CN116332908A - 一种shp2变构抑制剂及其制备方法和应用 - Google Patents
一种shp2变构抑制剂及其制备方法和应用 Download PDFInfo
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- CN116332908A CN116332908A CN202310275813.0A CN202310275813A CN116332908A CN 116332908 A CN116332908 A CN 116332908A CN 202310275813 A CN202310275813 A CN 202310275813A CN 116332908 A CN116332908 A CN 116332908A
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- methylpiperidin
- carboxamide
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- pyrimidine
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Abstract
Description
技术领域
本发明涉及药物化合物合成领域,尤其涉及一种SHP2变构抑制剂及其制备方法和应用。
背景技术
含Src同源性2的蛋白酪氨酸磷酸酶2(SHP2)是一种由PTPN11编码的非受体型蛋白酪氨酸磷酸酶,是蛋白酪氨酸磷酸酶(PTP)家族的重要成员。SHP2是Ras-Raf-MEK-ERK、PI3K-AKT-mTOR、JAK-STAT和PD-1/PD-L1多种信号通路的关键衔接蛋白。前期研究发现SHP2的表达水平或者活性异常与细胞增殖、分化、凋亡、粘附迁移以及发育性疾病密切有关。同时,抑制SHP2的酶活功能可有效克服酪氨酸激酶靶向药物治疗后产生的耐药性。因而SHP2是一个重要的癌症治疗潜在靶点,用小分子抑制剂抑制SHP2活性是治疗酪氨酸激酶受体驱动的癌症的有效治疗方法。
SHP2结构包含两个SH2结构域和PTP结构域。在自抑制构象中,SHP2的N-SH2区域与PTP域相互作用,从而阻断底物结合。在底物结合后,PTP域的催化位点进入激活状态。传统的SHP2正构抑制剂(例如NSC-8787711和NAT6-2977756)直接结合PTP结构域的活性位点并抑制酶活性。然而,由于催化位点高度保守的极性化学环境,开发有效的SHP2酶活位点抑制剂具有较大挑战性。目前研究证明,SHP2的变构抑制剂有望用于临床研究。因此发现新型变构抑制剂是SHP2靶向研究的主要方向。
目前,针对SHP2靶点的变构抑制剂尚无药物获批上市,多款SHP2小分子抑制剂获批临床。其中包括I期临床药物约11个,II期临床药物4个。此外,多个医药企业正在开展SHP2降解剂的开发。其中,诺华研发的SHP099衍生物TNO155也是首个进入临床测试的SHP2抑制剂,也是目前进展最快小分子,目前处于Ⅱ期临床。
值得注意的是,由于SHP2包含多个变构位点,因此在特定变构结合位点识别抑制剂仍然具有挑战性。
发明内容
本发明提供了一种式(I)化合物及其药物组合物,该化合物或其药物组合物不仅具有优异的SHP2酶活抑制能力和抗癌活性,还能克服酪氨酸激酶抑制剂的耐药,可用于制备抗肿瘤药物。
本发明的技术方案如下:
一种式(I)化合物,或其立体异构体、互变异构体、前药、药学上可接受的盐、水合物、溶剂化物:
其中,R为任选取代的芳基或杂芳基。
本发明式(I)化合物,或其立体异构体、互变异构体、前药、药学上可接受的盐、水合物、溶剂化物通过结构SHP2的经典变构位点,稳定SHP2的自抑制构象,从而选择性抑制SHP2酶活能力,具有较好的抗肿瘤活性。
优选的,R为4-甲氧基苯基、3,4-二甲氧基苯基、2,6-二甲氧基苯基、4-硝基苯基、4-氟苯基、3,4-二氯苯基、3,5-二氯苯基、苯基、萘基、联苯基、芴基或吡啶基。优选的化合物具有较好的SHP2抑制活性和抗肿瘤活性。
最优选的,R为4-甲氧基苯基、3,4-二甲氧基苯基、2,6-二甲氧基苯基、4-硝基苯基、4-氟苯基、3,4-二氯苯基、3,5-二氯苯基、萘基或吡啶基。优选的化合物具有更好的诱导SHP2抑制活性和抗肿瘤活性。
本发明所述的化合物还包括所示的化合物的立体异构体。本发明化合物的所有立体异构体,包括但不限于非对映异构体、对映异构体和阻转异构体以及他们的混合物(如外消旋物),均包括在本发明的范围内。
本发明所述的化合物还包括式(I)所示的化合物的互变异构体。属于“互变异构体”或“互变异构形式”是指经由低能垒相互转化的不同能量的结构异构体。
本发明所述的化合物还包括式(I)所示的化合物的衍生物的前药,式(I)所示的化合物的衍生物自身可能具有较弱的活性甚至没有活性,但是在给药后,在生理条件下(例如通过代谢、溶剂分解或另外的方式)被转化成相应的生物活性形式。
式(I)化合物的药学上可接受的盐包括与下列酸形成的加成盐:盐酸、氢溴酸、硫酸、磷酸、甲磺酸、乙磺酸、对甲苯磺酸、苯磺酸、茶二磺酸、乙酸、丙酸、乳酸、三氟乙酸、马来酸、柠檬酸、富马酸、草酸、酒石酸或苯甲酸。
本发明还提供了式(I)所示的化合物的制备方法,包括如下步骤:
(1)将式(Ⅰ)所示的化合物与式(Ⅱ)所示的化合物惰性气体条件下,在干燥双颈瓶中依次加入Pd2(dba)3(228.9mg,0.25mmol)、Ph3P(131.1mg,0.5mmol)和NaOtBu(1.44g,15mmol),并用10mL二氧六环溶解。搅拌15min形成催化剂体系后,通过注射器加入化合物Ⅰ(504.1mg,2.5mmol)和化合物Ⅱ(642.9mg,3mmol)的二氧六环悬浊液各7mL。用绝缘胶密封后在100℃下反应18h,随后将反应液冷却至室温,用20mL乙酸乙酯稀释,饱和NaHCO3溶液洗涤,水层用乙酸乙酯(20mL)萃取,合并有机层,饱和食盐水洗涤,无水Na2SO4干燥,旋干,硅胶柱层析。获得透明胶状液体化合物Ⅲ,产率74%。
