CN114213416B - 一种恶唑并[4,5-b]吡啶结构的不可逆BTK抑制剂及其应用 - Google Patents
一种恶唑并[4,5-b]吡啶结构的不可逆BTK抑制剂及其应用 Download PDFInfo
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Abstract
Description
技术领域
本发明涉及小分子药物领域,具体地,本发明提供一种具有不可逆性抑制蛋白酪氨酸激酶(BTK)活性的化合物,及此类化合物的合成和使用方法。
背景技术
据世界卫生组织统计,淋巴瘤发病率的年增长率为5%~7%,每年死亡人数超过20万,目前,我国淋巴瘤发病率的年增长率为3%~5%,每年的新发病例约10万,已经成为第八大高发性恶性肿瘤。
布鲁顿酪氨酸激酶(Bruton’s tyrosine kinase,BTK)属于非受体酪氨酸激酶Tec家族,是一种膜结合蛋白,存在于除T细胞和自然杀伤细胞外的所有造血细胞中。BTK是B细胞受体通路的重要信号分子,在B细胞的各个发育阶段表达,参与调控B细胞的增殖、分化与凋亡,在恶性B细胞的生存及扩散中起着重要作用。
B细胞淋巴瘤发生的主要原因是B细胞抗原受体(BCR)信号通路的过度活化,BTK激酶是BCR信号通路中的关键过程蛋白,广泛分布于淋巴、造血及血液***中,是一种膜结合蛋白,在B细胞、肥大细胞和巨噬细胞等免疫细胞中表达。BTK大量表达会使BCR信号通路异常激活,影响B细胞的增殖、分化和凋亡,从而引发各种B细胞恶性肿瘤;还会使B细胞功能失调、免疫耐受状态改变,并转化为自身反应性B细胞,分泌大量自身抗体诱发自身免疫性疾病。因此,BTK是目前临床治疗B细胞类肿瘤及B细胞类免疫疾病的研究热点。
BTK抑制剂可通过抑制BCR信号通路的过度激活从而抑制淋巴细胞增殖,对B细胞恶性肿瘤及自身免疫性疾病的治疗展现出极好的治疗前景。2013年,依鲁替尼被FDA批准为第一个有效的BTK选择性抑制剂,突破性治疗用于慢性淋巴细胞白血病、套细胞淋巴瘤、小淋巴细胞淋巴瘤、华氏巨球蛋白血症、边缘区淋巴瘤和移植物宿主病的治疗,具有划时代的意义,使BTK成为一个有前途的治疗靶点。在此背景下,第二代BTK抑制剂阿卡替尼、替拉鲁替尼、泽布替尼和奥布替尼,提高了对BTK的选择性,并且改善耐药性和降低药物毒性,均已被FDA批准上市。上市的BTK抑制剂的结构如下所示:
根据与BTK催化结构域结合模式的不同,小分子BTK抑制剂被分成两类:可逆抑制剂和不可逆抑制剂。不可逆的BTK抑制剂保留末端的亲电中心,如丙烯酰胺基团和2-丁炔酰胺基团,可以通过Michael加成、亲核加成、加成-消除或亲核取代等反应,与BTK蛋白的保守非催化半胱氨酸残基(Cys481)形成共价键,实现不可逆性强结合。
但目前存在的不可逆性BTK抑制剂仍然存在选择性差、水溶性不佳等缺陷,在抑制BTK的同时还可以抑制Tec家族的其他激酶,导致了出血、腹泻、皮疹等副作用并产生耐药性,因而,开发新的BTK抑制药物仍然是一个亟待解决的问题。
发明内容
本发明的目的是针对现有技术的不足,提供一种具有不可逆性抑制蛋白酪氨酸激酶(BTK)活性的化合物及其药学上可用的盐、其合成方法及其在抑制BTK蛋白的药物中的应用。
本发明的目的是通过以下技术方案来实现的:一种具有BTK抑制活性的恶唑并[4,5-b]吡啶结构的小分子化合物,一种具有不可逆性抑制BTK活性的化合物及其药学上可用的盐,具有如式A的结构:
其中,X、Y、Z分别具以下结构:
Z=H、F,
其中R具有以下结构:
进一步地,所述的一种具有BTK抑制活性的恶唑并[4,5-b]吡啶结构的小分子化合物,X、Y、Z分别具以下结构:
其中R具有以下的结构:
或者,X、Y、Z分别具以下结构:
Z=H,F,
其中R具有以下结构:
更进一步地,所述的具有BTK抑制活性的恶唑并[4,5-b]吡啶结构的小分子化合物,选自以下结构:
或为如下结构:
或为如下结构:
本发明所述的一种具有不可逆性抑制BTK活性的化合物可以单独使用,也可以用常规的方法制备成药学上可接受的盐使用,所述药学上可接受的盐为盐酸盐、氢溴酸盐、氢碘酸盐、硫酸盐、硫酸氢盐、磷酸盐、乙酸盐、丙酸盐、丁酸盐、草酸盐、酒石酸盐、甲磺酸盐、对甲苯磺酸盐、富马酸盐、牛磺酸盐、柠檬酸盐、琥珀酸盐,或其混合盐。
本发明同时提供一种具有不可逆性抑制BTK活性的化合物的制备方法,举例如下:
以对溴苯甲酰氯为起始原料,与氨基吡嗪在三乙胺碱性条件下通过亲核取代得到4-溴-N-(吡嗪-2-基)苯甲酰胺;随后,4-溴-N-(吡嗪-2-基)苯甲酰胺与联硼酸频那醇酯通过Suzuki偶联得到相应的硼酸酯产物;接着,硼酸酯产物再与(R)-苯基2-(8-氨基-1-溴咪唑[1,5-A]吡嗪-3-基)吡咯烷-1-羧酸酯经铃木反应得到偶联产物;偶联产物在33%的HBr-AcOH溶液条件下脱去苄氧羰基保护基得到吡咯烷类化合物;吡咯烷类化合物与2-甲基丙烯酸、2-丁炔酸、丙烯酸在缩合剂HATU的作用下得到目标产物。反应过程如下:
或者采用以下方法:
以对溴苯甲酸为起始原料,与苯胺在缩合剂EDC·HCl/HOBt和N,N-二异丙基乙胺作用下通过缩合反应得到N-苯基-4-溴苯甲酰胺;接着,N-苯基-4-溴苯甲酰胺与联硼酸频那醇酯通过铃木偶联得到相应的硼酸酯产物;然后,原料4-氨基-3-碘-1H-唑咯并[3,4-D]嘧啶与原料(S)-1-叔丁氧羰基-3-羟基哌啶经Mitsunobu反应得到(3R)-1-Boc-3-(4-氨基-3-碘-1H-吡唑并[3,4-D]嘧啶-1-基)哌啶;之后硼酸酯产物再与(3R)-1-Boc-3-(4-氨基-3-碘-1H-吡唑并[3,4-D]嘧啶-1-基)哌啶经Suzuki反应得到偶联产物;偶联产物在3mol的HCl-EA溶液条件下脱去Boc保护基得到哌啶类化合物;哌啶类化合物与α,β-不饱和酸在缩合剂HATU的作用下得到目标产物。反应过程如下:
本发明还提供所述具有BTK抑制活性的恶唑并[4,5-b]吡啶结构的小分子化合物及其在药学上可接受的盐在药学上的应用,该应用具体为:用于制备预防或治疗因BTK异常所引起的疾病的药物制剂。所述的疾病为淋巴瘤,包括慢性淋巴细胞白血病、B细胞淋巴瘤、套细胞淋巴瘤、淋巴浆细胞淋巴瘤、弥漫性大B细胞淋巴瘤、非霍奇金淋巴瘤、滤泡中心淋巴瘤、边缘区B细胞淋巴瘤,或者为自体免疫疾病,包括类风湿关节炎、***性红斑狼疮、强直性脊柱炎、银屑病,等。
与现有技术相比,本发明具有如下优点:
本发明所述具有BTK抑制活性的恶唑并[4,5-b]吡啶结构的小分子化合物及其在药学上可接受的盐一般具有良好的BTK抑制活性,同时也提高了水溶性,其药代动力学参数与上市阳性药物依鲁替尼基本一致,因而可以在制备预防或治疗因BTK异常所引起的疾病的药物制剂中广泛应用。
附图说明
图1为依鲁替尼、Ⅳ-2和Ⅶ-2在SD大鼠体内的血药浓度时间曲线图。
具体实施方式
下面结合实施例对本发明的结构、制备方法及在制备预防或治疗因微管蛋白表达过多所引起的疾病的药物制剂方面的应用作进一步阐述,但不限制本发明。
样品的分析数据由以下仪器测定:
温度计未经校正;Bruker DRX400核磁共振仪;安捷伦5975型质谱仪;BrukerVector 22红外光谱仪。
实施例1系列Ⅰ化合物的合成:
1.1中间体1-3的合成:
称取2-氨基吡嗪1-1 1.00g(10.53mmol),置于25mL三颈瓶中,N2保护,注射器加入5mL二氯甲烷,冰浴条件下加入DIPEA 1.41g(10.94mmol),最后滴加4-溴苯甲酰氯1-22.00g(9.11mmol)的二氯甲烷溶液,保持冰浴5min后置于室温下反应过夜。经TLC确定反应完全,用NaOH调节反应液至中性,后用乙酸乙酯(40mL×3)萃取,有机相合并,饱和食盐水100mL洗一次,经无水硫酸钠干燥,旋干后经过柱层析纯化得到1.2g淡黄色固体1-3,收率约为48.2%。MS(ESI):m/z=278.01[M+H]+;1H NMR(400MHz,CDCl3)δ9.71(d,J=1.3Hz,1H),8.57(s,1H),8.41(d,J=2.5Hz,1H),8.28(dd,J=2.4,1.6Hz,1H),7.87–7.76(m,2H),7.74–7.62(m,2H).
