Indolylarylsulfones as HIV-1 non-nucleoside reverse transcriptase inhibitors: New cyclic substituents at indole-2-carboxamide

Article abstract of DOI:10.1021/jm101614j

New indolylarylsulfone derivatives bearing cyclic substituents at indole-2-carboxamide linked through a methylene/ethylene spacer were potent inhibitors of the WT HIV-1 replication in CEM and PBMC cells with inhibitory concentrations in the low nanomolar range. Against the mutant L100I and K103N RT HIV-1 strains in MT-4 cells, compounds 20, 24-26, 36, and 40 showed antiviral potency superior to that of NVP and EFV. Against these mutant strains, derivatives 20, 24-26, and 40 were equipotent to ETV. Molecular docking experiments on this novel series of IAS analogues have also suggested that the H-bond interaction between the nitrogen atom in the carboxamide chain of IAS and Glu138:B is important in the binding of these compounds. These results are in accordance with the experimental data obtained on the WT and on the mutant HIV-1 strains tested.

Full text of DOI:10.1021/jm101614j

ARTICLE
pubs.acs.org/jmc
Indolylarylsulfones as HIV-1 Non-Nucleoside Reverse Transcriptase
Inhibitors: New Cyclic Substituents at Indole-2-carboxamide
Giuseppe La Regina,† Antonio Coluccia,† Andrea Brancale,‡ Francesco Piscitelli,† Valerio Gatti,†
Giovanni Maga,§ Alberta Samuele,§ Christophe Pannecouque,^ Dominique Schols,^ Jan Balzarini,^
Ettore Novellino,|| and Romano Silvestri*,†
†Istituto Pasteur - Fondazione Cenci Bolognetti, Dipartimento di Chimica e Tecnologie del Farmaco, Sapienza Universitꢀa di Roma,
Piazzale Aldo Moro 5, I-00185 Roma, Italy
‡Welsh School of Pharmacy, Cardiff University, King Edward VII Avenue, Cardiff CF10 3NB, U.K.
§Istituto di Genetica Molecolare, Consiglio Nazionale delle Ricerche, Via Abbiategrasso 207, I-27100 Pavia, Italy
Dipartimento di Chimica Farmaceutica e Tossicologica, Universitꢀa di Napoli Federico II, Via Domenico Montesano 49, I-80131,
Napoli, Italy
^Rega Institute for Medical Research, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium
S Supporting Information
b
ABSTRACT: New indolylarylsulfone derivatives bearing cyclic
substituents at indole-2-carboxamide linked through a methy-
lene/ethylene spacer were potent inhibitors of the WT HIV-1
replication in CEM and PBMC cells with inhibitory concentra-
tions in the low nanomolar range. Against the mutant L100I and
K103N RT HIV-1 strains in MT-4 cells, compounds 20, 24-
26, 36, and 40 showed antiviral potency superior to that of NVP
and EFV. Against these mutant strains, derivatives 20, 24-26,
and 40 were equipotent to ETV. Molecular docking experiments on this novel series of IAS analogues have also suggested that the
H-bond interaction between the nitrogen atom in the carboxamide chain of IAS and Glu138:B is important in the binding of these
compounds. These results are in accordance with the experimental data obtained on the WT and on the mutant HIV-1 strains tested.
