CN116162093B - 一种tyk2抑制剂化合物及其用途 - Google Patents
一种tyk2抑制剂化合物及其用途 Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
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Abstract
本发明提供了一种结构新颖的可作为TYK2抑制剂的新化合物,以及新的化合物在制备治疗和/或预防炎性疾病和自身免疫疾病中的应用。所述新的化合物相比先导化合物BMS‑986165具有更高的选择性和药用安全性,表现出低的心脏毒性风险和动物死亡率,以及更好的药代动力学性质,有望成为新一代具有高选择性,低副作用的TYK2抑制剂药物。
Description
技术领域
本发明属于药物化学领域,具体涉及一种TYK2抑制剂化合物或其药学可接受的盐,含有它们的药物组合物以及作为TYK2抑制剂在预防和/或治疗由该激酶介导的相关疾病、特别是自身免疫性疾病、炎性疾病和癌症的药物中的用途。
背景技术
Janus激酶(JAK)是一种细胞内非受体酪氨酸激酶,主要负责调控由各种细胞因子受体介导的信号传导通路,进而调控多种细胞的生长、活化、分化、凋亡,血管生成和免疫调节等重要的生理过程。JAK激酶家族由JAK1、JAK2、JAK3和TYK2四个亚型组成,其各亚型分别介导不同类型的细胞因子信号通路,JAK1、JAK2和TYK2在人体各组织细胞中均有表达,JAK3主要表达于各造血组织细胞中。TYK2是JAK家族最早发现的一个亚型,和同族其它激酶一样也由7个通源结构域组成四个保守结构域,即C端的假激酶区和激酶区,以及N端的FERM区和SH2结构域。TYK2在细胞内与JAK1形成二聚体介导I型干扰素的应答以及与JAK2形成二聚体介导IL-23和IL-12的信号传导并活性下游STAT(信号转导及转录激活因子)信号通路。研究表明这些细胞因子与多种炎性疾病、自身免疫性疾病和癌症的发病机制相关,通过对TYK2的抑制,可以达到治疗银屑病、炎症性肠病、系统性红斑狼疮、克莱恩病等多种炎症和自身免疫性疾病。
早期的TYK2抑制剂如Tofacitinib都属于JAK非选择性抑制剂,是首个口服JAK抑制剂,对JAK1、2、3亚型均有显著的抑制活性。对其它亚型如JAK1、JAK2和JAK3的活性抑制增加了Tofacitinib的疗效,但同时也带来了较为严重的副作用,不良反应包括感染、结核、肿瘤、贫血、肝损伤及胆固醇增加等。早期JAK抑制剂主要是竞争激酶结构域与ATP的结合而发挥作用,因此普遍存在选择性不高的问题,而百时美施贵宝已经上市的TYK2选择性抑制剂Deucravacitinib (BMS986165)为变构抑制剂,结合在保守的假激酶位点,相对于JAK家族的其它激酶位点中正构抑制剂ATP结合位点,选择性高具有更大的安全性。鉴于JAK非选择性抑制剂的良好疗效和多种靶点相关性严重副作用,开发安全性更高的TYK2选择性抑制剂药物用于银屑病等炎症性疾病的治疗具有巨大临床应用潜力。专利WO2014/074661公开了一种TYK2的高选择性JH2结合剂,具有如下结构式,其只抑制TYK2介导的生理功能,而不与JAKs的激酶区(JH1)的结合。
目前仍然需要开发新的化合物,其选择性地结合TYK2的伪激酶结构域(JH2),同时具有更好的药效、选择性、药用安全性、药物代谢结果的高选择性TYK2抑制剂全新分子结构。
本发明针对性的对先导化合物BMS-986165进行结构修饰,通过在其三氮唑上引入杂环基,特别是螺环基、并环基、桥环基等复杂双环基,获得了一系列新的化合物,所述新的化合物相比先导化合物BMS-986165具有更高的体外选择性和药用安全性,表现出较低的心脏毒性风险和动物死亡率,以及更好的药代动力学性质,特别是引入并环基、桥环基时,表现出令人预料不到的药理化性质,有望成为新一代具有高选择性,低副作用的TYK2抑制剂药物。
发明内容
