Novel N1-substituted 1,3-dihydro-2H-benzimidazol-2-ones as potent non-nucleoside reverse transcriptase inhibitors

Article abstract of DOI:10.1016/j.bmc.2008.06.012

Several N₁-substituted 1,3-dihydro-2H-benzimidazol-2-ones were synthesized and evaluated as anti-HIV agents. Some of them proved to be highly effective in inhibiting HIV-1 replication at nanomolar concentration as potent non-nucleoside HIV-1 RT

Full text of DOI:10.1016/j.bmc.2008.06.012

Bioorganic & Medicinal Chemistry 16 (2008) 7429–7435
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Novel N1-substituted 1,3-dihydro-2H-benzimidazol-2-ones as potent
non-nucleoside reverse transcriptase inhibitors
a
a
a
a
Anna-Maria Monforte a, , Angela Rao , Patrizia Logoteta , Stefania Ferro , Laura De Luca ,
Maria Letizia Barreca b, Nunzio Iraci a, Giovanni Maga c, Erik De Clercq d, Christophe Pannecouque d,
Alba Chimirri a
*
a Dipartimento Farmaco-Chimico, Università di Messina, Viale Annunziata, 98168 Messina, Italy
b Dipartimento di Chimica e Tecnologia del Farmaco, Via del Liceo 1, 06123 Perugia, Italy
c Istituto di Genetica Molecolare IGM-CNR, via Abbiategrasso 207, 27100 Pavia, Italy
d Rega Institute for Medical Research, Katholieke Universiteit Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
Several N1-substituted 1,3-dihydro-2H-benzimidazol-2-ones were synthesized and evaluated as anti-HIV
agents. Some of them proved to be highly effective in inhibiting HIV-1 replication at nanomolar concen-
tration as potent non-nucleoside HIV-1 RT inhibitors (NNRTIs) with low cytotoxicity. SAR studies high-
lighted that the nature of the substituents at N1 and on the benzene ring of benzimidazolone moiety
significantly influenced the anti-HIV activity of this class of potent antiretroviral agents.
Ó 2008 Elsevier Ltd. All rights reserved.
Received 24 April 2008
Revised 30 May 2008
Accepted 6 June 2008
Available online 13 June 2008
Keywords:
RT inhibitors
Benzimidazolones
Molecular modeling
Microwave-assisted synthesis
1. Introduction
nificantly decreases although this occurs after two or more
mutations in the HIV-1 RT.5
Human immunodeficiency virus (HIV) is the primary cause of
acquired immunodeficiency syndrome (AIDS). The replication of
HIV-1 in infected patients can be reduced considerably by HAART,
a highly active combination of drugs with multiple viral targets.
Officially approved drugs for anti-HIV treatment belong to the
class of nucleoside/nucleotide and non-nucleoside reverse trans-
criptase inhibitors (NRTIs and NNRTIs), to protease inhibitors
(PIs) and more recently to viral entry (Enfuvirtide)1,2 and integrase
inhibitors (Isentress).3
Despite the successes with such treatments, the permanent use
of anti-AIDS drugs induces drug-resistant viral variants and the
emergence of unwanted metabolic side effects.4
NNRTIs are a structurally diverse group of compounds, which
inhibit the enzyme in an allosteric mode by binding at about
10 Å from the polymerase active site causing a distortion of the
catalytic aspartate triad in a non-competitive fashion.5
As a consequence, there is a great need for additional drugs to
further optimize and improve the efficacy of long term HIV
treatment.
In previous papers, we combined different computational meth-
ods in order to obtain information for rational drug design6 and in
connection with these investigations, additional molecular model-
ing and synthetic approaches were applied to discover potent and
selective HIV-1 RT inhibitors.
In particular, we reported a 3D pharmacophore model for a sec-
ond generation of NNRTIs, built by using a combined ligand- and
structure-based molecular modeling approach, consisting of five
features: three hydrophobic groups, one hydrogen bond acceptor,
and one hydrogen bond donor (Fig. 1).7
We used this model for molecular modeling studies which led
to the discovery of N1-substituted 1,3-dihydro-2H-benzimidazol-
2-ones and some derivatives of this series proved to be potent
HIV-1 RT inhibitors.7
First-generation NNRTIs, such as nevirapine and delavirdine,
easily lose their inhibitory potential against mutant virus strains
that contain single amino acid mutations in their RT. Also the anti-
viral potency of second-generation NNRTIs, such as efavirenz, sig-
Considering 6-chloro-1-(2,6-difluorobenzyl)-substituted deriv-
ative (1a) (Scheme 1) as a starting point for lead optimization strat-
egy we designed and synthesized other new benzimidazolone
analogues such as derivative 2a, characterized by the presence of
a 3,5-dimethylbenzyl moiety and its sulfonyl derivative 2b, which
showed lower toxicity and antiretroviral activity similar to that of
* Corresponding author. Tel.: +39 90 6766477; fax: +39 90 6766402.
E-mail address: ammonforte@pharma.unime.it (A.-M. Monforte).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.06.012

7430
A.-M. Monforte et al. / Bioorg. Med. Chem. 16 (2008) 7429–7435
R''
Y
i
R
R
NH2
NO2
NH
R'
NO2
12a - 22a
R'
ii
R = Cl, CH3, CF3
R' = F, H,
8b-14b
iii
R''
R''
Y
N
Y
R
R
NH
iv
O
N
H
R'
R'
NH2
23a -33a
15b -21b
1a - 11a
1b -7b
vi
v
R''
R''
Y
Y
R
R
N
N
O
S
N
N
H
Figure 1. 3D pharmacophore model for second-generation NNRTIs aligned to
compound 1a. HY1–HY3: hydrophobic groups; HBA: hydrogen bond acceptor; HBD:
hydrogen bond donor.
R'
R'
COCH3
1c-3c
1d-3d
Compd
R
R'
H
H
H
H
H
H
H
H
H
H
H
H
F
Y
R''
efavirenz and greater than that of nevirapine, two of the four NNR-
TIs currently available in antiretroviral therapy.8
1a*, 1c, 1d Cl
CH2 2,6- difluorophenyl
SO2 2,6- difluorophenyl
CH2 3,5-dimethylphenyl
SO2 3,5-dimethylphenyl
CH2 3,5-difluorophenyl
SO2 3,5-difluorophenyl
CH2 2,6- difluorophenyl
SO2 2,6- difluorophenyl
CH2 3,5-dimethylphenyl
SO2 3,5-dimethylphenyl
CH2 3,5-difluorophenyl
SO2 3,5-difluorophenyl
CH2 3,5-dimethylphenyl
SO2 3,5-dimethylphenyl
CH2 3,5-difluorophenyl
CH2 2,6- difluorophenyl
CH2 3,5-dimethylphenyl
CH2 2,6- difluorophenyl
1b
Cl
Starting from these promising results, on which we have re-
cently published some preliminary findings,8 we report here the
synthesis of new N1-substituted 1,3-dihydro-2H-benzimidazol-2-
ones in which different structural modifications have been intro-
duced (Fig. 2). Docking and SAR studies have also been performed.