1.2中间体1-4的合成:
依次称取中间体1-3 1.00g(3.61mmol),联硼酸频那醇酯1.10g(4.33mmol),[1,1'-双(二苯基膦)二茂铁]二氯化钯二氯甲烷络合物(Pd(dppf)Cl2)79mg(0.11mmol),醋酸钾1.06g(10.83mmol)置于25mL三颈瓶中,N2保护,之后注射器加入四氢呋喃溶液,回流过夜。点板原料反应完毕后,反应液冷却至室温后用硅藻土抽滤除去Pd(dppf)Cl2,并用四氢呋喃冲洗2~3次,滤液浓缩后加水加乙酸乙酯萃取,有机层用水洗2~3次,减压回收后得到粗品,经柱层析分离纯化后得到1.04g淡黄色固体1-4,收率约为89.2%,送核磁并转投下一步。1H NMR(400MHz,CDCl3)δ9.87(d,J=1.3Hz,1H),8.63(s,1H),8.57(d,J=2.5Hz,1H),8.39(dd,J=2.4,1.6Hz,1H),7.83(d,J=7.8Hz,2H),7.49(d,J=7.9Hz,2H),1.36(s,12H).
1.3中间体1-5的合成:
依次称取中间体1-4 110mg(0.34mmol),(R)-苯基2-(8-氨基-1-溴咪唑[1,5-a]吡嗪-3-基)吡咯烷-1-羧酸酯133mg(0.31mmol),Pd(dppf)Cl2 22mg(0.03mmol),K2CO3 128mg(0.93mmol)加入到10mL三颈瓶中,N2保护,冰浴条件下加入1,4-二氧六环:水(3:1)的混合溶液,然后转移至105℃开始反应。8h后TLC监测反应进展,反应毕反应液先冷却至室温,用硅藻土抽滤除去Pd(dppf)Cl2,并用乙酸乙酯冲洗2~3次,然后滤液加水(100mL×4)和100mL乙酸乙酯萃取,有机相用无水硫酸钠干燥,浓缩滤液进行柱层析分离得到110mg淡棕色固体1-5,收率约为61.9%;MS(ESI):m/z=535.24[M+H]+;1H NMR(500MHz,DMSO-d6)δ7.98(s,1H),7.92(d,J=7.7Hz,2H),7.71(d,J=4.6Hz,1H),7.64–7.50(m,3H),7.35(s,1H),7.24(s,3H),7.11–7.03(m,1H),7.04–6.95(m,2H),6.67(d,J=7.3Hz,1H),6.01(s,2H),5.33(d,J=19.2Hz,1H),4.95(s,2H),3.54–3.47(m,2H),1.91(d,J=6.6Hz,2H).
1.4中间体1-6的合成:
称取中间体1-5 300mg(0.56mmol)置于10mL单颈瓶,置于0℃环境中,缓慢滴加33%HBr-AcOH(6.6mmol),5min后转移至室温反应2h。TLC监测反应毕后,反应液加水50mL和二氯甲烷(70mL×2)萃取,二氯甲烷层舍去,水层在冰浴条件下用NaOH水溶液调pH≈10,加二氯甲烷(60mL×4)萃取,有机层合并,无水硫酸钠干燥后浓缩得粗品,干法拌样过柱,柱层析梯度洗脱得到淡棕色粉末状固体206mg,即中间体1-6,收率约为91.6%。MS(ESI):m/z=401.21[M+H]+;1H NMR(400MHz,DMSO-d6)δ11.26(s,1H),9.51(d,J=1.4Hz,1H),8.60–8.45(m,2H),8.28(d,J=8.4Hz,2H),7.95(d,J=5.0Hz,1H),7.87(d,J=8.4Hz,2H),7.32(d,J=5.0Hz,1H),6.47(s,2H),5.35(t,J=7.7Hz,1H),3.14(q,J=7.3Hz,2H),2.54–2.49(m,2H),2.26(dd,J=11.9,5.6Hz,2H),2.16–2.10(m,1H).
1.5化合物Ⅰ-1的合成:
将2-甲基丙烯酸1-7 8mg(0.09mmol)溶于二氯甲烷,依次加入EDC·HCl14mg(0.08mmol),三乙胺21μL,搅拌5min后加入中间体1-6 30mg(0.08mmol),室温反应。反应3h后,TLC监测反应毕,减压除去溶剂二氯甲烷,后加乙酸乙酯50mL溶解,加水70mL萃取,后有机层用1N的稀HCl溶液(70mL×2)萃取,饱和NaCl溶液70mL萃取一次,乙酸乙酯层用无水硫酸钠干燥后旋出溶剂得粗品,干法拌样过柱。梯度洗脱得到27mg黄色半固体,即化合物Ⅰ-1,收率约为77.1%;MS(ESI):[M+H]+m/z=469.21[M+H]+;1H NMR(500MHz,CDCl3)δ9.74(d,J=1.2Hz,1H),8.40(d,J=2.5Hz,1H),8.35–8.26(m,1H),8.05(d,J=8.0Hz,2H),7.80(d,J=8.3Hz,3H),7.07(d,J=5.0Hz,1H),5.50(t,J=6.6Hz,1H),5.29(s,2H),3.89–3.74(m,2H),2.58(dd,J=13.2,7.3Hz,2H),2.44–2.30(m,2H),1.24(s,3H).13C NMR(126MHz,CDCl3)δ171.38,165.20,151.38,148.43,142.06,141.62,140.70,140.42,139.12,137.44,134.15,132.61,130.06,127.95,127.64,118.04,114.66,107.99,58.94,49.49,31.02,25.66,19.68.
1.6化合物Ⅰ-2的合成:
操作同1.5,用2-丁炔酸1-8代替2-甲基丙烯酸1-7,得淡棕色半固体Ⅰ-2,收率约为49.6%;MS(ESI):m/z=467.20[M+H]+;1H NMR(400MHz,DMSO-d6)δ11.13(s,1H),8.55–8.37(m,3H),8.18(dd,J=8.2,2.7Hz,2H),8.11(dd,J=14.2,7.2Hz,2H),8.03–7.90(d,J=8.4Hz,1H),7.81–7.64(d,J=5.6Hz,1H),5.04(t,J=6.9Hz,1H),3.17–3.10(m,2H),2.01(dd,J=6.7,3.9Hz,2H),1.96(d,J=5.1Hz,2H),1.87(s,1H).13C NMR(101MHz,DMSO-d6)δ164.05,158.26,149.28,148.12,144.77,143.04,141.93,140.63,137.96,136.59,129.32,128.36,127.78,126.19,121.14,110.20,91.89,80.94,58.30,48.55,33.21,24.86.
1.7化合物Ⅰ-3的合成:
化合物Ⅰ-3为合成化合物Ⅰ-2时的副产物,12mg淡黄色半固体,MS(ESI):m/z=443.20[M+H]+;1H NMR(400MHz,DMSO-d6)δ11.16(s,1H),9.42(s,1H),8.56–8.40(m,2H),8.18(d,J=8.2Hz,2H),7.94(d,J=15.3Hz,1H),7.80(dd,J=29.5,6.3Hz,2H),7.13(d,J=4.6Hz,1H),5.45(t,J=6.3Hz,1H),3.73(t,J=5.3Hz,2H),2.35-2.12(m,2H),2.02-1.92(m,2H),1.81(s,3H).
1.8化合物Ⅰ-4的合成:
操作同1.5,用丙烯酸1-9代替2-甲基丙烯酸1-7,得到淡黄色粉末状固体Ⅰ-4,收率约为78.3%;MS(ESI):m/z=455.20[M+H]+;1H NMR(400MHz,DMSO-d6)δ11.18(s,1H),9.46(s,1H),8.56–8.40(m,2H),8.18(d,J=8.2Hz,2H),7.80(dd,J=29.5,6.3Hz,2H),7.13(d,J=4.6Hz,1H),6.95(t,J=15.3Hz,1H),6.05(d,J=7.9Hz,2H),5.25(t,J=6.4Hz,1H),4.73(t,J=5.3Hz,1H),3.23(t,J=4.8Hz,2H),2.27–2.13(m,2H),1.96(dd,J=11.9,6.7Hz,2H).13CNMR(101MHz,DMSO-d6)δ166.39,164.81,156.66,148.62,142.99,141.32,139.98,137.98,136.63,135.33,134.34,131.10,129.69,129.48,127.17,126.63,123.07,121.87,109.56,58.05,48.36,31.09,24.76.
实施例2系列Ⅱ化合物的合成:
2.1中间体2-3的合成:
称取对溴苯甲酸2-1 1.31g(6.52mmol)置于25mL单颈瓶中,加入10mL二氯甲烷,冰浴条件下依次加入EDC·HCl 1.25g(6.52mmol),DIPEA2.81mL(16.14mmol)搅拌5min,保持0℃加入苯胺2-2 0.50g(5.38mmol)后置于室温下反应过夜。经TLC监测反应完全,减压除去二氯甲烷,加饱和食盐水(200mL×3)和乙酸乙酯(150mL)萃取,有机相合并,经无水硫酸钠干燥,旋干后干法拌样过柱得到1.1g白色粉末状固体2-3,收率约为73.6%。MS(ESI):m/z=275.98[M+H]+;1H NMR(400MHz,DMSO-d6)δ10.33(s,1H),7.90(s,2H),7.76(dd,J=8.0,3.7Hz,4H),7.36(t,J=7.9Hz,2H),7.12(t,J=7.4Hz,1H).
2.2中间体2-4的合成:
操作同1.2,用中间体2-3代替中间体1-3,得到1.3g浅棕色粉末状固体,收率约为89.9%;1H NMR(400MHz,CDCl3)δ7.92(d,J=8.2Hz,2H),7.86(d,J=8.2Hz,3H),7.65(d,J=7.8Hz,2H),7.38(t,J=7.9Hz,2H),1.37(s,12H).
2.3中间体2-7的合成:
分别称取4-氨基-3-碘-1H-吡唑[3,4-d]嘧啶2-5 500mg(1.92mmol),(R)-1-叔丁氧羰基-3-羟基哌啶2-6 774mg(3.83mmol),三苯基膦755mg(2.88mmol)加入到三颈瓶中,N2保护,冰浴条件下用注射器加入无水THF,再慢慢滴加DIAD的无水THF溶液,后转移至室温反应过夜。TLC点板原料反应完后,减压除去THF,干法拌样过柱,PE润柱可冲出黄色的DIAD,之后梯度洗脱得到708mg白色粉末状固体,即中间体2-7,收率约为83.1%。MS(ESI):m/z=467.24[M+Na]+;1H NMR(400MHz,DMSO-d6)δ8.21(s,1H),4.65–4.53(m,1H),3.88(t,J=61.6Hz,2H),2.98(s,1H),2.19–2.08(m,1H),2.07–1.98(m,1H),1.87(s,1H),1.54(dt,J=21.8,7.5Hz,2H),1.32(s,9H).
