多巴胺D3受体选择性拮抗剂的设计与合成
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摘要
多巴胺受体分为5个亚型,包括D1、D2、D3、D4以及D5,其中,多巴胺D3受体(D3R)主要分布在与情感、认知密切相关的边缘脑区,是精神分裂症、帕金森氏症、运动障碍及药物成瘾等相关疾病的潜在治疗靶点,高亲和力和高选择性D3R配体的研究是药物研究热点之一。一些具有较高亲和力和选择性的D3R配体,例如,BP 897、NGB 2904、SB-277011-A等已完成了临床前研究,初步显示以D3R为靶点治疗相关疾病的功效和优势。然而,现有选择性D3R配体普遍存在水溶性差、生物利用度不高的缺点,结合药理实验和新药研发需要,本论文设计合成了一系列具有自主知识产权的高亲和力和选择性且类药性较好的D3R拮抗剂。
     本论文研究工作和结果如下:
     1.本课题以已报道的18个具有较高亲和力和选择性的D3R配体为基础,建立了D3R选择性配体的药效团模型,模型表明该类化合物共有的药效特征包括头尾2个芳环作用端(A与B)、1个氢键受体及1个质子化氮作用中心,其中,芳环A与B之间距离约10?。在此基础上,我们将该药效特征作为D3R定向库设计的限定条件,以期提高筛选命中率。同时,考虑“类药性”改善是当前D3R拮抗剂研究的重要方向,在组合库设计过程中,我们以“优势结构方法”(Privileged Structure Approach)为指导,在芳环A端选择“类药性”极佳的苯并噁(噻)唑啉-2-酮类作为构建单元。此外,在保证重要药效特征的基础上,还选择了4-羟基-喹啉、喹啉和二茂铁类作为芳环A端的构建单元。芳环B端首次选择了已商品化的1-(5-氯-2-甲基-苯基)哌嗪作为构建单元。中间连接链结构相对保守,主要选择易于合成的四亚甲基,并在其中引入了顺式、反式双键作为空间限制以考察空间构型改变对化合物活性的影响。
     2.在合成路线设计和方法选择中,我们主要以定向多样性合成(Diversity-Oriented Synthesis)策略为指导,以期通过单一反应实现多个目标化合物的衍生合成,从而提高合成效率和组合库分子的多样性。本研究完成了苯并噁(噻)唑啉-2-酮、羟基-喹啉、喹啉和二茂铁4个系列目标化合物的合成工作,共得到37个全新目标化合物,结构经核磁共振(1H NMR,13C NMR和1D NOESY)、质谱(MS)、红外光谱(IR)确证,HPLC对其纯度进行了分析,所有化合物纯度达98%以上。CA网络版(SciFinder)检索证实为新化合物,已对“系列4”苯并噁(噻)唑啉-2-酮类化合物申请专利保护(专利申请号:200810167089.5)。
     3.油水分配系数(LogP)是反映化合物水(脂)溶性大小的重要参数,针对现有多巴胺D3受体选择性配体水溶性差、生物利用度不高的缺点,本课题在D3R选择性拮抗剂的设计过程中,着重强调目标化合物水溶性的提高。因此,建立快速、可靠的实验方法以对目标化合物LogP值进行早期、快速、可靠的测定对指导后期化合物的设计与优化具有重要意义。本研究根据“非经典”阴离子对试剂—三氟乙酸的特殊性质,建立了一种反相离子对色谱方法(RP-IPC),该方法测定范围广,满足了结构中同时含有碱性可质子化氮原子和酸性酰胺官能团的目标化合物测定要求,并成功应用于计算方法不能预测的二茂铁类化合物LogP值快速测定。测定结果显示,除“系列3”二茂铁甲酰胺衍生物“类药性”较差(LogP>5),其余3个系列化合物LogP值均在2-5范围内。
     4.在D3R和D2R百分抑制率初步筛选基础上,以稳定转染的D3R-CHO和D2R-HEK-293细胞株为载体,通过放射性配体([3H]spiperone)竞争结合实验,分别测定了目标化合物对D3R、D2R的亲和力和选择性。结果表明,“系列4”中所测定化合物大部分显示极佳的D3R亲和力(Ki,D3R)和选择性(Rs=Ki,D2R/Ki,D3R),其中2个化合物Y-QA14和Y-QA33对D3R具有皮摩尔级亲和力和选择性,远远高于所有已报道亲和力和选择性最高的化合物。Y-QA14对D3受体Ki值为5.2×10-5 nM,对D2受体Ki值为134.5 nM,Rs=2.58×106;Y-QA33对D3R、D2R的亲和力和选择性分别为8.25×10-5 nM,3.063 nM和3.7×104,远远高于阳性对照NGB 2904(Ki,D3R=1.26 nM,Ki,D2R =149.2 nM;Rs= 118)。值得注意的是,Y-QA14和Y-QA33均显示对D3R具有高、低两个亲合位点,药理功能性实验研究表明,Y-QA14可以剂量依赖的拮抗激动剂喹吡罗(Quinpirole,10μM)的激动活性,为D3R选择性拮抗剂,且拮抗作用主要对应于高亲和力位点。
     5.药效学评价方面,考察了Y-QA14对小鼠条件性位置偏爱的影响,在吗啡点燃的小鼠条件性位置偏爱实验中,与盐水组相比,YQA14在0.1mg/kg和5mg/kg两个剂量时可以显著对抗吗啡点燃的条件位置偏爱的重现(*P<0.01),表明YQA14对吗啡造成的精神依赖有确定的抑制作用。
     6.重点对“系列4”苯并噁(噻)唑啉-2-酮类化合物进行了初步的构效关系分析,结合同源模建和分子对接研究,对得到的活性筛选数据进行了合理性的阐述,结果表明构效关系和分子对接模拟对部分活性筛选结果的阐述具有一定的一致性。在两者联合应用下,确定了该类化合物结构中一个重要的活性官能团(内酰胺),分别在受体结合水平和配体电性特征方面对其进行了较为详细的分析和讨论。此外,确定了该系列化合物以反式构型或自由度较大的直链烷烃作为连接部分对保持高亲和力和选择性有利。
