CN117024445A - 用于制备hpk1激酶抑制剂的化合物及其合成方法 - Google Patents
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Classifications
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- C07D471/04—Ortho-condensed systems
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- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
- A61K31/4375—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a six-membered ring having nitrogen as a ring heteroatom, e.g. quinolizines, naphthyridines, berberine, vincamine
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Abstract
本发明提供了用于制备具有抑制HPK1激酶活性化合物的中间体化合物Int‑9,该中间体化合物的制备方法及其用在制备具有抑制HPK1激酶活性的化合物33,25,70,76和77的用途。
Description
技术领域
本发明涉及一种杂环化合物,具体地涉及一种高活性的HPK1激酶抑制剂及其用途。
背景技术
HPK1是MAP4K家族的成员之一,主要在造血系统细胞中表达,并且充当T细胞增殖和信号传导的细胞内负调节物。抗原刺激T细胞后促使胞浆中的接头蛋白SLP-76被招募到脂膜TCR复合体上,为信号转导相关激酶提供结合位点来实现TCR介导的信号传递而诱导T细胞激活。在这一过程中HPK1被酪氨酸激酶Lck和Zap70磷酸化而激活,参与调节T细胞受体蛋白相互作用。HPK1通过磷酸化接头蛋白SLP-76的Ser376位点,使得SLP-76与支架蛋白14-3-3ε结合进而通过蛋白酶体被降解,而这一效应使得SLP-76与信号转导相关激酶结合减少而阻断了TCR信号转导,继而抑制T细胞激活和增值。另一方面,HPK1还参与了调控树突状细胞(DCs)的成熟及激活,特别是抑制了DCs细胞中协助T细胞激活相关蛋白如CD80,CD86及MHC复合物等的表达,进而影响DCs调节T细胞激活的作用;而活化的DCs对肿瘤抗原的呈递及DCs和T细胞的相互协作是抗肿瘤免疫系统中最重要的环节之一。此外在肿瘤微环境中存在大量免疫抑制性的分子如PGE2和TGF-β,这些因子介导的免疫抑制作用也与HPK1有重要联系。总体而言,特异性靶向抑制HPK1的小分子化合物可通过以改善T细胞功能为主,增强DCs细胞功能并同时逆转肿瘤免疫抑制微环境等多途径发挥增强抗肿瘤免疫效应来发挥抑制肿瘤生长的作用。
发明内容
在一个方面,本发明提供了一种用于制备具有抑制HPK1激酶活性化合物的中间体,具有以下结构:
在另一个方面,本发明提供了上述的中间体Int-9的制备方法,其特征在于包括如下步骤:
将化合物Int-6(230mg,1.04mmol)溶于无水DMF(10mL)中,冰浴下加入NaH(42mg,含量60%,1.04mmol)。混合物在室温下搅拌30分钟后,冷却至0℃,滴加Int-8(244mg,1.14mmol)的DMF(3mL)溶液。滴加完毕室温反应2小时,LCMS检测原料反应完全。将0.1N的NaOH溶液(1mL)加入到反应溶液中,室温搅拌1小时。将反应液倒入水(40mL)中,有黄色固体析出,过滤收集固体,干燥得到Int-9
优选地,int-8通过以下方法制备:
将化合物Int-8a(300mg,1.42mmol)溶于二氯甲烷(10mL)中,冰浴下加入m-CPBA(604mg,含量85%,2.98mmol),加料完毕后冰浴下继续反应4小时,LCMS检测原料反应完全。反应液浓缩,残余物硅胶柱层析纯化得到淡黄色固体Int-8;
优选地,int-6通过以下方法制备:
将甲酸(2.14g,46.57mmol,1.76mL)滴加至乙酸酐(3.17g,31.05mmol,2.93mL)中,然后升至室温搅拌1小时。接着将该混合液重新冷却至0℃,滴加至Int-2(500mg,2.59mmol)的四氢呋喃(10mL)溶液(0℃)中,然后升至室温搅拌30分钟。用二氯甲烷稀释反应液,用饱和碳酸氢钠溶液洗涤三次。有机相用无水硫酸钠干燥,过滤,浓缩。残留物用硅胶柱层析纯化(二氯甲烷/甲醇=10/1)得到白色固体Int-6
更优选地,int-2通过以下方法制备:
将化合物Int-1(100mg,0.61mmol)溶于醋酸(3mL)中,加入N-溴代丁二酰亚胺(109mg,0.61mmol),反应混合物在室温下搅拌1小时。加入饱和碳酸氢钠水溶液淬灭反应直至不产生气泡,水相用甲醇/二氯甲烷(1/20,50mL×2)萃取,合并有机相,无水硫酸钠干燥,过滤浓缩得到化合物Int-2a.
