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

Bioorganic & Medicinal Chemistry 18 (2010) 2937–2953

Contents lists available at ScienceDirect

Bioorganic & Medicinal Chemistry

journal homepage: w ww.elsevier.com/locate/bmc

Antiviral activity of benzimidazole derivatives. II. Antiviral activity of

2-phenylbenzimidazole derivatives ^q

a

Michele Tonelli ^a,, Matteo Simone , Bruno Tasso , Federica Novelli , Vito Boido , Fabio Sparatore ,

*

Giuseppe Paglietti ^b, Sabrina Pricl ^c, Gabriele Giliberti ^d, Sylvain Blois ^d, Cristina Ibba ^d,

Giuseppina Sanna ^d, Roberta Loddo ^d, Paolo La Colla ^d,

*

^aDipartimento di Scienze Farmaceutiche, Università di Genova, Viale Benedetto XV 3, 16132 Genova, Italy

^bDipartimento Farmaco Chimico Tossicologico, Università di Sassari, Via Muroni, 23a, 07100 Sassari, Italy

^cDipartimento di Ingegneria Chimica, dell’Ambiente e delle Materie prime, Università di Trieste, Piazzale Europa 1, 34127 Trieste, Italy

^dDipartimento di Scienze e Tecnologie Biomediche, Università di Cagliari, Cittadella Universitaria, 09042 Monserrato (Cagliari), Italy

a r t i c l e i n f o

a b s t r a c t

Article history:

Seventy-six 2-phenylbenzimidazole derivatives were synthesized and evaluated in cell-based assays for

cytotoxicity and antiviral activity against a panel of 10 RNA and DNA viruses. The most commonly

affected viruses were, in decreasing order, CVB-2, BVDV, Sb-1, HSV-1, and YFV, while HIV-1 and VSV were

not affected, and RSV, VV and Reo-1 were only susceptible to a few compounds. Thirty-nine compounds

Received 4 December 2009

Revised 17 February 2010

Accepted 21 February 2010

Available online 4 March 2010

exhibited high activity (EC₅₀= 0.1–10

for their high and selective activity against VV (24, EC₅₀= 0.1

EC₅₀= 1.5, 0.8, and 1.0 M, respectively). The last compounds inhibited at low micromolar concentrations

lM) against at least one virus, and four of them were outstanding

l

M) and BVDV (50, 51, and 53 with

In memory of Anna Bennicelli

l

the NS5B RdRp of BVDV and also of HCV, the latter sharing structural similarity with the former. The con-

sidered compounds represent attractive leads for the development of antiviral agents against poxviruses,

pestiviruses and even HCV, which are important human and veterinary pathogens.

Keywords:

2-Phenylbenzimidazole derivatives

Antiviral activity

RNA and DNA viruses

NS5B RdRp inhibition

1. Introduction

1.69 l

M against Coxsackie Virus B3,⁵while 1-cyclohexyl-2-hetero-

arylbenzimidazole-5-carboxylic acids and their amido derivatives

inhibit the HCV NS5B polymerase at nanomolar concentrations.^6,7

Several derivatives of 1-cyclohexyl-2-phenylbenzimidazole-5-car-

boxylic acid behave similarly; among them, 2-{4-[4⁰-chloro-4-(2-

oxopyrrolidin-1-yl)biphenyl-2-ylmethoxy]-2-ﬂuorophenyl}-1-cyclo-

hexyl-1H-benzimidazol-5-carboxylic acid (JTK-109) inhibits the

HCV polymerase with IC₅₀= 17 nM, and the replication of subge-

Benzimidazole is isosteric with indole and purine nuclei, which

are present in a number of fundamental cellular components and

bioactive compounds.¹This heterocycle may represent a kind of

privileged substructure, which may interact with different proteins

and enzymes. Indeed, a number of important drugs used in different

therapeutic areas contain the benzimidazole ring,²as proton pump

inhibitors (omeprazole), antihypertensives (candesartan, telmisar-

tan), antihistaminics (astemizole), antihelmintics (albendazole,

mebendazole), as well as several other kinds of still investigational

therapeutic agents, including antitumorals and antivirals.^3,4

Among the antiviral benzimidazoles, an important position is

held by 2-aryl/heteroarylbenzimidazole derivatives, either en-

dowed with activity against Coxsackie Virus B3 or targeting spe-

ciﬁc hepatitis C virus enzymes. Thus, N-(2-ﬂuorophenyl)-2-(2-

pyridyl)benzimidazol-4-carboxamide shows an IC₅₀value of

nomic HCV RNA in a replicon cell system with EC₅₀= 0.32

Lastly, N,N⁰-bis[4-(2-benzimidazolyl)phenyl]-

-alkandicarboxa-

mides inhibit the HCV helicase with an IC₅₀= 0.7

M⁹(see Fig. 1).

l

M.⁸

ax

l

It is worth noting that derivatives of 2-phenylbenzimidazole-4-car-

boxamide and of 2-(4-acyl/aroylaminophenyl)benzimidazole, closely

related to some of the above antivirals, present a growing interest as

anticancer agents. Indeed, the former¹⁰inhibit the DNA repair enzyme

(PARP) and synergistically increase the cytotoxicity of temozolamide

and topotecan, while the latter¹¹potently inhibit the heparanase with

consequent angiogenetic and antimetastatic activity.

Our interest in the chemistry and biological activities of benz-

imidazole derivatives dates back to the early sixties; since then, hun-

dreds of compounds endowed with several pharmacological

activities have been described. Recently, 1-substituted-2-[(ben-

q

For note I, see Ref. 4.

* Corresponding authors. Tel.: +39 010 3538378; fax: +39 010 3538358 (M.T.);

tel.: +39 070 6754147; fax: +39 070 6754210 (P.L.).

E-mail addresses: michele.tonelli@unige.it (M. Tonelli), placolla@unica.it (P. La

Colla).

doi:10.1016/j.bmc.2010.02.037

2938

M. Tonelli et al. / Bioorg. Med. Chem. 18 (2010) 2937–2953

R'

R

O

C

NH

N

O

C

Heteroaryl

N

X

N

H

Y

X = N ; Y = CN; R = H, 2-Me, 3-Br

X = CH; Y = N; R = H, 2-F

(Coxsackie virus B3)

Heteroaryl = furan-3-yl; pyridin-2-yl

R’ = OH, NH-R”

(HCV NS5B polymerase)

O

N

OH

C

F

N

O

NH CO CH₂

N

H

2

Cl

JTK-109

(HCV NS5B-polymerase)

(HCV replicon)

(HCV helicase)

O

NH₂

C

R'

R

N

NH

NH CO

R"

N

H

N

H

N

H

R'

(PARP)

(heparanase)

Figure 1. 2-Aryl/heteroaryl-benzimidazoles with antiviral or antitumoral activity (and corresponding target).

zotriazol-1/2-yl)methyl]benzimidazoles have demonstrated to po-

tently inhibit the multiplication of RSV (Respiratory Syncytial Virus)

in in vitro assays, with EC₅₀values as low as 20 nM.⁴Moreover, in a

preliminaryscreeningof asetof2-phenylbenzimidazolespreviously

prepared with other pharmacological aims,¹²the simple 2-(4-

methoxyphenyl)-5-triﬂuoromethylbenzimidazole proved to inhibit

the multiplication of BVDV (bovine viral diarrhea virus) and CVB-2

2. Chemistry

Fifteen of the 76 tested compounds (1–4, 9, 20, 24, 25, 29, 30,

33, 34, 50–52) were already known, and were prepared according

to the references indicated in Supplementary data.

The remaining 61 novel compounds were synthesized accord-

ing to Schemes 1–5. Three different approaches were followed

for the benzimidazole ring closure reaction, namely:

(Coxsackie virus type B2), with EC₅₀values of 1

respectively.

lM and 7 lM,

ꢀ Method a:¹³the appropriate diamino compounds and aldehydes

were reacted in the presence of FeCl₃/O₂; in one case (13), adding

chloranil was found useful.

Basing ourselves on this, we found it interesting to investigate

the antiviral activity of a larger number of 2-phenylbenzimida-

zole derivatives. On the whole, 76 compounds, incorporating

several kinds of functionalities, have been evaluated in cell-

based assays for cytotoxicity and antiviral activity against a pa-

nel of ten RNA and DNA viruses (see Fig. 2), in order to select

hits active against individual viruses to be developed later on.

In several compounds a nitro group is incorporated, in position

5 of the benzimidazole nucleus or in the 2-phenyl substituent,

assuming that it could improve the binding on viral proteins

through hydrogen bonds formation, and/or its electron with-

ꢀ Method b:¹⁴the diamino compounds were condensed with the

aldehyde–sodium bisulﬁte addition compounds, which were pre-

pared extemporaneously as indicated by Shriner and Land.¹⁵

ꢀ Method c: compound 7 was obtained by Phillips cyclization¹⁶of

the amido compound formed by reaction of the diamine and acyl

chloride.

The required diamino compounds F, with Z = H, were commer-

cially available (R = R⁰= H; R = H, R⁰= NO₂; R = R⁰= Cl), or prepared

according to the literature (R = H, R⁰= CF₃¹⁷and COCH₃¹⁸), as were

those with R = H, R⁰= CF₃, and Z = Me¹⁹or cyclohexyl.²⁰

drawing properties, which may result in

p-interactions between

the electron deﬁcient ring of the drug and the electron rich ring

of an aromatic aminoacid.

Indeed, despite some potential for toxicity, the nitro group dis-

plays fundamental role in the pharmacophore of several classes of

chemotherapeutic agents, and of CNS, cardiovascular, anti-inﬂam-

matory and anti-androgen drugs.

The diamino compound with Z = 1-adamantyl, R = H, and

R⁰= CF₃was unknown and was prepared by reacting 2-nitro-4-(tri-

ﬂuoromethyl)chlorobenzene with 1-adamantylamine, followed by

the catalytic reduction of the nitro group (see Scheme 1).

M. Tonelli et al. / Bioorg. Med. Chem. 18 (2010) 2937–2953

2939

N

R"

NH R"'

R'

N

H

N

H

A (1 - 25)

C (29 - 58)

CF₃

N

NO₂

NH R"'

N

Z

N

Z

B (26 - 28)

D (59 - 76)

Z = Me; Cyclohexyl; 1-Adamantyl

Figure 2. Structures of the investigated 2-phenylbenzimidazole derivatives.

R₂

R₃

R₁

R

NO₂

R₁

N

a

R₄

N

Z

Cl

R

R₆

R₅

A: 12, 15, 22; B: 26-28; C: 37-53

R₂

R₃

R₁

R

NO₂

R₁

R

NH₂

R₁

N

b

R₄

N

H

NH

Z

NH

Z

R

E

F

A: 5, 6, 8, 10, 13, 14, 16-19, 21

A: and C: Z= H

B: Z= Me, Cyclohexyl, 1-Adamantyl

OCH₃

F₃C

N

c, d

R= H, R₁= H, CF , NO₂, COCH₃;

3

N

R= R₁= Cl

OCH₃

H

A: 7

Scheme 1. Reagents and conditions: (a) arylaldehyde, MeCN, FeCl₃/O₂, reﬂux; (b) arylaldehyde–sodium bisulﬁte, EtOH, 3 h reﬂux; (c) aroylchloride, dioxane, 7 h reﬂux; (d)

4 N HCl, 4 h reﬂux.

F₃C

N

a

NO₂

NH₂

N

Z

N

Z

A and C: Z= H

B and D: Z= Me, Cyclohexyl,

C: 36; D: 59, 69, 72

A: 12; B: 26-28

1-Adamantyl

NH₂

N

NO₂

R₁

R

N

a/b

OCH₃

N

H

N

H

R

A: 11, 23

A: 10, 22

R= H; R₁= CF₃; R= R₁= Cl

Scheme 2. Reagents and conditions: (a) H₂/Pd–C, EtOH, rt; (b) for R = R1 = Cl, SnCl₂+ concd HCl, EtOH, 1 h reﬂux.

The nitro-substituted benzimidazole derivatives were con-

The 2-(4-aminophenyl)benzimidazoles were acylated with the

appropriate anhydride (acetic, propionic, succinic, see Scheme 3)

or acylchloride (see Scheme 4). It is worth noting that the treat-

ment of 2-(4-aminophenyl)-5-triﬂuoromethylbenzimidazole with

ethyl chloroformate, instead of the expected ethyl carbamate, gave

verted into the corresponding amino compounds by catalytic

hydrogenation. However, compound 22 was better reduced by

using SnCl₂and concentrated HCl, thus avoiding the hydrogenoly-

sis of chlorine on the aromatic ring (see Scheme 2).

2940

M. Tonelli et al. / Bioorg. Med. Chem. 18 (2010) 2937–2953

R₁

R

R₁

O

C

N

a or b

NH₂

NH

W

N

Z

N

C: Z = H

R

D: Z= Me. Cyclohexyl,

1-Adamantyl

Z

C: 29, 36, 52

D: 59, 69, 72

C: 21, 38, 54 (W=Et); 48 (W= CH₂CH₂COOH)

D: 60, 70, 73 (W = Me)

61, 71, 74 (W = Et)

Scheme 3. Reagents and conditions: (a) Ac₂O or Pr₂O, benzene, 2 h reﬂux; (b) succinic anhydride, benzene, 18 h reﬂux.

R₁

O

C

N

a

NH

CH₂

N

H

N

R₁

N

C: 35, 47

NH₂

N

H

F₃C

O

C

N

CF₃

N

C: 29, 36

b

NH

N

H

N

H

C: 49

Scheme 4. Reagents and conditions: (a) homolupinanoyl chloride hydrochloride, Et₃N, CHCl₃, 6 h reﬂux; (b) diphosgene or EtOCOCl, benzene, 5 h reﬂux.

R₁

R

R₁

O

C

N

a

NH₂

NH

CH₂Cl

N

Z

N

Z

R

C: Z = H

D: Z = Me. 1-Adamantyl

C: 29, 36, 52

D: 59, 72

C: 29**, 39, 52**

D: 59**, 75

R₁

O

C

N

R₂

CH₂N

b

NH

R₃

N

Z

R

C: 32-34; 40-46; 55-58

D: 62-68; 76

Scheme 5. Reagents and conditions: (a) chloroacetyl chloride, Et₃N, benzene, 6 h reﬂux; (b) aminocompound (ratio 2:1), benzene (or EtOH), 18 h reﬂux (for 55–57 no solvent,

but excess of the aminocompound). (The N-chloroacetylderivatives 29**, 52** and 59** were not included in the antiviral screening.)

the same urea derivative that was obtained in higher yield by the

action of trichloromethyl chloroformate (diphosgene). The homo-

lupinanoyl chloride hydrochloride, required for the synthesis of

35 and 47, was prepared as previously described.^21,22

3. Results and discussion

3.1. Biological activity—general considerations

Finally, in order to obtain the 2-[4-(aminoacetyl)amino]phenyl-

benzimidazoles of structure C and D (see Tables 3 and 4), the suitable

amines were reacted with the 2-[4-(chloroacetylamino)phenyl]

benzimidazoles (see Scheme 5); some of the latter (29**, 52**, and

59**) were purposely prepared in order to obtain compounds 32–

34, 55–58, and 62–68, but were not considered for antiviral activity

screening.

The known 2-[4-(chloroacetylamino)phenyl]benzimidazole

(29**)²³was prepared by acylation of 2-(4-aminophenyl)benzimid-

azole which, in turn, was obtained according to the literature.^24,25

The structures of all novel compounds were supported by ele-

mental analyses and ¹H NMR spectra that are fully consistent with

the described structures.

