Supplement to Discovery of a high affinity, selective and beta-arrestinbiased 5-HT7R Agonist Grant

5-羟色胺7受体（5-HT 7 R）是G蛋白的一个成员， 偶联5-HT受体家族。许多研究表明，5-HT 7 R在各种疾病中起着重要作用。 行为和心理生理功能，如情绪稳定性、认知和运动功能、行为和心理功能， 抑制、成瘾行为、疼痛耐受性、睡眠模式、食欲、激素变化和体温调节。 临床前研究结果已经确定了5-HT 7 R在自闭症谱系障碍、脆性X综合征中的作用 (FXS)癫痫、睡眠障碍、神经性疼痛和偏头痛。因此，5-HT 7 R正在成为一个潜在的目标， 用于治疗各种精神疾病和其他相关疾病。同时，几种5-HT 7 R激动剂， 包括AS-19、E-55888、LP-12、LP-44、LP-211、AGH-192和化合物1g， 在各种CNS病症（疼痛、记忆和认知），神经性疼痛，睡眠障碍， 正在调查酗酒和吸毒问题。然而，激活或阻断该受体的有益效果并不明显。 在这些条件中的每一种中的5-HT 7 R通常不清楚地确定，主要是由于缺乏选择性的5-HT 7 R。 HT 7 R特工更关键的是缺乏有偏见的配体，可以澄清几个有争议的 与5-HT 7 R有关的观察。因此，我们的目标是设计，合成和重复评估 具有偏向G蛋白或β-抑制蛋白信号通路的信号传导的新药物在 了解5-HT 7 R及其在治疗各种CNS疾病中的应用。 在本申请中提出了三个具体目标。具体目标1将侧重于扩大我们的 先导化合物（44080、55933和57544）的药物样性质研究，包括脑 渗透、药代动力学研究、代谢特征分析、生物利用度评估和心血管 毒性预测（HERG，5-HT 2BR）。根据我们对铅化合物55933的代谢评价， 初步研究，具体目标2中提出了新化合物的合成和筛选。该特定 aim还将重点关注新制剂的优化和设计，以解决代谢稳定性问题， 如在初步研究中观察到的芳构化和葡萄糖醛酸化。因此，芳构化 敏感的四氢异喹啉（THI）部分将被不能进行 芳构化和CH 2 OH基团将被取代基如-F、-CONH 2取代，以限制 葡萄糖醛酸化，同时保持良好的药物样特征。此外，生物电子等排置换 将进行具有二氢萘酮（DNO）部分茚满酮。此外，探索电子 THI/异吲哚啉周围的供/吸（σ值）和亲水/疏水（pi值）空间 具有取代基如Cl、OMe、CN、SOCH 3和NMe 2的环系统将揭示它们的性能的任何改进。 药物特征。同时，对接研究将使用同源模型进行，以确定 与参与诱导β-抑制蛋白相关构象的关键氨基酸残基的相互作用 5-HT 7 R的招募具体目标3将涵盖铅5-HT 7 R配体的功能选择性研究， 它们的激动剂/拮抗剂性质和G蛋白或β-抑制蛋白信号传导偏倚，然后评价它们的 对体内条件下睡眠结构和NREM/REM睡眠模式的影响。最后，选择测试 将化合物与SB 269970（5-HT 7 R拮抗剂）和化合物1g（5-HT 7 R部分激动剂）进行比较 (as阳性对照）对NREM/REM睡眠模式的影响和β-arrestin偏性活性的相关性 使用小鼠模型。 项目摘要/摘要第6页

Project Summary/Abstract: The 5-hydroxytryptamine 7 receptor (5-HT7R) is a member of the G protein- coupled 5-HT receptor family. Numerous studies have indicated that 5-HT7R plays a significant role in various behavioral and psychophysiological functions such as mood stability, cognitive and motor functions, behavioral inhibition, addictive behaviors, pain tolerance, sleep patterns, appetite, hormonal changes and thermoregulation. Pre-clinical findings have established the role of 5-HT7R in autism spectrum disorders, Fragile X syndrome (FXS), epilepsy, sleep disorders, neuropathic pain and migraine. Thus, 5-HT7R is emerging as a potential target for the treatment of various psychiatric and other associated disorders. Meanwhile, several 5-HT7R agonists, including AS-19, E-55888, LP-12, LP-44, LP-211, AGH-192 and compound 1g have been reported and their potential use in various CNS conditions (pain, memory and cognition), neuropathic pain, sleep disorders, alcohol and drug abuse are being investigated. However, the beneficial effects of activation or blockade of the 5-HT7R in each of these conditions is not often clearly established, primarily due to the lack of selective 5- HT7R agents. Even more critical is the absence of biased ligands that could clarify several controversial observations that relate to the 5-HT7R. Thus, our goal to design, synthesize and pharmacologically evaluate new agents with biased signaling towards G-Protein or β-arrestin signaling pathways hold great promise in understanding the 5-HT7R and its application to the treatment of various CNS disorders. There are three specific aims proposed in this application. Specific aim 1 will focus on extending our studies on the lead compounds (44080, 55933 and 57544) for their drug-like properties including brain penetration, pharmacokinetic studies, metabolic profiling, bioavailability assessments and cardiovascular toxicity predictions (HERG, 5-HT2BR). Based on the metabolic evaluations of lead compound 55933 from our preliminary studies, the synthesis and screening of new compounds is proposed in specific aim 2. This specific aim will also focus on the optimization and design of new agents to address the metabolic stability issues such as aromatization and glucuronidation observed in the preliminary studies. For this reason, aromatization susceptible tetrahydroisoquinoline (THI) moiety will be replaced with isoindoline which could not undergo aromatization and the CH2OH group will be replaced with substituents such as -F, -CONH2 to restrict glucuronidation while maintaining good drug-like characteristics. In addition, bioisosteric replacement of indanone with dihydronaphthalenone (DNO) moiety will be carried out. Furthermore, exploring the electron donating/withdrawing (σ values) and hydrophilic/hydrophobic (pi values) space around the THI/isoindoline ring systems with substituents such as Cl, OMe, CN, SOCH3 and NMe2, will reveal any improvements in their drug-like characteristics. Simultaneously, docking studies will be carried out using homology models to identify interactions with the key amino acid residues involved in inducing conformations associated with β-arrestin recruitment to the 5-HT7R. Specific Aim 3 will cover functional selectivity studies of lead 5-HT7R ligands for their agonist/antagonist properties and G-Protein or β-arrestin signaling bias followed by evaluation of their effect on sleep architecture and NREM/REM sleep pattern under in vivo conditions. Finally, selected test compounds will be compared with SB269970 (5-HT7R antagonist) and compound 1g, a 5-HT7R partial agonist (as a positive control) for their effect on NREM/REM sleep pattern and correlation of β-arrestin biased activity using a mice model. Project Summary/Abstract Page 6