将化合物Int-2a(37mg,0.15mmol)溶于甲醇(1mL)中,加入碘化亚铜(3mg,0.015mmol),1,10-菲啰啉(3mg,0.03mmol)和碳酸铯(99mg,0.30mmol)。反应混合物置换氮气后用微波加热至100℃搅拌2小时。反应冷却至室温,浓缩反应液,残余物用制备型薄层层析纯化(甲醇/二氯甲烷/三乙胺=1/10/0.1)得到黄色固体Int-2.
更优选地,int-1通过以下方法制备:
将1-甲基-3,5-二硝基吡啶-2-酮Int-1a(1.0g,5.02mmol)溶于甲醇(50mL)中,依次加入氨甲醇溶液(7mol/L,8.61mL,60.27mmol)和1-甲基哌啶-4-酮Int-1b(625mg,5.52mmol)。反应混合物加热至50℃搅拌5小时。冷却至室温后静置48小时,减压浓缩反应液,残余物加入乙酸乙酯(50mL)后过滤。滤液减压浓缩后得到红色固体Int-1c(1.0g),直接用于下一步反应。
上一步得到的化合物Int-1c(1.0g)溶于甲醇(30mL)中,加入10% Pd-C(400mg),在氢气氛围下室温反应6小时。过滤除去钯碳,滤液浓缩得到黄色固体Int-1.
在又一个方面,本发明提供了上述中间体Int-9在制备具有抑制HPK1激酶活性的化合物的用途,其中所述具有抑制HPK1激酶活性的化合物选自如下结构:
本发明描述示例性实施方案的过程中,本发明的其它特征将变得显而易见,给出所述实施方案用于说明本发明而不意欲成为其限制,以下实施例使用本发明所公开的方法制备、分离和表征。
可以用有机合成领域的技术人员已知的多种方式来制备本发明的化合物,可使用下述方法以及有机合成化学领域中已知的合成方法或通过本领域技术人员所了解的其变化形式来合成本发明化合物。优选方法包括但不限于下文所述的这些。在适用于所使用试剂盒材料和适用于所实现转变的溶剂或溶剂混合物中实施反应。有机合成领域的技术人员将理解,分子上存在的官能性与所提出的转变一致。这有时需要加以判断改变合成步骤的顺序或原料以获得期望的本发明化合物。
具体实施方式
实施例
通用过程
未包括制备途径时,本发明所用原料与试剂均为已知产品,可以按照本领域已知的方法合成,或者可通过购买市售产品获得。使用的市售试剂均不需进一步纯化。室温是指20-30℃。
反应实施例中无特殊说明,反应均在氮气氛下进行。氮气氛是指反应瓶连接一个约1L的氮气气球。
氢化反应通常抽真空,充入氢气,反复操作3次。氢气氛是指反应瓶连接一个约1L的氢气气球。
微波反应使用Initiator+微波反应器。
本发明化合物的结构是通过核磁共振(NMR)和质谱(MS)来确定的。NMR位移(δ)以10-6(ppm)的单位给出。NMR的测定是用(Bruker AscendTM 500型)核磁仪,测定溶剂为氘代二甲基亚砜(DMSO-d6),氘代氯仿(CDCl3),氘代甲醇(CD3OD),内标为四甲基硅烷(TMS)。以下简写用于NMR信号的多重性:s=单峰,brs=宽峰,d=二重峰,t=三重峰,m=多重峰。耦合常数以J值列出,以Hz测量。
LC-MS的测定使用Thermo液质联用仪(UltiMate 3000+MSQ PLUS)。HPLC的测定使用Thermo高压液相色谱仪(UltiMate 3000)。反相制备色谱使用Thermo(UltiMate 3000)反相制备色谱仪。快速柱层析使用艾杰尔(FS-9200T)自动过柱机,硅胶预装柱使用三泰预装柱。薄层层析硅胶板用烟台黄海HSGF254或青岛GF254硅胶板,薄层层析分离纯化产品采用的规格是0.4mm~0.5mm。
本发明中一些中间体的合成方法如下:
中间体1
中间体1由以下步骤制备:
第一步:将1-甲基-3,5-二硝基吡啶-2-酮Int-1a(1.0g,5.02mmol)溶于甲醇(50mL)中,依次加入氨甲醇溶液(7mol/L,8.61mL,60.27mmol)和1-甲基哌啶-4-酮Int-1b(625mg,5.52mmol)。反应混合物加热至50℃搅拌5小时。冷却至室温后静置48小时,减压浓缩反应液,残余物加入乙酸乙酯(50mL)后过滤。滤液减压浓缩后得到红色固体Int-1c(1.0g),直接用于下一步反应。ESI-MS(m/z):194.4[M+H]+;1HNMR(500MHz,DMSO-d6)δ9.14(d,J=2.5Hz,1H),8.36(d,J=2.5Hz,1H),3.64(s,2H),3.02(t,J=6.0Hz,2H),2.74(t,J=6.0Hz,2H),2.39(s,3H)。
第二步:将上一步得到的化合物Int-1c(1.0g)溶于甲醇(30mL)中,加入10% Pd-C(400mg),在氢气氛围下室温反应6小时。过滤除去钯碳,滤液浓缩得到黄色固体Int-1(800mg,收率94.70%)。ESI-MS(m/z):164.2[M+H]+。
中间体2
中间体2由以下步骤制备:
第一步:将化合物Int-1(100mg,0.61mmol)溶于醋酸(3mL)中,加入N-溴代丁二酰亚胺(109mg,0.61mmol),反应混合物在室温下搅拌1小时。加入饱和碳酸氢钠水溶液淬灭反应直至不产生气泡,水相用甲醇/二氯甲烷(1/20,50mL×2)萃取,合并有机相,无水硫酸钠干燥,过滤浓缩得到化合物Int-2a(38mg,收率25%)。ESI-MS(m/z):242.3[M+H]+;1HNMR(500MHz,DMSO-d6)δ6.77(s,1H),5.25(s,2H),3.37(s,2H),2.69(t,J=6.0Hz,2H),2.60(t,J=6.0Hz,2H),2.32(s,3H)。
第二步:将化合物Int-2a(37mg,0.15mmol)溶于甲醇(1mL)中,加入碘化亚铜(3mg,0.015mmol),1,10-菲啰啉(3mg,0.03mmol)和碳酸铯(99mg,0.30mmol)。反应混合物置换氮气后用微波加热至100℃搅拌2小时。反应冷却至室温,浓缩反应液,残余物用制备型薄层层析纯化(甲醇/二氯甲烷/三乙胺=1/10/0.1)得到黄色固体Int-2(20mg,收率67%)。ESI-MS(m/z):194.5[M+H]+;1HNMR(500MHz,DMSO-d6)δ6.54(s,1H),4.68(s,2H),3.80(s,3H),3.30(s,2H),2.64(t,J=5.6Hz,2H),2.59(t,J=5.7Hz,2H),2.31(s,3H)。
中间体6
中间体6由以下步骤制备:
第一步:在0℃冰浴条件下,将甲酸(2.14g,46.57mmol,1.76mL)滴加至乙酸酐(3.17g,31.05mmol,2.93mL)中,然后升至室温搅拌1小时。接着将该混合液重新冷却至0℃,滴加至Int-2(500mg,2.59mmol)的四氢呋喃(10mL)溶液(0℃)中,然后升至室温搅拌30分钟。用二氯甲烷稀释反应液,用饱和碳酸氢钠溶液洗涤三次。有机相用无水硫酸钠干燥,过滤,浓缩。残留物用硅胶柱层析纯化(二氯甲烷/甲醇=10/1)得到白色固体Int-6(550mg,收率96%)。ESI-MS(m/z):222.5[M+H]+。
中间体8
中间体8由以下步骤制备:
第一步:将化合物Int-8a(300mg,1.42mmol)溶于二氯甲烷(10mL)中,冰浴下加入m-CPBA(604mg,含量85%,2.98mmol),加料完毕后冰浴下继续反应4小时,LCMS检测原料反应完全。反应液浓缩,残余物硅胶柱层析纯化得到淡黄色固体Int-8(300mg,收率86%)。ESI-MS(m/z):244.3[M+H]+。
中间体9
中间体9由以下步骤制备:
第一步:将化合物Int-6(230mg,1.04mmol)溶于无水DMF(10mL)中,冰浴下加入NaH(42mg,含量60%,1.04mmol)。混合物在室温下搅拌30分钟后,冷却至0℃,滴加Int-8(244mg,1.14mmol)的DMF(3mL)溶液。滴加完毕室温反应2小时,LCMS检测原料反应完全。将0.1N的NaOH溶液(1mL)加入到反应溶液中,室温搅拌1小时。将反应液倒入水(40mL)中,有黄色固体析出,过滤收集固体,干燥得到Int-9(230mg,产率62%)。ESI-MS(m/z):357.2[M+H]+。
本发明中实施例化合物的合成方法如下:
实施例25
N-(2-甲氧基-6-甲基-5,6,7,8-四氢-1,6-萘啶-3-基)-8-(2-甲氧基苯基)吡啶并[3,4-d]
嘧啶-2-胺
化合物25由以下步骤制备:
第一步:将Int-9(50mg,0.14mmol)和2-甲氧基苯硼酸(32mg,0.21mmol)溶于THF(10mL)和水(2mL)的混合溶液中,加入[1,1'-双(二苯基膦)二茂铁]二氯化钯二氯甲烷络合物(11mg,14umol),碳酸钠(29mg,0.28mmol)。反应体系置换氮气后加热至60℃搅拌过夜,LCMS检测产物生成。反应液浓缩,残余物用Prep-HPLC纯化得到黄色固体25(24mg,收率41%)。ESI-MS(m/z):429.1[M+H]+;1HNMR(500MHz,DMSO-d6)δ9.49(s,1H),8.55(d,J=5.3Hz,1H),8.33(s,1H),8.05(s,1H),7.85(d,J=5.4Hz,1H),7.58-7.50(m,1H),7.34(dd,J=7.4,1.6Hz,1H),7.23(d,J=8.3Hz,1H),7.14(t,J=7.4Hz,1H),3.90(s,3H),3.59(s,3H),3.08(s,2H),2.71(t,J=5.9Hz,2H),2.61(t,J=5.9Hz,2H),2.40(s,3H)。
实施例33
N-(2-甲氧基-6-甲基-5,6,7,8-四氢-1,6-萘啶-3-基)-8-(哌啶-1-基)吡啶并[3,4-d]嘧啶
-2-胺
化合物33由以下步骤制备:
第一步:将Int-9(50mg,140umol)溶于N-甲基吡咯烷酮(5mL)中,加入哌啶(178mg,2.1mmol)。反应液加热至120℃搅拌2小时,LCMS检测反应完全。反应液浓缩,残余物用Prep-HPLC纯化得到黄色固体33(31mg,收率55%)。ESI-MS(m/z):406.5[M+H]+;1HNMR(500MHz,DMSO-d6)δ9.23(s,1H),8.59(s,1H),8.01-7.90(m,2H),7.12(d,J=5.4Hz,1H),3.87(s,3H),3.69(br s,4H),3.48(s,2H),2.80(t,J=6.0Hz,2H),2.68(t,J=6.0Hz,2H),2.38(s,3H),1.59(br s,6H)。
实施例76
(R)-6-甲基-3-((8-(2-甲基哌啶-1-基)吡啶并[3,4-d]哌啶-2-基)氨基)-5,6,7,8-四氢
-1,6-萘啶-2(1H)-酮
用R-2-甲基哌啶替换实施例70中第一步的3-氨基-2,2-二甲基-1-丙醇,用类似的方法和反应步骤,可以得到化合物76。ESI-MS(m/z):405.4[M+H]+;1H NMR(500MHz,DMSO-d6)δ11.97(s,1H),9.30(s,1H),8.41(s,1H),8.08(s,1H),8.03(d,J=5.3Hz,1H),7.19(d,J=5.3Hz,1H),5.13(br s,1H),4.15-4.06(m,1H),3.28-3.22(m,2H),2.65-2.55(m,4H),2.35(s,3H),2.02-1.94(m,1H),1.80-1.75(m,2H),1.70-1.57(m,3H),1.10(d,J=6.8Hz,3H)。
