Novel 1,3-dihydro-benzimidazol-2-ones and their analogues as potent non-nucleoside HIV-1 reverse transcriptase inhibitors

Article abstract of DOI:10.1016/j.bmc.2009.12.059

A series of novel benzimidazolones and their analogues, characterized by the presence of one or more methyl groups or other bioisosteric moieties at different positions of the phenyl ring at N-1, were synthesized and evaluated as inhibitors of human immunodeficiency virus type-1 (HIV-1). Most of the new compounds proved to be highly effective in inhibiting both HIV-1 replication in MT4 cells with minimal cytotoxicity and RT enzyme at nanomolar concentrations. Some derivatives were also tested against RTs containing single amino acid mutations responsible for resistance to non-nucleoside reverse transcriptase inhibitors (NNRTIs). The different potencies displayed by the new compounds were studied using molecular modeling.

Full text of DOI:10.1016/j.bmc.2009.12.059

Bioorganic & Medicinal Chemistry 18 (2010) 1702–1710
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Novel 1,3-dihydro-benzimidazol-2-ones and their analogues as potent
non-nucleoside HIV-1 reverse transcriptase inhibitors
a
a
b
a
Anna-Maria Monforte a, , Patrizia Logoteta , Laura De Luca , Nunzio Iraci , Stefania Ferro ,
*
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, Università di Perugia, 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:
A series of novel benzimidazolones and their analogues, characterized by the presence of one or more
methyl groups or other bioisosteric moieties at different positions of the phenyl ring at N-1, were synthe-
sized and evaluated as inhibitors of human immunodeficiency virus type-1 (HIV-1). Most of the new
compounds proved to be highly effective in inhibiting both HIV-1 replication in MT4 cells with minimal
cytotoxicity and RT enzyme at nanomolar concentrations. Some derivatives were also tested against RTs
containing single amino acid mutations responsible for resistance to non-nucleoside reverse transcrip-
tase inhibitors (NNRTIs). The different potencies displayed by the new compounds were studied using
molecular modeling.
Received 2 September 2009
Revised 18 December 2009
Accepted 23 December 2009
Available online 4 January 2010
Keywords:
1,3-Dihydro-benzimidazol-2-ones
HIV-1 reverse transcriptase
Molecular modeling
Synthesis
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
(nevirapine, delavirdine, efavirenz, and etravirine) have been ap-
proved for clinical use to date.8
Standard therapy for human immunodeficiency virus type 1
(HIV-1) infection was based, for many years, on potent cocktails
of drugs targeting the viral protease (PR) and reverse transcriptase
(RT).1 More recently, two viral entry inhibitors, enfuvirtide2 and
maraviroc,3 and a viral integrase inhibitor (IN), raltegravir4 have
been added to these regimens.
Although this treatment has curtailed the number of AIDS
deaths, it is often associated with severe and long term side effects.
Moreover, the incomplete suppression of HIV replication is respon-
sible for the emergence of drug-resistant HIV strains. Therefore, a
continued research effort is required to develop more potent and
less toxic antiviral compounds to cope with the rapidly emerging
resistant strains.5
In recent papers, we reported a 3D-pharmacophore model
which led to the rational discovery of N1-substituted 1-3-dihy-
dro-2H-benzimidazol-2-ones and their sulfones as potent HIV-1
RT inhibitors.9–11
The most active derivative (1) (Fig. 1) of this class of NNRTIs,
characterized by the presence of a 3,5-dimethylphenylsulfonyl
moiety at N1 of the benzimidazolone system and a chlorine atom
Non-nucleoside reverse transcriptase inhibitors (NNRTIs) have
become very important components in antiretroviral combination
therapies due to their unique mechanism of action, low toxicity
and favorable pharmacokinetics properties.6,7
Consequently, many efforts have been directed in recent years
to the development of novel broad spectrum NNRTIs that inhibit
both wild-type and drug-resistant variants of RT and four NNRTIs
Figure 1. 3D-pharmacophore model for second-generation NNRTIs aligned to
compound 1 (cyan: hydrophobic groups; green: hydrogen bond acceptor; magenta:
hydrogen bond donor).
* Corresponding author. Tel.: +39 0906766477; fax: +39 0906766402.
E-mail address: ammonforte@pharma.unime.it (A.-M. Monforte).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.12.059

A.-M. Monforte et al. / Bioorg. Med. Chem. 18 (2010) 1702–1710
1703
at C-6, showed very low toxicity and potent antiretroviral activity
similar to that of efavirenz and higher than nevirapine against both
wild-type and in some mutant strains of HIV-1.10
The desired products were obtained in good yield and high pur-
ity (more than 95%). Both analytical and spectral data (1H NMR) of
all synthesized compounds were in full agreement with the pro-
posed structures.
SAR studies highlighted that the nature and the position of the
substituents at N1 and on the benzene ring of the benzimidazolone
moiety significantly influenced the anti-HIV activity.11 Moreover,
the opening or the replacement of the imidazol-2-one nucleus with
a homologous six-membered ring decreased the potency.12
Our molecular modeling studies performed on this class of com-
pounds highlighted the positive contribution to antiviral activity of
both a chlorine atom on the benzimidazole system (Fig. 1) and, in
particular, the electron-rich sulfonyl group, which is able to form
intermolecular interactions with important residues of the non-
nucleoside inhibitor binding pocket (NNIBP). We also reported that
3,5-dimethylphenyl-substituted benzimidazolones showed the
greatest activity levels, and docking studies suggested that this
might be due to the ability of the methyl groups to create addi-
tional intermolecular interactions with hydrophobic residues pres-
ent within the NNIBP space.10,11
Supported by these promising results and with the aim of
obtaining more potent compounds and to establish further SAR
on this class of NNRTIs, we designed and synthesized new ben-
zimidazolones and their analogues in which the chlorine atom
and the sulfonyl group were maintained in view of their para-
mount importance.
More specifically, we decided to prepare other 3,5-dimethyl-
phenyl-substituted derivatives in which the benzene ring of the
benzimidazolone system was replaced by a suitably heteroaro-
matic nucleus (as pyridine, 2) or where the carbonyl group was
converted into the bioisosteric thiocarbonyl moiety (3).
We also designed new derivatives in which other structural
modifications had been made to the aromatic portion at the N1
atom, such as the introduction of one or more methyl groups or
other bioisosteric substituents in different positions of the phenyl
ring (Fig. 2).
2.2. Biological activity
The new synthesized compounds 1–16 were evaluated by enzy-
matic tests for their ability to inhibit RT activity as well as HIV-1
(IIIB) replication in MT-4 cell cultures in parallel with cytotoxic
activity, and compared with nevirapine and efavirenz which were
used as reference drugs (Table 1).
Selected compounds were also tested against RTs containing
the K103N, Y181I, and L100I single amino acid mutations respon-
sible for resistance to NNRTIs (Table 2).
The biological activity data show that most of the new com-
pounds inhibit RT and prevent the cytopathic effect of HIV-1 IIIB
at nanomolar concentrations and with minimal toxicity to MT-4
cells, resulting in remarkably high selectivity indices (SI).
Among all the tested compounds, derivatives 3, 5, 8, 9 and 16,
proved to be highly potent and showed inhibitory potencies supe-
rior to that of nevirapine and in some cases were comparable to
that of efavirenz and our lead compound 1.
