CN113967214A - 一种咪唑并吡嗪环丙烷甲酰胺类化合物的应用 - Google Patents
一种咪唑并吡嗪环丙烷甲酰胺类化合物的应用 Download PDFInfo
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Abstract
本发明属于生物医药技术领域,具体涉及一种咪唑并吡嗪环丙烷甲酰胺类化合物的应用。本发明通过实验证明,咪唑并吡嗪环丙烷甲酰胺类化合物可以显著抑制炎症因子,达到显著的抗炎作用;在斑马鱼体内抗炎研究中,咪唑并吡嗪环丙烷甲酰胺类化合物安全性明显高于阳性对照药物,进一步控制其使用浓度,抗炎效果显著优于阳性对照药物,具有显著的进步,有较好的开发潜力。
Description
技术领域
本发明属于生物医药技术领域。更具体地,涉及一种咪唑并吡嗪环丙烷甲酰胺类化合物的应用。
背景技术
炎症反应是人体正常的免疫反应过程,在机体受到病原体或组织损伤的侵袭时,免疫系统被激活,机体做出的一系列防御反应行为,这对于维持机体的稳定具有积极的作用。但是,当炎症过度表达时,急性免疫持续发生,免疫系统会毫无差别的攻击自身完整的组织,形成过度的免疫保护行为,对身体造成损伤,进而转化成慢性免疫疾病,如类风湿性关节炎、牛皮癣等,对人体造成危害。
炎症信号通路的激活是炎症反应过程中最早的免疫应答之一,信号分子通过诱导和调节不同程度的趋化和细胞增殖来修复组织,即急性炎症。当炎症过度表达时,巨噬细胞对相应激活信号产生应答,并分泌出一氧化氮和多种促炎细胞因子等炎症介质,其中白细胞介素6(IL-6)作为一种促炎细胞因子参与到相关炎症过程中,加剧炎症反应。可见,抑制相关信号分子的传导、抑制相关炎症因子的表达、抑制炎症效应细胞的聚集等对于炎症反应的消退,机体恢复平衡至关重要。有中国专利申请公开了一种具有抗炎作用的化合物,具有如式A结构,可选范围较大,但经实验证明,只有其中的化合物7、9能够较好的抑制炎症因子IL-6的释放水平。
可见,开发一种新的化合物需要花费较长的时间和精力物力,并且有效的化合物较为缺少,因此,迫切需要提供多几种可供选择的化合物在制备抗炎药物中的应用。
发明内容
本发明要解决的技术问题是克服现有研发的抗炎药物数量有限的缺陷和不足,提供一种咪唑并吡嗪环丙烷甲酰胺类化合物在制备抗炎药物中的应用。
本发明上述目的通过以下技术方案实现:
一种咪唑并吡嗪环丙烷甲酰胺类化合物在制备抗炎药物中的应用,所述咪唑并吡嗪环丙烷甲酰胺类化合物具有式(I)结构:
其中,R为取代或非取代氨基、取代或非取代酰胺基、取代或非取代五元氮杂环、取代或非取代五元氮氧杂环、取代或非取代五元氮硫杂环、取代或非取代六元氮杂环、取代或非取代六元氮氧杂环、取代或非取代六元氮硫杂环;
所述取代的基团选自C1~6烷基、卤素、磺酰基中的一种或多种。
优选地,所述R为取代或非取代氨基、取代或非取代酰胺基、取代或非取代五元氮杂环、取代或非取代五元氮氧杂环、取代或非取代五元氮硫杂环、取代或非取代六元氮杂环、取代或非取代六元氮氧杂环、取代或非取代六元氮硫杂环;
所述取代的基团选自C1~3烷基、氟或磺酰基。
更优选地,所述R为二甲基氨基、三氟乙酰胺基、4,4-二氟哌啶基、吗啉环或1,1-二氧化物硫代吗啉环。
进一步地,所述咪唑并吡嗪环丙烷甲酰胺类化合物还包括式(I)化合物药学上可接受的盐、酯或溶剂化物。
优选地,所述式(I)化合物药学上可接受的盐为式(I)化合物与酸反应得到的产物,所述酸选自盐酸,磷酸,琥珀酸,马来酸。
