CN108299428B - 8-胺基-7-甲酸甲酯-吡嗪骈吡啶酮衍生物及其制备方法与应用 - Google Patents
8-胺基-7-甲酸甲酯-吡嗪骈吡啶酮衍生物及其制备方法与应用 Download PDFInfo
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- CN108299428B CN108299428B CN201810300833.8A CN201810300833A CN108299428B CN 108299428 B CN108299428 B CN 108299428B CN 201810300833 A CN201810300833 A CN 201810300833A CN 108299428 B CN108299428 B CN 108299428B
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- pyrazine
- reaction
- biphenyl
- benzyloxy
- dichloromethane
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- 238000002360 preparation method Methods 0.000 title claims abstract description 17
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- 239000003112 inhibitor Substances 0.000 claims abstract description 11
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 102
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 63
- -1 malonic acid monomethyl ester acyl chloride Chemical class 0.000 claims description 57
- 238000006243 chemical reaction Methods 0.000 claims description 55
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 claims description 44
- 125000000051 benzyloxy group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])O* 0.000 claims description 39
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 33
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 claims description 27
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- ZUOUZKKEUPVFJK-UHFFFAOYSA-N diphenyl Chemical group C1=CC=CC=C1C1=CC=CC=C1 ZUOUZKKEUPVFJK-UHFFFAOYSA-N 0.000 abstract 2
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- 235000010290 biphenyl Nutrition 0.000 abstract 1
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Abstract
本发明提供了一种8‑胺基‑7‑甲酸甲酯‑吡嗪骈吡啶酮衍生物,具有如下通式I所示的结构。其中,R为4'‑氰基‑[1,1'‑联苯]‑4‑基、4'‑氰基‑[1,1'‑联苯]‑3‑基、3'‑氰基‑[1,1'‑联苯]‑4‑基、4‑(嘧啶‑5‑基)苯基、3‑(嘧啶‑5‑基)苯基、3',4'‑二甲氧基‑[1,1'‑联苯]‑4‑基、3',4'‑二甲氧基‑[1,1'‑联苯]‑3‑基、萘‑1‑基、萘‑2‑基、[1,1'‑联苯]‑4‑基。本发明还涉及该类衍生物的制备方法及其作为HIV抑制剂在制备抗艾滋病药物中的应用。
Description
技术领域
本发明涉及一种衍生物及其制备方法,具体涉及8-胺基-7-甲酸甲酯-吡嗪骈吡啶酮衍生物的制备及其在抗HIV药物领域的应用,属于有机合成与医药应用技术领域。
背景技术
艾滋病,又名获得性免疫缺陷综合征(Acquired Immunodeficiency Syndrome,AIDS),是严重威胁人类生命健康的重大传染性疾病,其主要病原体是I型人类免疫缺陷病毒(Human Immunodeficiency Virus Type I,HIV-1)。由于HIV遗传物质的整合特质以及高度变异性,自上世纪八十年代首次发现以来,HIV感染的治疗一直是全球医药化学领域的一大难题。九十年代起出现的多种抗HIV药物联用的高效抗转录病毒联合疗法(HighlyActive Antiretroviral Therapy,HAART)的应用使HIV-1感染引起的发病率和死亡率大大降低,然而,随着HAART的普及,剂量复杂性、病人依从性以及药物相互作用等问题日益突出。同时,大量耐药突变株依旧不断涌现,耐药性向更加复杂的方向发展,使得具有新靶标、新作用机制的抗逆转录病毒药物的研制迫在眉睫。
HIV-1逆转录酶(Reverse Transcriptase,RT)是病毒生命周期的关键酶,它主要发挥RNA及DNA依赖的DNA聚合酶活性和核糖核酸酶H(Ribonuclease H,RNase H)活性。目前上市的HIV-1逆转录酶抑制剂药物均是靶向其聚合酶活性。RNase H水解活性在逆转录过程中同样扮演了十分重要的角色:它可以水解逆转录过程中的RNA/DNA杂合链上的RNA链,并清除tRNA前体,利于DNA双链的合成。RNase H结构域位于p66亚基的C末端,其水解活性位点包括4个高度保守的氨基酸残基(D443/E478/D498/D549,即DEDD序列),通过鳌合两个二价的镁离子发挥催化活性,是RNase H功能所必需。目前尚无RNase H抑制剂上市。因此RNaseH抑制剂将是抗HIV药物领域一个充满前景的研究方向。
整合酶(Integrase,IN)作用于HIV-1生命周期中病毒基因组整合入宿主DNA的过程,是近几年抗艾滋病药物研究的新靶点。其功能结构主要分为N端区、催化核心区和C端区。该酶主要催病毒DNA的3’端加工反应(3’-P)及链转移反应(ST)。其中,核心区含有3个非常保守的氨基酸残基(Asp64/Asp116/Glu152,即DDE序列),是内核酶和核酸转移酶的位点。该部位与辅助因子一个或两个二价阳离子(Mg2+或Mn2+)结合,发挥IN的催化功能。目前上市的整合酶抑制剂Raltegravir(RAL)、Elvitegravir(EVG)、Dolutegravir(DTG)均出现了耐药性问题。因此,亟需发现具有新作用机制、新结合模式的HIV整合酶抑制剂。
近年来,双靶点药物设计策略在抗HIV-1药物研发中的应用逐渐成为热点,即设计一种能够同时抑制两种病毒靶标的单一化合物(双靶点抑制剂),有望克服耐药性、降低药物相互作用、缓解毒性等。根据上文分析,HIV-1逆转录酶RNase H与整合酶的水解活性位点三维结构及催化机制的类似性,为设计RNase H和整合酶双靶点抑制剂提供了结构基础。
为发现新一代HIV抑制剂,本发明公开了一类全新结构的8-胺基-7-甲酸甲酯-吡嗪骈吡啶酮类HIV-1RNase H-IN双靶点抑制剂,现有技术中未见相关报道。
发明内容
本发明提供了8-胺基-7-甲酸甲酯-吡嗪骈吡啶酮衍生物及其制备方法,本发明还提供了上述化合物的部分活性筛选结果及其用途。
本发明的技术方案如下:
一、8-胺基-7-甲酸甲酯-吡嗪骈吡啶酮衍生物
本发明的8-胺基-7-甲酸甲酯-吡嗪骈吡啶酮衍生物,具有如下通式I所示的结构:
其中,
R为4'-氰基-[1,1'-联苯]-4-基、4'-氰基-[1,1'-联苯]-3-基、3'-氰基-[1,1'-联苯]-4-基、4-(嘧啶-5-基)苯基、3-(嘧啶-5-基)苯基、3',4'-二甲氧基-[1,1'-联苯]-4-基、3',4'-二甲氧基-[1,1'-联苯]-3-基、萘-1-基、萘-2-基、[1,1'-联苯]-4-基。
根据本发明优选的,8-胺基-7-甲酸甲酯-吡嗪骈吡啶酮衍生物,是下列之一:
8-((4'-氰基-[1,1'-联苯]-4-基)氨基)-5-羟基-6-氧代-5,6-二氢吡啶并[2,3-b]吡嗪-7-甲酸甲酯(7a)、
8-((4'-氰基-[1,1'-联苯]-3-基)氨基)-5-羟基-6-氧代-5,6-二氢吡啶并[2,3-b]吡嗪-7-甲酸甲酯(7b)、
