CN113816936B - 倒捻子素衍生化合物及其制备方法和应用 - Google Patents
倒捻子素衍生化合物及其制备方法和应用 Download PDFInfo
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Abstract
本发明涉及一种倒捻子素衍生化合物及其制备方法和应用。所述倒捻子素衍生化合物具有如下所示结构特征。该倒捻子素衍生化合物具有较倒捻子素更佳的RORγt抑制活性,为治疗免疫性疾病的临床新药研发提供了活性更高的先导结构。
Description
技术领域
本发明涉及药物化学技术领域,特别是倒捻子素衍生化合物及其制备方法和应用。
背景技术
T细胞亚群-Th17细胞和人体自身免疫调节密切相关。在类风湿性关节炎、银屑病、炎症性肠炎、多发性硬化症、系统性红斑狼疮等免疫性疾病中,Th17的特征细胞因子IL-17表达上调,而抑制IL-17A表达和Th17细胞分化可以减轻相关免疫性疾病的发生或病变程度。Th17细胞功能抑制作为免疫性疾病治疗的重要靶标已经获得学术界及医药企业的认同和广泛关注。多个以Th17细胞和IL-17A通路为靶标的单克隆抗体药物已经获得相关国家药监部门的审批用于临床治疗中,包括诺华的Cosentyx,礼来的Taltz(ixekizumab)等IL-17A的抗体药物。
RORγt是调控Th17细胞分化的关键转录因子,抑制其活性可以阻碍Th17细胞正常分化,进而抑制自身免疫性疾病的发生和进展。在RORγt基因敲除的小鼠中,Th17细胞的分化能力显著下降,数量减少,并延缓实验性脑膜炎的进展。Th17细胞介导的其它自身免疫性疾病在RORγt基因缺失或者活性抑制情况下,同样出现疾病程度减轻或者缓解的变化,表明RORγt可以作为免疫性疾病治疗的药物研发靶标。RORγt活性抑制剂的研究已经在全球各大药企纷纷推进。但是目前只有Arrien、Vitae、GSK和Japan Tobacco等少数几家公司进入到临床1-2期实验。这表明RORγt活性抑制剂在免疫性疾病治疗中的应用仍然有待突破。
在我们的前期研究中发现倒捻子素也具有一定的RORγt抑制活性,并在狼疮性肾炎小鼠模型中表型出良好的治疗效果,可以作为免疫性疾病治疗的候选潜力药物。但是倒捻子素的成药尚不确定,其物化特性也限制了关于其的临床药物研发,药效和成药性均有待进一步提升。
发明内容
基于此,有必要提供一种倒捻子素衍生化合物。该倒捻子素衍生化合物具有较倒捻子素更佳的RORγt抑制活性,为治疗免疫性疾病的临床新药研发提供了活性更高的先导结构。
具有如下所示结构特征的倒捻子素衍生化合物:
其中,
R1选自C2~C10烯基,R2选自-H、C1~C5烷基,且R1与R2连接成环或不连接成环;
R3选自-H或C1~C10烷基,且R2、R3不同时为-H;
当R2、R3选自所述烷基时,分别独立地被至少一个R0取代,各R0独立地选自:乙酯基、羧基、-Br、-N3、-NH2或取代或未取代的C2~C5含氮杂环基。
在其中一个实施例中,所述的倒捻子素衍生化合物具有如下式(I-1)所示结构特征:
在其中一个实施例中,
R2选自C2~C4烷基;
R3选自C2~C4烷基;
在其中一个实施例中,所述的倒捻子素衍生化合物具有如下式(I-2)所示结构特征:
在其中一个实施例中,R3选自-H或C2~C8烷基;
当R3选自所述烷基时,被至少一个R0取代,各R0独立地选自:乙酯基、羧基、-Br、-N3或-NH2。
在其中一个实施例中,所述的倒捻子素衍生化合物选自如下化合物1~9:
本发明还提供所述的倒捻子素衍生化合物的制备方法,包括如下步骤:
于式A所示化合物的2-C位点的-OH和/或12-C位点的-OH进行取代反应,引入R3和/或R2;R1与R2进行或不进行分子内成环反应;R2、R3的含义同上所述。
在其中一个实施例中,
当R2与R3相同,且均不为-H时,所述制备方法包括如下步骤:
于α-倒捻子素的2-C位点的-OH和12-C位点的-OH进行取代反应,引入R2和R3;
当R2与R3不相同,且均不为-H时,所述制备方法包括如下步骤:
对α-倒捻子素的2-C位点的-OH和12-C位点的-OH中的一个-OH上引入保护基团,然后进行取代反应,于另一个的-OH上引入相应的R2或R3,制备中间体;
对所述中间体进行脱保护基团处理,然后进行取代反应,引入相应的R2或R3;
当R2为-H时,所述制备方法包括如下步骤:
α-倒捻子素在氧化剂的作用下进行分子内成环反应,制备化合物B;
于化合物B的2-C位点的-OH进行取代反应,引入R3;
当R3为-H时,所述制备方法包括如下步骤:
于α-倒捻子素的12-C位点的-OH进行取代反应,引入R2;
R2、R3的含义同上所述。
本发明还提供如上所述的倒捻子素衍生化合物在制备具有治疗Th17细胞活性失调介导的免疫性疾病功效的药物中的应用。
本发明还提供如上所述的倒捻子素衍生化合物在制备RORγt活性抑制剂中的应用。
与现有技术相比较,本发明具有如下有益效果:
本发明在倒捻子素的基础上进行特定的结构修饰,获得的倒捻子素衍生化合物具有更高的RORγt抑制活性,有效提升了药效和成药性,为治疗免疫性疾病的临床新药研发提供了活性更高的先导结构。
附图说明
图1为化合物1的1H NMR图谱;
图2为化合物1的13C NMR图谱;
