CN117069686A - 一种京尼平衍生物、其制备方法及应用 - Google Patents
一种京尼平衍生物、其制备方法及应用 Download PDFInfo
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- CN117069686A CN117069686A CN202310914497.7A CN202310914497A CN117069686A CN 117069686 A CN117069686 A CN 117069686A CN 202310914497 A CN202310914497 A CN 202310914497A CN 117069686 A CN117069686 A CN 117069686A
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- genipin derivative
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Abstract
本发明公开了一种京尼平衍生物或其可药用盐,所述京尼平衍生物为式(Ia)或(Ib)所示的化合物:。本发明具有较现有抑郁症临床用药氟西汀、京尼平更优的抗抑郁以及神经保护作用,且起效快,副作用小,临床应用前景广阔。
Description
技术领域
本发明属于医药技术领域,特别涉及一种京尼平衍生物、其制备方法及应用。
背景技术
抑郁症是一种常见的心理精神障碍,以明显而持久的心境低落作为主要临床特征。研究发现,近年抑郁症在全球范围内的发病率正呈现上升趋势。
目前临床常用抗抑郁药有单胺氧化酶抑制剂、三环类抗抑郁药、选择性5-羟色胺和去甲肾上腺素再摄取抑制剂等,其中,单胺氧化酶抑制剂是20世纪50年代最早发现的抗抑郁药,通过提高脑内去甲肾上腺素、多巴胺及5-HT的水平,改善患者的情绪,提高对事物的兴趣,达到抗抑郁的效果。曾被广泛应用,但因为与多种药物合用都有潜在危险,所以逐渐被三环类抗抑郁药物取代。三环类抗抑郁药物自60年代就开始应用于治疗抑郁症,常用品种包括阿米替林、丙米嗪、氯丙嗪、多塞平及氯米帕明,目前应用最广泛的是氯米帕明。该类药物抗抑郁效果明显,但是服用剂量偏大,副作用也比较多,包括口干、便秘、排尿困难、视线模糊、头晕、嗜睡等,所以应用得越来越少。选择性五羟色胺(5-HT)再摄取抑制剂是目前临床应用较多的新型抗抑郁药,可选择性地抑制5-HT转运体,阻断突触前膜对5-HT的再摄取,延长和增加5-HT的作用,从而产生抗抑郁作用,对肾上腺素能、组胺能、胆碱能受体的亲和力低,作用较弱,因而产生的不良反应少,代表药物有氟西汀等。以上现有临床治疗药物均存在起效时间延迟(2-6周)、对部分难治性抑郁症有效率不高甚至无效等明显缺陷,临床应用效果并不理想的问题。
发明内容
针对上述问题,本发明提供一种抗抑郁疗效提高的,副作用少、起效快的快速抗抑郁药物京尼平衍生物、其制备方法及应用。
本发明的第一方面,提供一种京尼平衍生物或其可药用盐,所述京尼平衍生物为式(Ia)或(Ib)所示的化合物:
其中:X、Y各自独立的代表CH或N;
Z代表O或N-R2;
R1、R2各自独立的代表C1~C3直链或支链烷基;
R3代表H、C1~C6烷基、卤素、C1~C3烷氧基、卤代烷基或硝基中的任意一种;
n代表0、1、2或3。
作为本发明的具体实施方式,所述Z代表O。
作为本发明的具体实施方式,所述R3代表H、C1~C3烷基、甲氧基中的任意一种。
作为本发明的具体实施方式,所述可药用盐选自盐酸盐、硫酸盐、磷酸盐、氢溴酸盐、马来酸盐、富马酸盐、枸橼酸盐、甲磺酸盐、对甲苯磺酸盐、酒石酸盐或醋酸盐中的任意一种,优选为盐酸盐。
作为本发明的具体实施方式,其选自以下W-1~W-8所示的任一化合物:
本发明的第二方面,提供前述京尼平衍生物或其可药用盐的制备方法,所述制备方法包括:将化合物1、化合物2a或化合物2b、反应助剂和有机溶剂混合,常温使反应完全,所述化合物1、化合物2a、化合物2b的结构式如下:
其中:X、Y各自独立的代表CH或N;
Z代表O或N-R2;
R1、R2各自独立的代表C1~C3直链或支链烷基;
R3代表H、C1~C6烷基、卤素、C1~C3烷氧基、卤代烷基或硝基中的任意一种;
n代表0、1、2或3;
优选地,所述化合物1、化合物2a、反应助剂的摩尔比为1:1:2;所述化合物1、化合物2b、反应助剂的摩尔比为1:1:2;
优选地,所述反应助剂包括碳酸钾;
优选地,所述有机溶剂包括N,N-二甲基甲酰胺;
优选地,所述反应的时间为4h;
优选地,还包括对反应产物进行有机溶剂萃取,洗涤所得有机相并进行干燥,浓缩以及纯化。
本发明的第三方面,提供一种药物组合物,其是由治疗有效量的前述京尼平衍生物或其可药用盐和一种或多种药学上可接受的载体组成。
本发明的第四方面,提供前述京尼平衍生物或其可药用盐在制备预防或治疗抑郁症的药物中的用途。所述抑郁症包括轻度、中度或重度的单相抑郁症、双相抑郁症、精神性抑郁症、反应性抑郁症、继发性抑郁症、季节性抑郁症、产后抑郁症及更年期抑郁症中的任意一种。
本发明的第五方面,提供前述京尼平衍生物或其可药用盐在制备具有神经保护作用的药物中的用途。
本发明的有益效果为:
本发明提供的京尼平衍生物或其可药用盐,具有较现有抑郁症临床用药氟西汀、京尼平更优的抗抑郁以及神经保护作用,且起效快,副作用小,临床应用前景广阔。
附图说明
图1为各组行为绝望小鼠强迫游泳不动时间结果对比;
图2为各组行为绝望小鼠悬尾不动时间结果对比;
图3为CUMS造模、给药以及行为学测试时间计划表;
图4A为旷场实验观察各组小鼠自主活动旷场路线代表图结果对比;
图4B为旷场实验观察各组小鼠自主活动移动总路程和平均速度结果对比;
图5为强迫游泳实验观察各组对CUMS小鼠平衡能力的影响(n=10);
图6为悬尾实验观察各组对CUMS小鼠平衡能力的影响(n=10);
图7为W-3对脑组织中PKA-CREB-BDNF信号通路的影响(n=3)(#P<0.05,与正常组比较;*p<0.05,**p<0.01,与CUMS组比较);
图8A为小鼠海马各组色带颜色对比;
图8B为小鼠海马各组5HT1A含量对比;
图8C为小鼠海马各组GluN2B含量对比;
图8D为小鼠海马各组GluN2A含量对比;
图9A为小鼠海马各组色带颜色对比;
图9B为小鼠海马各组PKA含量对比;