8-((3'-氰基-[1,1'-联苯]-4-基)氨基)-5-羟基-6-氧代-5,6-二氢吡啶并[2,3-b]吡嗪-7-甲酸甲酯(7c)、
8-((3'-氰基-[1,1'-联苯]-3-基)氨基)-5-羟基-6-氧代-5,6-二氢吡啶并[2,3-b]吡嗪-7-甲酸甲酯(7d)、
5-羟基-6-氧代-8-((4-(嘧啶-5-基)苯基)氨基)-5,6-二氢吡啶并[2,3-b]吡嗪-7-甲酸甲酯(7e)、
5-羟基-6-氧代-8-((3-(嘧啶-5-基)苯基)氨基)-5,6-二氢吡啶并[2,3-b]吡嗪-7-甲酸甲酯(7f)、
8-((3',4'-二甲氧基-[1,1'-联苯]-4-基)氨基)-5-羟基-6-氧代-5,6-二氢吡啶并[2,3-b]吡嗪-7-甲酸甲酯(7g)、
8-((3',4'-二甲氧基-[1,1'-联苯]-3-基)氨基)-5-羟基-6-氧代-5,6-二氢吡啶并[2,3-b]吡嗪-7-甲酸甲酯(7h)、
5-羟基-8-(萘-1-基氨基)-6-氧代-5,6-二氢吡啶并[2,3-b]吡嗪-7-甲酸甲酯(7i)、
5-羟基-8-(萘-2-基氨基)-6-氧代-5,6-二氢吡啶并[2,3-b]吡嗪-7-甲酸甲酯(7j)、
8-([1,1'-联苯]-4-基氨基)-5-羟基-6-氧代-5,6-二氢吡啶并[2,3-b]吡嗪-7-甲酸甲酯(7k)。二、8-胺基-7-甲酸甲酯-吡嗪骈吡啶酮衍生物的制备方法
本发明8-胺基-7-甲酸甲酯-吡嗪骈吡啶酮衍生物的制备方法,以3-氯吡嗪-2-羧酸甲酯(1)为原料,与O-苄基羟胺经芳香性亲核取代反应,再与丙二酸单甲酯酰氯亲核取代反应,后经环合得到中间体5-(苄氧基)-8-羟基-6-氧代-5,6-二氢吡啶并[2,3-b]吡嗪-7-甲酸甲酯(4),再依次经与对甲基苯磺酰氯的亲核取代反应、与取代芳胺的亲核取代反应、脱苄基反应得到目标化合物。
合成路线如下:
反应试剂与反应条件:i)O-苄基羟胺,N,N-二异丙基乙胺,二甲基亚砜,微波反应100℃,60min;ii)丙二酸单甲酯酰氯,三乙胺,二氯甲烷,0℃转45℃;iii)甲醇钠/甲醇,室温,过夜;iv)对甲基苯磺酰氯,N,N-二异丙基乙胺,二氯甲烷,乙腈,室温,过夜;v)相应的取代芳香胺,N,N-二异丙基乙胺,N,N-二甲基甲酰胺,50℃,2h;vi)H2,10%Pd●C,二氯甲烷/甲醇,室温;
其中,R如上述通式I中所述。
所述的相应的取代芳香胺选自:4'-氨基-[1,1'-联苯基]-4-腈、3'-氨基-[1,1'-联苯基]-4-腈、4'-氨基-[1,1'-联苯基]-3-腈、3'-氨基-[1,1'-联苯基]-3-腈、4-(嘧啶-5-基)苯胺、3-(嘧啶-5-基)苯胺、3',4'-二甲氧基-[1,1'-联苯]-4-胺、3',4'-二甲氧基-[1,1'-联苯]-3-胺、1-萘胺、2-萘胺、[1,1'-联苯]-4-胺。
本发明8-胺基-7-甲酸甲酯-吡嗪骈吡啶酮衍生物的制备方法,具体步骤如下:
(1)将3-氯吡嗪-2-羧酸甲酯(1),O-苄基羟胺,二甲基亚砜和N,N-二异丙基乙胺加入到微波反应管,100℃条件下微波反应60min;反应完毕,将反应液移入分液漏斗,加入乙酸乙酯、饱和氯化钠溶液,分出有机相,水层用乙酸乙酯萃取,合并有机相,加入无水硫酸镁干燥,过滤,滤液减压浓缩,所得粗品经硅胶柱层析分离纯化后,重结晶得中间体3-((苄氧基)氨基)吡嗪-2-羧酸甲酯(2);
(2)将3-(苄氧基)氨基)吡嗪-2-羧酸甲酯(2),三乙胺和40mL二氯甲烷加入到100mL的茄型瓶中,并将其置于冰浴中搅拌;在冰浴下,向反应瓶中缓慢滴加丙二酸单甲酯酰氯;滴毕,撤去冰浴,将反应转移至45℃油浴回流搅拌;24小时后,将反应液移入分液漏斗,加入二氯甲烷,饱和氯化钠溶液进行萃取,水相用二氯甲烷萃取两次,合并有机相,加入无水硫酸镁干燥,过滤,滤液减压浓缩,所得粗品经硅胶柱层析分离纯化后,重结晶得中间体3-(N-(苄氧基)-3-甲氧基-3-氧代丙酰胺基)吡嗪-2-甲酸甲酯(3);
(3)将3-(N-(苄氧基)-3-甲氧基-3-氧代丙酰胺基)吡嗪-2-甲酸甲酯(3),甲醇钠和甲醇加入到50mL茄型瓶中,室温搅拌,过夜。反应完毕,向反应体系中滴加4N的HCl溶液,调节pH至3-4,此过程中有絮状白色不溶物生成,抽滤,滤饼干燥后,所得粗品重结晶得中间体5-(苄氧基)-8-羟基-6-氧代-5,6-二氢吡啶并[2,3-b]吡嗪-7-甲酸甲酯(4);
(4)将5-(苄氧基)-8-羟基-6-氧代-5,6-二氢吡啶并[2,3-b]吡嗪-7-甲酸甲酯(4),对甲基苯磺酰氯,N,N-二异丙基乙胺加入到50mL茄型瓶中;并向反应瓶中加入混合溶剂乙腈/二氯甲烷;室温搅拌,过夜;反应完毕,将反应液移入分液漏斗,加入饱和氯化钠溶液,二氯甲烷萃取,水相用二氯甲烷萃取,合并有机相,并用无水硫酸镁干燥;过滤,滤液减压浓缩,所得粗品经硅胶柱层析分离纯化后,重结晶得中间体5-(苄氧基)-6-氧代-8-(甲苯磺酰氧基)-5,6-二氢吡啶并[2,3-b]吡嗪-7-甲酸甲酯(5);
(5)将5-(苄氧基)-6-氧代-8-(甲苯磺酰氧基)-5,6-二氢吡啶并[2,3-b]吡嗪-7-甲酸甲酯(5),相应的取代芳香胺,N,N-二异丙基乙胺加入到100mL茄型瓶中,加入溶剂N,N-二甲基甲酰胺,50℃油浴加热,加盖干燥管,内装无水CaCl2,反应两小时;反应完毕,反应液用油泵尽量抽干,加入二氯甲烷溶解,然后将其转移入分液漏斗,加入饱和氯化钠溶液萃取,水相用二氯甲烷萃取两次,合并有机相,加入无水硫酸镁干燥;过滤,滤液减压浓缩,所得粗品经硅胶柱层析分离纯化后,重结晶得相应中间体(6a-6k);所述相应取代芳香胺选自:4'-氨基-[1,1'-联苯基]-4-腈、3'-氨基-[1,1'-联苯基]-4-腈、4'-氨基-[1,1'-联苯基]-3-腈、3'-氨基-[1,1'-联苯基]-3-腈、4-(嘧啶-5-基)苯胺、3-(嘧啶-5-基)苯胺、3',4'-二甲氧基-[1,1'-联苯]-4-胺、3',4'-二甲氧基-[1,1'-联苯]-3-胺、1-萘胺、2-萘胺、[1,1'-联苯]-4-胺;
(6)将中间体(6a-6k)加入到25mL茄型瓶中,加入甲醇和二氯甲烷使原料溶解,再加入10%Pd·C,氢气置换三次,在氢气球保护下,室温搅拌两小时;反应完毕,加硅藻土过滤,滤液蒸干,加入乙酸乙酯,超声,过滤收集滤饼,所得粗品重结晶得目标化合物(7a-7k)。
所得目标化合物7a-7k结构见表1。
表1目标化合物的结构式
三、8-胺基-7-甲酸甲酯-吡嗪骈吡啶酮衍生物的应用
活性测试结果表明,8-胺基-7-甲酸甲酯-吡嗪骈吡啶酮衍生物是一系列结构新颖的HIV-1RNase H-IN双靶点抑制剂,大部分化合物表现出显著的HIV-1RNase H-IN双靶点抑制活性,其中化合物7a(IC50 RNase H=1.77μM,IC50 IN=1.18μM)活性最优,说明该类化合物具有进一步的研究价值。
因此,本发明所提供的8-胺基-7-甲酸甲酯-吡嗪骈吡啶酮衍生物可作为HIV-1RNase H-IN双靶点抑制剂用于制备抗艾滋病药物。
一种抗HIV-1的药物组合物,含有上述的8-胺基-7-甲酸甲酯-吡嗪骈吡啶酮衍生物及其药学上可接受的盐与药用辅料,制成不同剂型的药物。
具体实施方式
下面结合实施例对本发明做进一步说明,所有目标化合物的编号与表1相同,所述百分比数均为质量百分比。
实施例1:中间体3-((苄氧基)氨基)吡嗪-2-羧酸甲酯(2)的制备
将3-氯吡嗪-2-羧酸甲酯(504mg,2.9mmol,1eq.),O-苄基羟胺(1073mg,8.72mmol,3eq.),二甲基亚砜(3mL)和N,N-二异丙基乙胺(1.44mL,8.72mmol,3eq.)加入到微波反应管,100℃条件下微波反应一小时。反应完毕,将反应液移入分液漏斗,加入乙酸乙酯30mL,饱和氯化钠溶液30mL,分出有机相,水层用乙酸乙酯萃取两次,合并有机相,加入无水硫酸镁干燥,过滤,滤液浓缩后用100-200目硅胶拌样,经200-300目硅胶柱层析分离(洗脱剂EA:PE=1:4)纯化后得中间体2粗品,粗品用石油醚/正己烷重结晶得中间体3-((苄氧基)氨基)吡嗪-2-甲酸甲酯(2,收率:16%),白色针状晶体,熔点:60-62℃。
波谱数据:
1H NMR(400MHz,DMSO-d6)δ10.33(s,1H,N-H),8.50(d,J=2.3Hz,1H,pyrazine-H),8.13(d,J=2.3Hz,1H,pyrazine-H),7.47(dd,J=8.2,1.6Hz,2H,Ph-H),7.42–7.33(m,3H,Ph-H),4.96(s,2H,CH2),3.82(s,3H,CH3).13C NMR(100MHz,DMSO-d6)δ165.51,155.09,146.75,136.19,134.63,128.69(2×C),128.30(2×C),128.18,125.53,76.85,52.44.ESI-MS:m/z260.3(M+1),282.4(M+23).C13H13N3O3[259.10].
