Design, synthesis, and structure-activity relationships of 1,3-dihydrobenzimidazol-2-one analogues as anti-HIV agents

Article abstract of DOI:10.1016/j.bmc.2009.06.068

Non-nucleoside reverse transcriptase inhibitors (NNRTIs) have become very important components in the antiretroviral combination therapies used to treat HIV. Recently, our group identified some 1,3-dihydrobenzimidazol-2-one derivatives and their sulfones as a potent and novel class of NNRTIs. We herein report the synthesis and biological evaluation of the new compounds in which different structural modifications have been introduced in order to investigate their effects on RT inhibition.

Full text of DOI:10.1016/j.bmc.2009.06.068

Bioorganic & Medicinal Chemistry 17 (2009) 5962–5967
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis, and structure–activity relationships of
1,3-dihydrobenzimidazol-2-one analogues as anti-HIV agents
a
a
a
a
Anna-Maria Monforte a, , Patrizia Logoteta , Stefania Ferro , Laura De Luca , Nunzio Iraci ,
*
Giovanni Maga b, Erik De Clercq c, Christophe Pannecouque c, Alba Chimirri a
a Dipartimento Farmaco-Chimico, Università di Messina, Viale Annunziata, 98168 Messina, Italy
b Istituto di Genetica Molecolare IGM-CNR, via Abbiategrasso 207, 27100 Pavia, Italy
c 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:
Non-nucleoside reverse transcriptase inhibitors (NNRTIs) have become very important components in
the antiretroviral combination therapies used to treat HIV. Recently, our group identified some 1,3-dihy-
drobenzimidazol-2-one derivatives and their sulfones as a potent and novel class of NNRTIs. We herein
report the synthesis and biological evaluation of the new compounds in which different structural mod-
ifications have been introduced in order to investigate their effects on RT inhibition.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 31 March 2009
Revised 22 June 2009
Accepted 28 June 2009
Available online 3 July 2009
Keywords:
RT
HIV
Synthesis
Dihydrobenzimidazol-2-ones
1. Introduction
However, only four NNRTIs (nevirapine, delavirdine, efavirenz,
and recently etravirine) have been approved for clinical use by
Since the identification of HIV-1 as the cause of acquired im-
mune deficiency syndrome (AIDS), considerable medical advances
have been made and nowadays a variety of drugs are available for
the clinical treatment of HIV-1 infection.1,2 These drugs mainly in-
hibit the following key enzymes in the HIV-1 life cycle: reverse
transcriptase (RT), protease (PR), and more recently integrase
(IN) as well as viral entry.3–5
In highly active antiretroviral therapy (HAART), potent combi-
nations of three or more reverse transcriptase and protease inhib-
itors are used to suppress replication of HIV.6 HAART combination
regiments have proven to be effective in controlling disease pro-
gression and prolonging the survival of HIV-infected patients.
However, the therapeutic situation is challenged by the rapid
mutation of the virus to yield resistant strains and by the emer-
gence of unpleasant side effects.7
the FDA. Unfortunately, treatment with these drugs results in the
rapid emergence of RT mutants.8,9 Therefore, development of novel
NNRTIs with enhanced therapeutic spectra and different resistance
mutation profiles is urgently required to successfully employ this
class of drugs in combination therapy.
In recent papers, aimed at the discovery of new NNRTIs, we re-
ported a 3D-pharmacophore model for a second generation of
NNRTIs. We used this model for molecular modeling studies which
led to the rational discovery of N1-substituted 1,3-dihydro-2H-
benzimidazol-2-ones and their sulfones and some of them have
proven to be potent HIV-1 RT inhibitors (Fig. 1).10–12
SAR studies highlighted that compounds containing a sulfonyl
moiety were more potent than the analogues with a methylene lin-
ker. Moreover, 3,5-phenylsubstituted derivatives with a chlorine
atom at 6 position of the benzimidazolone system proved to be less
Non-nucleoside reverse transcriptase inhibitors (NNRTIs) are a
variety of non-competitive inhibitors that bind specifically to a
hydrophobic pocket in proximity to the DNA polymerase active
site of the RT enzyme; most of them are highly specific against
HIV-1 RT and are characterized by low toxicity and favorable phar-
macokinetic properties.8
R''R'''
X
N
Y
RR'
N
X=CH2, SO2
Y= O, S
H
* Corresponding author.
E-mail address: ammonforte@pharma.unime.it (A.-M. Monforte).
Figure 1. Structure of N1-substituted 1,3-dihydro-2H-benzimidazol-2-ones.
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.06.068

A.-M. Monforte et al. / Bioorg. Med. Chem. 17 (2009) 5962–5967
5963
toxic and more active than nevirapine and in some cases than
R
efavirenz, against both wild-type and mutant strains of HIV-1.
Supported by these promising results, we planned the synthesis
of new benzimidazolone analogues and their upper homologue
quinoxalinones with the aim of establishing further SARs on this
class of NNRTIs.
SO2
SO2
Cl
NH2
NO2
Cl
R
Cl
NH
i
NO2
10-18
2. Results and discussion
ii
R
R
As part of a project devoted to structural optimization and to
provide further explanation of the structure–activity relationships
of 1,3-dihydrobenzimidazol-2-one derivatives, different structural
modifications were introduced on both the benzimidazol-2-one
system as well as on the aromatic portion at the N-1 atom.