’ INTRODUCTION
K103N and Y181C were the most prevalent mutations in clinical
HIV-1 isolates.7 Therefore, new NNRTIs that display a broad
spectrum of activity against clinically relevant HIV-1 mutant
strains are needed. New generation NNRTIs are currently on-
going clinical trials.8 Among them, etravirine (ETV) was ap-
proved for drug combination treatment of HIV-1 infected people
who experienced drug resistance to other drugs of this class.9
Our efforts have been focused toward the development of
indolylarylsulfone (IAS) NNRTIs correlated to the carboxamide
1.10 Structure-activity relationship (SAR) studies led to the
identification of three regions of the IAS scaffold: (region A) the
limited spectrum of activity of 1 against mutant HIV-1 strains was
significantly expanded by introduction of two methyl groups at
positions 3 and 5 of the 3-phenylsulfonyl moiety (2);11 (region
B) new potent HIV-1 inhibitors were obtained by coupling the
indole-2-carboxamide with either natural or unnatural amino
acids; against the mutant L100I, K103N, and Y181C RT HIV-1
strains in CEM cells, IASs 3-5 were comparable to efavirenz
(EFV);12,13 (region C) IAS 6 bearing the 5-chloro-4-fluoro
substitution pattern at the indole ring was a potent inhibitor of
Human immunodeficiency virus (HIV) was identified as the
causative agent of acquired immunodeficiency syndrome (AIDS)
in 1983.1 Although significant progress has been made in the
prevention and treatment of AIDS/HIV infection over the past
20 years, the number of people living with HIV is constantly
growing. Worldwide, AIDS-related diseases are still one of the
leading causes of death and are predicted to cause significantly
premature mortality in the coming decades.2
HIV drugs currently approved by Food and Drug Adminis-
tration (FDA) fall into seven target classes: nucleoside reverse
transcriptase inhibitors (NRTIs), nucleotide reverse transcrip-
tase inhibitors (NtRTIs), non-nucleoside reverse transcriptase
inhibitors (NNRTIs), protease inhibitors (PIs), fusion inhibitors
(FIs), CCR5 co-receptor inhibitors (CRIs), and integrase in-
hibitors (INIs).3 Highly active antiretroviral therapy (HAART)
combines three (recommended) or four different antiretroviral
drugs. HAART regimes slow viral replication and reduce plasma
viremia below the detection level, resulting in a substantial delay
of disease progression.4 However, long-term HAART treatments
lead to the emergence of complications due to drug resistance,5
toxicity, and adverse effects.6 A major concern of earlier NNRTIs
was the rapid development of drug resistance; in particular,
Received: August 3, 2010
Published: March 02, 2011
r
2011 American Chemical Society
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ARTICLE
Chart 1. Structure of New IASs 12-47 and Reference Compounds 1-11
RT WT and RTs carrying the K103N, Y181I, and L100I
mutations14 (Chart 1).
In the design of new NNRTIs, the strategy to add a hetero-
cyclic motif to the parent compound conveniently broadened the
spectrum of activity against the mutant strains. For example,
Janssen synthesized diaryltriazine (DATA) NNRTIs with very
good potency against HIV-1 WT and a panel of HIV-1 mutants
by intramolecular cyclization of the thiourea moiety of ITU
derivatives.15 BIRL 355 BS is a new NNRTI in advanced
development designed by Boehringer Ingelheim on the dipyr-
idodiazepinone scaffold of nevirapine; the addition of the func-
tionalized side tail provided a NNRTI endowed with potent
activity against drug resistant isolates of HIV-1.16 Merck gener-
ated NNRTIs with excellent HIV-1 potency (7) by replacing the
3-phenylsulfonyl of 1 with a pyrrolidin-1-ylsulfonyl moiety and
introducing an additional heterocycle at position 2 of the indole.17
Previously, we synthesized potent IAS derivatives bearing a
N-(2-hydroxyethyl)carboxamide/carbohydrazide (8, 9) func-
tionality at position 2 of the indole (B region) and two methyl
groups at the positions 3 and 5 of the 3-benzenesulfonyl moiety
(A region).18 As further development of that research project, we
synthesized new hydrazide derivatives (10, 11) that proved to be
more active than EFV against the viral RT carrying the K103N
mutation.19
Docking studies inside the non-nucleoside binding site (NNBS)
of the RT showed that the poses of 8 and 9 (not shown) presented
the indole ring rotated by about 180ꢀ compared to 2, while the
3-(3,5-dimethylphenyl) groups overlapped well with the corre-
sponding group of 2 (Figure 1). This new orientation allowed the
2-hydroxyethylamide/hydrazide moieties to fit into the NNBS
entrance channel formed by Val106, Pro225, Phe227, Leu234,
His235, Pro236, and Tyr318. Furthermore, 8 and 9 established a
H-bond between the terminal OH of the ethanolamide/hydrazide
chains and the carbonyl group of Leu234.18 In the case of 11 (not
shown), the docking results showed that the isopropyl group
occupied a pocket formed by Val106, Pro225, Phe227, Leu234,
and Pro236.19 The latest IASs series reported13 (compounds 3-5)
Figure 1. Binding mode of derivatives 2 (magenta), 4 (blue), 8
(yellow), and 16 (cyan). Binding site residues are also reported.
