CN113620956A - 转化生长因子受体拮抗剂、其制备方法和应用 - Google Patents
转化生长因子受体拮抗剂、其制备方法和应用 Download PDFInfo
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- CN113620956A CN113620956A CN202010374368.XA CN202010374368A CN113620956A CN 113620956 A CN113620956 A CN 113620956A CN 202010374368 A CN202010374368 A CN 202010374368A CN 113620956 A CN113620956 A CN 113620956A
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- methylpyridin
- pyridin
- pyrrolo
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- 238000010998 test method Methods 0.000 description 1
- ZZRPJWCNCLSOLR-UHFFFAOYSA-N trimethyl(prop-2-ynoxy)silane Chemical compound C[Si](C)(C)OCC#C ZZRPJWCNCLSOLR-UHFFFAOYSA-N 0.000 description 1
- 230000005760 tumorsuppression Effects 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
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Abstract
本发明涉及一种转化生长因子β受体的小分子拮抗剂,制备该小分子拮抗剂的方法,以及所述小分子拮抗剂在制备药物方面的应用。本发明的转化生长因子β受体的小分子拮抗剂具有治疗和/或预防由ALK5介导的多种疾病的用途,具有极大的临床应用潜力。
Description
技术领域
本发明涉及医药化学领域,具体涉及一种转化生长因子β受体的小分子拮抗剂,制备该小分子拮抗剂的方法,以及所述小分子拮抗剂在制备药物方面的应用。
背景技术
转化生长因子-β(transforming growth factorβ,TGF-β)是一种多功能细胞因子,以自分泌、旁分泌和内分泌的方式通过细胞表面复杂的受体信号传导途径参与调节细胞的增殖,分化和凋亡。TGF-β通路由超过33个TGF-β超家族游离配体和13个TGF-β跨膜激酶受体组成。根据下游信号转导所涉及的效应分子的不同,又分为基于Smad的经典途径和非经典途径两类。TGF-β具有至少3种亚型,分别为TGF-β1、TGF-β2和TGF-β3。TGF-β有3个主要的细胞受体:I型、II型和III型受体。I型和II型受体是跨膜丝氨酸/苏氨酸激酶,二者同时传导信息,III型受体不传递信息,功能主要是将TGF-β传递给II型受体,通过为受体II提供配体而间接地影响信号传导。
研究表明,异常的TGF-β信号和许多种疾病相关,比如癌症、肾纤维化、肝纤维化、肺纤维化、病毒感染、慢性肾炎、急性肾炎、糖尿病肾病、骨质疏松症、关节炎、伤口愈合、溃疡、角膜创伤、心脏瓣膜狭窄、充血性心脏坏死、神经功能损伤、阿尔兹海默综合征、腹膜或皮下粘连、动脉硬化症和肿瘤转移生长等;而TGF-β信号通路中的重要节点TGF-β R1(ALK5)是治疗这些疾病的理想靶点,通过抑制ALK5对其下游信号Smad2或Smad3的磷酸化,阻断或部分阻断TGF-β信号向细胞内的传播,从而纠正异常的TGF-β信号,可以治疗和预防各种ALK5介导的疾病(Nat Rev Drug Discov.2012October,11(10):790-811; Pharmacology&Therapeutics 147(2015)22-31)。
在肿瘤发生过程中,TGF-β通路与肿瘤相关的功能是复杂的。在癌前细胞中抑制肿瘤,而在恶化的癌细胞中促进肿瘤。一方面,在癌变过程中,TGF-β突变不断积累,失去了抑制肿瘤增殖的作用,癌变的细胞不再触发凋亡。另一方面,在特定肿瘤微环境中,如一些消化道癌,TGF-β相关基因表达上调,创造了一种免疫抑制性肿瘤微环境,促进癌细胞的进展和转移。
本发明人意外的发现了一类新的杂环化合物,可作为TGF-β拮抗剂,具有治疗和/或预防由ALK5介导的多种疾病的用途。
发明内容
本发明的目的之一是提供一种新的转化生长因子β受体拮抗剂,具体的,是TGF-β的I 型受体拮抗剂。本发明通过提供如下方案实现:
本发明提供式I结构的化合物,其异构体、混合物、溶剂合物、水合物、前药或其药学上可接受的盐:
其中,Ar是吡啶基,其被0-2个Rx取代;
A选自
Y选自O、-NH-、-N(CH3)-;
X选自
Rb选自氢、氧、硝基、羟基、氰基、卤素、C1-C6烷基、C1-C6卤代烷基、C1-C6烷氧基、 C3-C6环烷基、C3-C6环烯基、羟烷基、羟基取代的烯基、羟基取代的炔基;在某些具体的实施方案中,Rb选自氢、氧、卤素、C1-C6卤代烷基、羟烷基、C3-C6环烯基或羟基取代的炔基;在某些具体的实施方案中,Rb选自氢、氧、氯、氟、CF3、-CH2CH2CH2OH、-CH2CH2CH(CH3)OH、 -C≡CCH(CH3)OH、-C≡CCH2OH、
Rx选自氢、卤素、任选取代的C1-C6烷基、任选取代的C1-C6烷氧基、任选取代的C3-C6环烷基、-NH2、-CHF2、CF3或NHSO2(C1-C6)烷基;
在某些具体的实施方案中,本发明式I化合物中所述X选自
其中Ra、Y分别与前述定义一致。
在某些具体的实施方案中,本发明化合物具有如下式III所示的结构:
其中Ra、Y分别与前述定义一致。
在某些具体的实施方案中,本发明化合物具有如下式IV所示的结构:
在某些具体的实施方案中,本发明化合物具有如下式V所示的结构:
在某些具体的实施方案中,本发明所述Rx为甲基;
在某些具体的实施方案中,本发明所述Ar为2-甲基-吡啶-4-基;
在某些具体的实施方案中,本发明所述X1、X2仅有一个为-N(CH3)-。
在某些具体的实施方案中,本发明化合物具有下述的结构:
本发明的化合物包括其所有异构体(如构象异构体、互变异构体、对映异构体等)、混合物、溶剂合物、水合物、前药或其药学上可接受的盐。
本发明的另一目的是提供一种药物组合物,其包含治疗有效剂量的本发明所述的一种或多种转化生长因子受体拮抗剂,以及药学上可接受的辅料。
本发明的再一目的是提供所述化合物或药物组合物在制备治疗、预防或减少由TGF-β过度表达介导的疾病的药物中的应用。其中,所述TGF-β过度表达介导的疾病包括:癌症、肾纤维化、肝纤维化、肺纤维化、病毒感染、慢性肾炎、急性肾炎、糖尿病肾病、骨质疏松症、关节炎、伤口愈合、溃疡、角膜创伤、心脏瓣膜狭窄、充血性心脏坏死、神经功能损伤、阿尔兹海默综合征、腹膜或皮下粘连、动脉硬化症和肿瘤转移生长等。
本发明进一步提供一种通式II化合物的制备方法:
其中,Rx、Ra、Y的定义如前所述。
1)以化合物II-a为原料,在诸如甲醇钠、乙醇钠、叔丁醇钠等碱的作用下与乙酸乙酯反应生成化合物II-b,反应温度在100℃以上;
2)以化合物II-b为原料,与1H-吡唑-5-胺在冰乙酸和水的混合溶剂中关环得到化合物 II-c,反应由微波引发,并在100℃下进行;
3)以化合物II-c为原料,在卤化试剂诸如三氯氧磷和碱诸如N,N-二异丙基乙胺、三乙胺的作用下,发生卤化反应生成化合物II-d,反应温度在80℃下进行;
4)以化合物II-d为原料,在诸如碳酸钾、氢化钠等碱的作用下与化合物II-e反应生成通式II化合物,反应溶剂常选择丙酮、四氢呋喃等。
本发明进一步提供一种通式IV化合物的制备方法:
其中,Rx、Rb、Y的定义如前所述,Rc为N保护基。
1)当Y选自O时,以化合物II-d为原料,在诸如碳酸钾、氢化钠等碱的作用下与保护的化合物IV-a反应生成IV-1化合物,反应溶剂常选择丙酮、四氢呋喃等。
当Y选自NH时,以化合物II-d为原料,与保护的化合物IV-a经Buchwald偶联生成化合物IV-1;
2)以化合物IV-1为原料,根据不同保护基选择适宜的酸或碱进行去保护,得到通式IV 化合物。酸常选择三氟乙酸,碱常选择氢氧化锂、碳酸钾等。
具体实施方式
术语定义
“烷基”是指直链或含支链的饱和脂族烃基,“C1-C6烷基”是指含有1至6个碳原子的直链或支链饱和脂肪族烃基,示例性的C1-C6烷基包括但不限于甲基、乙基、正丙基、异丙基、正丁基、叔丁基、异丁基、戊基、己基及它们的各种异构体。
“烯基”是指含有指定数目碳原子和至少一个碳碳双键的直链、支链或环状非芳香烃基。
“炔基”是指含有指定数目碳原子和至少一个碳碳三键的直链或支链烃基。
“卤素”是指F、Cl、Br或I;“卤代”是指被选自F、Cl、Br或I的原子所取代。
“羟烷基”是指烷基基团被一个或多个羟基基团所取代,其中烷基基团具有如本发明所述的定义,这样的实例包括,但并不限于羟乙基,2-羟基丙基,羟甲基,1-羟基-1甲基乙基-1- 基等。
“环烷基”指饱和或部分不饱和的单环或多环环状烃取代基。例如,“C3-6环烷基”指包括3 至6个碳原子的环烷基,示例的C3-6环烷基包括但不限于:环丙基、环丁基、环戊基、环戊烯基、环己基、环己烯基等。
“环烯基”是指至少含有一个碳碳双键的环状烃取代基(非芳香族),例如“C3-C6环烯基”是指具有3-6个环原子,且至少含有一个碳碳双键的环状烃基,但不是芳香族。
“烷氧基”是指通过氧桥连接的具有所述碳原子数目的环状或者非环状烷基,“烷氧基”包含以上烷基和环烷基的定义。例如,“C1-6烷氧基”是指通过氧桥链接的具有1至6个碳原子的环状或非环状烷基,示例的C1-6烷氧基包括但不限于:-OMe、-OEt、-O-环烷基等。
“杂环基”是指饱和或部分不饱和的单环或多环状烃取代基,其中一个或多个环原子被选自N、O、S的杂原子取代,其余环原子为碳。例如:“C3-6杂环基”是指包括3至6个环原子的环基,包括但不限于:吡咯烷基、呋喃基、哌啶基、哌嗪基、吗啉基等。
“硝基”是指-NO2;
“羟基”是指-OH;
“氨基”是-NH2;
“氰基”是指-CN。
除非特别说明,本发明所描述的结构式意在包括所有的同分异构形式(如对映异构体、非对应异构体和几何异构体(或构象异构体));例如含有不对称中心的R、S构型,双键的(Z)、(E)异构体等。因此本发明化合物的单个立体化学异构体或其对映异构体、非对映异构体或几何异构体(或构象异构体)的混合物都属于本发明的范围。
本发明所述“溶剂合物”是指本发明化合物与溶剂分子配位形成特定比例的配合物。
本发明所述“水合物”是指本发明化合物与水进行配位形成的配合物。
本发明中所述“药学上可接受的盐”是指本发明化合物与酸或碱所形成的适合用作药物的盐。药学上可接受的盐包括无机盐和有机盐。示例性的酸加成盐包括但不限于:盐酸盐、氢溴酸盐、硫酸盐、硝酸盐、磷酸盐、氢氟酸盐、甲酸盐、乙酸盐、丙酸盐、草酸盐、丙二酸盐、琥珀酸盐、富马酸盐、马来酸盐、乳酸盐、苹果酸盐、酒石酸盐、柠檬酸盐、苦味酸盐、甲磺酸盐、苯甲磺酸盐、苯磺酸盐、天冬氨酸盐、谷氨酸盐等。示例性的碱式盐包括但不限于:碱金属盐、碱土金属盐、有机胺盐等。
本发明中的的缩写均为本领域技术人员已知的,除另有说明外,均代表本领域所通知的含义。
下面通过举例说明本发明的化合物和中间体的合成方法,下述举例仅作为本发明的示例,而不应作为对本发明范围的限制。除特殊说明外,本发明中所涉及的原料和试剂均可通过商业化渠道获得。
制备例1:7-氯-5-(6-甲基吡啶-2-基)吡唑并[1,5-a]嘧啶
步骤1:3-(6-甲基吡啶-2-基)-3-氧代丙酸乙酯的制备
氮气氛下,向甲苯(100mL)中加入乙酸乙酯(20mL),室温下分批加入乙醇钠(9g),反应 1.5h,加入6-甲基吡啶甲酸甲酯(10g),回流过夜。TLC监测原料消耗完毕,浓缩,加水淬灭,用冰乙酸调节pH至7,乙酸乙酯萃取,有机相分别用饱和碳酸氢钠溶液、水和饱和氯化钠溶液洗涤,无水硫酸钠干燥,浓缩得标题化合物12.0g。
MS(ESI)m/z(M+H)+=208.1.
