NOVEL PROBES FOR THE DOPAMINE TRANSPORTER

多巴胺转运蛋白的新型探针

• 批准号: 6103909
• 负责人: Amy Hauck Newman
• 金额: --
• 依托单位: NATIONAL INSTITUTE ON DRUG ABUSE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6103909
• 关键词: benztropine; binding proteins; chemical structure function; cocaine; dopamine antagonists; dopamine receptor; dopamine transporter; drug abuse; drug design /synthesis /production; drug interactions; muscarinic receptor; norepinephrine; protein structure; psychopharmacology; radiotracer; receptor binding; reinforcer; serotonin transporter; stereochemistry; transport proteins

Benztropine is a dopamine uptake inhibitor, equipotent to cocaine, but without appreciable abuse in humans or a cocaine-like behavioral profile in animal models. Benztropine's interesting neurochemical and behavioral profile, as well as its chemical structure, which shares some components (tropane-based ring) with cocaine and yet is different (lacks a 2-substituent and possesses a 3-diphenylmethoxy substituent) provided a promising lead compound. The goal of the initial studies was to identify optimum structural components for high affinity binding at the DAT, improve selectivity over the other monoamine transmitter transporters and reduce or eliminate muscarinic receptor binding, which for the parent compound was 100-fold more potent than at the DAT. The design, synthesis and in vitro evaluation of over 100 3-substituted and N-substituted BZT analogs led to the discovery of compounds with high affinity at the DAT (Ki=10-30 nM), selectivity over serotonin and norepinephrine transporters (100-2000-fold) and selectivity over muscarinic receptors (<50-fold). Molecular modeling studies using Conformational Molecular Field Analysis (CoMFA) have been performed that provide models for both the DAT and muscarinic receptors and demonstrate a divergence in the optimal structural components for these molecular targets as well as clues to further separability. These studies have also led to the synthesis of a radioiodinated photoaffinity ligand which has been demonstrated to covalently attach to the DAT. Proteolytic and immunological peptide mapping studies have shown that this ligand labels a binding domain on the DAT that is near transmembrane spanning regions 1-2. This site appears to be similar to that labeled by the GBR 12935-based photoaffinity label [125I]DEEP and in contrast, is different from the binding domain of the cocaine-related photoaffinity label [125I]RTI 82. These studies are the first to demonstrate that these tropane-based photoaffinity labels covalently link to different binding sites on the DAT and support the divergent SAR reported by our group and others between these classes of dopamine uptake inhibitors. Behavioral evaluation of many of the BZT analogs have all demonstrated that, with few exceptions, despite high affinity and selectivity for the DAT, the BZTs are not efficacious locomotor stimulants. In vivo microdialysis studies on 4'Cl-BZT demonstrated that this compound, at a dose equivalent to cocaine (40 mg/kg, i.p.) significantly increased dopamine efflux over baseline, demonstrating that the drug penetrates the blood brain barrier and blocks dopamine uptake, in vivo. Despite this neurochemical profile, 4'Cl BZT demonstrated a behavioral profile that was distinctive from cocaine. Upon further behavioral evaluation, 3'Cl and 4'Cl BZT were found to be self administered in rhesus monkeys, trained to self administer cocaine. However, these compounds did not appear to be as strongly reinforcing as cocaine. The 3',4"-diCl and the N-butylphenyl-4',4"diF analogs were not self administered in this model. A second class of dopamine uptake inhibitors, based on rimcazole, were discovred to bind to DAT equipotently to cocaine (Ki=100 nM) but these compounds are not efficacious locomotor stimulants. In order to better characterize the structural features that were required for DAT binding and to further assess whether or not these compounds were accessing a low affinity binding site on the DAT, an expanded series of rimcazole analogs were prepared where chemical modifications were made at the terminal piperazine nitrogen and at the carbazole ring structure. These compounds were evaluated in vitro for binding at the monoamine transporters and sigma receptors. SAR revealed that increased potency at the DAT, in general, resulted as the compounds more closely resembled GBR 12909. In addition, the rimcazole analogs, like the parent compound, monophasically displaced [3H]WIN 35,428 from the DAT and inhibited dopamine uptake. Further, the behavioral profile of the two most potent compounds, in this series, closely resembled the parent compound rimcazole, in animal models of cocaine abuse, despite structural similarity to GBR 12909. At this time it is unclear whether or not these rimcazole analogs bind to a binding domain on the DAT which is accessed by the GBR analogs. The role of sigma receptors and other neurochemical substrates for these analogs in their behavioral profile is currently under study. In an effort to further characterize the binding site of these rimcazole analogs, an N-alkylisothiocyanato-derivative of rimcazole has been discovered to be a novel irreversible ligand for the DAT. In addition, it appears that this analog has selectivity for the low affinity binding site, labeled by [3H]WIN 35,428. This finding suggests that this rimcazole analog may serve to discriminate functional correlates to the high and low affinity binding sites on the DAT. Meperidine, a mu-opioid receptor agonist, binds to the DAT and exhibits exaggerated biphasic inhibition of dopamine uptake in a chopped tissue preparation, as compared to cocaine. Based on the neurochemical and behavioral profile of meperidine, it was suggested that this drug may be interacting primarily with a high affinity component or conformational state of the DAT that is responsible for its psychomotor stimulant actions. It was recognized that meperidine shared some structural features of WIN 35,065, a cocaine analog. Based on the observation that the tropane ring in WIN 35,065 provides a conformationally rigid piperidine, the structurally rigid analogs of meperidine were initially targeted as starting points for SAR at the DAT. The tropane-ring based meperidine analogs, where the piperidine ring is set in a chair conformation, were obtained commercially. However, the azabicyclo[2.2.1]heptane system, where the piperidine is set in a boat conformation, had to be prepared. Thus, a stereoselective and high yielding synthesis of one of the stereoisomers was achieved and the synthesis of the opposite stereoisomer is underway.

