**1. Introduction**

Influenza A virus (IAV), the causative agent of influenza, is a major pathogen that causes public health problem and socioeconomic burden world-wide. With waterfowl as the primary reservoir, the virus is able to infect a wide variety of birds and mammals, including humans. Due to this trait, zoonotic spillovers occur occasionally and can lead to pandemics with severe consequences for the human population [1,2]. The avian-origin H5N1 and H7N9 subtypes of influenza viruses are recent examples of animal viruses that acquired the potential to infect and cause disease in humans. H5N1 IAV are often highly pathogenic (HP) avian influenza viruses. In 1997 in Hongkong, six people died out of 18 confirmed human cases with HP H5N1 virus infection [3]. In 2003, novel H5N1 IAV genetic variants circulated in Southeast Asian countries, which led innumerable poultry to death and caused sporadic human infections in the following years. By the end of 2017, 860 laboratory confirmed cases of H5N1 IAV infection from 16 different countries, resulting in 454 deaths, had been reported to the World Health Organization (WHO) [2]. Influenza A viruses mutate easily because of their segmented RNA genome, making it difficult to produce a timely and sufficiently effective vaccine to prevent the potential epidemic outbreaks. Therefore, using anti-influenza agents could be a more efficient approach for prevention and treatment at the beginning of outbreak. To date, two types of anti-influenza drugs have been approved. One type pertains to the M2 ion channel inhibitors, including amantadine and rimantadine, which block the release of viral RNA into the cytoplasm [4]. The others are neuraminidase (NA) inhibitors, including zanamivir, oseltamivir and peramivir, which prevent progeny virus from being released by their host cells [5,6]. However, it has been reported that most of the circulating IAV strains are resistant to M2 inhibitors and may rapidly develop resistance to NA inhibitors, which limits the use of those licensed drugs [7–9]. Therefore, it is urgently required to develop novel anti-influenza agents preferentially with novel mechanisms of action to combat influenza, especially the HP influenza.

The first and most critical step of IAV infection is viral entry mediated by the interaction of viral envelope protein hemagglutinin (HA) and its receptor on host cell surface [10]. HA forms a trimer including three HA1 and three HA2 subunits. Each HA2 contains a fusion peptide, a soluble ectodomain (SE) and a transmembrane domain [11]. After binding to receptor through HA1, IAV is endocytosed. Within the endosome, increasing acidity, pH 5–6, induces HA2 to undergo an irreversible conformational change, leading to the fusion peptide extruding toward the endosomal membrane, and ultimately fusion of the viral and endosomal membranes [10]. HA2, as a major component of the stem region of HA, is a highly conserved subunit. Consequently, blocking HA2- s conformational change could abolish viral membrane fusion and infection. Hence, HA2 is considered an attractive antiviral target [12,13]. The first discovered compound of this class was tertiary butylhydroquinone [14]. Later, several compounds that act in a similar way were described: BMY-27709, CL-385319, stachyflin and 4c [15–18]. However, these small molecular compounds were reported to display inhibitory effects on H1, H2 and H3 IAV subtypes rather than H5 subtype. Recently, guided by structural knowledge on the interactions of HA and anti-stem broadly neutralizing antibodies, Maria and colleagues successfully developed a small molecule JNJ4796 with the ability to inhibit HA-mediated fusion and protect mice against lethal and sublethal influenza challenges [19]. The potent anti-IAV activity of JNJ4796 further reinforces that HA2 could be a promising drug target for blocking IAV infection.

Pentacyclic triterpenoids (PTs), an abundant natural product in plants, have received considerable attention due to their wide spectrum of antiviral activities against various viruses, such as HIV, IAV and HCV [20–23], and some are already marketed as therapeutic agents or dietary supplements. For example, glycyrrhizin (glycyrrhizic acid) has been used for treating chronic hepatic diseases for over 40 years in China and Japan clinically.

Oleanane acid (OA) triterpene is probably the most well-known member of PTs with noticeable antiviral activities. Echinocystic acid (EA), an oleanane-type triterpene, was reported to have substantial inhibitory activity on HCV entry with EC50 at nanomolar range [24,25]. Zhou reported that a different chemical modification to the C-17-COOH of OA led to Y3 (an OA-acetyl galactose conjugate), which showed strong antiviral activity against H1N1 and H3N2 IAV infections in vitro [13]. Further study indicated that Y3 was able to bind tightly to HA protein, thereby disrupting the interaction of HA with its sialic acid receptor [13]. In our previous study, we used an efficient HIV-based pseudotyping system to screen a semisynthetic saponin library, and discovered that OA saponins with β-chacotriosyl residue at the C-3-OH of OA exhibited excellent inhibitory activity against H5N1 IAV entry [26]. Based on this finding, we further designed, synthesized and evaluated a series of 3-O-β-chacotriosyl oleanolic acid analogs as H5N1 IAV entry inhibitors. We found that the introduction of a disubstituted amide structure at the 17-COOH of OA could significantly improve the selective index while maintaining their antiviral activities in vitro [27]. However, as the previous antiviral evaluation of 3-O-β-chacotriosyl oleanolic acid analogs was based on a pseudotyping system, their inhibitory effects on IAV and mechanisms of action are unconfirmed. In the present study, antiviral activities of OA and its eleven analogs, including four newly synthesized derivatives, against H5N1 virus in A549 cells, were investigated, and their mechanisms of action and antiviral activities against other IAV subtypes based on a representative novel compound OA-10 were further investigated. To our knowledge, this is the first report on anti H5N1 IAV activity of OA derivatives.