步骤2:5-(6-甲基吡啶-2-基)吡唑并[1,5-a]嘧啶-7-醇的制备
将1H-吡唑-5-胺(2.1g),3-(6-甲基吡啶-2-基)-3-氧代丙酸乙酯(2.0g)溶于冰乙酸(6mL)和水(2mL)中,于微波100℃反应2h,(另外12.0g原料3-(6-甲基吡啶-2-基)-3-氧代丙酸乙酯分6批进行平行反应,合并后处理)。冷却至室温,浓缩,加水和乙酸乙酯后抽滤,滤饼用水、乙酸乙酯洗涤,收集滤液,滤液用乙酸乙酯萃取,有机相用饱和氯化钠溶液洗涤,无水硫酸钠干燥,浓缩除去大部分乙酸乙酯,冰浴下加入石油醚打浆,抽滤,固体合并,干燥,得标题化合物15.0g。
MS(ESI)m/z(M+H)+=227.1.
步骤3:7-氯-5-(6-甲基吡啶-2-基)吡唑并[1,5-a]嘧啶的制备
氮气氛下,将5-(6-甲基吡啶-2-基)吡唑并[1,5-a]嘧啶-7-醇(7g)溶于三氯氧磷(50mL)和 N,N-二异丙基乙胺(15mL)溶液中,体系升温至80℃反应过夜。体系冷至室温,浓缩,浓缩物加入冰水中,用碳酸氢钠粉末调节pH约至7,乙酸乙酯萃取,有机相无水硫酸钠干燥,浓缩,粗品通过柱层析纯化,得标题化合物4.5g。
MS(ESI)m/z(M+H)+=245.1.
制备例2:2-((2-(2-羟基丙烷-2-基)吡啶-4-基)氨基)吡啶-4-醇
氮气氛下,向二氧六环(5mL)中,加入2-(4-氨基吡啶-2-基)丙-2-醇(100mg),2-溴吡啶-4- 醇(136mg),磷酸钾(429mg),三(二亚苄基丙酮)二钯(31mg)和4,5-双二苯基膦-9,9-二甲基氧杂蒽(38mg),于110℃反应8h。加水淬灭,乙酸乙酯萃取,浓缩,粗品经反相制备,冻干得标题化合物75mg。
MS(ESI)m/z(M+H)+=246.1.
制备例3:4-氨基噻唑-5-羧酸乙酯
步骤1:(Z)-氰基碳亚氨基二硫代钾盐的制备
室温下,称取二硫化碳(20g)溶于甲醇(200mL)中,加入50%的氰胺水溶液(22g),在0℃下加入氢氧化钾(29.5g)的水溶液(100mL),恢复至室温反应8h,冷却至0℃,加入碘甲烷(37 g),于室温下反应过夜。浓缩,加入丙酮(350mL),搅拌5min,过滤,滤液浓缩,加入乙酸乙酯,搅拌5min,过滤,滤液浓缩,得标题化合物36g。
MS(ESI)m/z(M+H)+=170.9.
步骤2:4-氨基-2-(甲硫基)噻唑-5-羧酸乙酯的制备
将(Z)-氰基碳亚氨基二硫代钾盐(12g)溶于无水乙醇(200mL)中,冷却至0℃,缓慢加入 2-溴乙酸乙酯(11.7g),80℃反应1h,加入三乙胺(1.43g),于80℃反应3h。TLC显示原料消耗完毕,浓缩,冰水淬灭,搅拌5min,过滤,水洗,浓缩,得标题化合物10.5g。
MS(ESI)m/z(M+H)+=219.0.
步骤3:4-氨基噻唑-5-羧酸乙酯的制备
室温下,称取4-氨基-2-(甲硫基)噻唑-5-羧酸乙酯(10.5g)溶于甲醇(180mL)中,加入锌粉 (9.5g),滴加3mol/L的氯化氢溶液(32.1mL),室温反应6h。TLC显示原料消耗完毕,体系倒入饱和碳酸氢钠溶液(200mL)中,过滤,水洗,二氯甲烷萃取,浓缩,得标题化合物5.5g。
MS(ESI)m/z(M+H)+=173.0.
制备例4:6-甲基吡啶脒盐酸盐
步骤1:6-甲基吡啶甲基亚氨酸甲酯的制备
氮气氛下,将6-甲基吡啶啉(2.5g)溶于甲醇(30mL),加入5M甲醇钠的甲醇溶液(5mL),室温下反应过夜。TLC检测原料消耗完毕,浓缩,加入二氯甲烷溶解,水洗,有机相用无水硫酸钠干燥,浓缩得粗品。
MS(ESI)m/z(M+H)+=151.1.
步骤2:6-甲基吡啶脒盐酸盐的制备
室温下,将上述所得粗品溶于乙醇(40mL)与水(10mL)中,加入氯化铵(753mg),升温至80℃反应4小时,降至室温反应过夜。浓缩,粗品用乙酸乙酯重结晶得标题化合物3.5g。
MS(ESI)m/z(M+H)+=136.1.
制备例5:4-溴-3-氟-1-((2-(三甲基甲硅烷基)乙氧基)甲基)-1H-吡咯并[2,3-b]吡啶的制备
称取4-溴-1-((2-(三甲基甲硅烷基)乙氧基)甲基)-1H-吡咯并[2,3-b]吡啶(2g)溶于N,N-二甲基甲酰胺(10mL)中,加入1-氯甲基-4-氟-1,4-重氮化二环2,2,2辛烷双(四氟硼酸)盐(3.2g)于 60℃下反应4h。加水淬灭,乙酸乙酯萃取,饱和氯化钠溶液洗涤,柱层析纯化得标题化合物385mg。
MS(ESI)m/z(M+H)+=345.1.
制备例6:3,7-二氯-5-(6-甲基吡啶-2-基)吡唑并[1,5-a]嘧啶的制备
称取7-氯-5-(6-甲基吡啶-2-基)吡唑并[1,5-a]嘧啶(250mg),N-氯代丁二酰亚胺(200mg),溶于氯仿(4mL)中,微波90℃反应2h。冷却至室温,加水淬灭,乙酸乙酯萃取,有机相依次用水、饱和氯化钠溶液洗涤,无水硫酸钠干燥,浓缩后柱层析纯化,得标题化合物200mg。
MS(ESI)m/z(M+H)+=279.0.
制备例7:7-氯-5-(6-甲基吡啶-2-基)噻唑并[4,5-d]嘧啶的制备
步骤1:6-甲基吡啶甲酸的制备
称取6-甲基吡啶甲酸甲酯(5g)溶于乙醇(15mL)中,加入5M的氢氧化钠水溶液(70mL),在80℃下反应1h。TLC监测反应完全,冷却至室温,浓缩,加丙酮(100mL),搅拌5min,过滤,滤饼加甲醇溶解,搅拌5min,抽滤,滤饼用甲醇洗涤,滤液浓缩得标题化合物3.3g。
MS(ESI)m/z(M+H)+=138.1.
步骤2:4-(6-甲基吡啶并氨基)噻唑-5-羧酸乙酯的制备
称取6-甲基吡啶甲酸(960mg),4-氨基噻唑-5-羧酸乙酯(1g)溶于三氯氧磷(10mL)中,加入N,N-二异丙基乙胺(2.9mL),在80℃下反应1h。TLC监测原料消耗完毕,冷却至室温,浓缩,浓缩物倒入冰水中,加入碳酸氢钠调节体系pH至8,乙酸乙酯萃取,有机相用无水硫酸钠干燥,浓缩得标题化合物1.5g。
MS(ESI)m/z(M+H)+=292.0.
步骤3:4-(6-甲基吡啶甲酰胺基)噻唑-5-羧酸的制备
称取4-(6-甲基吡啶并氨基)噻唑-5-羧酸乙酯(500mg)溶于甲醇(12mL)中,加入碳酸钾溶液(2.8g溶于水(12mL)中),室温反应过夜。浓缩,加入水和乙酸乙酯,用1M盐酸溶液调节 pH至1-2,浓缩,浓缩物甲醇搅拌,抽滤,滤饼用甲醇洗涤,滤液浓缩得标题化合物800mg。
MS(ESI)m/z(M+H)+=264.0.