2a#, 2c, 2d
Cl
2b#
3a, 3c, 3d
3b
Cl
Cl
Cl
2. Results and discussion
4a,
4b
CH3
CH3
CH3
CH3
CH3
CH3
Cl
In order to better determine the structural characteristics that
were able to improve the anti-HIV-1 activity of this class of NNRTIs
and to investigate the effects of different chemical modifications
on the RT inhibition, an extensive SAR was examined by varying
the nature and the position of the substituents both on the benzene
ring of the benzimidazolone moiety and on the aromatic portion at
the N-1 atom.
In addition, we changed the linker connecting the benzimid-
azole system with N-1 substituents in order to investigate the ef-
fects on RT inhibition induced by the modified conformational
disposition of the two butterfly wings, an important characteristic
of HIV-1 RT inhibitors.8
5a,
5b
6a
6b
7a
7b
Cl
F
8a
Cl
F
9a
Cl
F
Moreover, we have planned further structural modifications
such as the conversion of the carbonyl group of the benzimidazo-
lone nucleus into the bioisostere thiocarbonyl one thus increasing
the lipophilicity of the molecules and the introduction of an acetyl
group on NH of some synthesized compounds in order to confirm
the importance of the NH residue of the benzimidazolone ring in
the interaction with Lys101 of the RT enzyme.7
10a
11a
CF3
CF3
H
H
Scheme 1. Reagents and conditions: (i) DMF, K2CO3 , MW, 2 steps 250 W, 120 °C,
6 min; 250 W, 130 °C, 6 min; (ii) dioxane, NaH, 0–5 °C, 30 min; (iii) Zn/HCl, EtOH,
80 °C, 1 h; (iv) 20% toluene solution of COCl2, HCl 2 N,
D, 4 h; (v) acetone, CSCl2, 1 h;
The synthesis of the new molecules was achieved by following
the reaction sequence reported in Scheme 1. The 2-nitroanilines
were N-substituted by treatment with the appropriate substi-
tuted-benzylbromides in the presence of potassium carbonate; in
this step the microwave-irradiation shortened the reaction times,
giving the desired products (12a–22a) in high yields. The N-sulfox-
ide derivatives were obtained by reacting with arylsulfonylchloride
using sodium hydride as base. The intermediates (12a–22a, 8b–
14b) were reduced with Zn dust in acidic medium. The cyclization
of the aminoderivatives (23a–33a, 15b–21b) with phosgene
afforded compounds (1a–11a, 1b–7b), while using thiophosgene
(vi) CH2Cl2, ClCOCH3/TEA, 30 min. See Refs. 6,7.
as reagent, compounds (1c–3c) were obtained. The N-acetylated
derivatives (1d–3d) were synthesized using acetyl chloride with
a catalytic amount of TEA.
Both analytical and spectral data (1H NMR) of all synthesized
compounds are in full agreement with the proposed structures.
All compounds were evaluated in enzymatic tests for their abil-
ity to inhibit RT activity as well as HIV-1 (IIIB) replication in MT-4
cell cultures and also cytotoxic activity, and compared with nevira-
pine and efavirenz, which were used as reference drugs.

A.-M. Monforte et al. / Bioorg. Med. Chem. 16 (2008) 7429–7435
7431
R''R'''
X
N
Y
RR'
N
H
Figure 2. Structural modifications of new N1-substituted 1,3-dihydro-2H-ben-
zimidazol-2-ones.
As shown in Table 1, most of the new compounds inhibited RT
and in particular prevented the cytopathic effect of HIV-1 IIIB at
nanomolar concentrations with low toxicity to MT-4 cells resulting
in high selectivity indices. Several derivatives were more potent
and less toxic than nevirapine and, in some cases, than efavirenz.
Considering first of all the effect of the substituents on the phe-
nyl ring at N-1, it is interesting to note that the 3,5-difluorophenyl
derivatives were generally more active than the corresponding 2,6-
substituted compounds. This underlines the importance of hydro-
phobic contacts with the surrounding lipophilic HY1 region of the
pharmacophore model, more favorable by 3,5-substitution.
In particular the greatest activity levels of the 3,5-dimethyl
derivatives might be due to the ability of the 3,5-dimethyl moiety
to occupy the hydrophobic space near the ‘roof’ of the NNRTI bind-
ing pocket (consisting of P95, Y181, Y188, and W229),8,9 thus cre-
ating additional intermolecular interactions (Fig. 3).
Furthermore, the biological effects also confirm that com-
pounds containing a sulfonyl moiety are more potent and less toxic
than the analogues with a methylene linker, with the exception of
the 2,6-difluoro substituted derivative 1a. The greatest potency of
the arylsulfonyl derivatives might also be due to the electronic
characteristics of the sulfonyl groups that are able to make closer
intermolecular contacts with the NNIBP residues (V106, V179,
Figure 3. Intermolecular interactions between the 3,5-dimethyl groups of com-
pound 2b and NNIBP residues P95, Y181, Y188, and W229.
Y181, and Y188) with respect to the methylene linker, as we have
suggested in a previous paper (Fig. 4).8
Moreover, the biological data of the synthesized compounds
highlighted that the nature and position of the substituent on the
benzene ring of benzimidazolone moiety greatly influence both
the anti-HIV activity and the enzyme inhibition. In particular, the
presence of a chlorine atom or a methyl group at the 6 position
of the benzimidazolone system provided the most active com-
pounds. The introduction of the CF3 group in position 6 and the
disubstitution in 5 and 6 positions of the benzene fused ring gen-
erally led to a reduction of the anti-HIV activity.
The replacement of the carbonyl moiety with the isosteric thio-
carbonyl functionality does not lead to a substantial variation in
activity highlighting that a further increase in the lipophilicity
resulting from this structural modification has no influence on
the antiviral activity of the molecules but causes a substantial
reduction of the selectivity index.
Furthermore, the introduction of the acetyl group on NH was
detrimental to the RT inhibition, confirming the importance of
the NH residue of the benzimidazolone ring in the interaction with
the enzyme. In contrast the antiviral activity in cell assays was pre-
served thus suggesting that this discrepancy could be due to a
hydrolytic consequence in cell tests (compounds 1d–3d).
In conclusion, novel anti-HIV agents acting as RT inhibitors have
been obtained. Some derivatives are more potent and less toxic
than nevirapine, one of the only four NNRTIs approved by the FDA.
The SAR data collected will be useful to design and synthesize
new NNRT inhibitors with improved anti-HIV properties.
