2-Arylbenzimidazoles as Antiviral and Antiproliferative Agents-Part 1

Medicinal Chemistry, 2009, 5, 507-516

507

2-Arylbenzimidazoles as Antiviral and Antiproliferative Agents-Part 2 Gabriella Vitalea, Paola Corona a, Mario Lorigaa, Antonio Cartaa, Giuseppe Pagliettia*, Paolo La Collab,*, Bernardetta Busonerab, Esther Marongiub, David Collub and Roberta Loddob a

Dipartimento Farmaco Chimico Tossicologico, University of Sassari, Via Muroni,23-07100 Sassari, Italy; bDipartimento di Scienze e Tecnologie Biomediche, Sez. di Microbiologia e Virologia Generale e Biotecnologie Microbiche, Università di Cagliari, Cittadella Universitaria, SS 554 Km 4,500, 09042 Monserrato (CA), Italy Abstract: In prosecution of an anti-Flaviviridae project a new series of variously substituted 2-diphenyl-benzimidazoles were synthesized and tested in vitro for antiviral and antiproliferative activities. Compounds were tested in cell-based assays against viruses representative of: i) two of the three genera of the Flaviviridae family, i.e. Flaviviruses and Pestiviruses; ii) other RNA virus families, such as Retroviridae, Picornaviridae, Paramyxoviridae, Rhabdoviridae and Reoviridae; iii) two DNA virus families (Herpesviridae and Poxviridae). The 5Acetyl-2-(4’-nitrobiphenyl-4-yl)-1H-benzimidazole (24) emerged as potent active lead compound against Yellow Fever Virus (a Flavivirus) (EC50= 0.5
M) and CVB-2 at 1 μM and was not cytotoxic, whereas the other title benzimidazoles showed no antiviral activity at concentrations not cytotoxic for the resting cell monolayers. Among the examined series, the most cytotoxic derivatives (11,12,14,16,18,19,20,21,23,25-30) against mock-infected MT-4 cells (CC50 100

>100

15

>15

12

CH3

H

H

6

39

>39

2

>2

13

CH3

CH3

H

30


100

>100

2

>2

14

Cl

H

H

7

5

>5

2.5

>2.5

15

Cl

Cl

H

18

100

>100

1

>1

16

CF3

H

H

6

5

>5

0.2

>0.2

17

MeCO

H

H

8

9

>9

4

>4

18

H

H

NO2

2

30

>30

4

>4

19

CH3

H

NO2

7


100

>100

2

>2

20

CH3

CH3

NO2

3

68

>68

5

>5

21

Cl

H

NO2

2

38

>38

4

>4

22

Cl

Cl

NO2

48

75

>75

17

>17

23

CF3

H

NO2

2

15

>15

2

>2

24

MeCO

H

NO2

>100

49

0.5

13

1

25

H

H

NH

0.7

15

>15

1.7

>1.7

26

CH3

H

NH2

0.3

9

>9

0.4

>0.4

27

CH3

CH3

NH2

0.4

11

>11

0.5

>0.5

28

Cl

H

NH2

0.8

10

>10

0.8

>0.8

29

Cl

Cl

NH2

1

2

>2

3

>3

30

CF3

H

NH2

0.3

1

>1

0.4

>0.4

31

H

H

NHCOCH3

>100

100

>100

>100

>100

32

CH3

H

NHCOCH3

>100

>100

>100

>100

22

33

CH3

CH3

NHCOCH3

87

>100

>100

>100

>100

34

Cl

H

NHCOCH3

>100

>100

>100

>100

>100

35

Cl

Cl

NHCOCH3

>100

>100

>100

>100

>100

36

CF3

H

NHCOCH3

33


100

>100

20

>20

NM 108^

>100

90

1.8

>100

20

NM 176*


100

>100

>100

>100

27

Reference compounds

Data represent mean values for three independent determinations. Variation among duplicate samples was less than 15%. a Compd. concn. (μM) required to reduce the viability of mock-infected MT-4 (CD4+ Human T-cells containing an integrated HTLV-1 genome) cells by 50%, as determined by the MTT method. b Compound concentration (μ
) required to achieve 50% protection of BHK cells (Kidney fibroblast) from the YFV (Yellow Fever Virus), induced cytopathogenicity, as determined by the MTT method. c Compound concentration (μM) required to reduce the viability of mock-infected VERO 76 (Monkey normal kidney) monolayers by 50%. f Compound concentration (μM) required to reduce the plaque number of CVB-2 (Coxsackievirus B2) by 50% in VERO-76 monolayers. ^ 2’-ß-methyl-guanosine. * 2’-ethynyl-D-citidine. All the samples resulted totally inactive against HIV-1wt, BVDV, Reo-1, HSV-1, VV, VSV, RSV and Sb-1.