图3为化合物2的1H NMR图谱;
图4为化合物2的13C NMR图谱;
图5为化合物3的1H NMR图谱;
图6为化合物3的13C NMR图谱;
图7为化合物4的1H NMR图谱;
图8为化合物4的13C NMR图谱;
图9为化合物5的1H NMR图谱;
图10为化合物5的13C NMR图谱;
图11为化合物6的1H NMR图谱;
图12为化合物6的13C NMR图谱;
图13为化合物8的1H NMR图谱;
图14为化合物8的13C NMR图谱;
图15为化合物9的1H NMR图谱;
图16为化合物9的13C NMR图谱;
图17为化合物的体外Th17细胞抑制活性实验结果;
图18为化合物对牛皮癣小鼠模型治疗的临床病理打分结果;
图19为化合物对牛皮癣小鼠模型的真皮层炎症细胞浸润计数结果;
图20为化合物对牛皮癣小鼠模型的角质层增生厚度结果;
图21为化合物对牛皮癣小鼠模型的腹股沟淋巴结和脾脏中的Th17细胞总数结果。
具体实施方式
以下结合具体实施例对本发明的倒捻子素衍生化合物及其制备方法和应用作进一步详细的说明。本发明可以以许多不同的形式来实现,并不限于本文所描述的实施方式。相反地,提供这些实施方式的目的是使对本发明公开内容理解更加透彻全面。
除非另有定义,本文所使用的所有的技术和科学术语与属于本发明的技术领域的技术人员通常理解的含义相同。本文中在本发明的说明书中所使用的术语只是为了描述具体的实施例的目的,不是旨在于限制本发明。
本文所使用的术语“烯基”是指包含具有至少一个不饱和部位,即碳-碳sp2双键的正碳原子、仲碳原子、叔碳原子或环碳原子的烃基,可以为直链烃基、也可以为支链烃基。包含该术语的短语,例如,“C2~C10烯基”是指包含2~10个碳原子的烯基,每次出现时,可以互相独立地为C2烯基、C3烯基、C4烯基、C5烯基、C6烯基、C7烯基、C8烯基、C9烯基、C10烯基。合适的实例包括但不限于:-CH=CH2、CH2CH=CH2、-CH2CH=CHCH3、-CH2CH=C(CH3)2、环戊烯基-C5H7、-CH2CH2CH2CH2CH=CH2。
术语“烷基”是指包含伯(正)碳原子、或仲碳原子、或叔碳原子、或季碳原子、或其组合的饱和烃。包含该术语的短语,例如,“C1~C10烷基”是指包含1~10个碳原子的烷基,每次出现时,可以互相独立地为C1烷基、C2烷基、C3烷基、C4烷基、C5烷基、C6烷基、C7烷基、C8烷基、C9烷基、C10烷基。合适的实例包括但不限于:甲基(Me、-CH3)、乙基(Et、-CH2CH3)、1-丙基(n-Pr、n-丙基、-CH2CH2CH3)、2-丙基(i-Pr、i-丙基、-CH(CH3)2)、1-丁基(n-Bu、n-丁基、-CH2CH2CH2CH3)、2-甲基-1-丙基(i-Bu、i-丁基、-CH2CH(CH3)2)、2-丁基(s-Bu、s-丁基、-CH(CH3)CH2CH3)、2-甲基-2-丙基(t-Bu、t-丁基、-C(CH3)3)、1-戊基(n-戊基、-CH2CH2CH2CH2CH3)、2-戊基(-CH(CH3)CH2CH2CH3)、3-戊基(-CH(CH2CH3)2)、2-甲基-2-丁基(-C(CH3)2CH2CH3)、3-甲基-2-丁基(-CH(CH3)CH(CH3)2)、3-甲基-1-丁基(-CH2CH2CH(CH3)2)、2-甲基-1-丁基(-CH2CH(CH3)CH2CH3)、1-己基(-CH2CH2CH2CH2CH2CH3)、2-己基(-CH(CH3)CH2CH2CH2CH3)、3-己基(-CH(CH2CH3)(CH2CH2CH3))、2-甲基-2-戊基(-C(CH3)2CH2CH2CH3)、3-甲基-2-戊基(-CH(CH3)CH(CH3)CH2CH3)、4-甲基-2-戊基(-CH(CH3)CH2CH(CH3)2)、3-甲基-3-戊基(-C(CH3)(CH2CH3)2)、2-甲基-3-戊基(-CH(CH2CH3)CH(CH3)2)、2,3-二甲基-2-丁基(-C(CH3)2CH(CH3)2)、3,3-二甲基-2-丁基(-CH(CH3)C(CH3)3和辛基(-(CH2)7CH3)。
“含氮杂环基”是指在环烷基的基础上至少一个碳原子被N原子所替代,可以为饱和环或部分不饱和环。包含该术语的短语,例如,“C2~C5含氮杂环基”是指包含2~5个碳原子的杂环基,每次出现时,可以互相独立地为C2含氮杂环基、C3含氮杂环基、C4含氮杂环基、C5含氮杂环基。
本发明的实施例提供具有如下式(I)所示结构特征的倒捻子素衍生化合物:
其中,R1选自C2~C10烯基,R2选自-H、C1~C5烷基,且R1与R2连接成环或不连接成环;
R3选自-H或C1~C10烷基,且R2、R3不同时为-H;
当R2、R3选自所述烷基时,分别独立地被至少一个R0取代,各R0独立地选自:乙酯基、羧基、-Br、-N3、-NH2或取代或未取代的C2~C5含氮杂环基。
可以理解地,上述式(I)中,当R2为H时,所述“连接成环”是指R1与R2取代的O连接成环。另外,R1与R2连接成环可以是各自的末端基团连接成环,也可以是R1或R2的中间的亚基基团与R2或R1的中间亚基基或末端基团连接成环。
本发明在倒捻子素的基础上进行特定的结构修饰,获得的倒捻子素衍生化合物具有更高的RORγt抑制活性,有效提升了药效和成药性,为治疗免疫性疾病的临床新药研发提供了活性更高的先导结构。