步骤4:4-(6-甲基吡啶甲酰胺基)噻唑-5-甲酰胺的制备
称取4-(6-甲基吡啶甲酰胺基)噻唑-5-羧酸(1g)溶于N,N-二甲基甲酰胺(10mL)中,加入 2-(7-氮杂苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(1.74g),N,N-二异丙基乙胺(3.8mL),氯化铵(82mg),室温反应过夜。加水淬灭,二氯甲烷萃取,有机相用饱和氯化钠溶液洗涤,无水硫酸钠干燥,浓缩后柱层析纯化,得标题化合物150mg。
MS(ESI)m/z(M+H)+=263.0.
步骤5:5-(6-甲基吡啶-2-基)噻唑并[4,5-d]嘧啶-7-醇的制备
称取4-(6-甲基吡啶甲酰胺基)噻唑-5-甲酰胺(150mg)溶于三氯甲烷(5mL)中,加入1,8-二氮杂二环十一碳-7-烯(0.21mL),80℃下反应3h。TLC监测原料消耗完毕,冷却至室温,浓缩,柱层析纯化得标题化合物150mg。
MS(ESI)m/z(M+H)+=245.0.
步骤6:7-氯-5-(6-甲基吡啶-2-基)噻唑并[4,5-d]嘧啶的制备
称取5-(6-甲基吡啶-2-基)噻唑并[4,5-d]嘧啶-7-醇(30mg)溶于三氯氧磷(1mL)中,加入N,N- 二异丙基乙胺(44mg),于80℃下反应4h。TLC显示原料消耗完毕,浓缩,用饱和碳酸氢钠溶液淬灭,乙酸乙酯萃取,用无水硫酸钠干燥,浓缩得标题化合物2.0mg。
MS(ESI)m/z(M+H)+=263.0.
制备例8:2-氟-1-((2-(三甲基甲硅烷基)乙氧基)甲基)-1H-吡咯并[2,3-b]吡啶-4-胺的制备
步骤1:4-溴-2-氟-1-((2-(三甲基甲硅烷基)乙氧基)甲基)-1H-吡咯并[2,3-b]吡啶的制备
氩气氛下,称取4-溴-1-((2-(三甲基甲硅烷基)乙氧基)甲基)-1H-吡咯并[2,3-b]吡啶(2g)溶于四氢呋喃(20mL)中,冷却至-78℃,加入二异丙基氨基锂(2g)反应1h,加入N-氟代双苯磺酰胺(2.9g)于-78℃下反应3h。加水淬灭,乙酸乙酯萃取,无水硫酸钠干燥,柱层析纯化得标题化合物1.8g。
MS(ESI)m/z(M+H)+=345.1.
步骤2:N-(2-氟-1-((2-(三甲基甲硅烷基)乙氧基)甲基)-1H-吡咯并[2,3-b]吡啶-4-基)-1,1-二苯基甲胺的制备
氩气氛下,称取4-溴-2-氟-1-((2-(三甲基甲硅烷基)乙氧基)甲基)-1H-吡咯并[2,3-b]吡啶(1.6 g)溶于1,4-二氧六环(15mL)中,加入二苯甲胺(1g),碳酸铯(3g),加入醋酸钯(57mg),4,5- 双二苯基膦-9,9-二甲基氧杂蒽(269mg),于110℃下反应12h。TLC显示原料消耗完毕,过滤,浓缩,柱层析纯化得标题化合物890mg。
MS(ESI)m/z(M+H)+=446.1.
步骤3:2-氟-1-((2-(三甲基甲硅烷基)乙氧基)甲基)-1H-吡咯并[2,3-b]吡啶-4-胺的制备
室温下,称取N-(2-氟-1-((2-(三甲基甲硅烷基)乙氧基)甲基)-1H-吡咯并[2,3-b]吡啶-4- 基)-1,1-二苯基甲胺(880mg)溶于甲醇(8mL)中,加入稀盐酸(4.9mL),于室温下反应1h。TLC 显示原料消耗完毕,加水淬灭,用饱和碳酸氢钠溶液调节pH值至弱碱性,乙酸乙酯萃取,无水硫酸钠干燥,浓缩,柱层析纯化得标题化合物450mg。
MS(ESI)m/z(M+H)+=282.1.
实施例1:7-((1H-吡咯并[2,3-b]吡啶-4-基)氧基)-5-(6-甲基吡啶-2-基)吡唑并[1,5-a]嘧啶的制备
步骤1:1-甲苯磺酰基-1H-吡咯并[2,3-b]吡啶-4-醇的制备
氮气氛下,将1H-吡咯并[2,3-b]吡啶-4-醇(3.0g)溶于N,N-二甲基甲酰胺(80mL)中,冰浴下,分批加入60%氢化钠(2.2g),在冰浴下反应15min,加入对甲苯磺酰氯(5.5g),升至室温反应0.5h,在45℃油浴中反应3h。TLC监测原料消耗完毕,冷却至室温,加水淬灭,乙酸乙酯洗涤三次,保留水相,水相再用1M氯化氢溶液调节pH至5~6,乙酸乙酯萃取,有机相依次用饱和氯化锂溶液、水和饱和氯化钠溶液洗涤,无水硫酸钠干燥,浓缩,粗品通过柱层析纯化,得标题化合物2.5g。
MS(ESI)m/z(M+H)+=289.0.
步骤2:5-(6-甲基吡啶-2-基)-7-((1-甲苯磺酰基-1H-吡咯并[2,3-b]吡啶-4-基)氧基)吡唑并[1,5-a] 嘧啶的制备
氮气氛下,将1-甲苯磺酰基-1H-吡咯并[2,3-b]吡啶-4-醇(200mg)溶于丙酮(5mL)中,加入碳酸钾(210mg),室温反应10min,加入7-氯-5-(6-甲基吡啶-2-基)吡唑并[1,5-a]嘧啶,升温至 55℃反应过夜。冷却至室温,加水抽滤,滤饼依次用水、乙酸乙酯洗涤,干燥,得标题化合物170mg。
MS(ESI)m/z(M+H)+=497.0.
步骤3:7-((1H-吡咯并[2,3-b]吡啶-4-基)氧基)-5-(6-甲基吡啶-2-基)吡唑并[1,5-a]嘧啶的制备
将5-(6-甲基吡啶-2-基)-7-((1-甲苯磺酰基-1H-吡咯并[2,3-b]吡啶-4-基)氧基)吡唑并[1,5-a] 嘧啶(100mg)溶于四氢呋喃(3.6mL),加入2M氢氧化锂溶液(1.2mL),45℃反应22h。冷却至室温,浓缩,加水和乙酸乙酯,抽滤,滤饼用乙酸乙酯洗涤两次,干燥,浓缩得粗品,用反相制备纯化,冻干得标题化合物3.7mg。
MS(ESI)m/z(M+H)+=343.0.
1H NMR(400MHz,DMSO-d6)δ12.17(s,1H),8.38-8.36(m,2H),8.29-8.27(d,J=7.8Hz,1H), 7.90-7.86(t,J=7.8Hz,1H),7.58–7.57(t,J=4Hz,1H),7.38-7.36(d,J=7.6Hz,1H),7.28(s, 1H),7.23(s,0.3H),7.21-7.19(d,J=5.3Hz,1H),7.10(s,0.3H),6.98(s,0.3H),6.93-6.92(d,J= 2.3Hz,1H),6.41-6.40(dd,J=3.5,1.9Hz,1H),2.42(s,3H).
实施例2:5-(6-甲基吡啶-2-基)-N-(1H-吡咯并[2,3-b]吡啶-4-基)吡唑并[1,5-a]嘧啶-7-胺的制备
步骤1:1,1-二苯基-N-(1-((2-(三甲基甲硅烷基)乙氧基)甲基)-1H-吡咯并[2,3-b]吡啶-4-基)甲胺的制备
氩气氛下,称取4-溴-1-((2-(三甲基甲硅烷基)乙氧基)甲基)-1H-吡咯并[2,3-b]吡啶(300 mg),二苯甲胺(144mg),碳酸铯(431mg)溶于二氧六环(5mL)中,加入醋酸钯(8mg),4,5- 双二苯基膦-9,9-二甲基氧杂蒽(39mg),于110℃下反应18h。TLC显示原料消耗完毕,加水淬灭,乙酸乙酯萃取,无水硫酸钠干燥,浓缩,得粗品820mg。
MS(ESI)m/z(M+H)+=428.2.
步骤2:1-((2-(三甲基甲硅烷基)乙氧基)甲基)-1H-吡咯并[2,3-b]吡啶-4-胺的制备
室温下,称取1,1-二苯基-N-(1-((2-(三甲基甲硅烷基)乙氧基)甲基)-1H-吡咯并[2,3-b]吡啶 -4-基)甲胺(770mg)溶于甲醇(6mL)中,加入醋酸钾(707mg),盐酸羟胺(374mg)于室温反应1.5 h。TLC显示原料消耗完毕,加入水淬灭,二氯甲烷萃取,无水硫酸钠干燥,浓缩,粗品用柱层析纯化,得标题化合物230mg。
MS(ESI)m/z(M+H)+=264.1.
步骤3:5-(6-甲基吡啶-2-基)-N-(1-((2-(三甲基甲硅烷基)乙氧基)甲基)-1H-吡咯并[2,3-b]吡啶-4- 基)吡唑并[1,5-a]嘧啶-7-胺的制备
氩气氛下,称取1-((2-(三甲基甲硅烷基)乙氧基)甲基)-1H-吡咯并[2,3-b]吡啶-4-胺(220 mg),7-氯-5-(6-甲基吡啶-2-基)吡唑并[1,5-a]嘧啶(255mg),碳酸铯(565mg)溶于二氧六环(5mL) 中,加入三(二亚苄基丙酮)二钯(40mg),4,5-双二苯基膦-9,9-二甲基氧杂蒽(50mg),于110℃反应8h。加水淬灭,乙酸乙酯萃取,无水硫酸钠干燥,浓缩,粗品用柱层析纯化,得标题化合物230mg。
MS(ESI)m/z(M+H)+=472.2.
步骤4:5-(6-甲基吡啶-2-基)-N-(1H-吡咯并[2,3-b]吡啶-4-基)吡唑并[1,5-a]嘧啶-7-胺的制备
1)室温下,称取5-(6-甲基吡啶-2-基)-N-(1-((2-(三甲基甲硅烷基)乙氧基)甲基)-1H-吡咯并[2,3-b]吡啶-4-基)吡唑并[1,5-a]嘧啶-7-胺(220mg)溶于二氯甲烷(3mL),加入三氟乙酸(1.5mL) 于室温下反应2h。TLC显示原料消耗完毕,浓缩,得粗品130mg。
MS(ESI)m/z(M+H)+=372.1.
2)室温下,上一步得到的粗品溶于甲醇(3mL),加入碳酸钾溶液(控制体系pH在8-9) 于室温下反应3h。TLC显示原料消耗完毕,加水淬灭,析出固体,抽滤,过反相制备得标题化合物12mg。
MS(ESI)m/z(M+H)+=342.1.
