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CN109700804B - 四氢咔唑结构类型雄激素受体拮抗剂及其应用 - Google Patents

四氢咔唑结构类型雄激素受体拮抗剂及其应用 Download PDF

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CN109700804B
CN109700804B CN201910125838.6A CN201910125838A CN109700804B CN 109700804 B CN109700804 B CN 109700804B CN 201910125838 A CN201910125838 A CN 201910125838A CN 109700804 B CN109700804 B CN 109700804B
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李丹
周文方
侯廷军
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Abstract

本分案申请公开了四氢咔唑结构类型雄激素受体拮抗剂及其应用，属于生物化学技术领域。本发明提供的化合物对雄激素受体具有明显的拮抗活性，因此上述化合物可以应用到雄激素受体拮抗剂、前列腺癌细胞增殖抑制剂和抗前列腺肿瘤药物的制备当中。本发明还提供了上述化合物作为有效成分的药物组合物，为目前治疗前列腺癌的药物研究提供新的选择。

Description

四氢咔唑结构类型雄激素受体拮抗剂及其应用
本申请为申请号201710117698.9、申请日2017年3月1日、发明名称“雄激素受体拮抗剂及其应用”的分案申请。
技术领域
本发明涉及生物化学技术领域，具体涉及雄激素受体拮抗剂及其应用。
背景技术
前列腺癌在西方国家是男性第二大致死性肿瘤，而近年来随着我国人民生活水平的提高和饮食结构的变化，国内前列腺癌的发病率呈现明显增高的趋势。对于局限期(早期)前列腺癌患者，前列腺癌根治术、放疗能达到较好疗效。但由于前列腺癌早期临床症状不明显、病情隐匿，加上筛查并不普及，许多患者就诊时已经处于肿瘤已转移的晚期，此时前列腺癌根治术和化疗很难取得理想效果，必须另外使用内分泌治疗。针对激素敏感型晚期前列腺癌患者，内分泌治疗是主要治疗手段，包括手术去势、药物去势和使用以雄激素受体(Androgen Receptor,AR)拮抗剂为主的抗雄激素药物；对于已进展为去抵抗性的前列腺癌(Castration-resistant Prostate Cancer,CRPC)患者，AR拮抗剂是重要的治疗手段之一。
AR拮抗剂(抗雄激素)在前列腺癌的治疗上的应用已有较长时间的历史，最初与化学去势药物促性腺激素释放激素(Luteinizing Hormone Releasing Hormone,LHRH)类似物联用，作为雄激素阻断疗法(Androgen Deprivation Therapy,ADT)中的一种补充手段，其主要作用是阻滞药物去势初期患者体内睾酮水平短期增高而导致的疾病症状加剧。
AR拮抗剂按结构类型可分为甾体类和非甾体类；甾体类拮抗体由于存在肝毒性、干扰性欲、心血管副作用和低效等缺陷，临床使用受限；而非甾体类拮抗剂的临床使用自上世纪八十年代以来，已出现第一代拮抗剂氟他胺、羟基氟他胺、比卡鲁胺、尼鲁米特以及第二代拮抗剂恩杂鲁胺。研究表明，AR及其主导的信号传导通路在前列腺癌的进展过程中发挥着关键性的作用，晚期前列腺癌的内分泌治疗也主要是针对于此。去势治疗可以尽可能切断患者体内雄激素的主要来源，使AR缺乏天然配体的结合来抑制该通路；AR拮抗剂则可以通过与雄激素竞争性地结合AR，来降低患者体内其他来源的雄激素的通路激活作用，从而达到完全的激素阻断效果。正因为如此，AR拮抗剂后来也被批准作为单药疗法在ADT中使用。