2-Arylbenzimidazoles as Antiviral and Antiproliferative Agents-Part 2

Table 2.

Medicinal Chemistry, 2009, Vol. 5, No. 6

511

Antiproliferative Activity Against Leukaemia-/Lymphoma-Derived Cell Lines a

CC50 [μM]

Compounds b

c

MT4

d

CCRF-CEM

e

WIL-2NS

CCRF-SB

11

7±0.5

14±4

13±0.5

12±4

12

6±0.5

9.3±1

20±1

25±0.5

14

7±0.2

11±2

19±0.6

9.4±0.2

16

6±0.4

14±0.1

17±0.6

12±0.7

17

8±0.3

8.5±1

15±0.7

8.8±0.8

19

7±0.1

5.8±0.3

7.6±0.1

3.2±0.5

20

3±0.2

1.1±0.5

2.1±0.3

1.4±0.5

21

2±0.05

25±5

5±1

8.5±0.2

23

2±0.1

4.7±2

2.6±0.4

2.8±0.6

25

0.7±0.3

4.8±0.4

1.3±0.2

1.5±0.6

26

0.3±0.05

0.8±0.1

0.5±0.08

0.3±0.08

27

0.3±0.1

0.7±0.2

0.9±0.09

0.7±0.05

28

0.8±0.2

1±0.05

0.8±0.2

1±0.3

29

1±0.2

1.8±0.1

0.8±0.3

0.9±0.3

30

0.4±0.05

0.4±0.05

0.6±0.05

0.3±0.05

6-MP

0.1±0.02

1±0.2

3±0.05

1±0.1

Etoposide

0.09±0.01

0.09±0.01

0.2±0.02

0.1±0.01

a

Compound concentration required to reduce cell proliferation by 50%, as determined by the MTT method, under conditions allowing untreated controls to undergo at least three consecutive rounds of multiplication. Data represent mean values (±SD) for three independent determinations. b CD4+ human T-cells containing an integrated HTLV-1 genome. c CD4+ human acute T-lymphoblastic leukaemia. d Human splenic B-lymphoblastoid cells. e Human acute B-lymphoblastic leukemia.

Table 3.

Antiproliferative Activity Against Solid Tumour-Derived Cell Lines a

CC50[μ M]

Compounds b

SK-MEL-28

c

MCF7

d

SKMES-1

e

HepG2

f

DU145

11

>100

14±3

>100

9.5±0.7

38±3

12

>100

>100

>100

>100

>100

14

17±3

9±2

21±1

13±1

17±1

16

15±0.3

16±6

6±0.5

17±4

13±0.3

17

15±2

6±0.2

7.9±1

8.6±1

17±3

19

11±0.05

26±8

10±2

30±4

7.7±0.7

20

3.8±1

5.7±0.5

3.7±0.05

4±0.6

1.8±1

21

77±9

60±6

13±3

4.5±0.8

9.1±0.2

23

14±4

20±2

6±1

3±0.3

6.5±0.7

25

8.3±1

10±1

3.7±0.9

1.7±0.05

2.7±0.6

26

2.7±0.4

1.7±0.05

0.6±0.2

0.9±0.08

0.9±0.4

512 Medicinal Chemistry, 2009, Vol. 5, No. 6

Vitale et al.

(Table 3) contd….. a

CC50[μ M]

Compounds b

SK-MEL-28

c

d

MCF7

SKMES-1

e

HepG2

f

DU145

27

3.4±0.2

2±0.05

2.6±1

1.6±0.6

0.9±0.2

28

9.5±0.1

7±0.5

1.7±0.5

1.2±0.5

1.5±0.2

29

>100

40±10

3.5±0.2

1.5±0.3

2.2±0.5

30

1.5±0.4

0.9±0.05

0.6±0.3

0.7±0.1

0.8±2

6-MP

15±2

3±0.2

58±4

8±1

2±0.2

Etoposide

1.2±0.5

1±0.1

0.3±0.02

0.7±0.04

0.4±0.02

a Compound concentration required to reduce cell proliferation by 50%, as determined by the MTT method, under conditions allowing untreated controls to undergo at least three consecutive rounds of multiplication. Data represent mean values (±SD) for three independent determinations. b Human skin melanoma. c Human breast adenocarcinoma. d Human lung squamous carcinoma. e Human hepatocellular carcinoma. f Human prostate carcinoma.