Using the skeleton of the above molecules as scaffold, new po-
tential NNRTIs were designed in which the imidazol-2-one nucleus
was converted into a six-membered ring homologue, like pipera-
zine, keeping the functional groups that are able to act as hydrogen
bond acceptor and donor.
SO2
SO2
Cl
NH
Cl
NH
iv
N
H
NH2
Cl
O
19-27
1b-9b
v
iii
The chlorine atom on the bicycle system and the sulfonyl group
were also retained as they are of paramount importance in increas-
ing antiviral activity.
Furthermore, the substitution on the arylsulfonyl portion was
explored in order to investigate its effects on antiretroviral activity
and on RT inhibition (Fig. 2).
R
R
SO2
SO2
Cl
Cl
N
N
O
The new molecules were synthesized following the reaction se-
quence reported in Scheme 1.
N
H
N
H
O
The 5-chloro-2-nitroaniline was N-substituted by treatment
with the appropriate arylsulfonylchloride using sodium hydride
as base and the N-(5-chloro,2-nitrophenyl)-benzenesulfonamides
thereby obtained (10–18) were reduced with Zn dust in acidic
medium to give derivatives 19–27.
The cyclization of the aminoderivatives (19–27) with phosgene
gave compounds (1a–9a), while the synthesis of N1-substituted-
3,4-dihydroquinoxalin-2(1H)-ones (1c–9c) was carried out by
reacting the suitable aminoderivative with chloroacetyl chloride
by microwave-irradiation and subsequent cyclization of the inter-
mediate obtained (1b–9b), with a catalytic amount of ethyldiiso-
propylamine (EDIPA) as base.
1a-9a
1c-9c
Compd
R
1a*, 1b, 1c,
2a*, 2b, 2c
3a, 3b, 3c
4a, 4b, 4c
5a#, 5b, 5c
6a, 6b, 6c
7a, 7b, 7c
8a, 8b, 8c
9a, 9b, 9c
2,6-difluorophenyl
3,5-difluorophenyl
2,6-dichlorophenyl
3,5-dichlorophenyl
3,5-dimethylphenyl
2-fluorophenyl
3-fluorophenyl
Both analytical and spectral data (1H NMR) of all synthesized
compounds are in full agreement with the proposed structures.
All the designed compounds were evaluated for their inhibition
of the cytopathic effects of HIV-1 (IIIB) in MT-4 cells. Compound-in-
duced cytotoxcity was also measured in MT-4 cells in parallel with
antiviral activity. The obtained compounds were also tested for
their ability to inhibit the enzymatic activity of HIV-1 RT.
Some intermediates of the reaction (1b–9b) were tested for
anti-HIV assays, considering that they could be ‘open models’ of
the designed molecules (1c–9c) which maintain all of the key
structural requirements for RT enzyme inhibition analogous to
the corresponding closed form.
The results from the cell-based and RT assays are summarized
in Table 1 and are compared with nevirapine and efavirenz as ref-
erence drugs.
As shown in Table 1, several of the new compounds prevented
the cytopathic effects of HIV-1 IIIB at micromolar or nanomolar
4-fluorophenyl
3-cyanophenyl
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) ClCH2COCl,
mw: 250 W, 25 °C, 10 min; (v) THF anhydrous, EDIPA, rt, 45 min. *, #See Refs. 11,12.
concentrations and, in some cases, were minimally toxic to MT-4
cells, thus resulting in high selectivity indices.
It can be observedthat anti-HIVactivityisdependent on thenature
and position of the substituents on the arylsulfonyl portion. In partic-
ular, the more potent compounds turned out to be derivatives 5a, 5b,
and 5c which were active at 0.002, 0.047, and 0.1 lM concentration,
respectively, and which presented a 3,5-dimethylphenylsulfonyl sub-
stituent on the nitrogen atom of the bicycle system.
Also the presence of two chlorine atoms in 2,6 or 3,5 positions
of the phenyl ring provided compounds active against cell replica-
tion at good micromolar concentrations (compd 3a, 3b, 3c, and 4a).
The mono substitution with a fluorine atom in 2 or 3 position
seems to influence anti-HIV activity positively (compd 6a and
7a), whereas the 4-substituted derivative 8a is inactive.
The anti-HIV-1 activity in MT-4 cells reported in Table 1 shows
that the benzimidazolone nucleus provided the most active com-
pounds whereas the substitution of the imidazol-2-one nucleus
with a six-member ring homologue seems to have a negative influ-
ence on anti-HIV activity. In fact, compounds 1a–9a were more
R
SO2
R
SO2
Cl
N
Cl
N
O
N
H
N
H
O
Figure 2. Structure of benzimidazolone and quinoxalinone derivatives.

5964
A.-M. Monforte et al. / Bioorg. Med. Chem. 17 (2009) 5962–5967
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 chemi-
cal shifts are expressed in d (ppm).