图9C为小鼠海马各组BDNF含量对比;
图9D为小鼠海马各组CREB含量对比;
图10A为小鼠脑组织各组Glu含量对比;
图10B为小鼠脑组织各组BDNF含量对比;
图10C为小鼠脑组织各组5-HT含量对比;
图10D为小鼠脑组织各组DA含量对比;
图10E为小鼠脑组织各组NE含量对比;
图11为化合物W-1的MS谱图;
图12为化合物W-1的1H-NMR谱图;
图13为化合物W-1的13C-NMR谱图;
图14为化合物W-2的MS谱图;
图15为化合物W-2的1H-NMR谱图;
图16为化合物W-2的13C-NMR谱图;
图17为化合物W-3的MS谱图;
图18为化合物W-3的1H-NMR谱图;
图19为化合物W-3的13C-NMR谱图;
图20为化合物W-4的MS谱图;
图21为化合物W-4的1H-NMR谱图;
图22为化合物W-4的13C-NMR谱图;
图23为化合物W-5的MS谱图;
图24为化合物W-5的1H-NMR谱图;
图25为化合物W-5的13C-NMR谱图;
图26为化合物W-6的MS谱图;
图27为化合物W-6的1H-NMR谱图;
图28为化合物W-6的13C-NMR谱图;
图29为化合物W-7的MS谱图;
图30为化合物W-7的1H-NMR谱图;
图31为化合物W-7的13C-NMR谱图;
图32为化合物W-8的MS谱图;
图33为化合物W-8的1H-NMR谱图;
图34为化合物W-8的13C-NMR谱图。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚,下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地说明,显然,所描述的实施例是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。下述试验例中所述实验方法,如无特殊说明,均为常规方法;所述试剂和材料,如无特殊说明,均可从商业途径获得。
实施例1
化合物W-3:甲基(1S,4aR)-1-乙氧基-7-((4-((3,5,6-三甲基吡嗪-2-基)甲基)哌嗪-1-基)甲基)-1,4a,5,7a-四氢环戊[c]吡喃-4-羧酸酯盐酸盐
合成方法如下:
步骤1:1-乙氧基-7-(羟甲基)-1,4a,5,7a-四氢环戊[c]吡喃-4-羧酸甲酯的合成
在50mL圆底烧瓶中依次加入京尼平(500.0mg,2.21mmol)、对甲苯磺酸一水合物(504.5mg,2.65mmol)、乙醇3.87mL,80℃反应1h后,TLC[V(石油醚):V(乙酸乙酯)=1:1为展开剂]显示反应基本完全,加入20mL饱和NaHCO3水溶液淬灭反应,并用二氯甲烷(3×50mL)萃取,合并有机相,有机相用饱和食盐水(3×50mL)洗涤,无水硫酸钠干燥,减压浓缩得黄色油状物,经硅胶柱层析纯化[V(石油醚):V(乙酸乙酯)=4:1为洗脱剂]得淡黄色油状物497.8mg,产率88.6%。
步骤2:1-乙氧基-7-((甲基磺酰基)氧基)-1,4a,5,7a-四氢环戊基[c]吡喃-4-羧酸甲酯的合成
在50mL圆底烧瓶中依次加入7-(羟甲基)-1-乙氧基-1,4a,5,7a-四氢环戊二烯并[c]吡喃-4-羧酸甲酯(200.0mg,786.5μmol)、二氯甲烷5mL、Et3N(0.2mL,1.4mmol),冰浴条件下缓慢滴加甲磺酰氯(0.060mL,786.5μmol),滴完后,在冰浴条件下继续反应2h,TLC[V(石油醚):V(乙酸乙酯)=1:1为展开剂]显示反应基本完全,10%稀盐酸淬灭反应,并用二氯甲烷(3×50mL)萃取,合并有机相,有机相用饱和食盐水(3×50mL)洗涤,无水硫酸钠干燥,浓缩得淡黄色油状物250mg,产率为95.6%。
步骤3:2,3,5-三甲基-6-(哌嗪-1-基甲基)对吡嗪的合成
在100mL圆底烧瓶中加入川芎嗪(4.00g,29.37mmol),用30mL冰醋酸溶解,然后加入30%H2O2(5.93mL,58.74mmol),70℃反应4h,TLC[V(石油醚):V(乙酸乙酯)=1:1为展开剂]显示反应基本完全,加入20mL饱和NaHCO3水溶液淬灭反应,并用二氯甲烷(3×50mL)萃取,合并有机相,有机相用饱和食盐水(3×50mL)洗涤,无水硫酸钠干燥,减压浓缩得2.479g白色固体,产率为75%。
在100mL圆底烧瓶中加入2,3,5,6-川芎嗪1-氧化物(2.50g,16.41mmol),加入16mL乙酸溶解,然后加入10ml乙酸酐,110℃反应6h,TLC[V(石油醚):V(乙酸乙酯)=1:1为展开剂]显示反应基本完全,加入20mL饱和NaHCO3水溶液淬灭反应,并用二氯甲烷(3×50mL)萃取,合并有机相,有机相用饱和食盐水(3×50mL)洗涤,无水硫酸钠干燥,滤液减压浓缩得2.92g棕色油状液体,产率为91.5%。
在100mL圆底烧瓶中加入(3,5,6-三甲基吡嗪-2-基)乙酸甲酯(2.92g,15.03mmol),用15mL无水乙醇溶解,然后加入NaOH(3.61g,90.20mmol)水溶液,75℃反应2h,TLC[V(石油醚):V(乙酸乙酯)=1:1为展开剂]显示反应基本完全,加入20mL蒸馏水淬灭反应,并用二氯甲烷(3×50mL)萃取,合并有机相,有机相用饱和食盐水(3×50mL)洗涤,无水硫酸钠干燥,减压浓缩得1.587g黄色固体,产率为69.4%。
在50mL圆底烧瓶中加入(3,5,6-三甲基吡嗪-2-基)甲醇(1.59g,10.43mmol),用10mL无水二氯甲烷溶解,然后在冰浴下加入SOCl2(1.86g,15.64mmol),室温反应3h,TLC[V(石油醚):V(乙酸乙酯)=1:1为展开剂]显示反应基本完全,加入20mL冰水淬灭反应,并用二氯甲烷(3×50mL)萃取,合并有机相,有机相用饱和食盐水(3×50mL)洗涤,无水硫酸钠干燥,减压浓缩得淡黄色油状液体,经硅胶柱层析纯化[V(石油醚):V(乙酸乙酯)=6:1为洗脱剂]得无色油状液体900.0mg,产率为50.6%。
在50mL圆底烧瓶中加入无水哌嗪(2.73g,31.65mmol),用15mL二氯甲烷溶解,0℃条件下缓慢滴加2-(溴甲基)-3,5,6-三甲基吡嗪(900.0mg,5.27mmol)的二氯甲烷溶液,滴完后,在冰浴条件下继续反应6h,TLC[V(石油醚):V(乙酸乙酯)=1:1为展开剂]显示反应基本完全,加入20mL蒸馏水淬灭反应,并用二氯甲烷(3×50mL)萃取,合并有机相,有机相用饱和食盐水(3×50mL)洗涤,无水硫酸钠干燥,减压浓缩得白色固体粗产物,经硅胶柱层析纯化[V(二氯甲烷):V(甲醇)=10:1为洗脱剂]得432.0mg白色固体,产率为37.2%。
