Substituted benzimidazoles: antiviral activity and synthesis of nucleosides

The antiviral activity of a series of benzimidazole derivatives and substituted benzimidazole β -L- and β -D-2’-deoxyribonucleosides against selected RNA and DNA viruses including HIV-1, BVDV, YFV, DENV-2, WNV, HBV, HCV and human RSV was evaluated. In addition, the synthesis of several benzimidazole β -L-2’-deoxyribonucleosides ( 1 - 4 ) and 2’-deoxy-2’-fluoro-β - D-arabinofuranosyl nucleoside 5 is described. The stereoselective glycosylation of the anions of the functionalized benzimidazoles 6a , 12a and 28 with 3,5-di-O -(4-methylbenzoyl)-2-deoxy-α- L-erythro -pentofuranosyl chloride ( 29 ) furnished β -L-2’-deoxyribonucleosides 1 - 4 while the glycosylation of the anion of 2-bromobenzimidazole ( 12a ) with 3,5-di-O -benzoyl-2-deoxy-2- fluoro-α-D-arabinofuranosyl bromide 34 gave the 2’-deoxy-2-fluoro-β -D-arabinofuranosyl nucleoside 5 . Moreover, the crystal structure of the benzoylated 2-bromobenzimidazole 2’- deoxy-2’-fluoro-β -D-arabinofuranosyl nucleoside 35 is reported.