As shown in Table 1, the results obtained highlighted that mod-
ifications on the bicycle system influenced the activity profile of
the molecules. In particular, the replacement of the benzene ring
of the benzimidazolone system with a pyridine resulted in a reduc-
tion in the anti-HIV activity, whereas the conversion of the car-
bonyl group into the bioisosteric thiocarbonyl moiety caused a
substantial decrease of the selectivity index, even if both com-
pounds retained potent RT inhibitory effects.
Furthermore, the anti-HIV-1 activity was influenced by the nat-
ure and the position of the substituents introduced on the aromatic
portion at N1.
Considering first of all the effect of the monomethyl substitu-
tion, the best results were obtained by the presence of a methyl
function at 3 position of the phenyl ring at the N1. Moreover, the
insertion of two or more methyl groups on the same moiety also
provided potent compounds (8, 9, 10) as suggested by molecular
modeling studies (see Section 2.3).
The influence of these structural changes on the antiretroviral
activity of this class of compounds is reported and discussed here-
in. The different degrees of potency displayed by the new mole-
cules against wild-type and mutants of RTs were studied by
molecular modeling approaches.
In contrast, the replacement of the methyl with a trifluoro-
methyl moiety (13 and 14) or 3-OCF3 (15) led to a decrease in anti-
retroviral activity whereas its bioisostere (16) 3-OCH3 substituted
exhibited high potency and selectivity.
2. Results and discussion
2.1. Chemistry
Compound 9, characterized by a 3,4-dimethyl-phenyl substitu-
tion, showed the best activity profile both in enzymatic tests on wt
RT (IC50 = 0.004 lM) and in cellular assays (EC50 = 0.006 lM) as
well as the best selectivity index (>61857). Overall, 9 showed an
anti-HIV profile superior to that of nevirapine and comparable to
that of efavirenz.
All of the ten selected derivatives were potent inhibitors of the
L100I RT mutant at submicromolar concentration; some inhibited
the K103N RT mutant in the micromolar range, whereas they were
generally inactive against Y181I.
The synthesis of new benzimidazolone derivatives (4–16) and
analogues (2–3) is reported in Scheme 1. The 5-chloro-2-nitroani-
line or 2-amino-6-chloro-3-nitropyridine was N-substituted by
treatment with the appropriate arylsulfonylchloride in the pres-
ence of sodium hydride at 0–5 °C in dioxane .
The obtained N-sulfoxide derivatives 32–46 were reduced with
Zn dust in HCl and ethanol at 80 °C.
The subsequent cyclization of the aminoderivatives 17–31 with
phosgene at room temperature afforded compounds 1–2 and 4–
16, while compound 3 was achieved when using thiophosgene as
reagent.
The most active compounds (2, 3 and 9) were more active than
nevirapine. However, they were less active than reference 1 and
efavirenz both against K103N and L100I RT HIV-1.
RR'
O
2.3. Computational results
S
O
Cl
X
N
In order to explore the possible binding conformation of the de-
signed compounds and their interaction mode with RT, a molecular
modeling study was performed using the program AUTODOCK and
Y
N
H
13
the docking protocol that we successfully applied in our previous
Figure 2. Structural modifications of new N1-substituted 1,3-dihydro-2H-ben-
zimidazol-2-ones.
papers on RT.9,10,14

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A.-M. Monforte et al. / Bioorg. Med. Chem. 18 (2010) 1702–1710
R
SO2
R
R
SO2
SO2
SO2
Cl
X
NH2
NO2
Cl
X
NH
Cl
Cl
X
Cl
X
NH
R
N
iii or iv
ii
Y
i
NH2
N
H
NO2
32-46
17-31
X= CH, N
1-16
Compd
X
Y
O
R
Compd.
X
Y
O
R
CH
3,5-(CH3)2-C6H3
3,5-(CH3)2-C6H3
3,5-(CH3)2-C6H3
2-CH3-C6H4
CH
3,4-(CH3)2-C6H3
2,4,6-(CH3)3-C6H2
2,3,5,6-(CH3)4 -C6H
2,3,4,5.6-(CH3)5-C6
3,5-(CF3)2-C6H3
3-CF3-C6H4
1*
9
N
O
S
CH
CH
CH
CH
CH
CH
CH
O
O
O
O
O
O
O
2
3
4
5
6
7
8
10
11
12
13
14
15
16
CH
CH
CH
CH
CH
CH
O
O
O
O
O
3-CH3-C6H4
4-CH3-C6H4
2,4-(CH3)2-C6H3
2,5-(CH3)2-C6H3
3-OCF3-C6H4
3-OCH3-C6H4
Scheme 1. Reagents and conditions: (i) Dioxane, NaH, 0–5 °C, 30 min; (ii) Zn/HCl, EtOH, 80 °C, 1 h; (iii) 20% toluene solution of COCl2, HCl 2 N,
D, 4 h; (iv) acetone, CSCl2, 1 h.
*See Ref. 10.
Table 1
Table 2
Anti-RT and anti-HIV-1 activities, cytotoxicity and selectivity index in MT-4 cells
HIV-1 RT inhibitory activity of compounds 1–3, 5, 8–10 and 16 against mutant
enzymes carrying single amino acid substitutions
Compd
IC50
(l
M)a
EC50
(
l
M)b
CC50
(l
M)c
SId
a
Compd
IC50
(
lM)
1e
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
0.005 0.001
0.020 0.002
0.006 0.001
0.269 0.027
0.021 0.002
0.149 0.015
0.63 0.060
0.049 0.005
0.004 0.0004
0.0012 0.0001 1.002 0.645
0.040 0.004
0.047 0.005
0.31 0.030
1.72 0.170
0.305 0.031
0.052 0.005
0.0022 0.0003 39 8.2
17846
>10475
4250
>201
>20528
0.026 0.0015
0.0082 0.002
1.92 2.04
>272.35
34.85 4.42
>387.27
L100I
Y181I
K103N
1
2
3
5
8
9
10
11
0.009 0.001
0.07 0.006
0.05 0.006
0.07 0.005
0.38 0.04
0.03 0.003
0.01 0.001
0.18 0.02
0.08 0.01
0.33 0.03
>40
0.4 0.05
2.8 0.2
0.9 0.1
27
18
2
1
0.0151 0.004
0.046 0.015
0.12 0.026
0.0097 0.002
0.006 0.002
>307.92
133.53 109.73 2902
39.28 4.27
>371.14
>371.14
>356
>40
>40
>40
>40
>40
>40
>40
30
10
1
327
>40
2.61 0.26
>40
>40
6.7 0.7
10.92 1.10
>37114
>61857
>355
>5412
1826
104
>664
>35
>10543
>205
>6666
0.063 0.016
0.129 0.006
0.31 0.15
0.50 0.02
3.66 1.93
P342.60
235.6 26.81
32.31 11.93
>331.79
>127.56
>368.99
>15
12
16
Nevirapine
Efavirenz
7
1
3
4
0.5
0.05 0.006
0.5 0.05
0.2 0.03
a
0.035 0.014
0.073 0.015
Compound concentration IC50
the indicated strain.
(lM) required to inhibit by 50% the RT activity of
Nevirapine 0.18 0.02
Efavirenz 0.004 0.001
0.0009 0.0002 >6
a
Concentration required to inhibit by 50% the in vitro RNA-dependent DNA
The cluster analysis revealed a predominant ligand orientation
within the binding pocket and the most energetically favorable
conformation for each molecule was chosen for further analysis.