The dopamine receptors have been found five receptor subtypes so far, which are divided into two receptor families, D1-like (D1 and D5) and D2-like (D2, D3, and D4). Among them, dopamine D3 receptor (D3R) is preferentially located in limbic brain regions which have an impact on emotional and cognitive functions.Various pharmacological studies have investigated the D3 system as an interesting therapeutic target for the treatment of schizophrenia, Parkinson's disease, drug-induced dyskinesia and drug abuse (in particular cocaine addiction). D3R ligands with high affinity and selectivity have been an active field of drug research in recent years. During the last decade, some of the compounds (e.g. BP 897, NGB 2904, SB-277011-A) that demonstrate both high affinity and selectivity for D3R have shown the potential for development of D3R antagonists and partial agonists as medications for treatment of the diseases related to D3R dysfunction. However, the poor aqueous solubility and limited bioavailability is always a major limitation for these described potent and selective D3R ligands and an obstacle for evaluation on these novel agents in behavioral animal models and their therapeutic potential. In this research, a series of novel compounds, with high selectivity and affinity for D3R in combination with impressive“drug-likeness”, has been designed and synthesized to meet the requirements of pharmacological research and potential drug development.
     The major results of our work can be summarized as follows:
     1.To increase the hits of candidates in the D3R-oriented combinatory library, a pharmacophore mode, which has been developed based on eighteen reported compounds that showed high affinity and selectivity for D3R, was applied as the filter standards. According to the model, all of the tested compounds bear the common features: two aromatic or heteroaromatic, one hydrogen donor, one hydrogen acceptor and one protonated center (basic group), and can be formulated as A-L-B, wherein, group A is the head with an aromatic ring bearing a hydrogen acceptor, group B is the tail with an aromatic ring conjugated with a basic group, and L is the linker between A and B which represents an alkyl-spacer with length about four methylene. Moreover, to increase the“drug-likeness”of our target compounds, the 2(3H)-benzox(thi)azolinone scaffolds was selected as an aromatic ring of group A in the combinatory library design inspired by the privileged structure approach. In addition, other building blocks such as 4-hydroxy-quinoline, quinoline, and ferrocene were incorporated into group A for our combinatory library design, alternativly. Furthermore, 1-(5-chloro-2-methylphenyl)piperazine was first selected as the groug B in D3R ligand design. Finally, in search for the potential effects of conformational changes, the semi-rigided 2-trans-butene-1,4-diyl and 2-cis-butene- 1,4-diyl were introduced as the linker (-L-) in addition to butan-1,4-diyl.