Introduction
The benzimidazole system is an integral part of numerous antiparasitic, fungicidal, anthelmintic and anti-inflammatory drugs. 1,2 mong them, modified 2-trifluoromethyl-benzimidazoles are particular promising as they show appreciable herbicidal activity due to the inhibition of photosynthesis. 38][9] Originally, their synthesis was encouraged by the discovery that 5,6-dimethyl-1-(α-D-erythro-pentofuranosyl)-benzimidazole is a constituent of vitamin B12. 10 5,6-Dichlorobenzimidazole ribonucleoside (DRB) inhibits cellular and viral RNA synthesis. 11,12 owever, this activity is accompanied by a substantial cytotoxicity and therefore this compound has not found application as an antiviral drug. 13,14 teworthy is the recent finding that the L-ribonucleoside of 5,6-dichloro-2isopropylaminobenzimidazole (1263W94) shows increased activity against the herpes virus HCMV (human cytomegalovirus) in vitro compared to its parent compound 2-bromo-5,6dichloro-1-β-D-ribofuranosylbenzimidazole (BDCRB) and a low cytotoxicity. 15This is in line with the general observation that several L-nucleosides exhibit an antiviral activity comparable and sometimes greater than their D-enantiomers, due to a more favourable toxicological profile and a greater metabolic stability. 16,17 7][18][19][20] The first synthesis of a L-nucleoside (β-L-dT) was reported in 1964 by Šmekal and Šorm 21 followed by the description of β-L-adenosine by Acton, Ryan and Goodman in the same year. 22Furthermore, the anti-hepatitis B virus activity of the 2'-deoxyribonucleoside L-dT (Telbivudine, Tyzeka TM , Sebivo TM ) 21b against hepatitis B virus infection led to its approval as antiviral drug.
The closely related 4,5,6,7-tetrabromobenzimidazole derivatives 6d-g having a trifluoromethyl, pentafluoroethyl, heptafluoropropyl or nonafluorobutyl side chain attached to position-2 of the benzimidazole ring exhibit strong cytotoxicity against the host cell lines in the low micromolar range (0.5-2.0 µM).On the other hand compounds 7d and 7w are completely inactive against all the tested ssRNA + viruses and are also not cytotoxic.Table 2. Antiviral activity of halogenated benzimidazoles derivatives against selected ssRNA + viruses (part 2    1. Next, various non-halogenated, chlorinated and brominated benzimidazole derivatives as well as 2-carboxybenzimidazoles (Figure 4) were evaluated for their antiviral activity against ssRNA + viruses (HIV-1, BVDV, YFV, DENV-2, WNV).Unfortunately, the compounds of this series do not show selective antiviral activity against any of the tested ssRNA + viruses.The moderate antiviral activities of 13g, 14f, 16f, 16g, 16m and 16o are accompanied by cytotoxicity against the host cell lines within the same concentration range as it was also observed for the 4,5,6,7-tetra-brominated benzimidazoles shown above.
However, from the data given in Table 2 it can be seen that compounds 14d, 16c, 18, 19 and 21-23 are strongly cytotoxic within a range of 1.0-4.0µM in all tested cell lines whereas compounds 12a, 12c, 12d, 13f, 17g, 17k and 17l are completely inactive against all tested ssRNA + viruses and are also non-cytotoxic.Table 3. Antiviral activity of nitrobenzimidazole derivatives against selected ssRNA + viruses (part 3 a Retroviridae.b Pestivirus.c Flavivirus. j For details of the antiviral assays see Table 1. The data summarized in Table 3 show that none of the nitrobenzimidazole derivatives (Figure 5) exhibit selective antiviral activity against ssRNA + viruses.Compound 24g is cytotoxic against the cell line used in the HIV-1 assay while compound 24h is toxic against all tested cell lines.Then, the benzimidazole β-L-2'-deoxyribonucleosides 1-4 as well as the fluoroarabino nucleoside 5 (Figure 2) and closely related β-D-2'-deoxyribonucleosides (26a, 27; Figure 6) were screened for their antiviral activity against ssRNA + viruses as demonstrated by Table 4. 29 Among them, the 4,5,6,7-tetrabrominated benzimidazole β-L-2'-deoxyribonucleoside 4 as well as its corresponding β-D-enantiomer 27 show similar antiviral activity against the tested ssRNA + viruses.However, these compounds are also cytotoxic against the host cell lines within the same concentration range.For comparison, 4,5,6,7-tetrabromobenzimidazole 6a also exhibits antiviral activity accompanied by toxicity (see Table 1).The 4,5,6,7-tetrabrominated benzimidazole 2'deoxyribonucleosides 4 and 27 are more toxic than the heterocycle 6a itself.It can be assumed that the nucleosides can enter the cells more efficiently and function as a release system delivering the active benzimidazole derivative 6a.Unfortunately, the other benzimidazole L-and D-nucleosides show no antiviral activity and are non-toxic.Several benzimidazole derivatives were also tested for their activity against the Hepatitis B virus (HBV) which belongs to the DNA viruses.In this assay the 50% inhibitory concentration (EC50) was determined for the virion (Vir) and the replicative intermediate (RI) of HBV.Table 5 shows that within the tested compounds the 4,5,6,7-tetrabrominated benzimidazole derivatives 6j and 7j both carrying a dimethylamino side chain attached to position-2 of the heterocycle show anti-HBV activity against the virion and the replicative intermediate in a low micromolar concentration range (6j: EC50 (RI) = 5.0 µM, EC50 (Vir) = 1.0 µM and 7j: EC50 (RI) = 0.8 µM, EC50 (Vir) = 0.6 µM) while the cytotoxicity is less pronounced (6j: CC50 = 100 µM and 7j: CC50 >100 µM).Interestingly, among the 4,5,6,7-tetrabrominated benzimidazoles having different side chains attached to position-2 (Figure 3) several compounds (6a, h, i, k, o) are toxic in low concentration (<1 µM).Apart from the L-nucleoside 4 which is cytotoxic the tested nucleosides 1-3, 5 are without activity.