Figure 3A reports the docking results of 1–16 derivatives compared
to the crystallized position of efavirenz: compounds 1–16 were
bound in the NNIBP with an analogous binding mode and inter-
acted with RT residues of the allosteric pocket in a similar fashion
to efavirenz. The potent inhibitory effects of several synthesized
derivatives might also be due to the ability of the methyl groups
to occupy the hydrophobic space near the ‘roof’ of the NNRTI bind-
ing pocket (consisting of P95, Y181, Y188, and W229),10,16 thus cre-
ating additional intermolecular interactions as reported in Figure 4
for derivative 9, the most interesting of our compounds.
polymerase activity of recombinant RT.
b
Effective concentration required to reduce HIV-1-induced cytopathic effect by
50% in MT-4 cells.
c
Cytotoxic concentration required to reduce MT-4 cell viability by 50%.
d
Selectivity index: ratio CC50/EC50
See Ref. 10.
.
e
Docking of compounds 1–16 into the RT allosteric pocket, ex-
tracted from the structure of the complex RT/efavirenz (PDB code
1FK9)15 generated a number of possible binding conformations
with the corresponding AUTODOCK estimated free binding energies.

A.-M. Monforte et al. / Bioorg. Med. Chem. 18 (2010) 1702–1710
1705
Figure 4. Intermolecular interactions (shown as dashed magenta lines) between
the 3,4-dimethyl groups of compound 9 and NNIBP residues P95, Y181, Y188 and
W229. Hydrogen bonds (shown as dashed yellow lines) are formed between the
ligand and RT. This figure was prepared using the program PYMOL. PYMOL Molecular
Graphics System; v 0.99; DeLano Scientific: San Carlos, CA, USA, 2002.
Both efavirenz and our compounds showed a binding mode
similar to that assumed in wild-type; in fact the benzimidazol-2-
one moiety is oriented in the same position near the K103 and
the phenyl ring close to Y181 thus making hydrogen bonds possi-
ble between the amide group and K101 (Fig. 3B).
In fact, the activity of these compounds in the presence of the
L100I mutation remains comparable to that against wild-type RT,
probably due to the hydrophobic nature of both leucine and isoleu-
cine, as well as to the similar size of the two residues.
For the mutation Y181I, since the structure of RT with this
mutation is not available in the Protein Data Bank, we used the
crystal structure of Y181C mutant HIV-1 RT in complex with efavi-
renz18 (PDB code 1JKH), in which we changed the cysteine 181 in
isoleucine in order to obtain a plausible model (see Section 4).
Once again, the AUTODOCK program was run for derivatives 1–3, 5,
8–12, 16 and efavirenz obtaining a different binding mode for our
compounds whereas the efavirenz showed an orientation similar
to that observed with wild-type enzyme.
In fact, in the benzimidazolones, although the phenyl ring is ori-
ented near the I181, the remaining part of the structure is pointed
in a different direction thus losing the interaction with K101 in the
active site (Fig. 3C).
The Y181 ring is important to create
nyl ring at N-1, stabilizing the orientation of the remaining part of
the molecule. Thus, the mutation of Y181I causes the loss of this
interaction thereby creating new hydrophobic contacts between
p–p interactions with phe-
p–
p
I181 and lipophilic groups such as methyl substituents or chlorine
atom. However, these new hydrophobic interactions are not suffi-
cient to stabilize a suitable orientation of our compounds in the
binding pocket.
Nevertheless, there is also a decrease in the activity in the case
of efavirenz, but it is not so relevant because it maintains better its
orientation due to the presence of hydrophobic cyclopropyl nu-
cleus which is less influenced by the mutation of aromatic tyrosine
in the lipophilic isoleucine residue.
Molecular docking simulations on the K103N RT were not per-
formed as it has clearly been established that this mutation has
generally a minimal influence on the bound conformation of NNR-
TIs, while this mutation greatly affects the kinetics of the inhibitor-
binding process by stabilizing the unbound state of RT.19–23
Figure 3. (A) Binding mode of compounds 1–16 (cyan) compared to the crystallized
position of efavirenz (violet) in wild-type RT. (B) Binding mode of compounds 1–3,
5, 8–12, 16 (cyan) efavirenz (violet) in mutant L100I (yellow) RT. (C) Binding mode
of compounds 1–3, 5, 8–12, 16 (cyan) efavirenz (violet) in mutant Y181I (yellow)
RT. This figure was prepared using the program PYMOL
.
We carried out further docking studies in order to understand
the behavior of the most active compounds against different mu-
tant enzymes.
For the mutation L100I the crystal Structure of L100I mutant
HIV-1 reverse transcriptase in complex with GW420867X (PDB
code 2OPQ) was used17 for AUTODOCK docking experiments.
A docking study of GW420867X was performed to validate the
ability of the method. The program perfectly reproduced the exper-
imental position of the ligand with root-mean square deviation of
3. Conclusion
0
0.28 ÅA (1 cluster in 250 runs out of 250).
We docked derivatives 1–3, 5, 8–12, 16 and efavirenz, as refer-
ence compound.
In the context of our ongoing efforts to prepare novel potent
NNRTIs, a number of new derivatives have been synthesized. The

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A.-M. Monforte et al. / Bioorg. Med. Chem. 18 (2010) 1702–1710
results obtained, in both enzymatic tests on wt RT and in cellular
assays, showed high potency and very low cytotoxicity for the
majority of compounds.
An excellent HIV-1 inhibitory profile is shown by compound 9
with a potency superior to that of nevirapine and comparable to
that of compound 1 and efavirenz but with a higher selectivity in-
dex (>61857).
4.1.1.5. N-(5-Chloro-2-nitrophenyl)-2,4-dimethylbenzenesulf-
onamide (37). Mp: 125–127 °C, yield 100%. 1H NMR (DMSO-d6): d
2.31 (s, 3H, CH3, C-40), 2.51 (s, 3H, CH3, C-20), 7.17 (d, J = 7.14, 1H, H-
50), 7.23 (s, 1H, H-6), 7.32 (s, 1H, H-30), 7.38 (d, J = 8.51, 1H, H-4),
7.66 (d, J = 7.69, 1H, H-50), 7.96 (d, J = 8.51, 1H, H-3), 10.46 (br s,
1H, NH). Anal. Calcd for C14H13ClN2O4S: C, 49.34; H, 3.85; N,
8.22. Found: C, 49.78; H, 3.44; N, 8.57.
Furthermore, compounds 2, 3 and 9 also proved to be potent
inhibitors of RTs carrying the K103N and L100I mutations.
The molecular modeling studies helped us to rationalize the
relationships between the molecular structure of the inhibitors
and their activity toward RTs.
4.1.1.6. N-(5-Chloro-2-nitrophenyl)-2,5-dimethylbenzenesulf-
onamide (38). Mp: 259–261 °C, yield 100%. 1H NMR (DMSO-d6): d
2.27 (s, 3H, CH3, C-40), 3.55 (s, 3H, CH3, C-20), 6.44 (d, J = 7.42, 1H, H-
40), 6.98–7.08 (m, 2H, H-30,4), 7.27 (s, 1H, H-6), 7.35 (d, J = 8.79, 1H,
H-3), 7.68 (s, 1H, H-50). Anal. Calcd for C14H13ClN2O4S: C, 49.34; H,
3.85; N, 8.22. Found: C, 48.94; H, 3.52; N, 8.07.