2.4中间体2-8的合成:
依次称取中间体2-4 436mg(1.35mmol),中间体2-7 500mg(1.13mmol),Pd(dppf)Cl2 25mg(0.03mmol),K2CO3 468mg(3.39mmol)加入到10mL三颈瓶中,氮气保护,冰浴条件下加入1,4-二氧六环:水(3:1)的混合溶液,然后转移至105℃开始回流反应。8h后TLC监测反应进展,反应毕反应液先冷却至室温,用硅藻土抽滤除去Pd(dppf)Cl2,然后滤液加水(100mL×4)和100mL乙酸乙酯萃取,有机相用无水Na2SO4干燥,浓缩滤液进行柱层析分离得456mg浅棕色固体2-8,收率约为78.6%;MS(ESI):m/z=514.19[M+H]+;1H NMR(400MHz,DMSO-d6)δ10.36(s,1H),9.58–9.24(m,2H),8.46(s,1H),8.19(d,J=7.5Hz,2H),7.85–7.60(m,2H),7.24(t,J=7.5Hz,2H),7.09(t,J=7.2Hz,1H),4.36-4.27(m,1H),3.48(d,J=11.4Hz,1H),3.53–3.38(m,2H),3.32(d,J=12.8Hz,2H),3.12–2.95(m,1H),1.47(s,9H),1.89(d,J=4.6Hz,2H).
2.5中间体2-9的合成:
称取中间体2-8 300mg(0.58mmol)于单颈瓶中,加入2mL乙酸乙酯,冰浴条件下滴加3mL 3M HCl的乙酸乙酯溶液,滴加过程中反应液变浑浊,滴加完毕后反应液转移至室温反应2h。TLC监测反应毕后,反应液滤纸过滤,滤饼即为产物盐酸盐。后将滤饼置于100mL 0℃的K2CO3水溶液中,后用乙酸乙酯(120mL×4)萃取,有机相合并,无水硫酸钠干燥,浓缩后得到196mg淡棕色粉末状固体产物2-9,收率约为81.3%;MS(ESI):m/z=414.27[M+H]+;1HNMR(400MHz,DMSO-d6)δ10.44(s,1H),9.70–9.33(m,2H),8.55(s,1H),8.21(d,J=8.3Hz,2H),7.90–7.75(m,2H),7.38(t,J=7.9Hz,2H),7.13(t,J=7.4Hz,1H),5.23(ddd,J=14.7,10.2,4.3Hz,1H),3.53–3.38(m,2H),3.32(d,J=12.6Hz,2H),3.12–2.95(m,1H),2.19(dd,J=13.3,7.2Hz,2H),1.97(d,J=3.2Hz,2H).
2.6化合物Ⅱ-1的合成:
操作同1.5,用中间体2-9代替中间体1-6,得到白色半固体Ⅱ-1,收率约为69.8%。MS(ESI):m/z=482.23[M+H]+;1H NMR(400MHz,DMSO-d6)δ10.36(s,1H),8.30(s,1H),8.16(d,J=8.1Hz,2H),7.92–7.75(m,4H),7.38(t,J=7.8Hz,2H),7.13(t,J=7.4Hz,1H),5.10(d,J=49.1Hz,2H),3.80(d,J=9.8Hz,2H),4.11(d,J=40.3Hz,1H),3.55(d,J=17.6Hz,1H),3.17(d,J=54.7Hz,1H),2.29(t,J=11.8Hz,1H),2.16(d,J=15.3Hz,1H),1.93(s,3H),1.72–1.54(m,2H).13C NMR(101MHz,DMSO-d6)δ170.67,165.51,158.66,156.26,154.60,143.50,140.75,139.52,136.22,135.10,129.11,128.93,128.69,124.38,120.88,115.28,97.93,20.56.
2.7化合物Ⅱ-2的合成:
操作同1.5,用中间体2-9代替中间体1-6,丙烯酸2-10代替中间体1-7得到白色粉末状固体Ⅱ-2,收率约为67.4%。MS(ESI):m/z=467.21[M+H]+;1H NMR(400MHz,CDCl3)δ9.11(s,1H),8.42(d,J=8.4Hz,1H),8.37–8.27(m,2H),8.13(d,J=8.3Hz,2H),7.85(d,J=8.3Hz,2H),7.83–7.77(m,1H),7.12(dd,J=8.1,5.0Hz,1H),6.06(t,J=10.2Hz,1H),5.94(d,J=14.7Hz,1H),5.06(d,J=4.3Hz,1H),4.12–4.03(m,1H),3.58(d,J=7.3Hz,1H),2.37(d,J=11.1Hz,1H),2.29(d,J=3.6Hz,1H),2.06–1.94(m,2H),1.78(d,J=11.7Hz,1H),1.26(dd,J=7.8,6.5Hz,2H).13C NMR(101MHz,CDCl3)δ165.93,157.92,155.79,155.71,143.42,138.08,136.24,135.36,130.95,129.00,128.56,128.33,127.66,124.65,120.74,58.54,50.04,46.25,42.26,30.55,25.14,19.17.
2.8化合物Ⅱ-3的合成:
操作同1.5,用中间体2-9代替中间体1-6,2-氟丙烯酸代替中间体1-7,得到无色油状液体,收率约为81.6%。MS(ESI):m/z=482.23[M+H]+;1H NMR(400MHz,DMSO-d6)δ10.37(s,1H),8.62(s,1H),8.45(d,J=6.7Hz,2H),8.07(d,J=8.0Hz,2H),7.81(s,1H),7.64(dd,J=16.2,8.3Hz,1H),7.61–7.48(m,1H),7.37(d,J=14.7Hz,1H),7.12(t,J=7.0Hz,1H),6.23(d,J=30.7Hz,1H),6.07(d,J=15.0Hz,1H),4.61–4.49(m,1H),3.63(d,J=9.6Hz,2H),3.18–3.05(m,2H),2.21(dd,J=10.5,5.5Hz,1H),1.99(dd,J=13.6,7.5Hz,1H),1.80–1.60(m,2H).13C NMR(101MHz,DMSO-d6)δ165.50,165.08,153.38,151.16,149.62,146.55,139.62,135.26,132.63,131.90,129.17,129.08,127.81,124.38,120.94,120.88,102.04,54.07,46.25,38.69,29.49,18.52,17.16.
2.9化合物Ⅱ-4的合成:
操作同1.5,用中间体2-9代替中间体1-6,2-氯丙烯酸代替中间体1-7,反应液后处理得到白色半固体,收率约为71.4%。MS(ESI):m/z=502.31[M+H]+;1H NMR(400MHz,DMSO-d6)δ10.35(s,1H),8.30(s,1H),8.16(d,J=8.0Hz,2H),7.87–7.77(m,2H),7.64(dd,J=15.6,7.9Hz,1H),7.52(s,1H),7.38(t,J=7.7Hz,2H),7.13(t,J=7.3Hz,1H),6.03(d,J=9.5Hz,1H),5.86(d,J=5.4Hz,1H),4.97–4.55(m,1H),3.23(t,J=6.5Hz,2H),2.31(dd,J=24.0,13.9Hz,2H),2.24–2.13(m,1H),2.06–1.95(m,1H),1.38(dd,J=15.0,7.4Hz,2H).13CNMR(101MHz,DMSO-d6)δ167.41,163.85,158.63,143.62,139.55,136.19,135.16,132.17,131.84,129.11,128.70,120.95,65.50,54.06,46.23,38.83,23.04.
2.10化合物Ⅱ-5的合成:
操作同1.5,用中间体2-9代替中间体1-6,2-溴丙烯酸代替中间体1-7,反应液后处理得到棕色固体,收率约为75.3%。MS(ESI):m/z=546.12[M+H]+;1H NMR(500MHz,CDCl3)δ8.38(s,1H),8.07(d,J=8.3Hz,1H),7.82(d,J=8.1Hz,2H),7.72(dd,J=5.7,3.3Hz,2H),7.53(dd,J=5.7,3.3Hz,2H),7.39(t,J=7.9Hz,1H),7.18(t,J=7.0Hz,1H),6.63(d,J=11.5Hz,1H),6.08(d,J=5.7Hz,1H),3.97–3.85(m,1H),3.74(dd,J=7.5,1.9Hz,2H),3.56(d,J=2.1Hz,1H),3.52(d,J=2.7Hz,1H),2.12–1.87(m,2H),1.73–1.68(m,2H).13C NMR(126MHz,CDCl3)δ167.78,165.13,136.20,132.43,130.96,129.14,128.85,128.30,124.82,120.43,65.61,47.21,47.16,30.56,29.71,19.19.
实施例3系列Ⅲ化合物的合成:
3.1中间体3-3的合成:
取对溴苯甲酸4-1 2.43g(12.09mol)置于单颈烧瓶中,加入10mL二氯甲烷,冰浴条件下依次加入HATU 4.59g(12.09mmol),DIPEA5.14mL(30.30mmol)和环己胺4-2 0.98mL,然后转移至室温反应过夜。通过TLC(EA:PE=1:2)确定反应达到终点,减压除去二氯甲烷,加饱和食盐水(200mL×3)和乙酸乙酯(150mL)萃取,有机相合并,经无水硫酸钠干燥,浓缩后干法拌样过柱,PE润柱,EA:PE=1:5洗脱得到2.35g白色粉末状固体4-3,收率约为82.9%。MS(ESI):m/z=282.15[M+H]+;1H NMR(400MHz,CDCl3)δ7.65–7.59(m,2H),7.58–7.52(m,2H),3.95(tdt,J=11.7,8.0,3.9Hz,2H),2.07–1.97(m,2H),1.82–1.71(m,2H),1.48–1.35(m,2H),1.23(dd,J=13.3,2.5Hz,2H).
