Antiviral activity of benzimidazole derivatives. II. Antiviral activity of 2-phenylbenzimidazole...

Bioorganic & Medicinal Chemistry 22 (2014) 4893–4909

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Bioorganic & Medicinal Chemistry

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Antiviral activity of benzimidazole derivatives. III. Novel anti-CVB-5,anti-RSV and anti-Sb-1 agents

http://dx.doi.org/10.1016/j.bmc.2014.06.0430968-0896/� 2014 Elsevier Ltd. All rights reserved.

⇑ Corresponding authors. Tel.: +39 010 3538867; fax: +39 010 3538399(M. Tonelli), tel.: +39 070 6754202; fax: +39 070 6754210 (R. Loddo).

E-mail addresses: [email protected] (M. Tonelli), [email protected](R. Loddo).

Michele Tonelli a,⇑, Federica Novelli a, Bruno Tasso a, Iana Vazzana a, Anna Sparatore b, Vito Boido a,Fabio Sparatore a, Paolo La Colla c, Giuseppina Sanna c, Gabriele Giliberti c, Bernardetta Busonera c,Pamela Farci c, Cristina Ibba c, Roberta Loddo c,⇑a Dipartimento di Farmacia, Università di Genova, Viale Benedetto XV 3, 16132 Genova, Italyb Dipartimento di Scienze Farmaceutiche, Università di Milano, Via Mangiagalli 25, 20133 Milano, Italyc Dipartimento di Scienze Biomediche, Università di Cagliari, Cittadella Universitaria, 09042 Monserrato (CA), Italy

a r t i c l e i n f o

Article history:Received 16 March 2014Revised 19 June 2014Accepted 22 June 2014Available online 1 July 2014

Keywords:Benzimidazole derivativesRNA and DNA virusesAnti-CVB-5, anti-RSV, anti-Sb-1 activity

a b s t r a c t

A library of eighty-six assorted benzimidazole derivatives was screened for antiviral activity against apanel of ten RNA and DNA viruses.

Fifty-two of them displayed different levels of activity against one or more viruses, among whichCVB-5, RSV, BVDV and Sb-1 were the most frequently affected. In particular, fourteen compoundsexhibited an EC50 in the range 9–17 lM (SI from 6 to >11) versus CVB-5, and seven compounds showedan EC50 in the range 5–15 lM (SI from 6.7 to P20) against RSV, thus resulting comparable to or morepotent than the respective reference drugs (NM108 and ribavirin). Most of these compounds derivefrom 2-benzylbenzimidazole, but also other molecular scaffolds [as 1-phenylbenzimidazole (2),2-trifluoromethylbenzimidazole (69), dihydropyrido[30,20:4,5]imidazo[1,2-a][1,4]benzodiazepin-5-one(3), dibenzo[c,e]benzimidazo[1,2-a]azepine (22), and 2-(tetrahydropyran-2-yl)benzimidazole (81, 82and 86)] are related to interesting levels of activity against these or other viruses (BVDV, Sb-1). Thus,these scaffolds (some of which, so far unexplored), represent valid starting points to develop moreefficient agents against pathologies caused by CVB-5, RSV, BVDV and Sb-1 viruses.

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1. Introduction

Because of its isosterism with indole and purine nuclei, that arepresent in many fundamental cellular components and bioactivecompounds, the benzimidazole ring represents a kind of privilegedsubstructure from which several important drugs used in differenttherapeutic areas have been obtained (proton pump inhibitors,angiotensin receptor antagonist, antihistaminics and antihelmin-tics).1 An even greater number of still investigational therapeuticagents, including antitumorals and antivirals, contain a benzimid-azole nucleus.

Among the antiviral benzimidazoles, an important position isheld by compounds acting against HIV-1,2a HCV and RSV,2b but sev-eral other viruses resulted affected by varied benzimidazole deriva-tives even if in a more occasional way (influenza B virus, rhinovirus,poliovirus, coxsackie B3, cytomegalovirus, etc.). In the active

compounds, the fundamental heterocyclic ring is decorated with avariety of substituents, from simple atoms or small groups to highlycomplicated (even multicyclic) moieties, so that structure–activityrelationships may be obtained only within homogeneous subsetsof compounds, affecting a given molecular/cellular target.

Since many years we are interested in the chemistry and biolog-ical properties of benzimidazole derivatives and, pursuing variedpharmacological aims, we have deeply investigated this chemicalspace.

A number of 2-[(benzotriazol-1/2-yl)methyl]benzimidazoles,bearing at position 1 a dialkylaminoalkyl or quinolizidin-1-ylalkylmoiety, exhibited a selective and potent activity against RSV, withEC50 as low as 20–30 nM for the best compounds.3 Only moderateactivity against a few other viruses (BVDV, YFV and CVB-2) wasalso observed for some of these compounds.

In a set of seventy-six non-basic derivatives of 2-phenylbenz-imidazole, thirty-nine were found to exhibit high activity(EC50 = 0.1–10 lM) against at least one virus of a panel of tenRNA and DNA viruses.4 Five of these compounds were outstandingfor their high and selective activity against single viruses (VV:EC50 = 0.1 lM, BVDV: EC50 = 0.8–1.5 lM; CVB-2: EC50 = 4 lM) and

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Cl

N

N N

NN

N

H

Cl

N

N N

N

H

N

N

RSV: EC50 = 0.02 µMRSV: EC50 = 0.03 µM

NH

NNO2

Cl

NH

NNH-C-CH3

O2N

Cl

O

VV: EC50 = 0.1 µM BVDV: EC50 = 0.8 µM

NH

NNH-C-C2H5

F3C O

CVB-2: EC50 = 4 µM

Figure 1. Previously studied benzimidazole derivatives with potent and selectiveantiviral activity.

4894 M. Tonelli et al. / Bioorg. Med. Chem. 22 (2014) 4893–4909

low toxicity for the corresponding host cells (CC50 >100 lM) (seeFig. 1).

Based on these findings we evaluated, in cell based assays, theantiviral activity of a library of 86 assorted benzimidazole deriva-tives in order to find out some additional hits to be developedtogether with those previously described. In Figure 2 are high-lighted all the chemical features explored in the library.

2. Results and discussion

2.1. Chemistry

Structurally, the tested compounds may be grouped accordingto the nature of substituents on the position 1 and 2 of thebenzimidazole ring (1- or 2-phenyl, 2-benzyl and various 1- or

NH

N

NH

NBaR

R= H, Ccpd 60-6

N

Nnon-Ar substituents

Basic chains

F3C

N

NR

NH

NR

cpd 68-71

R= H, Clcpd 72-74

R= H, Cl, CF3cpd 75-86

non-Ar/non-basicsubstituents

non-Ar/non-basicsubstituents

Figure 2. Chemical feat

2-non-aromatic substituents), and also to the presence, or less, ofa linear or cyclic basic moiety, as it is indicated in Figure 3. Adetailed representation of all structures is found in Figures 4–6.

Many of the 86 tested compounds of Figures 4–6 have beenalready prepared by some of us for other pharmacological aims:3;5 6–11;6 12, 13, 16, 69–71;7 15, 34, 35, 39;8 20, 59; 9 22;10

23;11 29–31;12 37, 38, 41;13 47, 48;14 68;15 72–74;16 79, 82–86.17

Compounds 21;18a,b 26;19 45;20 51;21 75;22 77;23 7824 were alreadydescribed by others and reprepared according to cited references.

The novel compounds were prepared as follows.Most commonly the benzimidazole ring was formed by heating

(in acetic acid or chloroform solution) the properly substituted1,2-phenylenediamine with the hydrochloride of the iminoester,previously obtained from the corresponding nitrile, absolute etha-nol and dry HCl (27, 28, 32, 33, 40, 42–44, 52–54, 57, 58 and 60;Scheme 1).

Alternatively, the heterocyclic ring may be formed by refluxingwith 4 N HCl the previously formed N-acyl-1,2-phenylenediamine(1, 2, 14, 61, 62; Scheme 2) or the mixture of the substituted1,2-phenylenediamine with 4-aminophenylacetic acid (49, 55) orwith the diaminoacid dihydrochlorides obtained by the hydrolyticcleavage of tetrahydrocytisine (64, 65) and D,L-lupanine25 (66, 67;Scheme 3).

In a few cases the ring formation was achieved by heating at180 �C, without solvent, the mixture of the suitable 1,2-phenylene-diamine and the acid (76, 80, 81; Scheme 4).

For the preparation of 2-phenylbenzimidazole derivatives wasvaluable the condensation of the N-substituted-1,2-phenylenedi-amine with the arylaldehyde sodium bisulfite adduct (4, 5, 17–19; Scheme 5).

2-(4-Aminobenzyl)benzimidazoles 45,20 49 and 55 (Scheme 3)were acylated in benzene solution to give the acetylamino deriva-tives 46, 50 and 56 (Scheme 6).

2-(4-Bromobenzyl)-5,6-dichlorobenzimidazole 53 (Scheme 1)was converted to sodium salt with NaNH2 and then alkylated withchlorolupinane26 (Scheme 7).

By reacting commercial 2-chlorobenzimidazole with (1R,9aR)-(octahydro-2H-quinolizin-1-yl)methane thiole (thiolupinine)27

the 2-(S-lupinylthio)benzimidazole 63 was obtained (Scheme 8).Finally the benzoimidazo[1,2-c][1,2,3]benzotriazine derivatives

24 and 25 were obtained by diazotization of the 2-(2-amino-4-methoxy)phenylbenzimidazoles, previously described4 (Scheme 9).While from the 5,6-dichlorobenzimidazole derivative the singlecompound 25 is obtained, starting from the corresponding5-trifluoromethyl-derivative two isomeric compounds can be

X N

NBasic chains

Ar substituents

R= H, Cl, CF3, COCH3cpd 4-17; 26-44

N

NAr substituents

Basic chains

R

X= CH, Ncpd 1-3 (3 fused ring)

R= H, Cl, CF3, OCH3cpd 18-25; 45-59 (22-25 fused ring)

N

NAr substituentsR

sic chains

l7

unsubstituted or non-basicsubstituents

ures of the library.

Figure 3. Groups of benzimidazole derivatives tested for antiviral activity. (a) Including one pyridoimidazole derivative.

N

NR

N N

N N

H

3

N

N

22

O

N

R=N

H2CH

NH2C

H

1

2

N

N

R

R= CH2CH2N(CH3)2 4CH2CH2CH2N(C2H5)2 5

OCH3

F3C

N

NR"R'

N

H

R'= H, R"= H 66-CH3, F 76-Cl, H 85-Cl, OCH3 9

N

NR"R'

N

HR'= 6-Cl, R"= H 10

5-Cl, OCH3 11

N

NF3C

N

H

R'= H 124-Cl 132,6-diF 144-OH 154-OCH3 16

N

NOCH3

F3C

N

H 17

N

N

R

OCH3

F3C

R= C4H9(n) 18CH2CH2OCH3 19

A

B

N

N

RR= CH(CH3)CH2COOH 20

CH2-C6H5 21

N

N

N N

N

N

N NN

N

N N

OCH3OCH3 OCH3

Cl

ClCH3O

CF3

23 24 25

R

Figure 4. Structures of the investigated 1/2-phenylbenzimidazole derivatives. (A) Compounds with a basic side-chain. (B) Compounds without basic side-chain.

M. Tonelli et al. / Bioorg. Med. Chem. 22 (2014) 4893–4909 4895

formed. Indeed, the isolated compound exhibits splitted signals inthe 1H NMR with one isomer largely prevailing on the other (10:1).Compound 24 was used as such for antiviral tests.

For the synthesis of compounds 17 and 42–44 (Schemes 5 and 1,respectively), the novel N-[(1R,9aR)-(octahydro-2H-quinolizin-1-yl)methyl]-4-trifluoromethyl-1,2-phenylenediamine was preparedby reacting the [(1R,9aR)-(octahydro-2H-quinolizin-1-yl)methan]

amine (epi-lupinylamine)28 with 4-chloro-3-nitrobenzotrifluoride.The N-substituted 2-nitro-4-trifluoromethylaniline obtained wasreduced by catalytic hydrogenation (Scheme 10).

All new compounds were characterized by elemental analysesand 1H NMR spectra that are fully consistent with the describedstructure. The purity of compounds (elemental analyses and 1HNMR spectra) resulted in all cases P95%.

