Chronic hepatitis B virus (HBV) infection is a major global health problem and can lead to serious health issues such as cirrhosis or liver cancer [1]. WHO estimates that 296 million people were living with chronic hepatitis B infection in 2019, with 1.5 million new infections each year. In 2019, hepatitis B resulted in an estimated 820 000 deaths, mostly from cirrhosis and hepatocellular carcinoma (primary liver cancer) [2]. The current therapy strategy for chronic HBV infection includes six nucleos(t)ide analog viral DNA polymerase inhibitors (NUCs) and pegylated interferon-alpha (pgIFN-α) [3]. These two types of therapies can effectively suppress HBV replication, successfully delay liver disease progression, greatly reduce liver cancer incidence, and significantly improve long-term survival [4]. However, both present significant challenges: nucleos(t)ide therapies typically require lifetime treatment to prevent viral rebound, while IFNs-based therapies are associated with poor tolerance, limited responsiveness, and frequent adverse effects [5,6]. The failure to achieve a functional cure by the NUC therapy is attributed to the lack of direct effects on viral covalently closed circular (ccc) DNA in infected hepatocytes and the inability to restore a functional antiviral immune response [7]. cccDNA is very stable with a low turnover rate and can maintain a chronic infection when a few copies of cccDNA per liver can (re)initiate a full-blown infection even after prolonged viral suppression [8,9]. However, completely eliminating covalently closed circular DNA (cccDNA) in HBV-infected cells using currently available antiviral agents is difficult, and drug resistance is frequently caused by the long-term use of these drugs [10,11]. Identification of efficient and safe anti-HBV drugs with novel molecular targets is necessary to overcome these unmet medical needs.

HBV capsid assembly is an essential step in the HBV life cycle, which can be interrupted to block HBV core protein aggregation and efficiently inhibit the synthesis of HBV DNA production [[12], [13], [14]]. The core protein is conserved across all HBV genotypes and is thus considered as an attractive target for small molecule intervention [15,16]. Several chemotypes of CAMs have been developed, such as phenyl-propenamides (PPAs, e.g., AT-130) [17], sulfamoylbenzamides (SBAs, e.g., NVR3-778) [18,19] and heteroaryldihydro-pyrimidines (HAPs, e.g., BAY41–4109, GLS4) (Fig. 1) [20]. Although they each have different chemical structures, all these CAMs bind to the same binding pocket at the Cp dimer-dimer interface [21]. They act as molecular glues to enhance the interaction between the Cps, thus accelerating the self-assembly of Cps and decreasing the formation of functional capsids [22]. Both CAM-E (NVR3-778, Phase II trial) and CAM-A (GLS4, Phase II trial) have been evaluated in clinical trials [23]. However, no compounds have been approved. Therefore, discovery of new chemotypes of CAMs would provide additional candidates, promoting the development of CAMs for new CHB therapeutics.

In this report, we describe the development of novel potent modulators of capsid assembly starting from hit compound CDI (IC50 = 2.46 ± 0.33 μM) with a benzimidazole skeleton, which was identified by screening of an in-house compound library (Fig. 2). The optimization involved structure−activity and −property relationship studies to monitor druggable biological profiles for potential preclinical candidates. Extensive biological assays such as protein- and cell-based capsid assembly assays and quantification of the viral DNA were used to evaluate a series of pyrimidine-based analogues. Moreover, high in vivo efficacy was confirmed by HBV infection animal experiments performed in a hydrodynamic injection mouse model.