CN108017584A - A3腺苷受体的小分子拮抗剂 - Google Patents
A3腺苷受体的小分子拮抗剂 Download PDFInfo
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Abstract
本发明描述A3腺苷受体的小分子拮抗剂及其在药物合成方面的应用，A3腺苷受体的小分子拮抗剂以及所述A3腺苷受体的小分子拮抗剂药学上的可用盐作为用于治疗或预防预防A3腺苷受体介导的相关疾病或病症的药物的用途。
Description
技术领域
本发明描述A3腺苷受体的小分子拮抗剂及其在药物合成方面的应用。
背景技术
腺苷(adenosine)是腺嘌呤核苷酸的前体和代谢物，腺苷的诸多生理作用都是其受体介导的.腺苷能和四种类型的G蛋白偶联受体A1，A2A，A2B和A3相互作用，进而调节腺苷酸环化酶、离子通道和磷脂酶的活性。在对心血管疾病和肾脏疾病的研究中发现，腺苷对心肌细胞和肾基底膜细胞的钾离子通道具有调节作用，同时腺苷受体激动剂和拮抗剂对于治疗中枢神经系统神经退行性病变(如帕金森病和阿尔茨海默病等)的研究也显示了良好的前景。激活A3腺苷受体具有保护心脑，抗癌的作用；其拮抗剂在抗炎、治疗癌症和青光眼、糖尿病、调节细胞生长方面起着重要作用。
A3腺苷受体分布广泛，不同种族的A3受体转录体的组织分布和表达水平有显著差异。在人和绵羊的睾丸、肺、肾、胎盘、心脏、大脑、脾、肝、子宫、膀胱、空肠、近端的结肠和眼睛中都有A3腺苷受体。
人体肥大细胞对腺苷的应答很复杂，这可能涉及到几种腺苷受体亚型。腺苷A3受体可能是导致哮喘的病因，而且腺苷A3受体的激动也可能增强肥大细胞过敏介质的释放。
因此，腺苷A3受体可能是腺苷与哮喘发病机制和速发型过敏反应间的桥梁，临床上黄嘌呤茶碱能扩张支气管，部分是由于其是腺苷A3受体拮抗剂之故。
腺苷A3受体拮抗剂可阻断心肌预适应，在兔心肌细胞中APNEA(A1/A3选择性)可预防局部缺血，但R-PIA(A1选择性的)却没有这种作用。腺苷A3受体能介导心肌预适应和减少心肌损伤，在离体的心肌细胞中，激活腺苷A3和腺苷A1受体可诱发心肌保护的最大预适应。
青光眼为全球的第二大致盲眼病，也是第一位的不可逆性致盲性眼病。青光眼视神经损害的确切机制尚未完全明了，目前认为眼压升高所致视网膜神经节细胞(retinalganglion cells，RGCs)轴浆流受阻和机械压迫等因素导致的RGCs凋亡是其发病的重要病理改变。在青光眼视神经保护研究方面，以往主要集中在神经营养因子，一氧化氮(NO)合成代谢和自由基清除等方面。近年来，由于Müller细胞等神经胶质细胞在视网膜内特有的结构和功能以及它们对正常及病理状态下视网膜微环境的调控的重要作用而越来越受到重视。
腺苷及其受体对青光眼状态下视网膜Müller细胞的钾离子通道和视网膜谷氨酰胺合成酶(glutamine synthetase，GS)、谷氨酸天冬氨酸转运体(L-Glutamate/L-Aspartate Transporter，GLAST)可以进行调控。
发明内容
本发明的目的是提供A3腺苷受体的小分子拮抗剂及其在药物合成方面的应用。
所述小分子为A3-3，它的化学名称为：(E)-1，3-二苯基-1H-吡唑-4-甲醛肟，结构式为I；
上述小分子能够有效拮抗A3腺苷受体，其抑制活性为：A3-3_IC50＝5556nM。
本发明的又一个目的是提供所述小分子治疗或预防预防A3腺苷受体介导的相关疾病或病症的药物的用途。
其中所述疾病或病症包括(但不仅限于)：动脉粥样硬化、风湿性关节炎、风湿性肩周炎、中枢神经系统神经退行性疾病、哮喘、多器官功能性障碍综合症、青光眼、糖尿病。
所述青光眼包括继发性开角型青光眼、继发性闭角型青光眼及其组合。
实现上述用途的的药用组合，包含治疗有效量的小分子抑制剂和任意一种或几种药学上可接受的赋形剂。
上述药学上可接受的赋形剂可以是药物制剂领域中任何常规的赋形剂，特定赋形剂的选择将用于治疗特定患者的给药方式或疾病类型和状态，用于特定给药模式的合适药物组合物的制备方法在药物领域技术人员的知识范围内。例如，可以作为药学上可接受的赋形剂包括药学领域常规的稀释剂、载体、填充剂、粘合剂、湿润剂、崩解剂等。
附图说明
附图1 A3-3的两次实验测定的IC50曲线图
具体实施方式
为了使本领域的技术人员更好的理解本发明的技术方案，下面以实施例进一步说明本发明。
1.材料与消耗品
2.实验步骤
2.1培养细胞
2.1.1在37℃，5％CO2浓度的条件下培养表达人腺苷受体的稳定细胞
2.1.2准备实验的缓冲液
| 试剂 | 体积 | 最终浓度 |
| Hank’s平衡盐溶液 | 14ml | - |
| HEPES缓冲液(1M) | 75μl | 5mM |
| 7.5％的BSA稳定剂(pH 7.4) | 200μl | 0.1％ |
| 20mM的Rolipram | 7.5μl | 10μM |
2.1.3使用乙二胺四乙酸溶液分离细胞，收集200g细胞在室温条件下离心5min后，然后用测定缓冲液来悬浮细胞，然后Countess(Invitrogen#C10281)来计算细胞的密度和活性。细胞活力大于85％的细胞才用于实验测定。
2.1.4在394孔板中添加10μl处理过的细胞。
2.2化合物的准备
2.2.1在384孔聚微丙烯板中用DMSO三倍连续稀释化合物，用TECAN EVO液体处理工作站来进行操作，最高的浓度是100mM。
