s.2C),113.74(s),71.00(s),67.52(s),58.65(s),58.26(s),55.36(s),53.45(s),43.77(s),36.05(s),27.63(s),26.23(s),24.49(s),23.74(s)；HRMS(ESI):calcd for C36H43FN8O4[M+H]+671.3464,found 671.3489。
1-(6-((2-氨基苯基)氨基)-6-氧代己基)-N-(3-((5-氟-2-((4-(2-甲氧基乙氧基)苯基)氨基)嘧啶-4-基)氨基)苯基)哌啶-3-甲酰胺(化合物Ⅲ-11)
类白色固体133mg，产率82.5％；Mp:90-92℃；1H NMR(400MHz,DMSO-d6)δ8.06(d,J＝3.6Hz,1H),7.86(s,1H),7.54(d,J＝9.0Hz,2H),7.46(d,J＝7.6Hz,1H),7.33(d,J＝7.9Hz,1H),7.25(t,J＝7.9Hz,1H),7.15(d,J＝7.8Hz,1H),6.89(t,J＝7.1Hz,1H),6.76(d,J＝9.0Hz,2H),6.71(d,J＝7.7Hz,1H),6.53(t,J＝7.4Hz,1H),4.04–3.98(m,2H),3.64–3.61(m,2H),3.30(s,3H),2.94–2.84(m,1H),2.82–2.70(m,2H),2.40–2.21(m,4H),2.14–2.03(m,1H),1.95–1.87(m,1H),1.85–1.76(m,1H),1.72–1.65(m,1H),1.63–1.58(m,2H),1.55–1.39(m,4H),1.33–1.30(m,2H)；13C NMR(100MHz,DMSO-d6)δ172.73(s),171.59(s),156.13(d,J＝2.6Hz),153.43(s),150.21(d,J＝10.8Hz),142.33(s),142.01(s),141.24(d,J＝18.6Hz),139.69(s),139.53(d,J＝7.5Hz),134.66(s),129.00(s),126.17(s),125.73(s),124.04(s),120.60(s,2C),117.48(s),116.66(s),116.37(s),115.04(s),114.59(s,2C),113.75(s),70.99(s),67.48(s),58.65(s),55.37(s),53.40(s),36.18(s),31.73(s),31.43(s),27.56(s),27.03(s),25.69(s),22.53(s),14.44(s)；HRMS(ESI):calcd for C37H45FN8O4[M+H]+685.3620,found 685.3649。
1-(4-((2-氨基苯基)氨基)-4-氧代丁基)-N-(3-((5-氟-2-((4-(2-甲氧基乙氧基)苯基)氨基)嘧啶-4-基)氨基)苯基)吡咯烷-3-甲酰胺(化合物Ⅲ-12)
类白色固体105mg，产率60.2％；Mp:74-76℃；1H NMR(400MHz,MeOD)δ8.02(s,1H),7.86(d,J＝3.9Hz,1H),7.44(d,J＝9.0Hz,2H),7.35(d,J＝7.9Hz,1H),7.32(d,J＝8.4Hz,1H),7.25(t,J＝8.0Hz,1H),7.08(d,J＝9.0Hz,1H),7.02(t,J＝7.0Hz,1H),6.83(d,J＝9.1Hz,1H),6.80(d,J＝9.0Hz,2H),6.70(t,J＝8.2Hz,1H),4.09–4.02(m,2H),3.74–3.68(m,2H),3.41(s,3H),3.24–3.12(m,2H),3.02–2.91(m,2H),2.89–2.82(m,1H),2.82–2.73(m,2H),2.51(t,J＝7.1Hz,2H),2.28–2.19(m,1H),2.18