实施例77
6-甲基-3-((8-(((1-甲基环丙基)甲基)氨基)吡啶并[3,4-d]嘧啶-2-基)氨基)-5,6,7,8-四氢-1,6-萘啶-2(1H)-one
用1-甲基环丙基乙胺替换实施例70中第一步的3-氨基-2,2-二甲基-1-丙醇,用类似的方法和反应步骤,可以得到化合物77。ESI-MS(m/z):392.2[M+H]+;1H NMR(500MHz,DMSO-d6)δ11.95(s,1H),9.23(s,1H),8.42(s,1H),8.23(s,1H),7.83(d,J=5.6Hz,1H),6.90(d,J=5.7Hz,1H),6.74(br s,1H),3.45(d,J=5.7Hz,2H),3.34(s,2H),2.64-2.56(m,4H),2.34(s,3H),1.17(s,3H),0.63-0.57(m,2H),0.36-0.32(m,2H)。
HPK1抑制剂生物学筛选和结果
试验例1:化合物对HPK1激酶活性抑制能力的检测(方法1)
所需使用试剂如下
实验步骤
具体操作如下:配置酶促反应体系缓冲液(10mM MOPS,pH 7.2,5mMβ-glycerol-phosphate,10mM MgCl2,0.8mM EDTA,2mM EGTA,0.1mM DTT);将测试的化合物(配于DMSO中1mM的化合物储液)用缓冲液稀释至为60uM最高浓度(包含6% DMSO),并配置60μM浓度起始用包含6% DMSO的缓冲液进行5倍稀释共计8个点的梯度浓度的化合物;随后使用缓冲液将HPK1激酶稀释至30nM。在Greiner 384孔微孔板(货号:784075)中每孔加入2μl的HPK1激酶稀释液,对照孔中补充2μl缓冲液;短暂离心后在反应孔中加入1μl的稀释化合物,对照孔中加入1μl包含6% DMSO的缓冲液;短暂离心后置于25℃恒温孵育箱(上海一恒科学仪器有限公司,货号:LRH-150)中孵育20min。在每孔中加入3μl反应底物(溶解于蒸馏水中的10μMMBP和20μM ATP),短暂离心后置于25℃恒温孵育箱中孵育60min,采用ADP-Glo KinaseAssay Kit检测酶促反应活性,ADP-Glo Kinase Assay Kit检测都依据试剂盒的操作说明进行。数据采用化合物的半数抑制浓度IC50描述。
化合物编号 | IC50(nM) |
25 | 0.16 |
33 | 0.52 |
70 | 0.29 |
76 | <0.1 |
77 | <0.1 |
试验例2:化合物对Jurkat细胞分泌细胞因子白介素-2(IL-2)激动能力的检测(方法2)
所需使用试剂及细胞如下
实验试剂:
实验细胞:
实验步骤
具体操作如下:将化合物粉末用DMSO溶解至10mM,取2μl化合物加入到998μl RPMI1640培养基(此试验中均含10% FBS)中,涡旋混匀后为最高浓度点。用0.2%DMSO培养基3倍逐渐稀释化合物溶液,共8个浓度点。以含浓度为0.1% DMSO的RPMI 1640培养基溶液处理作为对照。在康宁96孔细胞培养板(货号:3599)中每孔加入1×105Jurkat E6-1细胞,随后加入等量体积的化合物稀释液,对照组加入含0.2% DMSO的RPMI 1640培养基,置于37℃细胞培养箱(Thermo Fisher Scientific,型号:3111)孵育1h。随后加入终浓度为1μg/mlAnti-human CD3Antibody和1μg/ml Anti-human CD28 Antibody抗体,置于37℃细胞培养箱孵育24h。采用Human IL-2DuoSet ELISA KIT检测细胞上清液中的IL-2含量,Human IL-2DuoSet ELISA检测依据试剂盒的操作说明进行。数据采用化合物的刺激信号与0.1%DMSO的信号的最高倍数比值描述。
