Liver Targeting Dihydroquinolizinone (DHQ) Molecules as Hepatitis B Virus Antivirals with Reduced Toxicity

摘要 B型肝炎病毒慢性感染全球2.58亿人，每年造成88万人死亡， 肝硬化、肝细胞癌（HCC）和肝功能衰竭。目前的标准治疗药物，包括 调节宿主抗病毒免疫应答的聚乙二醇化干扰素α和抑制 病毒DNA聚合酶，可以有效地抑制病毒复制，但不能诱导HBV表面抗原的丢失 乙型肝炎病毒表面抗原（HBsAg）是成功免疫控制或慢性B型肝炎功能性治愈的标志， 大多数接受治疗的患者。因此，开发具有新机制的新型抗病毒药物， 激活宿主抗病毒免疫应答是实现慢性B型肝炎功能性治愈的必要条件 （CHB）.罗氏发现的一种小分子二氢喹嗪酮（DHQ）RG-7834可以迅速减少 B型肝炎病毒（HBV）RNA水平，因此几乎所有的病毒基因产物，包括HBsAg。这代表 一种全新的HBV抗病毒药物化学型和一种控制CHB的新方法。然而，观察到 RG-7834的神经毒性禁止其全身用于CHB管理。我们假设这一边 可以通过生产肝选择性和CNS难治性DHQ抗病毒剂来最小化或消除这种影响。支持 基于这一假设，我们最近设计并合成了一系列新的基于双羧酸的DHQ 在本系列中首次显示具有有机阴离子转运多肽（OATP）的衍生物， 底物性质，其促进化合物选择性分布至作用的治疗部位 （肝脏），相对于血流。这一发现导致了一种早期铅的发现，称为DHQ-65042， 在生物化学和细胞测定中均有效（在HepG2.2.15细胞系中EC 50 = 23 nM），同时证明 与RG-7834相比，具有相当大的安全性改善潜力，例如血脑屏障（BBB）降低 渗透性（体外试验）和体内肝脏对血浆的高选择性（小鼠PK中肝脏/血浆比为37.8 研究）。这一新的肝靶向DHQ系列有望通过有限的剂量改善安全性。 尽管DHQ-65042在血液、大脑和其他身体组织中的暴露， 在一项药代动力学（PK）研究中，经口给药途径对肝脏的毒性为中度。在R21提案中，为了验证 开发新型肝靶向DHQ衍生物作为B型肝炎病毒表面抗原还原剂的可行性， 实际价值，我们将进行铅优化，以改善肝脏暴露到有效水平， 维持血浆中的暴露量，并确定肝选择性和GI/肝脏之间的平衡窗口 通过三个目标中提出的工作吸收新的线索。该项目的成功完成将 使我们能够开发新一代的DHQ，选择性地输送到肝脏，浓度足以 足以显著降低HBsAg并具有更好的安全性，并提供一种新的体内电极导线 毒性和功效研究。

ABSTRACT Hepatitis B virus chronically infects 258 million people worldwide and causes 880 thousand deaths annually due to cirrhosis, hepatocellular carcinoma (HCC) and liver failure. The current standard of care medications, including pegylated interferon alpha that regulates host antiviral immune response and nucleos(t)ide analogues that inhibit viral DNA polymerase, can potently suppress viral replication, but fail to induce the loss of HBV surface antigen (HBsAg), an indication of successful immune control or the functional cure of chronic hepatitis B, in the vast majority of the treated patients. Therefore, the development of novel antivirals with a new mechanism that can activate the host antiviral immune response is required to achieve the functional cure of chronic hepatitis B (CHB). A dihydroquinolizinone (DHQ) RG-7834, a small molecule discovered by Roche, can rapidly reduce hepatitis B virus (HBV) RNA levels, and hence almost all viral gene products, including HBsAg. This represents an entirely new chemotype of HBV antivirals and a new approach for CHB control. However, the observation of neurotoxicity in RG-7834 prohibits its systemic use for the management of CHB. We hypothesize that this side effect can be minimized or eliminated by producing liver selective and CNS refractory DHQ antivirals. In support of this hypothesis, we have recently designed and synthesized a novel series of bis-carboxylic acid based DHQ derivatives that are shown, for the first time in this series, to have organic anion transporting polypeptide (OATP) substrate properties, which facilitates selective distribution of the compounds to the therapeutic site of action (liver), relative to the blood stream. This effort led to the discovery of an early lead, called DHQ-65042, which is potent in both biochemical and cellular assays (EC50 = 23 nM in HepG2.2.15 cell line), while demonstrating considerable safety improvement potential in comparison to RG-7834, such as low blood-brain barrier (BBB) penetration (in vitro assay) and high in vivo liver selectivity over plasma (liver/plasma ratio is 37.8 in a mouse PK study). This novel series of liver targeting DHQs are promising for improving safety profiles through limited distribution to the blood stream, the brain, and other body tissues, though the exposure of DHQ-65042 in the liver from the oral route of a pharmacokinetic (PK) study is moderate. In this R21 proposal, to validate the feasibility of developing novel liver targeting DHQ derivatives as hepatitis B virus surface antigen reducers with practical value, we will perform lead optimization to improve the liver exposure to an efficacious level with low exposure in plasma maintained, and identify a balance window between hepatoselectivity and GI/ liver absorption of new leads through the works proposed in three aims. Successful completion of this project will allow us to develop a new generation of DHQs that are selectively delivered to livers with concentrations sufficient enough to significantly reduce HBsAg and have better safety profiles, and to provide a new lead ready for in vivo toxicity and efficacy studies.