N

N

CH2CH2N

R

C2H5

C2H5

R= Cl 26Br 27I 28 N

N

CH2CH2N

R

CH3

CH3

R= H 29Cl 30OCH3 31

N

N

R

Cl

Cl

R= CH2CH2N(CH3)2 32CH2CH2CH2N(C2H5)2 33

F3C

N

N

N

H

Cl

Cl

R

R= 4-Cl 343,4-Cl2 354-Br 36

R= H 374-Cl 383,4-Cl2 394-Br 404-OCH3 41

N

N

N

H

F3CR

N

N

N

H

F3CR

R= H 423,4-Cl2 434-OCH3 44

A

NH

NCH2

BR

R= NH2 45NH-COCH3 46

NH

NCH2 R

F3C

R= Cl 47OCH3 48NH2 49NH-COCH3 50

NH

NCH2

Cl

Cl

R

R= 4-Cl 513,4-Cl2 524-Br 534-OCH3 54

4-NH2 554-NH-COCH3 56

N

NCH2

F3C

N

NCH2 ClCl

R CHCH3

CH2COOHR= C4H9(n) 57

CH2CH2OCH3 5859

Cl

O

Figure 5. Structures of the investigated 2-benzylbenzimidazole derivatives. (A)Compounds with a basic side-chain. (B) Compounds without basic side-chain.


2.2. Biological activity—General considerations

The 86 benzimidazole derivatives considered in this work wereevaluated for antiviral activity against ten RNA and DNA viruses.Among single-stranded, positive RNA viruses (ssRNA+), we consid-ered a Retrovirus (Human Immunodeficiency Virus type 1, HIV-1)two Picornaviruses (Coxsackie Virus type-5, CVB-5 and Poliovirustype-1, Sabin strain, Sb-1), a Flavivirus (Yellow Fever Virus, YFV)and a Pestivirus (Bovine Viral Diarrhea Virus, BVDV). Among sin-gle-stranded, negative RNA viruses (ssRNA�) a Paramyxoviridae(Respiratory Syncytial Virus, RSV) and a Rhabdoviridae (VesicularStomatitis Virus, VSV) were selected as representatives. Amongdouble-stranded RNA (dsRNA) viruses, a Reoviridae family mem-ber (Reo-1) was included. Finally, two representatives of DNA virusfamilies were also included: Herpes Simplex Virus type-1, HSV-1(Herpesviridae) and Vaccinia Virus, VV (Poxviridae).

Cytotoxicity was evaluated in parallel with the antiviral activ-ity. As reference inhibitors were used: EFV (efavirenz) for HIV-1

only, NM 108 (20-C-methyl-guanosine), NM 176 (20-C-ethynylcyti-dine) and ribavirin for the other ssRNA+ and dsRNA viruses; NM299 (6-azauridine), ribavirin and M 5255 (mycophenolic acid) forthe ssRNA� viruses; M 5255 and ACG (acyclovir) for DNA viruses.

Fifty-two of the 86 tested compounds (34 with and 18 withoutbasic side-chain) exhibited antiviral activity (EC50 < CC50) againstone or more viruses; in particular, 28 compounds displayed aselective activity against a single virus, while 16, 7 and 1 moleculeswere active against two, three, and four viruses, respectively. Noneof the active compounds inhibited the replication of HIV-1, but anincreasing number of molecules exhibited antiviral activityagainst, in the order: VSV (1), VV (2), HSV-1 (2), Reo-1 (3), YFV(4), Sb-1 (9), BVDV (13), RSV (16), and CVB-5 (35) (see Table 1).

Thirty-four, out of 52 active compounds, showed an EC50 6

30 lM against at least one virus, and seven of them exhibited evenan EC50 6 10 lM. Thirty-four compounds [14 bearing a basic side-chain and 20 devoid of it] were not able to inhibit the replication ofany virus at concentrations up to the corresponding CC50 for theirhost cells, or up to the highest concentration tested (100 lM).

Cytotoxicity and antiviral activities of all the 86 consideredbenzimidazole derivatives and reference compounds are reportedin Tables 1S–3S. In Tables 2–4 only the results of the 52 activebenzimidazoles and reference compounds are illustrated.

2.2.1. Cytotoxicity on host cellsThe studied compounds showed only moderate degree of cyto-

toxicity against the confluent cell monolayer (in stationary growth)used to support the replication of the different viruses.

The exponentially growing lymphoblastoid human cells (MT-4)used to growth HIV-1 were the most susceptible to toxicity, whilethe non-human host cell lines exhibited a progressively reducedsensitivity in the order BHK-21 P MDBK > > Vero-76. Indeed, only46% of compounds showed no toxicity (CC50 >100 lM) againstMT-4 cells, versus 76% on Vero-76 cells. Vice versa 18.6% of com-pounds exhibited a CC50 6 30 lM for MT-4 cells, compared withonly 1.2% in the case of Vero-76 cells.

More importantly, it is observed that the sensitivity of each hostcell line is largely different versus the single structural group ofbenzimidazole derivatives, making very difficult the definition ofgeneral structure–toxicities relationships. The 2-benzylbenzimi-dazoles devoid of basic side-chain are the most toxic group of com-pounds for Vero-76 and, particularly, for MDBK cells, but, on thecontrary, they are the least toxic for the commonly more sensitivehuman MT-4 cells. The introduction of basic side-chains of what-ever kind, inverted the toxicity for the foresaid cell lines: it wasminimal for MDBK and Vero-76 cells, and maximal for MT-4 cells.In the case of 1-/2-(non-aromatic substituted)benimidazoles, thepresence of a basic side chain is always bond to a net increase oftoxicity on all the four host cell lines.

Some compounds (as 35, 38, 43, 51, 53, 71 and 82), indepen-dently from the inhibition of viral replication, may still deserveinterest for their high toxicity (CC50 6 10 lM) against one or morehost cell lines, that warrants their evaluation as possible antiprolif-erative agents. Indeed a strong antiproliferative activity has beenrecently described for some other analogous benzimidazole deriv-atives against a panel of 60 human tumor cell lines.8

2.2.2. Antiviral activity and structure–activity relationshipsAs indicated in Figure 7, the antiviral activity was found in all

the subsets of benzimidazole derivatives, but it was not evenlydistributed among them. The activity is the results of several inter-acting structural features, which may address it versus specificviruses, modulating also the potency. Considering the two moreremarkable features, it is observed that compounds bearing a basicside-chain were more frequently active (34/48; 70.8%) than thosedevoid of it (18/38; 47.4%). On the other hand 1/2-phenyl and

NH

N

NH

N

NR

F3C

N

H

R= CH3 68CF3 69

N

N

R"

R'R'= H, R"=

Cl,

A

B

N

N

CH2NH

N

NHCH2

NH

N

NH

R= H 60Cl 61

62 63

NH

N(CH2)3

HN

NH

R= H 64Cl 65 N

H

N(CH2)3

HN

N

R= H 66Cl 67

CH2

CH2

70

71

R R

R'O

O

72

73O

ClCl74

NH

NR

R= C2H5 75

C CHC2H5

C3H7 76

CH277

CH2OC2H5 78

NH

NR

O

R= H 795-CF3 805-Cl 815,6-Cl2 82

NH

N

O

Cl

Cl

83

NH

N

OR

R= CN 84CONH2 85

NH

N

OCH2OH

Cl

Cl

86

SCH2

R

Figure 6. Structures of the investigated non-aromatic substituted benzimidazole derivatives. (A) Compounds with a basic side-chain. (B) Compounds without basic side-chain.

R'''

RIV

CH2-CN a R'''

RIV

CH2

Cl

C

OC2H5

NH2 b/cN

N

R''

R

R'

27, 28, 32, 33, 4042-44, 52-54, 57, 58

+H2N

HN

R

R'

R''

N

CH2-CN

H a N

CH2

H

CNH2

OC2H5

ClH

Cl

H2N

H2N

+ bNH

N

NH

CH2

60

CH2

RIV

R'''

Scheme 1. Reagents and conditions: (a) EtOH, dry HCl, CHCl3, 0 �C, overnight; (b) AcOH, 45 �C, 12 h; (c) in the case of R = R0 = Cl and R00 = H: CHCl3, reflux 10 h.


2-benzylbenzimidazole derivatives (with and without side-chain)were more frequently active (38/59; 64.4%) than benzimidazolederivatives devoid of any aromatic moiety in position 1 or 2(14/27; 51.8%). The simultaneous lack of an aromatic moiety andof a basic side-chain did not abolish, but strongly reduced theincidence of activity (5/15; 33.3%). These values may be

underestimated, because some compounds of this subset werenot tested against all viruses and some active compounds couldhave been undetected.

Confining the comparison within the 34 compounds with thehigher activities (EC50 6 30 lM against at least one virus), it isobserved an increasing importance of the presence of an aromatic

NH2

NH

R

RI

NH2C

HC

O

Cl+

HCl a

NH

NH

R

RI

C

O

NCH2

H HCl

bN

N

NH

CH2

RI

R

1, 2, 61, 62

F3C

N

H2CH

N

N

F

F

14

NH2

NH

F3C

+

N

CH2H

F

F

CO

Cla

NH

NH

F3C

N

CH2H

C

O

F

F

c

Scheme 2. Reagents and conditions: (a) dioxane, reflux 4 h; (b) 4 N HCl, reflux 3 h; (c) 4 N HCl, reflux 12 h.

N

NH

O

H2N

NH2b'

O

HOCl

Cl

+R NH2

NH2

a

NH

N(CH2)3

HN

NH

R

64, 65

H2C NH2C

O

HOa

R

R' NH

NCH2

49, 55 NH2

+

R NH2

NH2R'

N

N

O

H2N

Nb''

O

HOCl

Cl

+R NH2

NH2

a

H

NH

N(CH2)3

HN

N

R

66, 67

Scheme 3. Reagents and conditions: (a) 4 N HCl, reflux 10 h; (b0) 6 N HCl, reflux 6 h; (b00) concd HCl, 150 �C, 72 h.

R NH2

NH2

+

OC

O

HO

C CH

C2H5

C3H7CHO

O

NH

N

O

R

NH

NR

C CH

C2H5

C3H7

76

80, 81

a

a

Scheme 4. Reagents and conditions: (a) 180 �C (without solvent), 2 h.


moiety, in respect to that of a basic side-chain for the expression ofantiviral activity (24/34 compounds; 70.6%), while the absence ofboth structural features reduced the incidence of activity from33.3% to 11.8%. All compounds with EC50 6 10 lM are derivatives

of phenyl-(2, 22) and benzyl- (35, 36, 37, 54 and 57) benzimid-azole; four of them contain a basic side-chain and three are devoidof it. Beyond the definition of a generic importance of the presenceof a basic side-chain for the expression of antiviral activity, it was

Cl

Cl NH

NCH2 Br

53

aCl

Cl NNa

NCH2 Br +

N

HCH2Cl

b

N

N

N

H

Cl

Cl

36

Br

Scheme 7. Reagents and conditions: (a) DMF, NaNH2, 45 �C, 30 min; (b) 140 �C, 3 h.

R NH2

NH

R'

+ CH

HO

NaO3S

aR

N

N

R'

OCH3OCH3

4, 5, 17, 18, 19

Scheme 5. Reagents and conditions: (a) EtOH, reflux 3 h.

R

R' NH

NCH2 NH2

a, bR

R' NH

NCH2 NH C

O

CH3

45, 49, 55 46, 50, 56

Scheme 6. Reagents and conditions: (a) Ac2O, benzene, reflux 2 h; (b) EtOH, H2O, reflux 2 h.

N

HCH2SH

NH

NCl +

NH

N

NHSCH2

63

a

Scheme 8. Reagents and conditions: (a) CH3CN, 100 �C, 4 h (closed tube).


difficult to analyze the significance of each particular basic chain,because of the limited availability of terms of comparison. Never-theless it was observed that the 1-dimethylaminoethyl-(4) andthe 1-diethylaminopropyl-(5) derivatives of 2-(4-methoxy-phenyl)-5-trifluoromethylbenzimidazole were inactive, while thecorresponding 1-lupinyl- and 1-epi-lupinyl-derivatives (16 and

F3C NO2

Cl N

HCH2NH2

+ a

F3C

Scheme 10. Reagents and conditions: (a) D

NH

N

H2N

OCH3

R

R'

a

Scheme 9. Reagents and conditions:

17) displayed some activity versus, respectively, Reo and CVB-5viruses. Vice versa, in the case of 2-(3,4-dichlorobenzyl)-5-trifluo-romethylbenzimidazole, the linear basic chain derivatives 32 and33 were active against CVB-5 and Sb-1 viruses, while the lupinylderivative 39 was inactive.

The comparison of the 1-unsubstituted benzimidazole deriva-tives 52, 53 and 47 with the corresponding 1-lupinyl derivatives35, 36 and 38 indicated that the latter were the only active or moreactive; however in the couple 48 and 41 the inactive compoundwas the 1-lupinyl-2-(4-methoxybenzyl)-5-trifluoromethylbenzim-idazole 41 (it is worth noting that compound 41 was foundendowed8 with potent antiproliferative activity versus a largenumber of human cancer cell lines).