1H NMR(400MHz,DMSO-d6)δ11.85(s,1H),10.33(s,1H),8.32–8.28(m,2H),8.25(d,J=7.8 Hz,1H),7.84(t,J=7.7Hz,1H),7.48(dd,J=3.5,2.5Hz,1H),7.44(s,1H),7.32(d,J=7.4Hz, 1H),7.23(d,J=5.2Hz,1H),6.72(d,J=2.3Hz,1H),6.44(dd,J=3.5,1.9Hz,1H),2.42(s,3H).
实施例3:3-氟-5-(6-甲基吡啶-2-基)-N-(1H-吡咯并[2,3-b]吡啶-4-基)吡唑并[1,5-a]嘧啶-7-胺的制备
步骤1:7-氯-3-氟-5-(6-甲基吡啶-2-基)吡唑并[1,5-a]嘧啶的制备
氩气氛下,将7-氯-5-(6-甲基吡啶-2-基)吡唑并[1,5-a]嘧啶(1g)溶于乙腈(40mL)中,加入 N-氟代双苯磺酰胺(2.6g),于60℃下反应8h。浓缩,加水淬灭,乙酸乙酯萃取,无水硫酸钠干燥,浓缩,柱层析纯化得标题化合物200mg。
MS(ESI)m/z(M+H)+=263.0.
步骤2:3-氟-5-(6-甲基吡啶-2-基)-N-(1-((2-(三甲基甲硅烷基)乙氧基)甲基)-1H-吡咯并[2,3-b] 吡啶-4-基)吡唑并[1,5-a]嘧啶-7-胺的制备
称取1-((2-(三甲基甲硅烷基)乙氧基)甲基)-1H-吡咯并[2,3-b]吡啶-4-胺(60mg)溶于N,N-二甲基甲酰胺(2mL)中,冷却至-5℃,加入7-氯-3-氟-5-(6-甲基吡啶-2-基)吡唑并[1,5-a]嘧啶(60 mg),搅拌5min后加入双(三甲基硅烷基)氨基钾(275mg)于-5℃下反应5h。TLC显示原料消耗完毕,加水淬灭,乙酸乙酯萃取,无水硫酸钠干燥,浓缩,得标题化合物35mg。
MS(ESI)m/z(M+H)+=490.1.
步骤3:3-氟-5-(6-甲基吡啶-2-基)-N-(1H-吡咯并[2,3-b]吡啶-4-基)吡唑并[1,5-a]嘧啶-7-胺的制备
1)室温下,称取3-氟-5-(6-甲基吡啶-2-基)-N-(1-((2-(三甲基甲硅烷基)乙氧基)甲基)-1H- 吡咯并[2,3-b]吡啶-4-基)吡唑并[1,5-a]嘧啶-7-胺(35mg)溶于三氟乙酸(1mL)中,室温下反应2 h。TLC显示原料消耗完毕,加水淬灭,乙酸乙酯萃取,无水硫酸钠干燥,浓缩,得粗品10mg。
MS(ESI)m/z(M+H)+=390.1.
2)室温下,将上述粗品(10mg)溶于甲醇(2mL)中,加入饱和碳酸钾溶液调节pH至8-9,于室温下反应4h。TLC显示原料消耗完毕,加水淬灭,乙酸乙酯萃取,无水硫酸钠干燥,浓缩,过反相制备得标题化合物5.89mg。
MS(ESI)m/z(M+H)+=360.1.
1H NMR(400MHz,DMSO-d6)δ11.82(s,1H),8.42(d,J=3.5Hz,1H),8.26(t,J=6.6Hz,2H), 7.83(t,J=7.7Hz,1H),7.46(d,J=3.4Hz,1H),7.34–7.30(m,2H),7.18(d,J=5.2Hz,1H),6.40 (d,J=3.4Hz,1H),2.40(s,3H).
实施例4:5-(5-氟-6-甲基吡啶-2-基)-N-(1H-吡咯并[2,3-b]吡啶-4-基)吡唑并[1,5-a]嘧啶-7-胺的制备
制备方法与前述制备例1和实施例3采用的制备方法类似。
MS(ESI)m/z(M+H)+=360.1.
1H NMR(400MHz,DMSO-d6)δ11.85(s,1H),10.33(s,1H),8.35–8.28(m,3H),7.77(t,J=9.0 Hz,1H),7.50–7.46(m,1H),7.34(s,1H),7.22(d,J=5.2Hz,1H),6.71(d,J=2.3Hz,1H),6.43 (dd,J=3.5,1.8Hz,1H),2.39(d,J=2.9Hz,3H).
实施例5:N-(3-氟-1H-吡咯并[2,3-b]吡啶-4-基)-5-(6-甲基吡啶-2-基)吡唑并[1,5-a]嘧啶-7-胺的制备
制备方法与前述制备例5和实施例2采用的制备方法类似。
MS(ESI)m/z(M+H)+=360.1.
1H NMR(400MHz,DMSO-d6)δ11.77(s,1H),10.31(s,1H),8.36(d,J=5.2Hz,1H),8.31(d,J= 2.4Hz,1H),8.25(d,J=7.8Hz,1H),7.87(t,J=7.8Hz,1H),7.50(t,J=2.5Hz,1H),7.44(s,1H), 7.36(d,J=7.6Hz,1H),7.26(d,J=5.3Hz,1H),6.73(d,J=2.3Hz,1H),2.46(s,3H).
实施例6:3-氯-5-(6-甲基吡啶-2-基)-N-(1H-吡咯并[2,3-b]吡啶-4-基)吡唑并[1,5-a]嘧啶-7-胺的制备
制备方法与前述制备例6和实施例3采用的制备方法类似。
MS(ESI)m/z(M+H)+=376.1.
1H NMR(400MHz,DMSO-d6)δ11.79(s,1H),9.98(br,1H),8.40(s,1H),8.29-8.26(m,2H), 7.87-7.83(t,J=7.7Hz,1H),7.46-7.45(m,1H),7.35-7.31(m,2H),7.16-7.15(d,J=5.2Hz,1H), 6.40-6.39(d,J=3.5Hz,1H),2.41(s,3H).
实施例7:5-(6-甲基吡啶-2-基)-N-(2-(三氟甲基)-1H-吡咯并[2,3-b]吡啶-4-基)吡唑并[1,5-a]嘧啶 -7-胺三氟乙酸盐的制备
步骤1:4-溴-1-((2-(三甲基甲硅烷基)乙氧基)甲基)-1H-吡咯并[2,3-b]吡啶的制备
氩气氛下,称取4-溴-1H-吡咯并[2,3-b]吡啶(3.0g)溶于干燥的N,N-二甲基甲酰胺(30mL) 中,冰浴下,加入60%氢化钠溶液(900mg),反应10min,加入2-(三甲基硅烷基)乙氧甲基氯(4mL),室温反应3h。TLC监测原料消耗完毕,加水淬灭,乙酸乙酯萃取,有机相依次用水、饱和氯化钠溶液洗涤,无水硫酸钠干燥,浓缩,柱层析纯化,得标题化合物4.3g。
MS(ESI)m/z(M+H)+=327.1.
步骤2:1,1-二苯基-N-(1-((2-(三甲基甲硅烷基)乙氧基)甲基)-1H-吡咯并[2,3-b]吡啶-4-基)甲胺的制备
氩气氛下,称取4-溴-1-((2-(三甲基甲硅烷基)乙氧基)甲基)-1H-吡咯并[2,3-b]吡啶(6.0g),二苯甲胺(3.5g),碳酸铯(12g)加入1,4-二氧六环(100mL)中,加入醋酸钯(210mg),4,5-双(二苯基膦)-9,9-二甲基氧杂蒽(1g),105℃反应过夜。TLC监测原料消耗完毕,冷却至室温,过滤,滤液浓缩,柱层析纯化,得标题化合物6.1g。
MS(ESI)m/z(M+H)+=428.1.
步骤3:N-(2-碘-1-((2-(三甲基甲硅烷基)乙氧基)甲基)-1H-吡咯并[2,3-b]吡啶-4-基)-1,1-二苯基甲胺的制备
称取1,1-二苯基-N-(1-((2-(三甲基甲硅烷基)乙氧基)甲基)-1H-吡咯并[2,3-b]吡啶-4-基)甲胺 (3g)溶于四氢呋喃(30mL)中,-70℃下滴入二异丙基氨基锂的2M四氢呋喃溶液(10.5mL),该温度下搅拌反应1h后,滴入碘(3.5g)的四氢呋喃(40mL)溶液,反应1h。加入饱和氯化铵溶液淬灭,加亚硫酸氢钠,再加水,乙酸乙酯萃取,有机相用饱和氯化钠溶液洗涤,无水硫酸钠干燥,浓缩,柱层析纯化,得标题化合物800mg。
MS(ESI)m/z(M+H)+=554.1.
步骤4:2-碘-1-((2-(三甲基甲硅烷基)乙氧基)甲基)-1H-吡咯并[2,3-b]吡啶-4-胺的制备
室温下,称取N-(2-碘-1-((2-(三甲基甲硅烷基)乙氧基)甲基)-1H-吡咯并[2,3-b]吡啶-4- 基)-1,1-二苯基甲胺(400mg)溶于甲醇(5mL)中,加入2M盐酸水溶液(1.8mL),室温反应2h。 LCMS监测原料消耗完毕,浓缩,加水淬灭,乙酸乙酯萃取,用饱和碳酸氢钠溶液调节pH 至8-9,分出有机相,水相用乙酸乙酯萃取,合并有机相,无水硫酸钠干燥,浓缩,柱层析纯化得标题化合物240mg。
MS(ESI)m/z(M+H)+=390.0.
步骤5:N-(2-碘-1-((2-(三甲基甲硅烷基)乙氧基)甲基)-1H-吡咯并[2,3-b]吡啶-4-基)-5-(6-甲基吡啶-2-基)吡唑[1,5-a]嘧啶-7-胺的制备
氩气氛下,称取2-碘-1-((2-(三甲基甲硅烷基)乙氧基)甲基)-1H-吡咯并[2,3-b]吡啶-4-胺(240 mg)溶于四氢呋喃(3mL)中,-10℃下,加入双(三甲基硅基)氨基钾的四氢呋喃溶液(1.24mL, 1mol/L),-10℃反应10min,加入7-氯-5-(6-甲基吡啶-2-基)吡唑并[1,5-a]嘧啶(158mg)的四氢呋喃溶液(3mL),-10℃反应10min。TLC监测原料消耗完毕,加入饱和氯化铵溶液淬灭,加水、乙酸乙酯萃取,有机相用饱和氯化钠溶液洗涤,无水硫酸钠干燥,浓缩,柱层析纯化得标题化合物340mg。
MS(ESI)m/z(M+H)+=598.1.