–2.09(m,1H),2.03–1.91(m,2H)；13CNMR(100MHz,DMSO-d6)δ172.73(s),171.32(s),156.13(d,J＝2.7Hz),153.45(s),150.20(d,J＝11.0Hz),142.41(s),142.04(s),141.12(d,J＝22.1Hz),139.81(s),139.62(d,J＝5.9Hz),134.72(s),128.97(s),126.17(s),125.80(s),124.00(s),120.64(s,2C),117.27(s),116.60(s),116.31(s),114.94(s),114.63(s,2C),113.60(s),71.01(s),67.53(s),58.65(s),57.32(s),55.24(s),54.01(s),43.77(s),34.06(s),28.40(s),24.37(s)；HRMS(ESI):calcd for C34H39FN8O4[M+H]+643.3151,found 643.3182。
1-(5-((2-氨基苯基)氨基)-5-氧代戊基)-N-(3-((5-氟-2-((4-(2-甲氧基乙氧基)苯基)氨基)嘧啶-4基)氨基)苯基)吡咯烷-3-甲酰胺(化合物Ⅲ-13)
类白色固体70mg，产率40.3％；Mp:66-68℃；1H NMR(400MHz,MeOD)δ8.00(s,1H),7.86(d,J＝3.8Hz,1H),7.44(d,J＝8.9Hz,2H),7.36(d,J＝8.1Hz,1H),7.32(d,J＝8.4Hz,1H),7.25(t,J＝8.0Hz,1H),7.07(d,J＝7.8Hz,1H),7.01(t,J＝7.6Hz,1H),6.84(s,1H),6.80(d,J＝9.0Hz,2H),6.70(t,J＝7.7Hz,1H),4.09–4.00(m,2H),3.75–3.67(m,2H),3.41(s,3H),3.19–3.13(m,1H),3.13–3.06(m,1H),2.92(dd,J＝14.0,8.6Hz,1H),2.89–2.81(m,1H),2.78–2.72(m,1H),2.72–2.63(m,2H),2.46(t,J＝7.1Hz,2H),2.27–2.17(m,1H),2.17–2.06(m,1H),1.81–1.72(m,2H),1.72–1.62(m,2H)；13C NMR(100MHz,DMSO-d6)δ172.81(s),171.52(s),156.14(d,J＝2.6Hz),153.46(s),150.21(d,J＝10.7Hz),142.35(s),142.05(s),141.13(d,J＝19.5Hz),139.82(s),139.62(d,J＝6.5Hz),134.72(s),128.97(s),126.14(s),125.74(s),124.08(s),120.65(s,2C),117.28(s),116.65(s),116.38(s),114.94(s),114.63(s,2C),113.60(s),71.01(s),67.53(s),58.65(s),57.40(s),55.38(s),54.01(s),43.82(s),36.01(s),28.33(s),27.93(s),23.68(s)；MS(ESI):calcd forC35H41FN8O4[M+H]+657.33,found 657.19。
1-(6-((2-氨基苯基)氨基)-6-氧代己基)-N-(3-((5-氟-2-((4-(2-甲氧基乙氧基)苯基)氨基)嘧啶-4-基)氨基)苯基)吡咯烷-3-甲酰胺(化合物Ⅲ-14)