针对现有技术的需求,本发明提供了一种结构新颖的可作为TYK2抑制剂的化合物,该类化合物表现出高活性和安全性以及良好的药代动力学参数。
本发明提供了一种TYK2抑制剂化合物,具有如式(I)所示的化合物、其溶剂化物、立体异构体、氘代化合物、或其药学可接受的盐;
其中,R1为3至12元环烷基、3至12元杂环基、C2-6的炔基;所述炔基可进一步被3至12元环烷基所取代;
R2为甲基或氘代甲基。
在本发明的一些实施例中,所述R1选自3至12螺环基、3至12并环基、3至12桥环基。
在本发明提供的一些实施中,所述TYK2抑制剂化合物选自下式化合物1,或化合物2,或化合物3,或化合物4或其药学上可接受的盐,
化合物1 化合物2
化合物3 化合物4。
在本发明的一些实施例中,TYK2抑制剂化合物,为化合物1,或化合物2,或化合物3或其药学上可接受的盐。
优选的,所述的TYK2抑制剂化合物,为化合物1或其药学上可接受的盐。
优选的,所述TYK2抑制剂化合物,为化合物2或其药学上可接受的盐。
优选的,所述TYK2抑制剂化合物,为化合物3或其药学上可接受的盐。
本发明的另一方面涉及一种药物组合物,其包含治疗有效剂量的上述所述的化合物或其药学上可接受的盐以及一种或多种药学上可接受的载体、稀释剂或赋形剂。
本发明的另一方面提供了一种上述所示的化合物或其药学上可接受的盐,或其药物组合物在制备治疗和/或预防炎性疾病和自身免疫疾病中的应用;其中所述炎性疾病和自身免疫疾病选自类风湿性关节炎、皮炎、银屑病或炎症性肠病。
本发明针对性的对先导化合物BMS-986165进行结构修饰,通过在其三氮唑上引入杂环基,特别是螺环基、并环基、桥环基等复杂双环基,获得了一系列新的化合物,所述新的化合物相比先导化合物BMS-986165具有更高的体外选择性和药用安全性,表现出较低的心脏毒性风险和动物死亡率,以及更好的药代动力学性质,有望成为新一代具有高选择性,低副作用的TYK2抑制剂药物。
具体实施方式
以下实施例详细说明本发明技术方案,但本发明保护范围包括但不限于此。
实施例1
6-环丙烷胺基-4-{[2-甲氧基-3-(1-甲基-5-{2-氧杂-6-氮杂螺[3.3]庚烷-6-基}-1H-1,2,4-三唑-3-基)苯基]氨基}-N-氘代甲基哒嗪-3-甲酰胺(化合物1)的合成
步骤1:中间体6-(3-溴-1-甲基-1H-1,2,4-三唑-5-基)-2-氧杂-6-氮杂螺[3.3]庚烷(1-2)的合成
将化合物1-1(2.5 g,10.38 mmol),2-氧杂-6-氮杂螺[3.3]庚烷(1.03 g,10.38mmol)溶于N,N-二甲基甲酰胺(DMF,40 mL),再加入碳酸钾(K2CO3,4.3 g,31.14 mmol)升温至150℃,反应16 h。反应完全后反应液中加入水(300 mL),二氯甲烷(DCM,500 mL×2)萃取,有机相无水硫酸钠干燥后浓缩得到产物粗品,柱层析分离纯化(VPE:VEA=3:1),得到中间体1-2(1.1 g,白色固体,产率41.0%),[M+H]+:259.1。
步骤2:中间体2-甲氧基-3-(1-甲基-5-{2-氧杂-6-氮杂螺[3.3]庚烷-6-基}-1H-1,2,4-三唑-3-基)苯胺(1-4)的合成
在玻璃封管中依次加入将中间体1-2(1.1 g,4.25 mmol),化合物1-3(1.16 g,4.67 mmol),磷酸三钾(K3PO4,1.8 g,8.50 mmol),1,1'-双二苯基膦二茂铁二氯化钯(Pd(dppf)Cl2,309 mg,0.425 mmol),二氧六环(dioxane,21 mL),水(7 mL),氮气置换,升温至120℃反应16 h。反应完全后过滤,旋干,柱层析纯化(VPE:VEA=1:1)得到中间体1-4(900 mg,产率70.4%),[M+H]+:302.4。
步骤3:中间体6-氯-4-{[2-甲氧基-3-(1-甲基-5-{2-氧杂-6-氮杂螺[3.3]庚烷-6-基}-1H-1,2,4-三唑-3-基)苯基]氨基}-N-氘代甲基哒嗪-3-甲酰胺(1-6)的合成