Table 1
Anti-RT and anti-HIV-1 activities, cytotoxicity, and selectivity index in MT-4 cells
SId
a
b
c
Compound
IC50
(l
M)
EC50
(l
M)
CC50
>424
(lM)
1a
1b
1c
1d
2a
2b
2c
2d
3a
3b
3c
3d
4a
4b
5a
5b
0.70 0.1
30 1.5
0.24 0.05
>1891
124
8
190
281
17,846
4
614
1467
2934
2
24
13
11
11
48,889
<1
360
438
310
12
1.566 0.174
0.74 0.345
0.534 0.39
0.032 0.007
0.002 0.0003
0.076 0.030
0.0117 0.015
0.289 0.1
194.12 9.90
5.95 0.4
101.80 11.31
8.9 0.8
—
>100
0.15 0.02
0.005 0.001
0.046 0.006
>100
0.1 0.01
0.1 0.01
0.160 0.02
38.5 2.5
>100
39 8.2
0.305 0.018
7.18 0.7
>424
202.47 83.45
1.204 0.508
40.388 1.217
236.41 23.12
2.22 0.185
0.856 0.4
>308
72.95 60.37
23.37 11.16
>410
139.60 50.76
>400
0.069 0.055
>0.737
1.663 0.148
18.56 5.58
0.195 0.143
0.075 0.034
0.0063 0.0037
>72.95
0.065 0.014
0.938 0.45
0.45 0.11
1,3 0.1
76.92 7.5
0.1 0.01
>400
0.2 0.02
>100
0.158 0.02
0.003 0.0004
0.14 0.02
0.28 0.03
0.017 0.002
0.18 0.02
0.004 0.001
6a
6b
7a
7b
8a
9a
>33.90
28.975 17.59
0.425 0.202
5.497 1.425
0.073 0.015
0.0009 0.0002
266.88 58.942
37.418 7.28
>424
9
88
77
>205
>6666
10a
11a
Nevirapine
Efavirenz
>15
>6
a
Concentration required to inhibit by 50% the in vitro RNA-dependent DNA
polymerase activity of recombinant RT.
b
Effective concentration required to reduce HIV-1-induced cytopathic effect by
50% in MT-4 cells.
Figure 4. Intermolecular interactions between the methylene linker of compound
2a and the sulfonyl group of compound 2b and NNIBP residues V106, V179, Y181,
and Y188.
c
Cytotoxic concentration required to reduce MT-4 cell viability by 50%.
d
Selectivity index: ratio CC50/EC50
.

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A.-M. Monforte et al. / Bioorg. Med. Chem. 16 (2008) 7429–7435
3.1.1.7. 5-Chloro,4-fluoro-(2,6-difluorobenzyl)-2-nitroaniline
3. Experimental
3.1. Chemistry
(20a). Mp: 102–104 °C, yield 58%. 1H NMR: 4.56 (d, 2H, CH2), 6.89–
8.00 (m, 5H, ArH), 8.23 (br s, 1H, NH). Anal. Calcd for
C13H8ClF3N2O2: C, 49.31; H, 2.55; N, 8.85. Found: C, 49.64; H,
2.33; N, 9.05.
All microwave-assisted reactions were carried out in a CEM
Focused Microwave Synthesis System, Model Discover working
at the potency necessary for refluxing under atmospheric condi-
tions. Melting points were determined on a Kofler hot stage appa-
ratus and are uncorrected. Elemental analyses (C, H, N) were
carried out on a C. Erba Model 1106 Elemental Analyzer, and
the results were within 0.4% of the theoretical values. Merck sil-
ica gel 60 F254 plates were used for TLC. 1H NMR spectra were
measured with a Varian Gemini 300 spectrometer in CDCl3 with
TMS as internal standard or in DMSO-d6. Coupling constants (J)
are reported in hertz, and chemical shifts are expressed in d
(ppm).
3.1.1.8. 5-Trifluoromethyl-1-(3,5-dimethylbenzyl)-2-nitroaniline
(21a). Mp: 102–105 °C, yield 91%. 1H NMR (CDCl3): 2.32 (s, 6H,
CH3), 4.46 (d, 2H, CH2), 6.86–8.31 (m, 6H, ArH), 8.37 (br s, 1H,
NH). Anal. Calcd for C16H15F3N2O2: C, 71.09; H, 6.71; N, 10.36.
Found: C, 71.25; H, 6.88; N, 10.27.
3.1.1.9. 5-Trifluoromethyl-1-(2,6-difluorobenzyl)-2-nitroaniline
(22a). Mp: 105–106 °C, yield 25%. 1H NMR (CDCl3): 4.64 (d, 2H,
CH2); 6.87–8.29 (m, 6H, ArH); 8.40 (br s, 1H, NH). Anal. Calcd for
C14H9F5N2O2: C, 50.61; H, 2.73: N, 8.43. Found: C, 51.03; H, 3.11;
N, 8.78.
3.1.1. General procedure for the synthesis of N-substituted-2-
nitroanilines (14a–22a)
3.1.2. General procedure for the synthesis of N-(2-nitrophenyl)-
benzenesulfonamides (8b, 10b–14b)
The appropriate benzyl bromide (3 mmol) and anhydrous
potassium carbonate (10 mmol) were added to a solution of 5(4)
substituted 2-nitroaniline (2 mmol) in DMF (5 ml) in a cylindrical
quartz tube (2 cm). The reaction mixture was then stirred and irra-
diated in a microwave oven for two steps (I step: W 250, 6 min,
120 °C; II step: W 250, 6 min, 130 °C), cooled, filtered and, after
addition of water (60 ml), extracted with chloroform (2ꢀ 50 ml).
After removal of the solvent under reduced pressure, the residue
was powdered by treatment with diethyl ether and recrystallized
from ethanol.
Dry sodium hydride (10 mmol) was added to a stirred solu-
tion of 5(4) substituted 2-nitroaniline (2 mmol) in dioxane
(6 ml) at 0 °C. The mixture was stirred for 10 min. The appropri-
ate aryl sulfonyl chloride (3 mmol) was added dropwise and a
saturated NaHCO3 solution was added after 30 min to quench
the reaction. The reaction mixture was extracted with chloro-
form and dried over Na2SO4. After removal of the solvent under
reduced pressure, the residue was powdered by treatment with
diethyl ether.
3.1.2.1. N-(5-Chloro-2-nitrophenyl)-2,6-difluorobenzenesulfon-
amide (8b). Mp: 103–105 °C, yield 55%. 1H NMR (DMSO-d6): 7.01–
8.19 (m, 6H, ArH), 10.49 (br s, 1H, NH). Anal. Calcd for
3.1.1.1. 5-Chloro-1-(3,5-difluorobenzyl)-2-nitroaniline (14a). Mp:
122–124 °C, yield 68%. 1H NMR (CDCl3): 4.53 (d, 2H, CH2), 6.67–
8.19 (m, 6H, ArH), 8.47 (br s, 1H, NH). Anal. Calcd for
C12H7ClF2N2O4S: C, 41.33; H, 2.02; N, 8.03. Found: C, 41.48; H,
C13H9ClF2N2O2: C, 52.28; H, 3.04; N, 9.38. Found: C, 51.95; H,
1.91; N, 8.23.