3.2中间体3-4的合成:
操作同1.2,用中间体3-3代替中间体1-3,得到白色粉末状固体3-4,收率约为89.6%;1H NMR(400MHz,DMSO-d6)δ7.85(d,J=8.0Hz,2H),7.73(d,J=8.0Hz,2H),3.82–3.67(m,2H),2.34–2.21(m,2H),1.76(m,2H),1.61(m,2H),1.37–1.26(dd,J=30.5,11.9Hz,2H),1.20(s,12H).
3.3中间体3-8的合成:
操作同2.4,用中间体4-4代替中间体2-4,得到白色粉末状固体3-8,收率约为62.6%;MS(ESI):m/z=520.46[M+H]+;1H NMR(400MHz,DMSO-d6)δ8.20(d,J=8.0Hz,1H),7.87(d,J=8.2Hz,2H),7.45(d,J=8.2Hz,2H),4.20–4.12(m,1H),3.81(t,J=7.5Hz,2H),3.34(d,J=10.1Hz,1H),3.14(d,J=10.7Hz,1H),3.01(d,J=11.5Hz,1H),3.02–2.54(m,2H),2.18(dd,J=15.7,7.1Hz,2H),1.95(m,4H),1.83(d,J=9.3Hz,1H),1.74–1.68(m,2H),1.62(d,J=12.5Hz,1H),1.46–1.33(m,2H),1.31(s,9H).
3.4中间体3-9的合成:
操作同2.5,用中间体3-8代替中间体2-8,得到淡黄色粉末状固体3-9,收率约为87.4%;MS(ESI):m/z=420.37[M+H]+;1H NMR(400MHz,DMSO-d6)δ8.40(d,J=7.9Hz,1H),8.07(d,J=7.9Hz,2H),7.75(d,J=8.0Hz,2H),4.30–4.18(m,1H),3.81(dd,J=7.5,9.5Hz,2H),3.54(t,J=9.5Hz,2H),3.44(dd,J=10.3,10.7Hz,2H),3.09–2.94(m,2H),2.17(dd,J=15.9,7.3Hz,1H),1.97(d,J=3.7Hz,1H),1.83(d,J=9.1Hz,1H),1.78–1.69(m,2H),1.63(d,J=12.7Hz,1H),1.43–1.23(m,2H),1.22–1.08(m,3H).
3.5化合物Ⅲ-1的合成:
操作同1.5,用中间体3-9代替中间体1-6,反应液后处理得到淡黄色粉末状固体Ⅲ-1,收率约为68.8%。MS(ESI):m/z=488.30[M+H]+;1H NMR(400MHz,Acetone-d6)δ8.28(s,1H),8.05(d,J=8.2Hz,2H),7.79(d,J=8.2Hz,2H),7.65(d,J=7.7Hz,1H),5.76(s,1H),5.56(s,1H),4.41–4.30(m,1H),4.02–3.87(m,2H),2.45–2.32(m,2H),2.27–2.17(m,2H),2.06(dt,J=4.3,2.2Hz,2H),2.02–1.95(m,3H),1.90(s,3H),1.83–1.71(m,4H),1.66(m,3H),1.21–1.11(m,2H).13C NMR(101MHz,Acetone-d6)δ170.47,165.13,158.41,155.89,154.67,143.43,141.03,135.91,135.27,128.26,127.97,114.19,98.06,48.88,32.74,25.50,19.73.
3.6化合物Ⅲ-2的合成:
操作同1.5,用中间体3-9代替中间体1-6,丙烯酸代替2-甲基丙烯酸1-7,得到白色粉末状固体Ⅲ-2,收率约为77.6%。MS(ESI):m/z=496.45[M+Na]+;1H NMR(400MHz,DMSO-d6)δ8.26(m,1H),8.02(d,J=8.1Hz,2H),7.74(d,J=7.7Hz,2H),6.66(d,J=16.6Hz,1H),6.10(d,J=5.6Hz,1H),4.83–4.65(m,1H),4.22(dd,J=14.0,7.6Hz,1H),3.79(dd,J=12.7,5.4Hz,1H),2.29(dd,J=11.6,2.8Hz,1H),2.14(dd,J=10.7,1.8Hz,1H),1.95(dd,J=13.3,2.6Hz,1H),1.84(d,J=7.7Hz,2H),1.80–1.70(m,2H),1.64(dd,J=14.3,7.1Hz,3H),1.33(dd,J=16.9,9.9Hz,4H),1.25(t,J=7.9Hz,5H).13C NMR(101MHz,DMSO-d6)δ167.41,165.87,165.05,158.67,156.31,155.21,149.07,136.62,136.01,135.71,134.10,132.17,131.98,129.28,129.13,128.62,125.63,116.52,98.01,65.49,45.60,30.47,19.12.
3.7化合物Ⅲ-3的合成:
操作同1.5,用中间体3-9代替中间体1-6,2-氟丙烯酸代替2-甲基丙烯酸1-7,得到黄色半固体化合物Ⅲ-3,收率约为80.3%。MS(ESI):m/z=492.25[M+H]+;1H NMR(400MHz,DMSO-d6)δ8.30(s,1H),8.03(d,J=8.1Hz,2H),7.74(d,J=8.1Hz,2H),6.25(s,1H),6.09(s,1H),3.84–3.71(m,1H),2.70(m,2H),2.31(dd,J=22.3,11.9Hz,2H),2.16(d,J=16.6Hz,2H),1.99(d,J=13.6Hz,2H),1.85(dt,J=8.2,10.9Hz,3H),1.75(dt,J=9.0,11.4Hz,3H),1.62(q,J=12.2Hz,3H),1.42–1.09(m,2H).13C NMR(101MHz,DMSO-d6)δ165.34,161.17,160.86,158.63,156.24,154.59,143.69,135.59,135.09,128.52,99.26,99.11,97.98,48.90,38.69,32.88,25.71,25.36.
3.8化合物Ⅲ-4的合成:
操作同1.5,用中间体3-9代替中间体1-6,2-氯丙烯酸代替2-甲基丙烯酸1-7,得到淡黄色粉末状固体Ⅲ-4,收率约为72.4%。MS(ESI):m/z=508.22[M+H]+;1H NMR(400MHz,DMSO-d6)δ8.35–8.27(s,1H),8.03(d,J=8.0Hz,2H),7.74(d,J=8.0Hz,2H),5.86(s,1H),5.61(s,1H),3.88–3.71(m,1H),3.62(dq,J=10.5,6.6Hz,2H),3.21–3.06(m,3H),2.38–2.23(m,2H),2.22–2.09(m,2H),1.98(d,J=12.6Hz,2H),1.84(m,2H),1.75(q,J=6.8Hz,2H),1.62(m,2H),1.39–1.31(m,2H).13C NMR(101MHz,DMSO-d6)δ165.39,163.80,158.62,156.22,154.59,143.73,135.57,135.10,128.50,97.99,54.06,48.89,42.30,32.89,25.72,18.52.
3.9化合物Ⅲ-5的合成:
操作同1.5,用中间体3-9代替中间体1-6,2-溴丙烯酸代替2-甲基丙烯酸1-7,得到棕色固体化合物Ⅲ-5,收率约为85.3%。MS(ESI):m/z=552.16[M+H]+;1H NMR(500MHz,CDCl3)δ8.37(s,1H),7.93(d,J=8.3Hz,2H),7.76(d,J=8.2Hz,2H),6.17(s,1H),6.05(s,1H),4.09–3.95(m,1H),3.22(q,J=7.3Hz,2H),2.42–2.31(m,2H),2.27(dd,J=13.0,3.8Hz,2H),2.11–2.00(m,3H),1.84–1.75(m,3H),1.29(dd,J=11.3,2.8Hz,4H),1.14–1.05(m,2H).13C NMR(126MHz,CDCl3)δ166.05,165.06,157.60,155.69,155.62,143.67,135.83,135.64,132.29,130.93,128.85,128.59,127.97,65.60,55.65,49.02,33.16,24.95,17.18.
实施例4系列Ⅳ化合物的合成:
4.1中间体4-3的合成:
操作同2.1,2-氨基吡啶代替化合物苯胺2-2,得到棕色粉末状固体4-3,收率约为89.1%。MS(ESI):m/z=276.99[M+H]+;1H NMR(400MHz,CDCl3)δ8.93(s,1H),8.63(d,J=1.3Hz,1H),8.46(d,J=2.5Hz,1H),8.35(dd,J=2.5,1.5Hz,1H),7.60–7.58(m,2H),7.55–7.53(m,2H).
4.2中间体4-4的合成:
操作同2.2,中间体4-3代替中间体2-3,得到淡棕色粉末状固体4-4,收率约为92.6%;1H NMR(400MHz,DMSO-d6)δ10.89(s,1H),8.40(d,J=4.8Hz,1H),8.19(d,J=8.4Hz,1H),8.02(d,J=8.2Hz,2H),7.88–7.82(m,1H),7.79(d,J=8.2Hz,2H),7.24–7.15(m,1H),1.32(s,12H).
4.3中间体4-8的合成:
操作同2.5,中间体4-4代替中间体2-4,得到淡棕色粉末状固体4-8,收率约为73.4%;MS(ESI):m/z=515.25[M+H]+;1H NMR(400MHz,CDCl3)δ9.10(s,1H),8.44(d,J=8.4Hz,1H),8.37(s,1H),8.32(d,J=4.9Hz,1H),8.14(d,J=8.3Hz,2H),7.87(d,J=8.2Hz,2H),7.85–7.77(m,1H),7.13(dd,J=7.3,5.0Hz,1H),4.87–4.73(m,1H),3.63(dd,J=13.5,6.4Hz,2H),2.90(t,J=11.5Hz,2H),2.26–2.17(m,2H),1.84–1.64(m,2H),1.46(s,12H).
