Novel Probes For The Dopamine Transporter

通过多巴胺转运体(DAT)抑制多巴胺再摄取已被认为是可卡因产生精神运动兴奋作用的主要机制。为了进一步了解可卡因药理作用的分子机制，以及滥用可卡因的机制，结构-功能研究一直致力于在分子水平上表征DAT蛋白。3-α-(二苯甲氧基)托烷(BZT)类似物的设计、合成和评价为DAT提供了有效和选择性的探针。结构-活性关系(SAR)已经被开发出来，尽管结构上有很大的相似之处，但与可卡因的描述形成了对比。此外，在可卡因滥用的动物模型中，对许多BZT类似物的行为评估表明，这两类基于托烷的多巴胺摄取抑制剂具有不同的药理学特征。总的来说，我们之前的研究表明，BZT类似物在小鼠身上没有显示出有效的运动刺激，不能完全取代可卡因的歧视性刺激，在恒河猴身上也不能明显地自我给药。在体外，这些化合物作为多巴胺摄取抑制剂通常比可卡因更有效，尽管它们在体内的作用与这一作用不一致。这些观察表明，5-羟色胺和去甲肾上腺素转运体以及M受体的不同结合特征可能对这些化合物的药理作用产生重大影响。此外，通过改变母体化合物的结构从而改变它们的物理性质，药代动力学(PK)以及药效学(PD)将受到直接影响。与可卡因相比，使用一系列N-取代的BZT类似物在体外和体内模型中评估这些化合物以获得这些药物上的PK和PD曲线，结果表明这些化合物很容易穿透血脑屏障，但与可卡因相比，它们的起效和持续时间更慢，这是一个适合作为药物治疗药物开发的曲线。此外，还合成了两个新的系列化合物(1)新的()-2-取代-4‘，4“-dif BZT和(2)N-取代的4’，4”-dif-BZT，它们的N-取代基比前几代化合物的亲油性要低。对这两个系列化合物的体外评价表明，许多化合物在DAT中具有良好的耐受性，并显示出对该靶标的高亲和力和选择性。DAT的SAR进一步支持了我们之前的发现，即与可卡因类基于托烷的DAT抑制剂相比，BZT没有相同的结构要求或相同的结构修饰来优化DAT结合。然而，第一次，()-2-取代的BZT在运动活动和药物识别方面确实显示出类似可卡因的作用。对这类化合物的相关结构和药理作用的进一步研究正在进行中，并将这些药物开发为潜在的可卡因滥用疗法。除了开发用于体内研究的试剂外，我们还合成了一些重要的放射性和/或不可逆配体形式的分子工具。我们已经合成了基于托烷的DAT抑制剂的叠氮(光激活)和异硫氰酸衍生物的放射性碘类似物，目前正在结合蛋白质分解和MALDI MS来阐明这些化合物与DAT和SERT共价结合的跨膜结构域。我们还合成了MFZ 2-12的第一个半胱氨酸选择性MTS和马来酰亚胺类似物，并与半胱氨酸导向的DAT突变研究结合使用，以表征这种可卡因样不可逆配体的结合区域。在另一系列化合物中，以Sigma受体拮抗剂利姆卡唑为基础，设计合成了[3-顺-3，5-二甲基-1-哌嗪基]双-(4‘-氟苯基)胺类化合物。我们发现用双-(4‘-氟苯基)胺取代利姆卡唑的咔唑环系可以提高DAT的结合亲和力和选择性。到目前为止，这一系列中最有效的化合物显示出与DAT的亲和力(Ki=17.5nM)，与GBR 12909相当。尽管这些化合物在DAT上具有高亲和力结合，并且结构类似于GBR 12909，但初步研究表明，这些化合物的行为更像利姆卡唑，而不是GBR 12909，并且在小鼠身上没有表现出可卡因样的行为特征。然后合成了另一系列类似物，其中亲脂性降低，先导化合物的结合亲和力提高到Ki=8.5 nM。这些化合物还被评估为sigma配体，并进行了使用CoMFA和比较DAT和sigma受体要求的3D-QSAR研究。这一系列化合物似乎在DAT和SIGMA上都有双重作用，这可能被用来开发治疗可卡因滥用的药物。目前，正在对选定的化合物在WT和Datko小鼠身上进行条件性位置偏爱评估，以进一步阐明它们的作用机制。除了经典的药物设计和合成，我们还继续我们的计算化学工作，利用比较分子场分析(CoMFA)方法和相应的DAT结合亲和力，对利马唑类似物以及一系列2-取代3-芳基托烷进行了三维定量构效关系(3D-QSAR)研究。这些研究导致了新的2-取代托烷和利姆卡唑类似物的设计，我们将继续用它们来阐明DAT结合部位的结构和功能。

The inhibition of dopamine reuptake via the dopamine transporter (DAT) has been characterized as the primary mechanism by which cocaine produces its psychomotor stimulant actions. In order to understand further the molecular mechanisms underlying the pharmacological actions of cocaine, as well as mechanisms that underlie its abuse, structure-function studies have been directed toward characterizing the DAT protein at a molecular level. The design, synthesis and evaluation of 3-alpha-(diphenylmethoxy)tropane (benztropine, BZT) analogs have provided potent and selective probes for the DAT. Structure-activity relationships (SAR) have been developed that contrast with those described for cocaine, despite significant structural similarity. Furthermore, behavioral evaluation of many of the BZT analogs, in animal models of cocaine abuse, has suggested that these two classes of tropane-based dopamine uptake inhibitors have distinct pharmacological profiles. In general, our previous studies have shown that the BZT analogs, do not demonstrate efficacious locomotor stimulation in mice, do not fully substitute for a cocaine discriminative stimulus and are not appreciably self-administered in rhesus monkeys. These compounds are generally more potent than cocaine as dopamine uptake inhibitors, in vitro, although their actions in vivo are not consistent with this action. These observations suggest that differing binding profiles at the serotonin and norepinephrine transporters as well as at muscarinic receptors might have significant impact on the pharmacological actions of these compounds. In addition, by varying the structures of the parent compounds and thereby modifying their physical properties, pharmacokinetics (PK) as well as pharmacodynamics (PD) will be directly affected. Evaluating these compounds in both in vitro and in vivo models to obtain PK and PD profiles on these agents, in comparison to cocaine, with a series of N-substituted BZT analogues has demonstrated that these compounds readily penetrate