**1. Introduction**

Orthohantaviruses are classified as emerging viruses that cause two life-threatening diseases: hemorrhagic fever with renal syndrome (HFRS) and orthohantaviruses pulmonary syndrome (HPS), also known as hantavirus cardiopulmonary syndrome (HCPS) [1]. Small mammals are natural hosts of orthohantavirus, mainly rodents but recently reptiles and fishes [2] have also been discovered as carriers of these viruses that are transmitted to humans through the aerosol route. They are responsible for persistent infections without evident illness signs in their hosts [3]. The two diseases that are orthohantaviruses-related both induce an impressive rise in blood vessel permeability, strong immune responses and inflammation and viruses such as the Hantaan virus (HTNV) and Sin Nombre virus

(SNV) are the causative agents. Although orthohantaviruses are distributed worldwide, HFRS and HCPS occur generally in Eurasia and the Americas, respectively [4].

Orthohantaviruses are members of the Hantaviridae family, order Bunyavirales. Their tripartite, single-stranded, negative sense RNA genome codes for four proteins. The L segment, S segment, and M segment encode an RNA-dependent RNA polymerase (RdRp), a nucleocapsid protein (N protein) and Gn and Gc glycoproteins, respectively. The two surface glycoproteins, before being exposed on the viral surface, are post-translationally processed via the endoplasmic reticulum and Golgi apparatus. These proteins interact with integrin receptors allowing viruses to enter new host cells [5].

The three genomic RNA molecules form a complex within the virion with N protein and, most probably, with RdRp. The viral RdRp mediates the genomic and anti-genomic viral RNAs and the transcription of viral mRNAs exclusively in the cytoplasm [6].

Over the last few years, the search for an effective treatment for orthohantaviruses infections has undergone a considerable increase [7]. Ribavirin (RBV), a broad-spectrum inhibitor, is the only antiviral with recognized in vitro and in vivo activity on hantavirus replication [8,9]. In China [10] and Russia [11], clinical trials have been conducted for the treatment of HFRS using post-exposure, intravenous RBV but while significant results have been obtained in the first case, the second resulted ineffective, as well as the trial conducted in patients with HPS. Furthermore, the use of RBV is limited by its myelosuppression and toxicity [12]. Besides RBV, the use of some nucleoside analogues resulted in being highly inhibitory in animal models [13]. A few other candidates have been evaluated: Favipiravir, a pyrazine derivative endowed of anti-influenza properties and Vandetanib, a tyrosine-kinase inhibitor. The use of corticosteroids, unfortunately, does not determine benefit and immunotherapy although it has given encouraging results; in preventing and treating human hantavirus infections, it remains challenging [14].

Nowadays, there are no U.S. Food and Drug Administration (FDA) granted antivirals [14], vaccines, or immunotherapeutic for the treatment of HFRS or HPS, and consequently, therapeutic approaches are usually based on supportive care. It is precisely for that reason that an effort to develop potential therapeutic agents is strongly desirable. At the present time, a very limited number of antivirals have been tested for orthohantavirus [14].

In recent years, our research group has published several 1(2)H-benzo[d][1,2,3]triazole, usually called benzotriazole, derivatives that have shown marked antiviral activity against many viruses [15–18]. The versatile biological behavior of benzotriazole and its derivatives have recently been described in an in-depth review [19]. Among these benzotriazole derivatives, the 5,6-dichloro1(2)phenyl-benzotriazole scaffold turned out to be endowed with high activity against several different viruses.

In recent times, we described a series of 5,6-dichloro1(2)phenyl-benzotriazole derivatives that are active against te human respiratory syncytial virus (HRSV) in low micromolar range with low cytotoxicity (CC50 >100 μM) and very high selectivity when analyzed on a wide panel of positive- and negative-sense single-stranded RNA, double-stranded RNA, and DNA viruses [20]. In order to further investigate if these derivatives are able to inhibit viral infection processes of other negative sense RNA virus families, we subjected them (Figure 1) to a broad antiviral screening including the Hantaan virus (HTNV), a segmented RNA virus of the family Hantaviridae, for which there is currently no antiviral or approved vaccine.

In general terms, these molecules showed little or no activity except for HTNV. Series 2 derivatives (Figure 1) showed most interesting EC50 values, so we performed the same broad antiviral screening on a series of 2-phenyl-benzotriazole from our library (3k-n and 4k-n) [21] or newly synthetized (3f, j and 4f, j), all showed in Figure 2, in order to better understand the role of substituent in position C5 and C6 on the benzotriazole moiety and describe structure-activity relationships (SARs).