4.4中间体4-9的合成:
操作同2.4,中间体4-8代替中间体2-8,得到淡棕色粉末状固体4-9,收率约为61.9%;MS(ESI):m/z=437.20[M+H]+;1H NMR(400MHz,DMSO-d6)δ11.66(s,1H),9.75(d,J=10.1Hz,1H),9.51(d,J=10.9Hz,1H),8.64(s,1H),8.49(d,J=4.8Hz,1H),8.36–8.26(m,2H),8.10(t,J=7.7Hz,1H),7.86(d,J=8.1Hz,1H),7.47–7.30(m,1H),5.26–5.10(m,1H),3.54(d,J=10.7Hz,1H),3.50–3.38(m,2H),3.31(d,J=11.5Hz,1H),3.12–2.93(m,1H),2.15(d,J=5.2Hz,2H),1.05(t,J=7.0Hz,1H).
4.5化合物Ⅳ-1的合成:
操作同1.5,中间体4-9代替中间体1-6,得到白色粉末状固体Ⅳ-1,收率约为81.4%;MS(ESI):m/z=483.23[M+H]+;1H NMR(500MHz,CDCl3)δ9.28(s,1H),8.42(d,J=8.4Hz,1H),8.29(d,J=4.8Hz,2H),8.12(d,J=8.0Hz,2H),7.84(d,J=7.2Hz,2H),7.79(t,J=7.9Hz,1H),7.14–7.07(m,1H),5.17(s,1H),5.11(s,1H),5.06–4.88(m,1H),3.44–3.29(m,2H),2.86(d,J=9.8Hz,2H),1.97(s,3H),1.80–1.63(m,2H),1.68-1.58(m,2H).13C NMR(126MHz,CDCl3)δ171.51,165.26,157.99,155.84,154.41,151.56,147.77,143.23,134.74,128.81,128.40,120.16,115.52,114.53,98.66,98.59,30.28,29.68,20.52.
4.6化合物Ⅳ-2的合成:
操作同1.5,中间体4-9代替中间体1-6,丙烯酸代替2-甲基丙烯酸1-7,得到白色粉末状固体Ⅳ-2,收率约为74.3%;MS(ESI):m/z=469.21[M+H]+;1H NMR(500MHz,CDCl3)δ9.30(s,1H),8.42(d,J=8.3Hz,1H),8.29(dd,J=28.2,24.2Hz,2H),8.12(d,J=5.9Hz,2H),7.84(d,J=7.9Hz,2H),7.79(t,J=7.4Hz,1H),7.15–7.05(m,1H),6.69(q,J=6.6Hz,1H),6.30(d,J=16.9Hz,1H),5.69(d,J=4.1Hz,1H),4.86–4.72(m,1H),3.42–3.31(m,1H),2.92(t,J=11.4Hz,1H),2.38(dd,J=26.1,12.0Hz,1H),2.26(d,J=9.3Hz,1H),2.03(dd,J=20.8,9.0Hz,2H),1.29(dt,J=31.4,7.1Hz,2H).
4.7化合物Ⅳ-3的合成:
操作同1.5,中间体4-9代替中间体1-6,2-氟丙烯酸代替2-甲基丙烯酸1-7,得到米白色粉末状固体Ⅳ-3,收率约为61.0%;MS(ESI):m/z=487.20[M+H]+;1H NMR(400MHz,CDCl3)δ9.23(s,1H),8.43(d,J=8.4Hz,2H),8.30(d,J=4.9Hz,4H),8.13(d,J=8.3Hz,4H),7.89–7.76(m,6H),7.15–7.07(m,2H),5.12(d,J=13.3Hz,2H),5.00–4.86(m,2H),2.48–2.17(m,4H),2.13–1.97(m,3H),1.85–1.67(m,2H),1.25(d,J=5.8Hz,4H).13C NMR(101MHz,CDCl3)δ165.19,161.62,161.32,157.73,155.47,154.35,151.49,147.62,143.43,138.82,136.82,134.77,128.81,128.45,120.19,114.56,99.81,99.65,98.55,30.20,29.69.
4.8化合物Ⅳ-4的合成:
操作同1.5,中间体4-9代替中间体1-6,2-氯丙烯酸代替2-甲基丙烯酸1-7,得到淡棕色粉末状固体Ⅳ-4,收率约为64.8%;MS(ESI):m/z=503.17[M+H]+;1H NMR(400MHz,CDCl3)δ9.29(s,1H),8.43(d,J=8.4Hz,1H),8.38–8.24(m,2H),8.14(d,J=8.3Hz,2H),7.82(dd,J=19.1,8.7Hz,3H),7.17–7.05(m,1H),5.68(s,1H),5.64(s,1H),3.95–3.82(m,1H),2.37(d,J=10.7Hz,1H),2.31–2.23(m,2H),2.03(d,J=11.1Hz,1H),1.85–1.72(m,2H),1.26(t,J=7.1Hz,2H).13C NMR(101MHz,CDCl3)δ165.22,164.52,157.75,155.44,154.31,151.50,147.53,143.52,138.88,136.76,134.76,128.81,128.48,120.19,117.61,114.62,98.67,53.42,30.16,21.38,13.90.
4.9化合物Ⅳ-5的合成:
操作同1.5,中间体4-9代替中间体1-6,2-溴丙烯酸代替2-甲基丙烯酸1-7,得到浅棕色粉末状固体Ⅳ-5,收率约为67.5%;MS(ESI):m/z=547.12[M+H]+;1H NMR(400MHz,CDCl3)δ9.11(s,1H),8.42(d,J=8.4Hz,1H),8.37–8.27(m,2H),8.13(d,J=8.3Hz,2H),7.85(d,J=8.3Hz,2H),7.83–7.77(m,1H),7.12(dd,J=8.1,5.0Hz,1H),6.43(s,1H),6.06(s,1H),4.12–3.98(m,1H),3.58(d,J=7.3Hz,1H),2.37(d,J=11.1Hz,1H),2.29(d,J=3.6Hz,1H),2.06–1.94(m,2H),1.78(d,J=11.7Hz,1H),1.26(dd,J=7.8,6.5Hz,2H).13CNMR(101MHz,CDCl3)δ165.14,165.00,157.85,155.86,151.48,147.76,143.34,134.75,128.84,128.40,120.21,114.48,98.62,30.18.
4.10化合物Ⅳ-6的合成:
操作同1.5,中间体4-9代替中间体1-6,2-丁炔酸代替2-甲基丙烯酸1-7,得到白色粉末状固体Ⅳ-6,收率约为45.3%;MS(ESI):m/z=481.21[M+H]+;1H NMR(400MHz,CDCl3)δ8.80(s,1H),8.42(d,J=9.6Hz,2H),8.36–8.31(m,1H),8.12(dd,J=8.1,5.7Hz,2H),7.86(t,J=8.3Hz,2H),7.80(t,J=7.9Hz,1H),7.12(t,J=5.9Hz,1H),5.00–4.82(m,1H),4.51(ddd,J=44.4,22.2,8.9Hz,2H),3.34(dd,J=14.5,7.3Hz,1H),2.34(dd,J=12.0,3.6Hz,1H),1.91(s,3H),1.74(dd,J=25.3,13.3Hz,2H),1.33(t,J=9.6Hz,2H).13C NMR(101MHz,CDCl3)δ165.00,157.73,155.94,153.45,153.35,151.38,147.93,138.66,136.94,134.65,128.90,128.88,128.35,128.29,120.27,114.33,89.83,53.35,52.54,46.91,41.30,30.14,25.06,23.76.
实施例5系列Ⅴ化合物的合成:
5.1中间体5-3的合成:
将2-氨基-3-羟基吡啶5-1 1.10g(10mmol)对溴苯甲醛5-2 1.10g(10mmol),多聚磷酸(30mL)置于100mL单颈瓶中,氮气保护,150℃加热1h。反应液倒入300mL水中,抽滤,水洗,干燥后得到1.90g橘黄色固体,即中间体5-3,收率约为69.3%;MS(ESI):m/z=274.98[M+H]+;1H NMR(400MHz,DMSO-d6)δ8.57(dd,J=4.9,1.4Hz,1H),8.27(dd,J=8.2,1.4Hz,1H),8.21–8.15(m,2H),7.91–7.84(m,2H),7.50(dd,J=8.2,4.9Hz,1H).
5.2中间体5-4的合成:
操作同1.2,中间体5-3代替中间体1-3,得到淡黄色半固体5-4,收率约为89.8%;1H NMR(400MHz,DMSO-d6)δ8.58(dd,J=4.8,1.4Hz,1H),8.27(dd,J=8.2,1.4Hz,3H),7.93(d,J=8.3Hz,2H),7.50(dd,J=8.2,4.9Hz,1H),1.34(s,12H).
5.3中间体5-8的合成:
操作同2.4,中间体5-4代替中间体2-4,得到淡棕色半固体5-8,收率约为76.3%;MS(ESI):m/z=513.42[M+H]+;1H NMR(500MHz,CDCl3)δ8.63(dd,J=4.9,1.4Hz,1H),8.50(d,J=8.3Hz,2H),8.41(s,1H),7.95–7.89(m,3H),7.35(dd,J=8.1,4.9Hz,1H),4.96–4.83(m,1H),3.51(d,J=7.1Hz,2H),2.89(t,J=12.0Hz,1H),2.35–2.17(m,3H),1.79–1.66(m,2H),1.45(s,9H).
5.4中间体5-9的合成:
操作同2.5,中间体5-8代替中间体2-8,得到淡棕色粉末状固体5-9,收率约为81.0%;MS(ESI):m/z=413.35[M+H]+;1H NMR(400MHz,DMSO-d6)δ8.59(dd,J=4.8,1.3Hz,1H),8.41(d,J=8.4Hz,2H),8.34–8.26(m,2H),7.95(d,J=8.4Hz,2H),7.51(dd,J=8.2,4.9Hz,1H),3.15–3.06(m,1H),2.95(dd,J=23.5,11.9Hz,2H),2.12(dd,J=28.1,18.2,7.4Hz,2H),1.69(d,J=2.7Hz,1H),1.58(dd,J=8.8,3.7Hz,2H),1.37(d,J=7.1Hz,1H).
