*2.4. Cytotoxicity Assays*

Vero E6 cells were seeded at an initial density of 4 <sup>×</sup> 105 cells/mL in 6-well plates, in culture medium (EMEM 25mM HEPES buffer) supplemented with 1% L-glutamine, 10% fetal bovine serum (FBS), 1% NaPy, 1% NEAA, and 0.1% gentamycin. Cell cultures were then incubated at 37 ◦C in a humidified, 5% CO2 atmosphere in the absence or presence of serial dilutions of test compounds. Cell viability was determined after 7 days at 37 ◦C by the crystal violet staining method.

#### *2.5. Orthohantavirus Antiviral Screening Assay*

Vero-E6 cells were seeded in 6-well plates at an initial density of 4 <sup>×</sup> 105 cells/mL. Cell cultures were then infected and incubated at 37 ◦C in a humidified, 5% CO2 atmosphere for 7 days in the absence or presence of serial dilutions of test compounds. The antiviral activity was determined by chemiluminescence focus reduction assay (C-FRA), as described previously [22] with small modifications. Briefly, after 7 days of incubation, overlay medium were discarded and cell monolayers were fixed with ice-cold methanol for 8 min at room temperature. After fixing, the methanol was removed, and the cells let dry. Cells were rinsed twice with washing buffer, and then a rabbit anti-Malacky antibody was added at 1:1000 dilution for 1 h at 37 ◦C in a humidified, 5% CO2 atmosphere. Cells were then washed five times with washing buffer and a secondary goat anti-rabbit IgG antibody conjugated to horseradish peroxidase (HRP) was added for an additional hour in 5% CO2 atmosphere. After washing five times with washing buffer cells were incubated with chemiluminescent substrate immediately before the detection. Infected cell foci were detected with the CCD camera for 3 min (or Chemidoc for 30 sec).

#### *2.6. Yield Reduction assay*

Vero E6 cells were inoculated with HTNV at an m.o.i. of 0.05 in maintenance medium and tested compounds at non-cytotoxic concentrations. Following a 1-h adsorption period at 37 ◦C and 5% CO2 on a rocking platform, the inoculum was removed and replaced with fresh medium containing 20 μM concentration of compounds (2j, 2l, 2n). After 3 days, the cell supernatant was harvested and centrifuged (at 3000 rpm, at 4 ◦C, for 10 min) to remove debris and measured for the presence of infectious orthohantavirus hantavirus. The infectious progeny virus in the cell supernatant was evaluated by chemiluminescence focus reduction assay, as described above [22]. RBV was used as the reference compound.

#### *2.7. Statistical Analysis*

Data are represented as mean ± standard deviation (SD) unless otherwise stated. For the yield reduction assay, statistical comparisons were performed using the unpaired *t*-test, and *p*-values less than 0.05 were considered to be statistically significant. All analyses were performed with GraphPad Prism 6.

#### **3. Results and Discussion**

In Figure 3, we report the effects of compounds 5,6-dichloro-2-phenyl-2H-benzo[d][1,2,3]triazoles (2n, 2l, 2j, 2f, 2k) on the replication of HTNV in comparison to RBV, a known broad-spectrum antiviral agent. Series 1, 3, and 4 derivatives turned out as completely inactive and are not reported here (see Table S1).

**Figure 3.** Anti-hantaviral effect of 2n, 2l, 2j, 2f, 2k, and RBV in vitro. Each symbol shows mean values for three independent determinations + SD.

Compounds 1a-c, 1e-j, 2a-n, 3f, 3j-n, and 4f, 4j-n, were evaluated for their potential inhibitory activity against HTNV using a chemiluminescence focus reduction assay (C-FRA). Three compounds (2j, 2l, 2n) showed interesting inhibitory activity (i.e., ≤5 μM; Figure 3). In parallel, moderate to low cytotoxicity was detected for almost all compounds, with CC50 values mostly in the high micromolar range (>30 μM) against all tested cell lines (see Table S1).

From a structure–activity relationship perspective, the most relevant results concerned the potent and selective activity of two urea derivatives (2l and 2n) and one amide derivative (2j), all belonging to the series of the 5,6-dichloro-2-phenyl-derivatives. By contrast, none of the molecules of the series 5,6-dichloro-1-phenyl-derivatives exhibited anti-HTNV activity, with only the exception of the compound 1h (EC50 = 21 μM). Regarding the presence of the two chlorine atoms in positions C5 and C6 of benzotriazole, their elimination (3f, j-n) or substitution with methyl groups (4f, j-n) results in total loss of antiviral activity. Moreover, we can highlight that derivative 2l (R= NHCONH-propyl) showed an EC50 value of 5 μM, but when decreasing or increasing the steric hindrance of the side chain with ethyl-group (2k) or butyl-group (2m), it turned out in a total loss of antiviral activity.

Derivative 2n, recently described by our research group as an interesting inhibitor of HRSV entry [16], showed a remarkable EC50 value of 4 μM against HTNV, resulting in being ten-fold more potent than RBV (EC50 = 37 μM); Figure 4.

**Figure 4.** Focus reduction assay for 2n and ribavirin.VeroE6 cells were infected with HTNV (m.o.i. 0.05). The infected cultures were treated with 2n (panel **A**) and RBV (panel **B**; used as reference) at indicated doses. Ci = untreated control; CC = uninfected control. Antiviral activity was determined by focus reduction assay at day 7 post-infection.

2n also resulted in being more active in in vitro assays against HTNV than two orthohantavirus candidate drugs: ETAR (1-beta-d-ribofuranosyl-3-ethynyl-[1,2,4] triazole) (EC50 = 10 μM) and Favipiravir (EC50 =150.8 μM), respectively 2- and 37-fold more potent [13,23].

Accordingly, to obtain a detailed insight on the efficacy of 2j, 2l, and 2n against HTNV, a yield reduction assay was performed. The reduction of virus titer in the presence of the active compound during a single round of viral infection was determined. Treatment with non-cytotoxic 20 μM concentration of 2j, 2l and 2n caused a significant reduction of viral titer (Figure 5). A significant decline in viral titer of HTNV was also observed at 50 μM treatment of RBV (\* *p*-value <0.05, unpaired *t*-test).

**Figure 5.** Yield of infectious Hantaan (HTNV) viruses produced in infected VeroE6 cells treated with selected benzotriazoles (2j, 2l, 2n) (20 μM) and RBV (50 μM). Results are expressed as means ± standard deviations from 3 separate experiments done in triplicate. \* indicates the *p*-value <0.05.

VeroE6 cells were infected with HTNV (m.o.i. 0.05). The infected cultures were treated with 2j, 2l, 2n at indicated doses, and RBV was used as a reference compound. Viral yields in the culture supernatant were determined by viral titer reduction assay at day 3 post-infection.

In our assay, benzotriazole derivatives 2n, 2l, 2j (20 μM) determined a very interesting reduction of viral titer compared to control cells infected in the absence of inhibitors. The production of the virus was significantly reduced upon treatment with 2l (3,08 log10). The same trend of reduction in viral loads was detected for 2n (2,41 log10) and 2j (2.14 log10). Conversely, in order to obtain a titer decrease, less than 2 logs (1,78 log10) if compared to untreated control, the reference molecule ribavirin needed to be added at 50 μM concentration.