步骤6:5-(6-甲基吡啶-2-基)-N-(2-(三氟甲基)-1-(2-(三甲基甲硅烷基)乙氧基)甲基)-1H-吡咯并 [2,3-b]吡啶-4-基吡唑并[1,5-a]嘧啶-7-胺的制备
氩气氛下,称取N-(2-碘-1-((2-(三甲基甲硅烷基)乙氧基)甲基)-1H-吡咯并[2,3-b]吡啶-4- 基)-5-(6-甲基吡啶-2-基)吡唑[1,5-a]嘧啶-7-胺(30mg)溶于N,N-二甲基甲酰胺(1mL)中,加入 2,2-二氟-2-(氟磺酰基)乙酸甲酯(30mg),碘化亚铜(3mg),90℃反应3h。LCMS监测原料消耗完毕,体系冷却至室温,过滤,滤液加水,乙酸乙酯萃取,合并有机相用饱和氯化钠溶液洗涤,无水硫酸钠干燥,浓缩,得标题化合物20mg。
MS(ESI)m/z(M+H)+=540.2.
步骤7:5-(6-甲基吡啶-2-基)-N-(2-(三氟甲基)-1H-吡咯并[2,3-b]吡啶-4-基)吡唑并[1,5-a]嘧啶-7- 胺三氟乙酸盐的制备
室温下,称取5-(6-甲基吡啶-2-基)-N-(2-(三氟甲基)-1-(2-(三甲基甲硅烷基)乙氧基)甲基)-1H-吡咯并[2,3-b]吡啶-4-基吡唑并[1,5-a]嘧啶-7-胺(20mg)溶于二氯甲烷(1mL)中,加入三氟乙酸(0.5mL),室温反应过夜。LCMS监测原料消耗完毕,浓缩,加水淬灭,乙酸乙酯萃取,用饱和碳酸氢钠溶液调节pH至8~9,分离出有机相,水相用乙酸乙酯萃取,合并有机相用饱和氯化钠溶液洗涤,无水硫酸钠干燥,浓缩,用反相制备纯化,得标题化合物2.1mg。
MS(ESI)m/z(M+H)+=410.1.
1H NMR(400MHz,DMSO-d6)δ13.13(s,1H),10.64(s,1H),8.48(s,1H),8.33(m,1H),8.27-8.25(d,J=7.8Hz,1H),7.88-7.84(t,J=7.7Hz,1H),7.58(s,1H),7.36-7.34(m,2H),7.22 (s,0.6H),7.19(s,1H),7.09(s,0.6H),6.97(s,0.6H),6.76(s,1H),2.44(s,3H).
实施例8:N-甲基-5-(6-甲基吡啶-2-基)-N-(1H-吡咯并[2,3-b]吡啶-4-基)吡唑并[1,5-a]嘧啶-7-胺的制备
步骤1:5-(6-甲基吡啶-2-基)-N-(1-((2-(三甲基甲硅烷基)乙氧基)甲基)-1H-吡咯并[2,3-b]吡啶-4- 基)吡唑并[1,5-a]嘧啶-7-胺的制备
称取7-氯-5-(6-甲基吡啶-2-基)吡唑并[1,5-a]嘧啶(160mg)溶于四氢呋喃(3mL)中,加入 1-((2-(三甲基甲硅烷基)乙氧基)甲基)-1H-吡咯并[2,3-b]吡啶-4-胺(172mg),将体系冷却至0℃,加双(三甲基硅烷基)氨基钾(261mg)于0℃下反应8h。加水淬灭,乙酸乙酯萃取,无水硫酸钠干燥,柱层析纯化得标题化合物200mg。
MS(ESI)m/z(M+H)+=472.2.
步骤2:N-甲基-5-(6-甲基吡啶-2-基)-N-(1-((2-(三甲基甲硅烷基)乙氧基)甲基)-1H-吡咯并[2,3-b] 吡啶-4-基)吡唑并[1,5-a]嘧啶-7-胺的制备
室温下,将5-(6-甲基吡啶-2-基)-N-(1-((2-(三甲基甲硅烷基)乙氧基)甲基)-1H-吡咯并[2,3-b] 吡啶-4-基)吡唑并[1,5-a]嘧啶-7-胺(180mg)溶于N,N-二甲基甲酰胺(3mL)中,冷却至0℃,加双(三甲基硅烷基)氨基钾(152mg),搅拌30min后加入碘甲烷(82mg),于室温下反应1.5h。TLC显示原料消耗完毕,加入水淬灭,乙酸乙酯萃取,无水硫酸钠干燥,Pre-TLC纯化得标题化合物140mg。
MS(ESI)m/z(M+H)+=486.2.
步骤3:N-甲基-5-(6-甲基吡啶-2-基)-N-(1H-吡咯并[2,3-b]吡啶-4-基)吡唑并[1,5-a]嘧啶-7-胺的制备
1)室温下,称取N-甲基-5-(6-甲基吡啶-2-基)-N-(1-((2-(三甲基甲硅烷基)乙氧基)甲基)-1H- 吡咯并[2,3-b]吡啶-4-基)吡唑并[1,5-a]嘧啶-7-胺(140mg)溶于三氟乙酸(1.5mL)中,于室温下反应3h。TLC显示原料消耗完毕,加水淬灭,乙酸乙酯萃取,无水硫酸钠干燥,得粗品80mg。
MS(ESI)m/z(M+H)+=386.2.
2)室温下,将上述粗品(80mg)溶于甲醇(1.5mL)中,加入饱和碳酸钾调节pH至8-9于室温下反应4h。TLC显示原料消耗完毕,加水淬灭,乙酸乙酯萃取,无水硫酸钠干燥,过反相制备得标题化合物30mg。
MS(ESI)m/z(M+H)+=356.2.
1H NMR(400MHz,DMSO-d6)δ12.47(s,1H),8.35(d,J=7.8Hz,1H),8.28(d,J=6.6Hz,1H), 8.16(d,J=2.3Hz,1H),8.08(s,1H),7.94(t,J=7.8Hz,1H),7.44(d,J=7.6Hz,1H),7.26(dd,J =3.7,2.1Hz,1H),7.01(d,J=6.6Hz,1H),6.94(d,J=2.3Hz,1H),5.48(dd,J=3.8,1.5Hz,1H), 3.86(s,3H),2.57(s,3H).
实施例9:3-(4-((5-(6-甲基吡啶-2-基)吡唑并[1,5-a]嘧啶-7-基)氨基)-1H-吡咯并[2,3-b]吡啶-2- 基)丙-2-炔-1-醇的制备
步骤1:3-(4-((5-(6-甲基吡啶-2-基)吡唑并[1,5-a]嘧啶-7-基)氨基)-1-((2-(三甲基甲硅烷基)乙氧基)甲基)-1H吡咯并[2,3-b]吡啶-2-基)丙-2-炔-1-醇的制备
氩气氛下,称取N-(2-碘-1-((2-(三甲基甲硅烷基)乙氧基)甲基)-1H-吡咯并[2,3-b]吡啶-4- 基)-5-(6-甲基吡啶-2-基)吡唑[1,5-a]嘧啶-7-胺(200mg),三甲基(丙-2-炔-1-基氧基)硅烷(132 mg),三乙胺(0.5mL)溶于干燥的四氢呋喃(2mL)中,加入双三苯基膦二氯化钯(30mg),碘化亚铜(8mg),45℃反应过夜。冷却至室温,抽滤,滤液浓缩,柱层析纯化,得标题化合物 170mg。
MS(ESI)m/z(M+H)+=526.3.
步骤2:3-(4-((5-(6-甲基吡啶-2-基)吡唑并[1,5-a]嘧啶-7-基)氨基)-1H-吡咯并[2,3-b]吡啶-2-基) 丙-2-炔-1-醇的制备
室温下,称取3-(4-((5-(6-甲基吡啶-2-基)吡唑并[1,5-a]嘧啶-7-基)氨基)-1-((2-(三甲基甲硅烷基)乙氧基)甲基)-1H吡咯并[2,3-b]吡啶-2-基)丙-2-炔-1-醇(170mg)溶于二氯甲烷(10mL) 中,加入三氟乙酸(5mL),室温搅拌2h。浓缩,加甲醇溶解,用饱和碳酸钾溶液调节pH至 8-9,室温反应2h。浓缩,加水、乙酸乙酯萃取,有机相用无水硫酸钠干燥,浓缩,经Prep-HPLC 分离,得标题化合物1.53mg。
MS(ESI)m/z(M+H)+=396.1.
1H NMR(400MHz,DMSO-d6)δ12.49(s,1H),10.61(s,1H),8.33–8.32(m,2H),8.26-.8.24(d, J=7.8Hz,1H),7.89-7.85(t,J=7.7Hz,1H),7.52(s,1H),7.38-7.36(d,J=7.6Hz,1H), 7.25-7.23(d,J=5.5Hz,1H),7.22(s,0.2H),7.09(s,0.2H),6.96(s,0.2H),6.79-6.76(dd,J= 10.5,2.1Hz,2H),4.34(s,2H),2.47(s,3H).
实施例10:3-(4-((5-(6-甲基吡啶-2-基)吡唑并[1,5-a]嘧啶-7-基)氨基)-1H-吡咯并[2,3-b]吡啶-2- 丙基-1-醇的制备
室温下,称取3-(4-((5-(6-甲基吡啶-2-基)吡唑并[1,5-a]嘧啶-7-基)氨基)-1H-吡咯并[2,3-b] 吡啶-2-基)丙-2-炔-1-醇(15mg)溶于甲醇(1mL)中,加入少许2M氯化氢水溶液,加入10%钯碳(5mg),室温反应1.5h。过滤,滤液经Prep-HPLC分离,得到标题化合物2.89mg。
MS(ESI)m/z(M+H)+=400.2.
1H NMR(400MHz,DMSO-d6)δ12.24(s,1H),10.79(s,1H),8.32(d,J=2.3Hz,1H),8.27-8.25 (d,J=7.9Hz,1H),8.24-8.22(d,J=6.0Hz,1H),7.89-7.86(t,J=7.8Hz,1H),7.58(s,1H), 7.38-7.37(d,J=7.6Hz,1H),7.22-7.21(d,J=5.9Hz,1H),6.79(d,J=2.3Hz,1H),6.38(s,1H), 3.45-3.41(t,J=6.3Hz,2H),2.80-2.76(t,J=7.6Hz,2H),2.47(s,3H),1.84–1.77(m,2H).
实施例11:(S)-4-(4-((5-(6-甲基吡啶-2-基)吡唑并[1,5-a]嘧啶-7-基)氨基)-1H-吡咯并[2,3-b]吡啶 -2-基)丁-3-炔-2-醇的制备
制备方法与前述实施例9采用的制备方法类似。
MS(ESI)m/z(M+H)+=410.1.
