Human infection with hepatitis B virus can induce chronic liver damage, including cirrhosis and hepatocellular carcinoma, and, despite effective vaccines being available, eradicating chronic infections can be challenging. The current therapeutic drugs aginst HBV are nucleotide analogues such as lamivudine, tibivudine (LdT), entecavir, adefovir diaxil (ADV), and tenofovir-DF, as well as interferons (IFNs, non-pegylated or pegylated). These drugs either inhibit the reverse transcription of HBV [1] or eliminate the forming of covalently-closed-circular DNA (cccDNA) [2]. However, the issues of the toxicity, side effects, drug resistance and rebound after drug withdrawal of both nucleosides and interferons in clinical treatment still require great attention [3], [4]. Therefore, it is imperative to develop novel HBV inhibitors, providing a new direction for HBV therapy research and potentially leading to more effective treatments for hepatitis B.

Sodium taurocholate cotransporting polypeptide (NTCP) has been found to be a specific receptor of hepatocytes for triggering HBV entry [5], which was a major breakthrough in the study of the mechanism of HBV infection. In recent years, there has been a surge of interest in developing novel anti-HBV drugs focused on HBV entry targeting NTCP [6]. Myrcludex B was a newly developed HBV and hepatitis D virus (HDV) entry inhibitor. It worked by mimicking the sequence of the HBV PreS1 domain and specifically binding to NTCP, thereby interrupting the interaction between preS1 and NTCP, and protecting normal liver cells from infection [7]. FDA-approved drugs, including Cyclosporine A, irbesartan, rosiglitazone, zafirlukast, salazosulfapyridine, and 3,3′,5-triiodothyroacetic acid, have been recognized as NTCP inhibitors and utilized in NTCP research studies [8], [9], [10], [11]. Notably, some natural products also exhibited good NTCP inhibitory activity, such as curcumin [12], ergosterol peroxide [13], betulin [14], ophiopogon D' [15], and epigallocatechin-3-gallate [16] (Fig. 1). However, despite their efficacy in inhibiting NTCP in HBV, these inhibitors have been linked to numerous side effects [17], [18], [19]. Hence, the novel and reliable NTCP inhibitors remain required in combating HBV effectively.

Natural products and modified natural products play an important role in discovering novel anti HBV inhibitors with distinct antiviral mechanism [20], [21], [22], [23]. Pentacyclic triterpenoids are significant natural products found in traditional Chinese medicines for protecting liver, such as the roots and rhizomes of Glycyrrhiza uralensis, wipe fruits of Ligustrum lucidum, and wipe seeds of Ziziphus jujuba. Research has shown that these compounds exhibited remarkable anti-HBV [24] and hepatoprotective [25] activities. We also found two new compounds with rare seco-ring-A pentacyclic triterpenoids from Viola diffusa, a folk medicine for treating hepatitis, which demonstrated potential anti-HBV inhibitory activity compared to lamivudine [26]. Furthermore, the seco-ring-A derivatives of oleanolic acid and glycyrrhetinic acid were synthesized and displayed inhibition on both HBeAg secretion, HBV DNA and HBV cccDNA (covalently closed circular DNA) replication [27]. To enhance the activity and improve bioavailability of these compounds, medicinal chemists have made extensive modifications to the pentacyclic triterpenoids and discovered that the pentacyclic triterpenoids conjugates [28], [29], [30] obtained through molecular hybridizations had shown promising prospects in antiviral activity compared to the parent compounds. Some conjugates also had the potential to act as viral entry inhibitors [31], [32], [33], [34]. Podophyllotoxin (PPT) and 4 'demethoxypodophyllotoxin (4 'PPT) are arylnaphthalene lignan lactones isolated from Podophyllum peltatum [35], exhibiting antiviral [36], [37], [38], such as human papilloma virus (HPV), herpes simplex virus (HSV), molluscum contagiosum virus (MCV) and human immunodeficiency virus (HIV). In addition, 4 'PPT had obvious inhibitory effect on the increase of serum enzymes caused by liver injury in mice [39]. Recently, PPT and 4 'PPT from Dysosma versipellis were fished out and regarded as the bioactive ligands to NTCP with high affinity [40].

In view of the potential of pentacyclic triterpenes as virus entry inhibitors and the interaction of PPT and 4 'PPT with NTCP, a series of conjugates were designed and synthesized via molecular hybridizations in this study. Furthermore, we evaluated the in vitro anti-HBV activity of conjugates and preliminarily explored the mechanism targeting NTCP in order to obtain high-efficacy and low-toxicity lead compounds for anti-HBV treatment.