Residues of the cleft between the p66 and p51 RT subunits are reported
in orange. Val179 (up) and Ile180 (down) are visible behind the docked
structures.
showed a different binding mode compared to 8 and 9. In fact, the
putative binding for 3-5 was very similar to the one observed for
compound 2. In particular, the side chain at position 2 of the indole
nucleus lay in a cleft of the NNBS located at the interface between
the p66 (chain A) and p51 (chain B) RT subunits, forming a strong
H-bond with Glu138:B (orange residue in Figure 1).13 Preliminary
docking studies showed that new IASs bearing (hetero)cyclic
substituents at the indole-2-carboxamide moiety (16, Figure 1)
could adopt binding poses similar to those of 3-5,13 establishing
novel binding interactions in the solvent accessible cleft of the
NNBS described above and in particular formed by residues
Arg172, Ile180, Val179 and Glu138:B, and Thr139:B. Start-
ing from these observations, we focused our interest on exploring
further the B region of IASs by new substitutions at the indole-2-
carboxamide. In particular, our attention was on the introduction of
a basic nitrogen atom in the side chain that could interact more
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Scheme 1. Synthesis of Compounds 12-23a
a Reagents and reaction conditions: (a) R = 5-Cl or 5-NO2, secondary amine, 37% formaldehyde, t-BuOH, reflux, 7 h; (b) R = 5-Br or 5-Cl,4-F, secondary
amine, paraformaldehyde, benzene, reflux, 3 h, Dean-Stark apparatus.
Scheme 2. Synthesis of Compounds 24-47a
a Reagents and reaction conditions: (a) R = 5-Cl, 5-Br, or 5-NO2, R0 = H, BOP reagent, primary amine, triethylamine, DMF, room temperature,
overnight; (b) R = 5-Cl, 5-Cl-4-F, R0 = H, amine, ethanol, closed vessel, 150 ꢀC, 150 W, Pmax = 250 PSI, ramp time 3 min, hold time 15 min.
efficiently with the Glu138:B acid function and the presence of small
hydrophobic groups that could establish nonpolar interactions with
the hydrophobic side chains of the residues in the cleft. Further-
more, such a chemical modification could lead to an improvement
of the IAS’s pharmacokinetic properties.20,21 We here describe the
synthesis and the antiretroviral activity of a new series of IAS
compounds (12-47) characterized by (a) a cycloalkylamino, aryl,
or heteroaryl nucleus linked through (b) a methylene, ethylene, or
ethoxy linker to the 2-carboxamide and (c) different electron-
withdrawing substituents at positions 4 and 5 of the indole
(Chart 1).
’ CHEMISTRY
The synthesis of new IASs 12-23 is depicted in Scheme 1.
Mannich reaction of 5-chloro- (5)11 or 5-nitro-3-[(3,5-
dimethylphenyl)sulfonyl]-1H-indole-2-carboxamide
(49)
with pyrrolidine, piperidine, or morpholine in tert-butanol at
reflux temperature for 7 h in the presence of 37% formalde-
hyde provided the corresponding amines 12, 14, 16, 18, 20,
and 22. On the other hand, amines 13, 15, 17, 19, 21, and 23 were
obtained from 5-bromo- (48)11 or 5-chloro-4-fluoro-3-[(3,5-
dimethylphenyl)sulfonyl]-1H-indole-2-carboxamide14 (6) and pyr-
rolidine, piperidine, or morpholine in boiling benzene for 3 h in the
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Table 1. Structure and Anti-HIV Activity of New IAS Derivatives 12-27 in CEM Cellsa
a Data are mean values of two experiments performed in triplicate. b EC50: effective concentration (nM) or concentration required to protect CEM cells
against the cytopathicity of HIV by 50%, as monitored by giant cell formation. c CC50, cytostatic concentration: compound concentration (μM) required
to reduce by 50% the number of viable cells in mock-infected CEM cultures. d Syncytia cell formation was examined microscopically. e Selectivity index
was calculated as the ratio CC50/EC50. f Compound precipitation was detected at higher compound concentrations.
presence of paraformaldehyde using a Dean-Stark apparatus
(Scheme 1).
assay, compound 16 was 1 order of magnitude more potent than
EFV against the WT HIV-1 RT and >160 times more potent than
EFV against K103N RT, which is the major mutation emerging
in patients treated with EFV, whose viral loads rebound after an
initial response to the drug.23 Such results prompted us to
synthesize new IASs by replacing the chloro atom at position 5
of the indole with bromo or nitro (in our previous studies,
5-nitro-IASs were less cytostatic than the corresponding mono-
halogenated counterparts)13 or with the 5-chloro-4-fluoroindole
substitution which was effective for the antiviral activity of
carboxamide 6.14
Replacement of the piperidin-1-yl nucleus of 16 with a
pyrrolidin-1-yl ring provided IASs 12-14 which showed potent
antiretroviral activity against the mutant panel of virus strains. On
the contrary, replacing of the piperidin-1-yl nucleus with a
morpholin-4-yl one (20-22) resulted in a general drop of the
activity against all the RTs. Therefore, we replaced the morpho-
lin-4-yl moiety with the more lipophilic phenyl ring (24-28).