The 2-phenylbenzimidazole derivatives synthesized in this

work were evaluated for antiviral activity against ten RNA and

DNA viruses. Among single-stranded, positive RNA viruses

(ssRNA⁺), we considered a Retrovirus (Human Immunodeﬁciency

Virus type 1, HIV-1) two Picornaviruses (Coxsackie Virus type-2,

CVB-2 and Poliovirus type-1, Sabin strain, Sb-1), a Flavivirus (Yel-

low Fever Virus, YFV) and a Pestivirus (Bovine Viral Diarrhea

Virus, BVDV). Among single-stranded, negative RNA viruses

(ssRNA^ꢁ) a Paramyxoviridae (Respiratory Syncytial Virus, RSV)

and a Rhabdoviridae (Vesicular Stomatitis Virus, VSV) were se-

lected as representatives. Among double-stranded RNA (dsRNA)

viruses, a Reoviridae family member (Reo-1) was included. Final-

ly, two representatives of DNA virus families were also included:

M. Tonelli et al. / Bioorg. Med. Chem. 18 (2010) 2937–2953

2941

Table 1

Number of active compounds on susceptible viruses and range of their EC₅₀

Virus^a

No. of active over 76 tested compounds^b

No. of active compounds (range of EC₅₀

1 (7)

, lM)

Reo-1^c

VV^d

2

5

6

10

12

17

31

41

1 (85)

1 (87)

1 (0.1)

3 (4–8)

4 (7–10)

5 (7–8)

1 (7)

RSV^e

1 (20)

1 (40)

YFV^f

3 (12–20)

5 (12–28)

8 (14–30)

3 (15–16)

19 (11–30)

1 (46)

1 (100)

HSV-1^d

Sb-1^f

3 (38–49)

6 (35–50)

3 (42–47)

4 (32–35)

3 (51–60)

2 (89–90)

3 (60–90)

2 (87–100)

1 (2)

BVDV^f

CVB-2^f

13 (0.8–3)

5 (1.5–3)

9 (4–10)

11 (4–10)

a

HIV-1 and VSV were unaffected by all tested compounds.

Compounds with EC₅₀>100 lM or higher than CC₅₀for host cells are considered inactive. Twenty over seventy-six compounds were not able to inhibit the multiplication

b

of any virus.

c

Double stranded RNA virus.

d

e

f

DNA virus.

Single stranded RNA^ꢁvirus.

Single stranded RNA⁺virus.

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

Virus, VV (Poxviridae).

several cases, they exert a strong activity as viral replication inhib-

itors and only a moderate or no toxicity on the speciﬁc host cells.

This observation is well exempliﬁed by the cases of compounds 3

and 4. The ﬁrst is active against six viruses with EC₅₀values in

AZT (3⁰-azido-thymidine), NM 108 (2⁰-C-methyl-guanosine),

NM 176 (2⁰-C-ethynyl-cytidine), Ribavirin, NM 299 (6-azauridine),

M 5255 (mycophenolic acid), and ACG (acyclovir) were used as ref-

erence inhibitors of ssRNA⁺, ssRNA^ꢁ, and DNA viruses, respectively.

Fifty-six of the 76 tested compounds exhibited antiviral activity

against one or more viruses; in particular, 17 compounds exhibited

a selective activity against a single virus, while 20, 11, 7, and 1 mol-

ecules were active against two, three, four, and six viruses, respec-

tively. None of the active compounds inhibited the replication of

HIV-1 and VSV, but an increasing number of molecules exhibited

antiviral activity against, in the order: Reo-1 (2), VV (5), RSV (6),

YFV (10), HSV-1 (12), Sb-1 (17), BVDV (31), and CVB-2 (41) (see Ta-

ble 1).

the range 5–25

lines is >100 M, the CC₅₀for the human cell line MT-4 is only

17 M. Moreover, compound exhibited high activity

(EC₅₀= 1 M) against BVDV and CC₅₀>100 M for the host cells

MDBK; however, the CC₅₀for MT-4 was as low as 2 M. Thus, it

lM. While its CC₅₀for the corresponding host cell

l

4

l

will be of fundamental importance to identify the determining fac-

tors of toxicity on human cells in order to improve the drug-like-

ness of these benzimidazole derivatives.

The very high cytotoxicity observed in some of these com-

pounds may warrant their evaluation as possible antiproliferative

agents. Even compound 49, which is devoid of toxicity on three

host cell lines, may be interesting for cancer therapy, since several

analogous 1,3-bis[4-(benzimidazol-2-yl)phenyl]ureas have shown

to strongly inhibit heparanase, exhibiting anti metastasis

efﬁcacy.¹¹

In any case, it must be observed that the structure–toxicity rela-

tionships appear rather intriguing. For example, the introduction of

a nitro group, contrary to common expectation, gives rise to toxic-

ity on the host cells only in a minority of cases, mainly when other

groups are simultaneously present. Moreover, the introduction of

the same 1-adamantyl residue at position 1 of the benzimidazole

Thirty-nine of 56 compounds (70%) showed an EC₅₀6 10

against at least one virus, and a subset of 17 had at least one

EC₅₀value in the range 0.1–3 M.

lM

l

Twenty compounds [12/55 1-unsubstituted (A–C) and 8/21 1-

substituted (B–D) derivatives] were not able to inhibit the replica-

tion of any virus at concentrations up to the corresponding CC₅₀for

their host cells, or up to the highest concentration tested (100 lM).

Cytotoxicity and antiviral activities of all the newly synthesized

and reference compounds are reported in Tables2–4. In each Table,

those viruses for which none or only one active compound has

been found are not listed, the relevant results being reported as

footnotes.

ring of compounds 39 (CC₅₀= 5

duces opposite effects; in the ﬁrst case, a 16-fold increase of cyto-

toxicity is observed (75: CC₅₀= 0.3 M), while in the second case

the cytotoxicity is drastically reduced (76; CC₅₀>100 M).

lM) and 46 (CC₅₀= 0.9 lM) pro-

l

3.2. Cytotoxicity on host cells

l

The title compounds showed different degrees of cytotoxicity

against the conﬂuent cell monolayers (in stationary growth) used

to support the replication of the different viruses.

The exponentially growing lymphoblastoid human cells (MT-4)

used to grow HIV-1 were found to be the most susceptible to tox-

icity. The nonhuman host cell lines exhibited a progressively re-

duced sensitivity in the order: MDBK > BHV > Vero-76. Only 21%

3.3. Antiviral activity: structure–activity relationships

On the whole, the considered 2-phenyl benzimidazole deriva-

tives appear particularly active against CVB-2 and BVDV viruses

(although interesting levels of activity against other viruses have

also been observed, though more randomly distributed).

Compounds of structure A and B (see Table 2), bearing a variety

of substituents (although mainly NO₂and OCH₃) on the 2-phenyl

residue, active against CVB-2 (16 over 28 compounds = 57%) are

also characterized by a large spectrum of activity, with 3–5 (11–

18%) compounds active against each of the following viruses: Sb-

1, YFV (10.7% each), BVDV, VV (14.3% each), HSV-1 and RSV (18%

each) and only one against Reo-1 virus. It is interesting to observe

that the activity against VV is conﬁned to the 2-(4-nitrophenyl)

derivatives, while no activity against BVDV was found among

2-(x-nitrophenyl)benzimidazoles, with the exception of compound

27 (Table 2). On the other hand, the 2-(4-aminophenyl) and

of compounds showed no toxicity (CC₅₀>100

versus ꢂ70% on Vero-76 cells; on the contrary 58% of compounds

exhibited a CC₅₀6 30 M for MT-4 compared with only 13% in

the case of Vero-76 cells. Moreover, 9% of compounds had

CC₅₀6 3 M (with a lowest value of 0.3 M) for MT-4 cells. The

lM) against MT-4,

l

BHK cell line was somewhat more susceptible to toxicity than

Vero-76 cell line, while MDBK was only slightly less susceptible

than MT-4 cell line.

The high cytotoxicity of so many compounds against the MT-4

human cells decreases their interest as antiviral agents even if, in

2942

M. Tonelli et al. / Bioorg. Med. Chem. 18 (2010) 2937–2953

Table 2

Cytotoxicity against MT-4, MDBK, BHK and Vero-76 cell lines and antiviral activity of 2-phenylbenzimidazole derivatives of structure A (1–25) and B (26–28)

Compd^a

R⁰

R⁰⁰

Z

MT-4

CC₅₀

MDBK

CC₅₀

BVDV

EC₅₀

BHK

CC₅₀

YFV

EC₅₀

Vero-76

CC₅₀

CVB-2

EC₅₀

Sb-1

EC₅₀

HSV-1

EC₅₀

RSV

EC₅₀

VV

EC₅₀

b

c

d

e

f

g

h

i

j

k

l

1

H

5-CF₃

5-NO₂

5-COCH₃

5,6-DiCl

5-CF₃

2-NO₂

2,4-DiNO₂

4-OH

4-OCH₃

3-OCH₃

2,4-DiOCH₃

3,5-DiOCH₃

2,3,4-TriOCH₃

3,4,5-TriOCH₃

2-NO₂,4-OCH₃

2-NH₂,4-OCH₃

4-NO₂

2,4-DiNO₂

4-F

2,6-DiF

60

7

17

2

19

>100

19

93

4

85

5

6

9

>100

20

51

>100

28

>100

18

55

4

6

16

>100

>20

>51

1

>28

>100

>18

>55

>4

>100

90

>100

32

31

100

P100

18

8

>100

12

>100

>90

>100

>32

>31

>100

20

>18

>8

>100

15

>100

70

29

20

50

45

25

60

25

>100

18

>100

40

5

7

4

32

3

16

11

>20

6

12

9

13

>25

>100

>18

10

>80

>75

>100

30

>100

24

>100

25

>15

12

40

>100

20

>15

7

>100

10

>100

>15

>100

>70

>29

>20

>50

4

>25

8

>25

>100

>18

>30

>80

>75

0.1

>100

87

>100

11

>100

1.8

2

3^m

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

>15

>100

42

>29

>20

18

>45

>25

>60

>100

>70

>29

>20

>50

>45

>25

>60

>25

>100

40

>70

>29

>20

>50

>45

>25

>60

>25

>100

7

>30

>80

>75

>100

1.2

>6

>16

>19

>100

45

>100

42

>100

>11

>2.5

>28

>6.5

>56

>43

>100

60

22

19

25

>25

100

>100

12

1

2

3

17

P100

49

P100

>100

2

>100

31

P100

>100

11

2.5

28

6.5

56

43

>100

68

>100

42

P100

>100

29

>100

>29

>25

2,4-DiOCH₃

2,3,4-TriOCH₃

2,4-DiOCH₃

2,3,4-TriOCH₃

4-OH

>100

>18

>30

>80

>75

>100

>20

>100

>13

>18

20

4-OCH₃

18

80

>18

>80

30

80

75

2-NO₂,4-OCH₃

2-NH₂,4-OCH₃

4-NO₂

2,6-DiF

4-NO₂

>80

>75

>100

>20

3

>100

90

>100

1.8

P100

>100

20

>100

P13

>100

Me

Chⁿ

Ad^o

>100

45

>100

20

>20

>100

>13

27

5-CF₃

4-NO₂

28

>100

1.7

>100

7

NM 108

NM 299

ACG

Ribavirin

M 5255

NM 176

26

>100

7

0.6

>100

>13

>100

0.2

P100

>42

38

>100

23

>100

CC₅₀and EC₅₀

(

l

M).

a

None of these compounds inhibited the multiplication of HIV-1 and VSV viruses.

b

Compound concentration (

l

M) required to reduce the viability of mock-infected MT-4 (CD4⁺human T cells containing an integrated HJLV-1 genome) cells by 50%, as

determined by the MTT method.

c

Compound concentration (

lM) required to reduce the viability of mock-infected MDBK (Bovine normal kidney) cells by 50%, as determined by the MTT method.

M) required to achieve 50% protection of MDBK cells from BVDV (Bovine Viral Diarrhea Virus) induced cytopathogenicity, as determined by

d

Compound concentration (

l

the MTT method.

e

Compound concentration (

lM) required to reduce the viability of mock-infected BHK (Hamster normal kidney ﬁbroblast) monolayers by 50%, as determined by the MTT

method.

f

Compound concentration (

l

M) required to achieve 50% protection of BHK cells (kidney ﬁbroblast) from YFV (Yellow Fever Virus) induced cytopathogenicity, as

determined by the MTT method.

g

Compound concentration (

Active also on Reo-1 virus (EC₅₀= 7

Ch = cyclohexyl.

Ad = 1-adamantyl.

l

M) required to reduce the viability of mock-infected VERO-76 (Monkey normal kidney) monolayers by 50%.

M) required to reduce the plaque number of CVB-2 (Coxsackie Virus B2) by 50% in VERO-76 monolayers.

M) required to reduce the plaque number of Sb-1 (Poliovirus type-1, Sabin strain) by 50% in VERO-76 monolayers.

M) required to reduce the plaque number of HSV-1 (Herpes Simplex Virus, type-1) by 50% in VERO-76 monolayers.

M) required to reduce the plaque number of RSV (Respiratory Syncytial Virus) by 50% in VERO-76 monolayers.

M) required to reduce the plaque number of VV (Vaccinia Virus) by 50% in VERO-76 monolayers.

h

i

j

k

l

m

n

o

lM; NM 108: EC₅₀= 2.4 lM).

2-(4-acylaminophenyl) derivatives (Table 3) have predominant

activity against BVDV (66.7%) and CVB-2 (43.3%), with the two

activities simultaneously present in 36.7% of cases. These com-

pounds exhibit additional activity against Sb-1 (23%), HSV-1

(17%), and YFV (13%), while RSV, VV and Reo-1 viruses are affected

by only one compound each.