具体地,通过荧光素酶活性报告系统和Th17细胞分化实验验证其抑制RORγt转录活性和功能,证实所述倒捻子素衍生化合物可以抑制Th17细胞分化的关键转录因子RORγt的转录作用,能够抑制RORγt的靶基因IL17A和IL17F分子的转录表达;用流式细胞仪检测蛋白水平的IL17A在细胞内的表达也得到明显抑制;同时在银屑病模型中证实所述倒捻子素衍生化合物可以用于疾病治疗,缓解病理进程,并且体内证实Th17细胞分化和IL-17A等细胞因子表达均有效下降,表明所述倒捻子素衍生化合物通过Th17通路发挥作用。
在其中一个具体的实施例中,R2、R3结构相同。
具体地,所述的倒捻子素衍生化合物具有如下式(I-1)所示结构特征:
在其中一个具体的实施例中,式(I-1)中,R2选自C2~C4烷基;R3选自C2~C4烷基;
在其中一个具体的实施例中,式(I-1)中,R2、R3结构相同。
在其中一个具体的实施例中,式(I-1)中,R2、R3为丙基。更具体地,R3为1-丙基。
在其中一个具体的实施例中,式(I-1)中,R0取代于R2、R3的末端基团。
在其中一个具体的实施例中,式(I-1)中,R0为-Br。
具体地,所述的倒捻子素衍生化合物具有如下式(I-2)所示结构特征:
在其中一个具体的实施例中,式(I-2)中,R3选自-H或C2~C8烷基;
当R3选自所述烷基时,被至少一个R0取代,各R0独立地选自:乙酯基、羧基、-Br、-N3或-NH2。
在其中一个具体的实施例中,式(I-2)中,R3选自-H。
在其中一个具体的实施例中,式(I-2)中,R3为丙基。更具体地,R3为1-丙基。
在其中一个具体的实施例中,式(I-2)中,R3为己基。更具体地,R3为1-己基。
在其中一个具体的实施例中,式(I-2)中,R0取代于R3的末端基团。
在其中一个具体的实施例中,式(I-2)中,R0为-Br。
在其中一个具体的实施例中,式(I-2)中,R0为乙酯基。
在其中一个具体的实施例中,式(I-2)中,R0为羧基。
在其中一个具体的实施例中,式(I-2)中,R0为-N3。
在其中一个具体的实施例中,式(I-2)中,R0为-NH2。
更为具体地,所述的倒捻子素衍生化合物选自如下化合物1~9中的一种:
本发明的实施例中还提供所述的倒捻子素衍生化合物的制备方法,包括如下步骤:
于式A所示化合物的2-C位点的-OH和/或12-C位点的-OH进行取代反应,引入R3和/或R2;R1与R2进行或不进行分子内成环反应;;R2、R3的含义同上所述。
可以理解地,所述“和/或”的含义为:当R2与R3均不为-H时,需要在式A所示化合物的2-C位点的-OH和12-C位点的-OH进行取代反应,引入R2和或R3;当R2和R3中的一个为-H时,对应在式A所示化合物的2-C位点的-OH或12-C位点的-OH进行取代反应,引入不为-H的R2或R3。
在其中一个具体的实施例中,R2与R3相同,且均不为-H,所述制备方法包括如下步骤:
于α-倒捻子素的2-C位点的-OH和12-C位点的-OH进行取代反应,引入R2和R3。R2、R3的含义同上所述。
在其中一个具体的实施例中,R2与R3不相同,且均不为-H,所述制备方法包括如下步骤:
对α-倒捻子素的2-C位点的-OH和12-C位点的-OH中的一个-OH上引入保护基团,然后进行取代反应,于另一个的-OH上引入相应的R2或R3,制备中间体;
对所述中间体进行脱保护基团处理,然后进行取代反应,引入相应的R2或R3。R2、R3的含义同上所述。
可以理解地,-OH上保护基团的引入可以按照本领域的现有方式进行,如将-OH制成醚类或酯类基团,具体如硅醚基、苄醚基、甲基醚基、乙酯基等。
在其中一个具体的实施例中,R2为-H且R3不为-H,所述制备方法包括如下步骤:
α-倒捻子素在氧化剂的作用下进行分子内成环反应,制备化合物B;
于化合物B的2-C位点的-OH进行取代反应,引入R3。R3的含义同上所述。
在其中一个具体的实施例中,R3为-H且R2不为-H,所述制备方法包括如下步骤:
于α-倒捻子素的12-C位点的-OH进行取代反应,引入R2。R2的含义同上所述。
可以理解地,在上述制备方法中,为了引入R0基团,可根据所需的R0进行合适的后续反应和/或前序反应。具体地,所述后续反应包括但不限于:取代反应、水解反应、还原反应、叠氮化反应等;所述前序反应可如,先制备或直接获取包含R0,以及R2或R3的可反应化合物,再进行所述取代反应。
本发明的实施例还提供如上所述的倒捻子素衍生化合物在制备具有治疗免疫性疾病功效的药物中的应用。所述免疫性疾病为Th17细胞活性失调介导的免疫性疾病。更为具体地,所述免疫性疾病包括但不限于银屑病、特征性皮炎、多发性硬化症、类风湿性关节炎、哮喘、系统性红斑狼疮或炎症性肠道病等疾病。
本发明的实施例还提供如上所述的倒捻子素衍生化合物在制备RORγt活性抑制剂中的应用。
如下为具体的实施例,如无特别说明,实施例中采用的原料均为市售获得。
α-倒捻子素,购置于陕西昊辰生物技术有限公司。
实施例中化合物1~9的合成路线汇总如下:
制备步骤如下:
化合物1
5,9-二羟基-8-甲氧基-2,2-二甲基-7-(3-甲基丁烯-1-基)-2H,6H-吡喃酮[3,2-b]呫吨酮