3.19; N, 9.13.
3.1.2.2. N-(5-Chloro-2-nitrophenyl)-3,5-difluorobenzenesulfon-
amide (10b). Mp: 105–107 °C, yield 60%. 1H NMR: 7.03–8.17
(m, 6H, ArH), 10.06 (br s, 1H, NH). Anal. Calcd for
3.1.1.2. 5-Methyl-1-(2,6-difluorobenzyl)-2-nitroaniline (15a). Mp:
96–98 °C, yield 72%. 1H NMR (CDCl3): 2.35 (s, 3H, CH3), 4.58 (d,
2H, CH2), 6.46–8.07 (m, 6H, ArH), 8.35 (br s, 1H, NH). Anal. Calcd
for C14H12F2N2O2: C, 60.43; H, 4.35; N, 10.07. Found: C, 60.33; H,
4.51; N, 10.11.
C12H7ClF2N2O4S: C, 41.33; H, 2.02; N, 8.03. Found: C, 41.52;
H, 2.00; N, 7.90.
3.1.2.3. N-(5-Methyl-2-nitrophenyl)-2,6-difluorobenzenesulfon-
amide (11b). Mp: 122–124 °C, yield 51%. 1H NMR: 2.39 (s, 3H,
CH3), 6.94–8.11 (m, 6H, ArH), 10.48 (br s, 1H, NH). Anal. Calcd for
3.1.1.3. 5-Methyl-1-(3,5-dimethylbenzyl)-2-nitroaniline (16a). Mp:
115–116 °C, yield 81%. 1H NMR (CDCl3): 2.30 and 2.31 (s, 9H,
CH3), 4.44 (d, 2H, CH2), 6.45–8.11 (m, 6H, ArH), 8.40 (br s, 1H,
NH). Anal. Calcd for C16H18N2O2: C, 71.09; H, 6.71; N, 10.36. Found:
C, 71.25; H, 6.88; N, 10.27.
C13H10F2N2O4S: C, 47.56; H, 3.07; N, 8.53. Found: C, 47.47; H,
2.98; N, 8.71.
3.1.2.4. N-(5-Methyl-2-nitrophenyl)-3,5-dimethylbenzenesulfon-
amide (12b). Mp: 120–122 °C, yield 74%. 1H NMR: 2.33 (s, 6H,
30,50-CH3), 2.41 (s, 3H, CH3), 6.92–8.04 (m, 6H, ArH), 9.93 (br s,
1H, NH). Anal. Calcd for C15H16N2O4S: C, 56.24; H, 5.03; N, 8.74;
Found: C, 56.37; H, 5.30; N, 8.60.
3.1.1.4. 5-Methyl-1-(3,5-difluorobenzyl)-2-nitroaniline (17a). Mp:
134 °C, yield 40%. 1H NMR (CDCl3): 2.28 (s, 3H, CH3), 4.54 (d,
2H,CH2), 6.47–8.09 (m, 6H, ArH); 9.07 (br s, 1H, NH). Anal. Calcd
for C14H12F2N2O2: C, 60.43; H, 4.35; N, 10.07. Found: C, 60.83; H,
4.75; N, 9.87.
3.1.2.5. N-(5-Methyl-2-nitrophenyl)-3,5-difluorobenzenesulfon-
amide (13b). Mp: 113–114 °C, yield 57%. 1H NMR: 2.45 (s, 3H,
CH3), 7.01–8.07 (m, 6H, ArH), 9.96 (br s, 1H, NH). Anal. Calcd for
3.1.1.5. 5-Chloro,4-fluoro-(3,5-dimethylbenzyl)-2-nitroaniline
(18a). Mp: 98–100 °C, yield 89%. 1H NMR (CDCl3): 2.35 (s, 6H,
CH3), 4.41 (d, 2H, CH2), 6.88–8.02 (m, 5H, ArH), 8.26 (br s, 1H,
NH). Anal. Calcd for C15H14ClFN2O2: C, 58.35; H, 4.57; N, 9.02.
Found: C, 58.49; H, 4.41; N, 9.22.
C13H10F2N2O4S: C, 47.56; H, 3.07; N, 8.53. Found: C, 47.65; H,
3.19; N, 8.31.
3.1.2.6. N-(5-Chloro-4-fluoro-2-nitrophenyl)-3,5-dimethylbenzene-
solfonamide (14b). Mp: 164–165 °C, yield 88%. 1H NMR (CDCl3):
2.35 (s, 6H, CH3), 7.21–7.99 (m, 5H, ArH), 9.69 (br s, 1H, NH). Anal.
Calcd for C14H12ClFN2O4S: C, 46.87; H, 3.37; N, 7.81. Found: C,
46.70; H, 3.52; N, 7.90.
3.1.1.6. 5-Chloro,4-fluoro-(3,5-difluorobenzyl)-2-nitroaniline
(19a). Mp: 125–126 °C, yield 42%. 1H NMR (CDCl3): 4.52 (d, 2H,
CH2); 6.74–8.05 (m, 5H, ArH); 8.92 (br s, 1H, NH). Anal. Calcd for
C13H8ClFN2O2: C, 49.31; H, 2.55; N, 8.85. Found: C, 48.97, H,
2.25: N, 8.76.

A.-M. Monforte et al. / Bioorg. Med. Chem. 16 (2008) 7429–7435
7433
3.1.3. General procedure for the synthesis of N1-(substituted-
3.1.4. General procedure for the synthesis of N-(2-
benzyl)-2-amino-anilines (25a–33a)
aminophenyl)-benzenesulfonamides (15b, 17b–21b)
The mixture of appropriate N-substituted-2-nitroanilines
(0.6 mmol) in 3 ml HCl and 4 ml EtOH was stirred vigorously,
then zinc dust (20 mmol) was added in several portions at
room temperature. After this addition was completed, the
reaction mixture was heated in a water bath for 1 h, cooled,
made alkaline with NaOH 2 N, and then extracted with ethyl
acetate. The extract was washed with water, dried over
Na2SO4 and evaporated. The residue was crystallized from
ethanol.
With a similar procedure for compounds 23a–33a, the N-(2-
aminophenyl)-benzenesulfonamides (15b–21b) were prepared
starting from the appropriate N-(2-nitrophenyl)-benzenesulfon-
amide (0.6 mmol).
3.1.4.1. N-(2-Aminophenyl-5-chloro)-2,6-difluorobenzenesulfon-
amide (15b). Mp: 133–135 °C, yield 99%. 1H NMR (CDCl3): 3.47 (br
s, 2H, NH2), 4.20 (br s, 1H, NH), 7.01–8.19 (m, 6H, ArH). Anal. Calcd
for C12H9ClF2N2O2S: C, 45.22; H, 2.85; N, 8.79; Found: C, 45.36; H,
2.99; N, 8.62.