Table 1
Anti-RT and anti-HIV-1 activities, cytotoxicity and selectivity index in MT-4 cells
SId
a
b
c
Compd
IC50
(l
M)
EC50
(
lM)
CC50 (lM)
1a
1b
1c
2a
2b
2c
3a
3b
3c
4a
4b
4c
5a
5b
5c
6a
6b
6c
7a
7b
7c
8a
8b
8c
30 1.5
30.36 12.65
>40
0.1 0.01
27.83 5.57
>40
1.566 0.174
6.96 2.38
20.68 5.52
0.069 0.055
11.21 0.30
13.99 4.40
1.515 0.16
1.50 0.85
1.09 0.58
0.048 0.005
8.99 3.83
26.66 18.43
0.002 0.0003
0.047 0.0073
0.1 0.059
0.23 0.02
NTe
6.10 2.34
0.043 0.009
NTe
7.04 0.73
35.99 12.67
NTe
>172.50
0.149 0.089
>155.89
194.12 9.90
174.59 20.11
179.98 12.96
202.47 83.45
179.40 16.17
187.03 7.44
>331
159.95 9.27
>319.15
179.27 16.07
>291.97
P205.80
39 8.2
35.11 5.65
80.58 50.45
>382.57
124
25
9
2934
16
13
3.1.1. General procedures for the synthesis of N-(5-chloro-2-
nitrophenyl)-benzenesulfonamides (12–13, 15–18)
The appropriate aryl sulfonyl chloride (3 mmol) was added
dropwise to a solution of 5-chloro,2-nitroaniline (2 mmol) in diox-
ane (6 ml) at 0 °C, using dry sodium hydride (10 mmol) as catalyst.
The mixture was stirred for 30 min. The reaction mixture was then
quenched with a saturated NaHCO3 solution, extracted with chlo-
roform, and dried over Na2SO4. After removal of the solvent under
reduced pressure, the residue was powdered by treatment with
diethyl ether.
>100
218
107
>293
3735
>32
8
17,846
747
782
>1663
NTe
32
2965
NTe
27
P10
NTe
<1
32.70 2.33
1.67 0.75
0.003 0.001
15.88 6.77
>40
0.005 0.001
3.36 0.52
>40
0.85
3.1.1.1. N-(5-Chloro-2-nitrophenyl)-2,6-dichlorobenzenesulf-
onamide (12). Mp: 176–178 °C, yield 46%. 1H NMR (CDCl3): 7.10–
8.16 (m, 6H, ArH), 10.77 (br s, 1H, NH). Anal. Calcd for
C12H7Cl3N2O4S: C, 37.77; H, 1.85; N, 7.34. Found: C, 37.56; H,
1.98; N, 7.57.
NTe
>40
NTe
196.70 20.63
127.50 87.07
NTe
189.19 4.19
P355.02
0.132
NTe
>40
>40
NTe
NTe
3.1.1.2. N-(5-Chloro-2-nitrophenyl)-3,5-dichlorobenzenesulf-
onamide (13). Mp: 111–115 °C, yield 69%. 1H NMR (CDCl3): 7.17–
8.17 (m, 6H, ArH), 10.07 (br s, 1H, NH). Anal. Calcd for
C12H7Cl3N2O4S: C, 37.77; H, 1.85; N, 7.34. Found: C, 37.55; H,
2.03; N, 7.08.
>40
172.49 36.68
205.41 23.70
>155.89
186.27 12.02
>15
9a
9b
9c
0.36
283.68 21
>40
0.18 0.02
0.004 0.001
1357
<1
64
>205
>6666
P47.73
0.073 0.015
0.0009 0.0002
Nevirapine
Efavirenz
>6
a
Concentration required to inhibit by 50% the in vitro RNA-dependent DNA
polymerase activity of recombinant rt.
3.1.1.3. N-(5-Chloro-2-nitrophenyl)-3-cyanobenzenesulfona-
mide (15). Mp: 160 °C dec, yield 65%. 1H NMR (CDCl3): 7.18–8.17
(m, 7H, ArH), 10.06 (br s, 1H, NH). Anal. Calcd for C13H8ClN3O4S: C,
46.23; H, 2.39; N, 12.44. Found: C, 46.31; H, 2.49; N, 12.17.
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
.
e
Not tested.
3.1.1.4. N-(5-Chloro-2-nitrophenyl)-2-fluorobenzenesulfona-
mide (16). Mp: 106–108 °C, yield 100%. 1H NMR (CDCl3): 7.13–
8.13 (m, 7H, ArH), 10.02 (br s, 1H, NH). Anal. Calcd for
C12H8ClFN2O4S: C, 43.58; H, 2.44; N, 8.47. Found: C, 43.74; H,
2.31; N, 8.79.
active than the corresponding quinoxalinone derivatives 1c–9c as
well as 1b–9b.
Furthermore, most of the compounds 1b–9b showed only weak
inhibitory activity against RT whereas derivatives 1c-9c were inac-
tive, with the exception of 2,6-dichloro substituted derivative (3c)
3.1.1.5. N-(5-Chloro-2-nitrophenyl)-3-fluorobenzenesulfona-
mide (17). Mp: 108–110 °C, yield 82%. 1H NMR (CDCl3): 7.10–
8.15 (m, 7H, ArH), 10.03 (br s, 1H, NH). Anal. Calcd for
C12H8ClFN2O4S: C, 43.58; H, 2.44; N, 8.47. Found: C, 43.22; H,
2.65; N, 8.13.
which was active at 1.67
Interestingly, the 3,5-dichlorophenyl substituted compound
(4a) presented inhibitory activity at 0.003 M thus confirming a
lM concentration.
l
previous hypothesis that the 3,5-phenylsubstituted moiety inter-
acted with the hydrophobic roof of the non-nucleoside inhibitors
binding site.11
In summary, new potent anti-HIV agents have been obtained
active at nanomolar concentration and with very low toxicity.