实施例2:中间体3-(N-(苄氧基)-3-甲氧基-3-氧代丙酰胺基)吡嗪-2-甲酸甲酯(3)的制备
将中间体2(1897mg,7.29mmol,1eq.),三乙胺(2.02mL,14.59mmol,2eq.)和40mL二氯甲烷加入到100mL的茄型瓶中,并将其置于冰浴中搅拌。在冰浴下,向反应瓶中缓慢滴加丙二酸单甲酯酰氯(3.12mL,29.18mmol,4eq.)。30min后,撤去冰浴,将反应转移至45℃油浴回流搅拌。24小时后反应完毕,将反应液移入分液漏斗,加入30mL二氯甲烷,30mL饱和氯化钠溶液进行萃取,水相用二氯甲烷萃取两次,合并有机相,加入无水硫酸镁干燥,过滤,滤液浓缩后用100-200目硅胶拌样,经200-300目硅胶柱层析分离(洗脱剂EA:PE=1:3)纯化后得粗品,用正己烷/乙酸乙酯重结晶得中间体3-(N-(苄氧基)-3-甲氧基-3-氧代丙酰胺基)吡嗪-2-甲酸甲酯(3,收率:90%),黄色簇状晶体,熔点:81-82℃。
波谱数据:
1H NMR(400MHz,DMSO-d6)δ8.80(d,J=2.3Hz,1H,pyrazine-H),8.71(d,J=2.3Hz,1H,pyrazine-H),7.42–7.35(m,5H,Ph-H),5.04(s,2H,CH2),3.81(s,3H,CH3),3.78(s,2H,CH2),3.63(s,3H,CH3).13C NMR(100MHz,DMSO-d6)δ166.82(2×C),163.85,145.65,142.72,140.07,133.84,129.68,128.92(2×C),128.34,77.76,52.74,52.08,40.73.ESI-MS:m/z360.2(M+1),382.3(M+23),398.4(M+39).C17H17N3O6[359.34].
实施例3:中间体5-(苄氧基)-8-羟基-6-氧代-5,6-二氢吡啶并[2,3-b]吡嗪-7-甲酸甲酯(4)的制备
将中间体3(212mg,0.557mmol,1eq.),甲醇钠(74mg,1.376mmol,2.47eq.)和9.8mL甲醇加入到50mL茄型瓶中,室温搅拌,过夜。反应完毕,向反应体系中滴加4N的HCl溶液,调节pH至3-4,此过程中有絮状白色不溶物生成,抽滤,滤饼干燥后,得中间体5-(苄氧基)-8-羟基-6-氧代-5,6-二氢吡啶并[2,3-b]吡嗪-7-甲酸甲酯(4,收率:66%),白色固体,熔点:195-197℃。
波谱数据:
1H NMR(400MHz,DMSO-d6)δ8.88(d,J=2.3Hz,1H,pyrazine-H),8.70(d,J=2.3Hz,1H,pyrazine-H),7.64-7.62(m,2H,Ph-H),7.46-7.39(m,3H,Ph-H),5.17(s,2H,CH2),3.84(s,3H,CH3).13C NMR(100MHz,DMSO-d6)δ164.33,158.58,156.48,146.58,144.82,139.24,134.32,129.51(2×C),128.94,128.37(2×C),127.15,108.94,77.78,52.38.ESI-MS:m/z328.4(M+1),350.4(M+23).C16H13N3O5[327.30].
实施例4:中间体5-(苄氧基)-6-氧代-8-(甲苯磺酰氧基)-5,6-二氢吡啶并[2,3-b]吡嗪-7-甲酸甲酯(5)的制备
将中间体4(101mg,0.306mmol,1eq.),对甲基苯磺酰氯(175mg,0.917mmol,3eq.),N,N-二异丙基乙胺(0.303ml,1.834mmol,6eq.)加入到50mL茄型瓶中。并向反应瓶中加入溶剂乙腈:二氯甲烷(3:1)共20mL。室温搅拌,过夜。反应完毕,将反应液移入分液漏斗,加入饱和氯化钠溶液40mL,二氯甲烷40mL萃取,水相用二氯甲烷萃取两次,合并有机相,并用无水硫酸镁干燥。过滤,滤液用100-200目硅胶拌样,经200-300目硅胶柱层析分离(洗脱剂EA:PE=1:3)纯化后得粗品,用乙酸乙酯重结晶得中间体5-(苄氧基)-6-氧代-8-(甲苯磺酰氧基)-5,6-二氢吡啶并[2,3-b]吡嗪-7-甲酸甲酯(5,收率:75%),浅黄色晶体,熔点:162-164℃。
波谱数据:
1H NMR(400MHz,DMSO-d6)δ8.90(d,J=2.3Hz,1H,pyrazine-H),8.64(d,J=2.3Hz,1H,pyrazine-H),7.90(d,J=8.4Hz,2H,Ph-H),7.64(dd,J=7.4,2.0Hz,2H,Ph-H),7.52(d,J=8.1Hz,2H,Ph-H),7.47–7.43(m,3H,Ph-H),5.24(s,2H,CH2),3.66(s,3H,CH3),2.46(s,3H,CH3).13C NMR(100MHz,DMSO-d6)δ161.10,155.06,149.49,146.91,146.51,145.06,140.90,133.97,132.08,130.17(2×C),129.67(2×C),129.15,128.49(2×C),128.47,127.47(2×C),123.42,78.12,53.00,21.25.ESI-MS:m/z 482.4(M+1),504.4(M+23),520.4(M+39).C23H19N3O7S[481.48].
实施例5:中间体6a-6k的制备
将中间体5(1eq.),相应的取代芳香胺(1.5eq.),N,N-二异丙基乙胺(3eq.)加入到100mL茄型瓶中,加入6mL N,N-二甲基甲酰胺,50℃油浴加热,加盖干燥管(,内装无水CaCl2,反应两小时。反应完毕,反应液用油泵尽量抽干,加入50mL二氯甲烷溶解,然后将其转移入分液漏斗,加入饱和氯化钠溶液50mL萃取,水相用二氯甲烷萃取两次,合并有机相,加入无水硫酸镁干燥。过滤,滤液用100-200目硅胶拌样,经200-300目硅胶柱分离(洗脱剂MeOH:DCM=1:100),得粗品,粗品用适当溶剂重结晶分别得到中间体6a-6k。
取代芳香胺选用4'-氨基-[1,1'-联苯基]-4-腈与5-(苄氧基)-6-氧代-8-(甲苯磺酰氧基)-5,6-二氢吡啶并[2,3-b]吡嗪-7-甲酸甲酯(5)反应制得5-(苄氧基)-8-((4'-氰基-[1,1'-联苯]-4-基)氨基)-6-氧代-5,6-二氢吡啶并[2,3-b]-7-甲酸甲酯(中间体6a)。异丙醇/三氯甲烷重结晶,黄色颗粒状晶体,收率:63%,熔点:225-226℃。
波谱数据:
1H NMR(400MHz,DMSO-d6)δ9.74(s,1H,N-H),8.89(d,J=2.4Hz,1H,pyrazine-H),8.68(d,J=2.4Hz,1H,pyrazine-H),7.92(s,4H,Ph-H),7.77(d,J=8.6Hz,2H),7.66–7.64(m,2H,Ph-H),7.47-7.40(m,3H,Ph-H),7.32(d,J=8.6Hz,2H,Ph-H),5.18(s,2H,CH2),3.13(s,3H,CH3).13C NMR(100MHz,DMSO-d6)δ164.32,156.97,146.45,144.99,144.60,143.84,139.81,138.51,134.73,134.48,132.87(2×C),129.51(2×C),128.90,128.37(2×C),127.42,127.24(2×C),127.21(2×C),124.54(2×C),118.89,109.82,102.85,77.65,51.32.ESI-MS:m/z 504.4(M+1),526.5(M+23),542.5(M+39).C29H21N5O4[503.52].