第一代非甾体拮抗剂均由氟他胺衍生而出，因此具有类似的结构骨架；比卡鲁胺作为其中耐受性最好、最稳定和最为广泛使用的佼佼者，除了能竞争性结合AR使其处于拮抗状态而难以聚集共激活因子、结合DNA外，还能通过降低AR的稳定性来发挥抑癌效果。如果说第一代AR拮抗剂在前列腺癌的治疗中仅起到有限的辅助效用，那么第二代拮抗剂恩杂鲁胺的出现则将AR拮抗剂推到了CRPC标准治疗的新的重要地位。与比卡鲁胺相比，恩杂鲁胺对AR具有更高的亲和力，从而带来更强的药效；作用机制上除具备第一代非甾体拮抗剂特性之外，它还能抑制AR的核转移，使之不能入核发挥转录因子的作用。恩杂鲁胺最初于2012年被批准用于经内分泌疗法及化疗后癌症已扩散的CRPC患者的治疗，于2014年进一步被批准用于ADT治疗失败但未接受化疗的无症状或者轻微症状的转移性CRPC的治疗；可见，对于已少有药物可用的晚期转移性CRPC，能单独用药的新一代AR拮抗剂恩杂鲁胺具有举足轻重的重磅药地位；且随着进一步的临床研究，其在前列腺癌中治疗范围将会继续扩大。
然而，每一种前列腺癌药物使用后，随着疾病的进展总会产生耐药性，AR拮抗剂产生耐药性的具体原因目前尚未完全阐明，大量的研究观察到AR蛋白的突变是其中非常关键的一点。AR蛋白的点突变不仅会使拮抗剂失效，而且会导致曾经发挥拮抗作用的小分子功能逆转而产生激动剂效果，即使疗效上有独特优势的二代拮抗剂使用一段时间后也不可避免的会发生逆转效应。因此，新一代的、对AR高亲和力的、具有不同于以往AR拮抗剂骨架结构的新型拮抗剂分子仍然是前列腺癌药物研究的重点方向，也存在着迫切的临床需求。
临床在研的新一代AR拮抗剂药物主要有ARN-509、ODM-201和AZD3514，前两者各自针对接受过不同疗法和处于不同阶段的前列腺患者目前已分别进展至三期临床试验阶段，非常有望在不久的将来获批而加入治疗行列。ARN-509具有与恩杂鲁胺非常相似的结构，目前的研究结果表明与恩杂鲁胺相比它具有更强的受体结合能力，相对需要服用的剂量较低，而具有更低的中枢神经系统渗透与致癫痫副作用，但正因为结构的过度类似性，针对恩杂鲁胺具有耐药性的F876L突变同样会对ARN-509产生耐药性。ODM-201及其体内代谢物ORM-15341具有更为新颖的化学结构，它的作用机理类似于二代拮抗剂，但对AR的亲和力甚至超过ARN-509和恩杂鲁胺。
总结国内外AR拮抗剂的研究状况可以发现，开发靶向HBP位点且具有新型骨架结构、高亲和力和高选择性的新一代拮抗剂仍是研究的重点，随着人口老龄化问题的加剧，其存在着巨大的临床需求。靶向AR蛋白其他区域的非HBP拮抗剂，可以克服传统拮抗剂耐药性的缺陷，该方面研究仍存在非常大的临床空白；新型AR拮抗剂的药物开发任重而道远。
发明内容
本发明的目的是提供具有雄激素受体拮抗活性的化合物，将其应用到雄激素受体拮抗剂、抗前列腺肿瘤药物的制备当中。
本发明通过如下技术方案实现上述目的：
本发明采用计算机辅助药物分子设计的手段来发现靶向雄激素受体的先导化合物，再对多个小分子化合物三维结构数据库进行基于分子对接的虚拟筛选，得到得分靠前的1000个化合物(能量越低，得分越靠前)。之后通过前列腺癌经典细胞株LNCaP的MTT细胞增殖实验、AR转录因子活性的抑制实验和采用试剂盒PolarScreenTM AR CompetitorAssay,Green(Thermo Fisher Scientific)考察化合物与AR的配体结合域LBP的结合情况，最终筛得1个具有代表性的活性化合物：N-(6-(三氟甲基)-2,3,4,9-四氢-1H-咔唑-1-基)甲酰胺，结构式如式(1)所示；
Figure BDA0001973549890000031
本发明对上述筛选到的化合物进行进一步生物学活性测定，发现上述化合物对雄激素受体具有明显的拮抗活性，因此，本发明提供了上述化合物或其可药用盐在制备雄激素受体拮抗剂中的应用。
本发明研究发现：上述化合物在蛋白水平和细胞水平的抗前列腺肿瘤实验中表现良好的效果，因此，本发明提供了上述化合物或其可药用盐在制备前列腺癌细胞增殖抑制剂中的应用。
本发明还提供了上述化合物或其可药用盐在制备抗前列腺肿瘤药物中的应用。
本发明还提供了一种药物组合物，包含作为有效成分的化合物或其可药用盐。
作为有效成分的化合物是雄激素受体拮抗剂，因此，本发明的药物组合物可作为与雄激素受体有关疾病的治疗药物。
所述可药用盐为盐酸盐、磷酸盐、硫酸盐、醋酸盐、马来酸盐、枸橼酸盐、苯磺酸盐、甲基苯磺酸盐、富马酸盐或酒石酸盐。
所述药物组合物还包括药学上可接受的赋形剂、稀释剂或载体。具体如糖浆、阿拉伯胶和淀粉等。该药物组合物可以通过静脉、口服、舌下、经肌肉或皮下、皮肤黏膜途径给药。
所述药物组合物的制剂形式为液体制剂或固体制剂。具体如片剂、胶囊剂和注射剂等。各剂型均可以以药学常规方法制备而成。