将α-倒捻子素(1.22mmol,500.0mg,1eq)溶于100mL无水苯中,氮气保护搅拌下加入DDQ(1.34mmol,304.0mg,1.1eq),85℃加热回流反应3h,TLC监控反应进程。反应结束后,趁热过滤,旋干滤液,得粗产物596.5mg(黄色固体)。然后采用硅胶柱色谱(200-300目)对粗产物进行分离纯化(乙酸乙酯-石油醚5:95→10:90进行梯度洗脱),最后得到化合物1(491.3mg),黄色油状体,紫外灯254处有荧光,产率约为99.1%。
1H NMR(400MHz,CDCl3)δ13.68(s,1H),6.76(s,2H),6.68(d,J=10.0Hz,1H),6.18(s,1H),5.52(d,J=10.0Hz,1H),5.24(s,1H),3.76(s,3H),2.04(s,2H),1.81(s,3H),1.66(s,3H),1.43(s,6H)。具体如图1所示。
13C NMR(100MHz,CDCl3)δ182.08,171.71,159.95,156.36,155.79,154.94,142.90,137.18,132.21,127.25,115.85,112.18,104.60,103.82,101.92,94.30,78.07,62.05,60.73,28.46,26.67,25.99,18.38,14.34。具体如图2所示。
化合物2
乙烷基-7-((5-羟基-8-甲氧基-2,2-二甲基-7-(3-甲基丁烯-1-基)-6-含氧-2H,6H-吡喃酮[3,2-b]呫吨酮-9-基)含氧)庚酸乙酯
将化合物1(0.25mmol,100.0mg,1eq)与催化剂K2CO3(0.98mmol,135.0mg,4eq)混合于15mL无水丙酮中,氮气保护搅拌下缓慢滴加过量的7-溴庚酸乙酯(0.49mmol,0.10mL,2eq),60℃加热回流反应4h,TLC监控反应进程。反应结束后,减压除去丙酮液,加蒸馏水10mL,乙酸乙酯萃取三次,每次10mL,合并有机相,无水硫酸钠干燥,减压除去溶剂,得粗产物110.3mg(黄色油状物)。然后采用硅胶柱色谱(200-300目)对粗产物进行分离纯化(乙酸乙酯-石油醚3:97→5:95进行梯度洗脱),最后得到化合物2(83.3mg),黄色固体,紫外灯254处有荧光,产率约为59.2%。
1H NMR(400MHz,CDCl3)δ13.73(s,1H),6.67(m,2H),6.17(s,1H),5.52(d,J=10.0Hz,1H),5.20(t,J=6.7Hz,1H),4.11(m,4H),4.02(t,J=6.4Hz,2H),3.76(s,3H),2.29(t,J=7.5Hz,2H),1.86(d,2H),1.81(s,3H),1.63(d,5H),1.51(t,J=8.0Hz,2H),1.42(s,8H),1.22(t,J=8.4Hz,3H)。具体如图3所示。
13C NMR(100MHz,CDCl3)δ182.14,173.80,159.78,158.09,157.72,156.34,155.42,144.24,137.14,131.84,127.16,123.38,115.92,111.75,104.55,103.97,99.01,94.11,78.02,68.87,60.95,60.39,34.35,28.94,28.92,28.47,26.32,26.08,25.93,24.97,18.33,14.42。具体如图4所示。
化合物3
7-((5-羟基-8-甲氧基-2,2-二甲基-7-(3-甲基丁烯-1-基)-6-含氧-2H,6H-吡喃酮[3,2-b]呫吨酮-9-基)含氧)庚酸
取盐酸羟胺(0.18mmol,12.0mg,2eq)溶于10mL的无水丙酮,置于25mL的圆底烧瓶中,分批加入NaOH(0.44mmol,18.0mg,5eq),5分钟内加入完毕,室温下反应10分钟。30分钟内分批加入化合物2(0.09mmol,50.0mg,1eq),升温至50℃反应5h,TLC监控反应进程。反应结束后,旋干丙酮液,加蒸馏水10mL,乙酸乙酯10mL,萃取三次,旋干乙酸乙酯,得粗产物63.1mg(黄色油状物)。然后采用硅胶柱色谱(200-300目)对粗产物进行分离纯化(乙酸乙酯-石油醚20:80→25:75→35:65进行梯度洗脱),最后得到化合物3(39.9mg),黄色固体,紫外灯254处有荧光,产率约为79.1%。
1H NMR(400MHz,CDCl3)δ13.75(s,1H),6.69(m,2H),6.19(s,1H),5.54(d,J=10.0Hz,1H),5.21(s,1H),4.06(m,4H),3.77(s,3H),2.36(t,J=7.4Hz,2H),1.88(m,2H),1.82(s,3H),1.66(s,5H),1.53(t,J=8.0Hz,3H),1.44(s,8H),1.22(s,1H)。具体如图5所示。
13C NMR(100MHz,CDCl3)δ182.22,179.83(OC=O),159.85,158.15,157.76,156.41,155.51,144.28,137.27,131.97,127.24,123.38,115.95,111.86,104.63,104.05,99.08,94.16,78.10,68.89,61.02,34.07,29.90,28.93,28.89,28.51,26.37,26.12,25.95,24.70,18.38。具体如图6所示。