Of course, the expression of activity is also associated to thegroups that decorate the aromatic nuclei, but it is observed that

NO2

NH

CH2

N

H

b

F3C NH2

NH

CH2

N

H

HCl ClH

MF, 170 �C, 90 min; (b) H2/Pd-C, EtOH.

N

N

N N

OCH3

R

R'

24, 25

(a) 2 N HCl, NaNO2, 0 �C, 30 min.

Table 1Number of compounds inhibiting the replication of the indicated viruses, and range of their EC50 (lM)

Virus N. tested compd N. active compd N. hit compd, SI P 5 N. of active compounds with EC50 (lM) in the range:

610 11–20 21–30 31–50 51–100

Aa Bb Aa Bb Aa Bb Aa Bb Aa Bb

HIV-1 85 0 / / / / / / / / / / /VSV 82 1 / / / / / / / / / 1 /VV 75 2 / / / / / 1 / / 1 / /HSV-1 81 2 / / / / / 1 / / / / 1Reo-1 82 3 / / / / / 1 / 1 / / 1YFV 85 4 / / / / / 2 / 1 / 1 /Sb-1 82 9 2 / / / 2 / 1 / / 4 2BVDV 85 13 1 / / 2 / / 1 4 1 5 /RSV 74 16 7 2 3 2 1 2 2 2 / 2 /CVB-5 75 35 13 2 / 7 6 2 2 7 1 6 2Total 86c 85d 23e 4 3 11 9 9 6 15 3 19 6

a A: compounds bearing a side-chain.b B: compounds devoid of basic side-chain.c Some compounds were not tested against all viruses.d 85 responses of activity from 52 distinct compounds, of which 28 were active against 1 virus, 16 against 2 viruses, 7 against 3 viruses and 1 against 4 viruses.e Data from 19 distinct compounds, 4 of which displayed SI P 5 versus 2 viruses.

Table 2Cytotoxicity and antiviral activity of 1-/2-phenylbenzimidazole derivatives and related compounds. CC50 and EC50, lM

Compd MT-4 MDBK BVDV BHK-21 Reo-1 Vero-76 CVB-5 Sb-1 RSV VSV SI for CVB-5h SI for RSVi

CC50a CC50

b EC50c CC50

d EC50e CC50

f EC50g EC50

g EC50g EC50

g

1 33 >100 89 30 >j >100 49 > > > 2.04 /2 42 90 44 29 > >100 17 73 7 > >5.88 >14.283 31 91 17 37 / >90 > > > > / /6 100 >100 > >100 > P100 80 > > 68 1.25 /7 100 >100 > >100 > >100 80 > 63 > 1.25 >1.598 56 86 80 48 > 90 14 > 45 > 6.43 2.009 >100 >100 36 >100 > >100 > > > > / /10 >100 >100 40 >100 > P100 32 > > > 3.12 /13 24 40 > 75 > 24 14 > > > 1.71 /14 57 36 > 18 > P100 > 60 > > / /15 21 48 > 60 32 78 26 > > > 3.00 /16 25 >100 > 65 27 75 > > > > / /17 42 57 > 30 > >100 54 > > > >1.85 /18 >100 22 > 35 > 80 > 25 > > / /21 47 54 > 36 > 87 > > 25 > / 3.4822 44 23 > 20 > 80 > > 9 > / 8.8923 >100 >100 > >100 > >100 71 > > > >1.4 /24 >100 >100 > >100 > >100 40 > nt > >2.5 /NM 108 >100 >100 1.7 >100 2.4 >100 20 > > > >5.0 /NM 176 >100 >100 38 >100 > >100 30 25 > > >3.33 /Ribavirin 31 >100 8 >100 > >100 > > 7 > / >14.28NM 299 2.0 >100 > >100 > 20 > > 1.2 > / 16.67M 5255 0.2 42 > >100 > 20 > > 0.6 nt / 33.33ACG >100 >100 > >100 > >100 > > > > / /

a Compound concentration (lM) required to reduce the viability of mock-infected MT-4 cells by 50%, as determined by the MTT method.b Compound concentration (lM) required to reduce the viability of mock-infected MDBK cells by 50%, as determined by the MTT method.c Compound concentration (lM) required to achieve 50% protection of MDBK cells from the BVDV-induced cytopathogenicity, as determined by the MTT method.d Compound concentration (lM) required to reduce the viability of mock-infected BHK monolayers by 50%, as determined by the MTT method.e Compound concentration (lM) required to achieve 50% protection of BHK cells from the Reo-induced cytopathogenicity, as determined by the MTT method.f Compound concentration (lM) required to reduce the viability of mock-infected VERO-76 monolayers by 50%.g Compound concentration (lM) required to reduce the plaque number of the indicated virus by 50% in VERO-76 monolayers.h Selectivity index for CVB-5: CC50/EC50.i Selectivity index for RSV: CC50/EC50.j The sign ‘>’ indicates that the EC50 is higher than the CC50 for the corresponding host cell line or, anyhow, beyond the highest tested concentration (100 lM). nt = not

tested.


a given group may play different roles, even opposite, in relation tothe specific molecular context. These considerations may havesomewhat different relevance when considering each specificvirus. Thus, for compounds active against CVB-5, RSV and BVDV(the most frequently affected viruses) the presence of an aromaticmoiety and of a basic side-chain play always an important role,fairly in line with the foregoing general considerations. However,an aromatic moiety is more frequently present in compoundsactive against RSV (87.5%) and on CVB-5 (82.8%) and less in those

active against BVDV (69.2%). Vice versa the presence of a basicchain is more frequent in compounds active against BVDV(84.6%) and less in those active against CVB-5 (68.6%) and againstRSV (62.5%). These structural features have almost the same rele-vance for the activity against CVB-5 and RSV, and indeed 12 outof the 16 compounds active against RSV were also active againstCVB-5.

On the other hand, the activity on YFV rely on the presence of abasic side chain (65, 66, 68 and 69), and activity against Sb-1 does

Table 3Cytotoxicity and antiviral activity of 2-benzylbenzimidazole derivatives. CC50 and EC50, lM

Compd MT-4 MDBK BVDV BHK-21 Reo-1 Vero-76 CVB-5 Sb-1 RSV VV SI for CVB-5h SI for RSVi

CC50a CC50

b EC50c CC50

d EC50e CC50

f EC50g EC50

g EC50g EC50

g

26 42 >100 85 >100 > j >100 73 > > > >1.37 /27 49 >100 76 87 > >100 53 > 75 > >1.89 >1.3328 25 >100 > >100 > >100 32 > 40 > >3.12 >2.532 >100 24 > 16 > >100 33 > > > >3.03 /33 40 26 > >100 > >100 17 63 > > >5.88 /34 17 >100 > 30 > >100 28 > > > >3.57 /35 9 46 > 52 > >100 9 > 12 > >11.11 >8.3336 12 58 > 70 > >100 50 > 9 > >2.00 >11.1137 >100 >100 > 15 > >100 10 60 > > >10.00 /38 6 54 > 20 > >100 13 > 15 > >7.69 >6.6740 13 40 > 20 > >100 100 > 25 > >1.00 >4.0042 100 28 > 25 > 93 75 > > > 1.24 /45 >100 >100 > >100 > >100 17 58 > > >5.88 /47 >100 16 > 20 > 70 26 > > > 2.69 /48 69 56 > 60 > 92 20 > > > 4.60 /49 >100 >100 > >100 > >100 11 > > > >9.09 /54 29 17 > 19 > P100 > > 5 > / P20.055 71 62 22 43 > 80 12 80 20 > 6.67 4.0057 >100 11 > 21 > >100 12 > 9 31 >8.33 >11.1158 >100 >100 > >100 54 >100 12 > > > >8.33 /NM 108 >100 >100 1.7 >100 2.4 >100 20 > > > >5.0 /NM 176 >100 >100 38 >100 > >100 30 25 > > >3.33 /Ribavirin 31 >100 8 >100 > >100 > > 7 > / >14.28NM 299 2.0 >100 > >100 > 20 > > 1.2 11 / 16.67M 5255 0.2 42 > >100 > 20 > > 0.6 2 / 33.33ACG >100 >100 > >100 > >100 > > > > / /

a Compound concentration (lM) required to reduce the viability of mock-infected MT-4 cells by 50%, as determined by the MTT method.b Compound concentration (lM) required to reduce the viability of mock-infected MDBK cells by 50%, as determined by the MTT method.c Compound concentration (lM) required to achieve 50% protection of MDBK cells from the BVDV-induced cytopathogenicity, as determined by the MTT method.d Compound concentration (lM) required to reduce the viability of mock-infected BHK monolayers by 50%, as determined by the MTT method.e Compound concentration (lM) required to achieve 50% protection of BHK cells from the Reo-induced cytopathogenicity, as determined by the MTT method.f Compound concentration (lM) required to reduce the viability of mock-infected VERO-76 monolayers by 50%.g Compound concentration (lM) required to reduce the plaque number of the indicated virus by 50% in VERO-76 monolayers.h Selectivity index for CVB-5: CC50/EC50.i Selectivity index for RSV: CC50/EC50.j The sign ‘>’ indicates that the EC50 is higher than the CC50 for the corresponding host cell line or, anyhow, beyond the highest tested concentration (100 lM).


not require, in one third of the cases (81, 82 and 86), neither thepresence of an aromatic moiety, nor that of a basic side-chain.

As far as the viral drug-sensitivity is concerned, it is worth not-ing the widespread sensitivity of CVB-5 versus 35 over 75 testedcompounds, with a mean EC50 of 34.5 lM. More importantly, 14of these compounds (2, 8, 13, 33, 35, 37, 38, 45, 49, 55, 57, 58,69 and 70) exhibited EC50 in the range 9–17 lM (mean EC50

12.9 lM), thus resulting more potent than the reference com-pounds NM 108 and NM 176 (EC50 = 20 and 30 lM, respectively).Also the selectivity indexes (SI = CC50/EC50) of these compounds(with the exception of 13) are better (from �6 up to >11) thanthose of the reference drugs (>5 and 3.33, respectively), so thatthey may represent interesting starting models to obtain improvedagents against coxsackie viruses.

Coxsackie viruses are associated with several human pathologies(as hand-foot and mouth disease, pleurodynia, myopericarditis,aseptic meningitis, etc.) and with damage of pancreas beta-cells,prodromal to type 1 diabetes, but effective drugs are not availa-bles.29 Ribavirin, considered a broad spectrum antiviral agent, hasbeen tentatively used in the treatment of coxsackie virus infections,but with poor results, and indeed, it is practically inactive in in vitroassays (EC50 >100 lM versus CVB-5, and, accordingly to Chenget al.,30 EC50 >1800 lM versus CVB-3). Three (13, 49 and 70) of the14 best compounds inhibited the growth of only CVB-5, while theremaining exhibited different levels of activity against otherviruses; particularly, six compounds (2, 35, 38, 55, 57 and 69)showed good activity also versus RSV.

As mentioned in the introduction,2b activity against RSV is apeculiarity of several chemotypes related to benzimidazole, and,indeed, has been observed also for sixteen of the presently

investigated compounds, with the EC50 in the range 5–75 lM(mean EC50 = 23.8 lM). More importantly, the seven best com-pounds (54, 2, 36, 57, 22, 35 and 38, in order of decreasing SI) dis-played EC50 values in the range 5–15 lM (SI from P20 to >6.7),that are comparable with that of the reference drug ribavirin(EC50 = 7 lM; SI P14.3). The latter is the only drug available to treatRSV infections, but, as a consequence of its limited efficacy, theneed of prolonged aerosol administration and the risk of toxicity,its use is restricted to children considered at high risk. Therefore,effective and safe drugs to treat the severe RSV-linked respiratorypathologies, affecting also adults and the elderly, are stronglyneeded. Since the pioneering work of Dubovi et al.,31 illustratingthe strong activity of bis(5-amidinobenzimidazolyl)methane(BABIM), a large number of benzimidazole derived anti-RSV agentshave been described, whose activity has been attributed to the inhi-bition of the viral fusion process. Some of them even reached nano-and pico-molar potency in vitro but so far none of them has foundclinical application, because of some negative properties.32a–d Thusthe novel chemotypes of the present RSV inhibitors, exhibiting goodthough not outstanding potency, might be interesting for the devel-opment of improved anti-RSV agents. The present 1-substituted-2-benzylbenzimidazoles 27, 28, 35, 36, 40 and 57, and, maybe, alsothe 1-substituted-2-phenylbenzimidazoles 7, 8 and 21, may inhibitthe RSV replication by blocking the viral fusion and entry processes,as it has been demonstrated32a,b for the structurally similar1-substituted-2-[(benzotriazol-1/2-yl)methyl]benzimidazoles andother analogous compounds.

