Antiviral activities of 5-chlorobenzotriazole derivatives

Monatshefte für Chemie - Chemical Monthly

https://doi.org/10.1007/s00706-018-2234-7(0123456789(). -, vo Vl

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ORIGINAL PAPER

Antiviral activities of 5-chlorobenzotriazole derivatives

Roberta Ibba¹

Paola Corona¹Antonio Carta¹Paolo Giunchedi¹Roberta Loddo²Giuseppina Sanna²

•

Ilenia Delogu²Sandra Piras¹

•

Received: 28 February 2018 / Accepted: 19 May 2018

Ó Springer-Verlag GmbH Austria, part of Springer Nature 2018

Abstract

In this study, we designed and synthesized a series of new 5-chlorobenzotriazole derivatives. Compounds were tested for

cytotoxicity and antiviral activity in cell-based assays against several positive single-stranded RNA viruses: BVDV, YFV,

CVB-5, and Sb-1; two negative single-stranded RNA viruses: RSV and VSV; a double-stranded RNA virus (Reo-1); and

two DNA viruses: VV and HSV-1. N-[4-(5-Chloro-2H-benzo[d][1,2,3]triazol-2-yl)phenyl]-3,4,5-trimethoxybenzamide

turned out to be the most potent compound against bovine viral diarrhea virus (BVDV). Its activity was shown to be

comparable to the activity of the reference drug NM 107 (EC₅₀3.0 vs. 1.7 lM). It is going to be used as a lead compound

for future antiviral drug developments.

Graphical abstract

Cl

N

NH

R

Keywords 5-Chloro-phenyl-benzotriazoles Á Antiviral activity Á Drug research Á Heterocycles Á Respiratory syncytial virus

Introduction

compounds, originally synthesized as potential anticancer

agents, proved to be active against different viruses. Later

on, the discovery of the antiviral activity of drugs like

ribavirin and acyclovir opened the way for antiviral drug

development research, whose efforts are principally

focused on searching for nucleotides or nucleosides ana-

logues, because most antiviral compounds are inhibitors of

viral helicase and/or polymerase enzymes [1].

Viruses are small infectious obligate parasites that need a

host cell to reproduce. Over the years, viruses have

developed many strategies to be able to elude the immune

response of the host, so that, notwithstanding the ever-

improving antiviral drug development, viruses continue

causing many diseases in humans.

The ﬁrst viruses were discovered in the last years of the

nineteenth century, but only 70 years later, it was possible

to begin to treat viral infections. In the 1960s, several

Nowadays, many DNA and RNA viruses are known to

infect humans and cause several serious diseases [2], but

there are still no fool proof drugs against them. Antivirals

can be differentiated into two groups: immunomodulators

and antiviral agents. Antiviral agents inhibit viral replica-

tion by targeting different stages of the viral life cycle, such

as viral adsorption; virus–host cell fusion; viral uncoating;

viral nucleic acids (DNA or RNA) synthesis. Antivirals

inhibit the enzymes involved in these processes and stop or

restrict viral maturation [3]. Antivirals can also target host

proteins which are relevant for viral replication, such as

endoplasmic reticulum (ER) alpha-glucosidases involved

& Antonio Carta

acarta@uniss.it

& Roberta Loddo

rloddo@unica.it

1

Department of Chemistry and Pharmacy, University of

Sassari, Via Muroni 23, 07100 Sassari, Italy

2

Department of Biomedical Sciences, University of Cagliari,

Cittadella Universitaria, Monserrato, 09042 Cagliari, Italy

123

R. Ibba et al.

in the viral glycoprotein folding quality control [4]. Both

virus- and host-targeting antiviral agents need to inhibit

viral replication without affecting the host organism, and

they need to be non-toxic; for example, they cannot be

carcinogenic, allergenic, toxic, or mutagenic.

as reported in Schemes 2 and 3, by condensation of com-

pound 4a with the suitable substituted benzoyl chlorides in

dimethylformamide (DMF), at reﬂux for 2 h (compounds 5–

10); or with substituted isocyanates in dichloromethane

(DCM) at room temperature for 24 h (compounds 11, 12); or

with dimethyl sulfate in DMF at 100 °C for 2 h (compound

13); or with diethyl sulfate in DMF at 100 °C for 5 h

(compound 14); or with acetic anhydride in DMF at 60 °C

for 1 h (compound 15), respectively.

In the last 10 years, we have established an antiviral drug

development research program, and have synthesized sev-

eral molecules with powerful antiviral activity [5–9]. Indeed,

as it is well known, scaffolds such as benzimidazoles [10, 11]

and benzotriazoles [12] allow developing molecules with

different biological activities, including antiviral activity.

Following on from these encouraging results, we tried to

streamline and simplify these molecules [13–15].

The puriﬁcation of compounds 5–15 was achieved by

ﬂash chromatography using an appropriate mixture of

solvents as eluents. The amide-BT derivatives 5–10 were

isolated in the range of 13–27.3% yield and the ureido-

BT derivatives 11, 12 were obtained in the range of

38–50% yield. The N-alkyl derivatives 13, 14 and the

acetamide-derivative 15 were obtained in the range of

76–66% yield.

Encouraged by these evidences, we have now prepared a

series of 5-chlorobenzotriazole-benzamides (compounds

5–10, Scheme 2; compound 15, Scheme 3); 5-chloroben-

zotriazole-phenyl-ureas (11, 12); and 5-chlorobenzotria-

zole-benzamines (compounds 13, 14, Scheme 3), with the

aim of evaluating the selectivity of the compounds against

representative DNA viruses, positive- and negative-sense

single-stranded RNA viruses, and double-stranded RNA

viruses. All compounds were also tested for cytotoxicity in

the cell lines sustaining the alternative virus replication.

Anti-viral assays

All derivatives considered in this work (Table 1) were eval-

uated for antiviral activity against RNA and DNA viruses

(Table 2). Among single-stranded, positive RNA viruses

(ssRNA^?), we considered a Flavivirus (Yellow Fever Virus,

YFV) and a Pestivirus (Bovine Viral Diarrhea Virus, BVDV);

and two Picornaviruses (Coxsackie virus type-5, CVB-5, and

Poliovirus type-1, Sabin strain, Sb-1). Among single-stranded,

negative RNA viruses (ssRNA^-), a paramyxoviridae (respi-

ratory syncytial virus, RSV) and a Rhabdoviridae (Vesicular

Stomatitis Virus, VSV) were selected as representatives.

Among double-stranded RNA (dsRNA) viruses, a Reoviridae

family member (Reo-1) was included. Finally, two repre-

sentatives of DNA virus families were also included: Herpes

Simplex Virus type-1, HSV-1 (Herpesviridae), and Vaccinia

Virus, VV (Poxviridae). NM 107 (2⁰-C-methylcytidine), NM

108 (2⁰-C-methylguanosine), ribavirin, 6-azauridine, ACV

(acyclovir), pleconaril, and mycophenolic acid were used as

reference inhibitors (Table 2).