4. Experimental section
4.1. Chemistry
4.1.1.7. N-(5-Chloro-2-nitrophenyl)-3,4-dimethylbenzenesulf-
onamide (39). Mp: 154–155 °C, yield 98%. 1H NMR (DMSO-d6): d
2.25 (s, 3H, CH3, C-40), 2.27 (s, 3H, CH3, C-30), 7.29 (s, 1H, H-6), 7.34
(d, J = 7.96, 1H, H-50), 7.41 (d, J = 9.06, 1H, H-60), 7.49 (d, J = 8.79,
1H, H-4), 7.56 (s, 1H, H-20), 7.96 (d, J = 8.79, 1H, H-3), 10.39 (br s,
1H, NH). Anal. Calcd for C14H13ClN2O4S: C, 49.34; H, 3.85; N,
8.22. Found: C, 49.65; H, 4.21; N, 8.34.
Melting points were determined on a BUCHI Melting Point B-
545 apparatus 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 and
purity of tested compounds was >95%. Merck Silica Gel 60 F254
plates were used for TLC; column chromatography was performed
on Merck Silica Gel 60 (230–400 mesh) and flash chromatography
(FC) on Biotage SP1 EXP. 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).
4.1.1.8. N-(5-Chloro-2-nitrophenyl)-2,4,6-trimethylbenzene-
sulfonamide (40). Mp: 117–119 °C, yield 82%. 1H NMR (CDCl3): d
2.22 (s, 3H, CH3), 2.48 (s, 6H, 2CH3), 6.46 (d, J = 9.06, 1H, H-4), 6.76
(s, 2H, H-30,50), 6.92 (s, 1H, H-6), 7.63 (d, J = 9.06, 1H, H-3), 10.19 (br
s, 1H, NH). Anal. Calcd for C15H15ClN2O4S: C, 50.78; H, 4.26; N, 7.90.
Found: C, 50.39; H, 4.51; N, 7.73.
4.1.1. General procedures for the synthesis of N-(5-chloro-2-
nitrophenyl)-benzenesulfonamides (32, 34–46)
4.1.1.9. N-(5-Chloro-2-nitrophenyl)-2,3,5,6-tetramethylben-
zenesulfonamide (41). Mp: 184–186 °C, yield 71%. 1H NMR
(DMSO-d6): d 2.21 (s, 6H, CH3, C-30,50), 2.39 (s, 6H, CH3, C-20,60),
7.21 (s, 1H, H-6), 7.29 (s, 1H, H-40), 7.42 (d, J = 8.79, 1H, H-4),
8.01 (d, J = 8.79, 1H, H-3), 10.41 (br s, 1H NH). Anal. Calcd for
C16H17ClN2O4S: C, 52.10; H, 4.65; N, 7.59. Found: C, 51.86; H,
4.23; N, 7.14.
The mixture of the appropriate arylsulfonyl chloride (3 mmol)
and the 5-chloro-2-nitroaniline or 2-amino-6-chloro-3-nitropyri-
dine (2 mmol) in dioxane (6 mL), was stirred for 30 min at 0 °C
using dry sodium hydride (10 mmol) as catalyst. The reaction mix-
ture was then quenched with a saturated NaHCO3 solution, ex-
tracted with chloroform and dried over Na2SO4. After removal of
the solvent under reduced pressure, the residue was crystallized
from diethyl ether.
4.1.1.10. N-(5-Chloro-2-nitrophenyl)-2,3,4,5,6-pentamethylben-
zenesulfonamide (42). Mp: 182–184 °C, yield 100%. 1H NMR
(DMSO-d6): d 2.17 (s, 6H, CH3, C-30,50), 2.23 (s, 3H, CH3, C-40),
2.43 (s, 6H, CH3, C-20,60), 7.21 (s, 1H, H-6), 7.39 (d, J = 8.79, 1H, H-
4), 8.01 (d, J = 8.79, 1H, H-3), 10.36 (br s, 1H, NH). Anal. Calcd for
C17H19ClN2O4S: C, 53.34; H, 5.00; N, 7.32. Found: C, 53.61; H,
5.33; N, 7.68.
4.1.1.1. N-(5-Chloro-2-nitrophenyl)-3,5-dimethylbenzenesulf-
onamide (32). Compound 32 was prepared as previously
reported.10
4.1.1.2. N-(5-Chloro-2-nitrophenyl)-2-methylbenzenesulfona-
mide (34). Mp: 142–144 °C, yield 100%. 1H NMR (DMSO-d6): d
2.56 (s, 3H, CH3), 7.30 (s, 1H, H-6), 7.35–7.59 (m, 4H, H-4,
30,40,50), 7.77 (d, J = 7.96, 1H, H-60), 7.96 (d, J = 8.79, 1H, H-3),
10.59 (br s, 1H, NH). Anal. Calcd for C13H11ClN2O4S: C, 47.79; H,
3.39; N, 8.57. Found: C, 48.03; H, 3.21; N, 8,69.
4.1.1.11. N-(5-Chloro-2-nitrophenyl)-3,5-bistrifluoromethyl-
benzenesulfonamide (43). Mp: 121–124 °C, yield 95%. 1H NMR
(CDCl3): d 7.17 (d, J = 9.06, 1H, H-4), 7.80 (s, 1H, H-6), 8.09–8.37
(m, 4H, H-3, 20,40,60), 10.07 (br s, 1H, NH). Anal. Calcd for
C14H7ClF6N2O4S: C, 37.47; H, 1.57; N, 6.24. Found: C, 37.15; H,
1.79; N, 6.61.
4.1.1.3. N-(5-Chloro-2-nitrophenyl)-3-methylbenzenesulfona-
mide (35). Mp: 127–130 °C, yield 99%. 1H NMR (CDCl3): d 2.41 (s,
3H, CH3), 7.10 (d, J = 9.06, 1H, H-4), 7.39–7.71 (m, 4H, H-6, 40,50,60),
7.87 (s, 1H, H-20), 8.09 (d, J = 9.06, 1H, H-3), 10.01 (br s, 1H, NH).
Anal. Calcd for C13H11ClN2O4S: C, 47.79; H, 3.39; N, 8.57. Found:
C, 47.43; H, 3.45; N, 8.84.
4.1.1.12. N-(5-Chloro-2-nitrophenyl)-3-trifluoromethylbenzene-
sulfonamide (44). Mp: 120–123 °C, yield 100%. 1H NMR (CDCl3):
d 7.13 (d, J = 8.51, 1H, H-4), 7.67–7.88 (m, 4H, H-6, 40,50,60), 8.08
(d, J = 8.24, 1H, H-3), 8.14 (s, 1H, H-20), 10.04 (br s, 1H, NH). Anal.
Calcd for C13H8ClF3N2O4S: C, 41.01; H, 2.12; N, 7.36. Found: C,
41.44; H, 2.50; N, 7.64.
4.1.1.4. N-(5-Chloro-2-nitrophenyl)-4-methylbenzenesulfona-
mide (36). Mp: 133–135 °C, yield 100%. 1H NMR (DMSO-d6): d
2.35 (s, 3H, CH3), 7.27 (s, 1H, H-6), 7.38 (d, J = 7.96, 2H, H-30,50),
7.44 (d, J = 8.79, 1H, H-4), 7.65 (d, J = 7.69, 2H, H-20,60), 7.96 (d,
J = 8.79, 1H, H-3), 10.47 (br s, 1H, NH). Anal. Calcd for
C13H11ClN2O4S: C, 47.79; H, 3.39; N, 8.57. Found: C, 47.35; H,
3.72; N, 8.26.