(3)氮气保护下,在干燥双颈瓶中加入化合物Ⅲ(113.8mg,0.3mmol),并用1mL二氯甲烷溶解,冰水浴,强搅拌,用注射器逐滴加入25%的TFA二氯甲烷溶液(6mL),室温反应3h后,加入饱和K2CO3溶液,调节pH值至10,用乙酸乙酯(20mL)萃取,饱和食盐水(20mL)洗涤,无水Na2SO4干燥后减压旋干,硅胶柱层析,得到化合物Ⅳ收率为82%。
(4)氮气保护下,在试管中加入化合物Ⅳ(27.9mg,0.1mmol),用1mL甲苯溶解,搅拌下依次加入苯胺(27uL,0.3mmol),LiHMDS(0.3mL,0.3mmol),室温反应15h后,加入1.0MNH4Cl溶液淬灭,用硅胶粉滤过后用乙酸乙酯淋洗(3×1mL),减压旋干,硅胶柱层析,得到化合物Ⅴ,收率为95%。
其中,R选自以下基团:
上述步骤的反应式如下所示:
其中,R选自以下基团:
(a)Pd2(dba)3,Ph3P,NaOtBu,dioxane,N2,18h,100℃;(b)TFA,DCM,室温搅拌,2h;(c)LiHMDS,toluene,N2,15h。
本发明还提供了一种药物组合物,包括式(I)化合物或其立体异构体、互变异构体、前药、药学上可接受的盐、水合物、溶剂化物;还包括药学上可接受的赋形剂。
在所述的药物组合物中,式(I)化合物,或其立体异构体、互变异构体、前药、药学上可接受的盐、水合物、溶剂化物作为活性成份,与药学上可接受的赋形剂混合制成药物组合物。所述的赋形剂为用于药学领域的稀释剂、辅助剂或载体。
在药物组合物中加入药学上可接受的辅料制成的临床上可接受的剂型。所述剂型为注射剂、片剂或胶囊剂。
本发明还提供了一种药物组合物,包括式(I)化合物或其立体异构体、互变异构体、前药、药学上可接受的盐、水合物、溶剂化物,以及抗肿瘤药剂。本发明所述的化合物或其药学上可接受的盐、水合物可作为抗肿瘤药剂单独使用,还可以与不同的抗肿瘤药剂联合使用,用于治疗或预防肿瘤。
本发明还提供了式(I)化合物或其立体异构体、互变异构体、前药、药学上可接受的盐、水合物、溶剂化物在制备SHP2变构抑制剂中的应用。
本发明还提供了式(I)化合物或其立体异构体、互变异构体、前药、药学上可接受的盐、水合物、溶剂化物在制备预防和/或治疗癌症的药物中的应用。
本发明还提供了所述的药物组合物在制备预防和/或治疗癌症的药物中的应用。
所述的癌症为多发性骨髓瘤、胃癌、肺癌、乳腺癌、食管癌、结肠癌、髓母细胞瘤、急性粒细胞白血病、慢性白血病、前列腺癌、肝细胞瘤、肾细胞瘤、宫颈癌、皮肤癌、卵巢癌、结肠癌、神经胶质瘤、甲状腺癌或胰腺癌。
与现有技术相比,本发明的有益效果为:
(1)本发明提供的式(I)化合物,或其立体异构体、互变异构体、前药、药学上可接受的盐、水合物、溶剂化物是一种有效的SHP2选择性抑制剂,该类抑制剂能选择性作用SHP2变构位点,比酶活位点的SHP2抑制剂有更高的抑制活性和靶点选择性,口服生物利用度好,能显著抑制裸鼠中的肿瘤生长和pERK水平;
(2)本发明提供的式(I)化合物,或其立体异构体、互变异构体、前药、药学上可接受的盐、水合物、溶剂化物可作为具有RTK致癌驱动因子和SHP2相关疾病的有前途的先导或候选药物。
附图说明
图1为实施例制得优选化合物对不同肿瘤细胞的pERK活性的影响;
图2为化合物LC-129浓度对抑制肺癌细胞ERK磷酸化的影响。
具体实施方式
下文中提供的实施例和制备例进一步阐明和举例说明本发明化合物及其制备方法。应当理解,下述实施例和制备例的范围并不以任何方式限制本发明的范围。本发明的原料可以从商业途径获得或通过本领域已知的方法制备。
化合物的结构通过核磁共振(1H-NMR)和高分辨质谱(HRMS)来确定,NMR测定是用ACF-400BRUK型核磁共振仪,测定溶剂为氘代氯仿(CDC13)或氘代二甲亚砜(DMSO-D6)。柱层析采用200-300目硅胶。
化合物(Ⅰ)的制备方法如下:将式(Ⅰ)所示的化合物与式(Ⅱ)所示的化合物惰性气体条件下,在干燥双颈瓶中依次加入Pd2(dba)3(228.9mg,0.25mmol)、Ph3P(131.1mg,0.5mmol)和NaOtBu(1.44g,15mmol),并用10mL二氧六环溶解。搅拌15min形成催化剂体系后,通过注射器加入化合物Ⅰ(504.1mg,2.5mmol)和化合物Ⅱ(642.9mg,3mmol)的二氧六环悬浊液各7mL。用绝缘胶密封后在100℃下反应18h,随后将反应液冷却至室温,用20mL乙酸乙酯稀释,饱和NaHCO3溶液洗涤,水层用乙酸乙酯(20mL)萃取,合并有机层,饱和食盐水洗涤,无水Na2SO4干燥,旋干,硅胶柱层析。获得透明胶状液体化合物Ⅲ,产率74%。
(3)氮气保护下,在干燥双颈瓶中加入化合物Ⅲ(113.8mg,0.3mmol),并用1mL二氯甲烷溶解,冰水浴,强搅拌,用注射器逐滴加入25%的TFA二氯甲烷溶液(6mL),室温反应3h后,加入饱和K2CO3溶液,调节pH值至10,用乙酸乙酯(20mL)萃取,饱和食盐水(20mL)洗涤,无水Na2SO4干燥后减压旋干,硅胶柱层析,得到化合物Ⅳ收率为82%。
(4)氮气保护下,在试管中加入化合物Ⅳ(27.9mg,0.1mmol),用1mL甲苯溶解,搅拌下依次加入苯胺(27uL,0.3mmol),LiHMDS(0.3mL,0.3mmol),室温反应15h后,加入1.0MNH4Cl溶液淬灭,用硅胶粉滤过后用乙酸乙酯淋洗(3×1mL),减压旋干,硅胶柱层析,得到化合物Ⅴ,收率为95%。
实施例1:
制备:4-氨基-2-(4-氨基-4-甲基哌啶-1-基)-N-苯基嘧啶-5-甲酰胺(LC-112)