Results and discussion

Chemistry

The mixture of intermediates 5-chloro-2-(4-nitrophenyl)-

2H-benzo[d][1,2,3]triazole

(3a),

5-chloro-1-(4-nitro-

phenyl)-1H-benzo[d][1,2,3]triazole (3b), 6-chloro-1-(4-ni-

trophenyl)-1H-benzo[d][1,2,3]triazole (3c) was prepared as

reported in the literature [16] (Scheme 1) starting from

the condensation of 4-chloronitrobenzene (2) with

5-chlorobenzotriazole (1) in DMF with the presence of

Cs₂CO₃used as a base, to obtain a mixture of the 1(2)(3)-p-

nitrophenyl benzotriazole derivatives 3a–3c. The reduction

of the mixture of three isomers 3a–3c by methylhydrazine

in ethanol (at 100 °C in autoclave for 46 h) gave the

mixture of compounds 4a–4c. Chromatography on silica

gel (petroleum ether/ethyl acetate 8/2) allowed the sepa-

ration of the three isomers. The N2 isomer derivatives

appeared to be the most promising molecules; therefore,

Conclusions

As far as the antiviral activity is concerned, none of

compounds turned out to be active against Reo-1, YFV,

CVB-5, Sb-1, VSV, VV, and HSV-1 viruses. Some com-

pounds showed activity against RSV (4a, 4b, 13, and 14)

and BVDV (4a and 6) in the range of EC₅₀10–35 and

3–14 lM, respectively.

4-(5-chloro-2H-benzo[d][1,2,3]triazol-2-yl)aniline

(4a)

was isolated in 43% yield. The isomers 4b and 4c, as well

as the isomer 4a, were puriﬁed and tested in antiviral cell

assays for their potential antiviral activity, as well.

The series of N-[4-(5-chloro-2H-benzo[d][1,2,3]triazol-2-

yl)phenyl]carboxamides, 1-[4-(5-chloro-2H-benzo[d][1,2,3]

triazol-2-yl)phenyl]-3-alkylureas, and 4-(5-chloro-2H-benzo

[d][1,2,3]triazol-2-yl)-N-alkylanilines and N-[4-(5-chloro-2H-

benzo[d][1,2,3]triazol-2-yl)phenyl]acetamides were prepared,

We recall that human respiratory syncytial virus (RSV)

is one of the most important causes of lower respiratory

tract infections in infants and young children and of

pneumonia and bronchiolitis in adults [17, 18]. Further-

more, RSV is one of the main causes of respiratory

123

Antiviral activities of 5-chlorobenzotriazole derivatives

Scheme 1

Cl

N

H

NO₂

2

1

Cs₂CO₃, DMF

140 °C, 5 h

Cl

N

NO₂

-

3a 3c

CH₃NHNH₂

EtOH

100 °C, 46 h

NH₂

4'

3'

5'

4

3

2'

3'

Cl

2'

5

3a

7a

N

1'

6'

1'

N

NH₂

7

4

4'

2

6

Cl

7a

N¹

N

6

6'

1

5'

7

N

2

4a

4

7

3

5

N

3

3a

Cl

5

3a

7a

N

N ²

4c

6

N

1

1'

2'

4b

6'

3'

4'

5'

NH₂

problems in the elderly and in immuno-compromised

patients [19, 20]. For these reasons, new molecules with

anti-RSV activity are especially desirable. Therefore,

compound 4b showing a good antiviral activity accompa-

nied by high selectivity and devoid of cytotoxicity towards

all the cell lines used is a very promising molecule. The

molecule has a simple chemical structure and will be used

as lead compound in future drug development efforts

centered around the insertion of alkyl groups on the amino

nitrogen (the latter modiﬁcations have proved useful in

derivatives of 4a as shown in this paper).

Bovine viral diarrhea virus (BVDV) causes a range of

clinical manifestations in bovines, including respiratory

problems, abortion, and teratogenesis [21, 22], and impacts

signiﬁcantly on the livestock industry. N-[4-(5-Chloro-2H-

benzo[d][1,2,3]triazol-2-yl)phenyl]-3,4,5-trimethoxyben-

zamide (6) shows an excellent activity against BVDV,

comparable to that of the reference drug NM 107 (EC₅₀3.0

vs. 1.7 lM), and displays complete selectivity of action

and lack of cytotoxicity. Compound 6, therefore, proves to

be an excellent candidate for further drug development, in

particular for in vivo assays.

123

R. Ibba et al.

Scheme 2

R¹

R³

R¹

O

R²

O

R²

Cl

N

NH

DMF, reflux, 2 h

R³

5-10

Cl

N

NH₂

4a

R⁴

NH

O

Cl

N

R⁴

DCM, from 0 °C to r.t., 24 h

O

C

N

NH

11-12

Scheme 3

O

S

Cl

N

NH

DMF, 100 °C

N

13

O

S

Cl

N

NH₂

N

DMF, 100 °C

N

4a

14

O

Cl

N

O

N

NH

60 °C

15

Experimental

Table 1 List of compounds 5–12

Compound R¹

R²

R³

Compound R⁴

NMR spectra were recorded on a Bruker AVANCE III 400

Nanobay instrument for ¹H at 400 MHz and for ¹³C at

100 MHz, using TMS as internal standard. The chemical

shifts values (d) are reported in parts per million (ppm) and

coupling constants (J) are expressed in Hertz (Hz). Signal

multiplicities are represented by: s (singlet), d (doublet), dd

(double doublet), t (triplet), q (quadruplet), and m (multiplet).

A Koﬂer heating bench was used to determine the melting

point of each compound. GC–MS analyses were performed

5

H

NO₂

H

11

Cyclohexyl

Propyl

6

OCH₃OCH₃OCH₃12

7

H

OCH₃

CF₃

H

8

9

CH₃

CN

10

123

Antiviral activities of 5-chlorobenzotriazole derivatives

Table 2 Antiviral activity and cytotoxicity of 5-chlorobenzotriazoles (4a–4c and 5–15)

Comps

^aMDBK

^bBVDV

^cBHK 21 ^dYFV ^dReo-1 ^eVero-76 ^fCVB-5 ^fSb-1

CC₅₀/lM EC₅₀/lM

^fVSV, VV, HSV-1 ^fRSV

CC₅₀/lM EC₅₀/lM CC₅₀/lM EC₅₀/lM

4a

54

14

30

[ 30

[ 77

[ 30

[ 77

[ 100

[ 100 [ 100

10

4b

[ 100

70

[ 100

3

77

[ 100 [ 100

30

4c

[ 100

46

[ 100 [ 100 [ 100

[ 100

21

5

6

7

[ 100

[ 70

[ 75

[ 80

8

9

10

11

12

13

[ 46

[ 94

[ 46

[ 94

[ 100

14

75

94

35

15

80

[ 100

[ 100 [ 100 [ 100

[ 32

References

NM 107

NM 108

Ribavirin

6-Azauridine

ACV

[ 100

55

1.8

15

[ 100

1.4

46

10

1.1

[ 100

80

HSV-1 (2.4)