取代芳香胺选用3'-氨基-[1,1'-联苯基]-4-腈与5-(苄氧基)-6-氧代-8-(甲苯磺酰氧基)-5,6-二氢吡啶并[2,3-b]吡嗪-7-甲酸甲酯(5)反应制得5-(苄氧基)-8-((4'-氰基-[1,1'-联苯]-3-基)氨基)-6-氧代-5,6-二氢吡啶并[2,3-b]-7-甲酸甲酯(中间体6b)。异丙醇/三氯甲烷重结晶,黄色粉末状固体,收率:59%,熔点:239-241℃。
波谱数据:
1H NMR(400MHz,DMSO-d6)δ9.72(s,1H,N-H),8.89(d,J=2.3Hz,1H,pyrazine-H),8.68(d,J=2.3Hz,1H,pyrazine-H),7.96(d,J=8.5Hz,2H,Ph-H),7.88(d,J=8.5Hz,2H,Ph-H),7.65(dd,J=7.7,1.6Hz,2H,Ph-H),7.59(d,J=8.0Hz,1H,Ph-H),7.53–7.50(m,2H,Ph-H),7.47–7.41(m,3H,Ph-H),7.31(d,J=8.0Hz,1H,Ph-H),5.18(s,2H,CH2),2.99(s,3H,CH3).ESI-MS:m/z 504.4(M+1),526.5(M+23).C29H21N5O4[503.52].
取代芳香胺选用4'-氨基-[1,1'-联苯基]-3-腈与5-(苄氧基)-6-氧代-8-(甲苯磺酰氧基)-5,6-二氢吡啶并[2,3-b]吡嗪-7-甲酸甲酯(5)反应制得5-(苄氧基)-8-((3'-氰基-[1,1'-联苯]-4-基)氨基)-6-氧代-5,6-二氢吡啶并[2,3-b]-7-甲酸甲酯(中间体6c)。甲醇/三氯甲烷重结晶,黄色针状晶体,收率:64%,熔点:219-220℃。
波谱数据:
1H NMR(400MHz,DMSO-d6)δ9.72(s,1H,N-H),8.89(d,J=2.4Hz,1H,pyrazine-H),8.68(d,J=2.4Hz,1H,pyrazine-H),8.20(s,1H,Ph-H),8.06(d,J=8.0Hz,1H,Ph-H),7.83(d,J=8.0Hz,1H,Ph-H),7.77(d,J=8.6Hz,2H,Ph-H),7.70–7.64(m,3H,Ph-H),7.47–7.40(m,3H,Ph-H),7.31(d,J=8.6Hz,2H,Ph-H),5.18(s,2H,CH2),3.13(s,3H,CH3).ESI-MS:m/z504.4(M+1),526.5(M+23),542.5(M+39).C29H21N5O4[503.52].
取代芳香胺选用3'-氨基-[1,1'-联苯基]-3-腈与5-(苄氧基)-6-氧代-8-(甲苯磺酰氧基)-5,6-二氢吡啶并[2,3-b]吡嗪-7-甲酸甲酯(5)反应制得5-(苄氧基)-8-((3'-氰基-[1,1'-联苯]-3-基)氨基)-6-氧代-5,6-二氢吡啶并[2,3-b]-7-甲酸甲酯(中间体6d)。甲醇/三氯甲烷重结晶,黄色簇状晶体,收率:78%,熔点:215-217℃。
波谱数据:
1H NMR(400MHz,DMSO-d6)δ9.70(s,1H,N-H),8.90(d,J=2.4Hz,1H,pyrazine-H),8.69(d,J=2.4Hz,1H,pyrazine-H),8.14(s,1H,Ph-H),8.03(d,J=8.5Hz,1H,Ph-H),7.86(d,J=7.7Hz,1H,Ph-H),7.70(t,J=7.8Hz,1H,Ph-H),7.65(dd,J=7.7,1.6Hz,2H,Ph-H),7.60(d,J=8.0Hz,1H,Ph-H),7.55(s,1H,Ph-H),7.50(t,J=7.8Hz,1H,Ph-H),7.47–7.40(m,3H,Ph-H),7.29(d,J=7.9Hz,1H,Ph-H),5.19(s,2H,CH2),2.99(s,3H,CH3).ESI-MS:m/z504.4(M+1),526.5(M+23),542.5(M+39).C29H21N5O4[503.52].
取代芳香胺选用4-(嘧啶-5-基)苯胺与5-(苄氧基)-6-氧代-8-(甲苯磺酰氧基)-5,6-二氢吡啶并[2,3-b]吡嗪-7-甲酸甲酯(5)反应制得5-(苄氧基)-6-氧代-8-((4-(嘧啶-5-基)苯基)氨基)-5,6-二氢吡啶并[2,3-b]吡嗪-7-甲酸甲酯(中间体6e)。甲醇/三氯甲烷重结晶,黄色块状晶体,收率:91%,熔点:198-199℃。
波谱数据:
1H NMR(400MHz,DMSO-d6)δ9.77(s,1H,N-H),9.18(s,3H,pyrimidine-H),8.89(d,J=2.4Hz,1H,pyrazine-H),8.68(d,J=2.4Hz,1H,pyrazine-H),7.83(d,J=8.5Hz,2H,Ph-H),7.65(dd,J=7.7,1.6Hz,2H,Ph-H),7.47–7.39(m,3H,Ph-H),7.34(d,J=8.5Hz,2H,Ph-H),5.18(s,2H,CH2),3.15(s,3H,CH3).13C NMR(100MHz,DMSO-d6)δ164.37,157.12,157.00,154.44(2×C),146.49,144.99,144.62,139.99,138.54,134.50,132.54,130.28,129.55(2×C),128.93,128.40(2×C),127.46,127.07(2×C),124.66(2×C),102.89,77.67,51.47.ESI-MS:m/z481.4(M+1),503.4(M+23),519.4(M+39).C26H20N6O4[480.48].
取代芳香胺选用3-(嘧啶-5-基)苯胺与5-(苄氧基)-6-氧代-8-(甲苯磺酰氧基)-5,6-二氢吡啶并[2,3-b]吡嗪-7-甲酸甲酯(5)反应制得5-(苄氧基)-6-氧代-8-((3-(嘧啶-5-基)苯基)氨基)-5,6-二氢吡啶并[2,3-b]吡嗪-7-甲酸甲酯(中间体6f)。甲醇/三氯甲烷重结晶,黄色簇状晶体,收率:70%,熔点:220-222℃。
波谱数据:
1H NMR(400MHz,DMSO-d6)δ9.76(s,1H,N-H),9.21(s,1H,pyrimidine-H),9.14(s,2H,pyrimidine-H),8.90(d,J=2.4Hz,1H,pyrazine-H),8.69(d,J=2.4Hz,1H,pyrazine-H),7.66–7.62(m,4H,Ph-H),7.54(t,J=7.8Hz,1H,Ph-H),7.47–7.39(m,3H,Ph-H),7.37–7.31(m,1H,Ph-H),5.19(s,2H,CH2),3.00(s,3H,CH3).13C NMR(100MHz,DMSO-d6)δ164.47,157.51,157.01,154.65(2×C),146.54,145.25,144.59,139.99,138.54,134.49,134.00,132.63,129.85,129.54(2×C),128.92,128.39(2×C),127.39,125.00,123.69,122.16,102.60,77.68,51.15.ESI-MS:m/z 481.5(M+1),503.4(M+23),519.4(M+39).C26H20N6O4[480.48].
取代芳香胺选用3',4'-二甲氧基-[1,1'-联苯]-4-胺与5-(苄氧基)-6-氧代-8-(甲苯磺酰氧基)-5,6-二氢吡啶并[2,3-b]吡嗪-7-甲酸甲酯(5)反应制得5-(苄氧基)-8-((3',4'-二甲氧基-[1,1'-联苯]-4-基)氨基)-6-氧代-5,6-二氢吡啶并[2,3-b]吡嗪-7-甲酸甲酯(中间体6g)。甲醇/三氯甲烷重结晶,黄色针状晶体,收率:60%,熔点:200-201℃。
波谱数据:
1H NMR(400MHz,DMSO-d6)δ9.65(s,1H,N-H),8.89(d,J=2.4Hz,1H,pyrazine-H),8.67(d,J=2.4Hz,1H,pyrazine-H),7.65(d,J=8.5Hz,4H,Ph-H),7.47–7.41(m,3H,Ph-H),7.25–7.21(m,4H,Ph-H),7.04(d,J=8.2Hz,1H,Ph-H),5.17(s,2H,CH2),3.86(s,3H,CH3),3.79(s,3H,CH3),3.11(s,3H,CH3).13C NMR(100MHz,DMSO-d6)δ164.40,157.08,149.10,148.53,146.42,145.05,144.55,138.46,137.62,137.15,134.52,132.17,129.53(2×C),128.92,128.39(2×C),127.36,126.34(2×C),124.91(2×C),118.63,112.23,110.11,102.07,77.65,55.60(2×C),51.29.ESI-MS:m/z 539.5(M+1),561.4(M+23),577.4(M+39).C30H26N4O6[538.56].