步骤4:1-乙氧基-7-((甲磺酰基)氧基)-1,4a,5,7a-四氢环戊[c]吡喃-4-羧酸甲酯的合成
取250mL干燥的单口烧瓶,依次加入1-乙氧基-7-((甲磺酰基)氧基)甲基)-1,4a,5,7a-四氢环戊[c]吡喃-4-羧酸甲酯(361.22mg,1.13mmol),2-(哌嗪甲基)-3,5,6-三甲基吡嗪(500mg,2.27mmol),K2CO3(313.65mg,2.27mmol),用15mLDMF溶解,常温反应4h,TLC[V(石油醚):V(乙酸乙酯)=1:1为展开剂]显示反应基本完全,加入20mL蒸馏水淬灭反应,并用二氯甲烷(3×50mL)萃取,合并有机相,有机相用饱和食盐水(3×50mL)洗涤,无水硫酸钠干燥,减压浓缩,经硅胶柱层析纯化[V(二氯甲烷):V(甲醇)=20:1为洗脱剂]得87.2mg黄色固体,产率为58%。m.p.273.8~274.9℃。1H NMR(300MHz,Chloroform-d)δ7.52(s,1H,3-H),5.74(s,1H,7-H),4.71(d,J=7.3Hz,1H,1-H),3.75(s,4H,CH2),3.63(d,J=7.1Hz,3H,COOCH3),3.19(q,J=8.5,8.1Hz,2H,CH2),3.04(d,J=14.2Hz,1H,9-H),2.85(dd,J=15.6,9.1Hz,1H,5-H),2.71–2.63(m,2H,CH2),2.60(s,8H,piperazine-H),2.52(s,9H,CH3),1.26(d,J=7.1Hz,3H,CH3).13C NMR(101MHz,Chloroform-d)δ168.01,152.31,149.87,149.47,148.03,129.52,110.95,101.29,65.21,61.76,57.93,53.22,51.14,46.60,38.78,35.11,21.55,21.45,20.97,15.13.ESI-HRMS(m/z):[M+H]+calcd for C25H36N4O4:457.2809found457.2804。
实施例2
化合物W-1:1-甲氧基-7-((4-(4-甲氧基苄基)哌嗪-1-基)甲基)-1,4a,5,7a-四氢环戊烷[c]吡喃-4-羧酸甲酯盐酸盐
参照实施例1步骤1~2的方法合成甲基1-甲氧基-7-((甲基磺酰基)氧基)-1,4a,5,7a-四氢环戊基[c]吡喃-4-羧酸盐,将其与1-(4-甲氧基苄基)哌嗪为原料,参照实施例1步骤4的方法制得W-1白色固体,m.p.226.8-227.9℃。1H NMR(300MHz,D2O)δ7.39(s,1H,3-H),7.32(d,J=8.1Hz,2H,ArH),6.94(d,J=8.5Hz,2H,ArH),6.18(brs,1H,7-H),5.11(d,J=2.7Hz,1H,1-H),4.31(brs,1H,ArCH2-Ha),4.29(s,3H,ArOCH3),4.02~3.82(m,1H,ArCH2-Hb),3.71(s,6H,COOCH3,OCH3),3.65–3.47(m,8H,piperazine-H),3.46(s,2H,10-H),3.41(brs,1H,9-H),3.30(brs,1H,5-H),3.18~2.98(m,1H,6-Ha),2.71(dd,J=17.6,7.9Hz,1H,6-Hb).13C NMR(75MHz,D2O)δ:169.9,160.5,151.8,143.1,132.9,128.6,119.7,114.7,111.3,99.1,59.9,56.5,55.4,51.8,47.8,47.7,45.8,38.7,33.0;IR(KBr,cm-1)υ:2975.1,2904.6,2834.2,1708.9,1613.3,1517.1,1465.2,1439.6,1381.9,1253.2,1182.5,1074.4,1032.0,848.9;ESI-Mass for C24H32N2O5:m/z 429.25(M++H)。
实施例3
化合物W-2:1-甲氧基-7-((4-(吡啶-3-基甲基)哌嗪-1-基)甲基)-1,4a,5,7a-四氢环戊烷[c]吡喃-4-羧酸甲酯盐酸盐
参照实施例1步骤1~2的方法合成甲基1-甲氧基-7-((甲基磺酰基)氧基)-1,4a,5,7a-四氢环戊基[c]吡喃-4-羧酸盐,将其与1-(吡啶-3-基甲基)哌嗪为原料,参照实施例1步骤4的方法制得W-2白色固体,m.p.197.1-198.3℃。1H NMR(300MHz,D2O)δ8.98(s,1H,Pyridine-H),8.84(d,J=5.7Hz,1H,Pyridine-H),8.74(d,J=8.2Hz,1H,Pyridine-H),8.11(t,J=7.8Hz,1H,Pyridine-H),7.45(s,1H,3-H),6.23(d,J=18.1Hz,1H,7-H),4.55(d,J=8.3Hz,1H,1-H),3.97(brs,2H,CH2),3.83~3.61(m,8H,piperazine-H),3.59(s,3H,COOCH3),3.46(s,2H,10-CH2),3.30(s,3H,OCH3),3.09(t,J=8.1Hz,1H,9-H),2.79(ddd,J=11.1,8.7,2.7Hz,1H,5-H),2.70~2.56(m,1H,6-Ha),2.04(dd,J=17.1,9.4Hz,1H,6-Hb).13C NMR(75MHz,D2O)δ:169.8,153.1,151.7,149.7,143.4,129.8,129.1,128.2,110.4,102.5,99.1,57.3,56.48,56.0,55.6,55.1,51.8,48.6,46.0,38.7,35.0,32.9.IR(KBr,cm-1)υ:2978.7,2956.3,2824.1,1701.8,1633.2,1532.7,1436.2,1460.2,1379.3,1289.1,1179.3,1083.5,946.7,891.0,686.1;ESI-Mass for C22H29N3O4:m/z 400.22(M++H).