NA:表示未检测到IL-2的释放增强。
试验例3:化合物对小鼠脾脏细胞分泌细胞因子白介素-2(IL-2)激动能力的检测(方法3)
所需使用试剂及细胞如下
实验试剂:
实验动物:
实验步骤
具体操作如下:将化合物粉末用DMSO溶解至10mM,取2μl化合物加入到998μl RPMI1640培养基(此试验中均含10% FBS)中,涡旋混匀后为最高浓度点。用0.2%DMSO培养基3倍逐渐稀释化合物溶液,共8个浓度点。以含浓度为0.1% DMSO的RPMI 1640培养基溶液处理作为对照。在康宁96孔细胞培养板(货号:3599)中每孔加入小鼠105脾脏细胞,随后加入等量体积的化合物稀释液,对照组加入含0.2% DMSO的RPMI 1640培养基,置于37℃细胞培养箱(Thermo Fisher Scientific,型号:3111)孵育1h。随后加入终浓度为0.4μg/mlConcanavalin A,置于37℃细胞培养箱孵育24h。采用Mouse IL-2DuoSet ELISAKIT检测细胞上清液中的IL-2含量,Mouse IL-2DuoSet ELISA检测依据试剂盒的操作说明进行。数据采用化合物的刺激信号与0.1% DMSO的信号的最高倍数比值描述。
NA:表示未检测到IL-2的释放增强。
试验例4:化合物对DC2.4细胞分泌细胞因子白介素-6(IL-6)激动能力的检测(方法3)
所需使用试剂及细胞如下
实验试剂:
实验细胞:
实验步骤
具体操作如下:将化合物粉末用DMSO溶解至10mM,取2μl化合物加入到998μl RPMI1640培养基(此试验中均含10% FBS)中,涡旋混匀后为最高浓度点。用0.2%DMSO培养基3倍逐渐稀释化合物溶液,共8个浓度点。以含浓度为0.1% DMSO的RPMI 1640培养基溶液处理作为对照。在康宁96孔细胞培养板(货号:3599)中每孔加入105DC2.4细胞,随后加入等量体积的化合物稀释液,对照组加入含0.2% DMSO的RPMI 1640培养基,置于37℃细胞培养箱(Thermo Fisher Scientific,型号:3111)孵育1h。随后加入终浓度为3.2ng/ml LPS,置于37℃细胞培养箱孵育24h。采用Mouse IL-6DuoSet ELISAKIT检测细胞上清液中的IL-2含量,Mouse IL-6DuoSet ELISA检测依据试剂盒的操作说明进行。数据采用化合物的刺激信号与0.1% DMSO的信号的最高倍数比值描述。
NA:表示未检测到IL-6的释放增强。
试验例5:化合物对PBMC分泌细胞因子白介素-2(IL-2)激动能力的检测(方法5)
所需使用试剂及细胞如下
实验试剂:
/>
实验细胞:
实验步骤
具体操作如下:将化合物粉末用DMSO溶解至10mM,取2μl化合物加入到998μl RPMI1640培养基(此试验中均含10% FBS)中,涡旋混匀后为最高浓度点。用0.2%DMSO培养基3倍逐渐稀释化合物溶液,共8个浓度点。以含浓度为0.1% DMSO的RPMI 1640培养基溶液处理作为对照。在康宁96孔细胞培养板(货号:3599)中每孔加入1×105PBMC细胞,随后加入等量体积的化合物稀释液,对照组加入含0.2% DMSO的RPMI 1640培养基,置于37℃细胞培养箱(Thermo Fisher Scientific,型号:3111)孵育1h。随后加入终浓度为0.1μg/ml Anti-human CD3Antibody和1μg/ml Anti-human CD28 Antibody抗体,置于37℃细胞培养箱孵育24h。采用Human IL-2DuoSet ELISAKIT检测细胞上清液中的IL-2含量,Human IL-2DuoSetELISA检测依据试剂盒的操作说明进行。数据采用化合物的刺激信号与0.1% DMSO的信号的最高倍数比值描述。