0.05

2

Pleconaril

20

VV (1.4)

Mycophenolic acid [ 100

1.7

[ 100

1.4

Data represent mean values SD for three independent determinations. For values where SD is not shown, variation among duplicate samples

was less than 15%

The bold values are the most interesting results of this antiviral screening, are highlighted the EC₅₀values that are comparable with the EC₅₀of

the reference drugs

^aCompound concentration (lL) required to reduce the viability of mock-infected MDBK cells by 50%, as determined by the MTT method

^bCompound concentration (lL) required to achieve 50% protection of MDBK cells from the BVDV-induced cytopathogenicity, as determined

by the MTT method

^cCompound concentration (lL) required to reduce the viability of mock-infected BHK monolayers by 50%, as determined by the MTT method

^dCompound concentration (lM) required to achieve 50% protection of BHK cells from YFV and Reo-1 induced cytopathogenicity, as deter-

mined by the MTT method

^eCompound concentration (lM) required to reduce the viability of mock-infected VERO-76 monolayers by 50%

^fCompound concentration (lM) required to reduce the plaque number of the indicated virus by 50% in VERO-76 monolayers

using an Agilent Model 6850 series gas chromatograph sys-

tem coupled to an Agilent Technologies 5973 network mass

selective detector. The chromatographic separations were

performed using an HP-5 (5% Phenyl, 95% methyl

polysiloxane), 30 m 9 0.20 mm i.d., fused-silica capillary

column of 0.25 lm ﬁlm thickness. The injector temperature

was 250 °C and the splitless injection mode was used. The

oven temperature program was 80 °C (held for 3 min) to

300 °C at 10 °C/min (held for 15 min). The MS operating

conditions were positive electron ionization mode (EI).

LC–MS analyses were performed on the AB SCIEX 4000

QTRAP^ÒLC–MS/MS system by direct injection (50 mm³)

and detected in positive polarity.

Flash chromatography was performed using 70–230

mesh (Merck silica gel 60). Light petroleum refers to the

fraction with b.p. 40–60 °C. The progress of the reactions,

the R_f, and the purity of the ﬁnal compounds were moni-

tored by TLC using Merck F-254 commercial plates. In all

cases, elemental chemical composition was within 0.4%

of the theoretical value.

The

intermediates

5-chloro-2-(4-nitrophenyl)-2H-

(3a) and 4-(5-chloro-2H-

benzo[d][1,2,3]triazole

benzo[d][1,2,3]triazol-2-yl)aniline (4a) were synthesized

according to procedure previously reported by us [13].

5-Chlorobenzotriazole (1) and 4-chloronitrobenzene (2)

were commercially available (Sigma-Aldrich).

123

R. Ibba et al.

1

5-Chloro-2-(4-nitrophenyl)-2H-benzo[d][1,2,3]triazole (3a,

C₁₂H₇ClN₄O₂) A mixture of 1.0 g p-chloro-nitrobenzene

(1, 6.35 mmol), 1.2 g p-chloro-benzotriazole (2,

7.62 mmol), and 3.10 g Cs₂CO₃(9.52 mmol) in 10 cm³

DMF was stirred and heated at 140 °C for 5 h. Progress of

the reaction was monitored by TLC (diethyl ether/petro-

leum ether, 7/3). The reaction mixture was poured onto

about 250 cm³of crushed ice. After all ice melted, the solid

was ﬁltered off and washed with H₂O. The product was

suspended in 200 cm³MeOH, stirred for 30 min, and left

in suspension overnight. The precipitate was ﬁltered off

and dried in an oven (45 °C). The crude 3 was a mixture of

three isomers. Compound 3a was isolated by ﬂash chro-

matography (petroleum ether/diethyl ether, 3/1) in 20%

yield (60 mg). Yellow solid; m.p.: 218.8–220.0 °C; R_f-

= 0.67 (petroleum ether/diethyl ether, 3/1); ¹H NMR

(400 MHz, DMSO-d₆): d = 7.59 (d, 1H, J = 12 Hz, CH-6),

8.16 (d, 1H, J = 8.0 Hz, CH-5), 8.28 (s, 1H, CH-3), 8.53

(dd, 4H, J = 8.0 Hz, J = 20 Hz, CH-2⁰,5⁰, CH-3⁰,6⁰) ppm;

¹³C NMR (100 MHz, DMSO-d₆): d = 117.52 (CH-

2⁰? CH 6⁰), 120.44 (CH-3⁰? CH 5⁰), 121.28 (CH-4),

125.62 (CH-6), 129.85 (CH-7), 133.17 (C–Cl), 143.24

(C-1⁰), 143.61 (C-7a), 145.28 (C-3a), 147.44 (C-NO₂) ppm;

GC–MS: calc. m/z = 274.02 (C₁₂H₇ClN₄O₂, [M]^?), found

m/z = 274 ([M]^?) at 23.472 min (Scan 6460).

(petroleum ether/ethyl acetate, 7/3); H NMR (400 MHz,

DMSO-d₆): d = 5.63 (s, 2H, NH₂), 6.78 (d, 2H, J = 8.8 Hz,

CH-3⁰,5⁰), 7.60 (d, 2H, J = 8.8 Hz, CH-2⁰,6⁰), 7.80 (d, 1H,

J = 8.8 Hz, CH-6), 8.17 (d, 1H, J = 8.8 Hz, CH-7), 8.27 (s,

1H, CH-4) ppm; ¹³C NMR (100 MHz, DMSO-d₆):

d = 115.64 (CH-2⁰? CH 6⁰), 117.43 (CH-3⁰? CH 5⁰),

120.83 (CH-4), 122.94 (CH-6), 124.12 (CH-7), 127.41 (C-

1⁰), 129.08 (C-7a), 135.33 (C–Cl), 146.65 (C-3a), 149.81

(C-NH₂) ppm; LC–MS: calc. m/z = 244.05 (C₁₂H₉ClN₄,

[M]^?), found m/z = 245.1 ([M?H]^?).

4-(6-Chloro-1H-benzo[d][1,2,3]triazol-1-yl)aniline (4c, C₁₂H₉-

ClN₄) Pale yellow solid; m.p.: 166.7–168.0 °C; R_f= 0.18

(petroleum ether/ethyl acetate, 7/3); H NMR (400 MHz,

1

DMSO-d₆): d = 5.66 (s, 2H, NH₂), 6.78 (d, 2H, J = 8.8 Hz,

CH-3⁰,5⁰), 7.43 (d, 2H, J = 8.8 Hz, CH-2⁰,6⁰), 7.49 (d, 1H,

J = 8.8 Hz, CH-4), 7.82 (s, 1H, CH-7), 8.16 (d, 1H,

J = 8.8 Hz, CH-5) ppm; ¹³C NMR (100 MHz, DMSO-d₆):

d = 115.78 (CH-2⁰? CH 6⁰), 118.18 (CH-3⁰? CH 5⁰),

120.61 (CH-7), 122.81 (CH-5), 125.04 (CH-4), 128.14 (C-

1⁰), 130.52 (C-7a), 134.90 (C–Cl), 142.19 (C-3a), 149.80

(C-NH₂) ppm; LC–MS: calc. m/z = 244.05 (C₁₂H₉ClN₄,

[M]^?), found m/z = 245.1 ([M?H]^?).