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Page 238  ARKAT USA, Inc. Regarding the activity in the HCV replicon system a series of benzimidazoles were selected for testing (Table 6).Their ability to inhibit the HCV replicon replication at the test concentration of 15µM was evaluated by an HCV replicon assay (ELISA) as previously described. 28The cellular toxicity was determined at the test concentration of 15µM of drug. 28For most compounds inhibition of the HCV replicon replication is accompanied by cytotoxicity.Only in case of 6c, 7w and 12e inhibition was observed without cytotoxicity.The Respiratory Syncytial Virus (RSV) is a major cause of respiratory illness in infants and young children.However, antiviral approaches are still required to treat this infection.From Table 7 it can be seen that none of the tested benzimidazole derivatives show selective antiviral activity against RSV.The compounds are either toxic or completely inactive.However, in the series of the halogenated benzimidazoles including the 4,5,6,7-tetrabrominated benzimidazole Lnucleoside 4 most compounds are cytotoxic.
For the 2-bromobenzimidazole 2'-deoxy-2'-fluoro-β-D-arabinofuranosyl nucleosides 35 and 5 the presence of the fluorine atome in the sugar moiety was confirmed by 19 F-NMR spectra (35: -194.2 ppm and 5: -194.0 ppm).Moreover, JC,F coupling constants are given in Table 9.Table 8. 13 C NMR chemical shifts of benzimidazole β-Land Furthermore, the solid state structure of the benzoylated 2-bromo-1-[2-deoxy-2-fluoro-β-Darabinofuranosyl]-benzimidazole 35 was confirmed by single-crystal x-ray analysis. 38rom the crystal structure of 35, the orientation of the nucleobase relative to the sugar moiety was determined to be syn with χ = (O1'-C1'-N11-C18) = 39.7(9)o .The natural 2'deoxyribonucleosides usually adopt an anti conformation.However, it is observed that the introduction of a bulky substituent in position-2 of the heterocyclic moiety shifts the conformation around the glycosylic bond from anti to syn as it was recently reported for 2'deoxyimmunosine. 39The sugar moiety of 35 shows a pseudorotation phase angle P of 118.8° and a maximum amplitude of puckering τm of 41.8° which indicates a south (S) conformation.This result is in contrast to many other 2'-fluoroarabino nucleosides that are in N-conformation.However, the conformation of the sugar residue is also strongly influenced by the benzoyl protecting groups.

Conclusions
The antiviral activity of a series of benzimidazole derivatives and substituted benzimidazole β-Land β-D-nucleosides against selected RNA and DNA viruses including HIV-1, BVDV, YFV, DENV-2, WNV, HBV, HCV and human RSV was evaluated.Unfortunately, no selective antiviral activity was observed for the tested compounds.However, among the halogenated benzimidazoles including the 4,5,6,7-tetrabrominated benzimidazoles β-L-2'deoxyribonucleoside 4 several derivatives were found to be cytotoxic against the tested cell lines.These compounds are potential candidates for anti-cancer screening.

Figure 4 .
Figure 4. Halogenated and other benzimidazole derivatives for antiviral testing.

Figure 7 .
Figure 7.The crystal structure of compound 35 obtained from single crystal X-ray analysis.

Table 5 .
Activity of benzimidazole derivatives against HBV  ARKAT USA, Inc.

Table 5 .
Continued Compound concentration (µM) required to reduce the viability of heptatocellular carcinoma (HepG2) cells by 50%, as determined by the MTT method.
a Hepadnaviridae.b c Compound concentration (µM) required to reduce the intracellular HBV DNA (HBV replicative intermediates, RI).d Compound concentration (µM) required to reduce the intracellular HBV virion (HBV Vir).

Table 6 .
Activity of benzimidazole derivatives in the HCV a replicon system a Hepacivirus.bTheobservedinhibition of HCV replicon replication at the test concentration of 15µM of drug as determined by the HCV replicon (ELISA) assay.cTheobserved cellular toxicity at the test concentration of 15µM of drug.

Table 7 .
Activity of benzimidazole derivatives against RSV a a Paramyxoviridae.bCompoundconcentration (µM) required to reduce the Vero cell number determined by methylene blue staining.c Compound concentration (µM) required to reduce the virus plaque number by 50% in Vero cell lines.

Table 8 .
(continued) a Measured in DMSO-d6 at 298 K. b Systematic numbering.c Superimposed by the DMSO signal.d Tentative assignment.e The signals of C5 and C6 are superimposing.Table 9. JC,H and JC,F values (in Hz) of β-Land β-D-2'-deoxyribonucleosides a, b a Measured in DMSO-d6 at 298 K. b Systematic numbering is used.c The signals of C5 and C6 are superimposing.d Superimposed by the DMSO signal.e Superimposing multipletts.