取代芳香胺选用3',4'-二甲氧基-[1,1'-联苯]-3-胺与5-(苄氧基)-6-氧代-8-(甲苯磺酰氧基)-5,6-二氢吡啶并[2,3-b]吡嗪-7-甲酸甲酯(5)反应制得5-(苄氧基)-8-((3',4'-二甲氧基-[1,1'-联苯]-3-基)氨基)-6-氧代-5,6-二氢吡啶并[2,3-b]吡嗪-7-甲酸甲酯(中间体6h)。甲醇/三氯甲烷重结晶,黄色簇状晶体,收率:59%,熔点:159-161℃。
波谱数据:
1H NMR(400MHz,DMSO-d6)δ9.64(s,1H,N-H),8.89(d,J=2.4Hz,1H,pyrazine-H),8.67(d,J=2.4Hz,1H,pyrazine-H),7.65(dd,J=7.7,1.6Hz,2H,Ph-H),7.49(d,J=8.0Hz,1H,Ph-H),7.47-7.40(m,5H,Ph-H),7.24–7.22(m,2H,Ph-H),7.16(d,J=7.7Hz,1H,Ph-H),7.05(d,J=8.4Hz,1H,Ph-H),5.18(s,2H,CH2),3.85(s,3H,CH3),3.79(s,3H,CH3),3.02(s,3H,CH3).13C NMR(100MHz,DMSO-d6)δ164.40,157.07,149.03,148.70,146.45,145.03,144.58,140.50,139.27,138.45,134.52,132.28,129.53(2×C),129.21,128.91,128.38(2×C),127.35,123.44,122.94,122.04,118.85,112.16,110.37,102.04,77.65,55.58(2×C),51.16.ESI-MS:m/z539.6(M+1),561.4(M+23),577.4(M+39).C30H26N4O6[538.56].
取代芳香胺选用1-萘胺与5-(苄氧基)-6-氧代-8-(甲苯磺酰氧基)-5,6-二氢吡啶并[2,3-b]吡嗪-7-甲酸甲酯(5)反应制得5-(苄氧基)-8-(萘-1-基氨基)-6-氧代-5,6-二氢吡啶并[2,3-b]吡嗪-7-甲酸甲酯(中间体6i)。甲醇/三氯甲烷重结晶,黄色簇状晶体,收率:89%,熔点:213-214℃。
波谱数据:
1H NMR(400MHz,DMSO-d6)δ9.71(s,1H,N-H),8.92(d,J=2.4Hz,1H,pyrazine-H),8.71(d,J=2.4Hz,1H,pyrazine-H),8.01–7.94(m,2H,Ph-H),7.90(d,J=8.2Hz,1H,Ph-H),7.64(dd,J=7.7,1.7Hz,2H,Ph-H),7.59–7.49(m,3H,Ph-H),7.47–7.41(m,3H,Ph-H),7.38(d,J=7.3Hz,1H,Ph-H),5.17(s,2H,CH2),2.59(s,3H,CH3).13C NMR(100MHz,DMSO-d6)δ164.15,157.10,146.39,146.10,144.40,138.53,134.65,134.51,133.78,130.40,129.53(2×C),128.92,128.39(2×C),127.91,127.23,127.13,126.39,126.26,125.29,124.68,123.53,101.93,77.68,50.69.ESI-MS:m/z 453.5(M+1),475.4(M+23).C26H20N4O4[452.47].
取代芳香胺选用2-萘胺与5-(苄氧基)-6-氧代-8-(甲苯磺酰氧基)-5,6-二氢吡啶并[2,3-b]吡嗪-7-甲酸甲酯(5)反应制得5-(苄氧基)-8-(萘-2-基氨基)-6-氧代-5,6-二氢吡啶并[2,3-b]吡嗪-7-甲酸甲酯(中间体6j)。甲醇/三氯甲烷重结晶,黄色簇状晶体,收率:87%,熔点:185-186℃。
波谱数据:
1H NMR(400MHz,DMSO-d6)δ9.79(s,1H,N-H),8.90(d,J=2.3Hz,1H,pyrazine-H),8.69(d,J=2.3Hz,1H,pyrazine-H),7.92–7.90(m,2H,Ph-H),7.83(d,J=7.7Hz,1H,Ph-H),7.67–7.65(m,3H,Ph-H),7.54–7.38(m,6H,Ph-H),5.19(s,2H,CH2),2.80(s,3H,CH3).13CNMR(100MHz,DMSO-d6)δ164.39,157.09,146.48,145.26,144.58,138.53,136.63,134.51,132.88,130.84(2×C),129.53,128.92,128.39(2×C),128.33,127.56,127.39(2×C),126.53,125.60,123.98,121.27,102.35,77.67,51.00.ESI-MS:m/z 453.5(M+1),475.4(M+23),491.4(M+39).C26H20N4O4[452.47].
取代芳香胺选用[1,1'-联苯]-4-胺与5-(苄氧基)-6-氧代-8-(甲苯磺酰氧基)-5,6-二氢吡啶并[2,3-b]吡嗪-7-甲酸甲酯(5)反应制得8-([1,1'-联苯]-4-基氨基)-5-(苄氧基)-6-氧代-5,6-二氢吡啶并[2,3-b]吡嗪-7-甲酸甲酯(中间体6k)。甲醇/三氯甲烷重结晶,棕色块状晶体,收率:69%,熔点:185-187℃。
波谱数据:
1H NMR(400MHz,DMSO-d6)δ9.69(s,1H,N-H),8.89(d,J=2.4Hz,1H,pyrazine-H),8.68(d,J=2.4Hz,1H,pyrazine-H),7.70–7.64(m,6H,Ph-H),7.49-7.41(m,5H,Ph-H),7.37(t,J=7.3Hz,1H,Ph-H),7.28(d,J=8.5Hz,2H,Ph-H),5.18(s,2H,CH2),3.12(s,3H,CH3).13C NMR(100MHz,DMSO-d6)δ164.38,157.05,146.43,145.08,144.56,139.45,138.48,138.34,137.12,134.51,129.53,128.99,128.91,128.38,127.40,127.38,126.76,126.46,124.92,102.28,77.65,51.27.ESI-MS:m/z 479.5(M+1),501.5(M+23),517.4(M+39).C28H22N4O4[478.51].
实施例6:8-胺基-7-甲酸甲酯-吡嗪骈吡啶酮衍生物7a-7k的制备
将中间体6(1eq)加入到25mL茄型瓶中,加入5mL甲醇和5mL二氯甲烷使原料溶解,再加入10%Pd·C(10%w/w),氢气置换三次,在氢气球保护下,室温搅拌两小时。反应完毕,加硅藻土过滤,滤液蒸干,加入乙酸乙酯,超声,过滤收集滤饼,滤饼用少量甲醇/二氯甲烷溶解,然后加入正己烷使析出,过滤收集滤饼,滤饼干燥得目标产物7a-7k。
反应物选用5-(苄氧基)-8-((4'-氰基-[1,1'-联苯]-4-基)氨基)-6-氧代-5,6-二氢吡啶并[2,3-b]-7-甲酸甲酯(中间体6a)制得8-((4'-氰基-[1,1'-联苯]-4-基)氨基)-5-羟基-6-氧代-5,6-二氢吡啶并[2,3-b]吡嗪-7-甲酸甲酯(7a)。黄色固体,收率:32%,熔点:262-264℃。
波谱数据:
1H NMR(400MHz,DMSO-d6)δ10.98(s,1H,OH),9.60(s,1H,N-H),8.82(d,J=1.6Hz,1H,pyrazine-H),8.68(d,J=1.6Hz,1H,pyrazine-H),7.94-7.89(m,4H,Ph-H),7.76(d,J=8.4Hz,2H,Ph-H),7.29(d,J=8.4Hz,2H,Ph-H),3.11(s,3H,CH3).13C NMR(100MHz,DMSO-d6)δ164.51,157.60,146.42,144.96,144.11,143.88,139.99,137.90,134.49,132.88(2×C),127.23(2×C),127.19(2×C),126.86,124.38(2×C),118.9,109.77,103.25,51.32.ESI-MS:m/z 414.5(M+1),436.5(M+23),452.5(M+39).C22H15N5O4[413.39].
反应物选用5-(苄氧基)-8-((4'-氰基-[1,1'-联苯]-3-基)氨基)-6-氧代-5,6-二氢吡啶并[2,3-b]-7-甲酸甲酯(中间体6b)制得8-((4'-氰基-[1,1'-联苯]-3-基)氨基)-5-羟基-6-氧代-5,6-二氢吡啶并[2,3-b]吡嗪-7-甲酸甲酯(7b)。黄色固体,收率:61%,熔点:239-241℃。
波谱数据:
1H NMR(400MHz,DMSO-d6)δ10.94(s,1H,OH),9.55(s,1H,N-H),8.83(d,J=2.2Hz,1H,pyrazine-H),8.61(d,J=2.2Hz,1H,pyrazine-H),7.94(d,J=8.4Hz,2H,Ph-H),7.88(d,J=8.4Hz,2H,Ph-H),7.57(d,J=7.8Hz,1H,Ph-H),7.52–7.49(m,2H,Ph-H),7.29(d,J=7.8Hz,1H,Ph-H),2.97(s,3H,CH3).13C NMR(100MHz,DMSO-d6)δ164.59,157.61,146.42,144.93,144.39,144.04,139.91,138.45,137.84,132.88(2×C),129.65,127.47(2×C),126.83,124.82,123.73,122.46,118.84,110.25,102.78,51.08.ESI-MS:m/z 414.5(M+1).C22H15N5O4[413.39].