     2.Aim to obtain diversity of our target compounds with a single efficient round, the“diversity-oriented synthesis”approach is applied during our design and synthesis. Guided by this concept, thirty-seven compounds that belong to four series, respectively incorporated 2(3H)-benzox(thi)azolinone, 4-hydroxyquinoline, quinoline and ferrocene in group A , were synthesized and evaluated selectivity and affinity for D3R. All of the compounds synthesized are new chemical entities according to the search results of SciFinder, and the 2(3H)-benzox(thi)azolinone derivates of the forth serie has applied for patent protection (Application No, 200810167089.5). All compounds synthesized were characterized by their 1H NMR, 13C NMR,1D NOESY, MS and IR spectrum and further submitted to the HPLC analysis, the found purity is above 98% for the test compounds.
     3.The n-octanol/water partition coefficient (LogP) is widely used to represent molecular hydrophobicity or lipophilicity. It is of most important to increase the hydrophilicity of the target compounds in our research for developing novel dopamine D3 receptor antagonists with regard to the poor aqueous solubility and limited bioavailability of the reported ligands. To predict and monitor the LogP of our target compounds rapidly, we established a reversed phase ion-pair chromatography (RP-IPC) method based on the exceptional attributes of trifluoroacetic acid behaved as nonclassic ion-pair agents, which is applicable to determine the LogP values of the target compounds with wide pKa range, including both acidic amide groups and basic species or incorporated metal Fe atom in the structure, some of them can not be predicted by computer method. All of the target compounds except for ferrocene derivates show optimal“druglike”profiles as monitered by their LogP values, fallen between 2-5.
     4. Competition binding assays were employed to analyze affinity and selectivity profiles of the target compounds to the dopamine D2 and D3 receptors. The binding data was generated by measuring their ability to compete with [3H]-spiperone binding to the cloned human dopamine receptor subtypes D2 and D3 stably expressed in HEK-293 and Chinese hamster ovary cells (CHO). Most of the compounds in serial four showed impressively high affinity (Ki,D3R) and selectivity (Rs= Ki,D2R/ Ki,D3R) for the D3 receptor as compared to the control compound NGB 2904 (Ki,D3R =1.26 nM, Ki,D2R =149.2 nM; Rs=118). To the best of our knowledge, two new compounds can be identified as the best D3R ligands so far, which showed pico-molar binding affinity for D3R. For example, Y-QA14 displayed Ki value of 5.2×10-5 nM and 134.5 nM at human dopamine D2 and D3 receptors, respectively, with Rs of 2.58×106. Y-QA33 was somewhat weaker with Ki,D3R of 8.25×10-5 nM, Ki,D2R of 3.063 nM and Rs of 3.7×104.
     It is interesting to note that either Y-QA14 or Y-QA33 has two binding sites referring to the high- and low-affinity binding site, respectively. Further evaluation showed that compound Y-QA14 is a potent D3R antagonist and its antagonist activity is most responded to the high-affinity binding site as tested in a quinpirole-stimulated mitogenesis assay for functional activity at expressed human D3R in CHO cells.
     5. Further investigation was carried out in a conditioned place preference (CPP) paradigm, in which Y-QA14 showed substantial antagonist effects for morphine induced CPP at both the concentration of 0.1mg/kg and 5mg/kg(*P<0.01) intraperitoneally (i.p.).
     6. In this article, a good agreement between the results of structure activity relationship (SAR) and the observation in molecular docking is obtained for some of the test 2(3H)-benzox(thi)azolinone derivates that belong to the fourth series, and an important pharmacophore is identified and further validated according to the potential model of receptor binding and the form of charge distribution. In addition, introduction of a semi-rigid cis-butene linker into the library decrease the binding affinity and selectivity at the D3R as compared to the trans-butene linker and flexible butylene linker counterpart.
引文
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