本发明具备的有益效果：
本发明基于分子对接的虚拟筛选方法以及生物学活性测定发现化合物N-(6-(三氟甲基)-2,3,4,9-四氢-1H-咔唑-1-基)甲酰胺对雄激素受体具有明显的拮抗活性，可将其作为雄激素受体拮抗剂应用到与雄激素受体有关的疾病治疗当中，为目前治疗前列腺癌的药物研究提供新的选择。
附图说明
图1为本发明的13个化合物在浓度为10μM条件下对AR结合能力实验结果。
图2为化合物1在一系列浓度梯度下对AR结合能力实验结果。
图3为化合物3在一系列浓度梯度下对AR结合能力实验结果。
图4为化合物4在一系列浓度梯度下对AR结合能力实验结果。
图5为拮抗剂在AR活性口袋中的结合构象及拮抗剂和AR活性口袋残基之间的相互作用模式，其中(a)为拮抗剂在AR活性口袋中的结合构象(蛋白采用条带形状，拮抗剂采用棍状模型显示)；(b)为拮抗剂和AR活性口袋残基之间的相互作用模式。
图6为13个化合物在浓度为10μM条件下对AR的拮抗活性实验结果。
图7为化合物5-7在一系列浓度梯度下对AR拮抗能力实验结果。
图8为化合物8-10在一系列浓度梯度下对AR拮抗能力实验结果。
图9为化合物11-13在一系列浓度梯度下对AR拮抗能力实验结果。
图10为化合物1-8在浓度为10μM条件下对前列腺癌细胞增殖的抑制能力结果。
图11为化合物9-13对前列腺癌细胞增殖的抑制能力结果。
具体实施方式
下面结合具体实施例对本发明作进一步说明。
实施例1
(1)基于分子对接的虚拟筛选
实验原理：利用分子对接方法，对化合物数据库中的化合物与AR之间的相互作用进行预测、分析和评估，从而确定能够和AR结合的拮抗剂分子。
实验方法：基于AR和雄激素形成复合物的晶体结构(PDB编号：2PNU、2Q7I和3V49)，采用Schrodinger分子模拟软件中的Glide模块进行了基于分子对接的虚拟筛选研究。虚拟筛选所采用的化合物库包括最新版本的Chembridge、ChemDiv以及申请人课题组开发的包含6万多个化合物的中草药有效成分三维结构数据库。我们对虚拟筛选打分最佳的2000个化合物采用Reos规则进行了评价，剔除了包含反应基团的分子。
实验结果：分子对接能够较为准确地确定能和AR能形成较强相互作用的有机小分子。基于分子对接的预测结果，我们从商业化合物库中购买了200多个化合物，并进行了后续的基于分子水平结合实验(PolarScreenTM AR Competitor Assay,Green，Thermo FisherScientific)的活性测试，从中发现了一批具有明显AR拮抗活性的小分子化合物，具体见表1。
表1
Figure BDA0001973549890000041
Figure BDA0001973549890000051
Figure BDA0001973549890000061
上述化合物的结构式如下：
Figure BDA0001973549890000062
Figure BDA0001973549890000071
(2)AR竞争性结合实验
实验原理：化合物AR结合能力的测量采用了Invitrogen公司(Thermo FisherScientific旗下)的荧光偏正实验。雄激素受体{AR-LBD(His-GST)}与一种具有荧光的雄激素配体(FluormoneTMAL Green)结合形成二元复合物(AR-LBD(His-GST)/FluormoneTMALGreen)，该复合物具有较高的荧光偏正值。将此复合物加入到含有测试化合物的微孔板中，被测试化合物作为一种竞争性配体会替换掉二元复合物中的荧光配体(FluormoneTMALGreen)，使荧光偏正值下降。如果加入的是不具备替换荧光化合物能力的非竞争性配体，其偏正值仍会维持在高水平。因此，加入被测试化合物后，荧光偏正值的改变可以用来定量测量被测试化合物对AR-LBD(His-GST)的相对亲和力。
实验方法：将AR LBD蛋白和高亲和力荧光配体在缓冲液中混合后，加入不同浓度的测试化合物(虚拟筛选化合物)以雄激素二氢睾酮(DHT)作为阳性对照)。若测试化合物对AR LBD有较高亲和力，作为一种竞争性配体会替换掉二元复合物中的荧光配体，使体系的荧光偏正值下降；如果加入的测试化合物对AR LBD基本没有结合能力，则体系的荧光偏正值仍会维持在较高值，我们使用多功能酶标仪测量体系的荧光偏振值的变化即可定量测量虚拟筛选化合物对AR的结合能力(binding affinity)。