化合物4
9-(3-丙氧基溴)-5-羟基-8-甲氧基-2,2-二甲基-7-(3-甲基丁烯-1-基)-2H,6H-吡喃酮[3,2-b]呫吨酮
将化合物1(1.10mmol,450.0mg,1eq)与催化剂K2CO3(4.41mmol,609.0mg,4eq)混合于40mL无水丙酮中,氮气保护搅拌下缓慢滴加过量的1,3-二溴丙烷(2.20mmol,0.22mL,2eq),50℃加热回流反应4h,TLC监控反应进程。反应结束后,减压除去丙酮液,加蒸馏水20mL,乙酸乙酯萃取三次,每次10mL,合并有机相,无水硫酸钠干燥,减压除去溶剂,得粗产物670.4mg(黄色油状物)。然后采用硅胶柱色谱(200-300目)对粗产物进行分离纯化(乙酸乙酯-石油醚1:99→5:95→10:90进行梯度洗脱),最后得到化合物4(404.3mg),黄色粉末,紫外灯254处有荧光,产率约为69.1%。
1H NMR(400MHz,CDCl3)δ13.69(s,1H),6.69(t,J=5.0Hz,2H),6.18(s,1H),5.54(d,J=10.1Hz,1H),5.21(m,1H),4.19(t,J=5.8Hz,2H),4.07(d,J=6.7Hz,2H),3.76(s,3H),3.63(t,J=6.3Hz,2H),2.40(p,J=6.1Hz,2H),1.81(s,3H),1.66(s,3H),1.43(s,6H)。具体如图7所示。
13C NMR(100MHz,CDCl3)δ182.10,159.88,158.07,157.21,156.33,155.35,144.16,137.43,131.98,127.25,123.30,115.88,112.12,104.61,103.98,99.24,94.18,78.09,66.32,65.73,61.08,32.03,30.74,29.75,28.51,27.08,26.34,26.10,18.37。具体如图8所示。
化合物5
9-(3-叠氮丙氧基)-5-羟基-8-甲氧基-2,2-二甲基-7-(3-甲基丁烯-1-基)-2H,6H-吡喃酮[3,2-b]呫吨酮
取化合物4(0.76mmol,400.0mg,1eq)和碘化钠(0.04mmol,5.7mg,0.05eq)溶于5mLDMF中,加入叠氮化钠(2.27mmol,148.0mg,3eq),氮气保护搅拌下60℃反应5h,TLC追踪反映进程。加入冰块继续反应1h,反应结束后,加二氯甲烷10mL,萃取三次,旋干二氯甲烷液,得粗产物439.7mg(黄色油状物)。然后采用硅胶柱色谱(200-300目)对粗产物进行分离纯化(乙酸乙酯-石油醚→1:99→2:98进行梯度洗脱),最后得到化合物5(353.7mg),黄色固体,紫外灯254处有荧光,产率约为95.2%。
1H NMR(400MHz,CDCl3)δ13.67(s,1H),6.65(m,2H),6.16(d,J=7.4Hz,1H),5.53(t,J=8.9Hz,1H),5.21(t,J=7.0Hz,1H),4.07(dq,J=12.9,6.5Hz,4H),3.75(s,3H),3.56(q,J=7.1Hz,2H),2.12(q,J=6.7Hz,2H),1.82(s,3H),1.66(s,3H),1.44(s,6H)。具体如图9所示。
13C NMR(100MHz,CDCl3)δ181.95,159.77,157.94,157.11,156.19,155.18,144.07,137.23,131.77,127.15,123.31,115.80,111.93,104.48,103.83,99.07,94.12,77.99,65.51,60.93,48.18,28.64,28.45,26.26,26.04,18.29。具体如图10所示。
化合物6
9-(3-氨丙氧基)-5-羟基-8-甲氧基-2,2-二甲基-7-(3-甲基丁烯-1-基)-2H,6H-吡喃酮[3,2-b]呫吨酮
取化合物5(0.61mmol,300.0mg,1eq)溶于4mLTHF中,缓慢加入三苯基膦(0.73mmol,192.0mg,1.2eq),氮气保护搅拌下55℃反应2h,TLC追踪反映进程。加入水继续反应6h,反应结束后,加蒸馏水10mL,乙酸乙酯萃取三次,每次10mL,合并有机相,无水硫酸钠干燥,减压除去溶剂,得粗产物535.2mg(黄色固体)。然后采用硅胶柱色谱(200-300目)对粗产物进行分离纯化(甲醇-二氯甲烷(+0.25%氨水)→1:99→2:98→3:97进行梯度洗脱),最后得到化合物6(264.6mg),黄色固体,紫外灯254处有荧光,产率约为93.2%。
1H NMR(400MHz,CDCl3)δ6.60(d,J=5.4Hz,1H),6.53(s,1H),6.08(s,1H),5.46(d,J=8.0Hz,1H),5.16(d,J=6.8Hz,1H),4.00(m,4H),3.69(s,3H),2.88(t,J=5.7Hz,2H),1.93(p,J=6.3Hz,2H),1.76(s,3H),1.61(s,3H),1.38(s,6H)。具体如图11所示。