Theintroductionof analiphatic/cycloaliphaticresidueat position

1 of the 2-(4-amino/acylaminophenyl)-5-triﬂuoromethylbenzimi-

dazoles results in different effects, depending on its chemical nature

(methyl, cyclohexyl, or 1-adamantyl), as well as on the presence and

the nature of the acyl residue on the 4-amino group.

pounds sharing the activity on BVDV and CVB-2, and another third

being active only on CVB-2. Indeed, comparing compounds with

the same substitution pattern, the 1-substitution tends, the only

exception being compound 66, to reduce or suppress the activity

against BVDV, while only occasionally does it interfere negatively

with the activity versus CVB-2. Ultimately, it may even convert

inactive into active compounds, as observed in the cases of 62,

63, 66, 68, and 76. All these compounds and the corresponding

unsubstituted compounds 41, 42, 45, and 46 bear a basic head

on the acyl moiety, and the onset of activity may be the conse-

quence of balancing the excessive hydrophilicity due to the prot-

onable nitrogen of the chain. Similar improvement of activity or

conversion to activity of the inactive basic compounds is also

observed for Sb-1 virus (59, 60, 62, 63, 68, and 71). However the

Taking into consideration all the compounds of structure D (see

Table 4), an inverted trend with respect to that exhibited by the 1-

unsubstituted compounds, is observed, with one-third of com-

M. Tonelli et al. / Bioorg. Med. Chem. 18 (2010) 2937–2953

2943

Table 3

Cytotoxicity and antiviral activity of 2-(4-aminophenyl)- and 2-(4-acylaminophenyl)-5-triﬂuoromethyl benzimidazole derivatives of structure C (29–58)

Compd^a

R⁰

R⁰⁰⁰

MT-4

CC₅₀

MDBK

CC₅₀

BVDV

EC₅₀

BHK-21

CC₅₀

YFV

EC₅₀

Vero-76

CC₅₀

CVB-2

EC₅₀

Sb-1

EC₅₀

HSV-1

EC₅₀

b

c

d

e

f

g

h

i

j

29^k

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

H

81

100

35

40

31

100

18

62

19

P100

5

15

8

18

6

>100

6

0.9

4

>100

100

>100

17

>100

8

54

18

>100

11

>100

2

>100

31

>100

70

18

77

36

82

50

>100

17

23

13

15

6

>100

32

16

90

47

10

7

>77

10

>100

P100

55

31

>100

28

100

53

>100

22

18

23

>100

19

>31

>100

8

>100

>53

>100

>22

>18

7

>100

50

>100

35

45

24

>100

31

19

87

9

11

>100

35

>100

60

>100

7

>100

>35

CH₃CO

CH₃CH₂CO

(CH₂)₄N–CH₂CO

(CH₂)₅N–CH₂CO

O(CH₂CH₂)₂N–CH₂CO

Homolupinanoyl

H

CH₃CO

CH₃CH₂CO

ClCH₂CO

1-Adamantyl-NH–CH₂CO

(C₂H₅)₂N–CH₂CO

(CH₂)₄N–CH₂CO

(CH₂)₅N–CH₂CO

O(CH₂CH₂)₂N–CH₂CO

S(CH₂CH₂)₂N–CH₂CO

C₆H₅N(CH₂CH₂)₂N–CH₂CO

Homolupinanoyl

HOOC(CH₂)₂CO

4-(5-CF₃-benzimidazol-2-yl)phenylcarbamoyl

H

CH₃CO

H

CH₃CO

(CH₂)₄N–CH₂CO

(CH₂)₅N–CH₂CO

O(CH₂CH₂)₂N–CH₂CO

CH₃N(CH₂CH₂)₂N–CH₂CO

90

17

>100

47

16

>100

>35

>100

P100

17

3

4

5-CF₃

5-NO₂

5,6-DiCl

15

1.3

>100

>17

>23

2

24

>45

>24

>100

9

>100

>40

>100

11

>100

1.5

>100

13

>100

20

>20

>100

27

>45

>24

45

>24

3

2

29

12

86

7

>100

>31

>100

14

>100

25

>100

89

>100

>31

>100

>40

>100

28

>100

38

>100

51

>100

>20

3

>100

P12

>86

>100

>15

>100

>17

>100

>30

>100

>64

>39

>51

>100

>41

1.8

>100

>32

2

>100

1.5

0.8

1

2.5

2

2.4

>100

4

1.7

>100

7

>100

40

>100

15

>100

17

>100

30

>100

64

39

51

>100

41

90

46

47

48

49

12

>100

79

>100

7.5

58

16

45

16

>100

P100

>100

P100

>100

20

50

51

52^l

53

54

55

56

57

>100

90

58^m

NM 108

>100

>20

>100

23

6-Azauridine

ACG

Ribavirin

NM 176

>100

26

>100

P100

>100

CC₅₀and EC₅₀

(

l

M).

b–j

For the meaning see Table 2.

a

k

None of these compounds inhibited the multiplication of HIV-1 and VSV viruses.

Active also on Reo-1 virus (EC₅₀= 85 M; NM-108: EC₅₀= 2.4 M).

M; 6-azauridine: EC₅₀= 1.2 M).

M; M 5255: EC₅₀= 1.8 M).

l

Active also on RSV virus (EC₅₀= 10

Active also on VV virus (EC₅₀= 6

l

m

l

1-substitution has variable effects on activity against HSV-1 and

YFV, and suppresses that on RSV, VV, and Reo-1 viruses.

dex (S.I.) values, the best of these compounds (with S.I. >4) are col-

lected in Table 5.

Of the 17 most active compounds with a EC₅₀<3

l

M, only three

As previously observed, eight compounds exhibit a large spec-

trum of activity regarding 4–6 viruses, most commonly BVDV,

CVB-2, Sb-1, HSV-1, and YFV. The degree of activity is generally

belong to group A, while all remaining molecules are 2-(4-amino or

4-acylaminophenyl)benzimidazoles, either 1-unsubstituted (12;

Table 3) or 1-methyl substituted (2; see Table 4).

On the other hand, selective activity against a single virus is

more commonly found among compounds of groups A and B

(10/28 = 35.7%; see Table 2) than among compounds of groups C

and D (7/48 = 14.6%; see Tables 3 and 4).

high, with EC₅₀in the range 1–20

ular set; however, toxicity versus the human cell line MT-4 (CC₅₀

<20 M) is frequently found.

lM for two thirds of the molec-

l

In order to determine the possible step(s) which is inhibited by

our compounds during the BVDV replication cycle, a time of addi-

tion experiment was performed with the most potent compound

51, that was added at different time points after infection of MDBK

cell cultures with the relevant virus. Compound 51 retained its

inhibitory activity when even added no later than 6 h after infec-

tion (see Fig. 3). The reference compound NM 108 (2⁰-C-methylgu-

anosine) is a nucleoside analog that interacts with the viral

polymerase (up to 4 h pi) either as competitive inhibitor or as sub-

strate, preventing further chain elongation. The comparison of the

two time of addition curves pointed to a post viral entry step, be-

fore the budding stage (approximately after 13 h pi), in the BVDV

replication cycle as possible antiviral target of our compound

series.

Combined high activity and selectivity are shown by com-

pounds 51, 53, and 50 against BVDV (EC₅₀= 0.8–1.5

lM), and by

compound 24 with respect to VV (EC₅₀= 0.1 M). Moreover, these

l

compounds are well tolerated by the proper host cell lines (MDBK,

Vero-76), and even more by the human cell line MT-4, and com-

pare favorably with their reference compounds NM 108

(EC₅₀= 1.7

Some interest also deserves compound 38. Its selective activity

against CVB-2 (EC₅₀= 4 M) compares well with that of the refer-

ence compound NM 108 (EC₅₀= 20 M). Several other compounds

exhibit a high, even if not selective, activity against BVDV or CVB-2

M), and only moderate toxicity on the relevant

lM) and M 5255 (EC₅₀= 1.8 lM).

l

viruses (EC₅₀6 4

l

host cells, but their selectivity versus the human MT-4 cell line

Selective compounds 50, 51, and 53, endowed with the highest

activity against BVDV and the lowest toxicity for both the host cells

was rather low. Taking into account the EC₅₀and the selectivity in-

2944

M. Tonelli et al. / Bioorg. Med. Chem. 18 (2010) 2937–2953

Table 4

Cytotoxicity and antiviral activity of 2-(4-aminophenyl)- and 2-(4-acylaminophenyl)-5-triﬂuoromethyl-1-substituted benzimidazoles derivatives of structure D (59–76)

Compd^a

Z

R⁰⁰⁰

MT-4

CC₅₀

MDBK

CC₅₀

BVDV

EC₅₀

BHK

CC₅₀

YFV

EC₅₀

Vero-76

CC₅₀

CVB-2

EC₅₀

Sb-1

EC₅₀

HSV-1

EC₅₀

b

c

d

e

f

g

h

i

j

59

60

61

62

63

64

65

66

67

68

69

70

71

72

73

74

CH₃

H

20

15

8

9

16

9

P100

10

22

1.5

44

24

17

19

74

46

>100

57

65

100

>100

44

1.4

>100

17

18

19

82

>100

18

>100

69

85

>85

>100

90

>100

30

>100

30

>100

60

>100

20

>100

14

20

14

>100

35

50

>100

41

>100

49

>100

>90

CH₃CO

CH₃CH₂CO

>46

>100

10

7

6

>100

8

15

>1.4

>100

>17

>18

7

>82

>100

>18

>100

1.7

>100

7

>100

16

>100

7

>27

>100

46

2.5

>100

21

13

32

>100

22

19

3

>100

9

7

11

>100

27

(C₂H₅)₂N–CH₂CO

(CH₂)₄N–CH₂CO

(CH₂)₅N–CH₂CO

O(CH₂CH₂)₂N–CH₂CO

S(CH₂CH₂)₂N–CH₂CO

CH₃N(CH₂CH₂)₂N–CH₂CO

C₆H₅N(CH₂CH₂)₂N–CH₂CO

H

CH₃CO

CH₃CH₂CO

H

CH₃CO

CH₃CH₂CO

ClCH₂CO

C₆H₅N(CH₂CH₂)₂N–CH₂CO

27

51

>100

16

100

>100

15

>100

>30

>100

>30

>100

>60

>100

>20

3

>100

CH₃

7

>100

>15

>16

>100

>51

>100

1.8

2

Cyclohexyl

1-Adamantyl

>100

>30

30

15

16

>60

>100

>20

>100

23

28

86

>100

51

>100

90

>100

75

76

0.3

>100

2

>100

31

NM 108

6-Azauridine

ACG

Ribavirin

NM 176

20

26

>20

>100

27

>100

P100

38

CC₅₀and EC₅₀

(

l

M).

b–j

For the meaning see Table 2.

a

None of these compounds inhibited the multiplication of HIV-1, Reo-1, RSV, VSV, and VV viruses.

Table 5

Most potent antiviral compounds against BVDV, CVB-2 and VV, in order of decreasing selectivity index (S.I.) versus human MT-4 cell line (S.I. >4)

Virus

BVDV

Compd

EC₅₀

(

lM)

S.I. for proper host cell lines

S.I. for MT-4 cell line

Other susceptible viruses

51

53

50

55

52

37

56

42

41

0.8

1.0

1.5

2.0

1.0

1.3

2.4

3.0

2.0

>125

58

53

23

7.5

39

6.7

5.0

6.5

>125

>100

67

27

17

Selective

HSV-1

CVB-2, HSV-1

CVB-2, Sb-1, HSV-1

CVBV-2

15

7.5

6.0

4.0

YFV

CVB-2

38

7

37

60

54

5

4.0

3.0

2.5

1.5

4.0

>25

>33

17

>40

>67

>25

P25

6.3

6.0

5.3

4.8

Selective

RSV

BVDV, Sb-1, HSV-1

Sb-1, HSV-1

BVDV, Sb-1

HSV-1, RSV

VV

24

0.1

P1000

170

Selective

1000000

100000

10000

1000

100

(RdRp) of BVDV. The enzymatic inhibition assays showed that

these compounds inhibit the RNA polymerase at micromolar

concentrations, in a dose-dependent way (see Table 6), indicating

that the RdRp is indeed their target. Since BVDV is often used

as a surrogate model to screen antiviral agents against HCV,

the three compounds were also assayed against HCV1b poly-

merase and found to inhibit this enzyme with similar IC₅₀(see

Table 6).

10

The molecular target of our compounds has been further sup-

ported by the generation of compounds 50, 51, and 53 BVDV resis-

tant mutants,²⁶observing that while NS3 protease is not affected,

the NS5B RdRp was modiﬁed by the three compounds even if with

some differences. Functional mapping and sequence analysis of

resistant cDNAs revealed a single (A392 and N264D mutations

for 50 and 51, respectively) or a double amino acid substitution

(N264D, I261M mutations for 53) in the NS5B polymerase. These

regions of NS5B are highly conserved among pestiviruses suggest-

ing that these sites play an important role in the function of the

1

0

2

4

6

8

10

12

14

hrs post infection

Figure 3. Effect of time of (drug)-addition on antiviral activity of 51 (open circles).

The same test was performed using the nucleoside analog NM 108 (ﬁlled circles) for

comparison. Infected control: continuous line.

MDBK and the human cells MT-4, were also assayed against the

surface binding site of NS5B RNA-dependent RNA polymerase

M. Tonelli et al. / Bioorg. Med. Chem. 18 (2010) 2937–2953

2945

Table 6

Comparison of antiviral activity (EC₅₀) versus BVDV, inhibitory activity (IC₅₀) on NS5B RdRp of BVDV and HCV viruses, and activity in HCV replicon assay

Compd

Anti-BVDV activity

IC₅₀

BVDV

(l

M) on NS5B RdRp

HCV replicon assay

M) EC₅₀(lM)

CC₅₀

(l

M)

EC₅₀

(

lM)

S.I.

HCV

CC₅₀

(l

50

51

53

79

>100

58

1.5

0.8

1

53

>125

58

18

12

3

33

30

5

>90

>75

38

32

enzyme; mutational analysis of this region abolished the RNA

synthesis in vitro, suggesting that this sequence is important for

replicase activity. Furthermore, docking studies upon pharmaco-

phoric constraints and mutational data were carried out,²⁶and

the binding afﬁnity of all active compounds for the BVDV RdRp

were estimated with excellent agreement between in silico and

in vitro data.

Thus compound 24 still represents an interesting hit for the

development of anti-poxvirus agents, and provided that it will

not exhibit unduly toxicity in vivo, may in itself deserve further

investigation for in vivo efﬁcacy against poxvirus infections, and

for identiﬁcation of its mechanism of action.

On the other hand, simple compounds such as 50, 51, and 53

exhibited a high and selective activity against BVDV with EC₅₀in

Finally compounds 51 and 53 were also evaluated in the HCV

subgenomic replicon system, conﬁrming activity against HCV, even

if lower than that against BVDV (see Table 6).

the range 0.8–1.5

drug NM 108 (EC₅₀= 1.7

es which are responsible of severe epidemic outbreaks in livestock

with high mortality, and the availability of effective and inexpen-

sive anti-pestivirus drugs is of importance to relieve such an heavy

economic burden.

l

M, thus comparing favorably with the reference

M). BVDV is the prototype of pestivirus-

l

4. Conclusions

In the last years several potent anti-BVDV agents have been

developed and shown to target the RNA-dependent RNA-polymer-

ase (RdRp),^31–37but the structural simplicity of compounds 50, 51,

and 53 make them an attractive model to develop ever better anti-

pestivirus agents. Moreover, BVDV is surrogate model for the eval-

uation of novel, urgently needed agents against HCV,³⁵an emerg-

ing threat to human health worldwide. Indeed, compounds 50,

51 and particularly 53 were found able to inhibit both the BVDV

and HCV NS5B RdRp at similar concentrations and, therefore,

may represent also an interesting starting point for the develop-

ment of anti-HCV agents. Indeed compound 51 proved moderately

active in the HCV subgenomic replicon system.

Seventy-six 2-phenylbenzimidazole derivatives have been syn-

thesized and assayed for antiviral activity against a panel of 10

RNA and DNA viruses. Fifty-six of the tested compounds exhibited

antiviral activity against one or more viruses, and 39 of them

showed EC₅₀6 10 lM against at least one virus. A subset of 17

compounds (mainly 2-(4-aminophenyl)- or 2-(4-acylaminophe-

nyl)benzimidazoles had at least one EC₅₀value in the range 0.1–

3

l

M.

The considered 2-phenylbenzimidazole derivatives resulted

particularly effective against BVDV (31/56) and CVB-2 (41/56)

viruses, but interesting levels of activity against other viruses

(Sb-1, HSV-1, YFV, RSV, VV, and Reo-1) have been also observed.

Despite the high antiviral activity and good selectivity index for

the proper host cell lines, the value of some compounds results

somewhat impaired by their cytotoxicity for the human MT-4 cell

line. Nevertheless, taking into account the EC₅₀and S.I. values, sev-

eral compounds appear as very interesting.

5. Experimental

5.1. General

Compound 24 [5,6-dichloro-2-(4-nitrophenyl)benzimidazole]

Melting points were taken in open glass capillaries on a Büchi

apparatus and were uncorrected. Purity of compounds was

checked by thin-layer chromatography that was performed on sil-

ica gel plates [Merck (G F₂₅₄)] and the spots were observed under

UV light. Column chromatography (CC) was performed using basic

alumina (Across). Elemental analyses were performed on a Carlo

Erba EA-1110 CHNS-O instrument in the Microanalysis Laboratory

of the Department of Pharmaceutical Sciences of Genoa University.