Table 4.

Cytotoxicity Against “Normal” Cell Lines a

CC50[μ M]

Compounds b

MRC-5

a

c

CRL7065

11

>100

>100

12

>100

>100

14

29±4

36±5

16

13±0.5

26±1

18

>100

>100

19

30±6

41±2

20

7.4±2

39±5

21

>100

>100

23

36±8

55±8

25

13±1

49±6

26

22±0.5

50±9

27

5.2±1

10±4

28

14±3

43±12

29

85±15

>100

30

4.6±0.6

6±2

6-MP

>100

>100

Etoposide

>100

>100

Compound concentration required to reduce cell proliferation by 50%, as determined by the MTT method, under conditions allowing untreated controls to undergo at least three consecutive rounds of multiplication. Data represent mean values (±SD) for three independent determinations. b Human lung fibroblasts. c Human foreskin fibroblasts.

2-Arylbenzimidazoles as Antiviral and Antiproliferative Agents-Part 2

instrument at Laboratorio di Microanalisi, Dipartimento di Chimica, Università di Sassari, Italy, and results were within ±0.4% of theoretical values. Chemistry Intermediates The diamines (2-6) were commercially available. The diamine (7) was prepared according to Walley [25] starting from the commercially available parent nitro compound (Aldrich), while the diamine 8 has been obtained according to the procedure of W.Borsche and J.Barthenheier [26]. The bisulphite compounds (9, 10) were obtained in high yields from both the commercially available 4-biphenylcarboxyaldehyde (Aldrich) and the purposed prepared 4’nitrobiphenylyl-4-carboxaldehyde as described [27] with Na2S2O5 in ethanol respectively. General Procedure for the Preparation of the 2-biphenylyl5 and 5,6 Substituted Benzimidazoles (11-24) To a solution of 0.2 g of 1,2-diaminobenzenes (2-8) in 10 mL of ethanol was added in 1:1 molar ratio the sodium hydroxy(biphenyl-4-yl)methanesulfonate (9) or the sodium hydroxy(4’-nitrobiphenyl-4-yl)methanesulfonate (10). The reaction mixture was refluxed from 2 to 4 h. After cooling compounds 11-24, precipitated as solid, were collected by filtration and washed with water. Further amounts of products were obtained by evaporation in vacuo of the mother liquors followed by washing with water. Compounds 11-24 were purified, if necessary, by recrystallization from EtOH/H2O. 2-[Biphenyl-4-yl]-1H-benzimidazole (11). Reaction time: 2.5 h. Yield 80%. M.p. 283.7-285.4 °C. [Lit. 291-292 °C ] [17]. 2-[Biphenyl-4-yl]-5-methyl-1H-benzimidazole (12). Reaction time: 2.5h. Yield 64%. M.p. 275.7-277 °C. Analysis for C20H16N2. max cm-1: 1610; 1112; 846.
max nm: 319; 207. 1
NMR (DMSO-d6) : 12.78 (1H, br s, NH), 8.25 (2H, d, J=8.6Hz, 2”, 6”), 7.83 (2H, d, J=8.6Hz, H-3”, 5”), 7.76 (2H, d, J=8.2Hz, H-2’, 6’), 7.53-7.39 (5H, m, H-4,5, 3’, 4’, 5’), 7.03 (1H, d, J=8.4Hz, H-6), 2.45 (3H, s, CH3). 2-[Biphenyl-4-yl]- 5,6-Dimethyl -1H-benzimidazole (13). Reaction time: 2h. Yield 73%. M.p. 286.6-287.9 °C . Analysis for C21H18N2. max cm-1: 1608; 1118.
max nm: 328; 209. 1
-NMR (CDCl3-DMSO-d6) : 8.24 (2H, d, J=8.4Hz, H-2”, 6”), 7.90 (2H, d, J=8.4Hz, H-3”, 5”), 7.79 (2H, d, J=7.0Hz, H-2’, 6’), 7.60-7.38 (5H, m, H-3’, 4’, 5’, 4,7), 2.35 (6H, s, CH3). 2-[Biphenyl-4-yl] 5-Chloro-1H-benzimidazole (14). Reaction time: 4h. Yield 95%. M.p. 235-240 °C. Analysis for C19H13ClN2. max cm-1: 1610; 1114; 845.
max nm: 328; 212. 1
-NMR (CDCl3) : 8.25 (2H, d, J=8.0Hz, H-2”, 6”), 7.75 (2H, d, J=8.0Hz, H-3”, 5”), 7.75-7.57 (4H, m, H- 2’, 6’, 4, 6), 7.57-7.30 (3H, m, H-3’, 4’, 5’), 7.19 (1H, d, J=8.6Hz, H7). 2-[Biphenyl-4-yl]-5,6-Dichloro-1H-benzimidazole (15). Reaction time: 2h. Yield 80%. M.p. 267-269 °C. Analysis for C19H12Cl2N2. max cm-1: 3626; 1608; 842; 728.
max nm: 328 ; 212. 1
-NMR (CDCl3-DMSO- d6) : 8.26 (2H, d,