N-[4-(5-Chloro-2H-benzo[d][1,2,3]triazol-2-yl)phenyl]-4-ni-

trobenzamide (5, C₁₉H₁₂ClN₅O₃) A mixture of 130 mg

4-nitrobenzoyl chloride (0.68 mmol) in 1.5 cm³DMF and

140 mg compound 4a (0.57 mmol) in 2.5 cm³DMF was

stirred at 120 °C. The reaction was complete after 2 h.

Cold H₂O (100 cm³) was poured to the reaction mixture.

The precipitate was ﬁltered off and dried in an oven

overnight. The crude obtained was puriﬁed by ﬂash chro-

matography (petroleum ether/ethyl acetate, 7.5/1.5) to give

5 in 27.3% yield (60 mg). Pale yellow solid; m.p.:

272.8–275.1 °C; R_f= 0.32 (petroleum ether/ethyl acetate,

7.5/1.5); ¹H NMR (400 MHz, DMSO-d₆): d = 7.54 (d, 1H,

J = 12 Hz), 8.09–8.12 (m, 3H), 8.22–8.24 (m, 3H), 8.34 (d,

2H, J = 8.0 Hz), 8.41 (d, 2H, J = 8.0 Hz), 10.90 (s, 1H,

NH) ppm; ¹³C NMR (100 MHz, DMSO-d₆): d = 117.15

(CH-2⁰? CH 6⁰), 120.01 (CH-3⁰? CH 5⁰), 120.93

(CH-4), 121.15 (CH-6), 123.59 (CH-3⁰⁰? CH 5⁰⁰), 128.61

(CH-2⁰⁰? CH 6⁰⁰), 129.31 (CH-6), 132.01 (C–Cl), 135.09

(C-1⁰), 140.06 (C-1⁰⁰), 140.21 (C-NH), 142.98 (C-7a),

144.70 (C-3a), 149.28 (C-NO₂), 164.19 (CO) ppm; GC–

MS: calc. m/z = 393.06 (C₁₉H₁₂ClN₅O₃, [M]^?), found m/

z = 393 ([M]^?) at 24.582 min (Scan 4520).

Synthesis of compounds 4a–4c A mixture of 1.5 g of

compounds 3a–3c (5.46 mmol) and 3.75 cm³methylhy-

drazine (71.65 mmol) in 120 cm³EtOH was stirred and

heated in an autoclave at about 100 °C for 46 h. The sol-

vent was removed under reduced pressure to obtain the

crude mixture 4a–4c as a yellow solid. The TLC (petro-

leum ether/ethyl acetate, 7/3) conﬁrmed that the crude 4a–

4c is a mix of three isomers. Their separation was per-

formed by ﬂash chromatography (petroleum ether/ethyl

acetate, 4/1) to give 4a in 43% yield, 4b in 22% yield, and

4c in 35% yield.

4-(5-Chloro-2H-benzo[d][1,2,3]triazol-2-yl)aniline (4a, C₁₂H₉-

ClN₄) Pale yellow solid; m.p.: 161.3–162.1 °C; R_f= 0.34

(petroleum ether/ethyl acetate, 7/3); H NMR (400 MHz,

1

DMSO-d₆): d = 5.77 (s, 2H, NH₂), 6.74 (d, 2H, J = 8.0 Hz,

CH-3⁰,5⁰), 7.46 (d, 1H, J = 8.0 Hz, CH-5), 7.96 (d, 2H,

J = 8.0 Hz, CH-2⁰,6⁰), 8.01 (d, 1H, J = 8.0 Hz, CH-6),

8.11 (s, 1H, CH-3) ppm; ¹³C NMR (100 MHz, DMSO-d₆):

d = 113.70 (CH-2⁰? CH 6⁰), 116.66 (CH-3⁰? CH 5⁰),

119.44 (CH-4), 121.61 (CH-6), 127.53 (C-1⁰), 128.56 (CH-

7), 131.01 (C–Cl), 142.56 (C-7a), 144.33 (C-3a), 150.52

(C-NH₂) ppm; GC–MS: calc. m/z = 244.05 (C₁₂H₉ClN₄,

[M]^?), found m/z = 244 ([M]^?) at 23.498 min (Scan 7198).

N-[4-(5-Chloro-2H-benzo[d][1,2,3]triazol-2-yl)phenyl]-3,4,5-

trimethoxybenzamide (6, C₂₂H₁₉ClN₄O₄) A mixture of

140 mg 3,4,5-trimethoxybenzoyl chloride (0.57 mmol) in

2.0 cm³DMF and 160 mg compound 4a (0.57 mmol) in

3.0 cm³DMF was stirred at 120 °C. The reaction was

complete after 2 h. Cold H₂O (100 cm³) was poured onto

4-(5-Chloro-1H-benzo[d][1,2,3]triazol-1-yl)aniline (4b, C₁₂H₉-

ClN₄) Pale yellow solid; m.p.: 125.6–126.2 °C; R_f= 0.21

123

Antiviral activities of 5-chlorobenzotriazole derivatives

the reaction mixture. The precipitate was ﬁltered off and

dried in an oven overnight. The crude obtained was puriﬁed

by ﬂash chromatography (petroleum ether/ethyl acetate,

6/3) to give 5 in 20% yield (50 mg). White solid; m.p.:

222.3–224.5 °C; R_f= 0.40 (petroleum ether/ethyl acetate,

6/3); ¹H NMR (400 MHz, DMSO-d₆): d = 3.75 (s, 3H,

OCH₃-4⁰⁰), 3.90 (s, 6H, 2 9 OCH₃-3⁰⁰,5⁰⁰), 7.32 (s, 2H,

2 9 CH-2⁰⁰, 6⁰⁰), 7.54 (d, 1H, J = 8 Hz, CH-5), 8.06 (d, 2H,

J = 8.0 Hz, 2 9 CH-2⁰,6⁰), 8.10 (d, 1H, J = 12 Hz, CH-6),

8.21 (s, 1H, CH-3), 8.32 (d, 2H, J = 12 Hz, 2 9 CH-3⁰,5⁰),

10.45 (s, 1H, NH) ppm; ¹³C NMR (100 MHz, DMSO-d₆):

d = 56.12 (2 CH₃), 60.13 (CH₃), 105.42 (CH-2⁰⁰? CH 6⁰⁰),

117.12 (CH-2⁰? CH 6⁰), 119.97 (CH-3⁰? CH 5⁰), 120.84

(CH-4), 121.15 (CH-6), 128.54 (CH-7), 129.68 (C-1⁰⁰),

131.95 (C-1⁰), 134.74 (C–Cl), 140.46 (C-NH), 140.53 (C-

7a), 142.95 (C-4⁰⁰), 144.68 (C-3a), 152.65 (CH-3⁰⁰? CH

5⁰⁰), 164.35 (CO) ppm; LC–MS: calc. m/z = 438.10 (C_22-

H₁₉ClN₄O₄, [M]^?), found m/z = 439.3 ([M?H]^?).