黄色固体195mg，产率46.9％；Mp:62-64℃；1H NMR(400MHz,MeOD)δ7.98(s,1H),7.86(d,J＝3.9Hz,1H),7.44(d,J＝8.9Hz,2H),7.34(t,J＝8.6Hz,2H),7.25(t,J＝8.0Hz,1H),7.07(d,J＝7.8Hz,1H),7.01(t,J＝7.1Hz,1H),6.84(s,1H),6.81(d,J＝9.0Hz,2H),6.70(t,J＝7.7Hz,1H),4.08–4.05(m,2H),3.74–3.70(m,2H),3.42(s,3H),3.17–3.10(m,1H),3.08–3.02(m,1H),2.91–2.83(m,1H),2.82–2.74(m,1H),2.72–2.64(m,1H),2.63–2.55(m,2H),2.42(t,J＝7.4Hz,2H),2.23–2.15(m,1H),2.15–2.06(m,1H),1.80–1.70(m,2H),1.68–1.57(m,2H),1.51–1.41(m,2H),1.36–1.26(m,2H)；13C NMR(100MHz,DMSO-d6)δ177.73(s),176.37(s),160.89(d,J＝2.7Hz),158.21(s),154.97(d,J＝10.9Hz),147.09(s),146.78(s),145.96(d,J＝19.7Hz),144.54(s),144.31(d,J＝4.7Hz),139.42(s),133.75(s),130.91(s),130.50(s),128.82(s),125.41(s,2C),122.14(s),121.41(s),121.13(s),119.77(s),119.37(s2C),118.48(s),75.75(s),72.26(s),64.98(s),63.40(s),62.30(s),60.40(s),58.80(s),48.64(s),40.97(s),33.04(s),31.82(s),30.46(s)；MS(ESI):calcdfor C36H43FN8O4[M+H]+671.34,found 671.27。
实验例
化合物对HDACs酶的抑制活性测定、HDACs亚型选择性实验及对套细胞淋巴瘤细胞株Jeko-1的生长抑制活性测定实验1、化合物对HDACs的抑制活性实验
1、化合物对组蛋白去乙酰化酶的抑制活性实验
实验原理：采用荧光分析法测定化合物对HDACs、HDAC1、HDAC6的抑制活性。首先将所需测定化合物分别同HDACs、HDAC1、HDAC6酶及荧光底物I共同孵育，HDACs酶可催化底物脱掉乙酰基生成II，再加入胰酶(Trypsin)水解物质II，得到荧光基团AMC。在激发波长355nm、发射波长460nm下检测荧光强度，计算化合物对HDACs、HDAC1、HDAC6的抑制活性。
实验材料：HDACs酶(Enzo，北京安诺伦科技公司)，人重组HDAC1、6酶(BPSBIOSCIENCE)，Boc-Lys(acetyl)-AMC(Hela细胞核提取荧光底物)，Tris-HCl，NaCl，KCl，MgCl2，牛血清白蛋白(BSA)，Trypsin(胰蛋白酶)，DMSO，伏立诺他，96孔板，缓冲液(25mMTris-HCl，pH 8.0，80mL；137mM NaCl；2.7mM KCl；1mM MgCl2；10mg BSA(sigma)补齐蒸馏水至100mL)，终止液(67μM Trypsin、200nM伏立诺他溶于1L DMSO中，使用时缓冲液稀释5倍)等。
实验方法：HDACs、HDAC1、HDAC6酶分别用缓冲液稀释至0.5μg/μL、0.05μg/μL。取96孔板同时设置实验组、100％对照组和空白对照组，向空白孔中加入25μL缓冲液，100％对照孔加入20μL缓冲液、实验孔加入15μL缓冲液，除空白孔外每孔加入5μL酶，实验孔再加入5μL待测化合物，室温避光反应10min，待药物与酶充分作用后所有孔分别加入5μL Boc-Lys(AC)-AMC底物，混匀后在培养箱37℃，5％CO2下孵育60min，所有孔加入25μL终止液，摇匀20min停止反应。应用酶标仪在激发波长355nm、发射波长460nm下检测荧光强度，计算化合物的HDACs抑制活性。