3.1.3.1. 2-Amino-5-chloro-1-(3,5-difluorobenzyl)-aniline (25a). Mp:
115–116 °C, yield 94%. 1H NMR (DMSO-d6): 3.50 (br s, 2H, NH2),
3.90 (br s, 1H, NH), 4.31 (s, 2H, CH2), 6.49–6.94 (m, 6H, ArH). Anal.
Calcd for C13H11ClF2N2: C, 58.11; H, 4.13; N, 10.43. Found: C, 58.36;
H, 3.90; N, 10.59.
3.1.4.2. N-(2-Aminophenyl-5-chloro)-3,5-difluorobenzenesulfon-
amide (17b). Mp: 136–138 °C, yield 99%. 1H NMR (CDCl3): 5.12 (br s,
3H, NH and NH2), 6.45–7.41 (m, 6H, ArH). Anal. Calcd for
C12H9ClF2N2O2S: C, 45.22; H, 2.85; N, 8.79. Found: C, 45.11; H, 2.88;
N, 8.95.
3.1.3.2. 2-Amino-5-methyl-1-(2,6-difluorobenzyl)-aniline(26a). Mp:
70–72 °C, yield 98%. 1H NMR (CDCl3): 2.26 (s, 3H, CH3), 3.28 (br
s, 3H, NH and NH2), 4.20 (s, 2H, CH2), 6.47–7.02 (m, 6H, ArH). Anal.
Calcd for C14H14F2N2: C, 67.73; H, 5.68; N, 11.28. Found: C, 67.86;
H, 5.77; N, 11.09.
3.1.4.3. N-(2-Aminophenyl-5-methyl)-2,6-difluorobenzenesulfon-
amide (18b). Mp: 176–178 °C, yield 86%. 1H NMR (CDCl3): 2.01 (s,
3H, CH3), 3.77 (br s, 3H, NH and NH2), 6.47–7.37 (m, 6H, ArH). Anal.
Calcd for C13H12F2N2O2S: C, 52.34; H, 4.05; N, 9.39. Found: C,
52.28; H, 4.19; N, 9.30.
3.1.3.3. 2-Amino-5-methyl-1-(3,5-dimethylbenzyl)-aniline (27a).
Mp: 60–62 °C, yield 99%. 1H NMR (CDCl3): 2.25, 2.32 (s, 9H, CH3),
3.38 (br s, 3H, NH and NH2), 4.37 (s, 2H, CH2), 6.49–7.29 (m, 6H,
ArH). Anal. Calcd for C16H20ClN2: C, 79.96; H, 8.39; N, 11.66. Found:
C, 79.84; H, 8.51; N, 11.49.
3.1.4.4. N-(2-Aminophenyl-5-methyl)-3,5-dimethylbenzensolf-
onamide (19b). Mp: 125–127 °C, yield 77%. 1H NMR (CDCl3): 2.08
(s, 3H, CH3), 2.34 (s, 6H, 30,50-CH3), 3.82 (br s, 2H, NH2), 5.88 (br s,
1H, NH), 6.35–7.36 (m, 6H, ArH). Anal. Calcd for C15H18N2O2S: C,
62.04; H, 6.25; N, 9.65. Found: C, 62.21; H, 6.33; N, 9.44.
3.1.3.4. 2-Amino-5-methyl-1-(3,5-difluorolbenzyl)-aniline(28a). Mp:
80–82 °C, yield 55%. 1H NMR (CDCl3): 2.21 (s, 3H, CH3); 3.40 (br
s, 3H, NH and NH2); 4.32 (s, 2H, CH2); 6.36–6.94 (m, 6H, ArH). Anal.
Calcd for C14H14F2N2: C, 67.73; H, 5.68; N, 11.28. Found: C, 67.65;
H, 5.54; N, 10.98.
3.1.4.5. N-(2-Aminophenyl-5-methyl)-3,5-difluorobenzensulfo-
namide (20b). Mp: 120–122 °C, yield 97%. 1H NMR (CDCl3): 2.06
(s, 3H, CH3), 2.42 (br s, 3H, NH and NH2), 6.44–7.28 (m, 6H, ArH).
Anal. Calcd for C13H12F2N2O2S: C, 52.34; H, 4.05; N, 9.39. Found:
C, 52.56; H, 4.08; N, 9.32.
3.1.3.5. 2-Amino-5-chloro,4-fluoro-1-(3,5-dimethylbenzyl)-
aniline (29a). Mp: 102–105 °C, yield 98%. 1H NMR (CDCl3): 2.29 (s,
6H, CH3), 3.47 (br s, 3H, NH and NH2), 4.13 (s, 2H, CH2), 6.52–7.00
(m, 6H, ArH). Anal. Calcd for C15H16CFlN2: C, 64.63; H, 5.79; N,
10.05. Found: C, 64.51; H, 5.96; N, 9.88.
3.1.4.6. N-(2-Aminophenyl-5-chloro-4-fluoro)-3,5-dimethylben-
zensulfonamide (21b). Mp: 142–143 °C, yield 96%. 1H NMR
(CDCl3): 2.24 (s, 6H, CH3), 3.50 (br s, 1H, NH), 4.13 (br s, 2H,
NH2), 6.38–7.31 (m, 5H, ArH). Anal. Calcd for C14H14ClFN2O2S: C,
51.14; H, 4.29; N, 8.52. Found: C, 51.48; H, 4.13; N, 8.61.
3.1.3.6. 2-Amino-5-chloro,4-fluoro-1-(3,5-difluorolbenzyl)-aniline
(30a). Mp: 75–77 °C, yield 90%. 1H NMR (CDCl3): 3.52 (br s, 3H, NH
and NH2), 4.26 (s, 2H, CH2); 6.50–6.98 (m, 5H, ArH). Anal. Calcd for
3.1.5. General procedure for the synthesis of N1-substituted 1,3-
dihydro-2H-benzimidazol-2-ones (3a–11a)
An excess of a 20% toluene solution of phosgene (1 ml) was
added to a solution of the appropriate N1-(substituted-benzyl)-2-
amino-anilines (0.25 mmol) in HCl 2 N (4 ml), and the resulting
mixture was heated for 4 h. After cooling, the reaction mixture
was neutralized with NaOH 2 N, extracted with ethyl acetate,
washed with water, and evaporated under reduced pressure. The
residue was crystallized from ethyl acetate.
C13H10ClF3N2: C, 54.47; H, 3.52; N, 9.77. Found: C, 54.30; H, 3.34, N,
9.67.
3.1.3.7. 2-Amino-5-chloro,4-fluoro-1-(2,6-difluorolbenzyl)-aniline
(31a). Mp: 71–73 °C, yield 95%. 1H NMR (CDCl3): 3.38 (br s, 1H,
NH), 3.56 (br s, 2H, NH2), 4.30 (s, 2H, CH2), 6.50–7.29 (m, 5H,
ArH). Anal. Calcd for C13H10ClF3N2: C, 79.96; H, 8.39; N, 11.66.
Found: C, 65.48; H, 5.64; N, 9.76.