更进一步地,所述抗炎药物通过抑制炎症因子表达来抗炎。因此,与炎症因子过度表达的相关疾病,如类风湿性关节炎、牛皮癣等免疫炎症疾病,均可以应用本发明的咪唑并吡嗪环丙烷甲酰胺类化合物进行治疗。
优选地,所述炎症因子为白细胞介素、肿瘤坏死因子α、转化生长因子β、缓激肽、氧自由基、一氧化氮中的一种或多种。优选地,所述白细胞介素为IL-1、IL-4、IL-6、中的一种或多种。更优选地,所述白细胞介素为IL-6。
进一步地,所述抗炎药物的剂型为口服剂、注射剂、吸入剂或外用剂。
与现有技术相比,本发明具有以下有益效果:
本发明提供了一种咪唑并吡嗪环丙烷甲酰胺类化合物在制备抗炎药物中的应用,通过实验证明,咪唑并吡嗪环丙烷甲酰胺类化合物可以显著抑制炎症因子,抑制作用呈现剂量依赖性;在斑马鱼体内抗炎研究中,咪唑并吡嗪环丙烷甲酰胺类化合物安全性明显高于阳性对照药物,进一步控制其使用浓度,抗炎效果显著优于阳性对照药物,具有显著的进步,有较好的开发潜力。
附图说明
图1为本发明实施例中待测化合物的结构图,其中,包括化合物A、B、C、D、E。
图2为本发明实施例1细胞增殖-毒性实验中待测化合物对RAW264.7细胞活性影响图,分别对应化合物A、B、C、D、E;图中***为与空白对照组相比,P<0.01,**为与空白对照组相比,P<0.03。
图3为本发明实施例2中所作白介素6的标准曲线图。
图4为本发明实施例2中待测化合物对炎症因子白介素6的抑制活性结果统计图,分别对应化合物A、B、C、D、E;图中***为与空白对照组相比,P<0.01,###为与脂多糖组相比,P<0.01,##为与脂多糖组相比,P<0.03,#为与脂多糖组相比,P<0.05。
图5为本发明实施例3中斑马鱼胚胎或幼鱼在不同浓度待测化合物(以化合物A为代表)作用下的死亡率统计图。
图6为本发明实施例3中斑马鱼胚胎或幼鱼在待测化合物(以化合物A为代表)、阳性对照作用下的活动抑制率统计图。
图7为本发明实施例3中待测化合物对炎症效应细胞的聚集抑制活性结果统计图;图中*为与空白切尾组相比,P<0.01,#为与地塞米松阳性对照组相比,P<0.03,##为与地塞米松阳性对照组相比,P<0.05。
具体实施方式
以下结合说明书附图和具体实施例来进一步说明本发明,但实施例并不对本发明做任何形式的限定。除非特别说明,本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。
除非特别说明,以下实施例所用试剂和材料均为市购。
实施例1 CCK-8法检测RAW264.7细胞增殖毒性
1、实验材料:
RAW264.7细胞:购于中国科学院上海细胞库。
培养基:DMEM高糖培养基,超级,购于美国Gibco。
培养液:冰箱中取出解冻的胎牛血清和双抗,用75%酒精喷洒,迅速放入超净台中,50mL胎牛血清和5mL双抗加入到500mL的培养基中,摇匀,放入冰箱待用。
10%CCK-8试剂:在超净台中,使用移液枪吸取10mL的CCK-8试剂,加入到90mL培养基中,摇匀,待用。
不同浓度待测化合物:分别称取20mg经过纯化干燥的待测化合物(待测化合物结构参见图1),加入1mL DMSO溶解,过微孔过滤膜(0.22μm),以除掉细菌;滤液根据半倍稀释法用培养液稀释成320μM、160μM、80μM、40μM、20μM、10μM 6个浓度梯度,待用。
2、实验方法:
(1)细胞培养:取出冻存RAW264.7细胞,迅速的放入到37℃的水浴中,晃动冻存管以期望在1min内溶解;喷洒75%的酒精,放入超净台中并转移到无菌离心管中,添加培养基,离心3min,弃去上清液,加入2mL的培养液后转移到培养瓶中并添加培养液,上下左右轻轻晃动,确保能在培养瓶中分散开,最后放入培养箱中培养。定期的对细胞进行换液操作:将培养瓶从培养箱中取出,喷洒75%酒精,迅速放入超净台,灭菌后打开盖子,弃掉旧的培养基,添加新的培养基,放入培养箱继续培养。进行细胞传代操作:从培养箱中取出培养瓶,喷洒75%酒精后迅速放入超净台中,用酒精灯灭菌,将旧的培养基弃掉,使用PBS溶液洗涤掉残留的旧培养基,再加入0.25%胰蛋白酶,在培养箱中消化1~2min,使用显微镜察看消化效果,观察到细胞脱落,成圆状的透明颗粒,轻晃培养瓶时细胞会随液体移动后,加入1mL的培养基阻止消化的进程;吹打瓶面使细胞变成悬浮液,离心3min,将瓶中液体弃去并加入培养基,按大约1:3至1:4的比例转移到培养瓶中继续培养。
(2)CCK-8法细胞增殖-毒性实验:选取生长较好的细胞,按照传代方法处理,加0.25%的胰蛋白酶,离心,弃去离心管中的液体,加培养基稀释细胞,用计数板计数,确保每孔细胞数目为5×103个;在超净台中,在孔板的四周加入100μL PBS溶液,从计数好的离心管中吸取100μL细胞悬浮液加到孔中,第二排作空白对照,只添加培养基,从第三排开始接种细胞。将96孔板置于培养箱中,24h后观察细胞密度,当达到60%~70%时开始给药:吸出旧的培养液,从第四排开始快速添加100μL稀释好的不同浓度待测化合物,第三排作为对照组,不添加药液,每个浓度设置六个复孔,培养24h,置显微镜下察看细胞生长状态。吸出旧的培养液,添加100μL配制好的10%CCK-8试剂,继续在培养箱中培养2.5h。在450nm波长下用酶标仪测定OD值,记录结果,依照基础公式计算细胞存活率;计算结果使用GraphPadPrism 8处理。
3、实验结果:
实验结果参见图2,由图可见,待测化合物浓度在10μmol/L~40μmol/L时,细胞存活率与空白对照组相比差异无显著性(p<0.05)。结果表明待测化合物的安全浓度范围为10μmol/L~40μmol/L。
实施例2 Elisa法测定化合物对白介素6含量的影响
1、实验材料:
标准品为人白细胞介素6(IL-6),由ELISA检测试剂盒提供,购自江苏酶免实业有限公司。
待测化合物稀释液:用培养液稀释成40μM、20μM、10μM三个浓度。
2、实验方法:
(1)细胞上清液样品的制备:将RAW264.7细胞制成悬浮液,参考实施例1方法稀释细胞,用计数板计数后接种到96孔板中,每孔接种100μL,使每孔的细胞数目为5×103个,将细胞培养24小时;当细胞密度达到60%~70%左右,弃去旧的培养液,将待测化合物稀释液从第四排开始加入,每孔加100μL,每个组有6个复孔,放培养箱中培养3小时;弃去旧的培养液,将含脂多糖LPS(200ng/mL)的培养液从第三排开始加,每孔100μL,加入完毕后放培养箱中继续培养24小时;取出96孔板,吸取上清液到离心管中,然后在4℃下,8000rpm转速下离心20分钟,取上清液,置于冰箱(-20℃)中保存,备用;如在保存过程中有沉淀产生,则需要再次离心,以去除沉淀。
(2)标准曲线的绘制:采用半倍稀释法,将标准品的浓度稀释成120pg/mL、60pg/mL、30pg/mL、15pg/mL、7.5pg/mL五个浓度,每个浓度设置两个复孔,进行标准曲线的绘制。标准曲线图见图3。