应用GraphPad Prism 5计算化合物的IC50值，如表1所示。
表1.化合物对HDAC酶的抑制活性
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ND＝Not Detected；SAHA：伏立诺他(Vorinostat)
由表1可知，在5μM浓度下Ⅱ类绝大多数化合物对HDACs的抑制活性大于80％；而Ⅲ类绝大多数化合物对HDACs的抑制活性小于50％；与Ⅱ类化合物相比可知，邻苯二胺作为ZBG基团对HDACs的抑制活性弱于异羟肟酸。在100nM浓度下化合物Ⅱ-2、Ⅱ-3、Ⅱ-7对HDACs、HDAC1及HDAC6的抑制活性均优于SAHA；其他部分化合物对HDAC6的抑制作用与SAHA相当，但其对HDAC6的选择性优于SAHA。
进一步，本实施例优选化合物Ⅱ-2、Ⅱ-3、Ⅱ-4、Ⅱ-7、Ⅱ-12、Ⅱ-16，测定其对HDACs的半数抑制浓度(IC50)，如表2所示。
表2.优选化合物对HDACs的抑制活性
SAHA：伏立诺他(Vorinostat)
由表2可知，化合物Ⅱ-7对HDACs表现出最强的抑制活性是SAHA的近5倍；化合物II-2和Ⅱ-3对HDACs的抑制活性与SAHA相当。
2、化合物对套细胞淋巴瘤细胞株Jeko-1的生长抑制活性实验
实验材料：人套细胞淋巴瘤细胞株Jeko-1(中国科学院细胞库)，RPMI 1640培养基(BioInd)，澳洲胎牛血清(FBS，Gibco)，CCK-8增强型试剂盒(大连美仑)，台盼蓝试剂，100X青霉素-链霉素混合溶液(Genview)，PBS磷酸盐缓冲粉剂(北京鼎国昌盛)，24/96孔培养板，细胞培养瓶等。CO2恒温培养箱(Panasonic)，SW-CJ-1FD型超净台，细胞计数仪(江苏卓微生物科技有限公司)，Infinite F50型吸收光酶标仪(Tecan)，Centrifuge 542R型离心机(Eppendorf)以及高压灭菌器(Panasonic)等。
实验方法：取96孔板同时设置实验组、100％对照组和空白对照组，向空白孔加200μL培养基，100％对照孔和实验孔分别加入100μL细胞悬液(取对数生长期细胞计算细胞浓度，每孔细胞1.5×104个)，随后100％对照孔加入100μL培养基，实验孔加入100μL待测化合物，设置3-5个复孔。放入培养箱继续孵育48h，每孔加入20μL的CCK-8，避光培养0.5～4h，在450nm处测定吸光度，计算生长抑制率，应用GraphPad Prism 6.0计算化合物的IC50值。
表3.化合物对套细胞淋巴瘤细胞株Jeko-1的生长抑制活性
由表3可知，绝大多数化合物在5μM浓度下对Jeko-1细胞抗增殖活性>80％，优于套细胞淋巴瘤的一线用药IBN，其中Ⅱ-1、Ⅱ-2、Ⅱ-3、Ⅱ-4、Ⅱ-6、Ⅱ-7、Ⅲ-8、Ⅲ-11、Ⅲ-14的活性与SAHA相当。优选化合物Ⅱ-1、Ⅱ-2、Ⅱ-3、Ⅱ-4、Ⅱ-5、Ⅱ-6、Ⅱ-7、Ⅲ-1、Ⅲ-2、Ⅲ-6、Ⅲ-8、Ⅲ-11、Ⅲ-14进一步测定其对Jeko-1的IC50值，如表4所示。
表4.优选化合物对套细胞淋巴瘤细胞株Jeko-1的生长抑制活性
由表4可知，优选化合物对Jeko-1细胞株的生长抑制活性均强于阳性药Ibrutinib。化合物Ⅱ-2、Ⅱ-3、Ⅱ-7对Jeko-1细胞株的生长抑制活性IC50值均＜1μM，其中对HDACs酶具有最强抑制活性的化合物Ⅱ-7对Jeko-1细胞也表现出最强的抗增殖活性，是Ibrutinib的22倍，稍强于SAHA；其余化合物对Jeko-1细胞的抗增殖活性也较强，相比Ibrutinib其抗增殖活性提升2-11倍。
以上所述仅为本发明的优选实施例而已，并不用于限制本发明，对于本领域的技术人员来说，本发明可以有各种更改和变化。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
Claims (16)