3.1.5.1. 6-Chloro-1-(3,5-difluorobenzyl)-1,3-dihydro-2H-benzimid-
azol-2-one (3a). Mp: 195 °C, yield 96%. 1H NMR (CDCl3): 5.01 (s,
2H, CH2), 6.70–6.69 (m, 1H, H-40), 6.81–6.82(m, 3H, H-7, H-20, H-
60), 7.00 (d, J = 8.24, 1H, H-5), 7.07 (d, J = 8.24, 1H, H-4), 8.73 (br
s, 1H, NH). Anal. Calcd for C14H9ClF2N2O: C, 57.06; H, 3.08; N,
9.51. Found: C, 56.91; H, 3.32; N, 9.62.
3.1.3.8. 2-Amino-5-trifluoromethyl-1-(3,5-dimethylbenzyl)-aniline
(32a). Mp: 106–109 °C, yield 91%. 1H NMR (CDCl3): 2.33 (s, 6H,
CH3), 3.60 (br s, 3H, NH and NH2), 4.21 (s, 2H, CH2), 6.70–7.02
(m, 6H, ArH). Anal. Calcd for C16H17F3N2: C, 65.29; H, 5.82; N,
9.52. Found: C, 79.84; H, 8.51; N, 11.49.
3.1.5.2. 6-Methyl-1-(2,6-difluorobenzyl)-1,3-dihydro-2H-benzimid-
azol-2-one (4a). Mp: 250–253 °C, yield 32%. 1H NMR (CDCl3): 2.33
(s, 3H, CH3), 5.14 (s, 2H, CH2), 6.61–6.95 (m, 5H, H-4, H-5, H-7, H-30,
H-50), 7.21–7.31 (m, 1H, H-40), 8.34 (br s, 1H, NH). Anal. Calcd for
3.1.3.9. 2-Amino-5-trifluoromethyl-1-(2,6-difluorolbenzyl)-aniline
(33a). Mp: 77–79 °C, yield 95%. 1H NMR (CDCl3): 3.59 (br s, 3H, NH
and NH2), 4.40 (s, 2H, CH2), 6.70–7.31 (m, 6H, ArH). Anal. Calcd for
C15H12F2N2O: C, 65.69; H, 4.41; N, 10.21. Found: C, 65.73; H,4.57;
C14H11F5N2: C, 55.63; H, 3.67: N, 9.27. Found: C, 55.40; H, 3, 80; N,
N, 10.40.
9.30.

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A.-M. Monforte et al. / Bioorg. Med. Chem. 16 (2008) 7429–7435
3.1.5.3. 6-Methyl-1-(3,5-dimethylbenzyl)-1,3-dihydro-2H-ben-
zimidazol-2-one (5a). Mp: 142 °C dec, yield 55%.1H NMR (CDCl3):
2.28 (s, 6H, CH3), 2.33 (s, 3H, CH3), 4.97 (s, 2H, CH2), 6.70 (s, 1H, H-
7), 6.83–6.95 (m, 5H, H-4, H-5, H-20, H-40, H-60), 8.29 (br s, 1H, NH).
Anal. Calcd for C16H15ClN2O: C, 76.66; H, 6.81; N, 10.52. Found: C,
76.70; H, 6.68; N, 10.44.
7.75 (s, 1H, H-7), 7.80–7.89 (m, 3H, H-20, H-40, H-60), 11.79 (br s,
1H, NH). Anal. Calcd for C13H7ClF2N2O3S: C, 45.30; H, 2.05; N,
8.13. Found: C, 45.52; H, 2.01; N, 7.97.
3.1.6.3. 6-Methyl 1-(2,6-difluorophenylsulfonyl)-1,3-dihydro-
2H-benzimidazol-2-one (4b). Mp: 198–200 °C, yield 39%. 1H
NMR (CDCl3): 2.42 (s, 3H, CH3), 6.91 (d, J = 7.96, 1H, H-5), 6.94–
7.07 (m, 3H, H-4, H-30, H-50), 7.56–7.63 (m, 1H, H-40), 7.70 (s, 1H,
H-7), 7.97 (br s, 1H, NH). Anal. Calcd for C14H10F2N2O3S: C, 51.85;
H, 3.11; N, 8.64. Found: C, 51.95; H, 2.98; N, 8.73.
3.1.5.4. 6-Methyl-1-(3,5-difluorobenzyl)-1,3-dihydro-2H-ben-
zimidazol-2-one (6a). Mp: 191–193 °C, yield 50%. 1H NMR
(CDCl3): 2.34 (s, 3H, CH3); 5.01 (s, 2H, CH2); 6.64 (s, 1H, H-7);
6.69–6.75 (m, 1H, H-40), 6.82–6.90 (m, 3H, H-5, H-20, H-60), 6.97
(d, J = 7.69, 1H, H-4), 8.29 (br s, 1H, NH). Anal. Calcd for
3.1.6.4. 6-Methyl 1-(3,5-dimethylphenylsulfonyl)-1,3-dihydro-
2H-benzimidazol-2-one (5b). Mp: 280 °C dec, yield 43%. 1H
NMR (CDCl3): 2.37 (s, 6H, CH3), 2.44 (s, 3H, CH3), 6.89 (d, J = 7.96,
1H, H-5), 6.99 (d, J = 7.96, 1H, H-4), 7.27–7.76 (m, 4H, H-7, H-20,
H-40, H-60), 8.01 (br s, 1H, NH). Anal. Calcd for C16H16N2O3S: C,
60.74; H, 5.10; N, 8.85. Found: C, 60.97; H, 5.00; N, 8.71.
C15H12F2N2O: C, 65.69; H, 4.41; N, 10.21. Found: C, 65.71; H,
4.60; N, 10.35.
3.1.5.5. 6-Chloro,5-fluoro-(3,5-dimethylbenzyl)-1,3-dihydro-2H-
benzimidazol-2-one (7a). Mp: 221–223 °C, yield 72%. 1H NMR
(CDCl3): 2.29 (s, 6H, CH3), 4.95 (s, 2H, CH2), 6.84–6.94 (m, 5H, H-4,
H-7, H-20, H.40, H-60),8.87(brs,1H, NH).Anal.CalcdforC14H8ClF3N2O:
C, 53.78; H, 2.58; N, 8.96. Found: C, 53.96; H, 2.37; N, 10.59.
3.1.6.5. 6-Methyl 1-(3,5-difluorophenylsulfonyl)-1,3-dihydro-
2H-benzimidazol-2-one (6b). Mp: 230–232 °C dec, yield 45%.