室温下,在反应瓶中依次加入中间体1-4(400 mg,1.32 mmol),化合物1-5(247mg,1.20 mmol),四氢呋喃(THF,30mL),六甲基二硅基胺基锂(LiHMDS,4 mL,1 M in THF),氮气置换,升温至80℃反应3h。反应完后,反应液用饱和氯化铵溶液淬灭,加水(20 mL),乙酸乙酯(EA,50 mL×2)萃取,有机相干燥浓缩得到产物粗品,柱层析分离纯化(VPE:VEA=1:1),得到中间体1-6(300 mg,黄色固体,产率48.4%),[M+H]+:474.0。
步骤4:化合物6-环丙烷胺基-4-{[2-甲氧基-3-(1-甲基-5-{2-氧杂-6-氮杂螺[3.3]庚烷-6-基}-1H-1,2,4-三唑-3-基)苯基]氨基}-N-氘代甲基哒嗪-3-甲酰胺(1)的合成
在玻璃封管中依次加入将中间体1-6(100 mg,0.213 mmol),化合物1-7(36 mg,0.426 mmol),碳酸铯(Cs2CO3,139 mg,0.426 mmol),三(二亚苄基丙酮)二钯(Pd2(dba)3,20mg,0.021mmol),4,5-双(二苯基膦)-9,9-二甲基氧杂蒽(Xantphos,25 mg,0.043 mmol),dioxane(3 mL),氮气置换,110℃反应16 h。反应完后过滤反应液,浓缩得到粗产物,柱层析分离纯化(VPE:VEA=1:1),得到化合物1(30 mg,白色固体,产率27.1%),[M+H]+:523.5。
1H NMR(400 MHz,DMSO-d6) δ 11.30(s,1H),10.96(s,1H),9.18(d,J=5.2 Hz,1H),8.12(s,1H),7.59(dd,J=8.0,1.6 Hz,1H),7.48(dd,J=8.0,1.6 Hz,1H),7.25(d,J=8.0 Hz,1H),4.00(d,J=4.4 Hz,2H),3.82(d,J=10.8 Hz,2H),3.79(s,3H),3.71(s,3H),3.60(dd,J=11.2,2.0 Hz,2H),2.09(s,1H),2.01-1.98(m,2H),0.82-0.79(m,4H).
实施例2
6-环丙烷胺基-4-{[2-甲氧基-3-(1-甲基-5-{2-氧杂-6-氮杂螺[3.3]庚烷-6-基}-1H-1,2,4-三唑-3-基)苯基]氨基}-N-氘代甲基哒嗪-3-甲酰胺(化合物2)的合成
步骤1:中间体3-溴-5-{六氢-1H-呋喃[3,4-c]吡咯-5-基}-1-甲基-1H-1,2,4-三唑(2-1)的合成
将化合物1-1(2.5 g,10.38 mmol),六氢-1H-呋喃并[3,4-C]吡咯(1.18 g,10.38mmol)溶于DMF(40 mL),再加入K2CO3(4.3 g,31.14 mmol)升温至150℃,反应16 h。反应完全后反应液中加入水(30 mL),DCM(40 mL×2)萃取,有机相干燥浓缩得到产物粗品,柱层析分离纯化(VPE:VEA=3:1),得到中间体2-1(1.5 g,白色固体,产率53.1%),[M+H]+:273.1。
步骤2:中间体3-(5-{六氢-1H-呋喃[3,4-c]吡咯-5-基}-1-甲基-1H-1,2,4-三唑-3-基)-2-甲氧基苯胺(2-2)的合成
在玻璃封管中依次加入将中间体2-1(1.1 g,4.04 mmol),化合物1-3(1.11 g,4.45 mmol),K3PO4(1.71 g,8.08 mmol),Pd(dppf)Cl2(293 mg,0.404 mmol),dioxane(21mL),水(7 mL),氮气置换,升温至120℃反应16 h。反应完全后过滤,旋干,柱层析纯化(VPE:VEA=1:1)得到中间体2-2(950 mg,产率74.6%),[M+H]+:316.4。