The analytical results are within 0.4% of calculated values. ¹H

NMR spectra were recorded in CDCl₃or DMSO-d₆on Varian Gem-

ini-200 spectrometer; d in ppm rel. to Me₄Si as internal standard. J

in Hz. Results of elemental analyses, TLC and NMR spectra indi-

cated that the purity of all compounds was P95%.

exhibited a high and selective activity against VV (EC₅₀= 0.1

resulting 18 and 110 times more potent than the reference drugs

mycophenolic acid and 6-azauridine (EC₅₀= 1.8 M and 11 M,

lM),

l

respectively). Moreover, these reference drugs are much more

toxic than 24 versus the proper host cell line (Vero-76) and the hu-

man MT-4 cell line.

Even if the natural smallpox infection has been declared eradi-

cated by WHO, some concern is raised by the possible terroristic

use of the virus, thus the acquisition of protective agents, possibly

acting by oral route, should be fostered. At present the only antivi-

ral agent considered for this use by the U.S. Centers for Disease

Control and Prevention (CDC) is the costly cidofovir (CDV), by iv

infusion, which, however, according to De Clercq,²⁷could be refor-

mulated as lipid prodrug for oral use.

5.2. Intermediates

A potent and speciﬁc inhibitor of orthopoxvirus replication

has recently been discovered²⁸(ST-246: 4-triﬂuoromethyl-N-

(3,3a,4,4a,5,5a,6,6a,-octahydro-1,3-dioxo-4,6-ethenocycloprop[f]

isoindol-2(1H)-yl)benzamide), which was effective when adminis-

tered orally in mice. However poxvirus strains that are resistant to

CDV or ST-246 have been generated in cell culture.²⁹

Chemicals, solvents and commercially available intermediates

were purchased from Aldrich (Milan). The noncommercially avail-

able intermediates were prepared according to the literature (see:

Section 2), or as follows, when not previously known.

More recently, through the high-throughput screening of about

50,000 compounds, 16 hit compounds have been identiﬁed and

found to block viral infection with low cytotoxicity.³⁰Comparing

to our results, it is observed that their EC₅₀for plaque reduction

5.2.1. N-(2-Nitro-4-triﬂuoromethylphenyl)-1-adamantaneamine

A solution of 1-adamantaneamine (20 mmol) and 4-chloro-3-

nitrobenzotriﬂuoride (10 mmol) in DMF (3 mL) was heated with

stirring (90 min; 140 °C) in a pressure tube (Aldrich). After cooling,

was in the range 10–150 lM.

2946

M. Tonelli et al. / Bioorg. Med. Chem. 18 (2010) 2937–2953

the residue was taken up with water, ﬁltered and washed with

water and ﬁnally crystallized from Et₂O. Yield: 88%. Mp 210–

213 °C. ¹H NMR (CDCl₃): 1.78 (br s, 6H, CH₂, adamantane nucleus);

2.13 (br s, 6H, CH₂, adamantane nucleus); 2.25 (br s, 3H, CH₂, ada-

mantane nucleus); 4.75 (br s, NH, collapses with D₂O); 7.29 (d,

J = 9.6, 1 arom. H); 7.55 (dd, J = 9.6, 1.1, 1 arom. H); 8.53 (d,

J = 10, 1 arom. H). Anal. Calcd for C₁₇H₁₉F₃N₂O₂: C, 59.99; H 5.63;

N, 8.23. Found: C, 60.02; H, 5.70; N, 8.24.

(dd, J = 8.8, 1.2, 1 arom. H); 8.20 (d, J = 9.0, 2 arom. H); 8.43 (d,

J = 9.0, 2 arom. H). Anal. Calcd for C₁₅H₁₀F₃N₃O₂: C, 56.08; H,

3.14; N, 13.03. Found: C, 56.15; H, 3.24; N, 13.08.

5.3.5. 1-Cyclohexyl-2-(4-nitrophenyl)-5-triﬂuoromethy-1H-

benzimidazole (27)

Yield: 62%. Mp 159–161 °C (EtOH). ¹H NMR (DMSO-d₆): 1.32–

2.43 (m, 10H, cyclohexane nucleus); 4.18–4.40 (m, 1H, cyclohex-

ane nucleus); 7.62 (d, J = 8.6, 1 arom. H); 7.95 (d, J = 8.9, 2 arom.

H); 8.08–8.22 (m, 2 arom. H); 8.43 (d, J = 8.9, 2 arom. H). Anal.

Calcd for C₂₀H₁₈F₃N₃O₂: C, 61.38; H, 5.15; N, 10.74. Found: C,

61.44; H, 4.75; N, 10.73.

5.2.2. N-(2-Amino-4-triﬂuoromethylphenyl)-1-adamantaneamine

A suspension of the above nitrocompound (10 mmol) in 50 mL

of EtOH was hydrogenated at rt and atmospheric pressure in the

presence of 10% Pd/C (0.4 g). After 2 h and 30 min the calculated

volume of H₂was absorbed. The catalyst was removed and the sol-

vent was evaporated in vacuo, leaving an almost quantitative yield

of product which was crystallized from pentane. Yield: 98%. Mp

114–116 °C (pentane). ¹H NMR (CDCl₃): 1.71 (br s, 6H CH₂, ada-

mantane nucleus); 1.95 (br s, 6H, CH₂, adamantane nucleus);

2.15 (br s, 3H CH₂, adamantane nucleus); 3.58 (br s, NH₂and NH,

collapse with D₂O); 6.92–7.05 (m, 3 arom. H). Anal. Calcd for

C₁₇H₂₁F₃N₂: C, 65.79; H, 6.82; N, 9.03. Found: C, 65.86; H, 6.46;

N, 8.87.

5.3.6. 1-(1-Adamantyl)-2-(4-nitrophenyl)-5-triﬂuoromethyl-1H-

benzimidazole (28)

Yield: 82%. Mp 266–269 °C (MeCN). ¹H NMR (DMSO-d₆): 1.57–

1.80 (m, 6H, adamantane nucleus); 2.21- 2.34 (m, 9 H, adamantane

nucleus); 7.56 (d, J = 8.6, 1 arom. H); 7.93 (d, J = 8.9, 2 arom. H);

8.06–8.20 (s, 1 arom. H); 8.20–8.43 (m, 1 arom. H and 8.33, d,

J = 9.0, 2 arom. H superimposed). Anal. Calcd for C₂₄H₂₂F₃N₃O₂: C,

65.30; H, 5.02; N, 9.52. Found: C, 65.13; H, 5.37; N, 9.80.

5.3.7. 2-(4-Acetylaminophenyl)-5-triﬂuoromethyl-1H-benzimidazole

(37)

5.3. 2-Phenylbenzimidazoles. Method ‘a’ of ring closure

Yield: 45%. Mp 281–282 °C (MeCN). ¹H NMR (DMSO-d₆): 2.12 (s,

CH₃CO); 7.67 (d, J = 8.5, 1 arom. H); 7.98 (d, J = 8.8, 2 arom. H);

8.09–8.20 (m, 2 arom. H); 8.43 (d, J = 8.8, 2 arom. H); 10.15 (s,

NHCO, collapses with D₂O); 13.38 (br s, NH benzim., collapses with

D₂O). Anal. Calcd for C₁₆H₁₂F₃N₃O: C, 60.19; H, 3.79; N, 13.16.

Found: C, 59.96; H, 4.08; N, 13.26.

To a solution of the suitable 1,2-phenylenediamine (5 mmol) in

15 mL of MeCN the appropriate benzaldehyde (5 mmol) dissolved

in 15 mL of MeCN was added. The mixture was reﬂuxed for 1 h

with stirring, and then FeCl₃ꢃ6H₂O (14 mg) in 2 mL of MeCN was

added. Bubbling O₂, the heating was maintained for 6 h with stir-

ring. After cooling, the precipitate was ﬁltered and washed with

MeCN, leaving generally a pure product. Compounds 22 and 27

were crystallized from EtOH.

5.3.8. 2-(4-Acetylaminophenyl)-5,6-dichloro-1H-benzimidazole

(53)

In the case of compound 15, it was necessary to remove the sol-

vent from the reaction mixture, and to purify the residue by CC

(Al₂O₃, CH₂Cl₂). Finally the oily product was crystallized from dry

Et₂O/pentane (1:1).

Yield: 78%. Mp >300 °C (MeCN). ¹H NMR (DMSO-d₆): 2.05 (s,

CH₃CO); 7.87 (s, 2 arom. H); 7.89 (d, J = 9.8, 2 arom. H); 8.16 (d,

J = 9.8, 2 arom. H); 10.11 (s, NHCO, collapses with D₂O); 12.57

(br s, NH benzim., collapses with D₂O). Anal Calcd for

C₁₅H₁₁Cl₂N₃O + 0.25H₂O: C, 55.49; H, 3.57; N, 12.94. Found: C,

55.77; H, 3.78; N, 13.11.

5.3.1. 2-(4-Nitrophenyl)-5-triﬂuoromethyl-1H-benzimidazole

(12)

Yield: 46%. Mp 216–218 °C (MeCN). ¹H NMR (DMSO-d₆): 7.67

(d, J = 8.6, 1 arom. H); 7.97 (d, J = 8.6, 2 arom. H); 8.10–8.23 (m, 2

arom. H); 8.41 (d, J = 8.8, 2 arom. H); 13.51 (br s, NH benzim., col-

lapses with D₂O). Anal. Calcd for C₁₄H₈F₃N₃O₂: C, 54.73; H, 2.62; N,

13.68. Found: C, 54.84; H, 2.29; N, 13.70.

5.4. 2-Phenylbenzimidazoles. Method ‘b’ of ring closure

To a solution of the suitable 1,2-phenylenediamine (3.5 mmol)

in 18 mL of EtOH the appropriate benzaldehyde–sodium bisulﬁte

adduct (3.5 mmol) (prepared according to the method of (Shriner

and Land¹⁴) was added. The mixture was reﬂuxed for 3 h with stir-

ring. The solvent was evaporated and the residue was taken up

with water and ﬁltered, affording a residue that was crystallized

from the suitable solvent.

In the case of compound 13, chloranil (0.8 mmol) was added tothe

reaction mixture that was reﬂuxed for 72 h. After cooling, a precipi-

tate was ﬁltered and the ethanolic solution was evaporated to dry-

ness, leaving a solid product that was puriﬁed by CC (Al₂O₃/CH₂Cl₂).

5.3.2. 2-(2,6-Diﬂuorophenyl)-5-triﬂuoromethyl-1H-benzimidazole

(15)

Yield: 55%. Mp 166–168 °C (Et₂O/pentane). ¹H NMR (DMSO-d₆):

7.38 (t, J = 8.6, 2 arom. H); 7.50–7.96 (m, 3 arom. H); 8.06 (s, 1

arom. H); 13.35 (br s, NH benzim., collapses with D₂O). Anal. Calcd

for C₁₄H₇F₅N₂: C, 56.39; H, 2.37; N, 9.39. Found: C, 56.03; H, 2.41;

N, 9.36.

5.3.3. 5,6-Dichloro-2-(4-Methoxy-2-nitrophenyl)-1H-benzimidazole

(22)

5.4.1. 2-(3-Methoxyphenyl)-5-triﬂuoromethyl-1H-benzimidazole

(5)

Yield: 77%. Mp 193–194 °C (EtOH). ¹H NMR (CDCl₃): 3.93 (s,

OCH₃); 7.13 (d, J = 8.2, 1 arom. H); 7.39 (s, 1 arom. H); 7.55 (s, 2

arom. H); 7.80 (d, J = 8.2, 1 arom. H); 9.56 (s, NH of benzim., col-

lapse with D₂O). Anal. Calcd for C₁₄H₉Cl₂N₃O₃: C, 49.73; H, 2.68;

N, 12.43. Found: C, 49.78; H, 2.51; N, 12.36.

Yield: 25%. Mp 186–187 °C (benzene). ¹H NMR (DMSO-d₆): 3.89

(s, OCH₃); 7.07–7.20 (m, 1 arom. H); 7.42–7.89 (m, 5 arom. H); 8.05

(s, 1 arom. H); 13.38 (br s, NH benzim., collapses with D₂O). Anal.

Calcd for C₁₅H₁₁F₃N₂O: C, 61.64; H, 3.79; N, 9.59. Found: C, 61.64;

H, 3.79; N, 9.56.

5.3.4. 1-Methyl-2-(4-nitrophenyl)-5-triﬂuoromethyl-1H-benzimidazole

(26)

5.4.2. 2-(2,4-Dimethoxyphenyl)-5-triﬂuoromethyl-1H-benzimidazole

(6)

Yield: 65%. Mp 218–221 °C (MeCN). ¹H NMR (DMSO-d₆): 4.0 (s,

NCH₃); 7.68 (d, J = 8.6, 1 arom. H); 7.93 (d, J = 8.8, 1 arom. H); 8.12

Yield: 87%. Mp 175–176 °C (EtOH/H₂O). ¹H NMR (DMSO-d₆):

3.86 (s, OCH₃); 4.03 (s, OCH₃); 6.65–6.76 (m, 2 arom. H); 7.41–

M. Tonelli et al. / Bioorg. Med. Chem. 18 (2010) 2937–2953

2947

7.82 (m, 2 arom. H); 7.91 (d, J = 7.8, 1 arom. H); 8.23–8.35 (m, 1

arom. H); 12.28 (br s, NH benzim., collapses with D₂O). Anal. Calcd

for C₁₆H₁₃F₃N₂O₂: C, 59.62; H, 4.47; N, 8.69. Found: C, 59.42; H,

4.41; N, 8.52.

7.00 (d, J = 8.8, 1 arom. H); 7.66 (d, J = 8.0, 1 arom. H); 7.82–8.30

(m, 3 arom. H); 12.35 (br s, NH benzim., collapses with D₂O). Anal.

Calcd for C₁₈H₁₈N₂O₄: C, 66.24; H, 5.56; N, 8.58. Found: C, 66.11; H,

5.59; N, 8.47.

5.4.3. 5-Triﬂuoromethyl-2-(2,3,4-trimethoxyphenyl)-1H-benz-

imidazole (8)

5.4.11. 5,6-Dichloro-2-(4-methoxyphenyl)-1H-benzimidazole

(21)

Yield:73%. Mp 77–78 °C (EtOH/H₂O). ¹H NMR (DMSO-d₆): 3.86 (s,

OCH₃); 3.91 (s, OCH₃); 4.00 (s, OCH₃); 7.04 (d, J = 9.0, 1 arom. H); 7.48

(d, J = 8.6, 1 arom. H); 7.75–8.10 (m, 3 arom. H); 12.42 (br s, NH ben-

zim., collapseswithD₂O). Anal. Calcdfor C₁₇H₁₅F₃N₂O₃+ 0.25H₂O:C,

59.44; H, 4.37; N, 7.85. Found: C, 59.43; H, 4.21; N, 8.00.

Yield: 55%. Mp 218–220 °C (CH₂Cl₂). ¹H NMR (DMSO-d₆): 3.98

(s, OCH₃); 6.93 (d, J = 8.0, 2 arom. H); 7.80 (s, 2 arom. H); 7.99 (d,

J = 8.0, 2 arom. H); 13.00 (br s, NH benzim., collapses with D₂O).

Anal. Calcd for C₁₄H₁₀Cl₂N₂O: C, 57.36; H, 3.44; N, 9.56. Found: C,

57.00; H, 3.44; N, 9.50.

5.4.4. 2-(4-Methoxy-2-nitrophenyl)-5-triﬂuoromethyl-1H-benz-

imidazole (10)

5.5. 2-Phenylbenzimidazoles. Method ‘c’ of ring closure

Yield: 42%. Mp 113–114 °C (CH₂Cl₂). ¹H NMR (CDCl₃): 3.92 (s,

OCH₃); 7.18 (dd, J = 8.6, 2.7, 1 arom. H); 7.43 (d, J = 2.7, 1 arom.