the blood brain barrier, but compared to cocaine, they have a slower onset and duration of action, which is a suitable profile for development as pharmacotherapeutics. In addition, the synthesis of two additional series (1) novel (+)-2-substituted-4',4"-diF BZTs and (2) novel N-substituted 4',4"-diF-BZTs, whose N-substituents were designed to be less lipophilic then previous generations of compounds were prepared. In vitro evaluation of both series of compounds demonstrated that many were well tolerated at the DAT and showed both high affinity and selectivity for this target. SAR at the DAT further supports our previous findings wherein the BZTs do not share structural requirements nor the same structural modifications to optimize DAT binding, as compared to the cocaine class of tropane-based DAT inhibitors. Nevertheless, for the first time, the (+)-2-substituted BZTs did demonstrate cocaine-like actions in both locomotor activity and drug discrimination. Further investigation into correlating structure and pharmacological action of this class of compounds and developing these agents as potential cocaine-abuse therapeutics is ongoing. In addition to developing agents for in vivo studies, we have also synthesized a number of important molecular tools in the form of radioactive and/or irreversible ligands. Radioiodinated analogues of both azido (photoactivated) and isothiocyanato-derivatives of our tropane based DAT inhibitors have been synthesized and are currently being utilized in combination with proteolysis and MALDI MS to elucidate transmembrane domains at which these compounds bind covalently to both DAT and SERT. We have also synthesized the first cysteine-selective MTS and maleimide analogs of MFZ 2-12, that are being used in combination with cysteine-directed mutagenesis studies of the DAT to characterize the binding domain of this cocaine-like irreversible ligand. In another series of compounds, based on the sigma receptor antagonist, rimcazole, [3-cis-3,5-dimethyl-1-piperazinyl)alkyl] bis-(4'-fluorophenyl)amines were designed and synthesized. We found that substitution of the carbazole ring system of rimcazole with bis-(4'-fluorophenyl)amine improves binding affinity and selectivity for the DAT. The most potent compound in this series to date showed a DAT binding affinity of (Ki=17.5 nM) which is comparable to GBR 12909. Despite high affinity binding at DAT, and structural similarity to GBR 12909, preliminary studies suggest that these compounds behave more like rimcazole than GBR 12909 and do not demonstrate a cocaine-like behavioral profile in mice. Another series of analogues were then prepared in which lipophilicity was decreased and binding affinity of the lead compound improved to Ki = 8.5 nM. These compounds have also been evaluated as sigma ligands and 3D-QSAR studies using CoMFA and comparing DAT and sigma receptor requirements have been undertaken. This series of compounds appear to have dual actions at both DAT and sigma, which may be exploited in the development of agents to treat cocaine abuse. Currently, selected compounds are being evaluated for conditioned place preference in WT and DATKO mice to further elucidate the mechanism of their actions. In addition to classical drug design and synthesis, we have continued our computational chemistry efforts with a three dimensional quantitative structure-activity relationship (3D-QSAR) study on both the rimcazole analogues as well as a series of 2-substituted 3-aryltropanes, using the comparative molecular field analysis (CoMFA) method with their corresponding DAT binding affinities. These studies have led to the design of novel 2-substituted tropane and rimcazole analogues with which we will continue to elucidate structure and function of the binding sites of the DAT.