反应物选用5-(苄氧基)-8-((3'-氰基-[1,1'-联苯]-4-基)氨基)-6-氧代-5,6-二氢吡啶并[2,3-b]-7-甲酸甲酯(中间体6c)制得8-((3'-氰基-[1,1'-联苯]-4-基)氨基)-5-羟基-6-氧代-5,6-二氢吡啶并[2,3-b]吡嗪-7-甲酸甲酯(7c)。黄色固体,收率:54%,熔点:237-238℃。
波谱数据:
1H NMR(400MHz,DMSO-d6)δ10.97(s,1H,OH),9.57(s,1H,N-H),8.83(d,J=1.8Hz,1H,pyrazine-H),8.61(d,J=1.8Hz,1H,pyrazine-H),8.18(s,1H,Ph-H),8.05(d,J=7.8Hz,1H,Ph-H),7.82(d,J=7.8Hz,1H,Ph-H),7.75(d,J=8.5Hz,2H,Ph-H),7.67(t,J=7.8Hz,1H,Ph-H),7.28(d,J=8.5Hz,2H,Ph-H),3.11(s,3H,CH3).13C NMR(100MHz,DMSO-d6)δ164.55,157.65,146.43,144.96,144.16,140.54,139.57,137.91,134.43,131.19(2×C),130.91(2×C),130.20,129.92,127.07,126.86,124.52,118.85,112.16,103.07,51.38.ESI-MS:m/z 414.4(M+1),436.4(M+23),452.5(M+39).C22H15N5O4[413.39].
反应物选用5-(苄氧基)-8-((3'-氰基-[1,1'-联苯]-3-基)氨基)-6-氧代-5,6-二氢吡啶并[2,3-b]-7-甲酸甲酯(中间体6d)制得8-((3'-氰基-[1,1'-联苯]-3-基)氨基)-5-羟基-6-氧代-5,6-二氢吡啶并[2,3-b]吡嗪-7-甲酸甲酯(7d)。黄色固体,收率:60%,熔点:226-227℃。
波谱数据:
1H NMR(400MHz,DMSO-d6)δ10.94(s,1H,OH),9.53(s,1H,N-H),8.83(d,J=2.3Hz,1H,pyrazine-H),8.61(d,J=2.3Hz,1H,pyrazine-H),8.13(s,1H,Ph-H),8.03(d,J=7.8Hz,1H,Ph-H),7.85(d,J=7.8Hz,1H,Ph-H),7.69(t,J=7.8Hz,1H,Ph-H),7.58–7.47(m,3H,Ph-H),7.27(d,J=8.5Hz,1H,Ph-H),2.97(s,3H,CH3).13C NMR(101MHz,DMSO-d6)δ164.62,157.64,146.43,144.93,144.39,140.67,139.86,138.17,137.85,131.37,131.26,130.20,130.18,129.59,126.83,124.48,123.60,122.34,118.78,112.11,102.74,51.09.ESI-MS:m/z 414.4(M+1),431.5(M+18),436.5(M+23),452.5(M+39).C22H15N5O4[413.39].
反应物选用5-(苄氧基)-6-氧代-8-((4-(嘧啶-5-基)苯基)氨基)-5,6-二氢吡啶并[2,3-b]吡嗪-7-甲酸甲酯(中间体6e)制得5-羟基-6-氧代-8-((4-(嘧啶-5-基)苯基)氨基)-5,6-二氢吡啶并[2,3-b]吡嗪-7-甲酸甲酯(7e)。黄色固体,收率:57%,熔点:249-250℃。
波谱数据:
1H NMR(400MHz,DMSO-d6)δ10.99(s,1H,OH),9.61(s,1H,N-H),9.17(s,3H,pyrimidine-H),8.83(d,J=2.0Hz,1H,pyrazine-H),8.62(d,J=2.0Hz,1H,pyrazine-H),7.82(d,J=8.4Hz,2H,Ph-H),7.32(d,J=8.4Hz,2H,Ph-H),3.13(s,3H,CH3).13C NMR(100MHz,DMSO-d6)δ164.54,157.60,157.07,154.39(2×C),146.42,144.96,144.07,140.15,137.88,132.54,130.01,127.02(2×C),126.87,124.47(2×C),103.30,51.43.ESI-MS:m/z 391.4(M+1),413.5(M+23).C19H14N6O4[390.36].
反应物选用5-(苄氧基)-6-氧代-8-((3-(嘧啶-5-基)苯基)氨基)-5,6-二氢吡啶并[2,3-b]吡嗪-7-甲酸甲酯(中间体6f)制得5-羟基-6-氧代-8-((3-(嘧啶-5-基)苯基)氨基)-5,6-二氢吡啶并[2,3-b]吡嗪-7-甲酸甲酯(7f)。黄色固体,收率:51%,熔点:223-224℃。
波谱数据:
1H NMR(400MHz,DMSO-d6)δ10.98(s,1H,OH),9.59(s,1H,N-H),9.20(s,1H,pyrimidine-H),9.13(s,2H,pyrimidine-H),8.83(d,J=1.3Hz,1H,pyrazine-H),8.62(d,J=1.1Hz,1H,pyrazine-H),7.64–7.60(m,2H,Ph-H),7.52(t,J=7.8Hz,1H,Ph-H),7.32(d,J=7.7Hz,1H,Ph-H),2.98(s,3H,CH3).13C NMR(100MHz,DMSO-d6)δ164.65,157.61,157.47,154.63(2×C),146.46,144.94,144.39,140.12,137.87,133.97,132.66,129.80,126.83,124.86,123.48,122.10,102.93,51.11.ESI-MS:m/z 391.4(M+1),413.5(M+23),429.4(M+39).C19H14N6O4[390.36].
反应物选用5-(苄氧基)-8-((3',4'-二甲氧基-[1,1'-联苯]-4-基)氨基)-6-氧代-5,6-二氢吡啶并[2,3-b]吡嗪-7-甲酸甲酯(中间体6g)制得8-((3',4'-二甲氧基-[1,1'-联苯]-4-基)氨基)-5-羟基-6-氧代-5,6-二氢吡啶并[2,3-b]吡嗪-7-甲酸甲酯(7g)。黄色固体,收率:52%,熔点:213-215℃。
波谱数据:
1H NMR(400MHz,DMSO-d6)δ10.92(s,1H,OH),9.46(s,1H,N-H),8.82(d,J=2.0Hz,1H,pyrazine-H),8.61(d,J=2.0Hz,1H,pyrazine-H),7.64(d,J=8.5Hz,2H,Ph-H),7.23–7.21(m,4H,Ph-H),7.03(d,J=8.2Hz,1H,Ph-H),3.86(s,3H,CH3),3.79(s,3H,CH3),3.10(s,3H,CH3).13C NMR(100MHz,DMSO-d6)δ164.56,157.70,149.10,148.50,146.35,144.90,144.19,137.80,137.77,136.95,132.20,126.77,126.31(2×C),124.80(2×C),118.59,112.24,110.12,102.44,55.60,55.59,51.25.ESI-MS:m/z 449.5(M+1),471.5(M+23),487.4(M+39).C23H20N4O6[448.44].
反应物选用5-(苄氧基)-8-((3',4'-二甲氧基-[1,1'-联苯]-3-基)氨基)-6-氧代-5,6-二氢吡啶并[2,3-b]吡嗪-7-甲酸甲酯(中间体6h)制得8-((3',4'-二甲氧基-[1,1'-联苯]-3-基)氨基)-5-羟基-6-氧代-5,6-二氢吡啶并[2,3-b]吡嗪-7-甲酸甲酯(7h)。黄色固体,收率:36%,熔点:165-166℃。
波谱数据:
1H NMR(400MHz,DMSO-d6)δ10.96(s,1H,OH),9.48(s,1H,N-H),8.82(d,J=1.2Hz,1H,pyrazine-H),8.61(d,J=1.2Hz,1H,pyrazine-H),7.47(d,J=7.7Hz,1H,Ph-H),7.42–7.38(m,2H,Ph-H),7.23–7.19(m,2H,Ph-H),7.14(d,J=7.7Hz,1H,Ph-H),7.04(d,J=8.2Hz,1H,Ph-H),3.84(s,3H,CH3),3.79(s,3H,CH3),3.00(s,2H,CH3).13C NMR(100MHz,DMSO-d6)δ164.58,157.70,149.03,148.66,146.40,144.93,144.21,140.44,139.42,137.80,132.32,129.18,126.79,123.21,122.82,121.89,118.82,112.16,110.34,102.38,55.57,55.55,51.11.ESI-MS:m/z449.5(M+1).C23H20N4O6[448.44].