步骤3:中间体6-氯-4-{[3-(5-{六氢-1H-呋喃[3,4-c]吡咯-5-基}-1-甲基-1H-1,2,4-三唑-3-基)-2-甲氧基苯基]氨基}-N-氘代甲基哒嗪-3-甲酰胺(2-3)的合成
室温下,在反应瓶中依次加入中间体2-2(400 mg,1.27 mmol),化合物1-5(238mg,1.15 mmol),THF(30mL),LiHMDS(4 mL,1 M in THF),氮气置换,升温至80℃反应3h。反应完后,反应液用饱和氯化铵溶液淬灭,加水(20mL),EA(20 mL×2)萃取,有机相干燥浓缩得到产物粗品,柱层析分离纯化(VPE:VEA=1:1),得到中间体2-3(350 mg,黄色固体,产率56.9%),[M+H]+:488.7。
步骤4:化合物6-环丙烷胺基-4-{[2-甲氧基-3-(1-甲基-5-{2-氧杂-6-氮杂螺[3.3]庚烷-6-基}-1H-1,2,4-三唑-3-基)苯基]氨基}-N-氘代甲基哒嗪-3-甲酰胺(2)的合成
在玻璃封管中依次加入将中间体2-3(150 mg,0.310 mmol),化合物1-7(53 mg,0.620 mmol),Cs2CO3(202 mg,0.620 mmol),Pd2(dba)3(28 mg,0.031mmol),Xantphos(36mg,0.062 mmol),dioxane(3 mL),氮气置换,110℃反应16 h。反应完后过滤反应液,浓缩得到粗产物,柱层析分离纯化(VPE:VEA=1:1),得到化合物2(55 mg白色固体,产率33.3%),[M+H]+:537.5。
1H NMR(400 MHz,DMSO-d6) δ 11.29(s,1H),10.96(s,1H),9.16(d,J=5.2 Hz,1H),8.10(s,1H),7.60(dd,J=8.0,1.6 Hz,1H),7.47(dd,J=8.0,1.6 Hz,1H),7.26(d,J=8.0 Hz,1H),4.01(d,J=4.4 Hz,2H),3.81(d,J=10.8 Hz,2H),3.76(s,3H),3.70(s,3H),3.62(dd,J=11.2,2.0 Hz,2H),2.10(s,1H),2.03-1.99(m,4H),0.85-0.81(m,4H)。
实施例3
6-环丙烷胺基-4-{[2-甲氧基-3-(1-甲基-5-{3-氧杂-8-氮杂双环[3.2.1]辛-8-基}-1H-1,2,4-三唑-3-基)苯基]氨基}-N-氘代甲基哒嗪-3-甲酰胺(化合物3)的合成
步骤1:中间体8-(3-溴-1-甲基-1H-1,2,4-三唑-5-基)-3-氧杂-8-氮杂双环[3.2.1]辛烷(3-2)的合成
将化合物1-1(2 g,8.30 mmol),化合物3-1(1.24 g,8.30 mmol)溶于DMF(40 mL),再加入K2CO3(3.4 g,24.91 mmol)升温至150℃,反应16 h。反应完全后反应液中加入水(30mL),DCM(100 mL×2)萃取,有机相无水硫酸钠干燥后浓缩得到产物粗品,硅胶柱层析分离纯化(VPE:VEA=3:1),得到中间体3-2(1 g,灰白色固体,产率44.25%),[M+H]+:273.1。
步骤2:中间体2-甲氧基-3-(1-甲基-5-{3-氧杂-8-氮杂双环[3.2.1]辛烷-8-基}-1H-1,2,4-三唑-3-基)苯胺(3-3)的合成
在玻璃封管中依次加入将中间体3-2(1g,3.66 mmol),化合物1-3(1g,4.03mmol),K3PO4(1.56g,7.33 mmol),Pd(dppf)Cl2(268 mg,0.36 mmol),Dioxane(20 mL),水(7mL),氮气置换,升温至120℃反应16 h。反应完全后过滤,旋干,柱层析纯化(VPE:VEA=1:1)得到中间体3-3(860 mg,产率74.8%),[M+H]+:316.4。
步骤3:中间体6-氯-4-{[2-甲氧基-3-(1-甲基-5-{3-氧杂-8-氮杂双环[3.2.1]辛-8-基}-1H-1,2,4-三唑-3-基)苯基]氨基}-N-氘代甲基哒嗪-3-甲酰胺(3-4)的合成