IASs 24 and 26 showed good activity against the mutant L100I
RT HIV1 strain, superior to NVP and EFV, but they were less
effective against both K103N and Y181I RTs.
Reaction in sequence of 5-chloro- (50),13 5-bromo- (51),13 or
5-nitro-3-[(3,5-dimethylphenyl)sulfonyl]-1H-indole-2-carboxy-
lic acid13 (52) with (benzotriazol-1-yloxy)tris(dimethylamino)
phosphonium hexafluorophosphate (BOP reagent) and then with
benzylamine, 1-(2-aminoethyl)pyrrolidine, 4-(2-aminoethyl)morp-
holine, 2-aminoethylbenzene, 1-(2-aminoethyl)pyrrole, or 1-(2-
aminoethyl-1-oxy)benzene in the presence of triethylamine in
DMF at room temperature overnight afforded carboxamides 24-
26, 29, 30, 32-34, 36-38, 40-42, and 44-47. Carboxamides 27,
28, 31, 35, 39, 43, and 47 were preferably obtained by microwave-
assisted (MW) reaction of 5-chloro-4-fluoro-3-[(3,5-dimethyl-
phenyl)sulfonyl]-1H-indole-2-carboxylate (57)13 or, in the case of
28, the corresponding 5-chloro derivative (54),13 with the appro-
priate amine in ethanol at 150 W for 15 min (Scheme 2).
’ RESULTS AND DISCUSSION
On the basis of our molecular modeling results, we first synthesized
Mannich base 16 which was more potent than 413 against mutant
L100I and Y181I RT HIV-1 strains22 and equipotent to 4 against
K103N RT HIV-1 (Table 2 and Figure 1). In the enzymatic
IAS derivatives 12-47 were evaluated against the HIV-1 WT
in human T-lymphocyte (CEM) cells. Against HIV-1 WT, the
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Table 2. HIV-1 RT Inhibitory Activity of Compounds 12-27
and Reference Compounds 4, NVP, and EFV against the WT
and Mutant Enzymes Carrying Single Amino Acid
Substitutionsa
On the basis of our know-how in pyrrole bioisosterism,26 we
decided to introduce a pyrrol-1-yl nucleus at position C2 of the
linker. Indeed, IASs 40-43 potently inhibited HIV-1 WT in
CEM cells at nanomolar concentrations. Notably, such com-
pounds were potent inhibitors of the HIV-1 WT RT and the RTs
carrying the L100I and K103 mutations, but they failed to inhibit
Y181I RT.
IC50 b (nM)
compd
WT
L1001
K103N
Y181I
Compounds 20, 24-26, 28, 36, 40, and 44 were evaluated in
CEM cells against mutant HIV-1 strains harboring L100I,
K103N, Y181C, Y188L, and E138K single amino acid mutations
in the RT (Table 5). Compounds 20, 24-26, 36, and 40 were
potent inhibitors of the mutant L100I and K103N RT HIV-1
strains, and they were comparable to 4 and superior to NVP and
EFV. Against these mutant strains, 20, 24-26, and 40 were
equipotent to ETV (data not shown). Compounds 20, 24-26,
and 40 were comparable to EFV against the mutant Y181C RT
HIV-1 strain. Three compounds, 20, 24, and 26, were equipotent
to EFV against the mutant Y188L RT HIV-1 strain. Finally, 20,
24-26, 36, and 40 were comparable to EFV and ETV (data not
shown) against the E138K RT HIV-1 mutant. Such results were
in agreement with the inhibitory effect observed against the
corresponding mutant RT enzymes (Tables 2 and 4). Further
information about cytotoxicity, SI, and relative factor (RF) of
compounds 20, 24-26, 28, 36, 40, and 44 against mutant HIV-1
strains is available in Table 2S and Table 3S of Supporting
Information.
To ascertain that the test compounds were also inhibitory in
primary T-lymphocyte cells using virus strains that belong to
different clades of HIV-1, compounds 12, 40, and 20 were
included in this study (Table 6). The compounds proved to be
exquisitely inhibitory against virus members that belonged to
clade A, clade B, clade C, clade A/E, and group O (EC50 in the
higher picomolar or lower nanomolar range). The clade viruses,
in contrast to HIV-1(IIIB), were R-tropic. The group O virus was
X-tropic. These data point to a broad spectrum of anti-HIV-1
activity of this class of compounds.