13C NMR(100MHz,CDCl3)δ181.78,159.56,157.78,157.35,156.02,155.06,143.94,136.76,131.48,126.95,123.30,115.69,111.46,104.27,103.63,98.83,93.98,77.82,66.75,60.74,60.72,39.11,32.65,28.32,26.12,25.93,18.17。具体如图12所示。
化合物7
3,6-双(3-丙氧基溴)-1-羟基-7-甲氧基-2,8-双(3-甲基-丁-2-烯基)-9H-氧杂蒽酮
将α-倒捻子素(0.12mmol,50.0mg,1eq)与催化剂K2CO3(0.73mmol,101.0mg,6eq)混合于10mL无水丙酮中,氮气保护搅拌下缓慢滴加过量的1,3-二溴丙烷(0.49mmol,0.05mL,4eq),60℃加热回流反应4h,TLC监控反应进程。反应结束后,减压除去丙酮液,加蒸馏水10mL,乙酸乙酯10mL,合并有机相,无水硫酸钠干燥,减压除去溶剂,得粗产物115.0mg(黄色油状物)。然后采用硅胶柱色谱(200-300目)对粗产物进行分离纯化(乙酸乙酯-石油醚3:97→5:95→10:90进行梯度洗脱),最后得到化合物7(54.3mg),淡黄色粉末,紫外灯254处有荧光,产率约为69.3%。
1H NMR(400MHz,CDCl3)δ13.46(s,1H),6.73(s,1H),6.30(s,1H),5.20(dt,J=10.6,5.3Hz,2H),4.19(dt,J=17.3,5.8Hz,4H),3.77(s,3H),3.63(dt,J=13.2,6.3Hz,4H),3.33(d,J=7.1Hz,2H),2.39(dt,J=20.7,6.0Hz,4H),1.81(s,3H),1.75(s,3H),1.64(s,6H)。
13C NMR(100MHz,CDCl3)δ182.17,162.53,160.08,157.19,155.42,155.29,144.17,137.63,132.03,131.71,123.37,122.64,112.43,111.72,104.27,99.12,89.52,66.30,65.96,61.15,32.35,32.04,29.92,29.82,26.39,26.13,26.01,21.65,18.40,18.05。
化合物8
1-羟基-7-甲氧基-2,8-双(3-甲基-丁-2-烯基)-3,6-双(3-(吡咯烷-1-基)丙氧基)-9H-呫吨酮
取吡咯烷(0.49mmol,0.04mL,4eq)溶于15mL无水丙酮中,加入无水碳酸钾(0.74mmol,101.0mg,6eq)和化合物7(0.12mmol,80.0mg,1eq),氮气保护搅拌下60℃反应10h,TLC追踪反映进程。反应结束后,减压除去丙酮液,加蒸馏水10mL,乙酸乙酯10mL,萃取三次,无水硫酸钠干燥,减压除去溶剂,得粗产物143.4mg(黄色油状物)。然后采用硅胶柱色谱(200-300目)对粗产物进行分离纯化(甲醇-二氯甲烷5:95→10:90→20:80进行梯度洗脱),最后得到化合物8(26.9mg),黄色固体,紫外灯254处有荧光,产率约为34.2%。
1H NMR(400MHz,CDCl3)δ13.47(s,1H),6.71(s,1H),6.27(s,1H),5.22(t,J=7.1Hz,2H),4.10(dq,J=11.7,6.3Hz,4H),3.77(s,3H),3.32(d,J=7.2Hz,2H),2.66(td,J=7.5,3.9Hz,4H),2.54(h,J=3.4Hz,8H),2.07(dp,J=20.6,6.7Hz,4H),1.81(s,4H),1.78(s,6H),1.75(s,4H),1.64(s,6H)。具体如图13所示。
13C NMR(100MHz,CDCl3)δ182.18,162.87,159.96,157.55,155.47,155.31,144.16,137.27,131.83,131.48,123.50,122.77,112.10,111.60,104.06,99.03,89.51,67.39,67.02,61.02,54.45,54.43,53.31,53.17,29.89,28.79,28.68,26.37,26.11,26.03,23.65,21.64,18.38,18.11。具体如图14所示。
化合物9
1-羟基-7-甲氧基-2,8-双(3-甲基-丁-2-烯基)-3,6-双(3-(L-脯氨醇-1-基)丙氧基)-9H-呫吨酮
取L-脯氨醇(0.37mmol,0.04mL,4eq)溶于3mL无水DMF中,加入无水碳酸钾(0.55mmol,76.0mg,6eq)和化合物7(0.12mmol,80.0mg,1eq),氮气保护搅拌下60℃反应15h,TLC追踪反映进程。反应结束后,旋干溶液,加蒸馏水10mL,乙酸乙酯萃取三次,每次10mL,合并有机相,无水硫酸钠干燥,减压除去溶剂,得粗产物72.7mg(黄色油状物)。然后采用硅胶柱色谱(200-300目)对粗产物进行分离纯化(甲醇-二氯甲烷3:97→10:90→15:85进行梯度洗脱),最后得到化合物9(19.0mg),黄色固体,紫外灯254处有荧光,产率约为30.2%。
