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.

苯甲衣是一种多巴胺摄取抑制剂， 对可卡因的等值，但在人类中没有明显的虐待或 动物模型中的可卡因样行为曲线。苯甲衣 有趣的神经化学和行为概况及其 化学结构，共享一些组件（基于Tropane 带有可卡因的环），但有所不同（缺少2-掩埋的且 拥有3-二苯基氧基取代基）提供了有前途的 铅化合物。最初研究的目的是确定 高亲和力结合的最佳结构成分 DAT，提高对其他单胺发送器的选择性 转运蛋白并减少或消除毒蕈碱受体结合， 对于父母而言，这比在 dat。超过100多个的设计，合成和体外评估 3-取代和n个取代的BZT类似物导致了发现 Dat（Ki = 10-30 nm）的高亲和力的化合物 对5-羟色胺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 已经进行了分子场分析（COMFA） 为DAT和毒蕈碱受体提供模型以及 证明最佳结构组件的分歧 这些分子靶标以及进一步可分离性的线索。 这些研究也导致了放射性约束的合成 光性配体已被证明是共价的 附加到dat。蛋白水解和免疫学肽图 研究表明，该配体在 DAT靠近跨膜区域1-2。这个网站 似乎与基于GBR 12935标记的相似 光性标签[125i]深，相反， 与可卡因相关的光性标签的结合结构域[125i] rti 82。这些研究是第一个证明这些研究 基于Tropane的光性标签共价链接到不同的 DAT上的绑定位点并支持Divergent SAR报告 由我们的小组和其他多巴胺摄取之间的其他人 抑制剂。 许多BZT类似物的行为评估 所有这些都表明，尽管有很高的亲和力，但除了少数例外 DAT的选择性，BZTS不是有效的运动 兴奋剂。 4'Cl-BZT的体内微透析研究证明了 该化合物的剂量相当于可卡因（40 mg/kg，i.p.） 在基线上显着增加了多巴胺外排 药物穿透了血脑屏障并阻塞 多巴胺摄取，体内。尽管有这种神经化学概况，但4'Cl BZT展示了与 可卡因。 进一步 发现行为评估，3'Cl和4'cl BZT是自我的 在恒河猴中管理，接受了自我管理可卡因的训练。 但是，这些化合物似乎并不那么强烈 加强可卡因。 3'，4“ -dicl和 N-丁基苯基-4'，4英寸的dif类似物在此中没有自我施用 模型。 第二类多巴胺摄取 基于利姆卡尔（Rimcazole）的抑制剂被裂解以与DAT结合 等级到可卡因（Ki = 100 nm），但这些化合物不是 有效的运动兴奋剂。为了更好地描述 DAT绑定所需的结构特征和 进一步评估这些化合物是否正在访问 DAT上的低亲和力绑定站点，一系列扩展 在化学修饰的情况下制备了利马唑类似物 是在末端哌嗪氮和甲状腺癌上制造的 环结构。在体外评估这些化合物的 在单胺转运蛋白和Sigma受体上结合。 SAR 表明，总体上，DAT的效力增加导致 这些化合物更像GBR 12909。此外， 利姆卡尔的类似物，像父元一样，单向上 流离失所[3H]从DAT中赢得35,428并抑制多巴胺 吸收。此外，这两个最有效的行为概况 在本系列中的化合物非常类似于父级化合物 尽管结构性 与GBR 12909的相似性。目前尚不清楚是否清楚 这些Rimcazole类似物与DAT上的绑定域结合 GBR类似物可以访问。 Sigma受体的作用 和其他这些类似物的神经化学底物 目前正在研究行为概况。 为了进一步 表征这些利姆卡尔类似物的结合位点，一个 已经发现了Rimcazole的N-烷基异硫代氨基氨基氨基氨基氨基利道衍生物 成为一种新颖的不可逆配体。此外，它似乎 该模拟对低亲和力结合位点具有选择性， 由[3H]赢得35,428。这一发现表明这一点 利姆卡尔类似物可能有助于区分功能相关 DAT上的高和低亲和力绑定位点。 Mepridine，a Mu-Apoid受体激动剂与DAT结合并展示 夸张的双相抑制在切碎的多巴胺摄取 与可卡因相比，组织制备。基于 神经化学和行为概况，是 建议这种药物可能主要与高 DAT的亲和力组件或构象状态 负责其精神运动刺激的行动。它被认可 Mepridine分享了Win 35,065的一些结构性特征， 可卡因类似物。基于观察到的Tropane环在 Win 35,065提供了构象上的刚性哌啶， 最初以结构性刚性类似物作为靶向 SAR在DAT的起点。基于Tropane环 Mepridine类似物，哌啶环在椅子上设置在椅子上 构象在商业上获得。但是， azabicyclo [2.2.1]亨普烷系统，其中哌啶设置为 船体构型必须做好准备。因此，立体选择性 实现了一个立体异构体的高屈服合成 相反的立体异构体的合成正在进行中。