ﬂash chromatography (petroleum ether/ethyl acetate, 1/1)

to give 8 in 20% yield (160 mg). White solid; m.p.:

262.6–264.2 °C; R_f= 0.15 (petroleum ether/ethyl acetate,

1

8/2); H NMR (400 MHz, DMSO-d₆): d = 7.54 (d, 1H,

J = 8 Hz, CH-5), 7.95 (d, 2H, J = 8 Hz, CH-3⁰, 5⁰),

8.09–8.10 (m, 3H, CH-2⁰,6⁰, CH-6), 8.18–8.21 (m, 3H, CH-

3, CH-3⁰⁰,5⁰⁰), 8.33 (d, 2H, J = 8 Hz, CH-2⁰⁰,6⁰⁰), 10.80 (s,

1H, NHCO) ppm; ¹³C NMR-APT (100 MHz, DMSO-d₆):

d = 117.15 (CH-2⁰? CH 6⁰), 120.00 (CH-3⁰? CH 5⁰),

120.91 (CH-4), 121.10 (CH-6), 125.43 (CH-7), 128.70

(CH-2⁰⁰? CH 6⁰⁰), 126,130.06 (CH-3⁰⁰? CH 5⁰⁰), 131.71

(CF₃), 132.00 (C-1⁰), 134.99 (C–Cl), 138.40 (C-4⁰⁰), 140.20

(C-1⁰⁰), 141.01 (C-NH), 142.98 (C-7a), 144.70 (C-3a),

164.69 (CO) ppm; GC–MS: calc. m/z = 416.06 (C₂₀H_12-

ClF₃N₄O, [M]^?), found m/z = 416 ([M]^?) at 28.777 min

(Scan 5402).

N-[4-(5-Chloro-2H-benzo[d][1,2,3]triazol-2-yl)phenyl]-4-

methylbenzamide (9, C₂₀H₁₅ClN₄O) 4-Toluoyl chloride

(0.44 cm³, 3.34 mmol) was added to the solution of

400 mg compound 4a (1.67 mmol) in 15.0 cm³DMF. The

reaction mixture was stirred at 120 °C. The reaction was

complete after 2 h. Cold H₂O (100 cm³) was poured onto

the reaction mixture. The precipitate was ﬁltered off and

washed with water several times. The ﬁltered was dried in

an oven overnight. The crude obtained was puriﬁed by

ﬂash chromatography in eluent gradient (from petroleum

ether/ethyl acetate, 1/1 to ethyl acetate) to give 9 in 15%

yield (50 mg). White solid; m.p.: 271.8–273.0 °C; R_f-

= 0.18 (petroleum ether/ethyl acetate, 4/1); ¹H NMR

(400 MHz, DMSO-d₆): d = 2.41 (s, 3H, CH₃), 7.37 (d, 2H,

J = 8.0 Hz, CH-3⁰⁰,5⁰⁰), 7.53 (d, 1H, J = 8.0 Hz, CH-5),

7.92 (d, 2H, J = 8.0 Hz, CH-3⁰,5⁰), 8.09 (m, 3H, CH-2⁰,6⁰,

CH-6), 8.20 (s, 1H, CH-3), 8.30 (d, 2H, J = 8.0 Hz, CH-

2⁰⁰,6⁰⁰), 10.50 (s, 1H, NHCO) ppm; ¹³C NMR (100 MHz,

DMSO-d₆): d = 21.02 (CH₃), 117.13 (CH-2⁰? CH 6⁰),

119.98 (CH-3⁰? CH 5⁰), 120.83 (CH-4), 120.91 (CH-6),

127.79 (CH-2⁰⁰? CH 6⁰⁰), 128.53 (CH-3⁰⁰? CH 5⁰⁰),

128.96 (CH-7), 131.70 (C-1⁰), 131.93 (C-4⁰⁰), 134.63 (C–

Cl), 140.67 (C), 141.92 (C-7a), 142.95 (C-NH), 144.68 (C-

3a), 165.64 (CO) ppm; GC–MS: calc. m/z = 362.09 (C_20-

H₁₅ClN₄O, [M]^?), found m/z = 362 ([M]^?) at 24.840 min

(Scan 5152).

N-[4-(5-Chloro-2H-benzo[d][1,2,3]triazol-2-yl)phenyl]-4-

methoxybenzamide (7, C₂₀H₁₅ClN₄O₂)

A mixture of

630 mg 4-methoxybenzoyl chloride (3.68 mmol) in

8.0 cm³DMF and 450 mg compound 4a (1.84 mmol) in

8.0 cm³DMF was stirred at 120 °C. The reaction was

complete after 1 h. Cold H₂O (100 cm³) was poured onto

the reaction mixture. The precipitate was ﬁltered off and

washed with water several times. The ﬁltered was dried in

an oven overnight. The crude obtained was puriﬁed by

ﬂash chromatography (petroleum ether/ethyl acetate, 1/1)

to give 7 in 13% yield (40 mg). White solid; m.p.:

259.2–261.8 °C; R_f= 0.32 (petroleum ether/ethyl acetate,

2/3); ¹H NMR (400 MHz, DMSO-d₆): d = 3.66 (s, 3H,

OCH₃), 7.08 (d, 2H, J = 8.0 Hz, CH-3⁰⁰, 5⁰⁰), 7.54 (d, 1H,

J = 8.0 Hz, CH-5), 8.01 (d, 2H, J = 8.0 Hz, CH-3⁰,5⁰),

8.10 (m, 3H, CH-2⁰,6⁰, CH-6), 8.21 (s, 1H, CH-3), 8.30 (d,

2H, J = 8.0 Hz, Ch-2⁰⁰,6⁰⁰), 10.43 (s, 1H, NHCO) ppm; ¹³C

NMR-APT (100 MHz, DMSO-d₆): d = 55.46 (CH₃),

113.68 (CH-3⁰⁰? CH 5⁰⁰), 117.13 (CH-2⁰? CH 6⁰),

119.97 (CH-3⁰? CH 5⁰), 120.82 (CH-4), 120.52 (CH-6),

126.56 (C-1⁰⁰), 128.86 (CH-2⁰⁰? CH 6⁰⁰), 129.74 (CH-7),

131.92 (C-1⁰), 134.53 (C–Cl), 140.80 (C-NH), 142.95 (C-

7a), 144.68 (C-3a), 162.12 (C-4⁰⁰), 165.16 (CO) ppm; GC–

MS: calc. m/z = 378.08 (C₂₀H₁₅ClN₄O₂, [M]^?), found m/

z = 378 ([M]^?) at 43.878 min (Scan 9661).