Medicinal Chemistry, 2009, Vol. 5, No. 6

513

J=8.2Hz, H-2”, 6”), 7.98-7.82 (4H, m, H-3”, 5”, 4,7), 7.77 (2H, d, J=8.6Hz, H-2’, 6’), 7.60-7.28 (3H, m, 3’, 4’, 5’). 2-[Biphenyl-4-yl]-5-Trifluoromethyl-1H-benzimidazole (16). Reaction time: 2.5h. Yield 35%. M.p. 165-167 °C. Analysis for C20H13F3N2 max cm-1: 3647; 1613; 1336; 1109; 840.
max nm: 317 ; 211. 1
-NMR (CDCl3-DMSO- d6) : 8.29 (2H, d, J=8.0Hz, H-2”, 6”), 8.00 (1H, s, H-4), 7.987.82 (3H, m, H-3”, 5”, 6), 7.78 (2H, d, J=8.2Hz, H-2’, 6’), 7.70-7.36 (4H, m, H-3’, 5’, 7). 2-[Biphenyl-4-yl]- 5-Acetyl-1H-benzimidazole (17). Reaction time: 3.5h. Yield 93%. M.p. 179.7-182°C. Analysis for C21H16N2O. max cm-1: 3485; 1651; 1618; 1350; 1113; 854.
max nm: 331; 207. 1
-NMR (CDCl3-DMSO- d6) : 8.41-8.21 (3H, m, H-2”, 6”, 4), 7.89 (3H, d, J=8.0Hz, H3”, 5”, 6), 7.77 (2H, d, J=6.8Hz, H-2’, 6’), 7.69 (1H, d, J=8.4Hz, H-7), 7.62-7.38 (3H, m, H-3’, 4’, 5’), 2.66 (3H, s, COCH3). 2-[4’-Nitrobiphenyl-4-yl)-1H-benzimidazole (18). Yield 83%. M.p. 279-281 °C. Analysis for C19H13N3O2. max cm-1: 1598; 1571; 1038; 830.
max nm: 330; 206. 1
-NMR (CDCl3DMSO- d6) : 8.34 (4H, d, J=7.2Hz, H-2’’, 6’’, 3’, 5’), 8.047.92 (4H, m, H-2’, 6’, 3’’, 5’’), 7.65-7.61 (2H, m, H-5,6), 7.26-7.22 (2H, m, H-4,7). 2-[4’-Nitrobiphenyl-4-yl]-5-Methyl-1H-benzimidazole (19). Yield 98%. M.p. 256-258 °C. Analysis for C20H15N3O2. max cm-1: 1597; 1570; 1038; 829.
max nm: 341; 206. 1