(3)Elisa法测定:在Elisa板上加50μL的标准品(60pg/ml),将图1所示待测化合物稀释液40μL分别添加到样品孔中,随后加入10μL的细胞上清液样品,每个浓度设置3个复孔,同时设置无需加样品和酶标试剂的空白对照孔,缓慢晃动,摇匀。扯下封板膜盖在96孔板上,在37℃温育0.5h,洗涤液用PBS溶液稀释30倍,备用。温育好后将96孔板中的液体弃去,拍干,每个孔加200μL洗涤液进行洗涤,30秒后弃去,甩干,重复操作5次。除空白对照外其余每孔加50μL的酶标试剂,再次进行温育,洗涤。按顺序在每孔中先加50μL的显色剂A,再加入50μL的显色剂B,晃动,摇匀,37℃条件下避光显色10min。在每个孔中加50μL终止液进行终止操作,颜色发生转变。用空白对照孔调零,用酶标仪在450nm的波长下测定吸光度值,根据标准曲线计算出IL-6的含量。
3、实验结果:
结果参见图4,由图可见,在安全实验浓度范围内,待测化合物组与脂多糖组比较具有显著性差异(p<0.05),并且化合物对炎症因子白介素6的抑制作用呈现剂量依赖性。
实施例3化合物在斑马鱼体内抗炎活性研究
当炎症发生时,中性粒细胞和巨噬细胞等炎症效应细胞会流向炎症部位,并参与主动防御过程;这种主动免疫反应在斑马鱼免疫系统中得到了很好的保存和呈现,因此本实施例采用斑马鱼炎症模型作为体内抗炎反应的研究模型。
实验材料:斑马鱼(具有荧光表达特性的转基因斑马鱼Tg(lyz:DsRed2),由广东实验动物监测所引种自国家斑马鱼资源中心的AB系;本研究选用5-8dpf的仔鱼作为实验用鱼。斑马鱼亲鱼均饲养于斑马鱼养殖系统中,质量控制参考本单位牵头制定的国家标准GB/T 39649-2020《实验动物实验鱼质量控制》进行。
1、安全性评价
(1)斑马鱼的饲养与产卵:)饲养在养殖箱中,维持28.5±1℃的水温,每天给予14h的光照,养殖水充分曝气并且保持pH值在6.5~7.5。将雌雄斑马鱼在交配产卵的前一天晚上按照数量比例1:1置于繁殖盒中,用隔板隔开不同性别的斑马鱼以便控制产卵时间。次日,给予光照并拿开隔板,斑马鱼自然交配,产生斑马鱼胚胎。搜集2hpf内的胚胎,养殖水冲洗3次,移入培养皿中,借助体视显微镜去除掉未受精的鱼胚胎。
(2)安全性评价:将待测化合物(以图1化合物A作为代表,其他化合物效果与化合物A效果类似)用DMSO溶解,用养殖水稀释,分别配置成4.9μg/mL、9.81μg/mL、19.6μg/mL、39.3μg/mL、78.5μg/mL、157μg/mL和314μg/mL七个浓度梯度的试样溶液。将收集的已经受精的胚胎继续培养6h,然后转移到96孔板中,设置空白对照组、DMSO溶剂对照组、地塞米松阳性对照组和待测化合物组,每组设置3个复孔,每孔放置10个斑马鱼胚胎,向每孔中添加相应浓度的溶液2.5mL,在24h、48h、72h和96h的时间点上对孔内斑马鱼的胚胎和孵化的幼鱼进行观察并记录,依据基本公式对斑马鱼胚胎或幼鱼的死亡率和活动抑制率(多次刺激后无躲避行为)进行计算。采用SPSS软件对数据进行统计分析,采用单因素方差分析,显著性水平设定为α=0.05,因变量用均值±标准差表示。
(3)安全性评价实验结果参见图5,由图可见,当化合物浓度为4.9μg/mL时无致死作用(该浓度条件下,对四个时间点进行了监测,对照组和实验组斑马鱼胚胎致死个数均为零,未做统计图),与阳性对照相比,待测化合物对斑马鱼胚胎的致死率较低;得到化合物A的活动抑制率EC50为159.782μg/ml。