实施例4
化合物W-4:1-异丙氧基-7-((4-(4-甲氧基苄基)哌嗪-1-基)甲基)-1,4a,5,7a-四氢环戊烷[c]吡喃-4-羧酸甲酯盐酸盐
参照实施例1步骤1~2的方法合成甲基1-异丙氧基-7-((甲基磺酰基)氧基)-1,4a,5,7a-四氢环戊基[c]吡喃-4-羧酸盐,将其与1-(4-甲氧基苄基)哌嗪为原料,参照实施例1步骤4的方法制得W-4白色固体,m.p.225.3-226.5℃。1H NMR(300MHz,D2O)δ7.45(s,1H,3-H),7.38(d,J=8.7Hz,2H,ArH),6.97(d,J=8.7Hz,2H,ArH),6.32(s,1H,7-H),4.68(d,J=8.4Hz,1H,1-H),4.35(s,2H,CH2),4.02(dd,J=12.7,6.3Hz,2H,10-CH2),3.94~3.85(m,1H,CH),3.75(s,3H,COOCH3),3.62(s,3H,OCH3),3.61~3.45(m,8H,piperazine-H),3.12(q,J=8.4Hz,1H,6-Ha),2.82(ddd,J=11.4,8.7,2.7Hz,1H,5-H),2.55(t,J=8.0Hz,1H,9-H),2.07(dd,J=17.4,9.4Hz,1H,6-Hb),1.17(d,J=6.0Hz,3H,CH3),1.07(d,J=6.0Hz,3H,CH3).13C NMR(75MHz,D2O)δ170.0,160.5,153.4,143.1,132.9,130.0,119.7,114.8,110.3,100.1,73.8,72.9,60.0,55.6,55.4,51.8,48.3,47.8,46.2,38.8,35.4,33.8,22.4,20.9;IR(KBr,cm-1)υ:2972.1,2830.7,1712.0,1631.0,1518.0,1437.8,1382.9,1251.4,1157.9,1104.3,945.2,857.9;ESI-Mass for C26H36N2O5:m/z 457.21(M++H)。
实施例5
化合物W-5:1-甲氧基-7-((4-(4-甲基苄基)哌嗪-1-基)甲基)-1,4a,5,7a-四氢环戊烷[c]吡喃-4-羧酸甲酯盐酸盐
参照实施例1步骤1~2的方法合成甲基1-甲氧基-7-((甲基磺酰基)氧基)-1,4a,5,7a-四氢环戊基[c]吡喃-4-羧酸盐,将其与1-(4-甲基苄基)哌嗪为原料,参照实施例1步骤4的方法制得W-5白色固体,m.p.231.3-232.4℃。1HNMR(300MHz,D2O)δ:7.40(s,1H,3-H),7.29(d,J=8.1Hz,2H,ArH),7.23(d,J=8.1Hz,2H,ArH),6.18(brs,1H,7-H),5.12(d,J=3.0Hz,1H,1-H),4.33(s,2H,CH2),3.97(d,J=13.8Hz,1H,10-Ha),3.88(d,J=13.8Hz,1H,10-Hb),3.60(s,3H,COOCH3),3.58~3.35(m,8H,piperazine-H),3.30(s,3H,OCH3),3.09(t,J=8.1Hz,2H,6-H),2.76~2.68(m,1H,9-H),2.24(s,3H,CH3),2.12~2.05(m,1H,5-H).13CNMR(75MHz,D2O)δ:169.8,151.7,143.2,141.3,131.1,130.0,128.5,124.3,111.3,99.1,60.2,56.4,55.1,51.8,48.3,47.8,45.8,38.7,33.0,20.3;IR(KBr,cm-1)υ:2981.3,2950.6,1712.5,1637.4,1433.6,1368.0,1315.7,1288.9,1271.8,1197.4,1167.2,1074.0,1024.6,954.1,890.0;ESI-Mass for C24H32N2O4:m/z 413.30(M++H)。
实施例6
化合物W-6:1-异丙氧基-7-((4-(3-甲氧基苄基)哌嗪-1-基)甲基)-1,4a,5,7a-四氢环戊烷[c]吡喃-4-羧酸甲酯盐酸盐
参照实施例1步骤1~2的方法合成甲基1-异丙氧基-7-((甲基磺酰基)氧基)-1,4a,5,7a-四氢环戊基[c]吡喃-4-羧酸盐,将其与1-(3-甲氧基苄基)哌嗪为原料,参照实施例1步骤4的方法制得W-6白色固体,m.p.208.0-209.2℃。1H NMR(300MHz,D2O)δ7.44(s,1H,3-H),7.36(t,J=8.3Hz,1H,ArH),7.07~6.97(m,3H,ArH),6.26(s,1H,7-H),4.67(d,J=8.4Hz,1H,1-H),4.38(s,2H,CH2),4.03(dd,J=13.5,7.8Hz,2H,10-CH2),3.96~3.85(m,1H,CH),3.74(s,3H,COOCH3),3.66(s,3H,OCH3),3.61~3.47(m,8H,piperazine-H),3.11(q,J=8.4Hz,1H,6-Ha),2.81(ddd,J=11.4,8.4,2.7Hz,1H,5-H),2.55(t,J=7.5Hz,1H,9-H),2.06(dd,J=17.6,9.5Hz,1H,6-Hb),1.16(d,J=6.0Hz,3H,CH3),1.06(d,J=6.0Hz,3H,CH3).13C NMR(75MHz,D2O)δ170.0,159.4,153.4,143.3,130.8,129.9,128.8,123.8,116.6,116.2,110.3,100.1,73.8,60.3,55.6,55.5,51.8,48.3,48.1,46.2,38.8,35.4,23.7,22.4,20.9;IR(KBr,cm-1)υ:2973.2,2947.8,2883.6,2831.9,1706.6,1629.8,1371.3,1437.1,1270.1,1160.3,1101.6,940.3,888.5,787.1,697.6;ESI-Mass for C26H36N2O5:m/z457.16(M++H)。