The good activity of 2-(epi-lupinyl)-1-phenylbenzimidazole 2versus RSV (EC50 = 7 lM) and the lower ones versus BVDV, CVB-5and Sb-1, were strongly influenced by the configuration of the

Table 4Cytotoxicity and antiviral activity of 1-/2-(non-aromatic substituted)benzimidazole derivatives. CC50 and EC50, lM

Compd MT-4 MDBK BVDV BHK-21 YFV Vero-76 CVB-5 Sb-1 RSV VV HSV-1 SI for CVB-5h SI for RSVi

CC50a CC50

b EC50c CC50

d EC50e CC50

f EC50g EC50

g EC50g EC50

g EC50g

60 17 >100 34 78 >j >100 > > > > > / /61 53 77 17 52 > >100 > > > > > / /63 100 >100 > >100 > >100 48 > > > > >2.08 /65 >100 >100 > >100 25 >100 > > > > > / /66 >100 >100 94 >100 87 >100 > > > > > / /68 >100 >100 > >100 27 >100 47 > > 23 > >2.13 /69 54 P100 > 80 33 90 11 > 22 > > 8.18 4.0970 11 35 > 32 > >100 11 > > > > 9.09 /71 5 50 > 30 > >100 > > > > 30 / /76 61 >100 > 67 > P100 90 > 22 > > 1.11 4.5477 >100 >100 > >100 > >100 21 > > > > >4.76 /81 >100 >100 > >100 > >100 nt 20 nt nt > / /82 6 >100 > >100 > >100 nt 60 nt nt > / /86 33 >100 46 >100 > >100 nt 12 nt nt 60 / /NM 108 >100 >100 1.7 >100 1.5 >100 20 > > > > >5.0 /NM 176 >100 >100 38 >100 > >100 30 25 > > > >3.33 /Ribavirin 31 >100 8 >100 > >100 > > 7 > > / >14.28NM 299 2.0 >100 > >100 26 20 > > 1.2 11 > / 16.67M 5255 0.2 42 > >100 > 20 > > 0.6 2 > / 33.33ACG >100 >100 > >100 > >100 > > > > 1.2 / /

a Compound concentration (lM) required to reduce the viability of mock-infected MT-4 cells by 50%, as determined by the MTT method.b Compound concentration (lM) required to reduce the viability of mock-infected MDBK cells by 50%, as determined by the MTT method.c Compound concentration (lM) required to achieve 50% protection of MDBK cells from the BVDV-induced cytopathogenicity, as determined by the MTT method.d Compound concentration (lM) required to reduce the viability of mock-infected BHK monolayers by 50%, as determined by the MTT method.e Compound concentration (lM) required to achieve 50% protection of BHK cells from the YFV-induced cytopathogenicity, as determined by the MTT method.f Compound concentration (lM) required to reduce the viability of mock-infected VERO-76 monolayers by 50%.g Compound concentration (lM) required to reduce the plaque number of the indicated virus by 50% in VERO-76 monolayers.h Selectivity index for CVB-5: CC50/EC50.i Selectivity index for RSV: CC50/EC50.j The sign ‘>’ indicates that the EC50 is higher than the CC50 for the corresponding host cell line or, anyhow, beyond the highest tested concentration (100 lM). nt = not

tested.

Figure 7. Distribution of active compounds (a) among the subsets of benzimidazole derivatives, with (A) and without (B) basic side-chain. Number and per cent of active overtested compounds (see Table 1) of each subset. (a) EC50 < CC50, independently from number and nature of inhibited viruses; (b) some compounds of this subset were nottested against all viruses, thus the reported number and % may be somewhat underestimated.


basic side-chain. In fact the corresponding lupinyl derivative 1 wascompletely devoid of activity versus RSV and Sb-1 and exhibitedreduced activity against BVDV and CVB-5. Among the other cou-ples of epimeric compounds (16 and 17; 37 and 42; 39 and 43;41 and 44; 60 and 62), higher activity was commonly observedin the lupinyl epimers (particularly versus CVB-5, the EC50 for 37was 10 lM and for 42 was 75 lM) or no difference could bedetected, being inactive both epimers (39 and 43; 41 and 44).

Another interesting chemotype is represented by the pentacy-clic compound 22, that is a rigid analog of 1-benzyl-2-phenylbenz-imidazole 21. Both compounds were selective inhibitors of thereplication of RSV, but the quasi planar 22 is about three timesmore potent than 21, whose aromatic substituents are free totwist, assuming variable orientations in the space. The ability toadopt ‘butterfly-like’ orientation is considered determinant forthe expression of high inhibitory activity on HIV-1 reverse


transcriptase and on the multiplication of that virus, as observedfor 1-(2,6-difluorobenzyl)-2-(2,6-difluorophenyl)benzimidazole andits analogs33a–c and also for 1-benzoyl-2-(2-nitrophenyl)benzimid-azole.34 However 1-benzyl-2-phenylbenzimidazole (21) itself isdevoid of activity against HIV-1 and the other viruses presentlyconsidered, with the exception of RSV, but it has been shown35

to possess antitumoral activity, by inhibiting, in vitro and in vivo,the human chondrosarcoma. An extended comparison of the anti-viral and antitumoral activities of novel derivatives of the flexible1-benzyl-2-phenylbenzimidazole and the rigid dibenzo[c,e]benz-imidazo[1,2-a]azepine (22) is surely worthwhile.

2-Trifluoromethylbenzimidazoles are an important class ofcompounds endowed with various biological activities, and partic-ularly, the antiparasitic one.36a–c However, when some derivativesof this scaffold were evaluated as antivirals, they resulted eitherinactive, or highly cytotoxic versus the host cell lines.37 Notwith-standing, the only one compound of this type we have tested(69) exhibited a non-negligible activity against CVB-5, RSV andYFV, with EC50 = 11, 22 and 33 lM, respectively, and correspondingSI = 8.2, 4.1, 2.4. The activity of compound 69 may be related to thepresence of the cumbersome lupinyl (quinolizidin-1-ylmethyl) res-idue on position 1 of benzimidazole ring, but the importance of thetrifluoromethyl group is confirmed by the narrower spectrum ofactivity and lower potency of the 2-methyl analog 68. Thus 69 rep-resents another hit for developing better antiviral agents againsteither one of the above viruses.

Even if activity against BVDV was shown by thirteen over eighty-five tested compounds, only two of them (3 and 61) exhibited aworthwhile potency (EC50 = 17 lM) and were selective for thatvirus. The former is the unique known derivative of an unusual tet-racyclic ring system (pyrido[30,20:4,5]imidazo[1,2-a][1,4]benzodi-azepine-5-one) and the latter is characterized by the connectionof a lupinyl residue to position 2 of the benzimidazole nucleus,instead of the usual N(1). Both compounds represent interestingstarting points for the preparation of better anti-BVDV agents.

Finally, among the nine compounds inhibiting the replication ofthe poliovirus Sb-1, three (81, 82 and 86) were devoid of aromaticsubstituent and of basic side-chain, thus deserving some comment.They derive from 2-(tetrahydropyran-2-yl)benzimidazole that rep-resent an unexplored scaffold for the preparation of antiviralagents. Some related compounds (in particular compound 82)were found, by some of us,17 to inhibit the growth of 19 humantumor cell lines at near micromolar concentration, while lackingany anti-HIV-1 activity (the only one sought-after by us at thattime). The tetrahydropyranyl moiety of these compounds (particu-larly when bearing a 6-hydroxymethyl group, as in 86) resemblesthe 2-glucopyranosyl moiety of some benzimidazole derivatives,that have been shown to inhibit the glycogen phosphorylase38a,b

and also the a-glycosidase.39 These activities, besides their impor-tance for the development of new anti-diabetic drugs, were alsoshown to impact on the multiplication of viruses.40

Comparing compounds 82 and 86, it is observed a 5-foldincrease of activity, related to the presence of an alcoholic groupin the latter. On the other hand, it is observed that the mono-chloroderivative 81 is three times more potent than the di-chloro deriv-ative 82, thus the mono-chloro analog of 86 could be expected todisplay an improved activity against Sb-1 virus. Therefore the syn-thesis and evaluation of antiviral activity (particularly against Sb-1) of other derivatives of 2-(tetrahydropyran-2-yl)benzimidazole[including 2-(glucopyranosyl)benzimidazole] may be worthwhile.Indeed, poliomyelitis has been practically eradicated in developedcountries by extensive vaccination, but cases still occur in sub-Sah-aran Africa and southern Asia, and a specific antiviral therapy isstill unavailable.41

In spite of the structural analogy with 2-(glucopyranosyl)benz-imidazole, all the 2-(tetrahydropyranyl)- and 2-(tetrahydrofuranyl)

benzimidazoles (79–86) exhibited, paradoxically, variable degree(sometime very high) of hyperglycemic activity when injected ipin normoglycemic rats (unpublished results). The existence of anyrelation between antiviral activity and the derangement of releaseand/or the metabolism of glucose is worthy of investigation.

3. Conclusions

A library of eighty-six assorted derivatives of benzimidazolewas screened for antiviral activity against a panel of ten RNA andDNA viruses, pointing out a number of novel hit compounds suit-able for future developments. Fifty-two of them (60.5%) displayeddifferent degrees of activity versus one or more viruses; moreimportantly, nineteen (22%) of them exhibited SI P 5, thus repre-senting interesting hit compounds (Table 1). Among the sensitiveviruses CVB-5, RSV, BVDV and Sb-1 were (in decreasing order)the more frequently (and effectively) affected, while YFV, Reo-1,HSV-1, VV and VSV viruses were only occasionally (and often mod-estly) affected; HIV-1 replication was not inhibited by any of thesecompounds. It is interesting that in several cases CVB-5 and RSVshare sensitivity versus the same compounds.

Activity was mainly found in compounds bearing in position 1or 2 of the benzimidazole ring an aromatic moiety (substitutedphenyl or benzyl) and a basic side chain (linear or cyclic) (48.1%)or, at least, either one of them (42.3%); only 9.6% of the active com-pounds is devoid of both these structural features.

Among the thirty-five compounds endowed with activityagainst CVB-5, fourteen exhibited EC50 in the range 9–17 lM(mean EC50= 12.9 lM), and among the sixteen compounds activeversus RSV seven exhibited EC50 in the range 5–15 lM (with themean EC50 = 9.4 lM), thus resulting comparable to or more potentthan the respective reference drugs (NM 108 and ribavirin).

Among the above most active compounds, 49 and 70 wereselective for CVB-5, while 22 and 54 were selective for RSV; com-pounds 2, 35, 38 and 57 shared good activity against both CVB-5and RSV. Most of the best compounds active against CVB-5 andRSV were 2-benzylbenzimidazole derivatives, bearing or not abasic side-chain. Thus, through the introduction of a variety of sub-stituents on the two aromatic nuclei of 2-benzylbenzimidazole,and the variation of the eventual basic chain, improved inhibitorsof CVB-5 and or RSV replication might be expected.

Besides 2-benzylbenzimidazole, other molecular cores havebeen identified as hits for developing better antiviral agents, bymolecular manipulation. Thus, taking as models compounds 2and 69, from 1-phenylbenzimidazole and 2-trifluoromethylbenz-imidazole large spectrum antivirals could be achieved. On theother hand, starting from the unexplored, rigid scaffolds of com-pounds 3 (pyrido[30,20:4,5]imidazo[1,2-a][1,4]benzodiazepine-5-one) and 22 (dibenzo[c,e]benzimidazo[1,2-a]azepine), improvedand selective inhibitors versus, respectively, BVDV and RSV couldbe obtained. Finally, on the 2-(tetrahydropyran-2-yl)benzimid-azole core of 81, 82 and 86, better antiviral molecules, particularlyversus Sb-1, could be constructed.

Effective therapeutic agents for the human pathologies relatedto CVB-5 and RSV viruses are unavailable, and hence urgentlyneeded, but, despite the extensive vaccination, also agents againstpoliovirus Sb-1 may be important in the underdeveloped countries.

4. Experimental

4.1. General

Chemicals, solvents and reagents used for the syntheses werepurchased from Sigma–Aldrich, Fluka or Alfa Aesar, and were usedwithout any further purification. Column chromatography (CC):


neutral alumina (Al2O3), activity 1 (Merck). Mps: Büchi apparatus,uncorrected. 1H NMR spectra: Varian Gemini-200 spectrometer;CDCl3 or DMSO-d6; d in ppm rel. to Me4Si as internal standard. Jin Hz. Elemental analyses were performed on a Carlo ErbaEA-1110 CHNS-O instrument in the Microanalysis Laboratory ofthe Department of Pharmacy of Genoa University.