室温下,在反应瓶中依次加入中间体3-3(320 mg,1.02 mmol),化合物1-5(189mg,0.924 mmol),THF(30 mL),LiHMDS(3.2 mL,1M in THF),氮气置换,升温至80℃反应3h。反应完后,反应液用饱和氯化铵溶液淬灭,加水(20 mL),EA(20 mL×2)萃取,有机相干燥浓缩得到产物粗品,柱层析分离纯化(VPE:VEA=1:1),得到中间体3-4(310 mg黄色固体,产率63.0%),[M+H]+:488.6。
步骤4:化合物6-环丙烷胺基-4-{[2-甲氧基-3-(1-甲基-5-{3-氧杂-8-氮杂双环[3.2.1]辛-8-基}-1H-1,2,4-三唑-3-基)苯基]氨基}-N-氘代甲基哒嗪-3-甲酰胺(3)的合成
在玻璃封管中依次加入将中间体3-4(80 mg,0.165 mmol),化合物1-7(28 mg,0.33 mmol),Cs2CO3(107 mg,0.33 mmol),Pd2(dba)3(15 mg,0.0165 mmol),xantphos(19mg,0.33 mmol),dioxane(3 mL),氮气置换,110℃反应16 h。反应完后过滤反应液,浓缩得到粗产物,柱层析分离纯化(VPE:VEA=1:1),得到40 mg白色固体,过反相得到化合物3(20 mg白色固体,产率22.73%,纯度>98%),[M+H]+:537.5。
1H NMR(400 MHz,DMSO-d6) δ 11.31(s,1H),10.95(s,1H),9.15(d,J=5.2 Hz,1H),8.13(s,1H),7.59(dd,J=8.0,1.6 Hz,1H),7.48(dd,J=8.0,1.6 Hz,1H),7.25(d,J=8.0 Hz,1H),4.00(d,J=4.4 Hz,2H),3.82(d,J=10.8 Hz,2H),3.78(s,3H),3.73(s,3H),3.59(dd,J=11.2,2.0 Hz,2H),2.08(s,1H),2.01-1.88(m,4H),0.82-0.78(m,4H)。
实施例4
6-环丙烷酰胺-4-({3-[5-(2-环丙基乙炔基)-1-甲基-1H-1,2,4-三唑-3-基]-2-甲氧基苯基}氨基)-N-氘代甲基哒嗪-3-甲酰胺(化合物4)的合成
步骤1:中间体3-溴-5-(2-环丙基乙炔基)-1-甲基-1H-1,2,4-三唑(4-2)的合成
将化合物1-1(1.26 g,12.45 mmol),化合物4-1(1 g,37.35 mmol)溶于dioxane(25 mL),再加入四(三苯基膦)钯(Pd(PPh3)4,302 mg,0.62 mmol),碘化亚铜(CuI,50 mg,0.62 mmol),三乙胺(Et3N,1 g,24.9 mmol)于封管中,氮气置换,升温至100℃,反应16 h。反应完全后,反应液过滤,浓缩,柱层析分离纯化VPE:VEA=20:1),得到中间体4-2(478 mg,灰白色固体,产率40.51%),[M+H]+: 227.1。
步骤2:中间体3-[5-(2-环丙基乙炔基)-1-甲基-1H-1,2,4-三唑-3-基]-2-甲氧基苯胺(4-3)的合成
在玻璃封管中依次加入将中间体4-2(587 mg,2.61 mmol),化合物1-3(715 mg,2.87 mmol),K3PO4(1.11 g,5.22 mmol),Pd(dppf)Cl2(190 mg,0.26 mmol),dioxane(20mL),水(4 mL),氮气置换,升温至120℃反应16 h。反应完全后过滤,浓缩,柱层析纯化(VPE:VEA=5:1),得到中间体4-3(314 mg,棕色油状物,产率44.92%),[M+H]+:269.3。
步骤3:中间体6-氯-4-({3-[5-(2-环丙基乙炔基)-1-甲基-1H-1,2,4-三唑-3-基]-2-甲氧基苯基}氨基)-N-氘代甲基哒嗪-3-甲酰胺(4-4)的合成