1.一种含二苯基氨基嘧啶类化合物的制备方法，其特征在于，所述化合物结构如下式Ⅰ所示：
式Ⅰ；
其中，X独立的选自亚甲基、苄基、羰基、对位羰基哌啶基、间位羰基哌啶基或羰基吡咯烷基；
Y独立的选自异羟肟酸基或邻苯二胺甲酰基；
R1独立的选自卤素；
R2是C原子个数为1~6的烷氧基；
n是0~7的任意整数；
所述制备方法中，式Ⅰ所示含二苯基氨基嘧啶类化合物的合成路线如下：
具体合成步骤如下：
以(3-氨基苯基)氨基甲酸叔丁酯（1）为起始原料，与中间体2经取代反应得中间体3；对氟硝基苯（4）与中间体5经取代反应得到中间体6；中间体6在Pd/C催化下与氢气发生还原反应得到中间体7；中间体3和中间体7经取代反应得中间体8；中间体8在浓盐酸下脱去Boc保护基，得到中间体9；中间体9通过不同溴取代的羧酸甲酯经亲核取代，或与N-Boc-3-哌啶甲酸或N-Boc-4-哌啶甲酸、N-Boc-3-吡咯烷甲酸经酰胺缩合得中间体10；含羧酸甲酯的中间体10在碱性条件下与羟胺反应或经水解反应并进一步与邻苯二胺反应得式Ⅰ化合物。
2.如权利要求1所述含二苯基氨基嘧啶类化合物的制备方法，其特征在于，所述Y为异羟肟酸基，R1为氟，R2为甲氧乙氧基，n为0~5，所述化合物结构如下式Ⅱ所示：
式II；
其中，所述X选自亚甲基、苄基、羰基、对位羰基哌啶基、间位羰基哌啶基或羰基吡咯烷基。
3.如权利要求2所述含二苯基氨基嘧啶类化合物的制备方法，其特征在于，式II所示含二苯基氨基嘧类化合物具体选自以下化合物：
4-((3-((5-氟-2-((4-(2-甲氧基乙氧基)苯基)氨基)嘧啶-4-基)氨基)苯基)氨基)-N-羟基丁酰胺；
5-((3-((5-氟-2-((4-(2-甲氧基乙氧基)苯基)氨基)嘧啶-4-基)氨基)苯基)氨基)-N-羟基戊酰胺；
6-((3-((5-氟-2-((4-(2-甲氧基乙氧基)苯基)氨基)嘧啶-4-基)氨基)苯基)氨基)-N-羟基己酰胺；
4-(((3-((5-氟-2-((4-(2-甲氧基乙氧基)苯基)氨基)嘧啶-4-基)氨基)苯基)氨基)甲基)-N-羟基苯甲酰胺；
N 1-(3-((5-氟-2-((4-(2-甲氧基乙氧基)苯基)氨基)嘧啶-4-基)氨基)苯基)-N 5-羟基戊二酰胺；
N 1-(3-((5-氟-2-((4-(2-甲氧基乙氧基)苯基)氨基)嘧啶-4-基)氨基)苯基)-N 6-羟基己二酰胺；
N 1-(3-((5-氟-2-((4-(2-甲氧基乙氧基)苯基)氨基)嘧啶-4-基)氨基)苯基)-N 8-羟基辛二酰胺；
N-(3-((5-氟-2-((4-(2-甲氧基乙氧基)苯基)氨基)嘧啶-4-基)氨基)苯基)-1-(4-(羟基氨基)-4-氧丁基)哌啶-4-甲酰胺；
N-(3-((5-氟-2-((4-(2-甲氧基乙氧基)苯基)氨基)嘧啶-4-基)氨基)苯基)-1-(5-(羟基氨基)-5-氧代戊基)哌啶-4-甲酰胺；
N-(3-((5-氟-2-((4-(2-甲氧基乙氧基)苯基)氨基)嘧啶-4-基)氨基)苯基)-1-(6-(羟基氨基)-6-氧己基)哌啶-4-甲酰胺；
N-(3-(5-氟-2-((4-(2-甲氧基乙氧基)苯基)氨基)嘧啶-4-基)氨基)苯基)-1-(4-(羟基氨基)-4-氧丁基)哌啶-3-甲酰胺；
N-(3-((5-氟-2-((4-(2-甲氧基乙氧基)苯基)氨基)嘧啶-4-基)氨基)苯基)-1-(5-(羟基氨基)-5-氧代戊基)哌啶-3-甲酰胺；
N-(3-((5-氟-2-((4-(2-甲氧基乙氧基)苯基)氨基)嘧啶-4-基)氨基)苯基)-1-(6-(羟基氨基)-6-氧己基)哌啶-3-甲酰胺；
N-(3-((5-氟-2-((4-(2-甲氧基乙氧基)苯基)氨基)嘧啶-4-基)氨基)苯基)-1-(4-(羟基氨基)-4-氧丁基)吡咯烷-3-甲酰胺；
N-(3-((5-氟-2-((4-(2-甲氧基乙氧基)苯基)氨基)嘧啶-4-基)氨基)苯基)-1-(5-(羟基氨基)-5-氧代戊基)吡咯烷-3-甲酰胺；