H); 7.54 (s, 2 arom. H); 7.66–7.83 (m, 1 arom. H); 7.89 (d, J = 8.6,

1 arom. H); 10.94 (br s, NH, collapses with D₂O). Anal. Calcd for

C₁₅H₁₀F₃N₃O₃: C, 53.42; H, 2.99; N, 12.46. Found: C, 53.12; H,

2.92; N, 12.31.

To

a

solution of 4-triﬂuoromethyl-1,2-phenylenediamine

(5 mmol) in 7 mL of anhyd dioxane, a solution of 3,5-dim-

ethoxybenzoyl chloride (5 mmol) in 8 mL of anhyd dioxane was

added dropwise and the mixture was reﬂuxed for 7 h with stirring.

The solvent was removed, and the residue was taken up in 4 N HCl

(20 mL) and reﬂuxed for 6 h. After cooling, the acidic solution was

basiﬁed with concd NH₃and extracted with CHCl₃. The organic layer

was dried (Na₂SO₄) and evaporated to afford the benzimidazole that

was puriﬁed by CC (Al₂O₃/Et₂O).

5.4.5. 2-(2,4-Dinitrophenyl)-5-triﬂuoromethyl-1H-benzimidazole

(13)

Yield: 48%. Mp 187–188 °C (CH₂Cl₂). ¹H NMR (DMSO-d₆): 7.64

(d, J = 8.6, 1 arom. H); 7.90 (d, J = 8.6, 1 arom. H); 8.09 (s, 1 arom.

H); 8.33 (d, J = 8.6, 1 arom. H); 8.74 (dd, J = 8.6, 2.3, 1 arom. H);

8.91 (d, J = 2.3, 1 arom. H); 13.84 (br s, NH, collapses with D₂O).

Anal. Calcd for C₁₄H₇F₃N₄O₄: C, 47.74; H, 2.00; N, 15.91. Found:

C, 47.41; H, 1.71; N, 15.97.

5.5.1. 2-(3,5-Dimethoxyphenyl)-5-triﬂuoromethyl-1H-

benzimidazole (7)

Yield: 30%. Mp 182–184 °C (Et₂O). ¹H NMR (DMSO-d₆): 3.87 (s,

6H, OCH₃); 6.82 (t, J = 2.2, 1 arom. H); 7.36–7.60 (m, 3 arom. H);

7.82 (d, J = 8.6, 1 arom. H); 7.97 (s, 1 arom. H); 13.28 (br s, NH ben-

zim., collapses with D₂O). Anal. Calcd for C₁₆H₁₃F₃N₂O₂: C, 59.63;

H, 4.06; N, 8.69. Found: C, 59.50; H, 4.22; N, 8.80.

5.4.6. 2-(4-Fluorophenyl)-5-triﬂuoromethyl-1H-benzimidazole

(14)

Yield: 53%. Mp 170–171 °C (pentane). ¹H NMR (DMSO-d₆):

7.34–7.60 (m, 3 arom. H); 7.78 (d, J = 8.4, 1 arom. H); 7.96 (s, 1

arom. H); 8.15–8.33 (m, 2 arom. H); 13.24 (br s, NH, collapses with

D₂O). Anal. Calcd for C₁₄H₈F₄N₂: C, 60.01; H, 2.88; N, 10.00. Found:

C, 60.14; H, 2.60; N, 9.93.

5.6. Reduction of 2-(nitrophenyl)benzimidazoles. Method ‘a’

A solution of the suitable nitro derivative (3 mmol) in 25 mL of

EtOH was hydrogenated at rt and atmospheric pressure in the

presence of 10% Pd/C (0.1 g). After 1 h the calculated volume of

H₂was absorbed. The catalyst was removed and the solvent was

evaporated in vacuo, leaving an almost quantitative yield of amino

compound that was crystallized from EtOH or dry Et₂O.

5.4.7. 2-(2,4-Dimethoxyphenyl)-5-nitro-1H-benzimidazole (16)

Yield: 75%. Mp 268–270 °C (EtOH/H₂O). ¹H NMR (DMSO-d₆): 3.90

(s, OCH₃); 4.06 (s, OCH₃); 6.72–6.84 (m, 2 arom. H); 7.75 (d, J = 9.0, 1

arom. H); 8.08 (dd, J = 8.0, 2.7, 1 arom. H); 8.30 (d, J = 8.6, 1 arom. H);

8.48 (s, 1 arom. H); 12.48 (br s, NH benzim., collapses with D₂O).

Anal. Calcd for C₁₅H₁₃N₃O₄+ 0.25H₂O: C, 59.30; H, 4.47; N, 13.83.

Found: C, 59.64; H, 4.37; N, 13.86.

5.6.1. 2-(2-Amino-4-methoxyphenyl)-5-triﬂuoromethyl-1H-

benzimidazole (11)

Yield: 95%. Mp 184–185 °C (EtOH). ¹H NMR (CDCl₃): 3.85 (s,

OCH₃); 6.20–6.57 (m, 4 arom. H); 7.44 (d, J = 8.5, 1 arom. H);

7.52 (s, 1 arom. H); 7.86 (br s, NH₂, collapse with D₂O); 9.42 (s,

NH of benzim., collapses with D₂O). Anal. Calcd for C₁₅H₁₂F₃N₃O:

C, 58.63; H, 3.94; N, 13.68. Found: C, 58.63; H, 3.83; N, 13.70.

5.4.8. 5-Nitro-2-(4-trimethoxyphenyl)-1H-benzimidazole (17)

Yield: 76%. Mp 174–175 °C (EtOH/H₂O). ¹H NMR (DMSO-d₆): 3.86 (s,

OCH₃); 3.92 (s, OCH₃); 4.00 (s, OCH₃); 7.04 (d, J = 8.8, 1 arom. H); 7.97 (d,

J = 8.6, 1 arom. H); 7.98–8.20 (m, 2 arom. H); 8.51 (s, 1 arom. H); 12.63

(br s, NH benzim., collapses with D₂O). Anal. Calcd for C₁₆H₁₅N₃O₅: C,

58.36; H, 4.59; N, 12.76. Found: C, 58.12; H, 4.61; N, 12.85.

5.6.2. 2-(4-Aminophenyl)-5-triﬂuoromethyl-1H-benzimidazole

(36)

Yield: 53%. Mp 210–212 °C (Et₂O). ¹H NMR (DMSO-d₆): 5.77 (br

s, NH₂, collapses with D₂O); 6.84 (d, J = 8.7, 2 arom. H); 7.58–8.10

(m, 3 arom. H and 7.65, d, J = 8.7, 2 arom. H superimposed);

12.57 (br s, NH benzim., collapses with D₂O). Anal. Calcd for

C₁₄H₁₀F₃N₃: C, 60.65; H, 3.64; N, 15.16. Found: C, 60.54; H, 3.80;

N, 15.47.

5.4.9. 5-Acetyl-2-(2,4-dimethoxyphenyl)-1H-benzimidazole (18)

Yield: 75%. Mp 162–165 °C (EtOH/H₂O). ¹H NMR (DMSO-d₆):

2.64 (s, CH₃CO); 3.87 (s, OCH₃); 4.04 (s, OCH₃); 6.69–6.90 (m, 2

arom. H); 7.68–7.88 (m, 2 arom. H); 8.18–8.34 (m, 2 arom. H);

10.22 (br s, NH benzim., collapses with D₂O). Anal. Calcd for

C₁₇H₁₆N₂O₃+ 0.25H₂O: C, 67.87; H, 5.52; N, 9.31. Found: C,

67.68; H, 5.35; N, 9.07.

5.6.3. 2-(4-Aminophenyl)-1-methyl-5-triﬂuoromethyl-1H-

benzimidazole (59)

Yield: 96%. Mp 184–186 °C (EtOH). ¹H NMR (DMSO-d₆): 4.05 (s,

CH₃); 5.71 (br s, NH₂collapses with D₂O); 6.88 (d, J = 10, 2 arom.

H); 7.64–8.17 (m, 3 arom. H and 7.69, d, J = 10, 2 arom. H superim-

posed). Anal. Calcd for C₁₅H₁₂F₃N₃: C, 61.85; H, 4.15; N, 14.43.

Found: C, 61.64; H, 4.21; N, 14.43.

5.4.10. 5-Acetyl-2-(2,3,4-trimethoxyphenyl)-1H-benzimidazole

(19)

Yield: 64%. Mp 106–109 °C (EtOH/H₂O). ¹H NMR (DMSO-d₆):

2.63 (s, CH₃CO); 3.82 (s, OCH₃); 3.86 (s, OCH₃); 3.92 (s, OCH₃);

2948

M. Tonelli et al. / Bioorg. Med. Chem. 18 (2010) 2937–2953

5.6.4. 2-(4-Aminophenyl)-1-cyclohexyl-5-triﬂuoromethyl-1H-

benzimidazole (69)

5.8.3. 2-(4-Acetylaminophenyl)-1-(1-adamantyl)-5-triﬂuoromethyl-

1H-benzimidazole (73)

Yield: 99%. Mp 151–153 °C (EtOH). ¹H NMR (DMSO-d₆): 1.15–

2.39 (m, 10H, cyclohexane nucleus); 4.20–4.45 (m, 1H, cyclohexane

nucleus); 5.45 (br s, NH₂collapses with D₂O); 6.72 (d, J = 8.9 Hz, 2

arom. H); 7.35–8.18 (m, 3 arom. H and 7.79, d, J = 8.9 Hz, 2 arom. H

superimposed). Anal. Calcd for C₂₀H₂₀F₃N₃: C, 66.84; H, 5.61; N,

11.69; Found: C, 66.60; H, 5.51; N, 11.49.

Yield: 15%. Mp 267–269 °C (EtOH). ¹H NMR (DMSO-d₆): 1.52–

1.82 (m, 6H, adamantane nucleus); 2.00–2.20 (m, 9H, adamantane

nucleus and 2.09, s, CH₃CO superimposed); 7.37–7.57 (m, 1 arom.

H and 7.43, d, J = 8.0, 2 arom. H superimposed); 7.67 (d, J = 8.0, 2

arom. H); 7.97 (s, 1 arom. H); 8.20 (d, J = 8.6, 1 arom. H); 10.15

(s, NHCO, collapses with D₂O). Anal. Calcd for C₂₆H₂₆F₃N₃O: C,

68.86; H, 5.78; N, 9.27. Found: C, 68.79; H, 6.09; N, 9.10.

5.6.5. 1-(1-Adamantyl)-2-(4-aminophenyl)-5-triﬂuoromethyl-

1H-benzimidazole (72)

5.8.4. 2–4-(Propionylaminophenyl)-1H-benzimidazole (31)

Yield: 72%. Mp 285–287 °C. ¹H NMR (DMSO-d₆): 1.12 (t, J = 7.6,

3H, CH₃CH₂CO); 2.38 (q, 2H, CH₃CH₂CO); 7.10–7.24 (m, 2 arom. H);

7.48–7.64 (m, 2 arom. H); 7.78 (d, J = 8.6, 2 arom. H); 8.11 (d,

J = 8.6, 2 arom. H); 10.12 (s, NHCO, collapses with D₂O); 12.80

(br s, NH benzim., collapses with D₂O). Anal. Calcd for

C₁₆H₁₅N₃O + 0.5H₂O: C, 70.05; H, 5.88; N, 15.32. Found: C, 70.48;

H, 5.46; N, 15.35.

Yield: 98%. Mp 223–225 °C (EtOH). ¹H NMR (DMSO-d₆): 1.49–

1.77 (m, 6H, adamantane nucleus); 2.06–2.37 (m, 9H, adamantane

nucleus); 4.95 (br s, NH₂collapses with D₂O); 7.41–7.56 (m, 1

arom. H and 7.46, d, J = 9.0, 2 arom. H superimposed); 7.66 (d,

J = 8.9, 2 arom. H); 7.95 (s, 1 arom. H); 8.19 (d, J = 9.6, 1 arom.

H). Anal. Calcd for C₂₄H₂₄F₃N₃: C, 70.06; H, 5.88; N, 10.21. Found:

C, 70.23; H, 5.81; N, 10.06.

5.7. Method ‘b’

5.8.5. 2–4-(Propionylaminophenyl)-5-triﬂuoromethyl-1H-

benzimidazole (38)

The nitro compound 22 (4.0 mmol) was dissolved in 35 mL of

EtOH, and treated with a solution of SnCl₂ꢃ2H₂O (14 mmol) in

20 mL of concd HCl and the mixture was reﬂuxed for 1 h with stir-

ring. On cooling to room temperature, the precipitate was ﬁltered

and taken up with lukewarm water. The solution was neutralized

with 2 M NaOH and the precipitate was collected by ﬁltration

and crystallized from EtOH.

Yield: 60%. Mp 280–281 °C. ¹H NMR (DMSO-d₆): 1.12 (t, J = 5.8,

3H, CH₃CH₂CO); 2.38 (q, 2H, CH₃CH₂CO); 7.48–7.60 (m, 1 arom. H);

7.71–7.97 (m, 4 arom. H); 8.14 (d, J = 8.8, 2 arom. H); 10.17 (s,

NHCO, collapses with D₂O); 12.72 (br s, NH benzim., collapses with

D₂O). Anal. Calcd for C₁₇H₁₄F₃N₃O + H₂O: C, 58.12; H, 4.59; N,

11.96. Found: C, 57.92; H, 4.56; N, 11.85.

5.8.6. 5,6-Dichloro-2-(4-propionylaminophenyl)-1H-benzimidazole

(54)

5.7.1. 2-[4-(2-Amino-4-methoxyphenyl]-5,6-dichloro-1H-benz-

imidazole (23)

Yield: 73%. Mp >300 °C. ¹H NMR (DMSO-d₆): 1.12 (t, J = 6.0, 3H,

CH₃CH₂CO); 2.38 (q, 2H, CH₃CH₂CO); 7.75 (s, 2 arom. H); 7.78 (d,

J = 10, 2 arom. H); 8.08 (d, J = 10, 2 arom. H); 10.08 (s, NHCO, col-

lapses with D₂O); 12.48 (br s, NH benzim., collapses with D₂O).

Anal. Calcd for C₁₆H₁₃Cl₂N₃O + H₂O: C, 54.56; H, 4.29; N, 11.93.

Found: C, 54.32; H, 4.44; N, 11.66.

Yield: 66%. Mp 222–223 °C (EtOH). ¹H NMR (CDCl₃): 3.76 (s,

OCH₃); 6.32–6.52 (m, 2 arom. H); 7.31 (br s, NH₂, collapse with

D₂O); 7.65–7.92 (m, 3 arom. H); 12.72 (br s, NH of benzim., col-

lapses with D₂O) Anal. Calcd for C₁₄H₁₁Cl₂N₃O: C, 54.57; H, 3.60;

N, 13.64. Found: C, 54.32; H, 3.50; N, 13.73.

5.8. Acylation of 2-(4-aminophenyl)benzimidazoles with

anhydrides. Method ‘a’

5.8.7. 1-Methyl-2-(4-propionylaminophenyl)-5-triﬂuoromethyl-

1H-benzimidazole (61)

Yield: 92%. Mp 241–243 °C. ¹H NMR (DMSO-d₆): 1.13 (t, J = 7.4,

3H, CH₃CH₂CO); 2.39 (q, 2H, CH₃CH₂CO); 3.95 (s, NCH₃); 7.58–7.70

(m, 1 arom. H); 7.78–7.94 (m, 5 arom. H); 8.03 (s, 1 arom. H); 10.19

(s, NHCO, collapses with D₂O). Anal. Calcd for C₁₈H₁₆F₃N₃O: C,

62.24; H, 4.64; N, 12.10. Found: C, 62.31; H, 4.89; N, 12.02.