实施例7
化合物W-7:1-甲氧基-7-((4-(3-甲基苄基)哌嗪-1-基)甲基)-1,4a,5,7a-四氢环戊烷[c]吡喃-4-羧酸甲酯盐酸盐
参照实施例1步骤1~2的方法合成甲基1-甲氧基-7-((甲基磺酰基)氧基)-1,4a,5,7a-四氢环戊基[c]吡喃-4-羧酸盐,将其与1-(3-甲基苄基)哌嗪为原料,参照实施例1步骤4的方法制得W-7白色固体,m.p.230.5-231.6℃。1H NMR(300MHz,D2O)δ:7.48(s,1H,3-H),7.36~7.18(m,4H,ArH),6.28(brs,1H,7-H),4.56(d,J=8.2Hz,1H,1-H),4.36(s,2H,CH2),4.08~3.90(m,2H,10-CH2),3.63(s,3H,COOCH3),3.61~3.50(m,8H,piperazine-H),3.48(s,3H,OCH3),3.12(dd,J=15.9,7.5Hz,1H,9-H),2.81(dd,J=17.0,8.5Hz,1H,6-Ha),2.64~2.49(m,1H,6-Hb),2.27(s,3H,CH3),2.06(dd,J=16.5,8.7Hz,1H,5-H).13C NMR(75MHz,D2O)δ:170.00,153.10,142.64,139.77,131.72,131.23,129.86,129.33,128.11,127.31,110.44,102.57,60.43,57.30,55.65,51.82,48.33,47.94,45.98,38.75,35.06,20.33.IR(KBr,cm-1)υ:2977.1,2947.6,2629.5,1708.2,1627.6,1438.2,1369.9,1287.6,1178.7,1107.6,957.8,939.5,890.1,766.6,700.3.ESI-Mass for C24H32N2O4:m/z 413.26(M++H)。
实施例8
化合物W-8:1-异丙氧基-7-((4-(4-甲基苄基)哌嗪-1-基)甲基)-1,4a,5,7a-四氢环戊烷[c]吡喃-4-羧酸甲酯盐酸盐
参照实施例1步骤1~2的方法合成甲基1-异丙氧基-7-((甲基磺酰基)氧基)-1,4a,5,7a-四氢环戊基[c]吡喃-4-羧酸盐,将其与1-(4-甲基苄基)哌嗪为原料,参照实施例1步骤4的方法制得W-8白色固体,mp 223.1-224.4℃。1H NMR(300MHz,D2O)δ7.42(s,1H,3-H),7.28(d,J=8.0Hz,2H,ArH),7.21(d,J=8.0Hz,2H,ArH),6.28(s,1H,7-H),4.67(d,J=8.4Hz,1H,1-H),4.33(s,2H,CH2),4.00(d,J=14.1Hz,1H,10-Ha),3.93(d,J=14.1Hz,1H,10-Hb),3.79(s,3H,COOCH3),3.63~3.39(m,8H,piperazine-H),3.14(m,1H,CH),2.77(ddd,J=11.1,8.1,2.7Hz,1H,5-H),2.52(t,J=8.1Hz,1H,9-H),2.22(s,3H,CH3),2.20~2.16(m,1H,6-Ha),2.04(dd,J=17.7,9.4Hz,1H,6-Hb),1.13(d,J=6.3Hz,3H,CH3),1.02(d,J=6.3Hz,3H,CH3).13C NMR(75MHz,D2O)δ170.0,153.4,143.3,141.3,131.2,130.0,124.2,110.3,100.1,95.6,73.8,60.2,55.6,51.8,47.9,47.0,46.2,38.8,35.4,23.7,22.4,20.9,20.4;IR(KBr,cm-1)υ:3031.5,2972.9,2945.6,2873.2,1712.3,1630.8,1436.1,1384.2,1371.4,1284.7,1242.9,1157.0,1103.4,941.9,849.0;ESI-Mass for C26H36N2O4:m/z 441.26(M++H)。
试验例
一、本发明京尼平衍生物对谷氨酸诱导的HT-22细胞损伤保护活力检测
选取小鼠海马神经元HT-22细胞进行消化、收集和离心,对细胞进行重悬并计数后,以2×104/孔的HT-22细胞接种于96孔细胞培养板,每孔细胞悬液的体积为100μL,每组设置6个复孔,设置正常对照组、模型组与药物干预组,将细胞培养板放置于37℃、5%CO2的培养箱中培养24h,24h后将小鼠海马神经元HT-22细胞进行Glu诱导建立损伤模型。用移液枪吸去96孔细胞培养板各孔中的上清后再对细胞分组处理:①正常对照组正常换液培养24h;②模型组加入150μL的5mM Glu药液后将细胞放置于37℃、5%CO2培养箱中孵育24h,孵育结束前4h,再向孔板中的每孔加入10μL CCK-8溶液;③给药组分别加入150μL含有5mM终浓度的Glu的化合物药液,化合物终浓度分为0.1μM、1μM和10μM,将孔板放置于37℃、5%CO2培养箱中孵育24h,孵育结束前4h,再向孔板的每孔中加入10μL CCK-8溶液;④氟西汀组加入150μL的0.1μM、1μM和10μM不同浓度的Fluoxetine药液放置于37℃、5%CO2培养箱孵育24h,孵育结束前4h,向每孔中加入10μL CCK-8溶液;加入CCK-8试剂后将96孔板包裹锡纸置于培养箱孵育4h后,放入多功能酶标仪检测570nm波长下每孔OD值,实验重复3次。