1H NMR(400MHz,CDCl3)δ13.46(s,1H),6.67(s,1H),6.26(s,1H),5.21(q,J=6.9Hz,2H),4.09(dq,J=12.0,6.7Hz,6H),3.77(s,3H),3.63(dd,J=11.1,3.4Hz,3H),3.43(dd,J=11.1,2.8Hz,2H),3.32(d,J=7.1Hz,2H),3.25(m,2H),3.18(m,2H),3.05(td,J=12.0,6.1Hz,3H),2.68(dt,J=8.9,4.4Hz,2H),2.53(dq,J=11.8,5.7Hz,2H),2.33(tt,J=11.7,5.7Hz,2H),2.10(d,J=6.4,4H),1.81(s,3H),1.75(s,6H),1.64(s,6H)。具体如图15所示。
13C NMR(100MHz,CDCl3)δ182.15,162.69,160.00,157.39,155.42,155.28,144.14,137.38,131.87,131.52,123.46,122.74,112.20,111.56,104.12,98.98,89.42,66.96,66.55,65.81,65.62,62.18,62.07,61.06,54.58,54.52,51.69,51.54,29.90,28.54,28.41,27.64,26.37,26.12,26.03,23.87,23.79,21.64,18.39,18.10。具体如图16所示。
抑制效果检测
1、对RORγt-LBD+-Jurkat细胞的抑制活性实验
材料:
细胞系Jurkat为本实验室保存;DH5α细菌菌株由北京中科院遗传所马润林教授馈赠;限制性内切酶购自美国fermentas公司;DNA连接酶购自美国的NEB公司;报告基因序列IRES-GFP为发明人自有保存(也可以采用其他现有的报告基因序列);报告质粒pGL4.31[luc2P/GAL4UAS/Hygro]质粒和pBIND质粒购自美国promega公司;DMEM培养基和RPMI1640培养基以及培养细胞用的丙酮酸钠、谷氨酰胺、β巯基乙醇、非必须氨基酸、双抗是购自美国life公司;胎牛血清(FBS)购自美国hyclone公司;荧光素酶报告基因检测试剂盒购自美国Promega公司;Lipo2000购自美国life公司;无内毒素质粒提取试剂盒购自Sigma公司;8~12周龄的C57小鼠购自中山大学实验动物中心(SPF级);T细胞电转试剂盒和电穿孔仪购自瑞士的lonza公司;潮霉素B购自瑞士Roche公司;逆转录试剂盒购自大连Takara公司;Realtime检测试剂盒购自美国promega公司;细胞因子hTGF-β和mIL-6购自美国RD公司;小鼠CD3和CD28抗体购自美国eBioscience公司;小鼠IL-17A的ELISA检测试剂盒购自武汉华美公司;其他常用化学试剂购自Sigma公司和上海生工公司。
方法:
质粒构建:将荧光蛋白基因IRES-GFP目的片段通过Not1单酶切位点插入到pBIND载体中完成pBIND-IRES-GFP的质粒构建;用PCR法从PBMC的cDNA中调取人的RORγt(hRORγt)目的基因,hRORγt调取时两端加上BamH1的单酶切位点;再将hRORγt基因克隆到pBIND-IRES-GFP质粒中,完成pBIND-Gal4DBD-hRORγt-IRES-GFP重组质粒的构建(Gal4DBD的序列是pBIND质粒自带的)。
细胞培养:Jurkat细胞培养在含10%FBS(胎牛血清)、1%双抗、2mM谷氨酰胺、1mM丙酮酸钠、50μM的β巯基乙醇的RPMI1640完全培养基中。细胞置于5%CO2,37℃培养箱中恒温培养,约每两天传代一次。
稳定细胞系的构建:pGL4.31+hRORγt+Jurkat稳定细胞系的构建:将20μg经Not1单酶切的质粒pGL4.31[luc2P/GAL4UAS/Hygro]电转入Jurkat细胞(1×107)中,再用200μg/mL的潮霉素B进行抗性筛选;筛选进行2~3周后,再将20μg用限制性内切酶Eam1105酶切的pBIND-Gal4DBD-hRORγt-IRES-GFP重组质粒电转至经潮霉素B筛选后的Jurkat细胞中,再次转染后的细胞继续培养2~3周后,用GFP荧光蛋白标记进行流式分选,并收集GFP+阳性Jurkat细胞。
EC50试验:
EC50的测定基于构建的Gal4/hRORγt的报告体系:将pGL4.31+hRORγt+Jurkat细胞铺板至96孔圆底板中,每孔2×105个细胞,按药物浓度梯度设置不同浓度值,在不同浓度的化合物下作用6h,之后收集细胞,进行luciferase活性检测以得到化合物的EC50的值,结果如表1所示。
CC50试验:
CC50的测定基于构建的Gal4/hRORγt的报告体系:将pGL4.31+hRORγt+Jurkat细胞铺板至96孔圆底板中,每孔2×105个细胞,按药物浓度梯度设置不同浓度值,在不同浓度的化合物下作用48h,之后加入dimethylthiazolyl-2-5-diphenyltetrazoliumbromide(MTT)作用4h后收集细胞,进行比色法检测以得到化合物的CC 50的值;结果如表1所示。
EC50及CC50检测:EC50(half maximal(50%)effective concentration)是对RORγt-LBD+-Jurkat细胞的半最大效应浓度,CC50(median(50%)cytotoxic concentration)是对RORγt-LBD+-Jurkat细胞的细胞毒性浓度中位数,结果见下表1。