苯托品是一种多巴胺摄取抑制剂， 与可卡因等效，但对人类或人类没有明显的滥用 动物模型中类似可卡因的行为特征。苯托品 有趣的神经化学和行为特征，以及它的 化学结构，共享一些成分（基于托烷的 环）与可卡因但不同（缺少 2 个取代基并且 具有3-二苯基甲氧基取代基）提供了一种有前途的 铅化合物。初步研究的目标是确定 用于高亲和力结合的最佳结构组件 DAT，提高了相对于其他单胺发射机的选择性 转运蛋白并减少或消除毒蕈碱受体结合， 其母体化合物的效力比原化合物强 100 倍 数据。超过100个的设计、合成和体外评价 3-取代和N-取代的BZT类似物导致了这一发现 在 DAT 处具有高亲和力的化合物 (Ki=10-30 nM)， 对血清素和去甲肾上腺素转运蛋白的选择性 （100-2000 倍）和对毒蕈碱受体的选择性 （<50 倍）。使用构象的分子建模研究 分子场分析 (CoMFA) 已进行 提供 DAT 和毒蕈碱受体模型 证明最佳结构组件的差异 这些分子目标以及进一步分离的线索。 这些研究还导致了放射性碘化物的合成。 光亲和配体已被证明可以共价 附加到 DAT。蛋白水解和免疫肽图谱 研究表明，该配体在 DAT 靠近跨膜跨越区域 1-2。这个网站 似乎与基于 GBR 12935 的标签相似 光亲和标签 [125I]DEEP 和相反，不同于 可卡因相关光亲和标记 [125I]RTI 的结合域 82. 这些研究首次证明这些 基于托烷的光亲和标签共价连接到不同的 DAT 上的结合位点并支持报告的不同 SAR 由我们小组和其他人在这些类别的多巴胺吸收之间进行 抑制剂。 许多 BZT 类似物的行为评估 所有这些都表明，除了极少数例外，尽管有很高的亲和力和 由于 DAT 的选择性，BZT 不是有效的运动 兴奋剂。 4'Cl-BZT 体内微透析研究表明 该化合物的剂量相当于可卡因（40 mg/kg，腹膜内注射） 与基线相比，多巴胺流出显着增加，表明 该药物可穿透血脑屏障并阻断 体内多巴胺的摄取。尽管有这种神经化学特征，4'Cl BZT 表现出与其他人不同的行为特征 可卡因。 经进一步 行为评估，发现 3'Cl 和 4'Cl BZT 是自我 在恒河猴中进行注射，经过训练可以自行注射可卡因。 然而，这些化合物似乎没有那么强烈 像可卡因一样增强。 3',4"-diCl 和 N-丁基苯基-4',4"diF类似物在本次试验中并未自行施用 模型。 第二类多巴胺摄取 发现基于 rimcazole 的抑制剂可与 DAT 结合 与可卡因等效 (Ki=100 nM)，但这些化合物不是 有效的运动兴奋剂。为了更好地表征 DAT 结合所需的结构特征 进一步评估这些化合物是否正在访问 DAT 上的低亲和力结合位点，一系列扩展 林卡唑类似物是通过化学修饰制备的 在末端哌嗪氮和咔唑处制备 环状结构。这些化合物在体外进行了评估 结合单胺转运蛋白和西格玛受体。 SAR 揭示了 DAT 效力的增加，一般来说，结果是 这些化合物更类似于 GBR 12909。此外， 林卡唑类似物，像母体化合物一样，单相 从 DAT 中置换 [3H]WIN 35,428 并抑制多巴胺 吸收。此外，两个最有影响力的人的行为特征 该系列中的化合物与母体化合物非常相似 在可卡因滥用的动物模型中，尽管结构 与GBR 12909相似。目前尚不清楚是否 这些 rimcodium 类似物与 DAT 上的结合域结合 由 GBR 类似物访问。西格玛受体的作用 以及这些类似物的其他神经化学底物 目前正在研究行为概况。 为了进一步 表征这些 rimcodium 类似物的结合位点， 发现了林卡唑的 N-烷基异硫氰酸酯衍生物 成为 DAT 的新型不可逆配体。另外，看来 该类似物对低亲和力结合位点具有选择性， 由 [3H]WIN 35,428 标记。这一发现表明，这 rimc唑类似物可用于区分功能相关性 DAT 上的高亲和力和低亲和力结合位点。 哌替啶，一种 mu-阿片受体激动剂，与 DAT 结合并表现出 切碎的多巴胺摄取的夸大双相抑制 组织制备，与可卡因相比。基于 哌替啶的神经化学和行为特征， 表明该药物可能主要与高 DAT 的亲和成分或构象状态是 负责其精神运动兴奋作用。被认可了 哌替啶与 WIN 35,065 具有一些结构特征， 可卡因类似物。根据观察，托烷环 WIN 35,065 提供了一种构象刚性的哌啶， 哌替啶的结构刚性类似物最初的目标是 DAT 的 SAR 起点。基于托烷环的 哌啶类似物，其中哌啶环设置在椅子上 构象，是商业获得的。然而， 氮杂双环[2.2.1]庚烷系统，其中哌啶设置在 船的构造，必须做好准备。因此，立体选择性和 实现了一种立体异构体的高产率合成 相反的立体异构体的合成正在进行中。