结果如表1显示,体外HT-22细胞保护活性实验表明,本发明各实施例制备的京尼平衍生物均对Glu诱导损伤的HT-22细胞具有保护活性,结果呈浓度依赖性增加,并且所筛选的京尼平衍生物体外神经细胞保护活性均优于对照药京尼平。其中,化合物W-3表现出较好的神经细胞保护活性,在10μM时可显著性提高Glu诱导的小鼠海马神经元HT-22细胞损伤模型中细胞存活率(细胞存活率75.06±1.12),结果优于阳性对照氟西汀(细胞存活率74.84±0.92)和对照药京尼平(57.03±1.26)。
表1京尼平衍生物对谷氨酸(Glu)诱导的HT-22细胞损伤的影响(n=6)
注:空白对照组细胞存活率(100±2.88),药物处理组与模型组对照组相比,*p<0.05,**p<0.01;药物处理组与阳性药氟西汀组相比,#p<0.05,##p<0.01。
二、本发明京尼平衍生物对强迫游泳实验及悬尾实验小鼠的不动时间的影响
实验动物为雄性ICR小鼠(购自安徽医科大学,动物生产许可证号scxk(皖)-2017-001);京尼平衍生物(W-3和W-6)溶解于DMSO(购买于上海碧云天生物科技有限公司),在使用前用5%DMSO的9%Tween80/生理盐水溶液稀释成所需浓度腹腔注射给药;氟西汀(购自阿拉丁生化科技有限公司)。
实验流程:实验小鼠于12h/12h光暗周期中饲养,自由饮水,适应饲养5天后给予药物;小鼠随机分为8组,每组6只;药物溶媒为9%Tween80的生理盐水溶液配制成的5%DMSO溶液;8组动物分别为(a)空白对照组;(b)W-6低中高剂量组(1mg/kg、10mg/kg和30mg/kg);(c)W-3低中高剂量组(1mg/kg、10mg/kg和30mg/kg);(d)氟西汀30mg/kg组(溶于生理盐水)。每组给予单次腹腔注射,给药后1h后进行5min强迫游泳测试以及悬尾实验并记录不动时间。
强迫游泳测试:强迫游泳装置由透明有机玻璃制成(高30cm,直径15cm,水深15±1cm,水温25±1℃),小鼠于测试前一天适应15min;测试时,将小鼠置于其中,记录6min内后4min小鼠漂浮不动时间。漂浮不动时间定义为小鼠微蜷躯体、呈漂浮状态的时间(强迫游泳实验为经典抗抑郁药物筛选模型,有效抗抑郁药物可以明显降低小鼠在强迫游泳中的不动时间)。
悬尾试验:实验操作参照Lucien Steru的方法。采用实验室自制悬尾箱,每个悬尾箱(25×25×25cm)顶部固定一小夹子,将小鼠用胶布粘贴距尾尖2cm处,然后悬挂6min,并视频录像。之后采用软件或人工分析小鼠在6min悬尾期间后4min的不动时间。
统计分析:采用GraphPadprism 9.0统计分析数据,计量资料均以平均值±标准差表示。对数据进行正态性检验和方差齐性检验,满足条件的计量资料采用t检验(两组间比较)或方差分析(多组间比较),方差不齐时采用Dunnett’s-T3进行两两比较。数据不符合正态分布,则采用非参数检验中的Kruskal-Wills H检验。p≤0.05为有统计学意义。
强迫游泳实验结果如图1所示,与空白对照组(160.0±12.98s)相比,W-610mg/kg单次给药组(115.3±25.14s)和氟西汀30mg/kg单次给药组(112.2±19.15s)小鼠强迫游泳不动时间显著降低(p<0.05),W-31mg/kg单次给药组(84.5±24.93s)和W-310mg/kg单次给药组(82±17.61s)以及W-330mg/kg单次给药组(79.33±14.34s)小鼠强迫游泳不动时间极显著降低(p<0.01)。
悬尾试验结果如图2所示,与溶媒对照组(153.5±17.36s)相比,W-61mg/kg单次给药组(93.83±20.85s)、W-610mg/kg单次给药组(63.67±14.61s)、W-31mg/kg单次给药组(107.2±17.00s)、W-310mg/kg单次给药组(60.83±16.87s)、W-330mg/kg单次给药组(56.33±10.27s)和氟西汀30mg/kg单次给药组(76±20.14s)小鼠悬尾实验不动时间极显著降低(p<0.01)。W-6和W-3以及阳性药氟西汀与模型对照组相比,各浓度组均能提高小鼠不动时间(p≤0.01),表现出一定的抗抑郁活性。
三、本发明京尼平衍生物对慢性不可预见性应激所致小鼠抑郁样行为的影响
实验流程如图3所示,3月龄的雄性C57BL/6J小鼠(购自杭州子源实验动物科技有限公司,动物使用许可证号:SCXK(苏)2018-0008)。实验开始前于安徽中医药大学动物房适应性饲养一周,饲养室温度和湿度分别为25±2℃和55±5%,12h昼夜更替,动物实验均经安徽中医药大学实验动物伦理委员会(中国安徽合肥)批准。适应饲养5天后给予慢性不可预见性应激(CUMS);小鼠随机分为6组,每组10只;药物溶媒为生理盐水;6组动物分别为(a)正常组:生理盐水(i.p);(b)模型组:生理盐水(i.p)+CUMS;(c)W-30.1mg/kg+CUMS组;(d)W-31mg/kg+CUMS组;(e)W-310mg/kg+CUMS组;(f)氟西汀20mg/kg组+CUMS。分组结束后,给予CUMS的组别进行为期28天的CUMS,空白对照组不做行为干预。CUMS结束后,每组给予单次相应药物腹腔注射,CUMS结束后第一天进行糖水偏爱测试,第二天进行旷场试验,第三天进行强迫游泳测试,第7天再次进行糖水偏爱测试。