制备方法:氮气保护下,在试管中加入4-氨基-2-(4-氨基-4-甲基哌啶-1-基)嘧啶-5-羧酸乙酯(27.9mmol,0.1mmol),用1mL甲苯溶解,搅拌下依次加入苯胺(0.3mmol),LiHMDS(0.3mL,0.3mmol),室温反应15h后,加入1.0M NH4Cl溶液淬灭,用硅胶粉滤过后用乙酸乙酯淋洗(3×1mL),减压旋干,硅胶柱层析,得到化合物。
产物的核磁共振表征如下:1H NMR(500MHz,DMSO-d6)δ10.06(s,1H),8.72(s,1H),7.73–7.67(m,2H),7.34–7.27(m,2H),7.09–7.02(m,1H),4.29–4.20(m,2H),3.55–3.46(m,2H),1.83–1.63(m,4H),1.39(s,3H).13C NMR(101MHz,DMSO-d6)δ166.1,163.4,161.0,158.4,139.6,128.9,123.6,121.0,99.2,52.6,40.5,40.3,40.0,39.8,39.6,39.4,39.2,34.9,22.7.LC-MS(ESI+):calcd for C17H23N6O=326.19,found;[M+H]+=327.36.
实施例2:
制备4-氨基-2-(4-氨基-4-甲基哌啶-1-基)-N-萘-1-基嘧啶-5-甲酰胺(LC-118)
制备方法:氮气保护下,在试管中加入4-氨基-2-(4-氨基-4-甲基哌啶-1-基)嘧啶-5-羧酸乙酯(27.9mmol,0.1mmol),用1mL甲苯溶解,搅拌下依次加入1-萘胺(0.3mmol),LiHMDS(0.3mL,0.3mmol),室温反应15h后,加入1.0M NH4Cl溶液淬灭,用硅胶粉滤过后用乙酸乙酯淋洗(3×1mL),减压旋干,硅胶柱层析,得到化合物。
产物的核磁共振表征如下:1H NMR(500MHz,DMSO-d6)δ10.08(s,1H),8.84(s,1H),7.99–7.91(m,2H),7.83(dd,J=6.7,2.8Hz,1H),7.57–7.49(m,4H),4.07(dd,J=13.4,6.4Hz,2H),3.73–3.62(m,2H),1.67–1.52(m,4H),1.28(s,3H).13C NMR(101MHz,DMSO-d6)δ167.0,163.5,161.2,158.4,134.3,134.2,129.9,128.4,126.5,126.4,126.3,125.9,124.5,123.9,98.6,50.9,40.5,40.3,40.1,39.9,39.7,39.5,39.3,36.4,25.3,23.4.LC-MS(ESI+):calcd for C21H25N6O=376.20,found;[M+H]+=377.29.
实施例3:
制备4-氨基-2-(4-氨基-4-甲基哌啶-1-基)-N-萘-2-基嘧啶-5-甲酰胺(LC-119)
制备方法:氮气保护下,在试管中加入4-氨基-2-(4-氨基-4-甲基哌啶-1-基)嘧啶-5-羧酸乙酯(27.9mg,0.1mmol),用1mL甲苯溶解,搅拌下依次加入2-萘胺(0.3mmol),LiHMDS(0.3mL,0.3mmol),室温反应15h后,加入1.0M NH4Cl溶液淬灭,用硅胶粉滤过后用乙酸乙酯淋洗(3×1mL),减压旋干,硅胶柱层析,得到化合物。
产物的核磁共振表征如下:1H NMR(500MHz,DMSO-d6)δ10.11(s,1H),8.71(s,1H),8.31(d,J=2.0Hz,1H),7.89–7.79(m,3H),7.76(dd,J=8.9,2.1Hz,1H),7.63–7.57(m,1H),7.51–7.44(m,1H),4.13–4.06(m,2H),3.69–3.60(m,2H),1.67–1.55(m,4H),1.30(s,3H).13CNMR(101MHz,DMSO-d6)δ166.3,163.4,161.1,158.4,137.3,133.8,130.2,128.4,127.9,127.7,126.7,125.0,121.7,116.9,99.2,51.9,40.6,40.5,40.4,40.3,40.2,40.1,39.9,39.7,39.5,39.3,35.5,23.7.LC-MS(ESI+):calcd for C21H25N6O=376.20,found;[M+H]+=377.29.
实施例4:
制备N-(1,1'-联苯)-3-基)-4-氨基-2-(4-氨基-4-甲基哌啶-1-基)嘧啶-5-甲酰胺(LC-120)
制备方法:氮气保护下,在试管中加入4-氨基-2-(4-氨基-4-甲基哌啶-1-基)嘧啶-5-羧酸乙酯(27.9mg,0.1mmol),用1mL甲苯溶解,搅拌下依次加入3-氨基联苯(0.3mmol),LiHMDS(0.3mL,0.3mmol),室温反应15h后,加入1.0M NH4Cl溶液淬灭,用硅胶粉滤过后用乙酸乙酯淋洗(3×1mL),减压旋干,硅胶柱层析,得到化合物。
产物的核磁共振表征如下:1H NMR(500MHz,DMSO-d6)δ9.95(s,1H),8.66(s,1H),7.99(t,J=2.0Hz,1H),7.72–7.66(m,1H),7.66–7.61(m,2H),7.48(t,J=7.7Hz,2H),7.44–7.32(m,3H),3.86–3.79(m,4H),3.17(s,2H),1.47(t,J=5.8Hz,4H),1.16(s,3H).13C NMR(101MHz,DMSO-d6)δ166.2,163.4,161.1,158.3,140.9,140.6,140.2,129.6,129.4,128.0,127.0,122.0,119.8,119.1,98.8,49.1,40.5,40.4,40.3,40.2,40.1,39.9,39.7,39.5,39.3,38.2.LC-MS(ESI+):calcd for C23H27N6O=402.22,found;[M+H]+=403.36.