The biological results suggest that the benzimidazolone nucleus
is the best scaffold of these series of derivatives as it provides
the most active compounds. The results obtained will be used as
basis strategy point for the design of new molecules with improved
anti-HIV properties.
3.1.1.6. N-(5-Chloro-2-nitrophenyl)-4-fluorobenzenesulfona-
mide (18). Mp: 95–98 °C, yield 65%. 1H NMR (CDCl3): 7.15–8.12
(m, 7H, ArH), 10.05 (sa, 1H, NH). Anal. Calcd for C12H8ClFN2O4S:
C, 43.58; H, 2.44; N, 8.47. Found: C, 43.15; H, 2.79; N, 8.68.
3.1.2. General procedures for the synthesis of N-(2-
aminophenyl-5-chloro)-benzenesulfonamides (21–22, 24–27)
The mixture of appropriate N-(5-chloro-2-nitrophenyl)-ben-
zenesulfonamide (0.6 mmol) in 3 ml HCl and 4 ml EtOH anhydrous
was stirred in an ice bath, then zinc dust (20 mmol) was gradually
added in several portions. Once this addition was complete, the reac-
tion mixture was heated in a water bath for 1 h; it was then cooled,
made alkaline with NaOH 2 N and extracted with ethyl acetate. The
extract was washed with water, dried over Na2SO4, and evaporated.
The residue was crystallized from ethanol or diethyl ether.
3. Experimental
3.1. Chemistry
All microwave-assisted reactions were carried out in a CEM Fo-
cused Microwave Synthesis System, Model Discover working at the
power necessary for refluxing under atmospheric conditions. Melt-
ing points were determined on a Kofler hot stage 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. Merck Silica Gel 60 F254 plates were
3.1.2.1. N-(2-Aminophenyl-5-chloro)-2,6-dichlorobenzenesulf-
onamide (21). Mp: 256–258 °C, yield 92%. 1H NMR (DMSO-d6):
5.36 (br s, 2H, NH2), 6.60–7.87 (m, 6H, ArH). Anal. Calcd for
C12H9Cl3N2O2S: C, 40.99; H, 2.58; N, 7.97. Found: C, 40.77; H,
2.70; N, 7.80.

A.-M. Monforte et al. / Bioorg. Med. Chem. 17 (2009) 5962–5967
5965
3.1.2.2. N-(2-Aminophenyl-5-chloro)-3,5-dichlorobenzenesulf-
onamide (22). Mp: 206–208 °C, yield 100%. 1H NMR (DMSO-d6):
5.39 (br s, 2H, NH2), 6.69–7.74 (m, 6H, ArH). Anal. Calcd for
C12H9Cl3N2O2S: C, 40.99; H, 2.58; N, 7.97. Found: C, 40.77; H,
2.70; N, 7.80.
3.1.3.5. 6-Chloro-1-(3-fluorophenylsulfonyl)-1,3-dihydro-2H-
benzimidazol-2-one (8a). Mp: 222–224 °C yield 70%. 1H NMR
(DMSO-d6): 7.52 (s, 1H, H-7), 7.63–7.81 (m, 6H, ArH). Anal. Calcd
for C13H8ClFN2O3S: C, 47.79; H, 2.47; N, 8.57. Found: C, 48.05; H,
2.18; N, 8.21.
3.1.2.3. N-(2-Aminophenyl-5-chloro)-3-cyanobenzenesulfona-
mide (24). Mp: 225–227 °C, yield 98%. 1H NMR (DMSO-d6): 5.15
(br s, 2H, NH2), 6.53–8.14 (m, 7H, ArH). Anal. Calcd for
C13H10ClN3O2S: C, 50.74; H, 3.28; N, 13.65. Found: C, 50.97; H,
3.48; N, 13.80.
3.1.3.6. 6-Chloro-1-(4-fluorophenylsulfonyl)-1,3-dihydro-2H-
benzimidazol-2-one (9a). Mp: 313–315 °C yield 47%. 1H NMR
(DMSO-d6): 6.88–6.96 (m, 7H, ArH), 10.75 (br s, 1H, NH). Anal.
Calcd for C13H8ClFN2O3S: C, 47.79; H, 2.47; N, 8.57. Found: C,
47.41; H, 2.84; N, 8.96.
3.1.2.4. N-(2-Aminophenyl-5-chloro)-2-fluorobenzenesulfona-
mide (25). Mp: 157–159 °C, yield 100%. 1H NMR (DMSO-d6): 5.15
(br s, 2H, NH2), 6.59–7.71 (m, 7H, ArH). Anal. Calcd for
C12H10ClFN2O2S: C, 47.93; H, 3.35; N, 9.31. Found: C,48.14; H,
3.52; N, 9.78.
3.1.4. General procedure for the synthesis of 2-chloro-N-[4-
chloro-2-(phenylsulfonamido)-phenyl]acetamides (1b–9b)
The appropriate N-(2-aminophenyl-5-chloro)-benzenesulfon-
amide (1 mmol) was added to an excess of 2-chloroacetyl chloride
and the resulting mixture was stirred and irradiated in a micro-
wave oven (W 100, 10 min, 50 °C) without solvent. After cooling,
the reaction mixture was evaporated under reduced pressure and
the obtained residue was crystallized from diethyl ether.