4.2. Benzimidazoles from iminoesters. General method ofScheme 1

Cooling at 0 �C, a solution of 4-substituted- or 3,4-disubsti-tuted-phenylacetonitrile or of cyanolupinane42 (6 mmol) and0.35 mL of abs. EtOH in 5 mL of CHCl3 was saturated withanhydrous HCl and left overnight at 0–5 �C. After evaporation ofthe solvent the residue was reacted with the proper 1,2-phenyl-enediamine or its N1-alkyl-, N1-dialkylaminoalkyl- or N1-[(octahy-droquinolizin-1yl)methyl]derivative (3 mmol) in glacial CH3COOH(8 mL) and the mixture was heated at 45 �C for 12 h. Then the reac-tion mixture was concentrated and the residue was taken up withdil aq HCl and extracted with Et2O. The acidic solution was basifiedwith 2 N NH3 and extracted with Et2O. The dried organic layer(Na2SO4) was evaporated to afford an oily residue that crystallizedfrom the suitable solvent or was purified by CC.

Alternatively, the residue of iminoester hydrochloride wasreacted with the 3,4-dichloro-1,2-phenylenediamine in anhydrousCHCl3 solution (10 mL). After refluxing the mixture for 10 h thechloroform solution was washed with water, dried (Na2SO4) andevaporated; the residue was crystallized from the indicatedsolvent.

Most of the required N1-substituted-1,2-phenylenediaminewere commercially available or previously described.19,13,43 Thepreparation of the only novel diamine is described at the end ofthe Section.

4.2.1. 2-(4-Bromobenzyl)-1-[(2-diethylamino)ethyl]benzimidazole (27)

Yield: 28%. Mp 85–87 �C (Et2O/pentane). H NMR (CDCl3): 1.08(t, J = 7.2, 6H, N(CH2CH3)2); 2.47–2.76 (m, 6H, CH2CH2N(CH2CH3)2);4.10–4.28 (m, 2H, CH2CH2N(C2H5)2)); 4.37 (s, 2H, CH2-Ar); 7.18 (d,J = 8.4, 2 arom. H); 7.23–7.41 (m, 3 arom. H); 7.46 (d, J = 8.4, 2arom. H); 7.73–7.84 (m, 1 arom. H). Anal. Calcd for C20H24BrN3:C, 62.18; H, 6.26; N, 10.88. Found: C, 62.40; H, 6.34; N, 10.62.

4.2.2. 1-[(2-Diethylamino)ethyl]-2-(4-iodobenzyl)benzimidazole (28)

Yield: 42%. Mp 79–82 �C (Et2O/pentane).1H NMR (CDCl3): 1.05(t, J = 7.2, 6H, N(CH2CH3)2); 2.46–2.77 (m, 6H, CH2CH2N(CH2CH3)2);4.10–4.43 (m, 2H, CH2CH2N(C2H5)2) and 4.38, s, 2H, CH2-Ar, super-imposed); 7.09 (d, J = 8.2, 2 arom. H); 7.17–7.45 (m, 3 arom. H);7.66 (d, J = 8.2, 2 arom. H); 7.73–7.83 (m, 1 arom. H). Anal. Calcdfor C20H24N3I: C, 55.43; H, 5.58; N, 9.70. Found: C, 55.22; H, 5.62;N, 9.59.

4.2.3. 2-[(3,4-Dichloro)benzyl]-1-[(2-dimethylamino)ethyl]-5-trifluoromethylbenzimidazole (32)

Yield: 30%. Mp 90–91 �C (Et2O). 1H NMR (CDCl3): 2.42 (s,N(CH3)2); 2.63 (t, J = 7.0, 2H, CH2CH2N(CH3)2)); 4.22–4.55 (m, 2H,CH2-Q and 4.41, s, 2H, CH2-Ar, superimposed); 7.14–7.26 (m, 1arom. H); 7.34–7.59 (m, 4 arom. H); 8.07 (s, 1 arom. H). Anal. Calcdfor C19H18Cl2F3N3: C, 54.82; H, 4.36; N, 10.09. Found: C, 54.50; H,4.62; N, 10.00.

4.2.4. 2-[(3,4-Dichloro)benzyl]-1-[(3-diethylamino)propyl]-5-trifluoromethylbenzimidazole (33)

Yield: 31%. Mp 53–54 �C (pentane). 1H NMR (CDCl3): 1.07 (t,J = 7.2, 6H, N(CH2CH3)2); 1.81–2.00 (m, 2H, CH2CH2CH2N(C2H5)2);

2.44 (t, J = 7.0, 2H, CH2CH2N(C2H5)2)); 2.59 (q, 4H, N(CH2CH3)2);4.18 (t, J = 7.3, 2H, CH2CH2CH2N(C2H5)2); 4.39 (s, 2H, CH2-Ar);7.08 (dd, J = 8.6, 2.2, 1 arom. H); 7.36–7.62 (m, 4 arom. H); 8.07(s, 1 arom. H). Anal. Calcd for C22H24Cl2F3N3: C, 57.65; H, 5.28; N,9.17. Found: C, 58.27; H, 5.37; N, 9.17.

4.2.5. 2-[(4-Bromo)benzyl]-1-[(1S,9aR)-(octahydro-2H-quinolizin-1-yl)methyl]-5-trifluoro-methylbenzimidazole (40)

Yield: 28%. Mp 148–151 �C (Et2O). 1H NMR (CDCl3): 1.18–2.58(m, 14H, Q); 2.84–3.30 (m, 2Ha near N of Q); 4.20–4.58 (m, 2H,CH2-Q and 4.34, s, 2H, CH2-Ar superimposed); 7.18 (d, J = 8.0, 2arom. H); 7.34–7.57 (m, 4 arom. H); 8.05 (s, 1 arom. H). Anal. Calcdfor C25H27BrF3N3+0.25H2O: C, 58.77; H, 5.42; N, 8.22. Found: C,58.71; H, 5.72; N, 8.16.

4.2.6. 2-Benzyl-1-[(1R,9aR)-(octahydro-2H-quinolizin-1-yl)methyl]-5-trifluoro-methylbenzimidazole (42)

Yield: 35%. Mp 99–101 �C (Et2O). CC (Al2O3/Et2O). 1H NMR(CDCl3): 0.78–2.45 (m, 14H, Q); 2.90–3.18 (m, 2Ha near N of Q);3.40–3.60 (m, 1H, CH2-Q); 4.16–4.29 (m, 1H, CH2-Q); 4.32–4.54(AB syst., 2H, CH2-Ar.); 7.20–7.57 (m, 7 arom. H); 8.07 (s, 1 arom.H). Anal. Calcd for C25H28F3N3: C, 70.24; H, 6.60; N, 9.83. Found:C, 70.22; H, 6.48; N, 9.91.

4.2.7. 2-[3,4-(Dichloro)benzyl]-1-[(1R,9aR)-(octahydro-2H-quinolizin-1-yl)methyl]-5-trifluoro-methylbenzimidazole (43)

Yield: 38%. Mp 125–126 �C (Et2O). 1H NMR (CDCl3): 0.93–2.62(m, 14H, Q); 3.10–3.38 (m, 2Ha near N of Q); 3.60–3.82 (m, 1H,CH2-Q); 4.18–4.51 (m, 1H, CH2-Q and AB syst., 2H, CH2-Ar., super-imposed); 7.13–7.62 (m, 5 arom. H); 8.07 (s, 1 arom. H). Anal. Calcdfor C25H26Cl2F3N3: C, 60.49; H, 5.28; N, 8.46. Found: C, 60.53; H,5.33; N, 8.34.

4.2.8. 2-[4-(Methoxy)benzyl]-1-[(1R,9aR)-(octahydro-2H-quinolizin-1-yl)methyl]-5-trifluoro-methylbenzimidazole (44)

Yield: 35%. Mp 116–117 �C (pentane/Et2O 2:1). CC (Al2O3/Et2O).1H NMR (CDCl3): 0.84–2.60 (m, 14H, Q); 3.04–3.35 (m, 2Ha near Nof Q); 3.57–3.84 (m, 1H, CH2-Q and 3.81, s, OCH3, superimposed);4.12–4.46 (m, 1H, CH2-Q and AB syst., 2H, CH2-Ar., superimposed);6.88 (d, J = 8.8, 2 arom. H); 7.20 (d, J = 8.8, 2 arom. H). 7.32–7.57 (m,2 arom. H); 8.07 (s, 1 arom. H). Anal. Calcd for C26H30F3N3O: C,68.25; H, 6.61; N, 9.18. Found: C, 68.59; H, 6.67; N, 9.26.

4.2.9. 2-[(3,4-Dichloro)benzyl]-5,6-dichloro-1H-benzimidazole(52)

Yield: 32%. Mp 204–207 �C (Et2O). 1H NMR (DMSO-d6): 4.25 (s,2H, CH2-Ar); 7.33 (dd, J = 8.0, 2.0, 1 arom. H); 7.54–7.70 (m, 2 arom.H); 7.79 (s, 2 arom. H); 12.67 (br. s, NH benzim., collapses withD2O). Anal. Calcd for C14H8Cl4N2: C, 48.59; H, 2.33; N, 8.09. Found:C, 48.40; H, 2.27; N, 8.06.

4.2.10. 2-[(4-Bromo)benzyl]-5,6-dichloro-1H-benzimidazole(53)

Yield: 31%. Mp 224–227 �C (CHCl3/Et2O 1:1). 1H NMR (DMSO-d6): 4.19 (s, 2H, CH2-Ar); 7.30 (d, J = 8.4, 2 arom. H); 7.54 (d,J = 8.4, 2 arom. H); 7.78 (s, 2 arom. H); 12.65 (br. s, NH benzim., col-lapses with D2O). Anal. Calcd for C14H9BrCl2N2+0.25H2O: C, 46.69;H, 2.66; N, 7.78. Found: C, 46.75; H, 2.88; N, 7.70.

4.2.11. 5,6-Dichloro-2-[(4-methoxy)benzyl]-1H-benzimidazole(54)

Yield: 73%. Mp 191–193 �C (CHCl3/Et2O 1:1). 1H NMR (DMSO-d6): 4.11 (s, 5H, CH2-Ar and OCH3, superimposed); 6.10 (d, J = 8.8,2 arom. H); 6.45 (d, J = 8.8, 2 arom. H); 6.86 (s, 2 arom. H); 12.58(br. s, NH-Benz., collapses with D2O). Anal. Calcd for C15H12Cl2N2O:C, 58.65; H, 3.94; N, 9.12. Found: C, 58.58; H, 4.11; N, 9.00.


4.2.12. 1-Butyl-2-(3,4-dichlorobenzyl)-5-trifluoromethylbenzim-idazole (57)

Yield: 45%. Mp 71–74 �C (pentane/Et2O 2:1). 1H NMR (CDCl3):0.95 (t, J = 7.4, 3H, CH2CH2CH2CH3); 1.26–1.46 (m, 2H, CH2CH2CH2-

CH3); 1.50–1.77 (m, 2H, CH2CH2CH2CH3); 4.11 (t, J = 7.6, 2H, CH2-

CH2CH2CH3); 4.41 (s, 2H, CH2-Ar); 7.12–7.64 (m, 5 arom. H); 8.12(s, 1 arom. H). Anal. Calcd for C19H17Cl2F3N2: C, 56.87; H, 4.27; N,6.98. Found: C, 56.60; H, 4.17; N, 6.87.

4.2.13. 2-(3,4-Dichlorobenzyl)-1-[(2-methoxy)ethyl]-5-trifluoromethylbenzimidazole(58)

Yield: 47%. Mp 104–105 �C (pentane/Et2O 2:1). 1H NMR (CDCl3):3.27 (s, 3H, CH2CH2OCH3); 3.63 (t, J = 5.2, 2H, CH2CH2OCH3); 4.30 (t,J = 5.4, 2H, CH2CH2OCH3); 4.46 (s, 2H, CH2-Ar); 7.12–7.63 (m, 5arom. H); 8.11 (s, 1 arom. H). Anal. Calcd for C18H15Cl2F3N2O: C,53.62; H, 3.75; N, 6.95. Found: C, 53.39; H, 3.54; N, 7.24.