**Figure 1.** 5,6-dichloro-1-phenyl-1*H*-benzo[*d*][1,2,3]triazoles (1a-c, e-j) and 5,6-dichloro-2-phenyl-2 *H*-benzo[*d*][1,2,3]triazoles (2a-n).

**Figure 2.** 2-phenyl-2*H*-benzo[*d*][1,2,3]triazoles (3f, j-n) and 5,6-dimethyl-2-phenyl-2*H*-benzo[*d*][1,2,3] triazoles (4f, j-n).

#### **2. Materials and methods**

#### *2.1. Chemistry*

5,6-dichloro-1-phenyl-1*H*-benzotriazoles (1a-c, e-j), 5,6-dichloro-2-phenyl-2*H*-benzotriazoles (2a-n), 1-(4-(2*H*-benzotriazol-2-yl)phenyl)-3-alkylureas (3k-n) and 1-(4-(5,6-dimethyl-2*H*-benzotriazol -2-yl)phenyl)-3-alkylureas (4k-n) were obtain as recently described [20,21].

#### *2.2. General Procedure for Preparation of Derivatives 3f, j and 4f, j.*

Scheme 1 represents the synthetic route for obtaining derivatives 3f, j and 4f, j.

To a stirred solution of 1.47 mmol of 4-(2*H*-benzo[*d*][1,2,3]triazol-2-yl)aniline (3a) or 4-(5,6-dimethyl-2*H*-benzo[*d*][1,2,3]triazol-2-yl)aniline (4a) in anhydrous *N*,*N*-dimethylformamide (8 mL), 4.98 mmol (ratio 1:3 for compound 3j) or 1.76 mmol (ratio 1:1 + 20% for compounds 3f, 4f, 4j) of proper benzoyl-chloride (5f, 5j) in anhydrous N,N-dimethylformamide (DMFa) were added. The mixture was stirred at 80 ◦C for 2 h (3j) or 24 h (3f, 4f, 4j). At the end, reaction mixture was cooled to room temperature and the solids were obtained by filtration. Mother liquor was dried by evaporation obtaining further solid. The crude solids were purified by flash chromatography, using a mixture of petroleum spirit/ethyl acetate in a ratio 7/3 as eluent, followed by crystallization from ethanol.

**Scheme 1.** Synthesis the amide derivatives (3f, j and 4f, j).

2.2.1. *N*-(4-(2H-benzo[d][1,2,3]triazol-2-yl)phenyl)-4-methylbenzamide (3f)

Compound was obtained in 80% of total yield; m.p. 266–268 ◦C; TLC (petroleum spirit/ethyl acetate 1/1), Rf 0,90. 1H-NMR (DMSO-*d*6) δ: (1H, s, NHCO), 8.32 (2H, d, J = 8.4 Hz, H-2- , 6- ), 8.09 (2H, d, J = 8.4 Hz, H-3- , 5- ), 8.05-8.00 (2H, m, H-4, 7), 7.92 (2H, d, J = 7.6 Hz, H-2", 6"), 7.53–7.50 (2H, m, H-5, 6), 7.37 (2H, d, J = 7.6 Hz, H-3", 5"). 13C-NMR (DMSO-*d*6) δ: 165.60 (CO), 144.34 (C), 141.86 (2C), 140.36 (C), 134.90 (C), 131.72 (C), 128.94 (2CH), 127.77 (2CH), 127.43 (2CH), 120.89 (2CH), 120.68 (2CH), 118.04 (2CH), 21.00 (CH3). Anal. Calcd for C20H16N4O, C, 73.15; H, 4.91; N, 17.06. Found C, 73.23; H, 4.94; N, 16.98. MW 328.37, LC/MS: 329 (M + H).

#### 2.2.2. *N*-(4-(2H-benzo[d][1,2,3]triazol-2-yl)phenyl)-3,4,5-trimethoxybenzamide (3j)

Compound was obtained in 30% of total yield; m.p. 227–229 ◦C; TLC (petroleum spirit/ethyl acetate 7/3), Rf 0,29. 1H-NMR (DMSO-*d*6) δ: 10.44 (1H, s, NHCO), 8.34 (2H, d, J = 8.4 Hz, CH-2- , 6- ), 8.04–8.00 (4H, m, CH-4, 7, 3- , 5- ), 7.53–7.50 (2H, m, CH-5, 6), 7.33-7.28 (2H, m, CH-2", 6"). 13C-NMR (DMSO-*d*6) δ: 165.17 (CO), 152.65 (2C), 144.35 (2C), 140.36 (C), 135.02 (C), 129.71 (2C), 127.48 (2CH), 121.15 (2CH), 120.72 (2CH), 118.03 (2CH), 105.41 (2CH), 60.12 (OCH3), 56.12 (2OCH3). Anal. Calcd for C22H20N4O4, C, 65.34; H, 4.98, N, 13.85. Found C, 65.46; H, 5.20; N, 13.58. MW 404.42, LC/MS: 405 (M + H).