A suspension of the appropriate 2-(4-aminophenyl)-1-substi-

tuted-benzimidazole (1.2 mmol) in 2 mL of anhyd benzene and of

Ac₂O or propionic anhydride (3.6 mmol) was reﬂuxed for 2 h. After

removing the solvent, the residue was taken up with 5 mL of EtOH

and 1 mL of water and reﬂuxed for 1–2 h. The solvent was evapo-

rated under vacuum and the solid was taken up with water, ﬁltered

and washed with water. Acetyl derivatives and compound 71 were

crystallized from the indicated solvents.

5.8.8. 1-Cyclohexyl-2-(4-propionylaminophenyl)-5-triﬂuoro-

methyl-1H-benzimida-zole (71)

Yield: 78%. Mp 121–122 °C (EtOH/H₂O, 3:1). ¹H NMR (DMSO-

d₆): 1.10 (t, J = 7.0, 3H, CH₃CH₂CO); 1.20–2.45 (m, 10H, cyclohexane

nucleus and 2H, CH₃CH₂CO); 4.21–4.40 (m, 1H, cyclohexane nu-

cleus); 7.50–7.67 (m, 1 arom. H and d, J = 8.6, 2 arom. H); 7.83

(d, J = 8.6, 2 arom. H); 8.00–8.17 (m, 2 arom. H); 10.07 (s, NHCO,

collapses with D₂O). Anal. Calcd for C₂₃H₂₄F₃N₃O: C, 66.49; H,

5.82; N, 10.11. Found: C, 66.10; H, 6.09; N, 9.98.

5.8.1. 2-(4-Acetylaminophenyl)-1-methyl-5-triﬂuoromethyl-1H-

benzimidazole (60)

Yield: 50%. Mp 240–243 °C (EtOH). ¹H NMR (DMSO-d₆): 2.10 (s,

CH₃CO); 3.94 (s, NCH₃); 7.62 (d, J = 8.8, 1 arom. H); 7.76 (m, 5 arom.

H); 8.05 (s, 1 arom. H); 10.42 (s, NHCO, collapses with D₂O). Anal.

Calcd for C₁₇H₁₄F₃N₃O: C, 61.26; H, 4.23; N, 12.61. Found: C, 61.41;

H, 4.35; N, 12.57.

5.8.9. 1-(1-Adamantyl)-2-(4-propionylaminophenyl)-5-triﬂuoro-

methyl-1H-benzimidazole (74)

5.8.2. 2-(4-Acetylaminophenyl)-1-cyclohexyl-5-triﬂuoromethyl-

1H-benzimidazole (70)

Yield: 25%. Mp 252–254 °C. ¹H NMR (DMSO-d₆): 1.12 (t, J = 7.4,

3H, CH₃CH₂CO); 1.50–1.81 (m, 6H, adamantane nucleus); 2.00–

2.30 (m, 9H, adamantane nucleus); 2.50 (q, 2H, CH₃CH₂CO);

7.39–7.60 (m, 1 arom. H and 7.45, d, J = 8.6, 2 arom. H superim-

posed); 7.71 (d, J = 8.6, 2 arom. H); 7.97 (s, 1 arom. H); 8.21 (d,

J = 8.9, 1 arom. H); 10.09 (s, NHCO, collapses with D₂O). Anal. Calcd

for C₂₆H₂₈F₃N₃O: C, 69.36; H, 6.04; N, 8.99. Found: C, 69.35; H,

6.36; N, 8.65.

Yield: 85%. Mp 240–242 °C (Et₂O). 1.22–2.45 (m, 10H, cyclohex-

ane nucleus and 2.08, s, CH₃CO, superimposed); 4.17–4.37 (m, 1H,

cyclohexane nucleus); 6.85 (d, J = 8.2, 1 arom. H); 7.40 (d, J = 8.8, 2

arom. H); 7.68 (d, J = 8.8, 2 arom. H); 7.83–8.20 (m, 2 arom. H);

10.21 (s, NHCO, collapses with D₂O). Anal. Calcd for C₂₂H₂₂F₃N₃O:

C, 65.82; H, 5.52; N, 10.47. Found: C, 65.78; H, 5.79; N, 10.27.

M. Tonelli et al. / Bioorg. Med. Chem. 18 (2010) 2937–2953

2949

5.9. Method ‘b’

(pseudo s, 2 arom. H); 8.18 (d, J = 9.0, 4 arom. H); 9.18 (s, 2H,

NHCO, collapses with D₂O); 13.23 (br s, 2H, NH benzimidazole, col-

lapses with D₂O). Anal. Calcd for C₂₉H₁₈F₆N₆O: C, 60.00; H, 3.13; N,

14.48. Found: C, 59.80; H, 3.69; N, 14.55.

To a suspension of 2-(4-aminophenyl)-5-triﬂuoromethylbenz-

imidazole (2 mmol) in 10 mL of anhyd benzene, succinic anhydride

(2 mmol) was added and the mixture was reﬂuxed for 18 h with

stirring. After cooling the precipitate was collected by ﬁltration

and crystallized from anhyd benzene.

5.12. Method ‘c’

To an ice-cooled suspension of the suitable 2-(4-aminophe-

nyl)benzimidazole derivative (2–6 mmol) in anhyd benzene (4–

22 mL) and of triethylamine (2–6 mmol), a solution of chloroacetyl

chloride (2–6 mmol) in 6 mL of anhyd benzene was added drop-

wise under N₂and with stirring. The mixture was reﬂuxed for

6 h and then evaporated to dryness; the residue was taken up with

water, ﬁltered and washed with water. Compound 75 was crystal-

lized from EtOH/H₂O.

5.9.1. 2-(Succinylaminophenyl)-5-triﬂuoromethyl-1H-benzimi-

dazole (48)

Yield: 88%. Mp >300 °C (benzene). ¹H NMR (DMSO-d₆): 2.44–

2.56 (m, 4H, CH₂); 7.49 (d, J = 9.6, 1 arom. H); 7.72 (pseudo s, 1

arom. H); 7.78 (d, J = 10.6, 2 arom. H); 7.86 (pseudo s, 1 arom.

H); 8.17 (d, J = 10.6, 2 arom. H); 10.33 (s, NHCO, collapses with

D₂O); 13.01 (br s, 2H, NH of benzimidazole and COOH, collapse

with D₂O). Anal. Calcd for C₁₈H₁₄F₃N₃O₃: C, 57.30; H, 3.74; N,

11.14. Found: C, 57.14; H, 3.87; N, 10.95.

5.12.1. 2-(4-Chloroacetylaminophenyl)-5-triﬂuoromethyl-1H-

benzimidazole (39)

5.10. Acylation of 2-(4-aminophenyl)benzimidazoles with

acylchlorides. Method ‘a’

Yield: 74%. Mp >300 °C. ¹H NMR (DMSO-d₆): 4.36 (s, CH₂Cl);

7.63 (d, J = 8.8, 1 arom. H); 7.8–8.31 (m, 6 arom. H); 10.80 (s, NHCO,

collapses with D₂O); 12.68 (br s, NH benzim., collapses with D₂O).

Anal. Calcd for C₁₆H₁₁ClF₃N₃O + H₂O: C, 51.70; H, 3.52; N, 11.30.

Found: C, 52.09; H, 4.05; N, 11.52.

To an ice-cooled solution of homolupinanoyl chloride hydro-

chloride^21,22(3 mmol) in 8 mL of anhyd CHCl₃, a solution of 2-(4-

aminophenyl)-5-substituted benzimidazole (3 mmol) in 22 mL of

anhyd CHCl₃and triethylamine (3 mmol) were added under N₂

and with stirring. The mixture was reﬂuxed for 6 h. After cooling,

the solvent was removed, water was added and the acidic solution

was neutralized with 2 M NaOH and then shaken with CH₂Cl₂. The

organic layer was dried (Na₂SO₄) and evaporated to afford an oily

residue that was crystallized from dry Et₂O.

5.12.2. 2-(4-Chloroacetylaminophenyl)-5,6-dichloro-1H-benz-

imidazole (52**)

Yield: 96%. Mp >300 °C. ¹H NMR (DMSO-d₆): 4.31 (s, CH₂Cl);

7.85 (d, J = 8.7, 2 arom. H and 7.87, s, 2 arom. H superimposed);

8.14 (d, J = 8.7, 2 arom. H); 10.08 (s, NHCO, collapses with D₂O);

12.48 (br s, NH benzim., collapses with D₂O). Anal. Calcd for

C₁₅H₁₀Cl₃N₃O: C, 50.80; H, 2.84; N, 11.85. Found: C, 51.12; H,

2.98; N, 11.62.

5.10.1. 2-{4-[(1S,9aR)-Octahydro-2H-quinolizin-1-ylacetylamino-

phenyl}-1H-benzimidazole (35)

Yield: 32%. Mp 265–270 °C (Et₂O). ¹H NMR (DMSO-d₆): 1.02–

2.76 (m, 16H, octahydro-2H-quinolizine and 2H, CH₂CO); 7.20–

7.36 (m, 2 arom. H); 7.45–7.76 (m, 2 arom. H); 7.82 (d, J = 8.8, 2

arom. H); 8.15 (d, J = 8.8, 2 arom. H); 10.18 (s, NHCO, collapses with

D₂O); 13.16 (br s, NH of benzim., collapses with D₂O). Anal. Calcd

for C₂₄H₂₈N₄O + 0.75H₂O: C, 71.70; H, 7.40; N, 13.94. Found: C,

71.76; H, 7.16; N, 14.33.

5.12.3. 2-(4-Chloroacetylaminophenyl)-1-methyl-5-triﬂuoro-

methyl-1H-benzimida- zole (59**)

Yield: 75%. Mp >300 °C. ¹H NMR (DMSO-d₆): 3.95 (s, CH₃); 4.33

(s, CH₂); 7.6 (d, J = 8.8, 1 arom. H); 7.78–8.08 (m, 6 arom. H); 10.63

(s, NHCO, collapses with D₂O). Anal. Calcd for C₁₇H₁₃ClF₃N₃O: C,

55.52; H, 3.56; N, 11.43. Found: C, 55.23; H, 3.76; N, 11.22.

5.12.4. 1-(1-Adamantyl)-2-(4-chloroacetylaminophenyl)-5-

triﬂuoromethyl-1H-benzimidazole (75)

5.10.2. 2-{4-[(1S,9aR)-Octahydro-2H-quinolizin-1-ylacetyl-

aminophenyl}-5-triﬂuoromethyl-1H-benzimidazole (47)

Yield: 19%. Mp 252–255 °C (Et₂O). ¹H NMR (DMSO-d₆): 1.04–

2.80 (m, 16H, octahydro-2H-quinolizine and 2H, CH₂CO); 7.55 (d,

J = 9.6, 1 arom. H); 7.73–8.0 (m, 2 arom. H and, 7.82, d, J = 9.0, 2

arom. H superimposed); 8.14 (d, J = 9.0, 2 arom. H); 10.24 (s, NHCO,

collapses with D₂O); 13.24 (br s, NH of benzim., collapses with

D₂O). Anal. Calcd for C₂₅H₂₇F₃N₄O: C, 65.78; H, 5.96; N, 12.27.

Found: C, 65.79; H, 6.33; N, 12.23.

Yield: 75%. Mp >300 °C (EtOH/H₂O). ¹H NMR (DMSO-d₆): 1.50–

1.81 (m, 6H, adamantane nucleus); 2.08- 2.41 (m, 9 H, adamantane

nucleus); 4.31, (s, CH₂CO); 7.40–7.58 (m, 1 arom. H and 7.46, d,

J = 8.2, 2 arom. H superimposed); 7.71 (d, J = 8.2, 2 arom. H); 7.98

(s, 1 arom. H); 8.10 (d, J = 8.6, 1 arom. H); 10.57 (s, NHCO, collapses

with D₂O). Anal. Calcd for C₂₆H₂₅ClF₃N₃O: C, 64.00; H, 5.16; N, 8.61.

Found: C, 64.18; H, 5.24; N, 8.32.

5.13. 2-[4-(Aminoacetyl)amino]phenylbenzimidazoles

5.11. Method ‘b’

A

solution of 2-(4-chloroacetylaminophenyl)benzimidazole

To a suspension of 2-(4-aminophenyl)-5-triﬂuoromethylbenz-

imidazole (2 mmol) in 10 mL of anhyd benzene and of triethylamine

(2 mmol), cooled in an ice bath, a solution of trichloromethyl chloro-

formiate (1 mmol) in 5 mL of anhyd benzene was added dropwise,

under N₂and with stirring. The mixture was reﬂuxed for 5 h with

stirring. After removing the solvent, the precipitate was taken up

withwater, ﬁlteredandwashedwithwater. Theresiduewaspuriﬁed

by CC (Al₂O₃/ethyl acetate+2% MeOH).

derivative (1.5 mmol) and the appropriate amine (3.0 mmol) in

15 mL of EtOH was reﬂuxed under N₂for 18 h with stirring. The

solvent was removed and the residue was taken up with water, ﬁl-

tered, dried and puriﬁed by CC (Al₂O₃/CH₂Cl₂). Compounds were

further puriﬁed by rinsing the eluate residue with Et₂O or CH₂Cl₂.

Compounds 32 and 43 were prepared using benzene as solvent, in-

stead of EtOH, while in the case of compounds 55, 56 and 57 the chlo-

roacetyl derivative was treated with a large excess of secondary amine.

5.11.1. 1,3-Bis-{4-[(5-triﬂuoromethyl)-1H-benzimidazol-2-

yl]phenyl}urea (49)

5.13.1. 2-[4-(1-Pyrrolidinyl)acetylaminophenyl]-1H-

benzimidazole (32)

Yield: 43%. Mp >300 °C. ¹H NMR (DMSO-d₆): 7.51 (d, J = 10.6, 2

arom. H); 7.69 (d, J = 9.0, 4 arom. H); 7.78 (m, 2 arom. H); 7.91

Yield: 38%. Mp 250–252 °C (toluene). ¹H NMR (DMSO-d₆): 170–

1.182 (m, 4H, CH₂of pyrrolidine nucleus); 2.61–2.78 (m, 4H, CH₂of

2950

M. Tonelli et al. / Bioorg. Med. Chem. 18 (2010) 2937–2953

pyrrolidine nucleus); 3.37 (s, CH₂CO); 7.10–7.32 (m, 2 arom. H);

7.40–7.76 (m, 2 arom. H); 7.83 (d, J = 8.8, 2 arom. H); 8.13 (d,

J = 8.8, 2 arom. H); 10.04 (s, NHCO, collapses with D₂O); 12.68 (s,

NH of benzim., collapses with D₂O). Anal. Calcd for C₁₉H₂₀N₄O: C,

71.22; H, 6.29; N, 17.49. Found: C, 70.94; H, 6.05; N, 17.84.

s, NH benzim., collapses with D₂O). Anal. Calcd for C₂₀H₁₉F₃N₄OS:

C, 57.13; H, 4.55; N, 13.33. Found: C, 56.86; H, 4.31; N, 12.99.

5.13.8. 2-[4-(4-Phenylpiperazin-1-yl)acetylaminophenyl]-5-

triﬂuoromethyl-1H-benzimidazole (46)

Yield: 44%. Mp 232–236 °C (Et₂O). ¹H NMR (DMSO-d₆): 2.72

(pseudo s, 4H, CH₂of piperazine); 3.23 (s, CH₂CO); 3.63 (pseudo

s, 4H, CH₂of piperazine); 6.68–7.32 (m, 5 arom. H); 7.53 (d,

J = 9.0, 1 arom. H); 7.65–8.04 (m, 2 arom. H and 7.90, d, J = 8.7, 2

arom. H superimposed); 8.18 (d, J = 8.7, 2 arom. H); 10.11 (s, NHCO,

collapses with D₂O); 13.31 (br s, NH benzim., collapses with D₂O).

Anal. Calcd for C₂₆H₂₄F₃N₅O + 0.25H₂O: C, 64.52; H, 5.10; N, 14.47.