实施例5:
制备:4-氨基-2-(4-氨基-4-甲基哌啶-1-基)-N-(9H-芴-2-基)嘧啶-5-甲酰胺(LC-121)
制备方法:氮气保护下,在试管中加入4-氨基-2-(4-氨基-4-甲基哌啶-1-基)嘧啶-5-羧酸乙酯(27.9mg,0.1mmol),用1mL甲苯溶解,搅拌下依次加入2-氨基芴(0.3mmol),LiHMDS(0.3mL,0.3mmol),室温反应15h后,加入1.0M NH4Cl溶液淬灭,用硅胶粉滤过后用乙酸乙酯淋洗(3×1mL),减压旋干,硅胶柱层析,得到化合物。
产物的核磁共振表征如下:1H NMR(500MHz,DMSO-d6)δ9.96(s,1H),8.65(s,1H),7.97(d,J=1.9Hz,1H),7.85–7.80(m,2H),7.64(dd,J=8.3,1.9Hz,1H),7.56(d,J=7.5Hz,1H),7.39–7.33(m,1H),7.30–7.23(m,1H),4.06–3.97(m,2H),3.91(s,2H),3.73–3.65(m,2H),1.56(t,J=5.8Hz,4H),1.25(s,3H).13C NMR(101MHz,DMSO-d6)δ166.1,163.4,161.1,158.2,143.9,143.2,141.5,138.7,136.8,127.2,126.5,125.4,120.3,119.9,119.7,117.7,99.2,50.9,40.5,40.3,40.1,39.9,39.7,39.5,39.2,36.9,36.4,25.3,24.0.LC-MS(ESI+):calcd for C24H27N6O=414.22,found;[M+H]+=415.22.
实施例6:
制备:4-氨基-2-(4-氨基-4-甲基哌啶-1-基)-N-(4-甲氧基苯基)嘧啶-5-甲酰胺(LC-122)
制备方法:氮气保护下,在试管中加入4-氨基-2-(4-氨基-4-甲基哌啶-1-基)嘧啶-5-羧酸乙酯(27.9mg,0.1mmol),用1mL甲苯溶解,搅拌下依次加入对氨基苯甲醚(0.3mmol),LiHMDS(0.3mL,0.3mmol),室温反应15h后,加入1.0M NH4Cl溶液淬灭,用硅胶粉滤过后用乙酸乙酯淋洗(3×1mL),减压旋干,硅胶柱层析,得到化合物。
产物的核磁共振表征如下:1H NMR(500MHz,DMSO-d6)δ9.78(s,1H),8.61(s,1H),7.58–7.51(m,2H),6.92–6.86(m,2H),4.07–3.99(m,2H),3.73(s,3H),3.68–3.60(m,2H),1.83(s,2H),1.62–1.52(m,4H),1.26(s,3H).13C NMR(101MHz,DMSO-d6)δ165.8,163.4,161.1,158.0,155.7,132.5,122.7,114.0,99.1,55.6,51.1,40.5,40.3,40.1,39.9,39.7,39.58,39.50,39.2,36.2,24.8,23.7.LC-MS(ESI+):calcd for C18H25N6O2=356.20,found;[M+H]+=357.23.
实施例7:
制备:4-氨基-2-(4-氨基-4-甲基哌啶-1-基)-N-(3,4-二甲氧基苯基)嘧啶-5-甲酰胺(LC-123)
制备方法:氮气保护下,在试管中加入4-氨基-2-(4-氨基-4-甲基哌啶-1-基)嘧啶-5-羧酸乙酯(27.9mg,0.1mmol),用1mL甲苯溶解,搅拌下依次加入4-胺-1,2-二甲氧苯(0.3mmol),LiHMDS(0.3mL,0.3mmol),室温反应15h后,加入1.0M NH4Cl溶液淬灭,用硅胶粉滤过后用乙酸乙酯淋洗(3×1mL),减压旋干,硅胶柱层析,得到化合物。
产物的核磁共振表征如下:1H NMR(500MHz,DMSO-d6)δ9.77(s,1H),8.61(s,1H),7.34(d,J=2.4Hz,1H),7.20(dd,J=8.7,2.4Hz,1H),6.90(d,J=8.8Hz,1H),4.25(d,J=13.1Hz,2H),3.73(d,J=4.3Hz,6H),3.50–3.43(m,2H),1.68(t,J=4.9Hz,4H),1.37(s,3H).13C NMR(101MHz,DMSO-d6)δ165.7,163.3,161.0,158.0,148.7,145.3,133.0,112.9,112.2,106.2,99.4,56.1,55.8,52.5,40.5,40.3,40.1,39.9,39.7,39.5,39.3,34.9,22.7.LC-MS(ESI+):calcd for C19H27N6O3=386.21,found;[M+H]+=387.29.
实施例8:
制备:4-氨基-2-(4-氨基-4-甲基哌啶-1-基)-N-(2,6-二甲氧基苯基)嘧啶-5-甲酰胺(LC-124)
制备方法:氮气保护下,在试管中加入4-氨基-2-(4-氨基-4-甲基哌啶-1-基)嘧啶-5-羧酸乙酯(27.9mg,0.1mmol),用1mL甲苯溶解,搅拌下依次加入2,6-二甲氧苯胺(0.3mmol),LiHMDS(0.3mL,0.3mmol),室温反应15h后,加入1.0M NH4Cl溶液淬灭,用硅胶粉滤过后用乙酸乙酯淋洗(3×1mL),减压旋干,硅胶柱层析,得到化合物。
产物的核磁共振表征如下:1H NMR(500MHz,DMSO-d6)δ8.99(s,1H),8.66(s,1H),7.23(t,J=8.4Hz,1H),6.70(d,J=8.5Hz,2H),4.24–4.16(m,2H),3.73(s,6H),3.60–3.45(m,2H),1.66(q,J=5.1,4.6Hz,4H),1.35(s,3H).13C NMR(101MHz,DMSO-d6)δ166.1,163.5,161.2,156.7,115.0,104.7,99.0,56.1,52.6,40.5,40.3,40.1,39.9,39.7,39.5,39.3,34.9,22.7.LC-MS(ESI+):calcd for C19H27N6O3=386.21,found;[M+H]+=387.29.