反应物选用5-(苄氧基)-8-(萘-1-基氨基)-6-氧代-5,6-二氢吡啶并[2,3-b]吡嗪-7-甲酸甲酯(中间体6i)制得5-羟基-8-(萘-1-基氨基)-6-氧代-5,6-二氢吡啶并[2,3-b]吡嗪-7-甲酸甲酯(7i)。黄色固体,收率:62%,熔点:211-213℃。
波谱数据:
1H NMR(400MHz,DMSO-d6)δ10.91(s,1H,OH),9.51(s,1H,N-H),8.85(d,J=1.4Hz,1H,pyrazine-H),8.64(d,J=1.4Hz,1H,pyrazine-H),7.98(d,J=7.7Hz,2H,Ph-H),7.88(d,J=8.2Hz,1H,Ph-H),7.76-7.47(m,3H,Ph-H),7.36(d,J=7.2Hz,1H,Ph-H),2.58(s,3H,CH3).13C NMR(100MHz,DMSO-d6)δ164.31,157.73,146.32,145.28,144.79,137.88,134.76,133.79,130.43,127.91,127.09,126.54,126.35,126.23,125.30,124.68,123.49,102.29,50.65.ESI-MS:m/z 363.4(M+1),385.4(M+23).C19H14N4O4[362.35].
反应物选用5-(苄氧基)-8-(萘-2-基氨基)-6-氧代-5,6-二氢吡啶并[2,3-b]吡嗪-7-甲酸甲酯(中间体6j)制得5-羟基-8-(萘-2-基氨基)-6-氧代-5,6-二氢吡啶并[2,3-b]吡嗪-7-甲酸甲酯(7j)。黄色固体,收率:25%,熔点:212-214℃。
波谱数据:
1H NMR(400MHz,DMSO-d6)δ10.97(s,1H,OH),9.63(s,1H,N-H),8.83(d,J=2.2Hz,1H,pyrazine-H),8.62(d,J=2.2Hz,1H,pyrazine-H),7.91-7.88(m,2H,Ph-H),7.81(d,J=7.7Hz,1H,Ph-H),7.64(s,1H,Ph-H),7.52–7.45(m,2H,Ph-H),7.37(dd,J=8.7,2.0Hz,1H,Ph-H),2.79(s,3H,CH3).13C NMR(100MHz,DMSO-d6)δ164.57,157.70,146.42,144.94,144.38,137.88,136.78,132.91,130.74,128.28,127.54,127.34,126.82,126.48,125.49,123.96,121.09,102.75,50.98.ESI-MS:m/z 363.4(M+1),385.3(M+23),401.4(M+39).C19H14N4O4[362.35].
反应物选用8-([1,1'-联苯]-4-基氨基)-5-(苄氧基)-6-氧代-5,6-二氢吡啶并[2,3-b]吡嗪-7-甲酸甲酯(中间体6k)制得8-([1,1'-联苯]-4-基氨基)-5-羟基-6-氧代-5,6-二氢吡啶并[2,3-b]吡嗪-7-甲酸甲酯(7k)。黄色固体,收率:35%,熔点:221-222℃。
波谱数据:
1H NMR(400MHz,DMSO-d6)δ10.93(s,1H,OH),9.50(s,1H,N-H),8.82(d,J=2.2Hz,1H,pyrazine-H),8.61(d,J=2.2Hz,1H,pyrazine-H),7.69-7.64(m,4H,Ph-H),7.47(t,J=7.6Hz,2H,Ph-H),7.36(t,J=7.3Hz,1H,Ph-H),7.26(d,J=8.4Hz,2H,Ph-H),3.10(s,3H,CH3).13C NMR(100MHz,DMSO-d6)δ164.54,157.67,146.36,144.91,144.19,139.47,138.48,137.82,136.91,128.96,127.34,126.78,126.72,126.42,124.80,102.64,51.23.ESI-MS:m/z 389.4(M+1),411.4(M+23),427.4(M+39).C21H16N4O4[388.38].
实施例7:核糖核酸酶H(RNase H)抑制活性测试实验
实验材料:
由大肠杆菌表达并经纯化的野生型HIV-1BH10逆转录酶;合成寡核苷酸31Trna(5′-UUUUUUUUUAGGAUACAUAUGGUUAAAGUAU-3′)和21P(5′-ATACTTTAACCATATG TATCC-3′)(购买自Sigma)。
实验步骤:
核糖核酸酶H活性经由模板-引物31Trna/21P测定。模板RNA 5’末端由[γ-32P]ATP((购买自Perkin Elmer)和T4多核苷酸激酶((购买自New England Biolabs)标记,然后经小型Quick SpinTM柱((购买自Roche)纯化。标记好的模板在含50mM NaCl的50mM Tris-HCl(pH 8.0)中退火至21-nt引物(21P)。在30μl的50mM Tris-HCl(pH 8.0),50mM NaCl,5mMMgCl2,25nM 32P-labeled模板-引物(31Trna/21P),及5%二甲基亚砜(DMSO)溶液中,20-40nM HIV-1逆转录酶在37℃下进行核糖核酸酶H裂解测定4分钟。化合物的稀释液在37℃下于逆转录酶缓冲液中与逆转录酶进行预孵育,之后加入标记的模板-引物使反应开始。在适当的时间取出少许并以等量样品缓冲液[10mM EDTA的90%甲酰胺(包含二甲苯蓝FF(3mg/mL)和溴酚蓝(3mg/mL))溶液]淬灭后,进行变性聚丙烯酰胺凝胶电泳分析。
实验结果:
对表1所示11个化合物进行抗HIV-1RNase H活性筛选,其HIV-1RNase H抑制活性数据见表2。阳性对照药为β-侧柏酚(β-thujaplicinol)。
表2目标化合物对野生型HIV-1RNase H的抑制活性
如表2所示,所有目标化合物对野生型HIV-1RNase H均表现出了不同程度的抑制活性,IC50在1.77~13.32μM区间内。其中两个化合物(7a、7f)对野生型HIV-1RNase H的抑制活性最高,IC50分别为1.77μM和1.85μM,均超过阳性对照β-侧柏酚(β-thujaplicinol)(IC50=2.50μM)。
实施例8:整合酶活性测试实验
测试原理:整合酶活性试验采用XpressBio公司的非放射性试剂盒XpressBioHIV-1Integrase Assay Kit,其测试原理为:链霉亲和素覆盖的96孔板上附上末端经生物素标记的双链HIV-1LTRU5供体(DS)DNA。全长重组HIV-1整合酶蛋白结合到DS DNA上。测试化合物加入到酶反应中,然后一个3’-末端修饰过的双链靶标(TS)DNA也被加入到反应混合物中。整合酶经化合物抑制后,不能发挥正常作用,经washing buffer洗涤除去未结合的TSDNA后,kit反应就此终止;在正常的kit流程中,HIV-1整合酶从HIV-1LTR DS DNA暴露的3’-末端裂解末端的两个碱基然后催化链转移重组反应,将DS DNA整合入TS DNA。接下来加入的HRP-标记的TS DNA 3’-末端修饰物的抗体就会结合到反应链上,加入HRP底物后即可通过酶标仪比色读数,经进一步计算得出抑制率。
实验材料:
XpressBio公司购买的HIV-1Integrase Assay Kit,14.3M的β-巯基乙醇,移液枪,枪头,一次性手套,纸巾,去离子水,37℃孵育器,37℃水浴,酶标仪(Thermo,VarioskanFlash)。
实验方法:
(1)预热试剂
实验前将reaction buffer和blocking solution置于37℃水浴10min,除HIV-1整合酶以外,将kit里所有其他成分放至室温条件预热。
(2)DS DNA覆盖
将需要量的DS DNA用reaction buffer稀释100倍(10μL100×DS DNA+990μLreaction buffer)。每孔加入100μL 1×DS DNA溶液,37℃孵育30分钟,将reaction buffer再放入37℃水浴。
(3)封板
将液体从96孔板中吸出,用300μL 1×wash buffer洗5次。每孔加入200μLblocking buffer并于37℃孵育30分钟。
(4)加入整合酶
用前将整合酶在湿冰上解冻(或2-8℃)5min并用离心机简单离心(10000rpm×5sec)。用reaction buffer将酶稀释300倍(2μL HIV-1整合酶+598μLreaction buffer)。