CUMS流程:小鼠每天随机接受两种不同慢性不可预见性应激,持续28天。所用应激方式如表2所述。
表2慢性不可预见性轻度应激(CUMS)方法
旷场试验:采用旷场实验视频分析系统Supermaze(上海欣软信息科技),把小鼠放置在等待测试的房间中先适应大约10min后,放入旷场立体室(400mm(W)×400mm(H)×400mm(D))的中央位置,允许小鼠自由移动5min,录像结束以后由系统自动进行小鼠的轨迹分析,得出5min内的总路程和平均速度。每次旷场试验后用10%乙醇溶液清理去味再进行下一次旷场试验。
强迫游泳测试:强迫游泳装置由透明有机玻璃制成(高30cm,直径15cm,水深15±1cm,水温25±1℃),小鼠于测试前一天适应15min;测试时,将小鼠置于其中,记录6min内后4min小鼠漂浮不动时间。漂浮不动时间定义为小鼠微蜷躯体、呈漂浮状态的时间(强迫游泳实验为经典抗抑郁药物筛选模型,有效抗抑郁药物可以明显降低小鼠在强迫游泳中的不动时间)。
悬尾试验:实验操作参照Lucien Steru的方法。采用实验室自制悬尾箱,每个悬尾箱(25×25×25cm)顶部固定一小夹子,将小鼠用胶布粘贴距尾尖2cm处,然后悬挂6min,并视频录像。之后采用软件或人工分析小鼠在6min悬尾期间后4min的不动时间。
统计分析:采用GrphPadprism 9.0统计分析数据,计量资料均以平均值±标准差 表示。对数据进行正态性检验和方差齐性检验,满足条件的计量资料采用t检验(两组间比较)或方差分析(多组间比较),方差不齐时采用Dunnett’s-T3进行两两比较。数据不符合正态分布,则采用非参数检验中的Kruskal-Wills H检验。p≤0.05为有统计学意义。
旷场试验结果如图4A~4B所示,旷场实验是通过测量小鼠的移动总路程和平均移动速度来评价小鼠的自主活动能力。结果显示:与正常组相比,CUMS组小鼠的移动总距离和移动平均速度相较于正常组明显减少(p<0.01),盐酸氟西汀和京尼平衍生物给药组能够延长小鼠的活动总距离和移动的平均速度(p<0.5)。
强迫游泳实验结果如图5所示,强迫游泳实验是通过对小鼠的强迫游泳时间以及动作进行记录和评分。与正常组相比,CUMS组不动时间延长(p<0.01),实验过程中可观察到:CUMS组小鼠漂浮在水面不动时间延长。京尼平衍生物给药组和盐酸氟西汀组小鼠在水中漂浮不动时间减小(p<0.01),结果如图6。
悬尾试验结果如图6所示,悬尾实验是通过对小鼠的悬挂时间以及动作进行记录和评分。与正常组相比,CUMS组悬挂评分降低(p<0.01),实验过程中可观察到:CUMS组小鼠倒置悬挂身躯不动时间延长。京尼平衍生物给药组和盐酸氟西汀组小鼠所获倒置悬挂不动时间减小(p<0.01)。
上述实验表明,单次给予W-3对抑郁症产生治疗作用,并且该作用起效快、维持时间七天或以上。
四、本发明京尼平衍生物对BDNF/PKA/CREB信号通路的影响
将不同分组的样品冰上放置6孔板,用预冷PBS缓冲液洗涤3次;RNA提取液裂解:取500μL预冷的RNA提取液,提取液加入小皿,冰上放置5min使其充分裂解,然后样品转移至EP管中,混匀,室温下静置5min;加氯仿:按比例加入氯仿(提取液体积的1/5量)于另一EP管中,震荡混匀,室温下静置10min,离心15min(4℃,12000rpm);异丙醇沉淀:在上层水相中加入等体积异丙醇,混匀,室温下静置20min,离心10min(4℃,12000rpm/min);乙醇洗脱:弃上清,加入预冷的75%乙醇(无水乙醇:DEPC水=3:1)1mL,离心15min(4℃12000rpm/min);提取总RNA:弃上清,干燥10-30min,加入20μLDEPC水进行溶解沉淀,得到总RNA,置于-80℃保存;测定RNA纯度(OD260/OD280)。
qRT-PCR结果如图7所示:与正常组相比,CUMS组小鼠促进PKA、CREB、BDNF的mRNA表达水平降低(p<0.05),与CUMS组相比,化合物W-3组的mRNA表达水平升高(p<0.05),且呈剂量依赖性增加。
五、本发明京尼平衍生物对小鼠海马GluN2A、GluN2B、5-HT1A、BDNF、CREB、p-CREB以及PKA蛋白表达的影响
在众多的NMDA受体亚基中,GluN2A和GluN2B亚基对受体功能影响最大,与抑郁症、认知功能障碍等神经系统疾病关联密切。研究表明,CUMS中小鼠海马的GluN2A和GluN2B的蛋白表达明显增加。NMDA受体激活G蛋白偶联受体使cAMP浓度增加,激活胞内钙离子浓度促进CREB磷酸化,进而调节与神经元生长、存活关联的基因表达,最终达到抗抑郁的目的。5-HT1A异源受体在海马(CA1和CA3区)的神经元轴突中高度表达。激活突触后5-HT1A异源受体将引起突触后膜的超极化,抑制神经元的兴奋性。PKA-CREB信号通路的激活也能上调BDNF的表达。通过增加海马区p-CREB蛋白的表达,调节抑郁模型大鼠CERB的BDNF信号转导通路。CREB能激活BDNF的产生,BDNF也能诱导CREB的磷酸化,两者相互作用形成影响神经可塑性的重要正反馈环,发挥快速抗抑郁作用。因此,本实验利用免疫蛋白印迹法考察W-3给药后小鼠海马GluN2A、GluN2B、5-HT1A、BDNF、CREB、p-CREB以及PKA蛋白表达变化情况。
小鼠海马GluN2A和GluN2B蛋白表达水平
结果如图8A、8C、8D所示,CUMS模型组小鼠海马中GluN2A和GluN2B蛋白表达上调,给予化合物W-3干预后,上述两种蛋白的表达被下调,并且化合物W-3下调GluN2A和GluN2B蛋白表达的作用在高剂量(10mg/kg)时明显强于阳性对照氟西汀。