表1
化合物 | <![CDATA[EC<sub>50</sub>(μM)]]> | <![CDATA[CC<sub>50</sub>(μM)]]> | <![CDATA[CC<sub>50</sub>/EC<sub>50</sub>比值]]> |
1 | 6.3 | 32.5 | 5.16 |
2 | 11.2 | 35.6 | 3.18 |
3 | 8.2 | 28.9 | 3.52 |
4 | 15.1 | 40.3 | 2.67 |
5 | 13.3 | 45.3 | 3.41 |
6 | 1.2 | 8.6 | 7.17 |
7 | 10.2 | 32.5 | 3.19 |
8 | 0.9 | 5.3 | 5.89 |
9 | 0.55 | 2.4 | 4.36 |
α-倒捻子素 | 1.02 | 2.4 | 2.35 |
表1结果显示,修饰后的化合物CC50活性明显优于α-倒捻子素,具有更好的安全性。尽管部分化合物的药效EC50比α-倒捻子素差,但其对细胞的毒性下降的比例更大,CC50/EC50比值优于α-倒捻子素,因此其成药性潜力更大。
2、体外Th17细胞抑制活性实验
选取上述抑制活性更佳的化合物1、3、6、8、9进行体外Th17细胞分化实验。
试验操作:实验前一天晚上用含有5μg/mL CD3抗体、1μg/mL CD28抗体的PBS溶液包被12孔板,每孔0.5mL,4℃包被过夜。第二天开展实验,首先用Miltenyi磁珠分选小鼠脾脏的CD4+T细胞,分选后的细胞按1×106个/mL的细胞密度重悬细胞,向包被后的12孔板的每个孔加1mL的细胞悬液,同时向溶液中加入终浓度为5μg/mL的hTGF-β、30μg/mL的mIL-6的细胞因子、20μg/mL的mIL-1β、5μg/mL的anti-mouse-IL-4的抗体和5μg/mL的anti-mouse-IFN-γ的抗体,体外诱导CD4+T细胞向Th17细胞分化。24h后,再向每孔添加终浓度为5μM的不同化合物(并设置DMSO对照组,每组按照3个孔重复),37℃、5%二氧化碳培养48h。48h后,每孔补加2mL RPMI1640完全培养基以及终浓度为5μg/mL的hTGF-β、30μg/mL的mIL-6的细胞因子、20μg/mL的mIL-1β、5μg/mL的anti-mouse-IL-4的抗体和5μg/mL的anti-mouse-IFN-γ的抗体和对应浓度的化合物,补加完成后再培养48h。至此,CD4+T细胞体外诱导Th17分化培养了约5天,5天后收集细胞,进行流式细胞仪检测细胞分化程度。
流式细胞仪检测:用IL-17A和CD4的抗体进行细胞染色,实验操作严格按照eBioscience细胞内染色试剂盒和流式细胞仪操作说明进行。
实验结果如图17所示,化合物1、3、6、8、9都有抑制活性。图中数据是Th17细胞的比例(单位%)。1-7依次代表分化培养基中加入溶剂(DMSO)、α-倒捻子素、化合物1,化合物3、化合物6、化合物8和化合物9。
3、对牛皮癣小鼠模型的治疗作用
将BalB/C小鼠分成10组,1为未做处理的健康对照组,2~10组分别用5%咪喹莫特连续涂抹小鼠皮肤7日,同时用不同的药物涂抹皮肤:2为只用5%咪喹莫特处理,3为阳性药物氢化可的松治疗组,4为溶剂无水乙醇处理组,5为α-倒捻子素处理组(剂量为0.1毫克每只动物每天),6~10分别为化合物8、化合物1、化合物3、化合物6和化合物9(剂量为0.1毫克每只动物每天)。
临床病理打分通过观察小鼠患处皮肤的银屑病病变情况并拍照,从红斑、鳞屑、皮肤厚度3项指标进行评价,0~4分进行记分,记分相加得到总分。实验结果如图18所示(N.S.无统计学差异,*p<0.05,**p<0.01,***p<0.001,****p<0.0001,n=6)。显示化合物1、3、6、8、9可以显著减少银屑病病变。
取炎症部位的皮肤做病理切片,然后观察真皮层炎症细胞浸润计数(细胞/mm2)。结果如图19所示,显示化合物1、3、6、8、9可以显著减少真皮层炎症细胞浸润的数量。
取炎症部位的皮肤做病理切片,然后观察其角质层厚度(单位μm)。结果如图20所示,显示化合物1、3、6、8、9可以显著降低角质层增生的厚度。
运用流式细胞分析和细胞计数方法获得小鼠腹股沟淋巴结和脾脏中的Th17细胞总数,结果如图21所示,显示化合物1、3、6、8、9可以显著减少Th17细胞的数量,表明化合物在模型动物体内对Th17细胞分化和功能有明显影响。
以上所述实施例的各技术特征可以进行任意的组合,为使描述简洁,未对上述实施例中的各个技术特征所有可能的组合都进行描述,然而,只要这些技术特征的组合不存在矛盾,都应当认为是本说明书记载的范围。
以上所述实施例仅表达了本发明的几种实施方式,其描述较为具体和详细,但并不能因此而理解为对发明专利范围的限制。应当指出的是,对于本领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干变形和改进,这些都属于本发明的保护范围。因此,本发明专利的保护范围应以所附权利要求为准。
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