Found: C, 64.54; H, 5.32; N, 14.54.

5.13.2. 2-[4-(1-Adamantylamino)acetylaminophenyl]-5-tri-

ﬂuoromethyl-1H-benzimidazole (40)

Yield: 41%. Mp 279–282 °C (CH₂Cl₂). ¹H NMR (DMSO-d₆): 1.44–

1.77 (m, 12H, adamantane nucleus); 1.92–2.15 (m, 3H, adaman-

tane nucleus); 3.25 (s, CH₂CO); 4.20 (br s, NH-adamantane, col-

lapses with D₂O); 7.50 (d, J = 8.9, 1 arom. H); 7.75 (d, J = 8.2, 1

arom. H); 7.80–7.96 (m, 1 arom. H and 7.84, d, J = 8.6, 2 arom.

H); 8.17 (d, J = 8.6, 2 arom. H); 10.20 (s, NHCO, collapses with

D₂O); 12.46 (s, NH of benzim., collapses with D₂O). Anal. Calcd

for C₂₆H₂₇F₃N₄O: C, 66.65; H, 5.81; N, 11.96. Found: C, 66.42; H,

6.11; N, 11.79.

5.13.9. 5,6-Dichloro-2-[4-(1-pyrrolidinyl)acetylaminophenyl]-

1H-benzimidazole (55)

Yield: 46%. Mp 253–256 °C (Et₂O). ¹H NMR (DMSO-d₆): 1.88 (t,

J = 6.8, 4H, CH₂of pyrrolidine nucleus); 2.70 (t, J = 6.8, 4H, CH₂of pyr-

rolidine nucleus); 3.29 (s, CH₂CO); 7.56–7.85 (m, 4 arom. H); 8.00 (d,

J = 8.6, 2 arom. H);9.98 (s, NHCO, collapseswithD₂O);12.51 (brs, NH

benzim., collapses with D₂O). Anal. Calcd for C₁₉H₁₈Cl₂N₄O + H₂O: C,

56.03; H, 4.64; N, 13.76. Found: C, 56.45; H, 4.77; N, 13.66.

5.13.3. 2-[4-(Diethylamino)acetylaminophenyl]-5-triﬂuoro-

met hyl-1H-benzimida-zole (41)

Yield: 45%. Mp 209–211 °C (CH₂Cl₂). ¹H NMR (DMSO-d₆): 1.07

(t, J = 7.2, 6H, CH₃of N(C₂H₅)₂); 2.58 (q, 4H, CH₂of N(C₂H₅)₂);

3.30 (s, CH₂CO); 7.48 (d, J = 8.6, 1 arom. H); 7.61–8.00 (m, 2 arom.

H and 7.83, d, J = 8.9, 2 arom. H); 8.08 (d, J = 8.9, 2 arom. H); 10.18

(s, NHCO, collapses with D₂O); 13.11 (s, NH of benzim., collapses

with D₂O). Anal. Calcd for C₂₀H₂₁F₃N₄O: C, 61.53; H, 5.42; N,

14.35. Found: C, 61.16; H, 5.60; N, 14.17.

5.13.10. 5,6-Dichloro-2-[4-(1-piperidinyl)acetylaminophenyl]-

1H-benzimidazole (56)

Yield: 39%. Mp 259–261 °C (CH₂Cl₂). ¹H NMR (DMSO-d₆): 1.38–

1.71 (m, 6H, CH₂of piperidine nucleus); 2.60 (t, J = 7.2, 4H, CH₂of

piperidine nucleus); 3.12 (s, CH₂CO); 7.64–7.96 (m, 4 arom. H);

8.05 (d, J = 8.8, 2 arom. H); 9.95 (s, NHCO, collapses with D₂O);

12.40 (br s, NH benzim., collapses with D₂O). Anal. Calcd for

C₂₀H₂₀Cl₂N₄O: C, 59.56; H, 5.00; N, 13.89. Found: C, 59.88; H,

4.92; N, 13.50.

5.13.4. 2-[4-(1-Pyrrolidinyl)acetylaminophenyl]-5-triﬂuoro-

methyl-1H-benzimida-zole (42)

Yield: 30%. Mp 242–243 °C (CH₂Cl₂). ¹H NMR (DMSO-d₆): 1.81

(t, J = 7.2, 4H, CH₂of pyrrolidine nucleus); 2.76 (t, J = 7.2, 4H, CH₂

of pyrrolidine nucleus); 3.40 (s, CH₂CO); 7.44 (d, J = 8.9, 1 arom.

H); 7.70–8.00 (m, 2 arom. H and 7.86, d, J = 8.6, 2 arom. H superim-

posed); 8.16 (d, J = 8.6, 2 arom. H); 10.08 (s, NHCO, collapses with

D₂O); 13.30 (br s, NH benzim., collapses with D₂O). Anal. Calcd for

C₂₀H₁₉F₃N₄O: C, 61.85; H, 4.93; N, 14.43. Found: C, 61.66; H, 4.97;

N, 14.06.

5.13.11. 5,6-Dichloro-2-[4-(4-morpholino)acetylaminophenyl]-

1H-benzimidazole (57)

Yield: 38%. Mp 274–276 °C (CH₂Cl₂). ¹H NMR (DMSO-d₆): 2.55

(t, J = 6.8, 4H, CH₂of morpholine nucleus); 3.18 (s, CH₂CO); 3.66

(t, J = 6.8, 4H, CH₂of morpholine nucleus); 7.77–8.03 (m, 4 arom.

H); 8.15 (d, J = 9.4, 2 arom. H); 10.04 (s, NHCO, collapses with

D₂O); 13.45 (br s, NH benzim., collapses with D₂O). Anal. Calcd

for C₁₉H₁₈Cl₂N₄O₂: C, 56.30; H, 4.48; N, 13.83. Found: C, 55.95;

H, 4.55; N, 13.58.

5.13.5. 2-[4-(1-Piperidinyl)acetylaminophenyl]-5-triﬂuoro-

methyl-1H-benzimidazole (43)

Yield: 37%. Mp 240–241 °C (CH₂Cl₂). ¹H NMR (DMSO-d₆): 1.35–

1.66 (m, 6H, CH₂of piperidine); 2.37–2.60 (m, 4H, CH₂of piperi-

dine); 3.14 (s, CH₂CO); 7.48 (d, J = 8.9, 1 arom. H); 7.61–8.05 (m,

2 arom. H and 7.84 d, J = 8.7, 2 arom. H); 8.16 (d, J = 8.7, 2 arom.

H); 9.97 (s, NHCO, collapses with D₂O); 13.30 (br s, NH benzim.,

collapses with D₂O). Anal. Calcd for C₂₁H₂₁F₃N₄O: C, 62.67; H,

5.26; N, 13.92. Found: C, 62.79; H, 5.41; N, 13.80.

5.13.12. 5,6-Dichloro-2-[4-(4-methylpiperazin-1-

yl)acetylaminophenyl]-1H-benzimidazole (58)

Yield: 32%. Mp 272–275 °C (CH₂Cl₂). ¹H NMR (DMSO-d₆): 2.15

(s, NCH₃of piperazine); 2.39 (pseudo s, 4H, CH₂of piperazine);

2.50 (pseudo s, 4H, CH₂of piperazine); 3.14 (s, CH₂CO); 7.80 (s, 2

arom. H); 7.83 (d, J = 9.6, 2 arom. H); 8.10 (d, J = 9.6, 2 arom. H);

9.92 (s, NHCO, collapses with D₂O); 13.17 (br s, NH benzim., col-

lapses with D₂O). Anal. Calcd for C₂₀H₂₁Cl₂N₅O + 0.5H₂O: C,

56.21; H, 5.19; N, 16.39. Found: C, 56.72; H, 5.11; N, 16.30.

5.13.6. 2-[4-(4-Morpholino)acetylaminophenyl]-5-triﬂuoro-

methyl-1H-benzimida-zole (44)

Yield: 46%. Mp 234–236 °C (CH₂Cl₂). ¹H NMR (DMSO-d₆): 2.56

(t, J = 6.5, 4H, CH₂of morpholine); 3.19 (s, CH₂CO); 3.68 (t, J = 6.5,

4H, CH₂of morpholine); 7.50 (d, J = 8.6, 1 arom. H); 7.64–8.05

(m, 2 arom. H and 7.84 d, J = 8.8, 2 arom. H); 8.14 (d, J = 8.7, 2 arom.

H); 10.03 (s, NHCO, collapses with D₂O); 13.22 (br s, NH benzim.,

collapses with D₂O). Anal. Calcd for C₂₀H₁₉F₃N₄O₂: C, 59.40; H,

4.74; N, 13.85. Found: C, 59.22; H, 4.87; N, 13.74.

5.13.13. 2-[4-(Diethylamino)acetylaminophenyl]-1-methyl-5-

triﬂuoromethyl-1H-benzimidazole (62)

Yield: 50%. Mp 116–118 °C (CH₂Cl₂). ¹H NMR (DMSO-d₆): 1.04

(t, J = 7.0, 6H, CH₃of N(C₂H₅)₂); 2.61 (q, 4H, CH₂of N(C₂H₅)₂);

3.22 (s, CH₂CO); 3.96 (s, NCH₃); 7.50 (d, J = 8.9, 1 arom. H); 7.78–

7.96 (m, 5 arom. H); 8.05 (s, 1 arom. H); 9.97 (s, NHCO, collapses

with D₂O). Anal. Calcd for C₂₁H₂₃F₃N₄O: C, 62.37; H, 5.73; N,

13.85. Found: C, 62.07; H, 5.72; N, 13.72.

5.13.7. 2-[4-(4-Thiomorpholino)acetylaminophenyl]-5-triﬂuoro-

methyl-1H-benzimidazole (45)

Yield: 35%. Mp 240–242 °C (Et₂O). ¹H NMR (DMSO-d₆): 2.63–

2.85 (m, 8H, CH₂of thiomorpholine); 3.22 (s, CH₂CO); 7.52 (d,

J = 9.0, 1 arom. H); 7.75 (d, J = 8.5, 1 arom. H); 7.82–8.00 (m, 1

arom. H and 7.88, d, J = 8.7, 2 arom. H superimposed); 8.16 (d,

J = 8.7, 2 arom. H); 9.97 (s, NHCO, collapses with D₂O); 13.26 (br

5.13.14. 1-Methyl-2-[4-(1-pyrrolidinyl)acetylaminophenyl]-5-

triﬂuoromethyl-1H-benzimidazole (63)

Yield: 38%. Mp 156–158 °C (CH₂Cl₂). ¹H NMR (CDCl₃): 1.84–1.95

(m, 4H, CH₂of pyrrolidine nucleus); 2.68–2.80 (m, 4H, CH₂of pyr-

M. Tonelli et al. / Bioorg. Med. Chem. 18 (2010) 2937–2953

2951

rolidine nucleus); 3.34 (s, CH₂CO); 3.93 (s, NCH₃); 7.46 (d, J = 9.0, 1

5.14. Cell-based assays

5.14.1. Compounds

Compounds were dissolved in DMSO at 100 mM and then di-

luted in culture medium.

arom. H); 7.54–7.63 (m, 1 arom. H); 7.72–7.91 (m, 4 arom. H); 8.10

(s, 1 arom. H); 9.38 (s, NHCO, collapses with D₂O). Anal. Calcd for

C₂₁H₂₁F₃N₄O: C, 62.68; H, 5.26; N, 13.92. Found: C, 62.77; H,

5.26; N, 13.54.

5.13.15. 1-Methyl-2-[4-(1-piperidinyl)acetylaminophenyl]-5-

triﬂuoromethyl-1H-benzimidazole (64)

5.14.2. Cells and viruses

Cell lines were purchased from American Type Culture Collec-

tion (ATCC). The absence of mycoplasma contamination was

checked periodically by the Hoechst staining method. Cell lines

supporting the multiplication of RNA and DNA viruses were the

following: CD4⁺human T-cells containing an integrated HTLV-1

genome (MT-4); Madin Darby Bovine Kidney (MDBK); Baby Ham-

ster Kidney (BHK-21) and Monkey kidney (Vero 76) cells. Viruses

were purchased from American Type Culture Collection (ATCC) ex-

cept Yellow Fever Virus (YFV), and Human Immunodeﬁciency

Virus type-1 (HIV-1). Viruses representative of positive-sense sin-

gle-strand RNA (ssRNA⁺) group used were: (i) Retroviridae family:

the laboratory strain HIV-1_IIIBwild-type, obtained from the super-

natant of the persistently infected H9/III_Bcells (NIH 1983); (ii) Fla-

viviridae family: YFV strain 17-D vaccine (Stamaril Pasteur J07B01)

and Bovine Viral Diarrhea Virus (BVDV) strain NADL (ATCC VR-

534); (iii) Picornaviridae family: Human Coxsackievirus type B2

(CVB-2) strain Ohio-1 (ATCC VR-29) and Human Poliovirus type-

1 Sabin (Sb-1) strain Chat (ATCC VR-1562). Viruses representative

of a negative-sense single-strand RNA (ssRNA^ꢁ) group used were:

Paramyxoviridae family: Vesicular Stomatitis Virus (VSV) strain

Indiana Lab (ATCC VR-158) and Human Respiratory Syncytial Virus

(RSV) strain A2 (ATCC VR-1540). A virus representative of a double-

strand RNA (dsRNA) group used was: Reovirus type-1 (Reo-1)

strain 3651 (ATCC VR-214). Viruses representatives of DNA group

used were: (i) Poxviridae family: Vaccinia Virus (VV) strain Els-

tree-Lister Vaccine (ATCC VR-1549); (ii) Herpesviridae family: Hu-

man Herpesvirus 1 (HSV-1) strain KOS (ATCC VR-1493).

Yield: 48%. Mp 161–163 °C (CH₂Cl₂). ¹H NMR (CDCl₃): 1.44–1.77

(m, 6H, CH₂of piperidine nucleus); 2.63 (t, J = 7.0, 4H, CH₂of piper-

idine nucleus); 3.14 (s, CH₂CO); 3.93 (s, CH₃); 7.44 (d, J = 9.2, 1

arom. H); 7.53–7.63 (m, 1 arom. H); 7.80 (pseudo s, 4 arom. H);

8.10 (s, 1 arom. H); 9.54 (s, NHCO, collapses with D₂O). Anal. Calcd

for C₂₂H₂₃F₃N₄O: C, 63.45; H, 5.57; N, 13.45. Found: C, 63.51; H,

5.46; N, 13.51.

5.13.16. 1-Methyl-2-[4-(4-morpholino)acetylaminophenyl]-5-

triﬂuoromethyl-1H-benzimidazole (65)

Yield: 49%. Mp 224–228 °C (Et₂O). ¹H NMR (CDCl₃): 2.70 (t,

J = 5.8, 4H, CH₂of morpholine); 3.22 (s, CH₂CO); 3.84 (t, J = 5.8,

4H, CH₂of morpholine); 3.93 (s, NCH₃); 7.44 (d, J = 8.9, 1 arom.

H); 7.59 (d, J = 8.8, 1 arom. H); 7.77–7.83 (m, 4 arom. H); 8.12

(dd, J = 9.0, 1.2, 1 arom. H); 9.38 (s, NHCO, collapses with D₂O).