实验结果：如图1所示，1-13号化合物的AR结合率均超过30％。我们测试不同浓度的化合物与AR的结合能力，发现系列化合物有很好的结合能力，其抑制AR的荧光配体结合的半抑制浓度IC50为均为微摩尔级别，如图2、3、4所示，其中1、3、4号化合物的IC50值分别为33μM、50-100μM和2.6μM。
(3)拮抗剂和AR之间相互作用模式的评价
实验原理：基于分子对接和分子动力学模拟，从原子尺度上预测AR拮抗剂和AR之间的相互作用模式。
实验步骤：基于分子对接预测得到的结果，对拮抗剂/AR进行50ns的分子动力学模拟，模拟采用了AMBER14。
实验结果：通过分子对接预测和分子动力学模拟得到的拮抗剂和AR之间的相互作用如图5所示。预测结构表明，拮抗剂和AR之间的分子识别主要通过范德华和氢键相互作用。拮抗剂上的羟基会和Ser110形成稳定的氢键；两个苯环会和周围的多个疏水性残基产生强的范德华相互作用。
(4)AR拮抗能力评价实验
实验原理：AR作为转录因子，需要与特定的序列，即ARE反应元件，结合才能发挥转录活性；因此向AR阳性的前列腺癌细胞LNCaP导入ARR2PB启动子控制的报告基因增强型绿色荧光蛋白EGFP，经不同浓度测试化合物给药处理后，测量细胞中EGPF的表达量的高低即可得到化合物对AR拮抗能力的强弱。
实验步骤：我们用事先构建好的、含对AR具有强响应的ARR2PB启动子控制的EGFP(增强型绿色荧光蛋白)报告基因质粒，采用慢病毒稳定转染LNCaP细胞的方法得到受AR调控的稳定表达EGFP前列腺癌细胞系(LN-ARR2PB-EGFP)。LN-ARR2PB-EGFP细胞先在不含雄激素的完全培养基中培养几天，使背景荧光值降至较低水平，然后将其以40000个/孔的密度接种至黑色底透的96孔板中，待细胞稳定贴壁后，同时给予雄激素和不同浓度测试化合物(虚拟筛选化合物、已上市拮抗剂药物恩杂鲁胺)，孵育24-48h后，用多功能酶标仪在波长485nm激发光下检测530nm波长附近荧光强度值，即可定量计算出测试化合物拮抗AR蛋白的抑制率。
实验结果：
如图6所示，1-13号化合物均能达到30％以上的抑制率。
如图7-9所示，在不同浓度化合物处理LN-ARR2PB-EGFP细胞36h后，化合物对报告基因EGFP的表达产生明显的下调作用，且呈现剂量依赖关系，这说明我们所列举的化合物都是活性良好潜在AR拮抗剂。
(5)MTT法检测化合物抗前列腺肿瘤细胞增殖活性
实验原理：活细胞线粒体中的琥珀酸脱氢酶能使外源性MTT((3-(4，5-二甲基噻唑-2)-2，5-二苯基四氮唑溴盐))还原为水不溶性的蓝紫色结晶甲瓒(Formazan)并沉积在细胞中，而死细胞无此功能，加入缓冲液溶解细胞中形成的的甲瓒，用酶联免疫检测仪在490nm波长处测定其光吸收值，可间接反映活细胞数量。
实验步骤：用不含雄激素完全培养基以3000个/孔的密度在96孔板接种培养癌细胞，待细胞稳定贴壁后，同时给予1nM DHT和不同浓度测试化合物(虚拟筛选化合物、已上市拮抗剂药物或者DMSO)，孵育4days后每孔加入10μL 5mg/ml MTT，培养箱中继续孵育3hours，然后每孔加入100μL SDS-HCl-PBS三联缓冲液，37度过夜孵育后，在酶标仪下检测570nM处各孔吸光度值，换算为存活率，即可得到给药化合物的IC50值。
实验结果：如图10、11所示，本发明中的化合物对前列腺癌细胞LNCaP有明显的增殖抑制能力，抑制IC50均能到达与上市药物恩杂鲁胺相近水平。

Claims (4)

1.结构式如式(1)所示的化合物或其可药用盐在制备抗前列腺肿瘤药物中的应用，其特征在于，所述化合物或其可药用盐具有雄激素受体拮抗活性，抑制前列腺癌细胞增殖，
Figure FDA0002911300100000011
2.如权利要求1所述的应用，其特征在于，所述可药用盐为盐酸盐、磷酸盐、硫酸盐、醋酸盐、马来酸盐、枸橼酸盐、苯磺酸盐、甲基苯磺酸盐、富马酸盐或酒石酸盐。
3.如权利要求1所述的应用，其特征在于，还包括药学上可接受的赋形剂或载体。
4.如权利要求1所述的应用，其特征在于，药物组合物的制剂形式为液体制剂或固体制剂。
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