N-[4-(5-Chloro-2H-benzo[d][1,2,3]triazol-2-yl)phenyl]-4-

cyanobenzamide (10, C₂₀H₁₂ClN₅O) A mixture of 550 mg

4-cyanobenzoyl chloride (3.34 mmol) in 7.0 cm³DMF and

400 mg compound 4a (1.67 mmol) in 8.0 cm³DMF was

stirred at 120 °C. The reaction was complete after 2 h.

Cold H₂O (100 cm³) was poured onto the reaction mixture.

The precipitate was ﬁltered off and washed with water

several times. The ﬁltered was dried in an oven overnight.

The crude obtained was puriﬁed by ﬂash chromatography

in gradient method of eluents (from petroleum ether/ethyl

N-[4-(5-Chloro-2H-benzo[d][1,2,3]triazol-2-yl)phenyl]-4-(tri-

fluoromethyl)benzamide (8, C₂₀H₁₂ClF₃N₄O) A mixture of

800 mg 4-(triﬂuoromethyl)benzoyl chloride (3.84 mmol)

in 9.0 cm³DMF and 470 mg compound 4a (1.92 mmol) in

8.0 cm³DMF was stirred at 120 °C. The reaction was

complete after 2 h. Cold H₂O (100 cm³) was poured onto

the reaction mixture. The precipitate was ﬁltered off and

washed with water several times. The ﬁltered was dried in

an oven overnight. The crude obtained was puriﬁed by

123

R. Ibba et al.

acetate, 1/1 to ethyl acetate) to give 10 in 25% yield

(150 mg). White solid; m.p.: 281.9–283.1 °C; R_f= 0.12

(petroleum ether/ethyl acetate, 4/1); H NMR (400 MHz,

(400 MHz, DMSO-d₆): d = 0.89 (t, 3H, J = 8.0 Hz, CH₃),

1.45 (m, 2H, CH₂CH₂CH₃), 3.08 (m, 2H, CH₂CH₂CH₃),

6.30 (m, 1H, CONH-propyl), 7.51 (d, 1H, J = 12.0 Hz,

CH-5), 7.67 (d, 2H, J = 12.0 Hz, CH-3⁰,5⁰), 8.07 (d, 1H,

J = 8.0 Hz, CH-6), 8.16 (m, 3H, CH-2⁰,6⁰, CH-3), 8.83 (s,

1H, NHCO) ppm;¹³C NMR-APT (100 MHz, DMSO-d₆):

d = 21.30 (CH₃), 22.88 (CH₂), 40.88 (CH₂), 117.00 (CH-

2⁰? CH 6⁰), 117.93 (CH-3⁰? CH 5⁰), 119.83 (CH-4),

121.01 (CH-6), 128.22 (CH-7), 131.65 (C-1⁰), 132.67 (C–

Cl), 142.18 (C-7a), 144.82 (C-3a), 144.56 (C4⁰-NH),

154.90 (CO) ppm; LC–MS (in MeOH): calc. m/z = 329.10

(C₁₆H₁₆ClN₅O, [M]^?), found m/z = 330.1 ([M?H]^?).

1

DMSO-d₆): d = 7.53 (d, 1H, J = 12 Hz), 8.05–8.11 (m,

5H), 8.15 (d, 2H, J = 8 Hz), 8.21 (s, 1H), 8.33 (d, 2H,

J = 8 Hz) ppm; ¹³C NMR (100 MHz, DMSO-d₆):

d = 114.06 (CN), 118.26 (C–CN), 117.15 (CH-2⁰? CH

6⁰), 120.00 (CH-3⁰? CH 5⁰), 120.92 (CH-4), 121.11 (CH-

6), 128.35 (CH-2⁰⁰? CH 6⁰⁰), 128.61 (CH-7), 132.51 (CH-

3⁰⁰? CH 5⁰⁰), 132.09 (C-1⁰), 135.04 (C–Cl), 138.60 (C-4⁰⁰),

140.11 (C-1⁰⁰), 140.88 (C-NH), 142.56 (C-7a), 144.71 (C-

3a), 164.46 (CO) ppm; GC–MS (EI): calc. m/z = 373.07

(C₂₀H₁₂ClN₅O, [M]^?), found m/z = 371 ([M-2]^?) at

24.840 min (Scan 5152).

4-(5-Chloro-2H-benzo[d][1,2,3]triazol-2-yl)-N-methylaniline

(13, C₁₃H₁₁ClN₄) A solution of 500 mg compound 4a

(2.05 mmol) in 5 cm³DMF was treated with 0.22 cm³

dimethyl sulfate (2.30 mmol) and heated at 100 °C and

strongly stirred. The reaction ﬁnished after 2 h. By cooling

down, a white solid suspension was produced. The pre-

cipitate was ﬁltered off and washed with diethyl ether

several times. The ﬁltered was dried in an oven overnight.

The pure product 13 was obtained in 76% yield (380 mg).

White solid; m.p.: 302.5–304.1 °C; R_f= 0.47 (petroleum

1-[4-(5-Chloro-2H-benzo[d][1,2,3]triazol-2-yl)phenyl]-3-cy-

clohexylurea (11, C₁₉H₂₀ClN₅O) A solution of 130 mg

compound 4a (0.531 mmol) in 6.5 cm³DCM at 0 °C was

treated with 90 cm³cyclohexyl isocyanate (0.69 mmol)

and strongly stirred as the temperature slowly rose to room

temperature. After stirring at room temperature for 24 h,

the reaction was ﬁnished and a white solid suspension was

produced. To the resulting suspension, hexane was added

to yield more precipitate. The solid material was collected

by ﬁltration to provide the compound 11 in 50% yield

(60 mg). White solid; m.p.: 297.0–298.5 °C; R_f= 0.56

1

ether/ethyl acetate, 3/2); H NMR (400 MHz, DMSO-d₆):

d = 2.67 (s, 3H, CH₃), 6.72 (s, 1H, NH), 6.81 (d, 2H,

J = 8.8 Hz, CH-3⁰, CH-5⁰), 7.46 (d, 1H, J = 8.8 Hz, CH-7),

8.05–7.99 (m, 3H, CH-6, CH-2⁰, CH-6⁰), 8.11 (s, 1H, H-4)

ppm;¹³C NMR-APT (100 MHz, DMSO-d₆): d = 28.96

(CH₃), 110.59 (CH-3⁰? CH 5⁰), 121.35 (CH-2⁰? CH 6⁰),

123.44 (CH-4), 125.25 (C-1⁰), 128.88 (CH-6), 132.65 (CH-

7), 135.12 (C–Cl), 144.46 (C-7a), 147.50 (C-3a), 151.20

(C-NH) ppm; LC–MS (in MeOH): calc. m/z = 258.07

(C₁₃H₁₁ClN₄, [M]^?), found m/z = 259.1 ([M?H]^?).