小鼠海马5-HT1A蛋白表达水平
结果如图8A、8B所示,CUMS模型组小鼠海马脑区5-HT1A受体蛋白表达下降,给予化合物W-3干预后,小鼠海马脑区5-HT1A受体蛋白表达提升(结果呈剂量依赖性增加),其中化合物W-3在高剂量组(10mg/kg)时干预效果优于阳性对照氟西汀。
小鼠海马BDNF蛋白表达水平
结果如图9A、9C所示,与正常组相比,模型组小鼠条带颜色浅,说明模型组小鼠海马BDNF蛋白表达水平降低,结果表明抑郁会使小鼠海马内BDNF蛋白减少;与模型组相比,给药组小鼠海马条带颜色更深,BDNF水平呈现明显上调趋势,说明新型京尼平衍生物的干预治疗能够逆转慢性应激引起的BDNF蛋白表达减少,可能通过上调海马BDNF蛋白水平发挥抗抑郁作用。
小鼠海马CREB蛋白表达水平
结果如图9A、9D所示,与正常组相比,模型组小鼠条带颜色浅,说明模型组小鼠海马CREB蛋白表达水平降低,结果表明抑郁会使小鼠海马内CREB蛋白减少;与模型组相比,给药组小鼠海马条带颜色更深,CREB水平呈现明显上调趋势,说明新型京尼平衍生物的干预治疗能够逆转慢性应激引起的CREB蛋白表达减少,可能通过上调海马CREB蛋白水平发挥抗抑郁作用。
小鼠海马PKA蛋白表达水平
结果如图9A、9B所示,与正常组相比,模型组小鼠条带颜色浅,说明模型组小鼠海马PKA蛋白表达水平降低,结果表明抑郁会使小鼠海马内PKA蛋白水平下降;与模型组相比,给药组小鼠海马条带颜色更深,PKA水平呈现明显上调趋势,说明新型京尼平衍生物的干预治疗能够逆转慢性应激引起的PKA蛋白表达下降,此外,小鼠抑郁样行为被逆转可能与通过上调海马PKA蛋白水平有关。
六、本发明京尼平衍生物对体内信号因子的影响
ELISA检测结果如图10A~10E显示,与正常组比较,模型组小鼠脑组织中Glu含量上升(p<0.05);与模型组比较,化合物W-3组小鼠脑组织Glu水平下降(p<0.05);与正常组比较,模型组小鼠脑组织5-HT、DA、NE和BDNF含量下降(p<0.05);与模型组比较,化合物W-3组小鼠脑组织5-HT、DA、NE和BDNF含量提高(p<0.05)。
以上试验结果表明,本发明京尼平衍生物具有较好的抗抑郁活性,其中部分化合物具有良好的神经细胞保护活性。部分化合物能够显著上调PKA、pCREB/CREB、BDNF以及5-HT1A蛋白表达,并下调GluN2A和GluN2B蛋白表达。其中,化合物W-3对PKA、CREB、BDNF蛋白表达的影响在10mg/kg时明显优于阳性对照氟西汀。因此,本发明京尼平衍生物可用作抑郁症的治疗药物。
综上,本发明京尼平衍生物对抑郁症具有较好的改善作用,其机制可能与调控PKA-CREB-BDNF信号通路有关,亦可通过影响5-HT1A、GluN2A和GluN2B受体靶蛋白发挥潜在的快速抗抑郁作用,使其具有良好的应用前景。
以上详细描述了本发明的优选实施方式,但是,本发明并不限于上述实施方式中的具体细节,在本发明的技术构思范围内,可以对本发明的技术方案进行多种简单变型,这些简单变型均属于本发明的保护范围。
Claims (10)
1.京尼平衍生物或其可药用盐,其特征在于,所述京尼平衍生物为式(Ia)或(Ib)所示的化合物:
其中:X、Y各自独立的代表CH或N;
Z代表O或N-R2;
R1、R2各自独立的代表C1~C3直链或支链烷基;
R3代表H、C1~C6烷基、卤素、C1~C3烷氧基、卤代烷基或硝基中的任意一种;
n代表0、1、2或3。
2.根据权利要求1所述京尼平衍生物或其可药用盐,其特征在于,所述Z代表O。
3.根据权利要求1所述京尼平衍生物或其可药用盐,其特征在于,所述R3代表H、C1~C3烷基、甲氧基中的任意一种。
4.根据权利要求1所述京尼平衍生物或其可药用盐,其特征在于,所述可药用盐选自盐酸盐、硫酸盐、磷酸盐、氢溴酸盐、马来酸盐、富马酸盐、枸橼酸盐、甲磺酸盐、对甲苯磺酸盐、酒石酸盐或醋酸盐中的任意一种,优选为盐酸盐。
5.根据权利要求1所述京尼平衍生物或其可药用盐,其特征在于,其选自以下W-1~W-8所示的任一化合物:
6.权利要求1~5之一所述京尼平衍生物或其可药用盐的制备方法,其特征在于,所述制备方法包括:将化合物1、化合物2a或化合物2b、反应助剂和有机溶剂混合,
常温使反应完全,所述化合物1、化合物2a、化合物2b的结构式如下:
其中:X、Y各自独立的代表CH或N;
Z代表O或N-R2;
R1、R2各自独立的代表C1~C3直链或支链烷基;
R3代表H、C1~C6烷基、卤素、C1~C3烷氧基、卤代烷基或硝基中的任意一种;
n代表0、1、2或3;
优选地,所述化合物1、化合物2a、反应助剂的摩尔比为1:1:2;所述化合物1、化合物2b、反应助剂的摩尔比为1:1:2;
优选地,所述反应助剂包括碳酸钾;
优选地,所述有机溶剂包括N,N-二甲基甲酰胺;
优选地,所述反应的时间为4h;
优选地,还包括对反应产物进行有机溶剂萃取,洗涤所得有机相并进行干燥,浓缩以及纯化。
7.一种药物组合物,其特征在于,所述药物组合物是由治疗有效量的权利要求1~5之一所述京尼平衍生物或其可药用盐和一种或多种药学上可接受的载体组成。
8.权利要求1~5之一所述京尼平衍生物或其可药用盐在制备预防或治疗抑郁症的药物中的用途。
9.权利要求1~5之一所述京尼平衍生物或其可药用盐在制备具有神经保护作用的药物中的用途。
10.根据权利要求8所述的用途,其特征在于,所述抑郁症包括轻度、中度或重度的单相抑郁症、双相抑郁症、精神性抑郁症、反应性抑郁症、继发性抑郁症、季节性抑郁症、产后抑郁症及更年期抑郁症中的任意一种。
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