1H NMR (DMSO-d6): 2.32 (s, 3H, CH3), 6.82 (d, J = 7.69, 1H, H-
5), 6.91 (d, J = 7.69, 1H, H-4), 7.49 (s, 1H, H-7), 7.73–7.79 (m,
3H, H-20, H-40, H-60), 11.51 (br s, 1H, NH). Anal. Calcd for
3.1.5.6. 6-Chloro,5-fluoro-(3,5-difluorobenzyl)-1,3-dihydro-2H-
benzimidazol-2-one (8a). Mp: 216–218 °C, yield 44%. 1H NMR
(CDCl3): 5.01 (s, 2H, CH2), 6.73–6.83 (m, 4H, H-7, H-20, H-40, H-
60), 6.98 (d, J = 8.51, 1H, H-4), 9.69 (br s, 1H, NH). Anal. Calcd for
C14H10F2N2O3S: C, 51.85; H, 3.11; N, 8.64. Found: C, 51.80; H,
2.96; N, 8.77.
C14H8ClF3N2O: C, 53.; H, 4.63; N, 9.19. Found: C, 63.55; H, 4.60;
N, 9.54.
3.1.6.6. 6-Chloro-5-fluoro-1-(3,5-dimethylphenylsulfonyl)-1,3-
dihydro-2H-benzimidazol-2-one (7b). Mp: 245 °C dec, yield 47%.
1H NMR (DMSO-d6): 2.30 (s, 6H, CH3), 6.65 (d, J = 10.44, 1H, H-4),
7.29 (s, 1H, H-40), 7.38 (d, J = 7.69, 1H, H-7), 7.52 (s, 2H, H-20, H-
60). Anal. Calcd for C13H6ClF3N2O3S: C, 43.05; H, 1.67; N, 7.72.
Found: C, 43.30; H, 1.52; N, 7.84.
3.1.5.7. 6-Chloro,5-fluoro-(2,6-difluorobenzyl)-1,3-dihydro-2H-
benzimidazol-2-one (9a). Mp: 221–223 °C, yield 75%. 1H NMR
(CDCl3): 5.12 (s, 2H, CH2), 6.88–6.97 (m, 4H, H-4, H-7, H,30, H-50),
7.26–7.31 (m, 1H, H-40), 8.87 (br s, 1H, NH). Anal. Calcd for
C14H8ClF3N2O: C, 63.06; H, 4.63; N, 9.19. Found: C, 63.44; H,
4.45; N, 9.43.
3.1.7. General procedure for the synthesis of N1-substituted 1,3-
dihydro-2H-benzimidazol-2-thiones (1c–3c)
3.1.5.8. 6-Trifluoromethyl-1-(3,5-dimethylbenzyl)-1,3-dihydro-
2H-benzimidazol-2-one (10a). Mp: 198–201 °C, yield 58%. 1H
NMR (CDCl3): 2.28 (s, 6H, CH3), 5.02 (s, 2H, CH2), 6.93 (s, 3H, H-
20, H-40, H-60), 7.13–7.16 (m, 2H, H-4, H-7), 7.34 (d, J = 7.41, 1H,
H-5), 8.91 (br s, 1H, NH). Anal. Calcd for C17H15F3N2O: C, 63.75;
H, 4.72; N, 8.75. Found: C, 63.48; H, 5.08; N, 8.91.
Thiophosgene (0.25 mmol) was added to a solution of N1-
substituted-2-amino-5-chloroaniline (0.25 mmol) in acetone, and
the resulting mixture was stirred for 1 h at room temperature.
The reaction solvent was evaporated under reduced pressure, and
the residue was crystallized from ethanol.
3.1.7.1. 6-Chloro-1-(2,6-difluorobenzyl)-1,3-dihydro-2H-ben-
zimidazol-2-thione (1c). Mp: 210–212 °C, yield 88%. 1H NMR
(CDCl3): 5.49 (s, 2H, CH2), 7.04–7.10 (m, 2H, H-30, H-50), 7.15–
7.22 (m, 2H, H-4, H-5), 7.39 (m, 1H, H-40), 7.49 (s, 1H, H-7),
12.95 (br s, 1H, NH). Anal. Calcd for C14H9ClF2N2S: C, 54.11; H,
2.92; N, 9.01. Found: C, 53.67; H, 3.04; N, 9.58.
3.1.5.9. 6-Trifluoromethyl-1-(2,6-difluorobenzyl)-1,3-dihydro-
2H-benzimidazol-2-one (11a). Mp: 243–245 °C, yield 60%. 1H
NMR (CDCl3): 5.19 (s, 2H, CH2); 6.91–6.97 (m, 2H, H-4, H-7);
7.12 (d, J = 8.24, 1H, H-5), 7.25–7.61 (m, 3H, H-30, H-40, H-50),
8.66 (br s, 1H, NH). Anal. Calcd for C15H9F5N2O: C, 54.89; H, 2.76;
N, 28.94. Found: C, 54.48; H, 2.47; N, 29.02.
3.1.7.2. 6-Chloro-1-(3,5-dimethylbenzyl)-1,3-dihydro-2H-ben-
zimidazol-2-thione (2c). Mp: 263–265 °C, yield 45%. 1H NMR
(CDCl3): 2.28 (s, 6H, CH3), 5.40 (s, 2H, CH2), 7.02 (s, 3H, H-20, H-
40, H-60), 7.10 (s, 1H, H-7), 7.13–7.18 (m, 2H, H-4, H-5), 9.69 (br
s, 1H,NH). Anal. Calcd for C16H15ClN2S: C, 63.46; H, 4.99; N, 9.25.
Found: C, 63.58; H, 5.13; N, 9.52.
3.1.6. General procedure for the synthesis of 1-arylsulfonyl-1,3-
dihydro-2H-benzimidazol-2-ones (1b, 3b–7b)
With a similar procedure for compounds 1a–11a the 1-aryl-
sulfonyl-1,3-dihydro-2H-benzimidazol-2-ones (1b–7b) were prepared
from the appropriate N-(2-aminophenyl)-benzenesulfonamides
(0.25 mmol).
3.1.7.3. 6-Chloro-1-(3,5-difluorobenzyl)-1,3-dihydro-2H-ben-
zimidazol-2-thione (3c). Mp: 223–225 °C, yield 56%. 1H NMR
(CDCl3): 5.46 (s, 2H, CH2), 6.73–6.86 (m, 3H, H-20, H-40, H-60),
6.98 (s, 1H, H-7), 7.16–7.23 (m, 2H, H-4, H-5), 10.34 (br s, 1H,
NH). Anal. Calcd for C14H9ClF2N2S: C, 54.11; H, 2.92; N, 9.01.
Found: C, 54.37; H, 2.53; N, 9.54.
3.1.6.1. 6-Chloro-1-(2,6-difluorophenylsulfonyl)-1,3-dihydro-
2H-benzimidazol-2-one (1b). Mp: 163–165 °C, yield 54%. 1H NMR
(DMSO-d6): 7.24 (d, J = 8.51, 1H, H-5), 7.34–7.40 (m, 3H, H-4, H-30,
H-50), 7.58 (s, 1H, H-7), 7.69–7.92 (m, 1H, ArH), 10.78 (br s, 1H,
NH). Anal. Calcd for C13H7ClF2N2O3S: C, 45.30; H, 2.05; N, 8.13.