5.5化合物Ⅴ-1的合成:
操作同1.5,中间体5-9代替中间体1-6,得到白色粉末状固体Ⅴ-1,收率约为74.2%;MS(ESI):m/z=481.21[M+H]+;1H NMR(400MHz,CDCl3)δ8.63(dd,J=4.9,1.2Hz,1H),8.50(d,J=8.3Hz,2H),8.36(s,1H),7.99–7.85(m,3H),7.36(dd,J=8.1,4.9Hz,1H),5.19(s,1H),5.10(s,1H),3.91–3.79(m,1H),2.23(t,J=6.6Hz,2H),2.10–1.98(m,2H),1.85(s,3H),1.73(d,J=10.5Hz,2H),1.49–1.25(m,2H).
5.6化合物Ⅴ-2的合成:
操作同1.5,中间体5-9代替中间体1-6,丙烯酸代替2-甲基丙烯酸1-7,得到白色粉末状固体Ⅴ-2,收率约为79.6%;MS(ESI):m/z=467.20[M+H]+;1H NMR(400MHz,CDCl3)δ8.62(d,J=4.7Hz,1H),8.49(d,J=8.0Hz,2H),8.39(s,1H),7.92(dd,J=7.7,4.6Hz,3H),7.35(dd,J=8.0,4.9Hz,1H),6.74(t,J=6.0Hz,1H),6.48(d,J=13.3Hz,1H),6.19(d,J=7.6Hz,1H),3.87–3.65(m,1H),3.22(d,J=16.2Hz,1H),3.03–2.79(m,1H),2.50–2.32(m,2H),2.29(dd,J=14.8,6.2Hz,1H),2.09–1.93(m,2H),1.75(dd,J=24.2,11.7Hz,1H).13CNMR(101MHz,CDCl3)δ165.78,162.29,157.73,156.23,155.84,147.03,143.27,137.90,134.85,134.73,129.03,128.12,127.60,123.84,120.45,118.38,98.59,61.92,48.66,29.70,20.48.
5.7化合物Ⅴ-3的合成:
操作同1.5,中间体5-9代替中间体1-6,2-氟丙烯酸代替2-甲基丙烯酸1-7,得到白色粉末状固体Ⅴ-3,收率约为73.8%;MS(ESI):m/z=485.19[M+H]+;1H NMR(400MHz,CDCl3)δ8.62(dd,J=4.9,1.2Hz,1H),8.49(d,J=8.3Hz,2H),8.40(s,1H),7.97–7.86(m,3H),7.35(dd,J=8.1,4.9Hz,1H),5.90(s,1H),5.12(s,1H),3.97–3.82(m,1H),3.38(d,J=11.2Hz,2H),2.29(dd,J=12.9,3.7Hz,1H),2.10–2.00(m,2H),1.79(dd,J=25.7,12.2Hz,1H),1.53–1.37(m,2H).13C NMR(101MHz,CDCl3)δ164.84,161.62,161.32,157.75,156.20,155.81,154.52,147.02,143.33,143.27,136.97,129.02,126.98,120.46,118.38,29.69.
5.8化合物Ⅴ-4的合成:
操作同1.5,中间体5-9代替中间体1-6,2-氯丙烯酸代替2-甲基丙烯酸1-7,得到淡棕色粉末状固体Ⅴ-4,收率约为61.2%;MS(ESI):m/z=501.16[M+H]+;1H NMR(400MHz,CDCl3)δ8.62(dd,J=4.9,1.4Hz,1H),8.49(d,J=8.4Hz,2H),8.37(s,1H),7.96–7.87(m,3H),7.35(dd,J=8.1,4.9Hz,1H),5.68(s,1H),4.97(s,1H),3.64–3.48(m,1H),3.26(d,J=5.2Hz,2H),2.46–2.35(m,2H),2.07(t,J=7.3Hz,1H),1.79(d,J=13.4Hz,1H),1.33–1.24(m,2H).13C NMR(101MHz,CDCl3)δ176.14,164.83,164.52,157.84,156.18,155.61,154.46,147.01,143.48,143.27,136.93,129.02,127.01,120.47,118.41,98.58,60.41,30.19,29.69,21.06.
5.9化合物Ⅴ-5的合成:
操作同1.5,中间体5-9代替中间体1-6,2-溴丙烯酸代替2-甲基丙烯酸1-7,得到淡棕色粉末状固体Ⅴ-5,收率约为57.4%;MS(ESI):m/z=545.10[M+H]+;1H NMR(400MHz,DMSO-d6)δ8.59(d,J=4.6Hz,1H),8.43(d,J=8.1Hz,2H),8.33(s,1H),8.29(d,J=8.1Hz,1H),7.96(d,J=7.8Hz,2H),7.51(dd,J=8.1,4.9Hz,1H),6.14(s,1H),5.92(s,1H),4.45–4.35(m,1H),3.25(t,J=7.3Hz,2H),2.39(d,J=15.0Hz,1H),2.25–2.12(m,2H),2.01(dd,J=10.9,4.2Hz,1H),1.33–1.16(m,2H).13C NMR(101MHz,DMSO-d6)δ165.03,162.87,160.29,158.05,155.93,149.01,147.23,143.64,143.37,138.69,137.00,129.66,128.93,126.28,121.54,119.63,106.77,79.71,62.65,47.15,44.91,27.07,21.53.
5.10化合物Ⅴ-6的合成:
操作同1.5,中间体5-9代替中间体1-6,2-丁炔酸代替2-甲基丙烯酸1-7,得到白色粉末状固体Ⅴ-6,收率约为45.6%;MS(ESI):m/z=479.19[M+H]+;1H NMR(400MHz,CDCl3)δ8.62(d,J=4.4Hz,1H),8.49(d,J=8.2Hz,2H),8.43–8.35(m,1H),7.92(t,J=7.3Hz,3H),7.35(dd,J=8.0,4.9Hz,1H),5.03–4.83(m,1H),3.96(dd,J=15.6,8.4Hz,2H),3.08(t,J=4.2Hz,2H),2.32–2.24(m,2H),1.98(s,3H),1.80–1.66(m,2H).13C NMR(101MHz,CDCl3)δ171.54,171.54,164.86,164.86,157.76,157.71,156.19,156.19,155.79,155.79,154.47,154.47,147.02,147.02,143.27,143.27,137.02,129.03,127.01,120.47,120.47,118.41,118.41,98.60,77.37,76.73,31.89,29.70,29.30,22.69,20.55,4.06.
实施例6系列Ⅵ化合物的合成:
6.1中间体6-3的合成:
称取对溴苯甲酸6-1 2.43g(12.09mol)置于单颈烧瓶中,加入10mL二氯甲烷,0℃条件下依次加入HATU 4.59g(12.09mmol),DIPEA 5.14mL(30.30mmol)和2-氨基-4-氟吡啶6-2 1.13g(10.08mmol),然后转移至室温反应过夜。通过TLC(EA:PE=1:5)确定反应达到终点,减压除去二氯甲烷,加饱和食盐水(400mL×3)和乙酸乙酯500mL萃取,有机相合并,经无水硫酸钠干燥,浓缩后干法拌样过柱,PE润柱,EA:PE=1:5洗脱得到2.27g白色粉末状固体,收率约为76.5%。MS(ESI):m/z=294.98[M+H]+;1H NMR(400MHz,DMSO-d6)δ10.99(s,1H),8.41(ddd,J=4.8,1.9,0.8Hz,1H),8.17(d,J=8.4Hz,1H),8.02(dd,J=9.8,1.9Hz,1H),7.93–7.79(m,3H),7.25–7.14(m,1H).
6.2中间体6-4的合成:
操作同1.2,中间体6-3代替中间体1-3,得白色固体6-4。收率约为89.6%;1H NMR(400MHz,DMSO-d6)δ10.93(d,J=32.2Hz,1H),8.47–8.35(m,2H),8.18(d,J=8.4Hz,1H),7.90–7.69(m,3H),7.24–7.14(m,1H),1.33(s,12H).
6.3中间体6-8的合成:
操作同2.4,中间体6-4代替中间体2-4,收率约为78.5%;MS(ESI):m/z=551.24[M+H]+;1H NMR(400MHz,DMSO-d6)δ11.05(s,1H),8.44(d,J=3.1Hz,1H),8.35–8.24(m,2H),8.20(d,J=8.3Hz,2H),7.91–7.73(m,3H),4.72–4.59(m,1H),3.72(dd,J=72.6,18.4Hz,2H),3.65(d,J=44.0Hz,2H),3.05(dd,J=46.4,21.1Hz,1H),2.24–2.09(m,2H),2.11(dd,J=12.7,3.7Hz,1H),1.26(s,9H).
6.4中间体6-9的合成:
操作同2.5,中间体6-8代替中间体2-8,得到淡棕色半固体6-9,收率约为82.7%;MS(ESI):m/z=451.20[M+H]+;1H NMR(400MHz,DMSO-d6)δ11.05(s,1H),8.76(s,1H),8.44(d,J=2.7Hz,1H),8.31–8.24(m,2H),8.20(d,J=8.1Hz,1H),7.89–7.76(m,3H),4.83–4.58(m,1H),3.09(dd,J=10.5,1.5Hz,1H),2.94(dd,J=22.0,10.9Hz,2H),2.23–2.00(m,2H),1.77(d,J=12.9Hz,1H),1.65–1.48(m,1H),1.19(dd,J=17.3,10.2Hz,1H).
6.5化合物Ⅵ-1的合成:
操作同1.5,中间体6-9代替中间体1-6,丙烯酸代替2-甲基丙烯酸1-7,得到淡黄色固体Ⅵ-1,收率约为77.9%;MS(ESI):m/z=505.31[M+H]+;1H NMR(400MHz,DMSO-d6)δ11.01(s,1H),8.92(s,1H),8.43(d,J=3.0Hz,1H),8.32–8.24(m,2H),8.21(d,J=8.2Hz,1H),7.81(d,J=8.5Hz,1H),7.71(dt,J=23.0,6.3Hz,2H),6.52(t,J=6.5Hz,1H),6.24(d,J=10.8Hz,1H),5.92(d,J=5.2Hz,1H),3.13–3.00(m,1H),2.36–2.23(m,2H),2.15(d,J=12.9Hz,1H),1.95(d,J=13.6Hz,1H),1.69–1.60(m,2H),1.38(dd,J=15.0,7.4Hz,1H),1.18(t,J=7.3Hz,1H).13C NMR(101MHz,DMSO-d6)δ167.41,165.87,165.05,158.67,156.31,155.21,149.07,136.62,136.01,135.71,134.10,132.17,131.98,129.28,129.13,128.62,125.83,125.63,116.52,98.01,65.49,45.60,30.47,19.12.