2.3激动剂的滴定
2.3.1准备实验的缓冲液
| 试剂 | 体积 | 最终浓度 |
| Hank’s平衡盐溶液 | 14ml | - |
| HEPES缓冲液(1M) | 75μl | 5mM |
| 7.5％的BSA稳定剂(pH 7.4) | 200μl | 0.1％ |
| 20mM的Rolipram | 7.5μl | 10μM |
2.3.2在384孔板中准备NECA的连续稀释，
| 稀释次数 | 最终的浓度(uM) | 稀释体积 | DMSO |
| 1 | 1 | 15μl of 1mM | 45μl |
| 2 | 3.33×10-1 | 15μl of dil 1 | 30μl |
| 3 | 1.11×10-1 | 15μl of dil 2 | 30μl |
| 4 | 3.70×10-2 | 15μl of dil 3 | 30μl |
| 5 | 1.23×10-2 | 15μl of dil 4 | 30μl |
| 6 | 4.12×10-3 | 15μl of dil 5 | 30μl |
| 7 | 1.37×10-3 | 15μl of dil 6 | 30μl |
| 8 | 4.47×10-4 | 15μl of dil 7 | 30μl |
| 9 | 1.52×10-4 | 15μl of dil 8 | 30μl |
| 10 | 5.08×10-5 | 15μl of dil 9 | 30μl |
2.3.3用Echo转移NECA的10μl连续滴定液到384孔板中，对于hADORA3激活剂的滴定，通过Echo转移1mM的forskolin(真核细胞腺苷酸环化酶(AC)激活剂)，forskolin最终的浓度是1μM。
2.3.4通过上述的步骤制作细胞悬浮液，每个孔板里面10μl，1min离心150g。
2.3.5依据下面的步骤制备Eu-cAMP示踪液和Ulight-anti-cAMP工作环境按照以下的表格：
2.3.6在每个板中加入5μl的Eu-cAMP追踪剂，然后再往每一个板中加入5μl的Ulight-anti-cAMP。
2.3.7在细胞板上旋转30秒，然后在常温下孵化30分钟。
2.3.8用EnVison(λex＝320nm，λem＝665nm&615nm)读取细胞板，plot
Ratio665nm/615nmvs.the NECA浓度的曲线来计算EC50和EC80值。
2.4拮抗剂的滴定
2.4.1准备实验的缓冲液
| 试剂 | 体积 | 最终浓度 |
| Hank’s平衡盐溶液 | 14ml | - |
| HEPES缓冲液(1M) | 75μl | 5mM |
| 7.5％的BSA稳定剂(pH 7.4) | 200μl | 0.1％ |
| 20mM的Rolipram | 7.5μl | 10μM |
2.4.2准备NECA Stock作为cAMP的诱导剂
将995μl的DMSO溶液倒入一个EP试管中；然后加入1mM的NECA不停摇晃，NECA的浓度是5μM。
2.4.3移除细胞培养基，然后在每一个板孔内加入10μl的测定缓冲液，在细胞板上旋转30秒。
2.4.4将从化合物源孔板中得到的这些化合物可以通过Echo方法放在试剂中。
2.4.5在细胞板上旋转30秒，然后在常温下孵化20分钟。
2.4.6通过Echo方法添加1.5nl NECA到化合物测试集中，诱导cAMP反应。
2.4.7在细胞板上旋转30秒，然后在常温下孵化30分钟。
2.4.8依据下面的步骤制备Eu-cAMP示踪液和Ulight-anti-cAMP工作环境按照以下的表格：
2.4.9在每个板中加入5μl的Eu-cAMP追踪剂，然后再往每一个板中加入5μl的Ulight-anti-cAMP)在细胞板上旋转30秒，然后在常温下孵化30分钟。
2.4.10用EnVison(λex＝320nm，λem＝665nm&615nm)读取细胞板，plotRatio665nm/615nmvs.the NECA浓度的曲线来计算EC50和EC80值。
Claims (4)
1.小分子化合物在制备A3腺苷受体拮抗剂药物方面的用途，所述小分子化合物为：
(E)-1，3-二苯基-1H-吡唑-4-甲醛肟(I)，
2.根据权利要求1所述的用途，其特征在于：所述化合物以及所述化合物药学上的可用盐作为用于治疗或预防A3腺苷受体介导的相关疾病或病症的药物的用途。
3.权利要求2的方法，其中所述疾病或病症包括(但不仅限于)：动脉粥样硬化、风湿性关节炎、风湿性肩周炎、中枢神经系统神经退行性疾病、哮喘、多器官功能性障碍综合症、青光眼、糖尿病。
4.实现权利要求1-2所述用途的药物组合，其特征在于：包含所述化合物中任意一种或几种和药学上可接受的赋形剂。
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| CN110483466A (zh) * | 2019-09-09 | 2019-11-22 | 南开大学 | 腺苷a3受体拮抗剂及其应用 |
| CN110882253A (zh) * | 2019-06-24 | 2020-03-17 | 南开大学 | 具有腺苷a1/a2a受体选择性和腺苷a1受体拮抗活性的分子 |
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