Found: C, 45.44; H, 2.19; N, 7.98.
3.1.8. General procedure for the synthesis of N1-substituted 1-
H-2H-3-acetyl-benzimidazol-2-one (1d–3d)
Triethylamine and then acetyl chloride (0.3 mmoli) were added
dropwise to a solution of 1H-2H-benzimidazolone (1a, 2a or 3a)
3.1.6.2. 6-Chloro-1-(3,5-difluorophenylsulfonyl)-1,3-dihydro-
2H-benzimidazol-2-one (3b). Mp: 250 °C dec, yield 75%. 1H NMR
(DMSO-d6): 7.05 (d, J = 8.51, 1H, H-4), 7.26 (d, J = 8.51, 1H, H-5),

A.-M. Monforte et al. / Bioorg. Med. Chem. 16 (2008) 7429–7435
7435
(0.2 mmol) in dichloromethane (3 ml); the mixture was stirred for
3.2.2. RT inhibition assays
30 min at room temperature. Successively, the reaction mixture
was diluted with chloroform, washed with water and evaporated
under reduced pressure. The residue was crystallized from ethanol.
Reactions were performed under the conditions described for
the HIV-1 RT RNA-dependent DNA polymerase activity assay.11
Incorporation of radioactive dTTP into poly(rA)/oligo(dT) was mon-
itored in the presence of increasing amounts of the inhibitors to be
tested. Data were then plotted according to Lineweaver-Burke and
Dixon. For Ki determinations an interval of inhibitor concentra-
tions between 0.2 Ki and 5 Ki was used. Experiments have been
done in triplicate. Experimental errors ( SD) were 610%.
3.1.8.1. 3-Acetyl-6-chloro-1-(2,6-difluorobenzyl)-1,3-dihydro-
2H-benzimidazol-2-one (1d). Mp: 155–157 °C, yield 84%. 1H NMR
(CDCl3): 2.77 (s, 3H, CH3), 5.11 (s, 2H, CH2), 6.93–7.33 (m, 5H, H-5,
H-7, H-30, H-40, H-50), 8.12 (d, J = 8.51, 1H, H-4). Anal. Calcd for
C16H11ClF2N2O2: C, 57.07; H, 3.29; N, 8.32. Found: C, 57.12; H,
3.18; N, 8.46.
3.2.3. In vitro anti-HIV assay
The methodology of the anti-HIV assays has been previously de-
scribed.10 Briefly, MT-4 cells were infected with HIV-1 (IIIB) at
ꢁ100-times the CCID50 (50% cell culture infective dose) per millili-
ter of cell suspension. One-hundred microliters of the infected cell
suspension were then transferred to microtiter plate wells, mixed
3.1.8.2. 3-Acetyl-6-chloro-1-(3,5-dimethylbenzyl)-1,3-dihydro-
2H-benzimidazol-2-one (2d). Mp: 152–154 °C, yield 99%. 1H NMR
(CDCl3): 2.30 (s, 6H, CH3), 2.80 (s, 3H, COCH3), 4.94 (s, 2H, CH2),
6.88–6.94 (m, 4H, H-7, H-20, HP-40, H-60), 7.10 (d, J = 8.51, 1H, H-
5), 8.13 (d, J = 8.51, 1H, H-4). Anal. Calcd for C18H17ClN2O2: C,
65.75; H, 5.21; N, 8.52. Found: C, 65.66; H, 5.46; N, 8.34.
with 100 ll of the appropriate dilutions of the test compounds,
and further incubated at 37 °C. After 5 days (MT-4) of incubation,
the number of viable MT-4 cells was determined. The 50% effective
concentration (EC50) was defined as the concentration of com-
pound required to reduce the virus-induced cytopathicity by 50%.
3.1.8.3. 3-Acetyl-6-chloro-1-(3,5-difluorolbenzyl)-1,3-dihydro-
2H-benzimidazol-2-one (3d). Mp: 121–123 °C, yield 23%. 1H NMR
(CDCl3): 2.79 (s, 3H, CH3), 4.99 (s, 2H, CH2), 6.75–6.92 (m, 4H, H-20,
H-40, H-60), 7.15 (d, J = 8.51, 1H, H-5), 8.17 (d, J = 8.51, 1H, H-4).
Anal. Calcd for C16H11ClF2N2O2: C, 57.07; H, 3.29; N, 8.32. Found:
C, 57.24; H, 3.40; N, 8.52.
Acknowledgment
Financial support for this research by Fondo Ateneo di ricerca
(2004, Messina, Italy), European TRIoH Consortium (LSHB-CT-
2003-503480) is gratefully acknowledged.
3.2. Anti-HIV activity assays
References and notes
3.2.1. HIV-1 RT RNA-dependent DNA polymerase activity assay
Poly(rA)/oligo(dT) was used as a template for the RNA-depen-
dent DNA polymerase reaction by HIV-1 RT. For the activity assay,
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a 25 ll final reaction volume contained TDB buffer (50 mM Tris–
3. www.fda.gov/bbs/topics/NEWS/2007/NEW01726.html.
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Höltje, M.; Monforte, A. M.; Monforte, P.; Pannecouque, C.; Rao, A.; Zappalà, M.
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7. Barreca, M. L.; Rao, A.; Balzarini, J.; De Luca, L.; Zappalà, M.; Monforte, A. M.;
Maga, G.; Pannecouque, C.; Balzarini, J.; De Clercq, E.; Chimirri, A.; Monforte, P.
J. Med. Chem. 2005, 48, 3433.
8. Barreca, M. L.; Rao, A.; De Luca, L.; Iraci, N.; Monforte, A. M.; Maga, G.; De
Clercq, E.; Pannecouque, C.; Balzarini, J.; Chimirri, A. Bioorg. Med. Chem. Lett.
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HCl (pH 8.0), 1 mM dithiothreitol (DTT), 0.2 mg/ml bovine serum
albumin (BSA), 2% glycerol), 10 mM MgCl2, 0.5 mg of poly(rA):oli-
go(dT)10:1 (0.3 mM 30-OH ends), 10 mM 3[H]dTTP 1 Ci/mmol and,
finally, introduced into tubes containing aliquots of different en-
zyme concentrations (5–10 nM RT). After incubation at 37 °C for
indicated time, 20 ll from each reaction tube was spiked on glass
fiber filters GF/C and, immediately, immersed in 5% ice-cold tri-
chloroacetic acid (TCA) (AppliChem GmbH, Darmstadt). Filters
were washed three times with 5% TCA and once with ethanol for
5 min, then dried and, finally, added with EcoLumeÒ Scintillation
cocktail (ICN, Research Products Division, Costa Mesa, CA, USA)
to detect the acid-precipitable radioactivity by PerkinElmerÒ Trilux
MicroBeta 1450 Counter.
9. Hopkins, A. L.; Ren, J.; Tanaka, H.; Baba, M.; Okamato, M., et al J. Med. Chem.
1999, 42, 4500.
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