NMR (CDCl3-DMSO- d6) : 8.40-8.21 (4H, m, H-2’’, 6’’, 3’, 5’), 8.12-7.97 (4H, m, H-3’’, 5’’, 2’, 6’), 7.56 (1H, d, J=8.6Hz, H-6), 7.54 (1H, s, H-4), 7.13 (1H, d, J=8.0Hz, H7), 2.46 (3H, s, CH3). 2-(4’-nitrobiphenyl-4-yl)-5,6-Dimethyl-1H-benzimidazole (20). Yield 100%; M.p. 277-282°C. Analysis for C21H17N3O2. max cm-1: 1595; 1566; 1036; 839.
max nm: 342; 206. 1
-NMR (CDCl3-DMSO- d6) : 8.40-8.27 (4H, m, H2’’, 6’’, 3’, 5’), 8.03-7.85 (4H, m, H-2’, 6’, 3’’, 5’’), 7.38 (2H, s, H-4,7), 2.37 (6H, s, CH3). 2-(4’-nitrobiphenyl-4-yl)- 5-Chloro- 1H-benzimidazole (21). Yield 93%. M.p. 263-265 °C. Analysis for C19H12ClN3O2. max cm-1: 1597; 1569; 1038; 829; 731;
max nm: 340; 207. 1
-NMR (CDCl3-DMSO- d6) : 8.34 (4H, d, J=8.8Hz, H-2’’, 6’’, 3’, 5’), 8.07-7.97 (4H, m, H-2’, 6’, 3’’, 5’’), 7.70-7.55 (2H, m, H-4, 6), 7.24 (1H, d, J=8.6Hz, H-7). 2-(4’-nitrobiphenyl-4-yl)-5,6-Dichloro-1H-benzimidazole (22). Yield 94%. M.p. >300 °C. Analysis for C20H15N3O2. Analysis for C19H11Cl2N3O2. max cm-1: 3361; 1593; 1572; 1044; 832; 732.
max nm: 340; 211. 1
-NMR (CDCl3DMSO- d6) : 8.32 (4H, d, J=8.2Hz, H-2’’, 6’’, 3’, 5’), 8.03 (4H, m, H-3’’, 5’’, 2’, 6’), 7.75 (2H, s, H-4,7). 2-(4’-nitrobiphenyl-4-yl)-5-Trifluoromethyl-1H-benzimidazole (23). Yield 98%. M.p. 262-265 °C. Analysis for C20H12F3N3O2. max cm-1: 1597; 1337; 828.
max nm: 330; 209. 1
-NMR (CDCl3-DMSO- d6) : 8.36 (4H, d, J=8.2Hz, H-2’’, 6’’, 3’, 5’), 8.11-7.92 (4H, m, H-2’, 6’, 3’’, 5’’), 7.80 (2H, d, J=8.2Hz, H-4,6), 7.55 (1H, d, J=8.8Hz, H-7). 2-(4’-nitrobiphenyl-4-yl)5-Acetyl-1H-benzimidazole (24). Yield 100%; M.p. >300 °C. Analysis for C21H15N3O3. max cm-1: 3277, 1666, 1594,1039, 829.
max nm: 340, 202.