4.2.14. 2-[(1S,9aR)-Octahydro-2H-quinolizin-1-yl]benzimidazole(60)

Yield: 41%. Mp 232–234 �C (CH2Cl2). 1H NMR (CDCl3): 1.20–2.40(m, 14H, Q); 2.56–3.42 (m, 2Ha near N of Q and 2H, CH2-Q); 7.22–7.37 (m, 2 arom. H); 7.55–7.86 (m, 2 arom. H); 10.05 (s, NH-Benz.,exchanges with D2O). Anal. Calcd for C17H23N3: C, 75.80; H, 8.61; N,15.60. Found: C, 75.92; H, 8.66; N, 15.70.

4.3. Benzimidazoles from acyl chlorides. General method ofScheme 2

To a solution of the suitable N-phenyl- or 4-R-1,2-phenylenedi-amine (2 mmol) in anhydrous dioxane (4 mL), a solution of homo-lupinanoyl or epi-homolupinanoyl chloride hydrochloride44

(2 mmol) in anhydrous dioxane (4 mL) was added drop by drop,and the mixture was refluxed for 4 h.

Similarly, a solution of N1-[(1S,9aR)-(octahydroquinolizin-1yl)methyl]-4-trifluoromethyl-1,2-phenylenediamine (3 mmol)obtained as described13 and 2,6-difluorobenzoyl chloride (3 mmol)in anhydrous dioxane (6 mL) was refluxed for 4 h. In both cases,the solvent was removed and the residue was taken up in 4 NHCl (15 mL) and refluxed for 3 h and 12 h, respectively. The acidicsolution was basified with 6 N NaOH and extracted with Et2O; thedried organic layer (Na2SO4) was evaporated and the residue puri-fied by CC (Al2O3/Et2O).

4.3.1. 2-[(1S,9aR)-Octahydro-2H-quinolizin-1-yl]-1-phenylbenz-imidazole (1)

Yield: 40%. Mp 106–108 �C (Et2O). 1H NMR (CDCl3): 1.10–2.50(m, 14H, Q); 2.68–3.30 (m, 4H, 2Ha near N of Q and 2H, CH2-Q);7.10–7.70 (m, 8 arom. H); 7.8 (d, J = 8.8, 1 arom. H). Anal. Calcdfor C23H27N3: C, 79.96; H, 7.88; N, 12.16. Found: C, 79.77; H,7.91; N, 12.15.

4.3.2. 2-[(1R,9aR)-Octahydro-2H-quinolizin-1-yl]-1-phenylbenz-imidazole (2)

Yield: 32%. Mp 102–103 �C (Et2O). 1H NMR (CDCl3): 0.86–2.20(m, 14H, Q); 2.44–2.68 (m, 1H, CH2-Q); 2.71–3.00 (m, 2Ha near Nof Q); 3.06–3.18 (m, 1H, CH2-Q); 7.10–7.67 (m, 8 arom. H); 7.78(d, J = 9.0, 1 arom. H). Anal. Calcd for C23H27N3: C, 79.96; H, 7.88;N, 12.16. Found: C, 80.09; H, 7.87; N, 12.23.

4.3.3. 2-[(2,6-Difluoro)phenyl]-1-[(1S,9aR)-(octahydro-2H-quinolizin-1-yl)methyl]-5-trifluoro-methylbenzimidazole (14)

Yield: 39%. Mp 118–121 �C (pentane). 1H NMR (CDCl3): 0.90–2.20 (m, 14H, Q); 2.63–3.18 (m, 2Ha near N of Q); 4.27–4.56 (m,2H, CH2-Q); 7.07–7.24 (m, 2 arom. H); 7.46–7.80 (m, 3 arom. H);8.16 (s, 1 arom. H). Anal. Calcd for C24H24F5N3+0.5H2O: C, 62.87;H, 5.50; N, 9.17. Found: C, 63.18; H, 5.81; N, 9.29.

4.3.4. 5-Chloro-2-[(1S,9aR)-octahydro-2H-quinolizin-1-yl]benzimidazole (61)

Yield: 45%. Mp 172–173 �C (toluene). 1H NMR (CDCl3): 1.15–2.42 (m, 14H, Q); 2.90–3.14 (m, 2Ha near N of Q); 3.24 (d, J = 6.0,2H, CH2-Q); 7.18 (d, J = 8.6, 1 arom. H); 7.40–7.58 (m, 2 arom. H);9.98 (s, NH-Benz., exchanges with D2O). Anal. Calcd for C17H22ClN3:C, 67.20; H, 7.30; N, 13.83. Found: C, 66.85; H, 6.95; N, 13.81.

4.3.5. 2-[(1R,9aR)-Octahydro-2H-quinolizin-1-yl]benzimidazole(62)

Yield: 42%. Mp 231–233 �C (CH2Cl2). 1H NMR (CDCl3): 1.10–2.40(m, 14H, Q); 2.63–2.78 (m, 1H, CH2-Q); 2.88–3.13 (m, 2Ha near N ofQ); 3.18–3.37 (m, 1H, CH2-Q); 7.17–7.30 (m, 2 arom. H); 7.48–7.65(m, 2 arom. H); 10.06 (s, NH-Benz., exchanges with D2O). Anal.Calcd for C17H23N3+ 0.25H2O: C, 74.56; H, 8.65; N, 15.34. Found:C, 74.85; H, 8.69; N, 15.57.

4.4. Benzimidazoles from acids. General method of Scheme 3

(a) A mixture of 4-aminophenylacetic acid (5.3 mmol) and 4-tri-fluoromethyl- or 4,5-dichloro-1,2-phenylenediamine (3.4 mmol)in 15 mL of 4 N HCl was refluxed for 10 h. After cooling, the solu-tion was neutralized with 2 N NaOH and extracted with Et2O.The dried organic layer (Na2SO4) was evaporated to afford a spongyresidue that was purified by CC and crystallized from dry Et2O.

(b) Tetrahydrocytisine, obtained by catalytic hydrogenation ofcytisine, 45a,b or D,L-lupanine (extracted from Lupinus albus seeds)were dissolved, respectively in 6 N HCl (1 g in 4 mL) and concdHCl (1 g in 3 mL). The former solution was refluxed for 6 h, whilethe latter required 72 h at 150 �C (closed tube).25 The acid concen-tration was adjusted with water to about 4 N, then the equimolaramount of the suitable 1,2-phenylenediamine was added and themixture refluxed for 10 h. After cooling the acidic solution wasworked up as in point (a).

4.4.1. 2-[4-(Amino)benzyl]-5-trifluoromethyl-1H-benzimidazole (49)

Yield: 58%. Mp 170–171 �C (Et2O). CC (Al2O3/Et2O). 1H NMR(DMSO-d6): 4.04 (s, 2H, CH2-Ar); 4.99 (s, NH2-Ar); 6.52 (d, J = 8.6,2 arom. H); 6.98 (d, J = 8.6, 2 arom. H); 7.44 (d, J = 8.2, 1 arom.H); 7.66 (d, J = 8.2, 1 arom. H); 7.83 (s, 1 arom. H); 12.58 (br. s,NH-Benz., exchanges with D2O). Anal. Calcd for C15H12F3N3: C,61.85; H, 4.15; N, 14.43. Found: C, 61.90; H, 4.18; N, 14.56.

4.4.2. 2-[(4-Amino)benzyl]-5,6-dichloro-1H-benzimidazole (55)Yield: 73%. Mp 113–115 �C (Et2O). 1H NMR (DMSO-d6): 3.37 (s,

NH2-Ar); 3.99 (s, 2H, CH2-Ar); 6.52 (d, J = 8.6, 2 arom. H); 6.98 (d,J = 8.4, 2 arom. H); 7.74 (s, 2 arom. H); 12.52 (br. s, NH-Benz., col-lapses with D2O). Anal. Calcd for C14H11Cl2N3+0.5H2O: C, 55.83; H,4.01; N, 13.95. Found: C, 55.59; H, 4.17; N, 13.75.

4.4.3. 2-[3-(3,7-Diazabicyclo[3.3.1]nonan-2-yl)propyl]-1H-benzimidazole (64)

Yield: 28%. Oil. 1H NMR (CDCl3): 1.12–2.40 (m, 8H and 1H, NHexchanges with D2O); 2.68–3.82 (m, 8H); 3.95–4.40 (m, 1H); 6.00(br. s, NH, exchanges with D2O); 7.10–7.30 (m, 2 arom. H); 7.43–7.72 (m, 2 arom. H); 9.96 (s, NH-Benz., collapses with D2O). Hydro-chloride, Mp 250 �C (EtOH/Et2O). Anal. Calcd for C17H24N4+3HCl: C,51.85; H, 6.91; N, 14.23. Found: C, 51.64; H, 7.25; N, 13.88.

4.4.4. 5-Chloro-2-[3-(3,7-diazabicyclo[3.3.1]nonan-2-yl)propyl]-1H-benzimidazole (65)

Yield: 27%. Oil. 1H NMR (CDCl3): 1.10–2.40 (m, 8H and 1H, NHexchanges with D2O); 2.72–3.88 (m, 8H); 4.00–4.42 (m, 1H); 5.60(br. s, NH, exchanges with D2O); 7.10 (dd, J = 8.6, 2.0, 1 arom. H);7.37–7.58 (m, 2 arom. H); 10.05 (s, NH-Benz., collapses with


D2O). Hydrochloride, Mp >250 �C (EtOH/Et2O). Anal. Calcd for C17-

H23ClN4+3HCl: C, 47.68; H, 6.12; N, 13.08. Found: C, 47.59; H,6.30; N, 12.76.

4.4.5. 4-[3-(1H-Benzimidazol-2-yl)propyl]decahydro-1H-1,5-methanopyrido[1,2-a] [1,5]diazocine (66)

Yield: 13%. Oil. 1H NMR (CDCl3): 0.96–2.20 (m, 15H); 2.50–3.00(m, 6H); 3.04–3.36 (m, 3H); 5.40 (br. s, NH exchanges with D2O);7.06–7.26 (m, 2 arom. H); 7.36–7.67 (m, 2 arom. H); 9.97 (s, NH-Benz., collapses with D2O). Hydrochloride, Mp >270 �C (EtOH/Et2O). Anal. Calcd for C21H30N4+3HCl+ H2O: C, 54.14; H, 7.57; N,12.03. Found: C, 54.00; H, 7.91; N, 11.46.

4.4.6. 4-[3-(5-Chloro-1H-benzimidazol-2-yl)propyl]decahydro-1H-1,5-methanopyrido[1,2-a] [1,5] diazocine (67)

Yield: 15%. Mp 113–116 �C (Et2O). 1H NMR (CDCl3): 1.00–2.20(m, 15H); 2.56–3.00 (m, 6H); 3.06–3.40 (m, 3H); 5.50 (br. s, NHexchanges with D2O); 7.14 (dd, J = 8.4, 1.8, 1 arom. H); 7.40–7.60(m, 2 arom. H); 10.00 (s, NH-Benz., collapses with D2O). Anal. Calcdfor C21H29ClN4: C, 67.63; H, 7.84; N, 15.02. Found: C, 67.53; H, 8.18;N, 15.09.

4.5. Benzimidazoles from acids. General method of Scheme 4

A mixture of 1,2-pheylenediamine or 4-chloro-1,2-phenylendi-amine (5 mmol) and 2-ethyl-2-hexenoic acid or tetrahydropyran-2-carboxylic acid (5 mmol) was heated in a sealed tube at 180 �Cfor 2 h. The residue was taken up with dil aq. HCl and filtered; thenthe acidic solution was washed with Et2O, made basic with 6 NNaOH and extracted with Et2O. The solvent was removed and theresidue crystallized from dry Et2O.

4.5.1. 2-(Hept-3-en-3-yl)-1H-benzimidazole (76)Yield: 51%. Mp 182–184 �C (Et2O). 1H NMR (CDCl3): 0.90 (t,

J = 6.4, 3H, C(7)); 1.02 (t, J = 6.4, 3H, C(1)); 1.44 (q, 2H, C(6)); 2.00(q, 2H, C(2)); 2.08–2.26 (m, 2H, C(5)); 5.80 (t, J = 7.0, 1H, C(4));7.16–7.28 (m, 2 arom. H); 7.40–7.68 (m, 2 arom. H); 10.12 (s,NH-Benz., exchanges with D2O). Anal. Calcd for C14H18N2: C,78.46; H, 8.47; N, 13.07. Found: C, 78.38; H, 8.60; N, 13.12.