3.1.2.5. N-(2-Aminophenyl-5-chloro)-3-fluorobenzenesulfona-
mide (26). Mp: 139–141 °C, yield 93%. 1H NMR (DMSO-d6): 5.09
(br s, 2H, NH2), 6.59–7.65 (m, 7H, ArH), 9.53 (sa, 1H, NH). Anal.
Calcd for C12H10ClFN2O2S: C, 47.93; H, 3.35; N, 9.31. Found: C,
47.59; H, 3.07; N, 9.63.
3.1.4.1. 2-Chloro-N-[4-chloro-2-(2,6-difluoro-phenylsulfonami-
do)-phenyl]acetamide (1b). Mp: 130–133 °C, yield 100%. 1H
NMR (DMSO-d6): 4.26 (s, 2H, CH2), 7.16 (s, 1H, H-6), 7.24–7.32
(m, 3H, ArH), 7.58 (d, J = 8.79, 1H, H-3), 7.71–7.78 (m, 1H, H-40),
10.31 and 10.65 (br s, 2H, NH and NH). Anal. Calcd for
C14H10Cl2F2N2O3S: C, 42.55; H, 2.55; N, 7.09. Found: C, 42.73; H,
2.28; N, 7.40.
3.1.2.6. N-(2-Aminophenyl-5-chloro)-4-fluorobenzenesulfona-
mide (27). Mp: 231–234 °C, yield100%. 1H NMR (DMSO-d6): 4.62 (br
s, 2H, NH2), 6.31–6.49 (m, 7H, ArH). Anal. Calcd for C12H10ClFN2O2S: C,
47.93; H, 3.35; N, 9.31. Found: C, 48.22; H, 3.71; N, 9.75.
3.1.3. General procedure for the synthesis of 1-arylsulfonyl-1,3-
dihydro-2H-benzimidazol-2-ones (3a–4a, 6a–9a)
3.1.4.2. 2-Chloro-N-[4-chloro-2-(3,5-difluoro-phenylsulfonami-
do)-phenyl]acetamide (2b). Mp: 148–150 °C, yield 100%. 1H
NMR (DMSO-d6): 4.23 (s, 2H, CH2), 7.11 (s, 1H, H-6), 7.31 (dd,
J = 8.79, 1H, H-3), 7.35–7.38 (m, 2H, ArH), 7.58 (d, J = 8.79, 1H, H-
4), 7.61–7.69 (m, 1H, H-40), 10.02 and 10.30 (br s, 2H, NH and
NH). Anal. Calcd for C14H10Cl2F2N2O3S: C, 42.55; H, 2.55; N, 7.09.
Found: C, 42.70; H, 2.33; N, 7.46.
An excess of a 20% toluene solution of phosgene (1 ml) was
added to a solution of the appropriate N-(2-aminophenyl-5-
chloro)-benzenesulfonamide (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 ace-
tate, washed with water, and evaporated under reduced pressure.
The residue was crystallized from ethyl acetate.
3.1.4.3. 2-Chloro-N-[4-chloro-2-(2,6-dichloro-phenylsulfonam-
ido)-phenyl]acetamide (3b). Mp: 154–157 °C, yield 50%. 1H NMR
(DMSO-d6): 4.32 (s, 2H, CH2), 7.08 (s, 1H, H-6), 7.25 (dd, J = 8.54,
1H, H-3), 7.50–7.90 (m, 4H, ArH), 9.72 and 9.92 (br s, 2H, NH and
NH). Anal. Calcd for C14H10Cl4N2O3S: C, 39.28; H, 2.35; N, 6.54.
Found C, 39.49; H, 2.62; N, 6.28.
3.1.3.1. 6-Chloro-1-(2,6-dichlorophenylsulfonyl)-1,3-dihydro-
2H-benzimidazol-2-one (3a). Mp: 187 °C dec., yield 40%. 1H NMR
(CDCl3): 6.96 (d, J = 8.42, 1H, H-4), 7.19 (dd, J = 8.42, 1H, H-5), 7.40–
7.52 (m, 3H, ArH), 7.91 (s, 1H, H-7). Anal. Calcd for C13H7Cl3N2O3S:
C, 41.35; H, 1.87; N, 7.42. Found: C, 41.15; H, 1.48; N, 7.76.
3.1.4.4. 2-Chloro-N-[4-chloro-2-(3,5-dichloro-benzenesulfony-
lamido)-phenyl]acetamide (4b). Mp: 149–151 °C, yield 51%. 1H
NMR (DMSO-d6): 4.23 (s, 2H, CH2), 7.08(s, 1H, H-6), 7.33 (d,
J = 8.54, 1H, H-3), 7.55–7.63 (m, 3H, ArH), 7.99 (s, 1H, H-40), 9.59
and 9.98 (br s, 2H, NH and NH). Anal. Calcd for C14H10Cl4N2O3S:
C, 39.28; H, 2.35; N, 6.54. Found: C, 39.59; H, 2.52; N, 6.21.