6.6化合物Ⅵ-2的合成:
操作同1.5,中间体6-9代替中间体1-6,2-氯丙烯酸代替2-甲基丙烯酸1-7,得到淡棕色固体Ⅵ-2,收率约为80.2%;MS(ESI):m/z=505.14[M+H]+;1H NMR(400MHz,DMSO-d6)δ11.02(s,1H),8.96(s,1H),8.43(d,J=3.0Hz,1H),8.33(s,1H),8.26(dd,J=9.2,4.2Hz,1H),8.21(d,J=8.2Hz,2H),7.83(dd,J=14.3,5.7Hz,2H),5.89(s,1H),5.64(s,1H),4.87–4.56(m,1H),3.31(t,J=10.0Hz,2H),3.18(d,J=12.6Hz,2H),2.00(d,J=14.6Hz,1H),1.91(t,J=7.4Hz,1H),1.77–1.59(m,2H).13C NMR(101MHz,DMSO-d6)δ165.89,163.87,158.07,149.08,143.82,136.43,136.02,135.68,134.22,132.17,129.30,128.66,125.83,125.64,116.58,116.54,65.49,46.22,30.46,19.11.
6.7化合物Ⅵ-3的合成:
操作同1.5,中间体6-9代替中间体1-6,2-溴丙烯酸代替2-甲基丙烯酸1-7,得到浅棕色固体Ⅵ-3,收率约为76.2%;MS(ESI):m/z=583.10[M+H]+;1H NMR(400MHz,DMSO-d6)δ11.01(s,1H),8.43(d,J=3.0Hz,1H),8.32–8.24(m,2H),8.21(d,J=8.2Hz,2H),7.88–7.77(m,3H),6.13(s,1H),5.76(s,1H),3.95–3.72(m,1H),2.49(dd,J=19.6,11.3Hz,1H),2.18(t,J=12.6Hz,2H),2.00(d,J=14.6Hz,1H),1.77–1.59(m,2H),1.39(t,J=7.4Hz,2H).13CNMR(101MHz,DMSO-d6)δ170.97,165.90,163.42,160.06,158.32,155.35,154.06,142.93,139.05,136.46,131.60,128.06,125.82,122.12,104.17,97.22,93.80,67.49,49.52,45.73,29.44,23.72.
实施例7系列Ⅶ化合物的合成:
7.1中间体7-3的合成:
称取3-氟4-溴苯甲酸1.00g(4.57mol)置于单颈烧瓶中,加入5mL二氯甲烷,反应瓶置于冰浴条件下后依次加入HATU 1.74g(4.57mmol),DIPEA 2.39mL(13.71mmol)和2-氨基吡啶1.13g(3.81mmol),然后转移至室温反应过夜。通过TLC确定反应毕,减压除去溶剂二氯甲烷,加饱和食盐水(200mL×3)和乙酸乙酯250mL萃取,有机相合并,经无水硫酸钠干燥,浓缩后干法拌样过柱,PE润柱,EA:PE=1:4洗脱得到1.12g淡棕色粉末状固体,收率约为74.6%。MS(ESI):m/z=294.98[M+H]+;1H NMR(400MHz,DMSO-d6)δ10.99(s,1H),8.41(ddd,J=4.8,1.9,0.8Hz,1H),8.17(d,J=8.4Hz,1H),8.02(dd,J=9.8,1.9Hz,1H),7.93–7.79(m,3H),7.25–7.14(m,1H).
7.2中间体7-4的合成:
操作同1.2,中间体7-3代替中间体1-3,得到白色固体,收率约为91.6%;1H NMR(400MHz,DMSO-d6)δ10.93(d,J=32.2Hz,1H),8.47–8.35(m,2H),8.18(d,J=8.4Hz,1H),7.90–7.69(m,3H),7.24–7.14(m,1H),1.33(s,12H).
7.3中间体7-8的合成:
操作同2.4,中间体7-4代替中间体2-4,收率约为73.2%;MS(ESI):m/z=583.10[M+H]+;1H NMR(400MHz,DMSO-d6)δ10.99(s,1H),9.08(s,1H),8.43(d,J=3.9Hz,1H),8.28(d,J=8.8Hz,2H),8.21(t,J=10.0Hz,2H),7.97–7.82(m,1H),7.70(t,J=7.8Hz,1H),4.82–4.64(m,1H),2.29–2.15(m,2H),2.15–2.04(m,2H),1.90(dd,J=9.3,5.7Hz,2H),1.60(dd,J=24.5,13.2Hz,2H),1.37(s,9H).
7.4中间体7-9的合成:
操作同2.5,中间体7-8代替中间体2-8,得到淡棕色固体,收率约为82.1%;MS(ESI):m/z=433.25[M+H]+;1H NMR(400MHz,DMSO-d6)δ8.44–8.41(m,2H),8.24(s,1H),8.05(dd,J=6.1,1.1Hz,2H),8.04–8.01(m,1H),7.89–7.86(m,2H),3.71–3.68(m,1H),3.17(d,J=7.0Hz,1H),2.96(t,J=7.3Hz,2H),2.65(d,J=3.3Hz,1H),2.39–2.16(m,2H),1.84–1.80(m,2H).
7.5化合物Ⅶ-1的合成:
操作同1.5,中间体7-9代替中间体1-6,丙烯酸代替2-甲基丙烯酸1-7,得到淡棕色固体Ⅶ-1,收率约为63.8%;MS(ESI):m/z=487.19[M+H]+;1H NMR(400MHz,Acetone-d6)δ9.47(s,1H),8.25–8.17(m,2H),8.09(s,1H),8.01(dd,J=8.0,1.2Hz,1H),7.92–7.81(m,1H),7.42(ddd,J=21.1,12.1,4.7Hz,2H),7.15(dd,J=7.2,4.9Hz,1H),6.62(d,J=11.2Hz,1H),5.58(t,J=9.0Hz,2H),4.72(d,J=4.1Hz,1H),3.42–3.31(m,1H),3.31–3.13(m,2H),2.92(t,J=11.4Hz,1H),2.38(dd,J=26.1,12.0Hz,2H),2.26(d,J=9.3Hz,1H),2.03(dd,J=20.8,9.0Hz,2H).13C NMR(101MHz,Acetone-d6)δ168.83,165.56,158.85,156.88,154.27,152.84,146.90,141.84,137.10,135.99,132.27,128.33,124.77,124.07,120.07,114.35,109.51,99.48,96.60,64.11,48.64,28.09,21.08.
7.6化合物Ⅶ-2的合成:
操作同1.5,中间体7-9代替中间体1-6,2-氯丙烯酸代替2-甲基丙烯酸1-7,得到棕色固体Ⅶ-2,收率约为63.8%;MS(ESI):m/z=487.19[M+H]+;1H NMR(400MHz,Acetone-d6)δ9.83(s,1H),8.36(d,J=8.3Hz,2H),8.29(s,1H),8.13(d,J=7.9Hz,1H),8.05(d,J=10.8Hz,1H),7.85(dt,J=15.5,7.7Hz,2H),7.18(dd,J=7.1,4.8Hz,1H),6.62(s,1H),6.04(s,1H),3.69–3.49(m,1H),3.31(d,J=26.2Hz,2H),2.48–2.34(m,1H),2.29(d,J=3.8Hz,1H),2.10(dd,J=8.6,4.8Hz,1H),1.80(dd,J=9.2,4.3Hz,2H),1.30(d,J=6.0Hz,1H).13CNMR(101MHz,Acetone-d6)δ163.65,161.06,158.60,158.23,155.82,155.81,154.39,148.15,138.15,137.58,132.09,132.06,124.10,124.07,120.03,116.93,116.06,115.83,114.29,80.13,64.63,44.52,39.44,37.84,21.48.
7.7化合物Ⅶ-3的合成:
操作同1.5,中间体7-9代替中间体1-6,2-溴丙烯酸代替2-甲基丙烯酸1-7,得到棕色固体Ⅶ-3,收率约为63.8%;MS(ESI):m/z=565.11[M+H]+;1H NMR(400MHz,Acetone-d6)δ9.95(s,1H),8.43–8.32(m,2H),8.28(s,1H),8.12(dd,J=8.0,1.2Hz,1H),8.08–8.01(m,1H),7.86(ddd,J=21.1,12.1,4.7Hz,2H),7.19(dd,J=7.2,4.9Hz,1H),6.62(s,1H),6.35(s,1H),4.46–4.25(m,1H),3.91(dq,J=13.2,6.6Hz,2H),3.41(q,J=7.4Hz,2H),2.25(d,J=11.6Hz,1H),2.03(d,J=3.4Hz,1H),1.82–1.64(m,2H).13C NMR(101MHz,Acetone-d6)δ158.37,155.98,152.09,148.19,138.21,132.09,132.06,128.18,124.77,124.11,124.07,120.07,116.05,115.81,114.35,54.82,42.83,37.86,22.32.
实施例8系列Ⅷ化合物的合成:
8.1化合物Ⅷ-1的合成:
操作同1.5,中间体7-9代替中间体1-6,(E)-4-(二甲基氨基)丁-2-烯酸代替2-甲基丙烯酸1-7,得到白色固体Ⅷ-1,收率约为63.8%;MS(ESI):m/z=565.11[M+H]+;1H NMR(400MHz,DMSO-d6)δ10.19(s,1H),9.03(s,1H),8.56(d,J=4.6Hz,1H),8.19(d,J=7.9Hz,1H),8.15(t,J=11.2Hz,1H),8.07–7.96(m,2H),7.85–7.72(m,1H),7.64(t,J=8.4Hz,1H),6.70(m,1H),6.52(d,J=10.6Hz,1H),4.43–4.32(m,1H),3.71(d,J=7.6Hz,2H),2.78(s,6H),3.29–3.15(m,2H),3.02(d,J=9.4Hz,2H),2.15–2.04(m,2H),1.90(dd,J=9.3,5.7Hz,1H),1.60(dd,J=23.7,12.6Hz,1H).13C NMR(101MHz,DMSO-d6)δ168.24,163.61,160.09,158.85,154.74,151.54,149.02,143.63,136.36,135.28,129.24,128.39,127.67,124.00,120.75,115.44,112.68,98.30,66.05,65.83,46.23,33.92,20.96.