1H NMR(400MHz,Methanol-d4)δ8.32(d,J=5.9Hz,1H),8.29(d,J=2.3Hz,1H),8.19-8.17 (d,J=7.9Hz,1H),7.99(t,J=7.8Hz,1H),7.53-7.51(m,2H),7.37-7.36(d,J=6.0Hz,1H),6.92 (s,1H),6.81(d,J=2.3Hz,1H),4.75-4.71(dd,J=6.7Hz,J=6.7Hz,1H),2.69(s,3H),1.50(d,J =6.7Hz,3H).
实施例12:(S)-4-(4-((5-(6-甲基吡啶-2-基)吡唑并[1,5-a]嘧啶-7-基)氨基)-1H-吡咯并[2,3-b]吡啶 -2-基)丁烷-2-醇的制备
制备方法与前述实施例10采用的制备方法类似。
MS(ESI)m/z(M+H)+=414.2.
1H NMR(400MHz,Methanol-d4)δ8.27(d,J=2.3Hz,1H),8.23-8.21(m,2H),7.96-7.92(t,J= 7.8Hz,1H),7.64(s,1H),7.48-7.46(d,J=7.7Hz,1H),7.41-7.40(d,J=6.4Hz,1H),6.81-6.80(d, J=2.3Hz,1H),6.58-6.57(m,1H),3.83-3.75(dtd,J=7.9,6.2,4.7Hz,1H),3.02–2.86(m,2H), 2.65(s,3H),1.92-1.78(tdd,J=9.2,7.3,5.3Hz,2H),1.19-1.17(d,J=6.2Hz,3H).
实施例13:(R)-4-(4-((5-(6-甲基吡啶-2-基)吡唑并[1,5-a]嘧啶-7-基)氨基)-1H-吡咯并[2,3-b]吡啶 -2-基)丁-3-炔-2-醇的制备
制备方法与前述实施例9采用的制备方法类似。
MS(ESI)m/z(M+H)+=410.1.
1H NMR(400MHz,Methanol-d4)δ8.33-8.32(d,J=6.0Hz,1H),8.30-8.29(d,J=2.3Hz,1H), 8.19-8.17(d,J=7.9Hz,1H),8.02-7.98(t,J=7.8Hz,1H),7.54-7.52(d,J=8.1Hz,2H), 7.37-7.36(d,J=6.0Hz,1H),6.93(s,1H),6.82-6.81(d,J=2.3Hz,1H),4.76-4.71(dd,J=6.6Hz, J=6.6Hz,1H),2.69(s,3H),1.51-1.50(d,J=6.7Hz,3H).
实施例14:(R)-4-(4-((5-(6-甲基吡啶-2-基)吡唑并[1,5-a]嘧啶-7-基)氨基)-1H-吡咯并[2,3-b]吡啶 -2-基)丁烷-2-醇的制备
制备方法与前述实施例10采用的制备方法类似。
MS(ESI)m/z(M+H)+=414.2.
1H NMR(400MHz,Methanol-d4)δ8.28-8.27(d,J=2.3Hz,1H),8.23-8.20(m,2H),7.96-7.92(t, J=7.8Hz,1H),7.64(s,1H),7.48-7.46(d,J=7.7Hz,1H),7.41-7.40(d,J=6.4Hz,1H), 6.81-6.80(d,J=2.3Hz,1H),6.58(d,J=1.1Hz,1H),3.84–3.74(m,1H),3.01-2.85(m,2H), 2.65(s,3H),1.92-1.79(m,2H),1.19-1.17(d,J=6.2Hz,3H).
实施例15:3-(4-((5-(6-甲基吡啶-2-基)吡唑并[1,5-a]嘧啶-7-基)氨基)-1H-吡咯并[2,3-b]吡啶-2- 基)环戊-3-烯-1-醇的制备
步骤1:N-(2-(4-((叔丁基二苯基甲硅烷基)氧基)环戊-1-烯-1-基)-1-((2-(三甲基甲硅烷基)乙氧基) 甲基)-1H-吡咯并[2,3-b]吡啶-4-基)-5-(6-甲基吡啶-2-基)吡唑并[1,5-a]嘧啶-7-胺的制备
氩气氛下,将N-(2-碘-1-((2-(三甲基甲硅烷基)乙氧基)甲基)-1H-吡咯并[2,3-b]吡啶-4- 基)-5-(6-甲基吡啶-2-基)吡唑[1,5-a]嘧啶-7-胺(100mg),叔丁基二苯基(3-(4,4,5,5-四甲基-1,3,2- 二氧杂硼硼烷-2-基)环戊-3-烯-1-基)氧基)硅烷(113mg),碳酸铯(270mg)溶于1,4-二氧六环(3 mL)和水(0.3mL)的混合溶剂中,加入[1,1'-双(二苯基膦)二茂铁]二氯化钯二氯甲烷络合物(14 mg),微波150℃反应6h,。冷却至室温,抽滤,浓缩,柱层析纯化得标题化合物55mg。
MS(ESI)m/z(M+H)+=792.3.
步骤2:3-(4-((5-(6-甲基吡啶-2-基)吡唑并[1,5-a]嘧啶-7-基)氨基)-1-((2-(三甲基甲硅烷基)乙氧基)甲基)-1H吡咯并[2,3-b]吡啶-2-基)环戊-3-烯-1-醇的制备
室温下,称取N-(2-(4-((叔丁基二苯基甲硅烷基)氧基)环戊-1-烯-1-基)-1-((2-(三甲基甲硅烷基)乙氧基)甲基)-1H-吡咯并[2,3-b]吡啶-4-基)-5-(6-甲基吡啶-2-基)吡唑并[1,5-a]嘧啶-7-胺(55 mg)溶于1M叔丁基氟化铵的四氢呋喃溶液(4mL)中,室温反应3h。TLC监测原料消耗完毕,加饱和氯化铵溶液淬灭,乙酸乙酯萃取,有机相用水洗涤,无水硫酸钠干燥,浓缩,柱层析纯化得标题化合物20mg。
MS(ESI)m/z(M+H)+=554.3.
步骤3:3-(4-((5-(6-甲基吡啶-2-基)吡唑并[1,5-a]嘧啶-7-基)氨基)-1H-吡咯并[2,3-b]吡啶-2-基) 环戊-3-烯-1-醇的制备
室温下,称取3-(4-((5-(6-甲基吡啶-2-基)吡唑并[1,5-a]嘧啶-7-基)氨基)-1-((2-(三甲基甲硅烷基)乙氧基)甲基)-1H吡咯并[2,3-b]吡啶-2-基)环戊-3-烯-1-醇(20mg)溶于二氯甲烷(2mL) 中,加入三氟乙酸(0.7mL),室温搅拌反应2h。浓缩,甲醇溶解,用饱和碳酸钾溶液调节 pH至8-9,室温反应2h。浓缩,甲醇溶解,抽滤,滤饼用甲醇洗涤,滤液浓缩,经Prep-HPLC 纯化,得标题化合物7.06mg。
MS(ESI)m/z(M+H)+=424.2.
1H NMR(400MHz,Methanol-d4)δ8.28(d,J=2.4Hz,1H),8.24-8.22(d,J=6.4Hz,1H), 8.20-8.18(d,J=7.8Hz,1H),7.97-7.93(t,J=7.8Hz,1H),7.62(s,1H),7.49-7.47(d,J=7.7Hz, 1H),7.40-7.38(d,J=6.3Hz,1H),6.81-6.80(d,J=2.4Hz,1H),6.72(s,1H),6.42(s,1H), 4.62-4.59(dt,J=6.4,3.7Hz,1H),3.07–2.86(m,2H),2.69-2.65(m,4H),2.58-2.53(d,J=18.4 Hz,1H).
实施例16:5-(6-甲基吡啶-2-基)-N-(吡啶-4-基)吡唑并[1,5-a]嘧啶-7-胺的制备
称取7-氯-5-(6-甲基吡啶-2-基)吡唑并[1,5-a]嘧啶(50mg)溶于四氢呋喃(2mL)中,加入吡啶-4-胺(24mg),氢化钠(10mg)于60℃反应2h。TLC显示原料消耗完毕,加水淬灭,乙酸乙酯萃取,浓缩,反相制备得标题化合物26mg。
MS(ESI)m/z(M+H)+=303.1.
1H NMR(400MHz,DMSO-d6)δ9.26(s,1H),8.61–8.57(m,2H),8.30–8.24(m,2H),7.86(t,J= 7.7Hz,1H),7.80(s,1H),7.59–7.53(m,2H),7.37(d,J=7.5Hz,1H),6.73(d,J=2.3Hz,1H), 2.55(s,3H).
实施例17:N4-(5-(6-甲基吡啶-2-基)吡唑并[1,5-a]嘧啶-7-基)吡啶-2,4-二胺的制备
步骤1:N-(2-溴吡啶-4-基)-5-(6-甲基吡啶-2-基)吡唑并[1,5-a]嘧啶-7-胺的制备
称取2-溴吡啶-4-胺(60mg)溶于四氢呋喃(1mL)中,加入氢化钠(12mg)反应30min,加入 7-氯-5-(6-甲基吡啶-2-基)吡唑并[1,5-a]嘧啶(47mg),于60℃反应1h。TLC显示原料消耗完毕,加水淬灭,乙酸乙酯萃取,无水硫酸钠干燥,浓缩,柱层析纯化得标题化合物57mg。
MS(ESI)m/z(M+H)+=381.0.
步骤2:N4-(5-(6-甲基吡啶-2-基)吡唑并[1,5-a]嘧啶-7-基)吡啶-2,4-二胺的制备
称取N-(2-溴吡啶-4-基)-5-(6-甲基吡啶-2-基)吡唑并[1,5-a]嘧啶-7-胺(30mg)溶于氨水(1 mL)中,加入铜粉(4mg)于100℃反应8h。TLC显示原料消耗完毕,加硫化钠溶液(3mL)淬灭,过滤,二氯甲烷萃取,无水硫酸钠干燥,浓缩,反相制备得标题化合物13.6mg。
MS(ESI)m/z(M+H)+=318.1.
1H NMR(400MHz,DMSO-d6)δ10.07(s,1H),8.29–8.24(m,2H),7.95(d,J=5.6Hz,1H),7.86 (t,J=7.7Hz,1H),7.71(s,1H),7.37(d,J=7.5Hz,1H),6.69(dd,J=4.3,2.1Hz,2H),6.58(d,J= 1.9Hz,1H),6.09(s,2H),2.56(s,3H).