514 Medicinal Chemistry, 2009, Vol. 5, No. 6 1


-NMR (CDCl3-DMSO- d6) : 8.35 (4H, d, J=8.34Hz, 2’’, 6’’, 3’, 5’), 8.70 (1H, s, H-4), 8.10-8.00 (4H, m, H-2’, 6’, 3’’, 5’’), 7.89 (1H, d, J=8.2Hz, H-6), 7.69 (1H, d, J=8.4Hz, H-7), 2.66 (3H, s, COCH3). General Procedure for the Preparation of the 2-(4’aminobiphenylyl)-5 and 5,6 Substitutedbenzimidazoles (2530) To a suspension of 0.8 g of 2-(4’-nitro-biphenylyl)benzimidazoles in 80 mL of ethanol was added hydrazine monohydrate in the molar ratio of 1:20 and 0.08 g of 10% palladium on charcoal. The mixture was heated at 80 °C between 1 and 1.5 hours, depending on the nitroderivative. After filtration of the catalyst the mother liquors were evaporated under vacuum and the solid products obtained were purified by flash chromatography using a mixture of petroleum ether/acetone in the ratio of 6.5:3.5 in all cases, and of 7:3 for compound 29). 2-(4’-Aminobiphenyl-4-yl)-1H-benzimidazole (25). Reaction time: 1h. Yield: 56%. M.p. 221-225 °C. Analysis for C19H15N3. max cm-1: 3403, 3292,1601, 1180, 822.
max nm: 338, 210. 1
-NMR (CDCl3-DMSO- d6) : 8.20 (2H, d, J=8.4Hz, H-2’’, 6’’), 7.66 (2H, d, J=8.4Hz, H-3’’, 5’’), 7.63 (2H, dd, J=6.0 and J=3.4Hz, H-5, 6), 7.45 (2H, d, J=8.6Hz, H-2’, 6’), 7.22 (2H, dd, J=6.0 and J=3.4Hz, H-4,7), 6.75 (2H, d, J= 8.4Hz, H-3’, 5’). 2-(4’aminobiphenyl-4-yl)-5-Methyl-1H-benzimidazole (26). Reaction time: 1h 20’. Yield: 55%. M.p. 107-110 °C. Analysis for C20H17N3. max cm-1: 3329; 3188; 1604; 1180; 822.
max nm: 340, 211; 1
-NMR (DMSO- d6) : 8.24 (2H, d, J=8.4Hz, H-2’’, 6’’), 7.75 (2H, d, J=8.4Hz, H-3’’, 5’’), 7.50 (3H, d, J=8.4Hz, H-2’, 6’ and H-6), 7.41 (1H, s, H-4), 7.07 (1H, d, J=8.4Hz, H-7), 6.71 (2H, d, J=8.4Hz, H-3’, 5’), 3.40 (2H, br s, NH2), 2.44 (3H, s, CH3). 2-(4’aminobiphenyl-4-yl)-5,6-Dimethyl-1H-benzimidazole (27). Reaction time: 1h. Yield: 53%. M.p. 257-259 °C. Analysis for C21H19N3. max cm-1: 3475, 3361, 3212, 1607, 1179, 823.
max nm: 340, 210. 1
-NMR (CDCl3-DMSO- d6) : 8.17 (2H, d, J=8.2Hz, H-2’’, 6’’), 7.64 (2H, d, J=8.2Hz, H3’’, 5’’), 7.46 (2H, d, J=8.6Hz, H-2’, 6’), 7.38 (2H, s, H-4,7), 6.77 (2H, d, J=8.6Hz, H-3’, 5’), 4.36 (2H, br s, NH2), 2.37 (6H, s, CH3). 2-(4’-aminobiphenyl-4-yl)-5-Chloro-1H-benzimidazole (28). Reaction time: 1h 30’. Yield: 51%. M.p. 212-214 °C. Analysis for C21H14ClN3. max cm-1: 3347, 3190, 1604, 1178, 823, 722.
max nm: 340, 212. 1
-NMR (CDCl3-DMSO- d6) : 8.18 (2H, d, J=8.2Hz, H-2’’, 6’’), 7.66 (2H, d, J=8.0Hz, H3’’, 5’’) 7.61-7.52 (2H, m, H-4,6), 7.45 (2H, d, J=8.0Hz, H2’, 6’) 7.16 (1H, d, J=7.4Hz, H-7), 6.77 (2H, d, J=8.2Hz, H3’, 5’), 4.35 (2H, br s, NH2). 2-(4’-aminobipheny-l4-yl)-5,6-Dichloro-1H-benzimidazole (29). Reaction time: 1h. Yield: 60%. M.p. 281-283 °C. Analysis for C19H13Cl2N3. max cm-1: 3624, 1602, 825,719.
max nm: 342, 212. 1
-NMR (CDCl3-DMSO- d6) : 8.17 (2H, d, J=8.6Hz, H-2’’, 6’’), 7.73 (2H, s, H-4,7), 7.67 (2H, d, J=8.6Hz, H-3’, 5’’), 7.47 (2H, d, J=8.4Hz, H-2’, 6’), 6.79 (2H, d, J=8.4Hz, H-3’, 5’). 2-(4’aminobiphenyl-4-yl)-5-Trifluoromethyl -1H-benzimidazole (30). Reaction time: 1h. Yield: 51%. M.p. 218-219

Vitale et al.