室温下,在反应瓶中依次加入中间体4-3(198 mg,0.74 mmol),化合物1-5(303mg,1.48 mmol),THF(15 mL),LiHMDS(3.7 mL,1 M in THF),氮气置换,升温至80℃反应3h。反应完后,反应液用饱和氯化铵溶液淬灭,加水(20 mL),EA(20 mL×2)萃取,有机相干燥浓缩得到产物粗品,柱层析分离纯化(VPE:VEA=2:1),得到中间体4-4(250 mg,米白色固体,产率77.64%),[M+H]+: 440.8。
步骤4:化合物6-环丙烷酰胺-4-({3-[5-(2-环丙基乙炔基)-1-甲基-1H-1,2,4-三唑-3-基]-2-甲氧基苯基}氨基)-N-氘代甲基哒嗪-3-甲酰胺(4)的合成
在玻璃封管中依次加入将中间体4-4(230 mg,0.53 mmol),化合物1-7(89 mg,1.05 mmol),Cs2CO3(342 mg,1.05 mmol),Pd2(dba)3(48 mg,0.05 mmol),Xantphos(61 mg,0.105 mmol),dioxane(15 mL),氮气置换,110℃反应16 h。反应完后过滤反应液,浓缩得到粗产物,柱层析分离纯化(VPE:VEA=1:1),得到154 mg白色固体,过反相得到化合物4(130mg,白色固体,产率50.98%),[M+H]+: 490.5。
1H NMR(400MHz,DMSO-d6) δ11.32(s,1H),10.96(s,1H),9.16(q,J=4.8Hz,1H),8.14(s,1H),7.62(dd,J=8.0,1.6Hz,1H),7.53(dd,J=8.0,1.6Hz,1H),7.27(t,J=8.0Hz,1H),3.94(s,3H),3.71(s,3H),2.15-2.02(m,1H),1.74(tt,J=8.4,5.2Hz,1H),1.06-0.99(m,2H),0.96-0.89(m,2H),0.87-0.78(m,4H)。
生物学评价
测试本发明化合物对细胞TYK2信号通路抑制作用
实验方法:
本实验采用表达TYK2的U266细胞系,通过INF-α刺激激活TYK2信号通路,检测化合物对其下游STAT3磷酸化的抑制活性,并得出化合物对TYK2信号通路活性的半数抑制浓度IC50。
实验操作:
384孔检测板中铺入U266细3-12μL,每孔细胞个数为100-300K,加入2μL梯度稀释好的化合物溶液,二氧化碳培养箱孵育2小时。2小时后加入2μL INF-α,INF-α终浓度1000U/mL,室温震荡20min。加入2-5μL(5X)LANCE Ultra Lysis Buffer 2溶液,室温度震荡2h。2h后加入5μL终浓度为2nM的LANCE Ultra Eu-labeled Anti-STAT5(Y694/Y699)Antibody(PerkinElmer)和终浓度为20nM的LANCE Ultra ULight-labeled Anti-TAT5 Antibody(PerkinElmer)溶液,室温孵育过夜。酶标仪(BioTek公司,SynergyH1型号)测定各板孔的665nm荧光信号值,通过荧光信号值计算抑制率,根据不同浓度的抑制率通过曲线拟合得出化合物的IC50。通过于板上阳性对照孔(DMSO对照孔)和阴性对照孔(不加细胞)计算使用化合物处理的孔的百分比抑制数据{%抑制率=100-[(测试化合物值-阴性对照值)]/(阳性对照值-阴性对照值)×100}。使用GraphPad prism拟合不同浓度和相应百分比抑制率数据至四参数非线性逻辑公式计算出IC50值。
实验结果:通过以上方案得出本发明所示的化合物在细胞TYK2信号通路抑制的活性试验数据如表一所示;
表一:体外TYK2细胞活性测定结果(IC50)
实验结论:本发明化合物对细胞TYK2信号通路有良好的抑制作用