3.1.3.2. 6-Chloro-1-(3,5-dichlorophenylsulfonyl)-1,3-dihydro-
2H-benzimidazol-2-one (4a). Mp: 239–240 °C, yield 62%. 1H
NMR (DMSO-d6): 6.85 (d, J = 8.24, 1H, H-4), 7.04 (dd, J = 8.24, 1H,
H-5), 7.53 (s, 1H, H-7), 8.01 (s, 2H, H-20,60), 8.05 (s, 1H, H-40). Anal.
Calcd for C13H7Cl3N2O3S: C, 41.35; H, 1.87; N, 7.42. Found: C, 41.51;
H, 1.73; N, 7.38.
3.1.3.3. 6-Chloro-1-(3-cyanophenylsulfonyl)-1,3-dihydro-2H-
benzimidazol-2-one (6a). Mp: 220–223 °C, yield 28%. 1H NMR
(DMSO-d6): 7.03 (d, J = 8.51, 1H, H-4), 7.24 (d, J = 7.69, 1H, H-5),
7.74 (s, 1H, H-7), 7.87–7.90 (m, 1H, H-50), 8.26–8.39 (m, 2H,
ArH), 8.59 (s, 1H, H-20), 11.64 (br s, 1H, NH). Anal. Calcd for
C14H8ClN3O3S: C, 50.38; H, 2.42; N, 12.59. Found: C, 50.71; H,
2.64; N, 12.27.
3.1.4.5. 2-Chloro-N-[4-chloro-2-(3,5-dimethyl-phenylsulfo-
namido)-phenyl]acetamide (5b). Mp: 149–150 °C, yield 61%. 1H
NMR (DMSO-d6): 3.54 (s, 6H, CH3), 4.41 (s, 2H, CH2), 7.22 (s, 1H,
H-6), 7.45–7.48 (m, 4H, ArH), 7.83 (d, J = 8.79, 1H, H-4), 9.73 and
9.87 (br s, 2H, NH and NH). Anal. Calcd for C16H16Cl2N2O3S: C,
49.62; H, 4.16; N, 7.23. Found: C, 49.37; H, 4.43; N, 7.08.
3.1.4.6. 2-Chloro-N-[4-chloro-2-(3-cyano-phenylsulfonamido)-
phenyl]acetamide (6b). Mp: 176–178 °C, yield 82%. 1H NMR
(CDCl3): 4.18 (s, 2H, CH2), 7.02 (s, 1H, H-6), 7.29 (dd, J = 8.51, 1H,
H-4), 7.54 (d, J = 8.51, 1H, H-3), 7.65 (t, 1H, H-50), 7.89–7.93 (m,
2H, H-40,60), 8.03 (s, 1H, H-20), 8.58 (br s, 2H, NH and NH). Anal.
Calcd for C15H11Cl2N3O3S: C, 46.89; H, 2.89; N, 10.94. Found: C,
46.51; H, 2.96; N, 10.75.
3.1.3.4. 6-Cloro-1-(2-fluorophenylsulfonyl)-1,3-dihydro-2H-
benzimidazol-2-one (7a). Mp: 192–197 °C, yield 72%. 1H NMR
(DMSO-d6): 7.03 (d, J = 8.51, 1H, H-4), 7.22 (d, J = 8.79, 1H, H-5),
7.44–7.53 (m, 2H, ArH), 7.60 (s, 1H, H-7), 7.81–8.09 (m, 2H, ArH).
Anal. Calcd for C13H8ClFN2O3S: C, 47.79; H, 2.47; N, 8.57. Found:
C, 47.33; H, 2.69; N, 8.78.

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A.-M. Monforte et al. / Bioorg. Med. Chem. 17 (2009) 5962–5967
3.1.4.7. 2-Chloro-N-(4-chloro-2-(2-fluoro-phenylsulfonamido)-
phenyl)acetamide (7b). Mp: 124–129 °C, yield 71%. 1H NMR
(DMSO-d6): 4.26 (s, 2H, CH2), 7.10 (s, 1H, H-6), 7.26–7.73 (m, 6H,
ArH), 9.73 and 10.05 (br s, 2H, NH and NH). Anal. Calcd for
C14H11Cl2FN2O3S: C, 44.58; H, 2.94; N, 7.43. Found: C, 44.83; H,
3.12; N, 7.71.
3.1.5.6. 6-Chloro-4-(3-cyanodimethylphenylsulfonyl)-3,4-dihy-
dro-1H-quinoxalin-2-one (6c). Mp: 217–220 °C, yield 17%. 1H
NMR (DMSO-d6): 4.30 (s, 2H, CH2), 6.79 (d, J = 8.79, 1H, H-2),
7.33–7.53 (m, 6H, ArH), 10.40 (br s, 1H, NH). Anal. Calcd for
C15H10ClN3O3S: C, 51.80; H, 2.90; N, 12.08. Found: C, 51.46; H,
3.20; N, 12.35.
3.1.4.8. 2-Chloro-N-[4-chloro-2-(3-fluoro-phenylsulfonamido)-
phenyl]acetamide (8b). Mp: 137–140 °C, yield 94%. 1H NMR
(DMSO-d6): 4.25 (s, 2H, CH2), 7.03 (s, 1H, H-6), 7.28 (dd, J = 8.51,
1H, H-3), 7.46–7.66 (m, 5H, ArH), 9.56 and 9.91 (br s, 2H, NH and
NH). Anal. Calcd for C14H11Cl2FN2O3S: C, 44.58; H, 2.94; N, 7.43.
Found: C, 44.26; H, 2.45; N, 7.86.