4.1.1.13. N-(5-Chloro-2-nitrophenyl)-3-trifluoromethoxyben-
zenesulfonamide (45). Mp: 95–97 °C, yield 94%. 1H NMR (CDCl3):
d 7.16 (d, J = 8.99, 1H, H-4), 7.47 (d, J = 8.51, 1H, H-40), 7.54–7.59
(m, 1H, H-50), 7.73 (s, 1H, H-6), 7.83 (d, J = 7.69, 1H, H-60), 7.87
(s, 1H, H-20), 8.09 (d, J = 9.06, 1H, H-3), 9.99 (br s, 1H, NH). Anal.

A.-M. Monforte et al. / Bioorg. Med. Chem. 18 (2010) 1702–1710
1707
Calcd for C13H8ClF3N2O5S: C, 39.36; H, 2.03; N, 7.06. Found: C,
39.62; H, 2.31; N, 6.95.
4.1.3.6. N-(2-Aminophenyl-5-chloro)-2,5-dimethylbenzenesulf-
onamide (23). Mp: 150–152 °C, yield 48%. 1H NMR (DMSO-d6): d
2.27 (s, 3H, CH3, C-40), 2.51 (s, 3H, CH3, C-20), 5.15 (br s, 2H, NH2),
6.61 (s, 1H, H-6), 6.64 (d, J = 9.06, 1H, H-3), 6.91 (d, J = 8.51, 1H,
H-4), 7.22–7.35 (m, 2H, H-30,40), 7.56 (s, 1H, H-60), 9.36 (br s, 1H,
NH). Anal. Calcd for C14H15ClN2O2S: C, 54.10; H, 4.86; N, 9.01.
Found: C, 54.34; H, 4.99; N, 9.41.
4.1.1.14. N-(5-Chloro-2-nitrophenyl)-3-methoxybenzenesulfona-
mide (46). Mp: 111–113 °C, yield 73%. 1H NMR (CDCl3): d 3.84 (s,
3H, CH3), 7.12 (d, J = 8.79, 1H, H-4), 7.27 (s, 1H, H-6), 7.37–7.48 (m,
3H, H-40,50,60), 7.89 (s, 1H, H-20), 8.09 (d, J = 8.79, 1H, H-3), 9.99 (br
s, 1H, NH). Anal. Calcd for C13H11ClN2O5S: C, 45.56; H, 3.23; N, 8.17.
Found: C, 45.29; H, 3.37; N, 8.40.
4.1.3.7. N-(2-Aminophenyl-5-chloro)-3,4-dimethylbenzenesulf-
onamide (24). Mp: 95–97 °C, yield 100%. 1H NMR (DMSO-d6): d
2.21 (s, 3H, CH3, C-40), 2.25 (s, 3H, CH3, C-30), 4.70 (br s, 1H, NH2),
6.41–6.76 (m, 3H, H-3, 4, 6), 7.16 (d, J = 7.96, 1H, H-50), 7.37 (d,
J = 7.96, 1H, H-20), 7.44 (s, 1H, H-60). Anal. Calcd for C14H15ClN2O2S:
C, 54.10; H, 4.86; N, 9.01. Found: C, 53.83; H, 5.25; N, 8.77.
4.1.2. General procedure for the synthesis of N-(6-chloro-3-
nitropyridin-2-yl)-3,5-dimethylbenzenesulfonamide (33)
Using a similar procedure for 32, 34–46, compound 33 was pre-
pared from 2-amino-6-chloro-3-nitropyridine (2 mmol) and the
appropriate aryl sulfonyl chloride (3 mmol).
4.1.3.8. N-(2-Aminophenyl-5-chloro)-2,4,6-trimethylbenzene-
sulfonamide (25). Mp: 146–148 °C, yield 45%. 1H NMR (DMSO-
d6): d 2.68 (s, 3H, CH3), 2.74 (s, 6H, CH3), 5.37 (br s, 2H, NH2),
6.76 (s, 1H, H-6), 6.88 (d, J = 8.54, 1H, H-3), 7.16 (d, J = 8.54, 1H,
H-4), 7.25 (s, 2H, H-30,50). Anal. Calcd for C15H17ClN2O2S: C,
55.46; H, 5.28; N, 8.62. Found: C, 55,14; H, 5.67; N, 8.23.
Mp: 215–219 °C, yield 77%. 1H NMR (DMSO-d6): d 2.34 (s, 6H,
CH3), 7.25–7.35 (m, 2H, H-4, H-40), 7.70 (s, 2H, H20,60), 8.42 (d,
J = 8.24, 1H, H-3). Anal. Calcd for C13H12ClN3O4S: C, 45.69; H,
3.54; N, 12.29. Found: C, 45.48; H, 3.81; N, 12.57.
4.1.3. General procedures for the synthesis of N-(2-aminophenyl-
5-chloro)-benzenesulfonamides (17, 19–31)
4.1.3.9. N-(2-Aminophenyl-5-chloro)-2,3,5,6-tetramethylbenzene-
sulfonamide (26). Mp: 161–163 °C, yield 63%. 1H NMR (DMSO-
d6): d 2.19 (s, 6H, CH3, C-30,50), 2.34 (s, 6H, CH3, C-20,60), 5.14 (br
s, 2H, NH2), 6.54 (s, 1H, H-6), 6.64 (d, J = 8.51, 1H, H-3), 6.92 (d,
J = 8.51, 1H, H-4), 7.23 (s, 1H, H-40), 9.34 (br s, 1H, NH). Anal. Calcd
for C16H19ClN2O2S: C, 56.71; H, 5.65; N, 8.27. Found: C, 56,38; H,
5.14; N, 8.45.
Zinc dust (20 mmol) was added in several portions to a solu-
tion of the appropriate N-(5-chloro-2-nitrophenyl)-benzenesul-
fonamide (0.6 mmol) in 3 mL HCl 2 N and 4 mL EtOH anhydrous
under stirring in an ice bath. The reaction mixture was heated
in a water bath for 1 h. After cooling, it was made alkaline with
NaOH 2 N and extracted with ethyl acetate. The extract was
washed with water, dried over Na2SO4 and evaporated under re-
duced pressure. The residue was powdered by treatment with
ethanol or diethyl ether.
4.1.3.10. N-(2-Aminophenyl-5-chloro)-2,3,4,5,6-pentamethylben-
zenesulfonamide (27). Mp: 171–173 °C, yield 89%. 1H NMR
(DMSO-d6): d 2.15 (s, 6H, CH3, C-30,50), 2.22 (s, 3H, CH3, C-40),
2.36 (s, 6H, CH3, C-20,60), 5.13 (br s, 2H, NH2), 6.63–6.66 (m, 2H,
H-3,6), 6.92 (d, J = 8.51, 1H, H-4), 9.47 (br s, 1H, NH). Anal. Calcd
for C17H21ClN2O2S: C, 57.86; H, 6.00; N, 7.94. Found: C, 57.43; H,
6.25; N, 7.59.