1

(petroleum ether/ethyl acetate, 3/2); H NMR (400 MHz,

DMSO-d₆): d = 1.21 (m, 3H, CH₂-3⁰⁰, CH₂-4⁰⁰a), 1.32 (m,

2H, CH₂-5⁰⁰), 1.55 (m, 1H, CH₂-4⁰⁰b), 1.68 (m, 2H, CH₂-

2⁰⁰), 1.82 (m, 2H, CH₂-6⁰⁰), 3.50 (s, 1H, CH-1⁰⁰), 6.23 (d,

1H, J = 8.0 Hz, CONH-cyclohexyl), 7.51 (d, 1H,

J = 8.0 Hz, CH-5), 7.65 (d, 2H, J = 8.0 Hz, CH-3⁰,5⁰),

8.07 (d, 1H, J = 8.0 Hz, CH-6), 8.16 (m, 3H, CH-2⁰,6⁰,

CH-3), 8.72 (s, 1H, NHCO) ppm; ¹³C NMR (100 MHz,

DMSO-d₆): d = 24.31 (2 CH₂), 25.19 (CH₂), 32.82 (2

CH₂), 47.69 (CH), 117.01 (CH-2⁰? CH 6⁰), 117.87 (CH-

3⁰? CH 5⁰), 119.83 (CH-4), 121.04 (CH-6), 128.23 (CH-

7), 131.65 (C-1⁰), 132.67 (C–Cl), 142.12 (C-7a), 142.82 (C-

3a), 144.57 (C4⁰-NH), 154.07 (C=O) ppm; LC–MS (in

MeOH): calc. m/z = 369.13 (C₁₉H₂₀ClN₅O, [M]^?), found

m/z = 369.1 ([M]^?).

4-(5-Chloro-2H-benzo[d][1,2,3]triazol-2-yl)-N,N-diethylani-

line (14, C₁₆H₁₇ClN₄) A solution of 500 mg compound 4a

(2.05 mmol) in 5 cm³DMF was treated with 0.38 g diethyl

sulfate (0.32 cm³, 2.50 mmol), heated at 100 °C and

strongly stirred. The reaction resulted ﬁnished after 5 h. By

cooling down, a brown solid suspension was produced. The

precipitate was ﬁltered off and washed with diethyl ether

several times. The ﬁltered was dried in an oven overnight.

The crude product was puriﬁed by crystallization with

acetone and pure product 14 was obtained in 66% yield

(400 mg). Brown solid; m.p.: 279.2–280.1 °C; R_f= 0.53

1-[4-(5-Chloro-2H-benzo[d][1,2,3]triazol-2-yl)phenyl]-3-

propylurea (12, C₁₆H₁₆ClN₅O) A solution of 150 mg

compound 4a (0.613 mmol) in 10 cm³DCM at 0 °C was

treated with 120 cm³propyl isocyanate (1.226 mmol) and

strongly stirred as the temperature slowly rose to room

temperature. After stirring at room temperature for 24 h,

the reaction resulted ﬁnished and a white solid suspension

was produced. To the resulting suspension, hexane was

added to yield more precipitate. The solid material was

collected by ﬁltration to provide the compound 12 in 38%

yield (60 mg). White solid; m.p.: 304.5–306.1 °C; R_f-

= 0.57 (petroleum ether/ethyl acetate, 3/2); ¹H NMR

1

(petroleum ether/ethyl acetate, 3/2); H NMR (400 MHz,

DMSO-d₆): d = 1.12 (s, 3H, CH₂-CH₃), 3.73 (m, 2H,

CH₂CH₃), 6.76 (d, 2H, J = 8.8 Hz, CH-3⁰-5⁰), 7.46 (d, 1H,

J = 8.8 Hz, CH-7), 8.11–7.96 (m, 3H, CH-6, CH-2⁰,6⁰),

8.16 (s, 1H, CH-4) ppm; ¹³C NMR-APT (100 MHz,

DMSO-d₆): d = 12.98 (2 CH₃), 46.23 (2 CH₂), 110.75

(CH-3⁰? CH 5⁰), 122.43 (CH-2⁰? CH 6⁰), 123.46 (CH-

4), 128.33 (C-1⁰), 130.55 (CH-6), 133.76 (CH-7), 136.12

(C–Cl), 146.68 (C-7a), 148.31 (C-3a), 150.10 (C-NH) ppm;

123

Antiviral activities of 5-chlorobenzotriazole derivatives

LC–MS (in MeOH): calc. m/z = 300.11 (C₁₆H₁₇ClN₄,

[M]^?), found m/z = 301.1 ([M?H]^?).

of only two of the three genera of the Flaviviridae family,

i.e., Flaviviruses (Yellow Fever Virus, YFV) and Pes-

tiviruses (Bovine Viral Diarrhea Virus, BVDV), as Hepa-

civiruses can hardly be used in routine cell-based assays);

(3) Picornaviridae family: Human Coxsackievirus type B5

(CVB-5) strain (ATCC^ÒVR-1036AS/HO^TM) and Human

Poliovirus type-1 Sabin (Sb-1) [strain Chat (ATCC VR-

1562)]. Virus representative of a negative-sense single-

strand RNA (ssRNA^-) group used was: Vesicular

Stomatitis Virus (VSV) [strain Indiana Lab (ATCC VR-

158)] and Human Respiratory Syncytial Virus (RSV)

[strain A2 (ATCC VR-1540)]. A virus representative of a

double-strand RNA (dsRNA) group used was: reovirus

type-1 [strain 3651 (SV-12, simian virus 12) (ATCC VR-

214)]. Viruses representatives of DNA group used were:

(1) Poxviridae family: Vaccinia Virus (VV) [strain Elstree-

Lister Vaccine (ATCC VR-1549)]; (2) Herpesviridae

family: Human Herpesvirus 1 (HSV-1) [strain KOS

(ATCC VR-1493)].