N-(3-((5-氟-2-((4-(2-甲氧基乙氧基)苯基)氨基)嘧啶-4-基)氨基)苯基)-1-(6-(羟基氨基)-6-氧己基)吡咯烷-3-甲酰胺。
4.如权利要求1所述含二苯基氨基嘧啶类化合物的制备方法，其特征在于，所述Y为邻苯二胺甲酰基，R1为氟，R2为甲氧乙氧基，n为0~5，所述化合物结构如下式Ⅲ所示：
式Ⅲ；
其中，所述X选自亚甲基、苄基、羰基、对位羰基哌啶基、间位羰基哌啶基或羰基吡咯烷基。
5.如权利要求4所述含二苯基氨基嘧啶类化合物的制备方法，其特征在于，式Ⅲ所示含二苯基氨基嘧啶类化合物具体选自以下化合物：
N-(2-氨基苯基)-6-((3-((5-氟-2-((4-(2-甲氧基乙氧基)苯基)氨基)嘧啶-4-基)氨基)苯基)氨基)己酰胺；
N-(2-氨基苯基)-4-(((3-((5-氟-2-((4-(2-甲氧基乙氧基)苯基)氨基)嘧啶-4-基)氨基)苯基)氨基)甲基)苯甲酰胺；
N 1-(2-氨基苯基)-N 5-(3-((5-氟-2-((4-(2-甲氧基乙氧基)苯基)氨基)嘧啶-4-基)氨基)苯基)戊二酰胺；
N 1-(2-氨基苯基)-N 6-(3-((5-氟-2-((4-(2-甲氧基乙氧基)苯基)氨基)嘧啶-4-基)氨基)苯基)己二酰胺；
N 1-(2-氨基苯基)-N 8-(3-((5-氟-2-((4-(2-甲氧基乙氧基)苯基)氨基)嘧啶-4-基)氨基)苯基)辛二酰胺；
1-(4-((2-氨基苯基)氨基)-4-氧代丁基)-N-(3-((5-氟-2-((4-(2-甲氧基乙氧基)苯基)氨基)嘧啶-4-基)氨基)苯基)哌啶-4-甲酰胺；
1-(5-((2-氨基苯基)氨基)-5-氧代戊基)-N-(3-((5-氟-2-((4-(2-甲氧基乙氧基)苯基)氨基)嘧啶-4-基)氨基)苯基)哌啶-4-甲酰胺；
1-(6-((2-氨基苯基)氨基)-6-氧代己基)-N-(3-((5-氟-2-((4-(2-甲氧基乙氧基)苯基)氨基)嘧啶-4-基)氨基)苯基)哌啶-4-甲酰胺；
1-(4-((2-氨基苯基)氨基)-4-氧代丁基)-N-(3-((5-氟-2-((4-(2-甲氧基乙氧基)苯基)氨基)嘧啶-4-基)氨基)苯基)哌啶-3-甲酰胺；
1-(5-((2-氨基苯基)氨基)-5-氧代戊基)-N-(3-((5-氟-2-((4-(2-甲氧基乙氧基)苯基)氨基)嘧啶-4-基)氨基)苯基)哌啶-3-甲酰胺；
1-(6-((2-氨基苯基)氨基)-6-氧代己基)-N-(3-((5-氟-2-((4-(2-甲氧基乙氧基)苯基)氨基)嘧啶-4-基)氨基)苯基)哌啶-3-甲酰胺；
1-(4-((2-氨基苯基)氨基)-4-氧代丁基)-N-(3-((5-氟-2-((4-(2-甲氧基乙氧基)苯基)氨基)嘧啶-4-基)氨基)苯基)吡咯烷-3-甲酰胺；
1-(5-((2-氨基苯基)氨基)-5-氧代戊基)-N-(3-((5-氟-2-((4-(2-甲氧基乙氧基)苯基)氨基)嘧啶-4基)氨基)苯基)吡咯烷-3-甲酰胺；
1-(6-((2-氨基苯基)氨基)-6-氧代己基)-N-(3-((5-氟-2-((4-(2-甲氧基乙氧基)苯基)氨基)嘧啶-4-基)氨基)苯基)吡咯烷-3-甲酰胺。
6.如权利要求1-5任一项所述含二苯基氨基嘧啶类化合物的制备方法，其特征在于，所述含二苯基氨基嘧啶类化合物除包含具有上述结构的化合物实体，还包括上述化合物药学上可接受的盐；其中，所述药学上可接受的盐包括上述化合物与有机酸或无机酸形成的盐。
7.如权利要求2-5任一项所述含二苯基氨基嘧啶类化合物的制备方法，其特征在于，式Ⅱ或式Ⅲ所示含二苯基氨基嘧啶类化合物的具体制备步骤如下：
上述式Ⅱ、式Ⅲ所示化合物的具体合成步骤如下：
（1）将原料1，原料2a，N, N-二异丙基乙胺溶于异丙醇中85~90℃油浴，加热回流反应4~5 h后，减压蒸除溶剂重新加入甲醇后降温，获取析出的白色固体部分，即为中间体3a；
（2）将原料4、原料5a，KOH溶于二甲基亚砜中, 55~65℃油浴，加热反应1.5~2.5 h后冷却反应体系，并加入乙酸乙酯萃取，获取有机相部分去除溶剂得到中间体6a；