4.1.3.1. N-(2-Aminophenyl-5-chloro)-3,5-dimethylbenzenesulf-
onamide (17). Compound 17 was prepared as previously
reported.10
4.1.3.2. N-(2-Aminophenyl-5-chloro)-2-methylbenzenesulfona-
mide (19). Mp: 163–165 °C, yield 100%. 1H NMR (DMSO-d6): d
2.55 (s, 3H, CH3), 5.10 (br s, 2H, NH2), 6.57 (d, J = 8.79, 1H, H-3),
6.62 (s, 1H, H-6), 6.79 (d, J = 8.51, 1H, H-4), 7.27–7.36 (m, 2H, H-
30,40), 7.44–7.49 (m, 1H, H-50), 7.71 (d, J = 7.69, 1H, H-60). Anal.
Calcd for C13H13ClN2O2S: C, 52.61; H, 4.44; N, 9.44. Found: C,
52,33; H, 4.87; N, 9.14.
4.1.3.11. N-(2-Aminophenyl-5-chloro)-3,5-bistrifluorometh-
ylbenzenesulfonamide (28). Mp: 182–185 °C, yield 44%. 1H NMR
(DMSO-d6): d 5.39 (br s, 2H, NH2), 6.70 (d, J = 8.24, 1H, H-3), 6.84
(d, J = 8.24, 1H, H-4), 6.93 (s, 1H, H-6), 8.26 (s, 1H, H-40), 8.35 (s,
2H, H-20,60). Anal. Calcd for C14H9ClF6N2O2S: C, 40.16; H, 2.17; N,
6.69. Found: C, 39.82; H, 2.44; N, 6.24.
4.1.3.3. N-(2-Aminophenyl-5-chloro)-3-methylbenzenesulfona-
mide (20). Mp: 116–118 °C, yield 44%. 1H NMR (DMSO-d6): d 2.66
(s, 3H, CH3), 5.25 (br s, 2H, NH2), 6.76 (d, J = 8.51, 1H, H-3), 6.82 (s,
1H, H-6), 7.06 (d, J = 8.51, 1H, H-4), 7.54–7.68 (m, 4H, H-20,40,50,60),
9.52 (br s, 1H, NH). Anal. Calcd for C13H13ClN2O2S: C, 52.61; H,
4.44; N, 9.44. Found: C, 52.97; H, 4.23; N, 9.79.
4.1.3.12. N-(2-Aminophenyl-5-chloro)-3-trifluoromethylben-
zenesulfonamide (29). Mp: 116–119 °C, yield 59%. 1H NMR
(DMSO-d6): d 5.12 (br s, 2H, NH2), 6.65 (d, J = 8.79, 1H, H-3), 6.70
(s, 1H, H-6), 6.93 (d, J = 8.79, 1H, H-4), 7.77–7.97 (m, 3H, H-6,
40,50), 8.03 (d, J = 7.69, 1H, H-60), 9.55 (br s, 1H, NH), 7.85 (d,
J = 7.41, 1H, H-60). Anal. Calcd for C13H10ClF3N2O2S: C, 44.52; H,
2.87; N, 7.99. Found: C, 44.91; H, 2.56; N, 8.24.
4.1.3.4. N-(2-Aminophenyl-5-chloro)-4-methylbenzenesulfona-
mide (21). Mp: 158–160 °C, yield 87%. 1H NMR (DMSO-d6): d 2.34
(s, 3H, CH3), 5.07 (br s, 2H, NH2), 6.60 (d, J = 8.51, 1H, H-3), 6.72 (s,
1H, H-6), 6.87 (d, J = 8.51, 1H, H-4), 7.33–7.57 (m, 4H, H-20,30,50,60),
9.37 (br s, 1H, NH). Anal. Calcd for C13H13ClN2O2S: C, 52.61; H,
4.44; N, 9.44. Found: C, 52.25; H, 4.18; N, 9.82.
4.1.3.13. N-(2-Aminophenyl-5-chloro)-3-trifluoromethoxyben-
zenesulfonamide (30). Mp: 121–124 °C, yield 87%. 1H NMR
(DMSO-d6): d 5.51 (br s, 2H, NH2), 6.68 (d, J = 7.96, 1H, H-3), 6.91
(s, 1H, H-6), 6.96 (d, J = 8.51, 1H, H-4), 7.46–7.55 (m, 3H, H-40,50),
7.75 (s, 1H, H-20), 7.85 (d, J = 7.41, 1H, H-60). Anal. Calcd for
C13H10ClF3N2O3S: C, 42.58; H, 2.75; N, 7.64. Found: C, 42.29; H,
3.11; N, 8.06.
4.1.3.5. N-(2-Aminophenyl-5-chloro)-2,4-dimethylbenzenesulf-
onamide (22). Mp: 115–117 °C, yield 34%. 1H NMR (DMSO-d6): d
2.28 (s, 3H, CH3), 2.47 (s, 3H, CH3), 5.12 (br s, 2H, NH2), 6.59 (d,
J = 8.51, 1H, H-3), 6.65 (s, 1H, H-6), 6.85 (d, J = 8.51, 1H, H-4),
7.11 (d, J = 8.24, 1H, H-50), 7.18 (s, 1H, H-30), 7.58 (d, J = 7.96, 1H,
H-60), 9.38 (br s, 1H, NH). Anal. Calcd for C14H15ClN2O2S: C,
54.10; H, 4.86; N, 9.01. Found: C, 54.46; H, 4.57; N, 9.29.
4.1.3.14. N-(2-Aminophenyl-5-chloro)-3-methoxybenzenesulf-
onamide (31). Mp: 117–120 °C, yield 66%. 1H NMR (DMSO-d6): d
5.11 (br s, 2H, NH2), 6.79 (d, J = 8.51, 1H, H-3), 6.71 (s, 1H, H-6),
6.91 (d, J = 8.79, 1H, H-4), 7.18–7.48 (m, 4H, H-20,40,50,60), 9.39
(br s, 1H, NH), 7.85 (d, J = 7.41, 1H, H-60). Anal. Calcd for

1708
A.-M. Monforte et al. / Bioorg. Med. Chem. 18 (2010) 1702–1710
C13H13ClN2O3S: C, 49.92; H, 4.19; N, 8.96. Found: C, 49.54; H, 4.53;
N, 9.25.
J = 7.96, 1H, H-40), 7.49 (d, J = 7.69, 1H, H-30), 7.68 (s, 1H, H-7),
7.90 (s, 1H, H-60), 11.71 (br s, 1H, NH). Anal. Calcd for
C15H13ClN2O3S: C, 53.49; H, 3.89; N, 8.32. Found: C, 53.85; H,
3.46; N, 8.61.
4.1.4. General procedure for the synthesis of N-(3-amino-6-
chloropyridin-2-yl)-3,5-dimethylbenzenesulfonamide (18)
Using a similar procedure for 17, 19–31, compound 18 was pre-
pared from N-(6-chloro-3-nitropyridin-2-yl)-3,5-dimethylben-
zenesulfonamide 33 (0.6 mmol).
4.1.5.7. 6-Chloro-1-(3,4-dimethylphenylsulfonyl)-1,3-dihydro-
2H-benzimidazol-2-one (9). Mp: 260–262 °C, yield 59%. 1H NMR
(DMSO-d6): d 2.29 (s, 6H, CH3), 7.02 (d, J = 8.51, 1H, H-4), 7.23 (dd,
J = 1.92, J = 8.51, 1H, H-5), 7.42 (d, J = 7.96, 1H, H-50), 7.72–7.80 (m,
3H, H-7, H-20,60). Anal. Calcd for C15H13ClN2O3S: C, 53.49; H, 3.89;
N, 8.32. Found: C, 53.11; H, 3.97; N, 8.74.