依据多次刺激后是否有回避行为为判断指标,计算最大效应浓度,进行非线性拟合分析,得到拟合曲线图6。由图可见,待测化合物的活动抑制率较阳性对照低,总体毒性小于阳性药物地塞米松,安全性更高。
2、抑制炎症细胞聚集实验
(1)设置空白对照组(未切尾),切尾对照组(不给药),阳性对照组(给地塞米松)以及实验组。依据安全性评价计算得出化合物A的EC50,在安全浓度范围内设置并配制了20μg/mL、40μg/mL、80μg/mL和160μg/mL的待测化合物组(以图1化合物A作为代表,其他化合物效果与化合物A效果类似)浓度梯度,并将地塞米松阳性对照的浓度设置为50μg/mL;同时将160mg的三卡因置于离心管中,加入养殖水40mL,转移0.5mL母液于培养皿中,添加9.5mL养殖水来配置0.02%的三卡因工作液。将转基因斑马鱼置于荧光显微镜下,挑选出已经有荧光表达的培养了72hpf的斑马鱼,置于96孔板中,每组设置4个复孔。添加相应的溶液进行预处理,1h后用0.02%的三卡因对经过预处理的斑马鱼进行麻醉,置于体视显微镜下进行切除尾鳍操作。将已经切断尾鳍的斑马鱼继续置于相应溶液中暴露培养6h,三卡因麻醉,运用荧光显微镜观察斑马鱼的尾部,通过人工对巨噬细胞和中性粒细胞的数量进行统计。
(2)实验结果参见图7,由图可见,在20μg/mL的浓度下,待测化合物A对炎症效应细胞有明显的聚集抑制作用,与去尾对照组比较有显著性差异(p<0.03);当浓度达到160μg/mL时,待测化合物的抑制活性优于阳性药物地塞米松(P<0.05)。说明本申请化合物具有显著的抗炎效果。
上述实施例为本发明较佳的实施方式,但本发明的实施方式并不受上述实施例的限制,其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化,均应为等效的置换方式,都包含在本发明的保护范围之内。
Claims (10)
2.根据权利要求1所述应用,其特征在于,所述R为取代或非取代氨基、取代或非取代酰胺基、取代或非取代五元氮杂环、取代或非取代五元氮氧杂环、取代或非取代五元氮硫杂环、取代或非取代六元氮杂环、取代或非取代六元氮氧杂环、取代或非取代六元氮硫杂环;
所述取代的基团选自C1~3烷基、氟或磺酰基。
3.根据权利要求2所述应用,其特征在于,所述R为二甲基氨基、三氟乙酰胺基、4,4-二氟哌啶基、吗啉环或1,1-二氧化物硫代吗啉环。
4.根据权利要求1~3任一所述应用,其特征在于,所述咪唑并吡嗪环丙烷甲酰胺类化合物还包括式(I)化合物药学上可接受的盐、酯或溶剂化物。
5.据权利要求4所述应用,其特征在于,所述式(I)化合物药学上可接受的盐为式(I)化合物与酸反应得到的产物,所述酸选自盐酸,磷酸,琥珀酸,马来酸。
6.根据权利要求1~3任一所述应用,其特征在于,所述抗炎药物通过抑制炎症因子表达来抗炎。
7.根据权利要求6所述应用,其特征在于,所述炎症因子为白细胞介素、肿瘤坏死因子α、转化生长因子β、缓激肽、氧自由基、一氧化氮中的一种或多种。
8.根据权利要求7所述应用,其特征在于,所述白细胞介素为IL-1、IL-4、IL-6中的一种或多种。
9.根据权利要求8所述应用,其特征在于,所述白细胞介素为IL-6。
10.根据权利要求1~3任一所述应用,其特征在于,所述抗炎药物的剂型为口服剂、注射剂、吸入剂或外用剂。
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