（3）将中间体6a溶于乙酸乙酯中，加入Pd/C，通入氢气，室温反应3~5h后，减压蒸除溶剂，后柱层析纯化，所述柱层析的洗脱液为石油醚：乙酸乙酯= 4~6:1，得中间体7a；
（4）将中间体3a，中间体7a溶于叔戊醇中，加入冰乙酸，95~105℃加热回流9~11h后冷却反应体系，并加入乙酸乙酯进行萃取，获取有机相部分去除溶剂，通过柱层析纯化产物，得到中间体8a，所述柱层析的洗脱液为石油醚：乙酸乙酯= 4~6:1；
（5）将中间体8a溶于乙酸乙酯中，逐滴加入浓盐酸，室温反应1.5~2.5h，析出白色固体，获取该白色固体部分洗涤并干燥得到中间体9a；
（6）将1-（叔丁氧羰基）-3-哌啶甲酸、1-（叔丁氧羰基）-4-哌啶甲酸或1-（叔丁氧羰基）-3-吡咯烷甲酸、O-苯并三氮唑-四甲基脲六氟磷酸酯、三乙胺，溶于N,N-二甲基甲酰胺中，冰浴条件下搅拌0.5~1.5 h，再加入中间体9a，在室温下反应10~14 h后；将反应体系加入冷水中，再加入乙酸乙酯萃取，合并有机相干燥后去除溶剂，通过柱层析对产物进行纯化，得到中间体11a-11c，所述柱层析采用的洗脱液为二氯甲烷：甲醇=90~110: 1；
（7）将中间体11a-11c溶于乙酸乙酯中，逐滴加入浓盐酸，室温反应1.5~2.5 h，保留反应析出的白色固体，洗涤并干燥得到中间体12a-12c；
（8）将中间体9a溶于DMF中，加入不同溴代羧酸甲酯、K2CO3、KI、90~110℃加热反应30~34h；反应结束后，将反应体系加入冷水中，再加入乙酸乙酯萃取并合并有机相，干燥并去除溶剂后通过柱层析纯化得到中间体10a-10d，所述柱层析的洗脱液为二氯甲烷：甲醇= 90~110: 1；
（9）将中间体9a溶于二氯甲烷中，在冰浴条件下加入三乙胺活化0.5~1.5 h，再逐滴加入戊二酸单乙酯酰氯，冰浴搅拌8~12min，移至室温下反应8~12h；反应结束后加入冷水中降温，通过乙酸乙酯萃取，获取有机相部分去除溶剂并通过柱层析纯化得中间体10e，所述柱层析洗脱液为二氯甲烷：甲醇 = 140~160: 1；
（10）将己二酸单甲酯或辛二酸单甲酯、HBTU、三乙胺溶于DMF中，冰浴条件下搅拌20-30min，加入中间体9a，室温下反应10~14h；结束后将反应体系加入冷水中降温，通过乙酸乙酯萃取，获取有机相部分去除溶剂并通过柱层析纯化得中间体10f-10g，所述柱层析洗脱液为二氯甲烷：甲醇 = 140~160: 1；
（11）将中间体12a-12c，K2CO3，KI溶于DMF中，加入不同溴代羧酸甲酯，室温反应5~7 h；结束后将反应体系加入冷水中降温，通过乙酸乙酯萃取，获取有机相部分去除溶剂并通过柱层析纯化得中间体10h-10p，所述柱层析洗脱液为二氯甲烷：甲醇 =25~35: 1；
（12）向氢氧化钾中加入无水甲醇超声溶解，得溶液a；取盐酸羟胺加入无水甲醇超声溶解，得溶液b；将溶液a缓慢滴入溶液b中，室温反应0.5~1.5小时，过滤，滤液备用；将中间体10a-10p加入上述滤液中，室温搅拌0.5~1.5 h，待反应溶液由浑浊变澄清后结束反应，去除反应体系中的溶剂，向剩余反应产物中加入蒸馏水溶解，使其呈肥皂沫样；加入盐酸调节反应体系pH为5-6，获取析出的固体洗涤、重结晶并干燥，得到目标化合物II-1至II-16；
（13）将中间体10a-10p溶于无水乙醇中，加入NaOH至溶液pH 9-10，室温反应3~4h；反应结束后，去除反应体系中的溶剂，加入盐酸调节反应产物pH至5-6使其析出固体，保留该固体部分并干燥，得到中间体13a-13n；
（14）将中间体13a-13n、HBTU、三乙胺溶于DMF中，冰浴条件下搅拌20-30 min；再反应结束后，加入邻苯二胺后避光室温反应5~7 h；待反应结束后将反应体系加入冷水中降温，并加入乙酸乙酯进行萃取，获取有机相部分减压蒸除溶剂并通过柱层析纯化得目标化合物Ⅲ-1至 Ⅲ-14，所述柱层析洗脱液为二氯甲烷：甲醇 = 8~9: 1。