Mp: 202–205 °C, yield 78%. 1H NMR (DMSO-d6): d 2.17 (s, 6H,
CH3), 5.38 (br s, 2H, NH2), 6.63 (d, J = 7.69, 1H, H-3), 7.01 (s, 1H,
H-40), 7.21 (d, J = 8.51, 1H, H-4), 7.39 (s, 2H, H-20,60). Anal. Calcd
for C13H14ClN3O2S: C, 50.08; H, 4.53; N, 13.48. Found: C, 50.51;
H, 4.18; N, 13.81.
4.1.5.8. 6-Chloro-1-(2,4,6-trimethylphenylsulfonyl)-1,3-dihy-
dro-2H-benzimidazol-2-one (10). Mp: 240–242 °C, yield 62%. 1H
NMR (CDCl3): d 2.31 (s, 3H, CH3), 2.62 (s, 6H, 2CH3), 6.92 (d,
J = 8.24, 1H, H-4), 6.99 (s, 2H, H-30,50), 7.17 (d, J = 8.24, H-5), 7.96
(s, 1H, H-7). Anal. Calcd for C16H15ClN2O3S: C, 54.78; H, 4.31; N,
7.98. Found: C, 54.31; H, 4.74; N, 7.53.
4.1.5. General procedure for the synthesis of 1-arylsulfonyl-1,3-
dihydro-2H-benzimidazol-2-ones (1, 4–16)
An excess of phosgene 20% toluene (1 mL) was added to a solu-
tion of the appropriate N-(2-aminophenyl-5-chloro)-benzenesul-
fonamide (0.25 mmol) in HCl 2 N (4 mL), and the resulting
mixture was stirred and heated for 4 h. The reaction mixture was
then cooled, neutralized with NaOH 2 N, extracted with ethyl ace-
tate, washed with water and dried over Na2SO4. After evaporation
under reduced pressure, the residue was crystallized by ethyl ace-
tate or diethyl ether.
4.1.5.9. 6-Chloro-1-(2,3,5,6-tetramethylphenylsulfonyl)-1,3-dihy-
dro-2H-benzimidazol-2-one (11). Mp: 241–243 °C, yield 86%. 1H
NMR (DMSO-d6): d 2.24 (s, 6H, CH3, C-30,50), 2.39 (s, 6H, CH3, C-
20,60), 7.10 (d, J = 8.51, 1H, H-4), 7.28 (d, J = 8.51, 1H, H-5), 7.40 (s,
1H, H-40), 7.66 (s, 1H, H-7), 11.70 (br s, 1H, NH). Anal. Calcd for
C17H17ClN2O3S: C, 55.96; H, 4.70; N, 7.68. Found: C, 55.58; H,
4.36; N, 7.88.
4.1.5.1. 6-Chloro-1-(3,5-dimethylphenylsulfonyl)-1,3-dihydro-
2H-benzimidazol-2-one (1). Compound 1 was prepared as previ-
ously reported.10
4.1.5.10. 6-Chloro-1-(2,3,4,5,6-pentamethylphenylsulfonyl)-
1,3-dihydro-2H-benzimidazol-2-one (12). Mp: 243–245 °C,
4.1.5.2. 6-Chloro-1-(2-methylphenylsulfonyl)-1,3-dihydro-2H-ben-
zimidazol-2-one (4). Mp: 272–274 °C, yield 81%. 1H NMR (DMSO-
d6): d 2.40 (s, 3H, CH3), 7.09 (d, J = 8.24, 1H, H-4), 7.27 (d, J = 8.24,
1H, H-5), 7.45–7.70 (m, 4H, H-7, H-30,40,50), 8.09 (d, J = 7.96, 1H, H-
60), 11.73 (br s, 1H, NH). Anal. Calcd for C14H11ClN2O3S: C, 52.10; H,
3.44; N, 8.68. Found: C, 52.57; H, 3.73; N, 8.24.
1
yield 85%. H NMR (DMSO-d6): d 2.20 (s, 6H, CH3, C-30,50), 2.27 (s,
3H, CH3, C-40), 2.42 (s, 6H, CH3, C-20,60), 7.10 (d, J = 8.51, 1H, H-4),
7.27 (d, J = 8.51, 1H, H-5), 7.65 (s, 1H, H-7), 11.68 (br s, 1H, NH).
Anal. Calcd for C18H19ClN2O3S: C, 57.06; H, 5.05; N, 7.39. Found:
C, 57.45; H, 5.27; N, 7.69.
4.1.5.11. 6-Chloro-1-(3,5-bistrifluoromethylphenylsulfonyl)-1,3-
dihydro-2H-benzimidazol-2-one (13). Mp: 238–241 °C, yield
16%. 1H NMR (DMSO-d6): d 7.04 (d, J = 8.51, 1H, H-4), 7.24 (d,
J = 8.24, 1H, H-5), 7.78 (s, 1H, H-7), 8.66 (s, 1H, H-40), 8.69 (s, 2H,
H-20,60), 11.76 (br s, 1H, NH). Anal. Calcd for C15H7ClF6N2O3S: C,
40.51; H, 1.59; N, 6.30. Found: C, 40.17; H, 1.88; N, 5.92.
4.1.5.3. 6-Chloro-1-(3-methylphenylsulfonyl)-1,3-dihydro-2H-
benzimidazol-2-one (5). Mp: 213–216 °C, yield 43%. 1H NMR
(DMSO-d6): d 2.36 (s, 3H, CH3), 6.80 (d, J = 8.51, 1H, H-4), 6.98
(dd, J = 2.19, J = 8.51, 1H, H-5), 7.44–7.52 (m, 3H, H-40,50,60), 7.74
(s, 1H, H-7), 7.77 (s, 1H, H-20). Anal. Calcd for C14H11ClN2O3S: C,
52.10; H, 3.44; N, 8.68. Found: C, 51.76; H, 3.80; N, 8.92.
4.1.5.12. 6-Chloro-1-(3-trifluoromethylphenylsulfonyl)-1,3-dihy-
dro-2H-benzimidazol-2-one (14). Mp: 208–211 °C, yield 15%. 1H
NMR (DMSO-d6): d 6.96 (d, J = 8.24, 1H, H-4), 7.17 (d, J = 8.51, 1H,
H-5), 7.67 (s, 1H, H-7), 7.87–7.92 (m, 1H, H-50), 8.18 (d, J = 7.96,
1H, H-40), 8.33–8.35 (m, 2H, H-20,60). Anal. Calcd for
C14H8ClF3N2O3S: C, 44.63; H, 2.14; N, 7.44. Found: C, 44.79; H,
1.88; N, 7.63.
4.1.5.4. 6-Chloro-1-(4-methylphenylsulfonyl)-1,3-dihydro-2H-
benzimidazol-2-one (6). Mp: 274–277 °C, yield 87%. 1H NMR
(DMSO-d6): d 2.38 (s, 3H, CH3), 7.02 (d, J = 8.51, 1H, H-4), 7.24 (d,
J = 8.51, 1H, H-5), 7.46 (d, J = 8.24, 2H, H-30,50), 7.72 (s, 1H, H-7),
7.92 (d, J = 8.24, 2H, H-20,60), 11.66 (br s, 1H, NH). Anal. Calcd for
C14H11ClN2O3S: C, 52.10; H, 3.44; N, 8.68. Found: C, 52.34; H,
3.15; N, 8.53.