4.5.2. 2-(Tetrahydropyran-2-yl)-5-trifluoromethyl-1H-benzimidazole (80)

Yield: 38%. Mp 213–215 �C (Et2O). 1H NMR (CDCl3): 1.68–1.94(m, 2H, C(5) and 1H, C(4), pyrane); 2.08–2.30 (m, 1H, C(4) and1H, C(3), pyrane); 2.52–2.73 (m, 1H, C(3), pyrane); 3.70–4.12 (m,2H, C(6)); 6.05 (dd, J = 8.4, 2.8, 1H, C(2)); 7.16 (d, J = 8.8, 1 arom.H); 7.40–7.56 (m, 2 arom. H); 10.82 (s, NH-Benz., exchanges withD2O). Anal. Calcd for C13H13F3N2O: C, 57.78; H, 4.85; N, 10.37.Found: C, 58.00; H, 5.01; N, 10.42.

4.5.3. 5-Chloro-2-(tetrahydropyran-2-yl)-1H-benzimidazole (81)Yield: 33%. Mp 166–167 �C (Et2O). 1H NMR (CDCl3): 1.62–1.95

(m, 2H, C(5) and 1H, C(4), pyrane); 2.05–2.32 (m, 1H, C(4) and1H, C(3), pyrane); 2.46–2.69 (m, 1H, C(3), pyrane); 3.75–4.20 (m,2H, C(6)); 6.05 (dd, J = 8.6, 2.8, 1H, C(2)); 7.26 (d, J = 9.0, 1 arom.H); 7.46–7.67 (m, 2 arom. H); 12.28 (s, NH-Benz., exchanges withD2O). Anal. Calcd for C12H13ClN2O: C, 60.89; H, 5.54; N, 11.84.Found: C, 60.68; H, 5.60; N, 11.72.

4.6. Benzimidazoles from aldehyde-sodium bisulfite adducts.General method of Scheme 5

To a solution of the proper N1-alkyl- or N1-(dialkylamino-alkyl)-13 or N1-[(1R,9aR)-(octahydroquinolizin-1-yl)methyl]-4-trifluoromethyl-1,2-phenylenediamine (3.5 mmol) in 18 mL ofEtOH the 4-methoxybenzaldheyde sodium bisulfite adduct

(3.5 mmol) (prepared according to Shriner and Land)46 was addedand the mixture was refluxed for 3 h with stirring. Afterwards, thesolvent was evaporated and the residue was taken up with waterand filtered, or extracted with Et2O. The solid compounds werecrystallized from dry Et2O; the oily 17 was converted into themonohydrochloride with 1 N ethanolic solution of HCl.

4.6.1. 1-[(2-Dimethylamino)ethyl]-2-(4-methoxyphenyl)-5-trifluoromethylbenzimidazole (4)

Yield: 64%. Mp 112–113 �C (Et2O). 1H NMR (CDCl3): 2.32 (s, 6H,N(CH3)2); 2.77 (t, J = 7.1, 2H, CH2CH2N(CH3)2)); 3.92 (s, OCH3); 4.48(t, J = 7.3, 2H, CH2CH2N(CH3)2); 7.08 (d, J = 8.8, 2 arom. H); 7.59 (s, 2arom. H); 7.08 (d, J = 8.8, 2 arom. H); 8.09 (s, 1 arom. H). Anal. Calcdfor C19H20F3N3O: C, 62.80; H, 5.55; N, 11.56. Found: C, 63.10; H,5.71; N, 11.72.

4.6.2. 1-[(3-Diethylamino)propyl]-2-(4-methoxyphenyl)-5-trifluoromethylbenzimidazole (5)

Yield: 64%. Mp 107–109 �C (Et2O). 1H NMR (CDCl3): 1.05 (t,J = 7.2, 6H, N(CH2CH3)2); 1.94–2.16 (m, 2H, CH2CH2CH2N(C2H5)2);2.41–2.77 (m, 6H, CH2CH2N(CH2CH3)2)); 3.93 (s, OCH3); 4.14 (t,J = 7.3, 2H, CH2CH2CH2N(C2H5)2); 7.08 (d, J = 8.6, 2 arom. H); 7.57(s, 2 arom. H); 7.72 (d, J = 8.4, 2 arom. H); 8.09 (s, 1 arom. H). Anal.Calcd for C22H26F3N3O: C, 65.17; H, 6.46; N, 10.36. Found: C, 65.51;H, 6.70; N, 10.50.

4.6.3. 4-(Methoxyphenyl)-1-[(1R,9aR)-octahydro-2H-quinolizin-1-yl]-5-trifluoromethyl-benzimidazole (17)

Yield: 35%. Oil. 1H NMR (CDCl3): 0.86–2.23 (m, 14H, Q); 2.42–2.64 (m, 1H, CH2-Q); 2.67–3.00 (m, 2Ha near N of Q); 3.05–3.16(m, 1H, CH2-Q); 3.87 (s, OCH3); 7.05 (d, J = 8.8, 2 arom. H); 7.62–7.94 (m, 4 arom. H); 8.08 (s, 1 arom. H). Hydrochloride, mp>178–202 �C (EtOH/Et2O). Anal. Calcd for C25H29ClF3N3O+HCl+H2-

O: C, 60.30; H, 6.43; N, 8.44. Found: C, 60.26; H, 6.13; N, 8.36.

4.6.4. 1-Butyl-2-(4-methoxyphenyl)-5-trifluoromethylbenzimidazole (18)

Yield: 48%. Mp 81–82 �C (Et2O/pentane 1:1). 1H NMR (CDCl3):0.92 (t, J = 7.4, 3H, CH2CH2CH2CH3); 1.24–1.42 (m, 2H, CH2CH2CH2-

CH3); 1.76–1.95 (m, 2H, CH2CH2CH2CH3); 3.93 (s, OCH3); 4.32 (t,J = 7.8, 2H, CH2CH2CH2CH3); 7.10 (d, J = 9.0, 2 arom. H); 7.44–7.64(m, 2 arom. H); 7.73 (d, J = 9.0, 2 arom. H); 8.16 (s, 1 arom. H). Anal.Calcd for C19H19F3N2O: C, 65.50; H, 5.50; N, 8.04. Found: C, 66.43;H, 5.42; N, 7.83.

4.6.5. 1-[(2-Methoxy)ethyl]-2-(4-methoxyphenyl)-5-trifluoromethylbenzimidazole (19)

Yield: 52%. Mp 110–112 �C (Et2O). 1H NMR (CDCl3): 3.32 (s, 3H,CH2CH2OCH3); 3.82 (t, J = 5.4, 2H, CH2CH2OCH3); 3.92 (s, OCH3);4.49 (t, J = 5.4, 2H, CH2CH2OCH3); 7.09 (d, J = 9.0, 2 arom. H);7.55–7.69 (m, 2 arom. H); 7.84 (d, J = 8.8, 2 arom. H); 8.16 (s, 1arom. H). Anal. Calcd for C18H17F3N2O2: C, 61.71; H, 4.89; N, 8.00.Found: C, 61.49; H, 4.63; N, 8.00.

4.7. Acetylation of 2-(4-aminobenzyl)benzimidazoles. Generalmethod of Scheme 6

A suspension of the 2-[(4-amino)benzyl]benzimidazole 4520 orof the above 2-[(4-amino)benzyl]-5-trifluoromethyl- or 5,6-dichlo-robenzimidazoles (49, 55) (1.2 mmol) in 2 mL of anh. benzene and3.6 mmol of Ac2O was refluxed for 2 h. After removing the solvent,the residue was taken up with 5 mL of EtOH plus 1 mL of water andrefluxed for 2 h, in order to hydrolyze any acetyl groups attached tobenzimidazole nitrogen. The solvent was evaporated under vac-uum and the residue taken up with water, filtered and washedwith water. The compounds were crystallized from EtOH/H2O.


4.7.1. 2-[4-(Acetylamino)benzyl]-1H-benzimidazole (46)Yield: 63%. Mp 243–245 �C (EtOH/H2O). 1H NMR (CDCl3): 2.19

(s, CH3CO); 4.56 (s, 2H, CH2-Ar); 7.16–7.55 (m, 6 arom. H); 7.75–8.20 (m, 2 arom. H); 9.88 (s, NHCO, collapses with D2O); 12.28(br. s, NH-Benz., collapses with D2O). Anal. Calcd for C16H15N3O:C, 72.43; H, 5.70; N, 15.84. Found: C, 72.64; H, 5.94; N, 16.09.

4.7.2. 2-[4-(Acetylamino)benzyl]-5-trifluoromethyl-1H-benzimidazole (50)

Yield: 82%. Mp 240–241 �C (EtOH/H2O). 1H NMR (DMSO-d6):2.09 (s, CH3CO); 4.19 (s, 2H, CH2-Ar); 7.26 (d, J = 8.2, 2 arom. H);7.41–7.60 (m, 2 arom. H); 7.67 (d, J = 8.2, 2 arom. H); 7.85 (s, 1arom. H); 9.94 (s, NHCO, collapses with D2O); 12.68 (br. s, NH-Benz., collapses with D2O). Anal. Calcd for C17H14F3N3O+0.25H2O:C, 60.45; H, 4.32; N, 12.43. Found: C, 60.09; H, 4.14; N, 12.50.

4.7.3. 2-[(4-Acetylamino)benzyl]-5,6-dichloro-1H-benzimidazole (56)

Yield: 75%. Mp 270–271 �C (EtOH/H2O). 1H NMR (DMSO-d6):2.03 (s, CH3CO); 4.14 (s, 2H, CH2-Ar); 7.24 (d, J = 8.4, 2 arom. H);7.53 (d, J = 8.2, 2 arom. H); 7.80 (s, 2 arom. H); 9.93 (s, NHCO, col-lapses with D2O); 12.57 (br. s, NH-Benz., collapses with D2O). Anal.Calcd for C16H13Cl2N3O+ 0.25H2O: C, 56.74; H, 4.02; N, 12.41.Found: C, 56.64; H, 3.93; N, 12.26.

4.8. 2-[(4-Bromo)benzyl]-5,6-dichloro-1-[(1S,9aR)-(octahydro-2H-quinolizin-1-yl)methyl)]-benzimidazole (36). (Scheme 7)

A solution of 3.5 mmol of the above described 2-[4-(bromo)-benzyl]-5,6-dichlorobenzimidazole (53) in anhydrous DMF (6 mL)was treated with finely ground NaNH2 (200 mg) and stirred for30 min. at 45 �C under a stream of dry N2. Then a solution of chlo-rolupinane26 (3.8 mmol) in DMF (3 mL) was added and the mixturewas heated at 140 �C for 3 h. The solvent was removed under vac-uum and the residue was taken up with diluted aqueous NaOH andextracted with Et2O. The dried (Na2SO4) organic solution was evap-orated and the spongy residue was crystallized from dry Et2O.

Yield: 49%. Mp 125–127 �C (Et2O). 1H NMR (CDCl3): 1.20–2.44(m, 14H, Q); 2.82–3.28 (m, 2Ha near N of Q); 4.09–4.52 (m, 2H,CH2-Q and, 4.30, s, 2H, CH2-Ar, superimposed); 7.07–7.70 (m, 5arom. H); 7.84 (s, 1 arom. H). Anal. Calcd for C24H26BrCl2N3: C,56.82; H, 5.17; N, 8.28. Found: C, 56.76; H, 5.24; N, 8.23.

4.9. 2-{[(1R,9aR)-(octahydro-2H-quinolizin-1-yl)methyl]thio}-1H-benzimidazole (63). (Scheme 8)

Commercially available 2-chloro-1H-benzimidazole (2.0 mmol)was dissolved in 5 mL of CH3CN in an Aldrich pressure tube. Asolution of (1R,9aR)-(octahydro-2H-quinolizin-1-yl)methanthiole(thiolupinine, 2.0 mmol)27 in 1 mL of CH3CN was rapidly added;the tube was flushed with dry N2 and thoroughly closed. The mix-ture was heated at 100 �C for 4 h. After evaporation of the solvent,the residue was crystallized from a mixture of CH3CN/dry Et2O(2:1).

Yield: 67%. Mp 172–173 �C (CH3CN/Et2O 2:1). 1H NMR (CDCl3):0.92–2.38 (m, 14H, Q); 2.71–3.06 (m, 2Ha near N of Q); 3.26–3.82(m, 2H, CH2-S); 7.04–7.37 (m, 3 arom. H); 7.41–7.72 (m, 1 arom. Hsuperimposed with NH, that collapses with D2O). Anal. Calcd forC17H23N3S: C, 67.74; H, 7.69; N, 13.94. Found: C, 67.50; H, 7.63;N, 13.91.