#### 2.2.3. *N*-(4-(5,6-dimethyl-2H-benzo[d][1,2,3]triazol-2-yl)phenyl)-4-methylbenzamide (4f)

This compound was obtained in 46% of total yield; m.p. 295–296 ◦C; TLC (petroleum spirit/ethyl acetate 7/3), Rf 0,53. 1H-NMR (DMSO-*d*6) δ: 10.46 (1H, s, NH), 8.25 (2H, d, J = 9.2 Hz, H-2- , 6- ), 8.05 (2H, d, J = 9.2 Hz, H-3- , 5- ), 7.91 (2H, d, J = 8.4 Hz, H-2", 6"), 7.76 (2H, s, H-4, 7), 7.37 (2H, d, J = 8.4 Hz, H-3", 5"), 2.51 (3H, s, CH3), 2.40 (6H, s, 2CH3). 13C-NMR (DMSO-*d*6) δ: 165.57 (CO), 143.79 (2C), 141.85 (C), 139.88 (C), 137.86 (2C), 135.06 (C), 131.73 (C), 128.95 (2CH), 127.74 (2CH), 121.40 (2CH), 120.90 (2CH), 116.17 (2CH), 29.96 (CH3), 20.99 (2CH3). Anal. Calcd for C22H20N4O, C, 74.14; H, 5.66; N, 15.72. Found C, 74.18; H, 5.82; N, 15.67. MW 356.42; LC/MS: 457 (M + H).

2.2.4. *N*-(4-(5,6-dimethyl-2H-benzo[d][1,2,3]triazol-2-yl)phenyl)-3,4,5-trimethoxybenzamide (4j)

This compound was obtained in 20% of total yield; m.p. 272–273 ◦C; TLC (petroleum spirit/ethyl acetate 6/4), Rf 0,34. 1H-NMR (DMSO-*d*6) δ: 10.40 (1H, s, NH), 8.28 (2H, d, J = 8.8 Hz, CH-2- , 6- ), 8.02 (2H, d, J = 9.2 Hz, CH-3- , 5- ), 7.76 (2H, s, CH-2", 6"), 7.32 (2H, s, CH-4, 7), 3.90 (6H, s, 2OCH3), 3.75 (3H, s, OCH3), 2.41 (6H, s, 2CH3). 13C-NMR (DMSO-*d*6) δ: 165.11 (CO), 152.64 (2C), 143.8 (2C), 140.47 (C), 139.68 (C), 137.88 (2C), 135.18 (C), 129.73 (C), 121.14 (2CH), 120.32 (2CH), 116.26 (2CH), 105.38 (2CH), 60.12 (OCH3), 56.12 (2OCH3), 20.41 (2CH3). Anal. Calcd for C24H24N4O4, C, 66.65; H, 5.59; N, 12.96. Found C, 69.46; H, 5.82; N, 12.58. MW 432.47; LC/MS: 433 (M + H).

#### *2.3. Cells, Viruses, Reagents*

All experimental work involving viruses was performed in a biosafety level 3 (BSL3) containment laboratory. Hantaan (HTNV) (strain 76–118) (kindly provided by Prof. Dr. D. H. Krüger and Dr. Boris Klempa, Institute of Virology, Charitè University of Berlin) was used for all experiments. Vero E6 cells (ATCC CRL 1586) were maintained in complete EMEM (minimum essential medium with Earle's salt, 25mM Hepes supplemented with 10% fetal bovine serum, 1% glutamine, 1% sodium pyruvate (NaPy), 1% non-essential amino acids NEAA, 0.1% gentamicyn). RBV was purchased from Sigma–Aldrich Co. (St. Louis, MO, USA). The primary antibody, rabbit anti-Malacky Ab, was obtained from Dr. Boris Klempa, Berlin. The secondary antibody was goat anti-rabbit IgG (H+L) HRP conjugate (Cat. No. 170-6515, Bio-Rad, Inc., CA, USA). SuperSignal West Pico Chemiluminescent Substrate (Cat. No. 34080, Thermo Scientific, MA, USA)