3.1.5.7. 6-Chloro-4-(2-fluorophenylsulfonyl)-3,4-dihydro-1H-
quinoxalin-2-one (7c). Mp: 183–186 °C, yield 100%. 1H NMR
(DMSO-d6): 4.37 (s, 2H, CH2), 6.83 (d, J = 8.51, 1H, H-2), 7.28–
7.44 (m, 3H, ArH), 7.49 (s, 1H, H-5), 7.61–7.77 (m, 2H, ArH),
10.57 (br s, 1H, NH). Anal. Calcd for C14H10ClFN2O3S: C, 49.35; H,
2.96; N, 8.22. Found: C, 49.67; H, 3.34; N, 8.56.
3.1.4.9. 2-Chloro-N-[4-chloro-2-(4-fluoro-phenylsulfonamido)-
phenyl]acetamide (9b). Mp: 167–169 °C, yield 59%. 1H NMR
(DMSO-d6): 4.26 (s, 2H, CH2), 7.03 (s, 1H, H-6), 7.26 (d, 1H, H-3),
7.40–7.98 (m, 5H, ArH), 10.01 and 10.04 (br s, 2H, NH and NH).
Anal. Calcd for C14H11Cl2FN2O3S: C, 44.58; H, 2.94; N, 7.43. Found:
C, 44.70; H, 2.69; N, 7.25.
3.1.5.8. 6-Chloro-4-(3-fluorophenylsulfonyl)-3,4-dihydro-1H-
quinoxalin-2-one (8c). Mp: 172–175 °C, yield 35%. 1H NMR
(DMSO-d6): 4.32 (s, 2H, CH2), 6.79 (d, J = 8.51, 1H, H-2), 7.24–
7.26 (m, 2H, ArH), 7.35 (dd, J = 8.51, 1H, H-3), 7.53 (s, 1H, H-5),
7.56–7.61 (m, 2H, ArH), 10.43 (br s, 1H, NH). Anal. Calcd for
C14H10ClFN2O3S: C, 49.35; H, 2.96; N, 8.22. Found: C, 49.70; H,
2.64; N, 8.53.
3.1.5. General procedure for the synthesis of 4-arylsulfonyl-3,4-
dihydro-1H-quinoxalin-2-ones (1c–9c)
An excess of ethyldiisopropylamine (EDIPA) was added drop-
wise to a solution of the appropriate 2-chloro-N-(4-chloro-phe-
nyl)-acetamide (1 mmol) in anhydrous THF (2 ml). The mixture
was stirred for 1 h at room temperature.
The reaction mixture was washed with a saturated NaHCO3 solu-
tion and then dried over Na2SO4. After removal of the solvent under
reduced pressure, the residue was crystallized from diethyl ether.
3.1.5.9. 6-Chloro-4-(4-fluorophenylsulfonyl)-3,4-dihydro-1H-
quinoxalin-2-one (9c). Mp: 189–191 °C, yield 86%. 1H NMR
(DMSO-d6): 4.34 (s, 2H, CH2), 6.79 (d, J = 8.51, 1H, H-2), 7.38 (dd,
J = 8.51, 1H, H-3), 7.54 (s, 1H, H-5), 7.64–8.22 (m, 4H, ArH), 10.41
(br s, 1H, NH). Anal. Calcd for C14H10ClFN2O3S: C, 49.35; H, 2.96;
N, 8.22. Found: C, 49.51; H, 2.66; N, 8.38.
3.2. Anti-HIV activity assays
3.1.5.1. 6-Chloro-4-(2,6-difluorophenylsulfonyl)-3,4-dihydro-
1H-quinoxalin-2-one (1c). Mp: 214–217 °C, yield 50%. 1H NMR
(DMSO-d6): 4.42 (s, 2H, CH2), 6.85 (d, J = 8.51, 1H, H-2), 7.23–
7.52 (m, 4H, ArH), 7.71–7.80 (m, 1H, H-40), 10.65 (br s, 1H, NH).
Anal. Calcd for C14H9ClF2N2O3S: C, 46.87; H, 2.53; N, 7.81. Found:
C, 46.30; H, 2.67; N, 7.53.
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,
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
albumine (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
was finally introduced into tubes containing aliquots of different
enzyme concentrations (5–10 nM rt). After incubation at 37 °C
3.1.5.2. 6-Chloro-4-(2,6-dichlorophenylsulfonyl)-3,4-dihydro-
1H-quinoxalin-2-one (2c). Mp: 204–207 °C, yield 86%. 1H NMR
(DMSO-d6): 4.37 (s, 2H, CH2), 6.86 (d, J = 8.51, 1H, H-2), 7.26 (dd,
J = 8.51, 1H, H-3), 7.42 (s, 1H, H-5), 7.50–7.95 (m, 3H, ArH), 10.68
(br s, 1H, NH). Anal. Calcd for C14H9Cl3N2O3S: C, 42.93; H, 2.32;
N, 7.15. Found: C, 42.75; H, 2.56; N, 7.38.
for the indicated time, 20 ll from each reaction tube were spiked
on glass fiber filters GF/C and immediately immersed in 5% ice-cold
trichloroacetic acid (TCA) (AppliChem GmbH, Darmstadt). Filters
were washed three times with 5% TCA and once with ethanol for
5 min, then dried and, lastly, EcoLumeÒ Scintillation cocktail
(ICN, Research Products Division, Costa Mesa, CA USA) was added
to detect the acid-precipitable radioactivity using a PerkinElmerÒ
Trilux MicroBeta 1450 Counter.