Anal. Calcd for C₂₁H₂₁F₃N₄O₂+ 0.5H₂O: C, 59.01; H, 5.19; N,

13.33. Found: C, 58.99; H, 5.49; N, 13.02.

5.13.17. 1-Methyl-2-[4-(4-thiomorpholino)acetylaminophenyl]-

5-triﬂuoromethyl-1H-benzimidazole (66)

Yield: 73%. Mp 178–180 °C (Et₂O). ¹H NMR (DMSO-d₆): 2.64–

2.87 (m, 8H, thiomorpholine nucleus); 3.21 (s, CH₂CO); 3.98 (s,

NCH₃); 7.60 (d, J = 8.7, 1 arom. H); 7.80–7.97 (m, 5 arom. H);

8.03 (s, 1 arom. H); 10.00 (s, NHCO, collapses with D₂O). Anal. Calcd

for C₂₁H₂₁F₃N₄OS: C, 58.05; H, 4.87; N, 12.90. Found: C, 58.12; H,

5.03; N, 12.84.

5.14.3. Cytotoxicity assays

5.13.18. 1-Methyl-2-[4-(4-methyl-1-piperazinyl)acetylamino-

phenyl]-5-triﬂuoromethyl-1H-benzimidazole (67)

Cytotoxicity assays were run in parallel with antiviral assays.

Exponentially growing MT-4 cells were seeded at an initial density

of 1 ꢄ 10⁵cells/mL in 96-well plates in RPMI-1640 medium sup-

plemented with 10% fetal calf serum (FCS), 100 units/mL penicillin

Yield: 52%. Mp 159–161 °C (Et₂O). ¹H NMR (DMSO-d₆): 2.17 (s,

NCH₃of piperazine); 2.26–2.40 (m, 8H, piperazine nucleus); 3.18

(s, CH₂CO); 3.97 (s, CH₃); 7.60 (d, J = 8.6, 1 arom. H); 7.80–7.96

(m, 5 arom. H); 8.02 (s, 1 arom. H); 10.02 (s, NHCO, collapses with

D₂O). Anal. Calcd for C₂₂H₂₄F₃N₅O: C, 61.24; H, 5.61; N, 16.23.

Found: C, 61.30; H, 5.49; N, 16.51.

G and 100 lg/mL streptomycin. Cell cultures were then incubated

at 37 °C in a humidiﬁed, 5% CO₂atmosphere in the absence or pres-

ence of serial dilutions of test compounds. Cell viability was deter-

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

diphenyl-tetrazolium bromide (MTT) method.³⁸

5.13.19. 1-Methyl-2-[4-(4-phenyl-1-piperazinyl)acetyl-

aminophenyl]-5-triﬂuoro-methyl-1H-benzimidazole (68)

Yield: 80%. Mp 165–167 °C (Et₂O). ¹H NMR (DMSO-d₆): 2.63–

2.80 (m, 4H, piperazine nucleus); 3.18–3.35 (m, 4H, piperazine nu-

cleus and 3.27, s, CH₂CO superimposed); 3.98 (s, NCH₃); 6.70–7.30

(m, 5 arom. H); 7.42 (d, J = 8.8, 1 arom. H); 7.81–7.98 (m, 5 arom.

H); 8.03 (s, 1 arom. H); 10.10 (s, NHCO, collapses with D₂O). Anal.

Calcd for C₂₇H₂₆F₃N₅O: C, 65.71; H, 5.31; N, 14.19. Found: C, 65.94;

H, 5.63; N, 14.24.

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

and 1 ꢄ 10⁶cells/mL in 96-well plates, respectively, in culture

medium (Minimum Essential Medium with Earle’s salts (MEM-E)

with

L-glutamine, supplemented with 10% horse serum and

1 mM sodium pyruvate (for MDBK cells) or with 10% fetal bovine

serum (FBS) (for BHK cells), 1% kanamycin). Cell cultures were then

incubated at 37 °C in a humidiﬁed, 5% CO₂atmosphere in the ab-

sence or presence of serial dilutions of test compounds. Cell viabil-

ity was determined after 48–96 h at 37 °C by the MTT method.

5.13.20. 1-Adamantyl-2-[4-(4-phenyl-1-piperazinyl)acety-

laminophenyl]-5-triﬂuoro-methyl-1H-benzimidazole (76)

Yield:46%. Mp 238–241 °C (Et₂O). ¹H NMR(CDCl₃): 1.70–1.85(m,

6H, adamantane nucleus); 2.16–2.41 (m, 9H, adamantane nucleus);

2.78–2.94 (m, 4H, piperazine nucleus); 3.20–3.45 (m, 4H, piperazine

nucleus and 3.28, s, CH₂CO superimposed); 6.85–7.38 (m, 5 arom.

H); 7.42–7.58 (m, 1 arom. H and 7.45, d, J = 8.8, 2 arom. H); 7.70 (d,

J = 8.8, 2 arom. H); 7.92 (d, J = 9.2, 1 arom. H); 8.04 (s, 1 arom. H);

9.35 (s, NHCO, collapses with D₂O). Anal. Calcd for C₃₆H₃₈F₃N₅O: C,

70.45; H, 6.24; N, 11.41. Found: C, 70.66; H, 6.38; N, 11.17.

5.14.4. Antiviral assay

Compounds activity against HIV-1 was based on inhibition of

virus-induced cytopathogenicity in MT-4 cells acutely infected

with a multiplicity of infection (m.o.i.) of 0.01. Brieﬂy, 50 lL of

RPMI containing 1 ꢄ 10⁴MT-4 cells were added to each well of

ﬂat-bottom microtitre trays containing 50 lL of RPMI, without or

with serial dilutions of test compounds. Then, 20 lL of a HIV-1 sus-

pension containing 100 CCID₅₀were added. After a 4-day incuba-

tion at 37 °C, cell viability was determined by the MTT method.

2952

M. Tonelli et al. / Bioorg. Med. Chem. 18 (2010) 2937–2953

Compounds activity against YFV and Reo-1 was based on inhi-

A₆₀₀reached 0.6–0.7. These cultures were then induced overnight

with 1 mM isopropyl-b- -thiogalactopyranoside. The cells were

bition of virus-induced cytopathogenicity in BHK-21 cells acutely

infected with a m.o.i. of 0.01. Compounds activity against BVDV

was based on inhibition of virus-induced cytopathogenicity in

MDBK cells acutely infected with a m.o.i. of 0.01. Brieﬂy, BHK

and MDBK cells were seeded in 96-well plates at a density of

5 ꢄ 10⁴and 3 ꢄ 10⁴cells/well, respectively, and were allowed to

form conﬂuent monolayers by incubating overnight in growth

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

D

harvested by centrifugation and stored at ꢁ80 °C until the

puriﬁcation.

5.14.7. Expression of HCV1b-NS5B

The gene coding for the C-terminal 21-amino-acid-deleted

NS5B polymerase (NS5B 21) of HCV BK strain (genotype 1b) C-ter-

D21 polymerase

D

minally fused with a 6xHis-tag was cloned between the BamHI and

XhoI cloning sites of the pET-21a(+) expression plasmid (Novagen).

The construct encoding the 6xHis-tagged HCV1b-NS5BD21 protein

under the control of the T7 RNA polymerase promoter was con-

ﬁrmed by dideoxynucleotide sequencing and transformed into

the E. coli strain Rosetta™ 2(DE3)pLysS (Novagene). A single colony

layers were then infected with 50

maintenance medium (MEM-E with

with 0.5% inactivated FBS and 1 mM sodium pyruvate, 1% kanamy-

cin) to give an m.o.i of 0.01. After 1 h, 50 L of maintenance med-

l

L of a proper virus dilution in

L-glutamine, supplemented

l

ium, without or with serial dilutions of test compounds, were

added. After a 3–4-day incubation at 37 °C, cell viability was deter-

mined by the MTT method.

Compounds activity against CVB-2, Sb-1, VSV, VV, HSV-1 and

RSV was determined by plaque reduction assays in infected Vero

76 cell monolayers. To this end, Vero 76 cells were seeded in 24-

well plates at a density of 2 ꢄ 10⁵cells/well and were allowed to

form conﬂuent monolayers by incubating overnight in growth

expressing the 6xHis-tagged HCV1b-NS5B

lected and cultured in 5 mL of LB medium supplemented with

100 g/mL ampicillin and 30 g/mL chloramphenicol at 30 °C

D21 protein was se-

l

overnight. The culture was diluted into 1 L of the same culture

medium and incubated at 30 °C until the absorbance reached

0.6–0.7 at 600 nm. The culture was then induced overnight at

25 °C with 1 mM isopropyl-b-D-thiogalactopyranoside. The cells

medium (Dulbecco’s Modiﬁed Eagle Medium (D-MEM) with

tamine and 4500 mg/L -glucose, supplemented with 10% fetal bo-

vine serum and 1% Kanamycin) at 37 °C in a humidiﬁed CO₂(5%)

atmosphere. Then, monolayers were infected for 2 h with 250

of proper virus dilutions to give 50–100 PFU/well. Following re-

moval of unadsorbed virus, 500 L of maintenance medium (D-

MEM medium with -glutamine and 4500 mg/L -glucose supple-

mented with 1% inactivated FBS and 0.75% methyl-cellulose), with-

out 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 and EC₅₀(50% effective concentration) was cal-

culated by linear regression technique. The cytotoxicity of test

compounds was determined in parallel on the same 24-well plate

used for the EC₅₀determination.

L

-glu-

were harvested by centrifugation and stored at ꢁ80 °C until the

D

puriﬁcation.

lL

5.14.8. Puriﬁcation of NS5B proteins

The cell pellets were thawed and immediately lysed by the

addition of 10 mL of CelLytic B (Sigma). Any insoluble material

was removed by centrifugation at 11,000 rpm 4 °C for 60 min.

The soluble extract was applied to a 5-mL column of nickel–nitrilo-

triacetic acid–agarose (Qiagen) previously equilibrated with the ly-

sis buffer (50 mM NaH₂PO₄, 300 mM NaCl, 10 mM imidazole

pH8.0). The column was washed extensively with the wash buffer

(50 mM NaH₂PO₄, 300 mM NaCl, 20 mM imidazole pH 8.0) and

then, the proteins were eluted stepwise with the elution buffer

containing increasing concentration of imidazole (50 mM NaH_2-

PO₄, 300 mM NaCl, 50–250 mM imidazole pH 8.0). The polypeptide

composition of the column fractions was monitored by Coomassie

stained SDS–PAGE analysis. Fractions enriched in pure 6xHis-

tagged NS5B proteins recovered in the 130–250 mM imidazole elu-

ates were pooled and dialyzed against the buffer containing 25 mM

Tris–HCl, pH 7.5, 2.5 mM MgCl₂, 1 mM dithiothreitol, 50% glycerol.

The protein concentration was determined by the micro-Bradford

method (Bio-Rad) with bovine serum albumin as the standard. Fol-

l

L

D

5.14.5. Time-of-addition experiment

A time-of-addition experiment was carried out with MDBK

cells. The conﬂuent monolayers of MDBK cells, seed in 96-well tis-

sue culture plates were inoculated at room temperature with 300

PFU of BVDV, corresponding to a multiplicity of infection of 0.01

PFU/cell. After adsorption for 60 min, the monolayers were washed

two times with MEM-E + 1 mM NaPyr + 0.5% FBSi (Maintenance

Medium in the presence of FBS inactivated) and incubated with

the same medium at 5% CO₂and 37 °C. The test medium containing

10ꢄ compound concentration was added at ꢁ1 to 0 (adsorption), 0

to 2, 2 to 4, 4 to 6, 6 to 8, 8 to 10, 10 to 12, or 12 to 14 h. After each

incubation period, the monolayers were washed two times with

maintenance medium and incubated with fresh medium until 14

h post-infection. Then, after 24–36 h post-infection the CPE was

evaluated and the monolayers were collected, centrifuged and fro-

zen at ꢁ80 °C. The viral titre was determined by a plaque reduction

assay.

lowing dialysis, the puriﬁed 6xHis-tagged HCV1b-NS5B

D

21 and

6xHis-tagged BVDV-NS5BD24 proteins were aliquoted and stored

at ꢁ80 °C.

5.14.9. In vitro RNA-dependent RNA polymerase activity assays

In vitro synthesis assays were performed in 96-well plates using

10

ences) as template and 0.1

mer and 80 M GTP (Invitrogen) as substrate in a 20

l

g/mL poly(rC) (GE Healthcare, formerly Amersham Biosci-

g/mL oligo (rG)₁₂(Invitrogen) as pri-

L reaction

l

mixture containing 20 mM Tris/HCl pH 7.0, 1 mM dithiothreitol,

25 mM NaCl, 20 U/mL RNasin (Promega), 0.5 mM MnCl₂or 5 mM

MgCl₂, 5% DMSO, 5% glycerol and 500–600 ng of each puriﬁed pro-

tein. After an enzyme/drug pre-incubation for 30 min at room tem-

perature, reactions were started by the addition of GTP. One

microliter of threefold serial dilutions of test compounds, or DMSO

alone (as a negative control of inhibition), or the nucleotide analog

3⁰-deoxyguanosine-5⁰-triphosphate (3⁰-dGTP) (tebu-bio) (as a posi-

tive control of inhibition), was added to the reactions and incu-

5.14.6. Expression of BVDV-NS5B

D

24 polymerase

Expression and puriﬁcation of BVDV-NS5B

D

have been done as previously described.³⁹Brieﬂy, the expression

plasmid encoding the N-terminal His-tagged C-terminal 24-ami-

no-acid-deleted BVDV-NS5B was transformed into the Escherichia

coli strain Rosetta™2 (DE3) pLysS (Novagene), and the transfor-

bated for 120 min at 37 °C (for BVDV-NS5B

D

24) or 25 °C (for

of

mants were then cultured in 5 mL of LB medium with 25

kanamycin and 30 g/mL chloramphenicol at 30 °C overnight. Cul-

tures were diluted into 1 L of LB medium with 25 g/mL kanamy-

cin and 30 g/mL chloramphenicol and incubated at 30 °C until the

l

g/mL

HCV1b-NS5B 21), then stopped by the addition of 2 lL

D

l

200 mM EDTA. One hundred thirty-eight microliters of PicoGreen

Quantitation Reagent (Molecular Probes), diluted 1/345 in TE, were

added into each sample and incubated for 5 min at room temper-

l

M. Tonelli et al. / Bioorg. Med. Chem. 18 (2010) 2937–2953

2953

ature protected from light. After an excitation at ꢂ480 nm, the

References and notes

ﬂuorescence was measured at ꢂ520 nm in a ﬂuorescence micro-

plate reader (VICTOR³Multilabel Plate Reader, PerkinElmer). ‘Rela-

tive ﬂuorescence’ was calculated by subtracting the mean

ﬂuorescence of the blank from all samples and converted into per-

cent of activity. Percent of residual activity was then plotted versus

the compound concentrations. Dose–response curves were ﬁt with

Kaleidagraph (Synergy Software) to obtain the drug concentration

that provides 50% of inhibition (IC₅₀).

1. Adler, T. K.; Albert, A. J. Med. Chem. 1963, 6, 480.

2. Brunton, L. L.; Lazo, J. S.; Parker, K. L. Goodman & Gilman’s The Pharmacological

Basis of Therapeutics, 11th ed.; Mc Graw-Hill: New York, 2006. passim.

3. Meanwell, N. A.; Krystal, M. Drugs Future 2007, 32, 441.

4. Tonelli, M.; Paglietti, G.; Boido, V.; Sparatore, F.; Marongiu, F.; Marongiu, E.; La

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pounds as described above and cellular viability was monitored

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lan software as described above.

Acknowledgments

Financial support from Italian MIUR (FIRB RBNE01J3SK01) and

BIOMEDICINE PROJECT is gratefully acknowledged. The authors

would like to thank O. Gagliardo for performing elemental

analyses.

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Supplementary data

Supplementary data (References for the preparation of already

known compounds (1–4, 9, 20, 24, 25, 29, 30, 33, 34, 50–52) asso-

ciated with this article can be found, in the online version, at

doi:10.1016/j.bmc.2010.02.037.

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

DOI: 10.1016/j.bmc.2010.02.037

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