°C. Analysis for C20H14F3N3. max cm-1: 1604, 1336, 1159, 820.
max nm: 345, 212. 1
-NMR (CDCl3-DMSO- d6) : 8.22 (2H, d, J=8.0Hz, H-2’’, 6’’), 7.91 (1H, s, H-4), 7.69 (3H, d, J=8.4Hz, H-3’’, 5’’ and H-6), 7.57-7.40 (3H, m, H-2’, 6’, 7), 6.78 (2H, d, J=8.2Hz, H-3’, 5’), 2.88 (2H, br s, NH2). General Procedure for the Preparation of the 2-(4’Acetylaminobiphenylyl)-5 and 5,6 Substituted –Benzimidazoles (31-36) 0.1 g of 2-(4’-aminobiphenyl)benzimidazoles were suspended in 1 mL of acetic anhydride and stirred at 100 °C for 15 minutes. After cooling the solids formed were collected and washed with ethyl ether. 2-(4’-Acetylaminobiphenyl-4-yl)-1H-benzimidazole (31). Yield: 87%. M.p. >300 °C. Analysis for C21H17N3O. max cm1 : 1672, 1598, 1181, 827.
max nm: 316, 205. 1
-NMR (CDCl3-DMSO-d6) : 12.92 (1H, br s, NH), 10.07 (1H, s, CONH), 8.25 (2H, d, J=8.0 Hz, H-2’’, 6’’), 7.83 (2H, d, J=8.0 Hz, H-3’’, 5’’), 7.72 (6H, s, H-5, 6, 2’, 3’, 5’, 6’), 7.25-7.18 (2H, m, H-4,7). 2-(4-’acetylaminobiphenyl-4-yl)-5-Methyl-1H-benzimidazole (32). Yield: 45%. M.p. >300 °C. Analysis for C22H19N3O. max cm-1 (KBr): 1673, 1598, 1184, 826.
max nm: 330, 209. 1
-NMR (CDCl3-DMSO- d6) : 10.06 (1H, s, NHCO), 8.22 (2H, d, J=8.2Hz, H-2’’, 6’’), 7.81 (2H, d, J=8.2 Hz, H-3’’, 5’’), 7.71 (4H, s, H-2’, 6’, 3’, 5’), 7.48 (1H, d, J=8.2 Hz, H-7), 7.38 (1H, s, H-4), 7.03 (1H, d, J=8.2 Hz, H6), 2.44 (3H, s, CH3), 2.08 (3H, s, COCH3). 2-(4’-acetylaminobiphenyl-4-yl)-5,6-Dimethyl-1H-benzimidazole (33). Yield: 63%. M.p. >300 °C. Analysis for C23H21N3O. max cm-1 (KBr): 1670, 1599, 1185, 827.
max nm: 330, 207. 1
-NMR (CDCl3-DMSO- d6) : 11.97 (1H, br s, NH), 10.07 (1H, s, NHCO), 8.20 (2H, d, J=8.0 Hz, H-2’’, 6’’), 7.82 (2H, d, J=8.0 Hz, H-3’’, 5’’), 7.71 (2H, s, H-2’, 6’, 3’, 5’), 7.37 (2H, s, H-4,7), 2.33 (6H, s, CH3), 2.05 (3H, s, COCH3). 2-(4’-acetylaminobiphenyl-4-yl)-5-Chloro-1H-benzimidazole (34). Yield: 98%. M.p. >300 °C. Analysis for C21H16ClN3O. max cm-1 (KBr): 1671, 1599, 1184, 826, 719.
max nm: 328, 211. 1
-NMR (CDCl3-DMSO- d6) : 10.08 (1H, s, NHCO), 8.24 (2H, d, J= 8.0 Hz, H-2’’, 6’’), 7.85 (2H, d, J=8.0 Hz, H-3’’, 5’’), 7.81-7.58 (6H, m, H-2’, 3’, 5’, 6’, 4,6), 7.23 (1H, d, J=7.23 Hz, H-7), 2.10 (3H, s, COCH3). 2-(4-’acetylaminobiphenyl-4-yl)-5,6-Dichloro-1H-benzimidazole (35). Yield: 73%. M.p. >300 °C. Analysis for C21H15Cl2N3O. max cm-1: 1668, 1597, 1184, 827, 720.
max nm: 330, 213. 1
-NMR (DMSO- d6) :13.31 (1H, br s, NH), 10.10 (1H, s, NHCO), 8.22 (2H, d, J=8.4 Hz, H-2’’, 6’’), 7.88 (2H, d, J=8.4 Hz, H-3’’, 5’’), 7.73 (6H, s, H-2’, 6’, 3’, 5’, 4,7), 2.08 (3H, s, COCH3). 2-(4’-acetylaminobiphenyl-4-yl)-5-Trifluoromethyl-1Hbenzimidazole (36). Yield: 82%. M.p. >300 °C. Analysis for C21H16F3N3O. max cm-1: 1670, 1598, 1331, 1185, 826.
max nm: 319, 211. 1
-NMR (CDCl3) : 10.11 (1H, s, NHCO), 8.28 (2H, d, J=8.0 Hz, H-2’’, 6’’), 8.03-7.80 (4H, m, H-3’’, 5’’, 4,6), 7.75 (4H, s, H-2’, 6’, 3’, 5’), 7.55 (1H, d, J=8.2Hz, H-7), 2.09 (3H, s, COCH3).