实施例18:N4-(3-氟-5-(6-甲基吡啶-2-基)吡唑并[1,5-a]嘧啶-7-基)吡啶-2,4-二胺的制备
室温下,称取N-(4-氨基吡啶-2-基)乙酰胺(15mg)溶于四氢呋喃(1mL)中,加入氢化钠(5mg) 于室温下搅拌20min,加入7-氯-3-氟-5-(6-甲基吡啶-2-基)吡唑并[1,5-a]嘧啶(15mg)于室温下反应1h。TLC显示原料消耗完毕,加水淬灭,乙酸乙酯萃取,无水硫酸钠干燥,浓缩得标题化合物21mg。
MS(ESI)m/z(M+H)+=378.1.
步骤2:N4-(3-氟-5-(6-甲基吡啶-2-基)吡唑并[1,5-a]嘧啶-7-基)吡啶-2,4-二胺的制备
称取N-(4-((3-氟-5-(6-甲基吡啶-2-基)吡唑并[1,5-a]嘧啶-7-基)氨基)吡啶-2-基)乙酰胺(21 mg)溶于甲醇(1mL)中,加入氢氧化钾(1mL,1mol/L)于60℃下反应5h。TLC显示原料消耗完毕,加水淬灭,乙酸乙酯萃取,无水硫酸钠干燥,浓缩,反相制备得标题化合物2mg。
MS(ESI)m/z(M+H)+=336.1.
1H NMR(400MHz,DMSO-d6)δ10.43(s,1H),8.45(d,J=3.5Hz,1H),8.28(d,J=7.8Hz,1H), 7.96(d,J=5.9Hz,1H),7.89(t,J=7.7Hz,1H),7.73(s,1H),7.40(d,J=7.6Hz,1H),6.76(dd,J =5.9,2.0Hz,1H),6.64(d,J=2.0Hz,1H),6.47(s,2H),2.56(s,3H).
实施例19:2-(4-((4-((5-(6-甲基吡啶-2-基)吡唑并[1,5-a]嘧啶-7-基)氨基)吡啶-2-基)氨基)吡啶-2- 基)丙-2-醇的制备
氩气氛下,称取N-(2-溴吡啶-4-基)-5-(6-甲基吡啶-2-基)吡唑并[1,5-a]嘧啶-7-胺(36mg), 2-(4-氨基吡啶-2-基)丙-2-醇(15mg),叔丁醇钠(20mg)溶于二氧六环(1mL)中,加入三(二亚苄基丙酮)二钯(5mg)和4,5-双二苯基膦-9,9-二甲基氧杂蒽(6mg),于100℃反应4h。TLC显示原料消耗完毕,加水淬灭,乙酸乙酯萃取,无水硫酸钠干燥,浓缩,反相制备得标题化合物3.4mg。
MS(ESI)m/z(M+H)+=453.2.
1H NMR(400MHz,DMSO-d6)δ10.39(s,1H),9.69(s,1H),8.31–8.26(m,3H),8.23–8.20(m, 1H),7.88(t,J=7.8Hz,1H),7.80–7.74(m,3H),7.38(d,J=7.5Hz,1H),7.10(d,J=4.6Hz,2H), 6.74(d,J=2.3Hz,1H),5.14(s,1H),2.54(s,3H),1.42(s,6H).
实施例20:5-(6-甲基吡啶-2-基)-7-(吡啶-4-基氧基)吡唑并[1,5-a]嘧啶的制备
称取7-氯-5-(6-甲基吡啶-2-基)吡唑并[1,5-a]嘧啶(50mg)溶于N,N-二甲基甲酰胺(2mL)中,加入吡啶-4-醇(24mg),碳酸钾(57mg)于80℃下反应1.5h。TLC显示原料消耗完毕,加水淬灭,固体析出,抽滤,旋干,过反相制备得标题化合物42mg。
MS(ESI)m/z(M+H)+=304.1.
1H NMR(400MHz,DMSO-d6)δ8.39(d,J=2.4Hz,1H),8.32(d,J=7.8Hz,1H),8.25–8.22(m, 2H),8.10(s,1H),7.95(t,J=7.8Hz,1H),7.47(d,J=7.6Hz,1H),7.04(d,J=2.4Hz,1H), 6.38–6.34(m,2H),2.62(s,3H).
实施例21:2-(4-((4-((5-(6-甲基吡啶-2-基)吡唑并[1,5-a]嘧啶-7-基)氧基)吡啶-2-基)氨基)吡啶 2-基)丙-2-醇的制备
制备方法与前述实施例20和实施例19采用的制备方法类似。
MS(ESI)m/z(M+H)+=454.1.
1H NMR(400MHz,DMSO-d6)δ9.73(s,1H),8.40(d,J=5.7Hz,1H),8.34(d,J=2.3Hz,1H), 8.30(d,J=7.9Hz,1H),8.2–8.24(m,1H),7.91(t,J=7.8Hz,1H),7.78–7.74(m,2H),7.52(s,1H), 7.42(d,J=7.6Hz,1H),7.01(dd,J=5.7,2.2Hz,1H),6.94(d,J=2.3Hz,1H),6.82(d,J=2.2Hz, 1H),5.15(s,1H),2.52(s,3H)1.42(s,6H).
实施例22:2-(4-((4-((5-(6-甲基吡啶-2-基)噻唑[4,5-d]嘧啶-7-基)氧基)吡啶-2-基)氨基)吡啶-2- 基)丙-2-醇的制备
制备方法与前述实施例20采用的制备方法类似。
MS(ESI)m/z(M+H)+=472.1.
1H NMR(400MHz,Methanol-d4)δ9.84(s,1H),8.41(d,J=5.7Hz,1H),8.20(d,J=5.8Hz,1H), 8.12(d,J=7.8Hz,1H),7.90(d,J=2.2Hz,1H),7.81(t,J=7.8Hz,1H),7.73(dd,J=5.8,2.3Hz, 1H),7.39(d,J=7.7Hz,1H),7.05–6.98(m,2H),2.64(s,3H),1.53(s,6H).
实施例23:5-(6-甲基吡啶-2-基)-N-(1H-吡唑-4-基)吡唑并[1,5-a]嘧啶-7-胺的制备
制备方法与前述实施例16采用的制备方法类似。
MS(ESI)m/z(M+H)+=292.1.
1H NMR(400MHz,DMSO-d6)δ9.95(s,1H),8.28–8.24(m,2H),7.89(d,J=7.1Hz,3H),7.41 (d,J=7.6Hz,1H),7.19(s,1H),6.64(d,J=2.3Hz,1H),2.56(s,3H).
实施例24:3-(4-((5-(6-甲基吡啶-2-基)吡唑并[1,5-a]嘧啶-7-基)氨基)-1H-吡咯并[2,3-b]吡啶-2- 基)环戊-3-烯-1-醇的制备
步骤1:N-(2-氟-1-((2-(三甲基甲硅烷基)乙氧基)甲基)-1H-吡咯并[2,3-b]吡啶-4-基)-5-(6-甲基吡啶-2-基)吡唑[1,5-a]嘧啶-7-胺的制备
将2-氟-1-((2-(三甲基甲硅烷基)乙氧基)甲基)-1H-吡咯并[2,3-b]吡啶-4-胺(120mg)溶于四氢呋喃(2mL)中,冷却至0℃,缓慢加入双(三甲基硅烷基)氨基钾(340mg)反应30min,加入 7-氯-5-(6-甲基吡啶-2-基)吡唑并[1,5-a]嘧啶(105mg)于0℃下反应2h。加水淬灭,乙酸乙酯萃取,无水硫酸钠干燥,浓缩,柱层析纯化得标题化合物175mg。
MS(ESI)m/z(M+H)+=490.1.
步骤2:4-((5-(6-甲基吡啶-2-基)吡唑并[1,5-a]嘧啶-7-基)氨基)-1,3-二氢-2H-吡咯并[2,3-b]吡啶 -2-酮的制备
室温下,称取N-(2-氟-1-((2-(三甲基甲硅烷基)乙氧基)甲基)-1H-吡咯并[2,3-b]吡啶-4- 基)-5-(6-甲基吡啶-2-基)吡唑[1,5-a]嘧啶-7-胺(30mg)溶于三氟乙酸(1mL)中,于室温下反应40 min。所得粗品经反相制备得标题化合物13.14mg。
MS(ESI)m/z(M+H)+=358.1.
1H NMR(400MHz,DMSO-d6)δ11.18(s,1H),10.32(s,1H),8.31(d,J=2.3Hz,1H),8.27(d,J= 7.8Hz,1H),8.15(d,J=5.8Hz,1H),7.89(t,J=7.7Hz,1H),7.39(d,J=10.9Hz,2H),7.18(d,J =5.8Hz,1H),6.74(d,J=2.3Hz,1H),3.67(s,2H),2.52(s,3H).
实施例25:6-甲基-2-(6-甲基吡啶-2-基)-N-(1H-吡咯并[2,3-b]吡啶-4-基)-5,6,7,8-四氢吡啶并 [4,3-d]嘧啶-4-胺的制备
步骤1:2-(6-甲基吡啶-2-基)-4-氧代叔丁基-3,5,7,8-四氢吡啶并[4,3-d]嘧啶-6(4H)-羧酸盐的制备
将1-(叔丁基)-3-乙基4-氧哌啶-1,3-二羧酸酯(2.7g)、6-甲基吡啶甲脒盐酸盐(1.7g)溶于50 mL甲醇中,加入5M甲醇钠的甲醇溶液(4mL),体系升温至80℃,反应过夜。TLC显示原料消耗完毕,体系冷却至室温,浓缩,粗品用柱层析纯化得标题化合物2.0g。
MS(ESI)m/z(M+H)+=343.2.
步骤2:2-(6-甲基吡啶-2-基)-5,6,7,8-四氢吡啶并[4,3-d]嘧啶-4(3H)-酮的制备
室温下,将2-(6-甲基吡啶-2-基)-4-氧代叔丁基-3,5,7,8-四氢吡啶并[4,3-d]嘧啶-6(4H)-羧酸盐(2.0g)溶于4M盐酸/二氧六环溶液(30mL)中,于室温下反应4h。TLC显示原料消耗完毕,浓缩,得粗品。
MS(ESI)m/z(M+H)+=243.1.
步骤3:6-甲基-2-(6-甲基吡啶-2-基)-5,6,7,8-四氢吡啶并[4,3-d]嘧啶-4(3H)-酮的制备
室温下,将上述所得粗品用甲醇(30mL)溶解,加入37%甲醛溶液(20mL),冰乙酸(1mL),氰基硼氢化钠(1.1g),于室温下反应1h。TLC显示原料消耗完毕,加入二氯甲烷稀释,用1M 氢氧化钠水溶液反洗,再用二氯甲烷萃取,有机相用无水硫酸钠干燥,浓缩,粗品经柱层析纯化得标题化合物1.1g。
MS(ESI)m/z(M+H)+=257.1.