8.2化合物Ⅷ-2的合成:
操作同1.5,中间体7-9代替中间体1-6,10-羟基-2-癸烯酸代替2-甲基丙烯酸1-7,得到淡黄色半固体Ⅷ-2,收率约为63.8%;MS(ESI):m/z=601.30[M+H]+;1H NMR(400MHz,Acetone-d6)δ8.37(d,J=8.1Hz,2H),8.28(s,1H),8.11(d,J=7.1Hz,1H),8.05(d,J=10.9Hz,1H),7.85(d,J=22.2Hz,2H),7.19(t,J=6.7Hz,1H),6.81(d,J=16.2Hz,1H),6.47(m,1H),3.91–3.77(m,1H),3.61–3.44(m,4H),3.30(t,J=6.5Hz,1H),3.19(t,J=10.5Hz,1H),2.38(m,2H),2.26–2.19(m,2H),1.96–1.71(m,4H),1.64–1.52(m,2H),1.43–1.35(m,2H),1.33–1.24(m,2H),1.18–1.02(m,2H).13C NMR(101MHz,Acetone-d6)δ165.18,164.05,156.12,155.74,154.33,152.04,148.34,148.02,138.33,138.06,124.67,123.94,115.90,114.41,114.11,101.90,81.00,78.79,61.43,56.73,54.23,53.67,46.98,42.25,32.77,32.33,29.68,25.27,17.77.
实施例9.体外BTK酶抑制实验
9.1化合物配制
称取以上化合物1mg溶解在100%DMSO-d6中,配制成10mM储存液,氮气柜中避光保存。
9.2激酶反应过程
(1)配制1×Kinase buffer。
(2)化合物浓度梯度的配制:受试化合物起始测试浓度为2400nM,7个浓度,复孔检测。在384source板中稀释成100倍终浓度的100%DMSO-d6溶液。使用分液器Echo 550向目的板3575板转移250nl 100倍终浓度的化合物。
(3)用1×Kinase buffer配制2.5倍终浓度的激酶溶液。
(4)在化合物孔和阳性对照孔分别加10μl的2.5倍终浓度的激酶溶液;在阴性对照孔中加10μl的1×Kinase buffer。
(5)1000rpm离心30秒,反应板振荡混匀后室温孵育10分钟。
(6)用1×Kinase buffer配制5/3倍终浓度的ATP和Kinase substrate 2的混合溶液。
(7)加入15μl的5/3倍终浓度的ATP和底物的混合溶液,起始反应。
(8)将384孔板1000rpm离心30秒,振荡混匀后室温孵育30分钟。
(9)加入30μl终止检测液停止激酶反应,1000rpm离心30秒,振荡混匀。
(10)用Caliper EZ Reader读取转化率。
结果见表1。
表1.化合物的BTK抑制活性结果
可见,本发明所述的小分子化合物具有良好的BTK抑制活性,部分化合物(VII-1、VII-2、VII-3)无论是抑制率还是IC50都优于上市药物阿卡替尼,且IC50与依鲁替尼相近。
实施例10化合物水溶性测试
10.1内标配制
精密称取氯雷他定(Loratadine)标准品1.00mg,精密移取1mL二甲亚砜(DMSO)溶解,摇匀,配制成浓度为1mg/mL的储备液,-20℃储存备用。精密移取储备液适量用乙腈稀释成浓度为5.0ng/mL的内标工作液。
10.2待测物储备液
精密称取待测物1.00mg溶于1mL DMSO配制成1.00mg/mL的待测物储备液A,用于标准曲线的配制,-20℃储存备用。
精密称取待测物1.00mg溶于1mL DMSO配制成1.00mg/mL的待测物储备液B,用于质控样品(QC)的配制,-20℃储存备用。
10.3标准曲线的配制:
取储备液A适量,乙腈梯度洗脱成浓度为0.05μg/mL,0.1μg/mL,0.2μg/mL,0.5μg/mL,1μg/mL,2μg/mL,5μg/mL,10μg/mL,20μg/mL,50μg/mL,100μg/mL和200μg/mL的工作液;
精密移取47.5μL超纯水,分别精密加入上述工作液2.5μL,涡旋1min混匀,配制成浓度为2.5ng/mL,5ng/mL,10ng/mL,25ng/mL,50ng/mL,100ng/mL,250ng/mL,500ng/mL和1000ng/mL的标准曲线样本,分别精密加入200μL含内标氯雷他定的乙腈,涡旋5min,4℃12000rpm离心10min,取上清液,LC-MS/MS进样分析。
10.4质控(QC)样本配制:
取储备液B适量,乙腈梯度洗脱成浓度为0.1μg/mL,2.0μg/mL和16μg/mL的工作液;
精密移取47.5μL超纯水,分别精密加入上述工作液2.5μL,涡旋1min混匀,配制成浓度为5ng/mL,100ng/mL和800ng/mL的QC样本,分别精密加入200μL含内标氯雷他定的乙腈,涡旋5min,4℃12000rpm离心10min,取上清液,LC-MS/MS进样分析。
10.5待测物水溶性样本配制
分别称取待测物适量,分别加入1mL纯水,配置成饱和水溶液,涡旋2h,室温静置1h,4℃12000rpm离心10min,分别取上清液50μL加200μL含内标氯雷他定的乙腈,涡旋5min,4℃12000rpm离心10min,取上清液,LC-MS/MS进样分析,每份样本3份平行实验。
结果见表2:
表2.1标准曲线方程表
表2.2化合物的水溶性测试结果
可见,本发明提供的化合物具有较好的水溶性,VII系列化合物的水溶性有所提高,其中VII-2较阳性对照药物依鲁替尼提高了15.8%,化合物VIII-2较阳性对照药物依鲁替尼提高了18.4%以上。
实施例11化合物PK测试
化合物依鲁替尼、Ⅳ-1和Ⅷ-2在SD大鼠体内静脉和口服分别单剂量给药后,于不同时间点采集血样,LC-MS/MS测定大鼠血浆中化合物依鲁替尼、Ⅳ-1和Ⅷ-2的浓度并计算相关药代参数,考察化合物依鲁替尼、Ⅳ-1和Ⅷ-2在大鼠体内的生物利用度及药代属性。
雄性SD大鼠9只,200g左右,随机分成3组,每组3只。给药前禁食12h,自由饮水。
每只动物每次通过眼眶取血0.100mL,EDTA-二钠抗凝,采集时间点为:IV组:给予受试物后5min、15min、30min、1h、2h、4h、6h、8h和24h;PO组:给予受试物后15min、30min、1h、2h、4h、6h、8h和24h。血液样本采集后置于冰上,并于30min内离心分离血浆(离心条件:3000rpm,10min,4℃)。离心完成后分别取上清液50μL加200μL含内标氯雷他定的乙腈,涡旋5min,4℃12000rpm离心10min,取上清液,LC-MS/MS进样分析,每份样本3份平行实验。
实验测试结果如下:
表3.1血浆中化合物线性范围、标准曲线方程表:
SD大鼠经静注(IV.,2.00mg/kg)给药依鲁替尼(Ibrutinib),Ⅳ-2和Ⅶ-2后随时间变化的血药浓度曲线图如附图1。
SD大鼠灌胃给药依鲁替尼、Ⅳ-1、Ⅶ-2(PO.,10mg/kg)后,根据血药浓度数据,使用WinNonlin V6.3非房室模型计算给药后的药代参数,见表3.2。
表3.2 SD大鼠灌胃给药后的主要药动学参数
可见,本发明提供的化合物具有较好的药代动力学参数,VII-2与阳性药物依鲁替尼基本一致,或略优于阳性药物。
Claims (7)
3.如权利要求1或2所述的恶唑并[4,5-b]吡啶结构的不可逆BTK抑制剂在制备预防或治疗因BTK异常所引起的疾病的药物制剂的应用。
4.如权利要求3所述的应用,其特征在于,所述的疾病为淋巴瘤、自体免疫疾病,包括类风湿关节炎、***性红斑狼疮、强直性脊柱炎或者银屑病,所述的淋巴瘤包括慢性淋巴细胞白血病、B细胞淋巴瘤、套细胞淋巴瘤、淋巴浆细胞淋巴瘤、弥漫性大B细胞淋巴瘤、非霍奇金淋巴瘤、滤泡中心淋巴瘤、边缘区B细胞淋巴瘤。
5.如权利要求1或2所述的恶唑并[4,5-b]吡啶结构的不可逆BTK抑制剂在药学上可接受的盐,其特征在于,所述药学上可接受的盐为盐酸盐、氢溴酸盐、氢碘酸盐、硫酸盐、硫酸氢盐、磷酸盐、乙酸盐、丙酸盐、丁酸盐、草酸盐、酒石酸盐、甲磺酸盐、对甲苯磺酸盐、富马酸盐、牛磺酸盐、柠檬酸盐、琥珀酸盐,或其混合盐。
6.如权利要求5所述的恶唑并[4,5-b]吡啶结构的不可逆BTK抑制剂在药学上可接受的盐在制备预防或治疗因BTK异常所引起的疾病的药物制剂的应用。
7.如权利要求6所述的应用,其特征在于,所述的疾病为淋巴瘤、自体免疫疾病,包括类风湿关节炎、***性红斑狼疮、强直性脊柱炎或者银屑病,所述的淋巴瘤包括慢性淋巴细胞白血病、B细胞淋巴瘤、套细胞淋巴瘤、淋巴浆细胞淋巴瘤、弥漫性大B细胞淋巴瘤、非霍奇金淋巴瘤、滤泡中心淋巴瘤、边缘区B细胞淋巴瘤。
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