测试本发明化合物对细胞JAK2信号通路抑制作用
实验方法:
本实验采用TF-1细胞系,通过IL6刺激激活JAK2信号通路,检测化合物对其下游STAT3磷酸化的抑制活性,并得出化合物对JAK2信号通路活性的半数抑制浓度IC50。
实验操作:
384孔检测板中铺入TF-1细胞3-12μL,每孔细胞个数为100-300K,加入2μL梯度稀释好的化合物溶液,二氧化碳培养箱孵育2小时。2小时后加入2μL IL6,IL6终浓度30ng/mL,室温震荡20min。加入2-5μL(5X)LANCE Ultra Lysis Buffer2溶液,4度震荡2h。2h后加入5μL终浓度为2nM的LANCE Ultra Eu-labeled Anti-STAT3(Tyr705)Antibody(PerkinElmer)和终浓度为20nM的LANCE Ultra ULight-labeled Anti-STAT3 Antibody(PerkinElmer)溶液,室温孵育过夜。酶标仪(BioTek公司,SynergyH1型号)测定各板孔的665nm荧光信号值,通过荧光信号值计算抑制率,根据不同浓度的抑制率通过曲线拟合得出化合物的IC50。通过于板上阳性对照孔(DMSO对照孔)和阴性对照孔(不加细胞)计算使用化合物处理的孔的百分比抑制数据{%抑制率=100-[(测试化合物值-阴性对照值)]/(阳性对照值-阴性对照值)×100}。使用GraphPad prism拟合不同浓度和相应百分比抑制率数据至四参数非线性逻辑公式计算出IC50值。
实验结果:通过以上方案得出本发明所示的化合物在细胞JAK2信号通路抑制的活性试验数据如表二所示:
表二:体外TYK2细胞、JAK2细胞活性测定结果(IC50)
实验结论:本发明化合物对TYK2细胞活性抑制与JAK2细胞活性抑制相比,有较高的选择性,特别是化合物2、化合物3,尤其是化合物3相比对照组BMS-986165选择性倍数高出6.4倍,表现出高选择性。
测试本发明化合物对hERG钾电流的阻断作用
试验系统
细胞:中国仓鼠卵巢(CHO)细胞系,CHO-hERG细胞用于本试验。
细胞培养液及培养条件:完全培养基为F12培养基,补充加入10% 胎牛血清,1%Geneticin®选择性抗生素(G418),89 µg/mL 潮霉素B (HB)。复苏培养基为F12培养基补充加入10%胎牛血清。CHO-hERG细胞生长在37℃(±2℃)、5% CO2(4%至8%)的高湿度培养箱中。细胞用复苏培养基复苏,完全培养基传代,用于膜片钳试验的细胞在最后一次传代时换成复苏培养基。
表三:细胞外液及内液成分:
试验方法
(1)将处于指数生长期的CHO-hERG细胞收集并重悬在ECS中备用。
(2)手动膜片钳试验
全细胞膜片钳技术下记录hERG电流,记录温度为室温。膜片钳放大器输出信号通过数模转换以及2.9 KHz低通滤波。数据记录用Patchmaster Pro软件采集。
细胞种在细胞记录槽中放置在倒置显微镜载物台上,随机选择记录槽中的一个细胞进行试验。灌流系统固定在倒置显微镜载物台上用ECS 持续灌流细胞。
用毛细玻璃管制备手动膜片钳试验记录微电极,其中充灌细胞內液。在膜片钳试验当天,使用硼硅酸盐玻璃管(BF150-117-10, SUTTER INSTRUMENT USA)制备电极。电极充灌ICS后电阻在2-5 MΩ之间。
钳制电压为-80 mV,第一步去极化至+60 mV并维持850 ms开放hERG通道。然后,电压设置为-50 mV并维持1275 ms,产生反弹电流或者称为尾电流,尾电流的峰值将被测量并用于分析。最后,电压恢复到钳制电压(-80 mV)。试验过程中,这个指令电压程序每间隔15s重复一次。