步骤4:4-氯-6-甲基-2-(6-甲基吡啶-2-基)-5,6,7,8-四氢吡啶并[4,3-d]嘧啶的制备
将6-甲基-2-(6-甲基吡啶-2-基)-5,6,7,8-四氢吡啶并[4,3-d]嘧啶-4(3H)-酮(1.0g)溶于三氯氧磷(15mL),于120℃反应4h。TLC显示原料消耗完毕,浓缩,所得粗品直接用作下一步。
MS(ESI)m/z(M+H)+=275.1.
步骤5:4-碘-6-甲基-2-(6-甲基吡啶-2-基)-5,6,7,8-四氢吡啶并[4,3-d]嘧啶的制备
将粗品用57%的氢碘酸水溶液溶解,加入碘化钠(645mg),于室温下反应过夜。TLC显示原料消耗完毕,体系倒入冰水中,加饱和碳酸氢钠溶液调pH至弱碱性,乙酸乙酯萃取,有机相用饱和氯化钠溶液反洗,无水硫酸钠干燥,浓缩,柱层析纯化得标题化合物900mg。
MS(ESI)m/z(M+H)+=367.0.
步骤6:6-甲基-2-(6-甲基吡啶-2-基)-N-(1-((2-(三甲基甲硅烷基)乙氧基)甲基)-1H-吡咯并[2,3-b] 吡啶-4-基)-5,6,7,8-四氢吡啶并[4,3-d]嘧啶-4-胺的制备
氮气氛下,将4-碘-6-甲基-2-(6-甲基吡啶-2-基)-5,6,7,8-四氢吡啶并[4,3-d]嘧啶(100mg)、 1-((2-(三甲基甲硅烷基)乙氧基)甲基)-1H-吡咯并[2,3-b]吡啶-4-胺(72mg)、[(4,5-双(二苯膦基)-9,9-二甲基黄嘌呤)-2-(2'-氨基-1,1'-联苯基)]甲磺酸钯(II)(26mg)、4,5-双(二苯基膦)-9,9-二甲基氧杂蒽(32mg)和碳酸铯(176mg)溶于二氧六环(10mL)中,于110℃反应24h。LC-MS检测原料消耗完毕,体系用二氯甲烷和甲醇稀释,过滤,滤液浓缩,用柱层析纯化得标题化合物20mg。
MS(ESI)m/z(M+H)+=502.2.
步骤7:6-甲基-2-(6-甲基吡啶-2-基)-N-(1H-吡咯并[2,3-b]吡啶-4-基)-5,6,7,8-四氢吡啶并[4,3-d] 嘧啶-4-胺的制备
将6-甲基-2-(6-甲基吡啶-2-基)-N-(1-((2-(三甲基甲硅烷基)乙氧基)甲基)-1H-吡咯并[2,3-b] 吡啶-4-基)-5,6,7,8-四氢吡啶并[4,3-d]嘧啶-4-胺(20mg)溶于三氟乙酸(5mL),体系于50℃反应过夜。TLC显示原料消耗完毕,浓缩。向浓缩体系中加入甲醇(5mL),用饱和碳酸钾水溶液调节反应体系的pH至碱性后,室温下继续反应。LC-MS检测原料消耗完毕,浓缩,粗品用 prep-HPLC纯化得标题化合物5.6mg。
MS(ESI)m/z(M+H)+=372.2.
1H NMR(400MHz,Methanol-d4)δ8.37(d,J=7.7Hz,1H),8.32(d,J=6.7Hz,1H),8.24(t,J= 7.9Hz,1H),7.95(d,J=6.7Hz,1H),7.78(d,J=7.7Hz,1H),7.54(d,J=3.6Hz,1H),6.94(d,J= 3.6Hz,1H),4.73(s,2H),3.82(t,J=6.4Hz,2H),3.47(t,J=6.3Hz,2H),3.21(s,3H),2.81(s, 3H).
实施例26:7-甲基-2-(6-甲基吡啶-2-基)-N-(1H-吡咯并[2,3-b]吡啶-4-基)-5,6,7,8-四氢吡啶并 [3,4-d]嘧啶-4-胺的制备
制备方法与前述实施例25采用的制备方法类似。
MS(ESI)m/z(M+H)+=372.2.
生物活性验证
1.1实验目的
利用ADP-Glo的方法,在ALK5(TGFβR1)激酶上进行受试化合物的筛选,起始浓度为1 μM,3倍稀释,10个浓度,单孔或复孔检测。
1.2实验材料
1.2.1试剂及耗材
试剂、材料名称 | 供货商 | 货号 | 批号 |
ALK5(TGFβR1) | Carna Biosciences,Inc. | 09-141 | 10CBS-0450L |
ADP-Glo Kit | Promega Corporation | V9102 | 0000319847 |
384-well white plate | PerkinElmer | 6008280 | 8310-17411 |
1.2.2仪器离心机(生产厂家:Eppendorf,型号:5430)
酶标仪(生产厂家:PerkinElmer,型号:Envision)
分液器Echo 550(生产厂家:Labcyte,型号:Echo 550)
1.3实验方法
1.3.1化合物配置
化合物溶解在100%DMSO中,配制成10mM储存液,氮气柜避光储存。
1.3.2激酶反应过程
(1)配制1×Kinase buffer。
(2)化合物浓度梯度的配制:
取受试化合物浓度为100μM的100%DMSO溶液,按3倍梯度稀释成10个浓度,每个浓度分别用分液器Echo 550转移50nL到384孔板的化合物孔(受试化合物的测试浓度为1μM起始);阴性对照孔和阳性对照孔中分别加50nL的DMSO。
(3)用1×Kinase buffer配制2倍终浓度的激酶溶液。
(4)在化合物孔和阳性对照孔分别加2.5μL的2倍终浓度的激酶溶液;在阴性对照孔中加 2.5μL的1×Kinase buffer。
(5)1000rpm离心30秒,振荡混匀后室温孵育10分钟。
(6)用1×Kinase buffer配制2倍终浓度的ATP溶液。
(7)加入2.5μL的2倍终浓度的ATP溶液,起始反应。
(8)将384孔板1000rpm离心30秒,振荡混匀后室温孵育120分钟。
(9)加入5μL ADP-Glo Reagent,1000rpm离心30秒,振荡混匀后室温孵育120分钟。
(10)加入10μL Kinase Detection Reagent,1000rpm离心30秒,振荡混匀后室温孵育30分钟。
(11)用Envision酶标仪读数。
1.3.3数据分析
其中:RLU:样品的化学发光值;Mean(NC):阴性对照孔均值;Mean(PC):阳性对照孔比值均值。
拟合量效曲线
以浓度的log值作为X轴,百分比抑制率为Y轴,采用分析软件GraphPad Prism 5的log(inhibitor)vs.response-Variable slope拟合量效曲线,从而得出各个化合物对酶活性的IC50值。
计算公式是Y=Bottom+(Top-Bottom)/(1+10^((LogIC50-X)*HillSlope))
1.4实验结果
本发明化合物对ALK5激酶抑制活性通过上述试验方法验证,并计算IC50值。在某些实施例中,本发明中的化合物对ALK5激酶活性的IC50值小于1000nM。在某些实施例中,本发明中的化合物对ALK5激酶活性的IC50值小于200nM。在某些实施例中,本发明中的化合物对ALK5激酶活性的IC50值小于100nM。在某些实施例中,本发明中的化合物对ALK5激酶活性的IC50值小于10nM。在某些实施例中,本发明中的化合物对ALK5激酶活性的IC50值在小于1nM。
本发明中部分化合物活性例举如下表1。
表1本发明化合物对ALK5激酶抑制活性
Claims (11)
1.式I结构的化合物,其异构体、混合物、溶剂合物、水合物、前药或其药学上可接受的盐:
其中,Ar是吡啶基,其被0-2个Rx取代;
A选自
Y选自O、-NH-、-N(CH3)-;
X选自
Rb选自氢、氧、硝基、羟基、氰基、卤素、C1-C6烷基、C1-C6卤代烷基、C1-C6烷氧基、C3-C6环烷基、C3-C6环烯基、羟烷基、羟基取代的烯基、羟基取代的炔基;在某些具体的实施方案中,Rb选自氢、氧、卤素、C1-C6卤代烷基、羟烷基、C3-C6环烯基或羟基取代的炔基;在某些具体的实施方案中,Rb选自氢、氧、氯、氟、CF3、-CH2CH2CH2OH、-CH2CH2CH(CH3)OH、-C≡CCH(CH3)OH、-C≡CCH2OH、
Rx选自氢、卤素、任选取代的C1-C6烷基、任选取代的C1-C6烷氧基、任选取代的C3-C6环烷基、-NH2、-CHF2、CF3或NHSO2(C1-C6)烷基;
2.根据权利要求1所述的化合物,其异构体、混合物、溶剂合物、水合物、前药或其药学上可接受的盐,其中X选自
Rb选自氢、氧、硝基、羟基、氰基、卤素、C1-C6烷基、C1-C6卤代烷基、C1-C6烷氧基、C3-C6环烷基、C3-C6环烯基、羟烷基、羟基取代的烯基、羟基取代的炔基;在某些具体的实施方案中,Rb选自氢、氧、卤素、C1-C6卤代烷基、羟烷基、C3-C6环烯基或羟基取代的炔基;在某些具体的实施方案中,Rb选自氢、氧、氯、氟、CF3、-CH2CH2CH2OH、-CH2CH2CH(CH3)OH、-C≡CCH(CH3)OH、-C≡CCH2OH、
7.根据权利要求1-6中任一项所述的化合物,其中,所述Rx为甲基;所述Ar为2-甲基-吡啶-4-基。
8.根据权利要求1、2或6所述的化合物,其中所述X1、X2仅有一个为-N(CH3)-。
10.一种药物组合物,其包含治疗有效剂量的权1-权9中任一项所述化合物,其异构体、混合物、溶剂合物、水合物、前药或其药学上可接受的盐,以及药学上可接受的辅料。
11.权利要求1-10中任一项所述的化合物,其异构体、混合物、溶剂合物、水合物、前药或药学上可接受的盐或药物组合物在制备治疗、预防或减少由TGF-β过度表达介导的疾病的药物中的应用;其中,所述TGF-β过度表达介导的疾病包括:癌症、肾纤维化、肝纤维化、肺纤维化、病毒感染、慢性肾炎、急性肾炎、糖尿病肾病、骨质疏松症、关节炎、伤口愈合、溃疡、角膜创伤、心脏瓣膜狭窄、充血性心脏坏死、神经功能损伤、阿尔兹海默综合征、腹膜或皮下粘连、动脉硬化症和肿瘤转移生长等。
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