4.1.5.13. 6-Chloro-1-(3trifluoromethoxyphenylsulfonyl)-1,3-
dihydro-2H-benzimidazol-2-one (15). Mp: 207–209 °C, yield
24%. 1H NMR (DMSO-d6): d 6.81 (d, J = 8.24, 1H, H-4), 7.02 (d,
J = 8.51, 1H, H-5), 7.51 (s, 1H, H-7), 7.72–7.99 (m, 4H, H-
20,40,50,60). Anal. Calcd for C14H8ClF3N2O4S: C, 42.82; H, 2.05; N,
7.13. Found: C, 42.57; H, 2.41; N, 7.78.
4.1.5.5. 6-Chloro-1-(2,4-dimethylphenylsulfonyl)-1,3-dihydro-
2H-benzimidazol-2-one (7). Mp: 206–208 °C, yield 40%. 1H
NMR (DMSO-d6): d 2.34 (s, 3H, CH3, C-40), 2.47 (s, 3H, CH3, C-
20), 7.06 (d, J = 8.24, 1H, H-4), 7.23 (s, 1H, H-30), 7.24 (d,
J = 8.51, 1H, H-20), 7.29 (d, J = 8.24, 1H, H-5), 7.66 (s, 1H, H-7),
7.96 (d, J = 7.96, 1H, H-60), 11.58 (br s, 1H, NH). Anal. Calcd for
C15H13ClN2O3S: C, 53.49; H, 3.89; N, 8.32. Found: C, 53.10; H,
3.61; N, 8.03.
4.1.5.14. 6-Chloro-1-(3-methoxyphenylsulfonyl)-1,3-dihydro-
2H-benzimidazol-2-one (16). Mp: 270–273 °C, yield 17%. 1H
NMR (DMSO-d6): d 3.81 (s, 3H, CH3), 7.03 (d, J = 8.24, 1H, H-4),
7.24 (d, J = 8.24, 1H, H-5), 7.36–7.72 (m, 5H, H-7, H-20,40,50,60),
11.7 (br s, 1H, NH). Anal. Calcd for C14H11ClN2O4S: C, 49.64; H,
3.27; N, 8.27. Found: C, 50.06; H, 2.92; N, 8.55.
4.1.5.6. 6-Chloro-1-(2,5-dimethylphenylsulfonyl)-1,3-dihydro-
2H-benzimidazol-2-one (8). Mp: 272–274 °C, yield 41%. 1H NMR
(DMSO-d6): d 2.33 (s, 3H, CH3, C-50), 2.37 (s, 3H, CH3, C-20), 7.08 (d,
J = 8.51, 1H, H-4), 7.26 (dd, J = 1.92, J = 8.51, 1H, H-5), 7.34 (d,

A.-M. Monforte et al. / Bioorg. Med. Chem. 18 (2010) 1702–1710
1709
4.1.6. General procedure for the synthesis of 6-chloro-1-(3,5-di-
methylphenylsulfonyl)-1H-imidazo[4,5-b]pyridin-2(3H)-one (2)
Using a similar procedure for 1, 4–16, compound 2 was pre-
pared from N-(3-amino-6-chloropyridin-2-yl)-3,5-dimethylben-
zenesulfonamide 18 (0.25 mmol).
concentration (EC50) was defined as the concentration of com-
pound required to reduce the virus-induced cytopathicity by 50%.
4.3. Computational procedures
Mp: 160–163 °C, yield 52%. 1H NMR (DMSO-d6): d 2.35 (s, 6H,
CH3), 7.27 (d, J = 8.24, 1H, H-4), 7.40–7.43 (m, 2H, H-5, H-40),
7.69 (s, 2H, H-20,60), 11.81 (br s, 1H, NH). Anal. Calcd for
C14H12ClN3O3S: C, 49.78; H, 3.58; N, 12.44. Found: C, 49.33; H,
3.95; N, 12.24.
The structures of ligands were constructed using the Schroding-
er Maestro26 and were then submitted to Polak–Ribiere conjugate
gradient minimization (0.0005 kJ/(Å mol) convergence). The target
macromolecules from 1FK9, 2OPS and 1JKH PDB complexes were
prepared using the protein preparation workflow in Maestro.26
1JKH was used as a template to build a Y181I RT model; prior to
the last step of the protein preparation workflow, cysteine 181
was mutated to isoleucine, and this sidechain was submitted to
an exhaustive conformational search, the lowest-energy conformer
was finally selected for the last step of the protein preparation
workflow (i.e. relaxation of the complex using the OPLS force-
field.27
4.1.7. General procedure for the synthesis of 6-chloro-1-(3,5-
dimethylphenylsulfonyl)-1,3-dihydro-2H-benzimidazol-2-
thione (3)
Thiophosgene (0.25 mmol) was added to a solution of N-(2-ami-
nophenyl-5-chloro)-3,5-dimethylbenzenesulfonamide
17
(0.25 mmol) in acetone and the resulting mixture was stirred for
1 h at room temperature. The reaction solvent was evaporated un-
der reduced pressure and the residue was crystallized from
ethanol.
All docking calculations were performed using AUTODOCK version
4.0.13
For each enzyme, a grid box of 60–60–60 points (0.375 spacing)
was centered on the allosteric site, using the crystallographic posi-
tions of the ligands as reference, and used to define the docking
space.
The GA-LS method was used with the default settings and 250
docking runs were performed for each ligand/enzyme docking. The
docking results were clustered using an rmsd tolerance of 1 Å and
the lowest-energy conformer was selected as binding conformation.
Autodock input files were prepared using AUTODOCKTools.28
The resulting complexes were then minimized using 200 steps
of steepest descent minimization followed by 200 steps of
Polak–Ribiere conjugate gradient minimization using the MACRO-
MODEL program29 (OPLS force-field27).
Mp: 234–237 °C, yield 14%. 1H NMR (DMSO-d6): d 2.35 (s, 6H,
CH3), 7.04 (d, J = 8.51, 1H, H-4), 7.24 (dd, J = 1.92, J = 8.51, 1H, H-
5), 7.43 (s, 1H, H-40), 7.68 (s, 2H, H-20,60), 7.73 (s, 1H, H-7), 11.67
(br s, 1H, NH). Anal. Calcd for C15H13ClN2O2S: C, 51.06; H, 3.71;
N, 7.94. Found: C, 51.47; H, 3.30; N, 7.58.
4.2. Anti-HIV activity assays
4.2.1. 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,
a 25 lL final reaction volume contained: TDB buffer (50 mM Tris–
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
lastly, was introduced into tubes containing aliquots of different
enzyme concentrations (5–10 nM RT). After incubation at 37 °C
Acknowledgment
Financial support for this research by the Fondo Ateneo di ric-
erca (2005, Messina, Italy).
for the indicated time, 20 lL from each reaction tube was spiked
References and notes
on glass fiber filters GF/C and immediately immersed in 5% ice-cold
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5 min, then dried and, lastly, EcoLumeÒ Scintillation cocktail
(ICN, Research Products Division, Costa Mesa, CA USA) was added
to detect acid-precipitable radioactivity using a PerkinElmerÒ Tri-
lux MicroBeta 1450 Counter.
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1710
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