实施例9:
制备4-氨基-2-(4-氨基-4-甲基哌啶-1-基)-N-(4-硝基苯基)嘧啶-5-甲酰胺(LC-125)
制备方法:氮气保护下,在试管中加入4-氨基-2-(4-氨基-4-甲基哌啶-1-基)嘧啶-5-羧酸乙酯(27.9mg,0.1mmol),用1mL甲苯溶解,搅拌下依次加入4-硝基苯胺(0.3mmol),LiHMDS(0.3mL,0.3mmol),室温反应15h后,加入1.0M NH4Cl溶液淬灭,用硅胶粉滤过后用乙酸乙酯淋洗(3×1mL),减压旋干,硅胶柱层析,得到化合物。
产物的核磁共振表征如下:1H NMR(500MHz,DMSO-d6)δ10.41(s,1H),8.68(s,1H),8.26–8.19(m,2H),7.99–7.92(m,2H),7.56(s,2H),3.95–3.77(m,4H),1.50(t,J=5.8Hz,4H),1.19(s,3H).13C NMR(101MHz,DMSO-d6)δ166.6,163.4,161.1,159.0,146.3,142.3,125.2,120.0,98.4,50.0,40.5,40.3,40.1,39.9,39.7,39.52 39.3,37.3,26.9.LC-MS(ESI+):calcd for C17H22N7O3=371.17,found;[M+H]+=372.23.
实施例10:
制备4-氨基-2-(4-氨基-4-甲基哌啶-1-基)-N-(4-氟苯基)嘧啶-5-甲酰胺(LC-126)
制备方法:氮气保护下,在试管中加入4-氨基-2-(4-氨基-4-甲基哌啶-1-基)嘧啶-5-羧酸乙酯(27.9mg,0.1mmol),用1mL甲苯溶解,搅拌下依次加入4-氟苯胺(0.3mmol),LiHMDS(0.3mL,0.3mmol),室温反应15h后,加入1.0M NH4Cl溶液淬灭,用硅胶粉滤过后用乙酸乙酯淋洗(3×1mL),减压旋干,硅胶柱层析,得到化合物。
产物的核磁共振表针如下:1H NMR(500MHz,DMSO-d6)δ9.91(s,1H),8.61(s,1H),7.70–7.62(m,2H),7.51(s,2H),7.20–7.11(m,2H),3.95–3.86(m,2H),3.80–3.72(m,3H),1.50(t,J=5.8Hz,4H),1.20(s,3H).13CNMR(101MHz,DMSO-d6)δ166.0,163.3,161.1,158.2,157.3,135.94,135.91,122.8,122.7,115.6,115.4,98.8,50.4,40.5,40.3,40.1,39.9,39.7,39.5,39.3,37.0,26.3.LC-MS(ESI+):calcd for C17H22FN6O=344.18,found;[M+H]+=345.36.
实施例11:
制备4-氨基-2-(4-氨基-4-甲基哌啶-1-基)-N-(3,4-二氯苯基)嘧啶-5-甲酰胺(LC-127)
制备方法:氮气保护下,在试管中加入4-氨基-2-(4-氨基-4-甲基哌啶-1-基)嘧啶-5-羧酸乙酯(27.9mg,0.1mmol),用1mL甲苯溶解,搅拌下依次加入3,4-二氯苯胺(0.3mmol),LiHMDS(0.3mL,0.3mmol),室温反应15h后,加入1.0M NH4Cl溶液淬灭,用硅胶粉滤过后用乙酸乙酯淋洗(3×1mL),减压旋干,硅胶柱层析,得到化合物。
产物的核磁共振波谱表征如下:1H NMR(500MHz,DMSO-d6)δ10.05(s,1H),8.60(s,1H),8.04(d,J=2.4Hz,1H),7.64(dd,J=8.8,2.4Hz,1H),7.57(d,J=8.8Hz,1H),7.50(s,2H),3.96–3.88(m,2H),3.77–3.68(m,2H),1.47–1.36(m,4H),1.12(s,3H).13C NMR(101MHz,DMSO-d6)δ166.3,163.3,161.1,158.5,139.9,131.1,130.8,124.8,121.7,120.6,98.3,48.9,40.5,40.4,40.3,40.2,40.1,39.9,39.7,39.5,39.3,38.4,28.8.LC-MS(ESI+):calcdfor C17H21Cl2N6O=394.11,found;[M+H]+=395.23.
实施例12:
制备4-氨基-2-(4-氨基-4-甲基哌啶-1-基)-N-(3,5-二氯苯基)嘧啶-5-甲酰胺(LC-128)
制备方法:氮气保护下,在试管中加入4-氨基-2-(4-氨基-4-甲基哌啶-1-基)嘧啶-5-羧酸乙酯(27.9mg,0.1mmol),用1mL甲苯溶解,搅拌下依次加入4,5-二氯苯胺(0.3mmol),LiHMDS(0.3mL,0.3mmol),室温反应15h后,加入1.0M NH4Cl溶液淬灭,用硅胶粉滤过后用乙酸乙酯淋洗(3×1mL),减压旋干,硅胶柱层析,得到化合物。
产物的核磁共振波谱表征如下:1H NMR(500MHz,DMSO-d6)δ10.59(d,J=4.4Hz,1H),8.83(d,J=2.0Hz,1H),7.93(d,J=1.9Hz,2H),7.23(t,J=1.9Hz,1H),4.24–4.17(m,2H),3.49(s,2H),1.78–1.62(m,4H),1.36(s,3H).13C NMR(101MHz,DMSO-d6)δ166.5,163.3,161.1,158.9,142.3,134.1,122.5,118.7,98.6,52.4,40.5,40.3,40.1,39.9,39.6,39.4,39.2,35.1,23.0.LC-MS(ESI+):calcd for C17H21Cl2N6O=394.11,found;[M+H]+=395.29.