4.10. Benzoimidazo[1,2-c][1,2,3]benzotriazines. (Scheme 9)

To a suspension of 2-[(2-amino-4-methoxy)phenyl]-5-trifluo-romethylbenzimidazole or 2-[(2-amino-4-methoxy)phenyl]-5,6-dichlorobenzimidazole4 (2.5 mmol) in 7 mL of 2 N HCl, cooled

in an ice bath, a solution of sodium nitrite (2.7 mmol) in 4 mL ofH2O was added. After 30 min of stirring, the precipitate was col-lected and washed with water. The solid, dissolved in EtOH, wasdecolorized with activated charcoal. Concentration of the ethanolicsolution afforded the crystalline compound.

4.10.1. 3-Methoxy-9/10-(trifluoromethyl)benzo[e]benzo[4,5]imidazo[1,2-c][1,2,3]triazine (24)

Yield: 58%. Mp 171–173 �C (EtOH). 1H NMR (CDCl3): 4.10 (s,OCH3); 7.64 (dd, J = 8.8, 2.4, 1 arom. H); 7.96–7.83 (m, 2 arom.H); 8.09 (d, J = 8.6, 1 arom. H); 8.61 (d, J = 8.8, 1 arom. H); 8.70(d, J = 2.4, 1 arom. H). The splotted signals indicate the presenceof two unresolvable isomers in the ratio 10:1. Anal. Calcd for C15H9-

F3N4O: C, 56.61; H, 2.85; N, 17.60. Found: C, 56.46; H, 2.71; N,17.59.

4.10.2. 9,10-Dichloro-3-methoxybenzo[e]benzo[4,5]imidazo[1,2-c][1,2,3]triazine (25)

Yield: 65%. Mp 255–257 �C (dioxane). 1H NMR (CDCl3): 4.10 (s,OCH3); 7.62 (dd, J = 9.0, 2.4, 1 arom. H); 7.88 (d, J = 2.4, 1 arom. H);8.09 (s, 1 arom. H); 8.49 (s, 1 arom. H); 8.57 (d, J = 9.0, 1 arom. H).Anal. Calcd for C14H8Cl2N4O: C, 52.69; H, 2.53; N, 17.56. Found: C,52.86; H, 2.77; N, 17.40.

4.11. 2-Nitro-N-[(1R,9aR)-(octahydro-2H-quinolizin-1-yl)methyl]-4-trifluoromethyl-benzeneamine monohydrochloride

A solution of [(1R,9aR)-(octahydro-2H-quinolizin-1-yl)metha-n]amine (epi-lupinylamine)28 (15 mmol) and 4-chloro-3-nitro-benzotrifluoride (15 mmol) in DMF (3 mL) was heated withstirring (90 min; 170�) in a pressure tube (Aldrich). At rt the resi-due was taken up with dry Et2O affording a crystalline solid (asmonohydrochloride) that was collected by filtration and washedthoroughly with dry Et2O.

Yield: 88%. Mp 220–224 �C. 1H-NMR (CDCl3): 1.04–2.08 (m,14H, Q); 2.66–2.84 (m, 2Ha near N of Q); 2.97–3.13 (m, 1H, CH2-Q); 3.25–3.40 (m, 1H of CH2-Q); 6.72 (d, J = 9.2, 1 arom. H,); 7.29(dd, J = 9.4, 2.4, 1 arom. H); 8.04 (s, NH, exchanges with D2O);8.10 (d, J = 2.6, 1 arom. H). Anal. Calcd for C17H22F3N3O2+HCl: C,51.84; H, 5.89; N, 10.67. Found: C 51.66, H 5.96, N 10.49.

4.12. N-[(1R,9aR)-(Octahydro-2H-quinolizin-1-yl)methyl]-4-trifluoromethyl-1,2-phenylenediamine monohydrochloride

A solution of the above nitrocompound (10 mmol) in EtOH(50 mL) was hydrogenated at rt and atmospheric pressure in thepresence of 10% Pd/C (0.4 g). After 1 h the calculated volume ofH2 was absorbed. The catalyst was removed and the solvent wasevaporated under vacuum, leaving the 1,2-phenylenediaminemonohydrochloride that was crystallized by absolute EtOH/dryEt2O and was used as such for the preparation of benzimidazolederivatives (17, 42–44).

Yield: 92%. Mp 160–163 �C (EtOH/Et2O). 1H-NMR (CDCl3): 1.04–2.06 (m, 14H, Q); 2.68–2.86 (m, 2Ha near N of Q); 3.06–3.18 (m, 2H,CH2-Q); 3.30 (s, 3H, NH2 and NH, exchange with D2O); 6.40–6.72(m, 3 arom. H). Anal. Calcd for C17H24F3N3+HCl: C, 56.12; H, 6.93;N, 11.55. Found C 56.00, H 6.67, N 11.70.

4.13. Cell-based assays

4.13.1. CompoundsCompounds were dissolved in DMSO at 100 mM and then

diluted in culture medium. The final DMSO concentration did notexceed 1% (commonly 0.1%) which did not affect the biologicalassay results.


4.13.2. Cells and virusesCell lines were purchased from American Type Culture Collec-

tion (ATCC). The absence of mycoplasma contamination waschecked periodically by the Hoechst staining method. Cell linessupporting the multiplication of RNA and DNA viruses were thefollowing: CD4+ human T-cells containing an integrated HTLV-1genome (MT-4); Madin-Darby Bovine Kidney (MDBK) [ATCC CCL22 (NBL-1) Bos Taurus]; Baby Hamster Kidney (BHK-21) [ATCCCCL 10 (C-13) Mesocricetus auratus] and Monkey kidney (Vero-76) [ATCC CRL 1587 Cercopithecus Aethiops]. Viruses werepurchased from American Type Culture Collection (ATCC) exceptYellow Fever Virus (YFV), and Human Immunodeficiency Virustype-1 (HIV-1). Viruses representative of positive-sense single-strand RNA (ssRNA+) group used were: (i) Retroviridae family:the laboratory strain HIV-1 IIIB wild-type, obtained from thesupernatant of the persistently infected H9/IIIB cells (NIH 1983);(ii) Flaviviridae family: YFV [strain 17-D vaccine (Stamaril PasteurJ07B01)] and Bovine Viral Diarrhea Virus (BVDV) [strain NADL(ATCC VR-534)] (title compounds were evaluated in vitro for anti-viral activity against viruses representative of only two of the threegenera of the Flaviviridae family, that is, Flaviviruses (Yellow FeverVirus, YFV) and Pestiviruses (Bovine Viral Diarrhea Virus, BVDV), asHepaciviruses can hardly be used in routine cell-based assays); (iii)Picornaviridae family: Human Coxsackievirus type B5 (CVB-5)strain (ATCC� VR-1036AS/HO™) and Human Poliovirus type-1Sabin (Sb-1) [strain Chat (ATCC VR-1562)]. Virus representativeof a negative-sense single-strand RNA (ssRNA�) group used were:Vesicular Stomatitis Virus (VSV) [strain Indiana Lab (ATCCVR-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, sim-ian virus 12) (ATCC VR-214)]. Viruses representatives of DNA groupused were: (i) Poxviridae family: Vaccinia Virus (VV) [strain Els-tree-Lister Vaccine (ATCC VR-1549)]; (ii) Herpesviridae family:Human Herpesvirus 1 (HSV-1) [strain KOS (ATCC VR-1493)].

As reference inhibitors were used: EFV (efavirenz) for HIV-1only, NM 108 (20-C-methyl-guanosine), NM 176 (20-C-ethynylcyti-dine) and ribavirin for the other ssRNA+ and dsRNA viruses; NM299 (6-azauridine), ribavirin and M 5255 (mycophenolic acid) forthe ssRNA� viruses; M 5255 and ACG (acyclovir) for DNA viruses.

4.13.3. Cytotoxicity assaysCytotoxicity assays were run in parallel with antiviral assays.

Exponentially growing MT-4 cells were seeded at an initial densityof 1 � 105 cells/mL in 96-well plates in RPMI-1640 mediumsupplemented with 10% fetal bovine serum (FBS), 100 units/mLpenicillin G and 100 lg/mL streptomycin. Cell cultures were thenincubated at 37 �C in a humidified, 5% CO2 atmosphere in theabsence or presence of serial dilutions of test compounds. Cell via-bility was determined after 96 hrs at 37 �C by the 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) method.47

MDBK and BHK cells were seeded at an initial density of 6 � 105

and 1 � 106 cells/mL in 96-well plates, respectively, in culturemedium (Minimum Essential Medium with Earle’s salts (MEM-E)with L-glutamine, supplemented with 10% horse serum and1 mM sodium pyruvate (for MDBK cells) or with 10% fetal bovineserum (FBS) (for BHK cells), 0.025 g/L kanamycin). Cell cultureswere then incubated at 37 �C in a humidified, 5% CO2 atmospherein the absence or presence of serial dilutions (from 100 lM, ratio1:5) of test compounds. Cell viability was determined after48–96 h at 37 �C by the MTT method. Vero-76 cells were seededat an initial density of 4 � 105 cells/mL in 24-well plates, in culturemedium (Dulbecco’s Modified Eagle Medium (D-MEM) withL-glutamine, supplemented with fetal bovine serum (FBS),0.025 g/L kanamycin). Cell cultures were then incubated at 37 �Cin a humidified, 5% CO2 atmosphere in the absence or presence

of serial dilutions of test compounds. Cell viability was determinedafter 48–96 h at 37 �C by the Crystal violet staining method.

4.13.4. Antiviral assaysCompounds activity against HIV-1 was based on inhibition of

virus-induced cytopathogenicity in MT-4 cells acutely infectedwith a multiplicity of infection (m.o.i.) of 0.01. Briefly, 50 lL ofRPMI containing 1 � 104 MT-4 cells were added to each well, offlat-bottom microtiter trays, containing 50 lL of RPMI, withoutor with serial dilutions (from 100 lM, ratio 1:5) of test compounds.Then, 20 lL of a HIV-1 suspension containing 100 CCID50 wereadded. After a 4-days incubation at 37 �C, cell viability was deter-mined by the MTT method. Compounds activity against YFV andReo-1 was based on inhibition of virus-induced cytopathogenicityin BHK-21 cells acutely infected with a m.o.i. of 0.01. Compoundsactivity against BVDV was based on inhibition of virus-inducedcytopathogenicity in MDBK cells acutely infected with a m.o.i. of0.01. Briefly, BHK and MDBK cells were seeded in 96-well platesat a density of 5 � 104 and 3 � 104 cells/well, respectively, andwere allowed to form confluent monolayers by incubating over-night in growth medium at 37 �C in a humidified CO2 (5%) atmo-sphere. Cell monolayers were then infected with 50 lL of aproper virus dilution in maintenance medium (MEM-E with L-glu-tamine, supplemented with 0.5% inactivated FBS, 1 mM sodiumpyruvate and 0.025 g/L kanamycin) to give a m.o.i of 0.01. After2 h, 50 lL of maintenance medium, without or with serial dilutions(from 100 lM, ratio 1:5) of test compounds, were added. After a3–4 days incubation at 37 �C, cell viability was determined by theMTT method.

Compounds activity against CVB-5, Sb-1, VSV, VV, HSV-1 andRSV was determined by plaque reduction assays in infectedVero-76 cell monolayers. To this end, Vero-76 cells were seededin 24-well plates at a density of 2 � 105 cells/well and wereallowed to form confluent monolayers by incubating overnight ingrowth medium (Dulbecco’s Modified Eagle Medium (D-MEM)with L-glutamine and 4500 mg/L D-glucose, supplemented with10% FBS and 0.025 g/L Kanamycin) at 37 �C in a humidified CO2

(5%) atmosphere. Then, monolayers were infected for 2 h with250 lL of proper virus dilutions to give 50–100 PFU/well. Follow-ing removal of unadsorbed virus, 500 lL of maintenance medium(D-MEM medium with L-glutamine and 4500 mg/L D-glucose sup-plemented with 1% inactivated FBS and 0.75% methyl-cellulose),without or with serial dilutions (from 100 lM, ratio 1:5) of testcompounds, 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 thenfixed with PBS containing 50% ethanol and 0.8% crystal violet,washed and air-dried. Plaques were then counted and EC50 (50%effective concentration) was calculated by linear regression tech-nique. The cytotoxicity of test compounds was determined in par-allel on the same 24-well plate used for the EC50 determination.

Linear regression analysis: viral and cell growth at each drugconcentration was expressed as percentage of untreated controlsand concentrations resulting in 50% (EC50 and CC50) growth inhibi-tion were determined by linear regression analysis.

Supplementary data

Supplementary data (cytotoxicity and antiviral activities of all86 tested benzimidazole derivatives and reference compoundsare depicted in Tables 1S-3S) associated with this article can befound, in the online version, at http://dx.doi.org/10.1016/j.bmc.2014.06.043.

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Antiviral activity of benzimidazole derivatives. II. Antiviral activity of 2-phenylbenzimidazole...

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