NA:表示未检测到IL-2的释放增强。
Claims (7)
1.用于制备具有抑制HPK1激酶活性化合物的中间体化合物Int-9,具有以下结构:
2.如权利要求1所述的中间体化合物Int-9的制备方法,其特征在于包括如下步骤:
将化合物Int-6(230mg,1.04mmol)溶于无水DMF(10mL)中,冰浴下加入NaH(42mg,含量60%,1.04mmol)。混合物在室温下搅拌30分钟后,冷却至0℃,滴加Int-8(244mg,1.14mmol)的DMF(3mL)溶液。滴加完毕室温反应2小时,LCMS检测原料反应完全。将0.1N的NaOH溶液(1mL)加入到反应溶液中,室温搅拌1小时。将反应液倒入水(40mL)中,有黄色固体析出,过滤收集固体,干燥得到Int-9。
3.如权利要求2所述的步骤,其特征在于,化合物int-8通过以下方法制备:
将化合物Int-8a(300mg,1.42mmol)溶于二氯甲烷(10mL)中,冰浴下加入m-CPBA(604mg,含量85%,2.98mmol),加料完毕后冰浴下继续反应4小时,LCMS检测原料反应完全。反应液浓缩,残余物硅胶柱层析纯化得到淡黄色固体Int-8。
4.如权利要求2所述的步骤,其特征在于,化合物int-6通过以下方法制备:
将甲酸(2.14g,46.57mmol,1.76mL)滴加至乙酸酐(3.17g,31.05mmol,2.93mL)中,然后升至室温搅拌1小时。接着将该混合液重新冷却至0℃,滴加至Int-2(500mg,2.59mmol)的四氢呋喃(10mL)溶液(0℃)中,然后升至室温搅拌30分钟。用二氯甲烷稀释反应液,用饱和碳酸氢钠溶液洗涤三次。
有机相用无水硫酸钠干燥,过滤,浓缩。残留物用硅胶柱层析纯化(二氯甲烷/甲醇=10/1)得到白色固体Int-6。
5.如权利要求4所述的步骤,其特征在于,化合物int-2通过以下方法制备:
(a)将化合物Int-1(100mg,0.61mmol)溶于醋酸(3mL)中,加入N-溴代丁二酰亚胺(109mg,0.61mmol),反应混合物在室温下搅拌1小时。加入饱和碳酸氢钠水溶液淬灭反应直至不产生气泡,水相用甲醇/二氯甲烷(1/20,50mL×2)萃取,合并有机相,无水硫酸钠干燥,过滤浓缩得到化合物Int-2a;
(b)将化合物Int-2a(37mg,0.15mmol)溶于甲醇(1mL)中,加入碘化亚铜(3mg,0.015mmol),1,10-菲啰啉(3mg,0.03mmol)和碳酸铯(99mg,0.30mmol)。反应混合物置换氮气后用微波加热至100℃搅拌2小时。反应冷却至室温,浓缩反应液,残余物用制备型薄层层析纯化(甲醇/二氯甲烷/三乙胺=1/10/0.1)得到黄色固体Int-2。
6.如权利要求5所述的步骤,其特征在于,化合物int-1通过以下方法制备:
(a)将1-甲基-3,5-二硝基吡啶-2-酮Int-1a(1.0g,5.02mmol)溶于甲醇(50mL)中,依次加入氨甲醇溶液(7mol/L,8.61mL,60.27mmol)和1-甲基哌啶-4-酮Int-1b(625mg,5.52mmol)。反应混合物加热至50℃搅拌5小时。
冷却至室温后静置48小时,减压浓缩反应液,残余物加入乙酸乙酯(50mL)后过滤。滤液减压浓缩后得到红色固体Int-1c(1.0g),直接用于下一步反应;
(b)上一步得到的化合物Int-1c(1.0g)溶于甲醇(30mL)中,加入10%Pd-C(400mg),在氢气氛围下室温反应6小时。过滤除去钯碳,滤液浓缩得到黄色固体Int-1。
7.如权利要求1所述的中间体化合物Int-9在制备具有抑制HPK1激酶活性的化合物的用途,其中所述具有抑制HPK1激酶活性的化合物选自如下结构:
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