吸出96孔板中的液体,用200μL reaction buffer洗3次。每孔加入100μL的reaction buffer(空白对照)或整合酶溶液(阴性对照和实验组)37℃孵育30min。空白对照做复孔。将reaction buffer重新置于37℃水浴。
(5)加入抑制剂
将要测试的化合物用reaction buffer稀释至终浓度的两倍。例如,如果实验需要10μM或10μg/mL,就准备20μM或20μg/mL的溶液并连续用reaction buffer稀释得到一系列所需的梯度浓度。化合物最多可含5%DMSO体积比的终浓度。
将液体从孔里吸出,用200μL reaction buffer洗三次。每孔加入50μL化合物的reaction buffer溶液(空白和阴性对照只有reaction buffer),室温下孵育5min。
(6)加入TS DNA
将需要量的100×TS DNA用reaction buffer稀释100倍(如:10μL 100×TS DNA+990μL reaction buffer),每孔加50μL 1×TS DNA,在桌上水平轻摇平板3-5次将反应液混匀,37℃孵育30min。
(7)加入HRP抗体
吸出孔里的液体,用300μL wash buffer洗五遍。每孔加入100μL HRP抗体溶液,37℃孵育30min。
(8)加入过氧化物酶底物TMB
吸出板孔中的液体,用300μL wash buffer洗五遍。每孔加入100μL过氧化物酶底物TMB溶液,室温下孵育10min。
(9)加入TMB终止液
向含有TMB底物的孔里直接加入100μL TMB终止液。用枪头把大气泡戳破。用酶标仪读取450nm下的吸光度值。读数应在加入TMB终止液后10min内完成。
(10)数据处理
①吸光度校正
计算空白对照的平均吸光度(一般小于0.25OD单位)作为背景吸光度(A0)。阴性对照与实验组每个孔的吸光度读数(A)都减去这个背景吸光度得到校正过的吸光度(A校正)。
A校正=A-A0
②吸光度平均值与误差计算
阴性对照及实验组经背景校正后,计算吸光度平均值以及SD值和CV值(SD/mean×100%)。
③抑制率计算
首先计算活性百分比
整合酶活性=测试化合物的平均吸光度/阴性对照的平均吸光度值×100%。
SD校正=整合酶活性×CV阴性对照
抑制率=1–整合酶活性±SD校正
④IC50计算
方法一:以终浓度为横坐标,相应的抑制率为纵坐标对所得数据进行对数回归,通过得到的回归方程计算出抑制率为50%时对应的待测样品浓度,即IC50。
方法二:以ln(终浓度)为横坐标,相应的抑制率为纵坐标对所得数据进行线性回归,通过得到的回归方程可以计算出抑制率为50%时对应的待测样品浓度,即IC50。
实验结果:
对表1所示11个化合物进行抗HIV-1整合酶活性筛选,其HIV-1整合酶抑制活性数据见表3。阳性对照药设为雷特格韦(RAL)。
表3目标化合物对野生型HIV-1IN的抑制活性
如表3所示,除7c,7g,7i,7k(IC50均大于25μM)外,所有化合物对野生型HIV-1整合酶表现出了显著的抑制活性,IC50在0.22~22.80μM区间内。其中,化合物7d,7h对野生型HIV-1整合酶的抑制活性最高,IC50分别为0.25μM和0.22μM,接近于整合酶抑制剂上市药物雷特格韦(RAL)(IC50=0.20μM)。
Claims (5)
1.8-胺基-7-甲酸甲酯-吡嗪骈吡啶酮衍生物,其特征在于,化合物为下列之一:
2.如权利要求1所述的8-胺基-7-甲酸甲酯-吡嗪骈吡啶酮衍生物的制备方法,其特征在于,包括步骤:
以3-氯吡嗪-2-羧酸甲酯(1)为原料,与O-苄基羟胺经芳香性亲核取代反应生成(2),再经环合得到中间体5-(苄氧基)-8-羟基-6-氧代-5,6-二氢吡啶并[2,3-b]吡嗪-7-甲酸甲酯(4),再依次经与对甲基苯磺酰氯的亲核取代反应、与相应的取代芳胺的亲核取代反应、脱苄基反应得到目标化合物;
合成路线如下:
反应试剂与反应条件:i)O-苄基羟胺,N,N-二异丙基乙胺,二甲基亚砜,微波反应100℃,60min;ii)丙二酸单甲酯酰氯,三乙胺,二氯甲烷,0℃转45℃;iii)甲醇钠/甲醇,室温,过夜;iv)对甲基苯磺酰氯,N,N-二异丙基乙胺,二氯甲烷,乙腈,室温,过夜;v)相应的取代芳香胺,N,N-二异丙基乙胺,N,N-二甲基甲酰胺,50℃,2h;vi)H2,10%Pd·C,二氯甲烷/甲醇,室温;
其中,R为4'-氰基-[1,1'-联苯]-4-基,4'-氰基-[1,1'-联苯]-3-基,3'-氰基-[1,1'-联苯]-3-基,4-(嘧啶-5-基)苯基,3-(嘧啶-5-基)苯基,3',4'-二甲氧基-[1,1'-联苯]-3-基,萘-2-基。
3.如权利要求2所述的制备方法,其特征在于,具体步骤如下:
(1)将3-氯吡嗪-2-羧酸甲酯(1),O-苄基羟胺,二甲基亚砜和N,N-二异丙基乙胺加入到微波反应管,100℃条件下微波反应60min;反应完毕,将反应液移入分液漏斗,加入乙酸乙酯、饱和氯化钠溶液,分出有机相,水层用乙酸乙酯萃取,合并有机相,加入无水硫酸镁干燥,过滤,滤液减压浓缩,所得粗品经硅胶柱层析分离纯化后,重结晶得中间体3-((苄氧基)氨基)吡嗪-2-羧酸甲酯(2);
(2)将3-(苄氧基)氨基)吡嗪-2-羧酸甲酯(2),三乙胺和40mL二氯甲烷加入到100mL的茄型瓶中,并将其置于冰浴中搅拌;在冰浴下,向反应瓶中缓慢滴加丙二酸单甲酯酰氯;滴毕,撤去冰浴,将反应转移至45℃油浴回流搅拌;24小时后,将反应液移入分液漏斗,加入二氯甲烷,饱和氯化钠溶液进行萃取,水相用二氯甲烷萃取两次,合并有机相,加入无水硫酸镁干燥,过滤,滤液减压浓缩,所得粗品经硅胶柱层析分离纯化后,重结晶得中间体3-(N-(苄氧基)-3-甲氧基-3-氧代丙酰胺基)吡嗪-2-甲酸甲酯(3);
(3)将3-(N-(苄氧基)-3-甲氧基-3-氧代丙酰胺基)吡嗪-2-甲酸甲酯(3),甲醇钠和甲醇加入到50mL茄型瓶中,室温搅拌,过夜;反应完毕,向反应体系中滴加4N的HCl溶液,调节pH至3-4,此过程中有絮状白色不溶物生成,抽滤,滤饼干燥后,所得粗品重结晶得中间体5-(苄氧基)-8-羟基-6-氧代-5,6-二氢吡啶并[2,3-b]吡嗪-7-甲酸甲酯(4);
(4)将5-(苄氧基)-8-羟基-6-氧代-5,6-二氢吡啶并[2,3-b]吡嗪-7-甲酸甲酯(4),对甲基苯磺酰氯,N,N-二异丙基乙胺加入到50mL茄型瓶中;并向反应瓶中加入混合溶剂乙腈/二氯甲烷;室温搅拌,过夜;反应完毕,将反应液移入分液漏斗,加入饱和氯化钠溶液,二氯甲烷萃取,水相用二氯甲烷萃取,合并有机相,并用无水硫酸镁干燥;过滤,滤液减压浓缩,所得粗品经硅胶柱层析分离纯化后,重结晶得中间体5-(苄氧基)-6-氧代-8-(甲苯磺酰氧基)-5,6-二氢吡啶并[2,3-b]吡嗪-7-甲酸甲酯(5);
(5)将5-(苄氧基)-6-氧代-8-(甲苯磺酰氧基)-5,6-二氢吡啶并[2,3-b]吡嗪-7-甲酸甲酯(5),相应的取代芳香胺,N,N-二异丙基乙胺加入到100mL茄型瓶中,加入溶剂N,N-二甲基甲酰胺,50℃油浴加热,加盖干燥管,内装无水CaCl2,反应两小时;反应完毕,反应液用油泵尽量抽干,加入二氯甲烷溶解,然后将其转移入分液漏斗,加入饱和氯化钠溶液萃取,水相用二氯甲烷萃取两次,合并有机相,加入无水硫酸镁干燥;过滤,滤液减压浓缩,所得粗品经硅胶柱层析分离纯化后,重结晶得相应中间体(6a,6b,6d,6e,6f,6h,6j);所述相应的取代芳香胺选自:4'-氨基-[1,1'-联苯基]-4-腈、3'-氨基-[1,1'-联苯基]-4-腈、3'-氨基-[1,1'-联苯基]-3-腈、4-(嘧啶-5-基)苯胺、3-(嘧啶-5-基)苯胺、3',4'-二甲氧基-[1,1'-联苯]-3-胺、2-萘胺;
(6)将中间体(6a,6b,6d,6e,6f,6h,6j)加入到25mL茄型瓶中,加入甲醇和二氯甲烷使原料溶解,再加入10%Pd·C,氢气置换三次,在氢气球保护下,室温搅拌两小时;反应完毕,加硅藻土过滤,滤液蒸干,加入乙酸乙酯,超声,过滤收集滤饼,所得粗品重结晶得目标化合物(7a,7b,7d,7e,7f,7h,7j)。
4.如权利要求1所述的8-胺基-7-甲酸甲酯-吡嗪骈吡啶酮衍生物在制备HIV-1RNaseH-IN双靶点抑制剂中的应用。
5.一种抗HIV药物组合物,其特征在于,包括权利要求1所述的8-胺基-7-甲酸甲酯-吡嗪骈吡啶酮衍生物或其可药用盐和一种或多种药学上可接受载体或赋形剂。
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