*3.1. Compound OA-10 Inhibits IAV Infections in A549 Cells with Minimal Cytotoxicity*

Chemical structures of oleanane acid (OA-0) and its 11 derivatives, including four new derivatives, are shown in Figure S1 of the Supplementary Material. The cytotoxicity of OA-0 and its derivatives on A549 cells was first evaluated using the 3-(4,5-dimethylthiozol-2-yl)-3,5-dipheryl tetrazolium bromide (MTT) assay. For each compound, CC50 value, the concentration required to reduce normal cell viability by 50% after 24 h of compound treatment, was determined, as shown in Table 2. Derivatives OA-1, OA-2, OA-4, OA-5, OA-7, OA-8 and OA-9 exhibited greater cytotoxicity on A549 cells with CC50 ≤ 20.5 μM, while other derivatives and oleanolic acid exhibited less cytotoxicity with CC50 ≥ 31.1 μM. Noticeably, OA-10 showed the least cytotoxicity with CC50 > 640 μM (Figure 1C). DMSO (0.4%, used as solvent) did not exhibit detectable cytotoxicity on A549 cells**.** No obvious cytotoxicity was observed

for OA-10 at concentrations ≤ 80 μM after 24, 48 or 72 h of treatment, as shown in Figure 1C. Thus, 80 μM of OA-10 was selected as the maximum concentration for further studies.


**Table 2.** Cellular toxicity and inhibitory activity of oleanane-type triterpenoid derivatives against H5N1 influenza A virus (IAV) replication in A549 cells.

<sup>a</sup> CC50, the concentration required to reduce normal, noninfected cell viability by 50%; <sup>b</sup> EC50, the concentration required to protect 50% cells from H5N1 IAV infection; <sup>c</sup> SI (selectivity index) is the ratio of CC50 to EC50. Data were presented as means ± SDs of results from three independent experiments.

The antiviral activities of synthesized OA derivatives against H5N1 IAV were evaluated via relative nucleoprotein (NP) expression level (%) detected by immunofluorescence microscopy at 24 hpi. The initial results showed that OA-0 and the 11 derivatives exhibited various antiviral activities against H5N1 IAV infection (Table 2). It was found that OA-0 and all other derivatives except OA-6 significantly reduced H5N1 IAV infection in A549 cells with EC50 ≤ 14.2 μM, while OA-6 showed the lowest antiviral activity with EC50 > 86.6 μM. However, most of the effective derivatives, including OA-1, OA-2, OA-3, OA-4, OA-5, OA-7, OA-8, OA-9 and OA-11, exhibited high cytotoxicity on A549 cells with CC50 ≤ 31.1 μM, while OA-0, OA-10 and OA-11 showed relatively lower cytotoxicity on A549 cells with CC50 ≥ 60.0 μM. Most interestingly, OA-10 showed significant inhibition on H5N1 IAV replication in a dose-dependent manner in concentrations ranging from 20 to 80 μM (EC50: 14.0 μM) with very low cytotoxicity, as shown in Figure 1B,C. Among the 12 evaluated compounds, OA-10 showed the highest select index (SI > 45). Thus, OA-10 was selected for further studies.

To explore whether OA-10 possesses a broad inhibitory effect on different IAV subtypes, three other IAV strains (PR8, H9N2 and H3N2) were evaluated using IFA and A549 cells at 24 hpi. As shown in Figure S2A and Table 3, OA-10 also exhibited significant inhibitions on PR8 (EC50: 6.7μM), H9N2 (EC50: 15.3 μM) and H3N2 (EC50: 19.6 μM) replications.

**Table 3.** Inhibitory activity of OA-10 against influenza A virus replication in A549 cells.


Data are presented as means ± SDs from three independent experiments.

To more accurately assess OA-10- s inhibitory role, we further examined its antiviral effects against H5N1 IAV infection using virus titration and RT-PCR at 48 hpi. As shown in Figure 2A, treatment with OA-10 resulted in a significant reduction of progeny virus titer in a dose-dependent manner. Treatment with 80 μM of OA-10 led to a 1.8 log reduction in progeny virus production compared to that in DMSO-treated control. In fact, OA-10 at concentrations from 20 to 80 μM significantly inhibited H5N1 IAV NP RNA levels in A549 cells in a dose-dependent manner (Figure 2B). We further studied the viral inhibition kinetics by OA-10 at 80 μM. In the H5N1 IAV-infected control, the levels of virus titer and virus mRNA expression increased continuously from 24 to 72 hpi (Figure 2C,D). The addition

of 80 μM OA-10 significantly reduced progeny virus titers and viral RNA levels at all time-points, as shown in Figure 2C,D. Simultaneously, treatment with OA-10 also reduced progeny virus titer on PR8, H9N2 and H3N2 infection, respectively, in a dose-dependent manner at 48 hpi, as shown in Figure S2B. Peramivir, a well-known neuraminidase inhibitor, was used as a positive antiviral drug control in this study. Our results showed that 15 μM of peramivir exhibited a significant inhibition on IAV infections in the same assays.

**Figure 2.** OA-10 inhibited H5N1 IAV infection in A549 cells. A549 cells grown in 24-well plates were infected with H5N1 IAV (0.1 MOI) for 1 h, and then cultured in fresh medium containing various concentrations of OA-10 or 15 μM peramivir. At 48 hpi (**A**,**B**) or indicated time-points post infection (**C**,**D**), cells and supernatants of each well were mixed and subjected to viral titer or RT-PCR analysis. H5N1 IAV expression of GAPDH was used as a loading control, and a DMSO-treated sample (H5N1 IAV infected and non-drug treated) at 48 hpi was used as treatment control (set as 1). Data are presented as means ± SDs of results from three independent experiments. \**P* < 0.05, \*\* *P* < 0.01 and \*\*\* *P* < 0.001 compared to the respective virus control.