在溶媒对照工作溶液灌流的记录开始阶段,监测尾电流峰值直至稳定3条以上扫描曲线后则可以灌流待测试的供试品/阳性对照工作溶液,直到供试品/阳性对照工作溶液对hERG电流峰值的抑制作用达到稳定状态。一般以最近的连续3个电流曲线峰值基本重合作为判断是否稳定状态的标准。达到稳定态势以后继续灌流下一浓度供试品。一个细胞上可以测试一个或多个供试品/阳性对照,或者同一种药物的多个浓度,不同供试品/阳性对照之间需用溶媒对照工作液冲洗直到hERG电流回复到加药物之前80%以上的大小。同一浓度下各记录细胞抑制率的标准差不超过15%。
阳性对照西沙必利的测试浓度为0.1 μM,重复测定两个细胞。根据科学文献报道,0.1 μM的西沙必利抑制hERG电流超过50%。(Milnes, J.T.,et al.)。
(3)手动膜片钳数据接受标准
封接标准:全细胞模式形成后,施加钳制电压(-80 mV),可以记录到细胞膜相关参数(Cm,Rm以及Ra)。一个好的的全细胞记录应该满足以下条件:路径电阻(Rs)小于10 MΩ;膜电阻(Rm)大于500 MΩ和膜电容(Cm)小于100 pF。
电流大小:供试品/阳性对照品作用前峰电流幅度在400 pA和5000 pA之间。否则,放弃该细胞。
漏电流:在-80 mV的钳制电压下,漏电流绝对值应该小于200 pA。电流幅度将会用-80 mV下的漏电流校正。漏电流绝对值大于200 pA的扫描曲线不能用于分析。
数据分析
对于每个细胞,每一个浓度的供试品及阳性对照的抑制百分比由记录到的电流反应用以下公式算出:(1–供试品/阳性对照灌流后记录到的尾峰值电流/溶媒对照灌流记录到的尾峰值电流(起始电流))×100%。
对于每一个浓度记录到所有的细胞抑制百分比取均值,IC50值由Hill拟合的方法由浓度效应曲线中得出。
试验结果:本发明部分化合物对hERG电流的抑制结果,具体见下表四;
表四:受试化合物对hERG电流的抑制结果
注:IC50>30μM为++,20μM>IC50+。
本发明实施例化合物相比对照组具有较高的hERG IC50值,具有显著差异,表现出对hERG抑制作用更弱,说明本发明化合物的心脏毒性风险较低。
药代动力学实验
化合物单次口服或者静脉给药(溶媒5%DMSO+10%Solutol(HS-15)+85%saline)于动物(例如小鼠、大鼠、犬或者猴子),在固定的时间点取血。血样采集后,立即温和地颠倒试管至少5次,保证混合充分后放置于冰上。血液用肝素抗凝,然后8000 rpm离心5分钟,将血清与红细胞分离。用移液器吸岀血清转移至2 mL的聚丙烯管,标明化合物的名称和时间点,在进行LC-MS分析前保存在-40℃冰箱,待测。髙浓度样品用空白血浆稀释测定时。样品处理后,用LCMS/MS对血浆中的物质进行定量分析。通过进行了验证的药动学计算机程序,用以这种方式获得的血浆浓度/时曲线来计算药动学参数。实验发现本发明化合物均具有较好的药代动力学性质。
SD雄性大鼠以表四组别剂量灌胃给药后(各组为等摩尔剂量给药,溶媒为5%DMSO+10%Solutol(HS-15)+85%saline,每组3只),在固定的时间点取血检测。本发明的部分化合物的在大鼠血浆中的原型化合物药代动力学参数如下表五;
表五:受试化合物药代动力学参数
本发明实施例化合物在大鼠体内展现出良好的药代动力学性质;与对照组相比,本发明化合物在血浆中游离碱的AUC(h*ng/mL)均有显著提高。
急性毒性实验
化合物单次静脉给药(溶媒5%DMSO+10%Solutol(HS-15)+85%saline)于SD大鼠(10只动物/组,雌雄各半),给药剂量为0.1mM/kg,给药后进行临床观察。临床观察第一天两次,第二天开始一天一次,连续14天。包括行为学观察、全身触摸检查、腔体观察:包括皮肤,粘膜,毛色,眼睛,呼吸,自主活动和神经系统行为和死亡情况,记录中毒体征和死亡情况。实验结果见表六。
表六:受试化合物大鼠急性毒性预实验结果
实验结果表明本发明化合物有良好的安全性。化合物2、化合物3在0.1mM/kg剂量下大鼠单次静脉给药的动物死亡百分比比BMS-986165低,表明本发明化合物具有良好的安全性。
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