3.1.5.3. 6-Chloro-4-(3,5-difluorophenylsulfonyl)-3,4-dihydro-
1H-quinoxalin-2-one (3c). Mp: 191–194 °C, yield 44%. 1H NMR
(DMSO-d6): 4.35 (s, 2H, CH2), 6.83 (d, J = 8.79, 1H, H-2), 7.15 (s, 2H,
H-20,60), 7.38 (dd, J = 8.51, 1H, H-3), 7.53 (s, 1H, H-5), 7.73–7.79 (m,
1H, H-40), 10.51 (br s, 1H, NH). Anal. Calcd for C14H9ClF2N2O3S: C,
46.87; H, 2.53; N, 7.81. Found: C, 46.49; H, 2.20; N, 7.63.
3.2.2. RT inhibition assays
Reactions were performed under the conditions described for
the HIV-1 RT RNA-dependent DNA polymerase activity assay.13
The incorporation of radioactive dTTP into poly(rA)/oligo(dT) was
monitored 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 con-
centrations of between 0.2 Ki and 5 Ki was used. Experiments were
done in triplicate. Experimental errors ( SD) were 610%.
3.1.5.4. 6-Chloro-4-(3,5-dichlorophenylsulfonyl)-3,4-dihydro-1H-
quinoxalin-2-one (4c). Mp: 167–170 °C, yield 55%. 1H NMR (DMSO-
d6): 4.35 (s, 2H, CH2), 6.83 (d, J = 8.79, 1H, H-2), 7.33 (s, 2H, H-20,60),
7.41(d, J = 8.79, 1H, H-3), 7.54 (s, 1H, H-5), 8.08 (s, 1H, H-40), 10.19 (br
s, 1H, NH). Anal. Calcd for C14H9Cl3N2O3S: C, 42.93; H, 2.32; N, 7.15.
Found: C, 42.65; H, 2.58; N, 7.36.
3.2.3. In vitro anti-HIV assay
3.1.5.5. 6-Chloro-4-(3,5-dimethylphenylsulfonyl)-3,4-dihydro-
quinoxalin-2(1H)-one (5c). Mp: 181–184 °C, yield 30%. 1H NMR
(DMSO-d6): 2.21 (s, 6H, CH3), 4.26 (s, 2H, CH2), 6.78 (d, J = 8.51,
1H, H-2), 6.99–7.50 (m, 5H, ArH), 10.35 (br s, 1H, NH). Anal. Calcd
for C16H15ClN2O3S: C, 54.78; H, 4.31; N, 7.98. Found: C, 54.56; H,
4.79; N, 7.47.
The methodology of the anti-HIV assays has been previously de-
scribed.14 Briefly, MT-4 cells were infected with HIV-1 (IIIB) at
ꢀ100-times the CCID50 (50% cell culture infective dose) per ml of
cell suspension. Hundred microliters of the infected cell suspen-
sion were then transferred to microtiter plate wells, mixed with

A.-M. Monforte et al. / Bioorg. Med. Chem. 17 (2009) 5962–5967
5. Opar, A. Nat. Rev. Drug Disc. 2007, 6, 258.
5967
100 ll of the appropriate dilutions of the test compounds, and fur-
6. Orkin, C.; Stebbing, J.; Nelson, M.; Bower, M.; Johnson, M.; Mandalia, S.;
Jones, R.; Moyle, G.; Fisher, M.; Gazzard, B. J. Antimicrob. Chemoth. 2005,
55, 246.
7. Vandamme, A. M.; Van Vaerenbergh, K.; De Clercq, E. Anti Viral Chem.
Chemother. 1998, 9, 187.
ther 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%.
8. Das, K.; Lewi, P. J.; Hughes, S. H.; Arnold, E. Prog. Biophys. Mol. Biol. 2005, 88,
209.
9. Wainberg, M. A. J. Acquired Immune Defic. Syndr. 2003, 34, S2.
10. 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.
11. 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.
2007, 17, 1956.
12. Monforte, A. M.; Rao, A.; Logoteta, P.; Ferro, S.; De Luca, L.; Barreca, M. L.; Iraci,
N.; Maga, G.; De Clercq, E.; Pannecouque, C.; Chimirri, A. Bioorg. Med. Chem.
2008, 16, 7429.
13. Balzarini, J.; Karlsson, A.; Perez-Perez, M. J.; Vrang, L.; Walbers, J., et al Virology
1993, 192, 246.
Acknowledgment
Financial support for this research by Fondo Ateneo di ricerca
(2005, Messina, Italy).
References and notes
1. De Clercq, E. J. Clin. Virol. 2004, 30, 115.
2. Struble, K.; Murray, J.; Cheng, B.; Gegeny, T.; Miller, V.; Gulick, R. AIDS 2005, 19,
747.
3. De Clercq, E. Exp. Opin. Emerg. Drugs 2005, 10, 241.
4. De Clercq, E. J. Med. Chem. 2005, 48, 1.
14. Maga, G.; Amacker, M.; Ruel, N.; Hubscher, U.; Spadari, S. J. Mol. Biol. 1997, 274,
738.