实施例13:
制备4-氨基-2-(4-氨基-4-甲基哌啶-1-基)-N-吡啶-3-基)嘧啶-5-甲酰胺(LC-129)
制备方法:氮气保护下,在试管中加入4-氨基-2-(4-氨基-4-甲基哌啶-1-基)嘧啶-5-羧酸乙酯(27.9mg,0.1mmol),用1mL甲苯溶解,搅拌下依次加入3-氨基吡啶(0.3mmol),LiHMDS(0.3mL,0.3mmol),室温反应15h后,加入1.0M NH4Cl溶液淬灭,用硅胶粉滤过后用乙酸乙酯淋洗(3×1mL),减压旋干,硅胶柱层析,得到化合物。
产物的核磁共振波谱表征如下:1H NMR(500MHz,DMSO-d6)δ10.07(s,1H),8.83(d,J=2.6Hz,1H),8.67(s,1H),8.26(dd,J=4.7,1.5Hz,1H),8.07(dt,J=8.3,2.1Hz,1H),7.35(dd,J=8.4,4.7Hz,1H),3.91–3.76(m,4H),1.49(t,J=5.7Hz,4H),1.18(s,3H).13C NMR(101MHz,DMSO-d6)δ166.4,163.3,161.1,158.6,144.5,142.6,136.3,127.9,123.8,98.7,98.7,52.5,40.5,40.3,40.1,39.9,39.7,39.5,39.3,34.9,22.7.LC-MS(ESI+):calcd forC16H21N7ONa=327.18,found;[M+Na]+=350.30.
实施例14:
制备4-氨基-2-(4-氨基-4-甲基哌啶-1-基)-N-吡啶-2-基甲基嘧啶-5-甲酰胺(LC-130)
制备方法:氮气保护下,在试管中加入4-氨基-2-(4-氨基-4-甲基哌啶-1-基)嘧啶-5-羧酸乙酯(27.9mg,0.1mmol),用1mL甲苯溶解,搅拌下依次加入2-胺甲基吡啶(0.3mmol),LiHMDS(0.3mL,0.3mmol),室温反应15h后,加入1.0M NH4Cl溶液淬灭,用硅胶粉滤过后用乙酸乙酯淋洗(3×1mL),减压旋干,硅胶柱层析,得到化合物。
产物的核磁共振波谱表征如下:1H NMR(500MHz,DMSO-d6)δ8.81(t,J=6.0Hz,1H),8.56(s,1H),8.52–8.47(m,1H),7.78–7.71(m,1H),7.32–7.22(m,2H),4.49(d,J=5.9Hz,2H),3.99–3.89(m,2H),3.72–3.64(m,2H),1.52(t,J=5.8Hz,4H),1.22(s,3H).13C NMR(101MHz,DMSO-d6)δ167.3,163.3,161.1,159.6,157.6,149.2,137.1,122.4,121.2,98.5,50.7,44.5,40.5,40.3,40.3,40.18,40.12,39.9,39.7,39.5,39.2,36.6,25.6.LC-MS(ESI+):calcd for C17H23N7ONa=341.20,found;[M+Na]+=364.29.
实施例15:
制备4-氨基-2-(4-氨基-4-甲基哌啶-1-基)-N-吡啶-2-基)嘧啶-5-甲酰胺(LC-131)
制备方法:氮气保护下,在试管中加入4-氨基-2-(4-氨基-4-甲基哌啶-1-基)嘧啶-5-羧酸乙酯(27.9mg,0.1mmol),用1mL甲苯溶解,搅拌下依次加入2-氨基吡啶(0.3mmol),LiHMDS(0.3mL,0.3mmol),室温反应15h后,加入1.0M NH4Cl溶液淬灭,用硅胶粉滤过后用乙酸乙酯淋洗(3×1mL),减压旋干,硅胶柱层析,得到化合物。
产物的核磁共振波谱表征如下:1H NMR(500MHz,DMSO-d6)δ10.38(s,1H),8.69(s,1H),8.37–8.32(m,1H),8.04–7.98(m,1H),7.82–7.75(m,1H),7.58(s,2H),7.14–7.08(m,1H),4.01–3.92(m,2H),3.76–3.68(m,2H),1.61–1.49(m,4H),1.24(s,3H).13C NMR(101MHz,DMSO-d6)δ166.6,163.5,161.0,159.0,152.7,148.2,138.3,119.7,115.3,98.5,51.7,40.5,40.3,40.1,39.9,39.7,39.5,39.3,35.7,24.0.LC-MS(ESI+):calcd for C16H21N7ONa=327.18,found;[M+Na]+=350.23.