2-Arylbenzimidazoles as Antiviral and Antiproliferative Agents-Part 2

Biological Assays 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 contamination was checked periodically by the Hoechst staining method. Cell lines supporting the multiplication of RNA viruses were the following: CD4 + human T-cells containing an integrated HTLV-1 genome (MT-4); Madin Darby Bovine Kidney (MDBK); Baby Hamster Kidney (BHK-21) and Monkey kidney (Vero 76) cells. Cytotoxic / Antiproliferative Assays For cytotoxicity tests, run in parallel with antiviral assays, MDBK, BHK and Vero 76 cells were resuspended in 96 multiwell plates at an initial density of 6x105, 1x106 and 5x105 cells/mL, respectively, in maintenance medium, without or with serial dilutions of test compounds. Cell viability was determined after 48-96 hrs at 37 °C in a humidified CO2 (5%) atmosphere by the MTT method. The cell number of Vero 76 monolayers was determined by staining with the crystal violet dye. For cytotoxicity evaluations, exponentially growing cells derived from human haematological tumors [CD4+ human Tcells containing an integrated HTLV-1 genome (MT-4); CD4+ human acute T-lymphoblastic leukaemia (CCRFCEM); Human splenic B-lymphoblastoid cells (WIL-2NS); Human acute B-lymphoblastic leukemia (CCRF-SB)] were seeded at an initial density of 1x105 cells/mL in 96 well plates in RPMI-1640 medium supplemented with 10% fetal calf serum (FCS), 100 units/mL penicillin G and 100
g/mL streptomycin. Human cell lines derived from solid tumours [skin melanoma (SKMEL-28); breast adenocarcinoma (MCF7); lung squamous carcinoma (SKMES-1); hepatocellular carcinoma (HepG2); prostate carcinoma (DU145)] or normal tissues [lung fibroblasts (MRC-5); foreskin fibroblasts CRL7065)] were also seeded at 1x105 cells/mL in 96 well plates in specific media supplemented with 10% FCS and antibiotics as above. 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 96 hrs at 37 °C by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) method [28]. 6-mercaptopurine (6-MP) and Eto-poside were used as reference drugs in antiproliferative assays. Antiviral Assay Activity of compounds against Human Immunodeficiency virus type-1 (HIV-1) was based on inhibition of virusinduced cytopathogenicity in MT-4 cells acutely infected with a multiplicity of infection (m.o.i.) of 0.01. Briefly, 50 4 μL of RPMI containing 1x10 MT-4 were added to each well of flat-bottom microtitre trays containing 50 μL of RPMI, without or with serial dilutions of test compounds. Then, 20 μL of an HIV-1 suspension containing 100 CCID50 were

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added. After a 4-day incubation, cell viability was determined by the MTT method. Activity of compounds against Yellow fever virus (YFV) and Reo virus type-1 (Reo-1) was based on inhibition of virus-induced cytopathogenicity in acutely infected BHK-21 cells. Activities against Bovine viral diarrhoea virus (BVDV), in infected MDBK cells, were also based on inhibition of virus-induced cytopathogenicity. BHK and MDBK cells were seeded in 96-well plates at a density of 5x104 and 3x104 cells/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
L of a proper virus dilution (in serum-free medium) to give an m.o.i = 0.01. 1 hr later, 50
L of MEM Earle’s medium, supplemented with inactivated foetal calf serum (FCS), 1% final concentration, without or with serial dilutions of test compounds, were added. After 3-4 days incubation at 37 °C, cell viability was determined by the MTT method. Activity of compounds against Coxsackie virus, B-2 strain (CVB-2), Polio virus type-1 (Polio-1), Sabin strain, Vesicular Stomatitis Virus (VSV), Vaccinia Virus (VV), Herpes Virus 1 (HSV-1) and against Respiratory syncytial virus (RSV), A-2 strain, in infected Vero 76 cells, was determined by plaque reduction assays in Vero 76 cell monolayers. To this end, Vero 76 cells were seeded in 24-well plates at a density of 2x105 cells/well and were allowed to form confluent monolayers by incubating overnight in growth medium at 37 °C in a humidified CO2 (5%) atmosphere. Then, monolayers were infected with 250
L of proper virus dilutions to give 50-100 PFU/well. Following removal of unadsorbed virus, 500
L of Dulbecco’s modified Eagle’s medium, supplemented with 1% inactivated FCS 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-2, 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. 50% effective concentrations (EC50) were calculated by linear regression technique. AZT (3’-azido-thymidine), NM 108 (2’-ß-methylguanosine), NM 176 (2’-ethynyl-D-citidine), NM 299 (6azauridine), M 5255 (Mycophenolic Acid) and ACG (acycloGuanosine) were used as reference inhibitors of ssRNA+, ssRNA- and DNA viruses, respectively. Linear Regression Analysis Viral and cell growth at each drug concentration was expressed as percentage of untreated controls and concentrations resulting in 50% (EC50 and CC50) growth inhibition were determined by linear regression analysis. AKNOWLEDGEMENT This work was supported by grants from Italian FIRB [(RBNE01J3SK 001)] and Cybersar (MIUR, PON) Projects. REFERENCES [1]

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Received: July 05, 2009

Revised: September 13, 2009

Accepted: September 13, 2009

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