N-[4-(5-Chloro-2H-benzo[d][1,2,3]triazol-2-yl)phenyl]acet-

amide (15, C₁₄H₁₁ClN₄O) A portion of 500 mg of com-

pound 4a (2.05 mmol) was added to 10 cm³of acetic

anhydride (90 mmol, 9.25 g), heated at 60 °C, and strongly

stirred. The reaction resulted ﬁnished after 1 h and moni-

tored by TLC (petroleum ether/ethyl acetate, 7/3). By

cooling down, a white solid suspension was produced. The

precipitate was ﬁltered off and washed with water several

times and diethyl ether. The ﬁltered was dried in an oven

overnight. The pure product was obtained in 66% yield

(510 mg). White solid; m.p.: 241.2–243.0 °C; R_f= 0.39

1

(petroleum ether/ethyl acetate, 3/2); H NMR (400 MHz,

DMSO-d₆): d = 2.11 (s, 1H, CH₃), 7.51 (d, 2H, J = 9.2 Hz,

CH-7), 7.86 (d, 1H, J = 8.4 Hz, CH-3⁰-5⁰), 8.08 (d, 1H,

J = 9.2 Hz, CH-6), 8.18 (s, 1H, CH-4), 8.23 (d, 2H,

J = 8.4 Hz, CH-2⁰,6⁰), 10.29 (s, 1H, NH) ppm; ¹³C NMR-

APT (100 MHz, DMSO-d₆): d = 23.88 (CH₃), 120.15

(CH-3⁰? CH 5⁰), 121.20 (CH-2⁰? CH 6⁰), 124.66 (CH-

4), 129.77 (CH-6), 131.61 (CH-7), 132.82 (C-1⁰), 133.99

(C–Cl), 145.51 (C-7a), 148.13 (C-3a), 148.76 (C-NH),

169.55 (CO) ppm; LC–MS (in MeOH): calc. m/z = 286.06

(C₁₄H₁₁ClN₄O, [M]^?), found m/z = 287.1 ([M?H]^?).

Cytotoxicity assays

Cytotoxicity assays were run in parallel with antiviral

assays. MDBK and BHK cells were seeded at an initial

density of 6 9 10⁵and 1 9 10⁶cells/cm³in 96-well

plates, respectively, in culture medium [Minimum Essen-

tial Medium with Earle’s salts (MEM-E) with ^L-glutamine,

supplemented with 10% horse serum and 1 mM sodium

pyruvate (for MDBK cells) or with 10% fetal bovine serum

(FBS) (for BHK cells), and 0.025 g/dm³kanamycin]. Cell

cultures were then incubated at 37 °C in a humidiﬁed, 5%

CO₂atmosphere in the absence or presence of serial dilu-

tions of test compounds. Cell viability was determined after

48–96 h at 37 °C by the 3-(4,5-dimethylthiazol-2-yl)-2,5-

diphenyltetrazolium bromide (MTT) method [23]. Vero-76

cells were seeded at an initial density of 4 9 10⁵cells/cm³

in 24-well plates, in culture medium [Dulbecco’s Modiﬁed

Eagle Medium (D-MEM) with ^L-glutamine, supplemented

with fetal bovine serum (FBS), and 0.025 g/dm³kanamy-

cin]. Cell cultures were then incubated at 37 °C in a

humidiﬁed, 5% CO₂atmosphere in the absence or presence

of serial dilutions of test compounds. Cell viability was

determined after 48–96 h at 37 °C by the crystal violet

staining method.

Test compounds

Compounds were dissolved in DMSO at 100 mM and then

diluted in culture medium.

Cells and viruses

Cell lines were purchased from American Type Culture

Collection (ATCC). The absence of mycoplasma contam-

ination was checked periodically by the Hoechst staining

method. Cell lines supporting the multiplication of RNA

and DNA viruses were the following: Madin-Darby Bovine

Kidney (MDBK) [ATCC CCL 22 (NBL-1) Bos taurus];

Baby Hamster Kidney (BHK-21) [ATCC CCL 10 (C-13)

Mesocricetus auratus]; Monkey kidney (Vero-76) (ATCC

CRL 1587 Cercopithecus aethiops). Viruses were pur-

chased from American Type Culture Collection (ATCC)

except Yellow Fever Virus (YFV), and Human Immun-

odeﬁciency Virus type-1 (HIV-1). Viruses representative of

positive-sense single-strand RNA (ssRNA^?) group used

were: (1) Retroviridae family: the laboratory strain HIV-1

IIIB wild type, obtained from the supernatant of the per-

sistently infected H9/IIIB cells (NIH 1983); (2) Flaviviri-

dae family: YFV [strain 17-D vaccine (Stamaril Pasteur

J07B01)] and Bovine Viral Diarrhea Virus (BVDV) [strain

NADL (ATCC VR-534)] (compounds were evaluated

in vitro for antiviral activity against viruses representative

Antiviral assays

Compound activity against YFV and Reo-1 was based on

inhibition of virus-induced cytopathogenicity in BHK-21

cells acutely infected with an m.o.i. of 0.01. Compounds

activity against BVDV was based on inhibition of virus-

induced cytopathogenicity in MDBK cells acutely infected

with an m.o.i. of 0.01. Brieﬂy, BHK and MDBK cells were

123

R. Ibba et al.

seeded in 96-well plates at a density of 5 9 10⁴and

3 9 10⁴cells/well, respectively, and were allowed to form

conﬂuent monolayers by incubating overnight in growth

medium at 37 °C in a humidiﬁed CO₂(5%) atmosphere.

Cell monolayers were then infected with 50 mm³of a

proper virus dilution in maintenance medium (MEM-E

with ^L-glutamine, supplemented with 0.5% inactivated

FBS, 1 mM sodium pyruvate and 0.025 g/dm³kanamycin)

to give an m.o.i of 0.01. After 2 h, 50 mm³of maintenance

medium, without or with serial dilutions of test com-

pounds, were added. After a 3–4 day incubation at 37 °C,

cell viability was determined by the MTT method.

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4. Alonzi DS, Scott KA, Dwek RA, Zitzmann N (2017) Biochem

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assays in infected Vero-76 cell monolayers. To this end,

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2 9 10⁵cells/well and were allowed to form conﬂuent

monolayers by incubating overnight in growth medium (D-

MEM with ^L-glutamine and 4500 mg/dm³D-glucose, sup-

plemented with 10% FBS and 0.025 g/dm³kanamycin) at

37 °C in a humidiﬁed CO₂(5%) atmosphere. Then,

monolayers were infected for 2 h with 250 mm³of proper

virus dilutions to give 50–100 PFU/well. Following

removal of unadsorbed virus, 500 mm³of maintenance

medium (D-MEM medium with ^L-glutamine and 4500 mg/

dm³^D-glucose supplemented with 1% inactivated FBS and

0.75% methyl-cellulose), without or with serial dilutions of

test compounds were added. Cultures were incubated at

37 °C for 2 (Sb-1 and VSV), 3 (CVB-5, VV and HSV-1),

or 5 days (RSV), and then ﬁxed with PBS containing 50%

ethanol and 0.8% crystal violet, washed, and air-dried.

Plaques were then counted and EC₅₀(50% effective con-

centration) was calculated by linear regression technique.

Linear regression analysis: viral and cell growth at each

drug concentration was expressed as percentage of

untreated controls and concentrations resulting in 50%

(EC₅₀and CC₅₀) growth inhibition.

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Acknowledgements The authors gratefully acknowledge the MIUR—

`

Ministero dell’Istruzione dell’Universita e della Ricerca—(Italy) for

the ﬁnancial support.

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123

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