实施例16:SHP2变构抑制酶活测定
SHP2通过双-酪氨酰-磷酰化的肽与其Src同源2(SH2)结构域的结合而变构活化。该在后的活化步骤导致SHP2的自动抑制界面的释放,这又使该SHP2蛋白酪氨酸磷酸酶(PTP)活化并可用于底物识别和反应催化。在迅速荧光测定版式中使用替代物DiFMUP监测SHP2的催化活性。
试验步骤:
(1)化合物配制:
用100% DMSO将本发明化合物(10mM储液)稀释成合适倍数,本发明化合物最终测试浓度为20μM、10μM、5μM、2.5μM、0.5μM、0.25μM、0.125μM、0.0625μM。
(2)准备酶反应工作液:
在室温下在384孔黑色聚苯乙烯板(不透明,平底,非结合表面)(Cat#6007270,Perkin Elmer)中,使用50uL的最终反应体积和以下测定缓冲条件进行SHP2酶活检测:60mMHEPES,75mM NaCl,75mM KCl,1mM EDTA,5mM DTT。
(3)酶催化反应及数据监测:
取本发明化合物加到对应的96孔板中,设置不加化合物和酶只加缓冲液的作为空白试验孔。将SHP2 Activating Peptide(bisphosphorylated IRS1 peptide(sequence:H2N-LN(pY)IDLDLV(dPEG8)LST(pY)ASINFQK-amide))置于冰上融化,每孔加入0.5μM,然后取0.5nM SHP2蛋白样品加到对应孔板中,室温孵育1小时。
DiFMUP(Invitrogen,Cat#D6567)加入反应,室温反应0.5小时后。采用分别使用340nm和450nm的激发波长和发射波长的酶标仪(SpectraMax iD5,Molecular Devices)监测荧光信号。
(4)数据分析:
计算公式:
抑制率%=[1-(Responsesample-Responsemin)(Responsemax-Responsemin)]×100%
其中:Responsesample是样品的转化率读数;Responsemin是空白对照孔均值,代表没有酶活孔的转化率读数;Responsemax是阳性对照孔比值均值,代表没有化合物抑制孔的转化率读数。采用分析软件GraphPad Prism的log(inhibitor)vs.res。测试结果如表1所示。
表1.化合物对SHP2酶活抑制筛选
Comp. | SHP2 IC50(μM) | Comp. | SHP2 IC50(μM) |
LC-112 | 1.4 | LC-125 | 2.0 |
LC-118 | >100 | LC-126 | 0.62 |
LC-119 | 8.0 | LC-127 | 0.25 |
LC-120 | >100 | LC-128 | 3.4 |
LC-121 | >100 | LC-129 | 0.083 |
LC-122 | 1.9 | LC-130 | 8.1 |
LC-123 | 2.3 | LC-131 | 0.80 |
LC-124 | 12.5 |
实施例17:SHP2抑制剂对不同肿瘤细胞ERK磷酸化的抑制活性评价
采用Western Blot建立了肿瘤细胞ERK磷酸化的抑制活性评价方法。
人非小细胞肺癌细胞NCI-H1975用含有10%胎牛血清(Bio-channel)的RPMI-1640(Bio-channel)培养基进行培养﹐培养条件是37℃,95%空气和5%的CO2,培养于100mm塑料细胞培养皿(Nest)中,一周传代培养2-3次,将细胞以2×106/孔的密度接种在6孔细胞培养板(Nest)中,并在37℃,95%空气和5%的CO2中进行培养。24小时细胞贴壁后加入10mM所试化合物与阳性对照的100% DMSO储液,细胞培养液中DMSO的终浓度为0.1%,所试化合物与阳性对照的终浓度是10μM。将上述细胞37℃孵育8小时后吸掉培养基,用预冷的PBS洗一次﹐然后加入含磷酸酶抑制剂(Cat#P1081,Beyotime Biotechnology)和PMSF(Cat#ST506,Beyotime Biotechnology)的RIPA裂解液(Cat#P0013C,Beyotime Biotechnology)裂解10分钟,收集细胞悬液,4℃12000rpm离心15分钟,取上清用BCA试剂盒(Cat#P0012C,BeyotimeBiotechnology)定量,然后用Western Blot免疫印迹法检测p-ERK和ERK表达量。
人急性髓系白血病细胞MV-411用含有10%胎牛血清的DMEM培养基进行培养﹐培养条件是37℃,95%空气和5%的CO2,培养于100mm塑料细胞培养皿中,一周传代培养2-3次,将细胞以2×106/孔的密度接种在6孔细胞培养板中,并在37℃,95%空气和5%的CO2中进行培养。24小时后加入10mM所试化合物与阳性对照的100% DMSO储液,细胞培养液中DMSO的终浓度为0.1%,所试化合物与阳性对照的终浓度是10μM。将上述细胞37℃孵育2小时后离心取细胞沉淀,用预冷的PBS洗一次﹐然后加入含磷酸酶抑制剂和PMSF的RIPA裂解液裂解10分钟,收集细胞悬液,4℃12000rpm离心15分钟,取上清用BCA试剂盒定量,然后用WesternBlot免疫印迹法检测p-ERK和ERK的表达量。
人非小细胞肺癌细胞NCI-H2228用含有10%胎牛血清的RPMI-1975培养基进行培养﹐培养条件是37℃,95%空气和5%的CO2,培养于100mm塑料细胞培养皿中,一周传代培养2-3次,将细胞以2×106/孔的密度接种在6孔细胞培养板中,并在37℃,95%空气和5%的CO2中进行培养。24小时后加入10mM所试化合物与阳性对照的100% DMSO储液,细胞培养液中DMSO的终浓度为0.1%,所试化合物与阳性对照的终浓度是10μM。将上述细胞37℃孵育12小时后吸掉培养基,用预冷的PBS洗一次﹐然后加入含磷酸酶抑制剂和PMSF的RIPA裂解液裂解10分钟,收集细胞悬液,4℃12000rpm离心15分钟,取上清用BCA试剂盒定量,然后用WesternBlot免疫印迹法检测p-ERK和ERK的表达量。