8.一种药物组合物，其特征在于，所述组合物中包括权利要求1-7任一项所述含二苯氨基嘧啶类化合物的制备方法制备得到的化合物。
9.如权利要求8所述的药物组合物，其特征在于，所述药物组合物中还包括其他具有抗肿瘤或辅助抗肿瘤效果的活性成分，所述活性成分为细胞毒类、激素类、生物反应调节剂或单克隆抗体。
10.如权利要求8所述的药物组合物，其特征在于，所述药物组合物中还包括依鲁替尼。
11.如权利要求8所述的药物组合物，其特征在于，所述药物组合物中，含有药物载体，包括离子交换剂、血清蛋白、缓冲物质、饱和植物脂肪酸的部分甘油酯混合物、水、盐、胶态氧化硅、三硅酸镁、聚乙烯吡咯烷酮、聚乙二醇、羧甲基纤维素钠、聚丙烯酸酯、蜂蜡、羊毛脂。
12.如权利要求8所述的药物组合物，其特征在于，所述药物组合物采用以下方式施与受试者，包括口服、喷雾吸入、直肠给药、鼻腔给药、阴道给药，以及皮下、静脉、肌内、腹膜内、鞘内、心室内、胸骨内或颅内注射或输入，或借助一种外植的储器用药。
13.如权利要求12所述的药物组合物，其特征在于，所述药物组合物采用以下方式施与受试者，为口服、肌注、腹膜内或静脉内用药方式。
14.权利要求1-7任一项所述含有二苯氨基嘧啶类化合物的制备方法制备得到的化合物、权利要求8-13任一项所述药物组合物在制备HDACs酶作为抑制靶点的套细胞淋巴瘤的治疗药物中的应用。
15.一种抗套细胞淋巴瘤药物，其特征在于，所述药物中，包括活性剂量的权利要求1-7任一项所述含二苯氨基嘧啶类化合物的制备方法制备得到的化合物、或权利要求8-13任一项所述药物组合物。
16.如权利要求15所述的抗套细胞淋巴瘤药物，其特征在于，所述含二苯基氨基嘧啶化合物选自((3-((5-氟-2-((4-(2-甲氧基乙氧基)苯基)氨基)嘧啶-4-基)氨基)苯基)氨基)-N-羟基丁酰胺、5-((3-((5-氟-2-((4-(2-甲氧基乙氧基)苯基)氨基)嘧啶-4-基)氨基)苯基)氨基)-N-羟基戊酰胺、6-((3-((5-氟-2-((4-(2-甲氧基乙氧基)苯基)氨基)嘧啶-4-基)氨基)苯基)氨基)-N-羟基己酰胺、4-(((3-((5-氟-2-((4-(2-甲氧基乙氧基)苯基)氨基)嘧啶-4-基)氨基)苯基)氨基)甲基)-N-羟基苯甲酰胺、N 1-(3-((5-氟-2-((4-(2-甲氧基乙氧基)苯基)氨基)嘧啶-4-基)氨基)苯基)-N 5-羟基戊二酰胺、N 1-(3-((5-氟-2-((4-(2-甲氧基乙氧基)苯基)氨基)嘧啶-4-基)氨基)苯基)-N 6-羟基己二酰胺、N 1-(3-((5-氟-2-((4-(2-甲氧基乙氧基)苯基)氨基)嘧啶-4-基)氨基)苯基)-N 8-羟基辛二酰胺、N-(2-氨基苯基)-6-((3-((5-氟-2-((4-(2-甲氧基乙氧基)苯基)氨基)嘧啶-4-基)氨基)苯基)氨基)己酰胺、N-(2-氨基苯基)-4-(((3-((5-氟-2-((4-(2-甲氧基乙氧基)苯基)氨基)嘧啶-4-基)氨基)苯基)氨基)甲基)苯甲酰胺、1-(4-((2-氨基苯基)氨基)-4-氧代丁基)-N-(3-((5-氟-2-((4-(2-甲氧基乙氧基)苯基)氨基)嘧啶-4-基)氨基)苯基)哌啶-4-甲酰胺、1-(6-((2-氨基苯基)氨基)-6-氧代己基)-N-(3-((5-氟-2-((4-(2-甲氧基乙氧基)苯基)氨基)嘧啶-4-基)氨基)苯基)哌啶-4-甲酰胺、1-(6-((2-氨基苯基)氨基)-6-氧代己基)-N-(3-((5-氟-2-((4-(2-甲氧基乙氧基)苯基)氨基)嘧啶-4-基)氨基)苯基)哌啶-3-甲酰胺、1-(6-((2-氨基苯基)氨基)-6-氧代己基)-N-(3-((5-氟-2-((4-(2-甲氧基乙氧基)苯基)氨基)嘧啶-4-基)氨基)苯基)吡咯烷-3-甲酰胺所示化合物。
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