人胃癌细胞KATO III用含有10%胎牛血清的RPMI-1975培养基进行培养﹐培养条件是37℃,95%空气和5%的CO2,培养于100mm塑料细胞培养皿中,一周传代培养2-3次,将细胞以2×106/孔的密度接种在6孔细胞培养板中,并在37℃,95%空气和5%的CO2中进行培养。24小时后加入10mM所试化合物与阳性对照的100% DMSO储液,细胞培养液中DMSO的终浓度为0.1%,所试化合物与阳性对照的终浓度是10μM。将上述细胞37℃孵育8小时后吸掉培养基,用预冷的PBS洗一次﹐然后加入含磷酸酶抑制剂和PMSF的RIPA裂解液裂解10分钟,收集细胞悬液,4℃12000rpm离心15分钟,取上清用BCA试剂盒定量,然后用Western Blot免疫印迹法检测p-ERK和ERK的表达量。
人胃癌细胞Huh7用含有10%胎牛血清的RPMI-1975培养基进行培养﹐培养条件是37℃,95%空气和5%的CO2,培养于100mm塑料细胞培养皿中,一周传代培养2-3次,将细胞以2×106/孔的密度接种在6孔细胞培养板中,并在37℃,95%空气和5%的CO2中进行培养。24小时后加入10mM所试化合物与阳性对照的100% DMSO储液,细胞培养液中DMSO的终浓度为0.1%,所试化合物与阳性对照的终浓度是10μM。将上述细胞37℃孵育24小时后吸掉培养基,用预冷的PBS洗一次﹐然后加入含磷酸酶抑制剂和PMSF的RIPA裂解液裂解10分钟,收集细胞悬液,4℃12000rpm离心15分钟,取上清用BCA试剂盒定量,然后用Western Blot免疫印迹法检测p-ERK和ERK的表达量。
人非小细胞肺癌细胞NCI-H1975用含有10%胎牛血清的RPMI-1640培养基进行培养﹐培养条件是37℃,95%空气和5%的CO2,培养于100mm塑料细胞培养皿中,一周传代培养2-3次,将细胞以2×106/孔的密度接种在6孔细胞培养板中,并在37℃,95%空气和5%的CO2中进行培养。24小时细胞贴壁后加入10mM LC-129与阳性对照的100% DMSO储液,细胞培养液中DMSO的终浓度为0.1%,LC-129的终浓度从30μM开始三倍梯度稀释至0.01μM。将上述细胞37℃孵育8小时后吸掉培养基,用预冷的PBS洗一次﹐然后加入含磷酸酶抑制剂和PMSF的RIPA裂解液裂解10分钟,收集细胞悬液,4℃12000rpm离心15分钟,取上清用BCA试剂盒定量,然后用Western Blot免疫印迹法检测p-ERK和ERK表达量。
本发明不同优选化合物对不同肿瘤的ERK磷酸化抑制活性如图1所示,优选化合物LC-129浓度对抑制肺癌细胞的ERK磷酸化影响如图2所示。
以上所述的实施例对本发明的技术方案和有益效果进行了详细说明,应理解的是以上所述仅为本发明的具体实施例,并不用于限制本发明,凡在本发明的原则范围内所做的任何修改、补充和等同替换等,均应包含在本发明的保护范围之内。
Claims (9)
2.根据权利要求1所述的式(I)化合物,或其立体异构体、互变异构体、前药、药学上可接受的盐、水合物、溶剂化物,其特征在于,R为4-甲氧基苯基、3,4-二甲氧基苯基、2,6-二甲氧基苯基、4-硝基苯基、4-氟苯基、3,4-二氯苯基、3,5-二氯苯基、苯基、萘基、联苯基、芴基或吡啶基。
3.根据权利要求1所述的式(I)化合物,或其立体异构体、互变异构体、前药、药学上可接受的盐、水合物、溶剂化物,其特征在于,所述式(I)化合物选自:
4-氨基-2-(4-氨基-4-甲基哌啶-1-基)-N-苯基嘧啶-5-甲酰胺、4-氨基-2-(4-氨基-4-甲基哌啶-1-基)-N-萘-1-基嘧啶-5-甲酰胺、4-氨基-2-(4-氨基-4-甲基哌啶-1-基)-N-萘-2-基嘧啶-5-甲酰胺、N-(1,1'-联苯)-3-基)-4-氨基-2-(4-氨基-4-甲基哌啶-1-基)嘧啶-5-甲酰胺、
4-氨基-2-(4-氨基-4-甲基哌啶-1-基)-N-(9H-芴-2-基)嘧啶-5-甲酰胺、4-氨基-2-(4-氨基-4-甲基哌啶-1-基)-N-(4-甲氧基苯基)嘧啶-5-甲酰胺、4-氨基-2-(4-氨基-4-甲基哌啶-1-基)-N-(3,4-二甲氧基苯基)嘧啶-5-甲酰胺、
4-氨基-2-(4-氨基-4-甲基哌啶-1-基)-N-(2,6-二甲氧基苯基)嘧啶-5-甲酰胺、
4-氨基-2-(4-氨基-4-甲基哌啶-1-基)-N-(4-硝基苯基)嘧啶-5-甲酰胺、4-氨基-2-(4-氨基-4-甲基哌啶-1-基)-N-(4-氟苯基)嘧啶-5-甲酰胺、4-氨基-2-(4-氨基-4-甲基哌啶-1-基)-N-(3,4-二氯苯基)嘧啶-5-甲酰胺、4-氨基-2-(4-氨基-4-甲基哌啶-1-基)-N-(3,5-二氯苯基)嘧啶-5-甲酰胺、4-氨基-2-(4-氨基-4-甲基哌啶-1-基)-N-吡啶-3-基)嘧啶-5-甲酰胺、4-氨基-2-(4-氨基-4-甲基哌啶-1-基)-N-吡啶-2-基甲基嘧啶-5-甲酰胺、4-氨基-2-(4-氨基-4-甲基哌啶-1-基)-N-吡啶-2-基)嘧啶-5-甲酰胺。
4.一种药物组合物,其特征在于,包括如权利要求1-3任一项所述的式(I)化合物或其立体异构体、互变异构体、前药、药学上可接受的盐、水合物、溶剂化物;还包括药学上可接受的赋形剂。
5.一种药物组合物,其特征在于,包括如权利要求1-3任一项所述的式(I)化合物或其立体异构体、互变异构体、前药、药学上可接受的盐、水合物、溶剂化物以及抗肿瘤药剂。
6.一种如权利要求1-3任一项所述的式(I)化合物或其立体异构体、互变异构体、前药、药学上可接受的盐、水合物、溶剂化物在制备SHP2变构抑制剂中的应用。
7.一种如权利要求1-3任一项所述的式(I)化合物或其立体异构体、互变异构体、前药、药学上可接受的盐、水合物、溶剂化物在制备预防和/或治疗癌症的药物中的应用。
8.一种如权利要求4或5所述的药物组合物在制备预防和/或治疗癌症的药物中的应用。
9.根据权利要求7-8任一项所述的应用,其特征在于,所述的癌症为多发性骨髓瘤、胃癌、肺癌、乳腺癌、食管癌、结肠癌、髓母细胞瘤、急性粒细胞白血病、慢性白血病、前列腺癌、肝细胞瘤、肾细胞瘤、宫颈癌、皮肤癌、卵巢癌、结肠癌、神经胶质瘤、甲状腺癌或胰腺癌。
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