12
12
26
96
442
13
8
14
67
418
14
34
ndc
54
ndc
287
ndc
1023
ndc
15
16
8
37
120
581
17
30
19
125
497
18
13
ndc
10
307
ndc
999
ndc
19
ndc
117
100
99
20
54
512
5481
4303
1602
10210
>40000
>40000
>40000
>40000
2493
>20000
400
21
168
150
80
863
22
864
23
241
15
1172
>40000
1852
459
24
21
25
1054
17
273
14
26
27
4d
NVPd
EFVd
40
40
144
23
112
9000
ndc
190
400
80
7000
>20000
a Data represent mean values of at least three separate experiments.
b Compound concentration (IC50, nM) required to inhibit by 50% the
RT activity of the indicated strain. c nd: no data. d Reference 13.
inhibitory activities (EC50 values) of 12-27 seemed only
marginally affected by the substitutions at the positions 4 and 5
of the indole nucleus, and no correlation between cLogP and
biological activity was observed (Table 1S of Supporting
Information).24,25 We previously observed similar results for
IASs bearing an amino acid unit at the indole-2-carboxamide.13
In contrast, a strong influence of the substituents at the positions
4 and 5 on the anti-HIV-1 activity of IASs was observed in the
presence of the primary 2-carboxamide functionality.11,13,14 As
expected, the compounds did not inhibit the HIV-2 strain
(Table 1).
Compounds 28-31 were 1 order of magnitude less active
than the corresponding Mannich bases 12-15 against HIV-1
WT in CEM cells; a similar behavior was observed against the
panel of the mutated RTs (Tables 3 and 4). The drop of antiviral
activity of 28-31 might be due to the suboptimal length of the
methylene linker.
We replaced the pyrrolidin-1-yl nucleus of 28-31 with a phenyl
ring to give IASs 36-39. Such a chemical modification improved
potency against the mutant L100I HIV-1 strain, but the activity
against K103N dropped. With the only exception of 33,
N-(morpholinyl)ethyl derivatives 32-35 were equipotent to the
corresponding methylenes 20-23 against HIV-1 WT in CEM cells.
Against L100I RT, 32-35 were comparable to 20-23, but they
were less potent against the mutant K103N and Y188I RT strains.
Neither introduction of a phenethoxy linker (44-47; 44 is the
pheneylther of IAS 818), nor further lengthening of the alkyl
chain (data not shown) resulted in effective inhibition of the
mutant HIV-1 strains.
’ MOLECULAR MODELING STUDIES
The binding mode of the IASs was extensively studied by
means of docking experiments into the RT NNBS. The aim of
the docking simulations was to evaluate our hypothesis that
targeting the cleft of the NNBS located at the interface between
the p66 and p51 subunits could improve the anti-HIV-1 activity
of the IASs.
In our previous studies,13,27 we reported the comparison
between four docking software packages (AutoDock3.0,28
PLANTS1.1,29 FlexX,30 and MOE docking algorithm31) in term
of ability to reproduce the co-crystallized binding pose of the
ligand for a training set of seven different crystal structures of the
RT (2rf2,32 1vrt,33 2zd1,34 1fk0,35 1s1t,36 2opq,37 and 1jkh38).
Our experiments showed that PLANTS gave the best results.
The docking experiments were carried out on the WT (2rf2,
1vrt, and 2zd1) and mutated RT (1fk0 K103N mutated RT; 1s1t
and 2opq L100I mutated RT; 1jkh Y181C mutated RT). Of the
best scored poses of the most active compounds docked into the
WT RT, it was possible to visualize a series of well-known
interactions: (i) the H-bond was formed between the indole
NH and the carbonyl oxygen of Lys101; (ii) the halogen atom at
position 5 of the indole established steric rather than electrostatic
interactions with the hydrophobic pocket formed by Val106 and
Leu234; (iii) an intramolecular H-bond between an oxygen of
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Table 3. Structure and Anti-HIV Activity of New IAS Derivatives 28-47 in CEM Cellsa
a Data are mean values of two experiments performed in triplicate. b EC50: effective concentration (nM) or concentration required to protect CEM cells
against the cytopathicity of HIV by 50%, as monitored by giant cell formation. c CC50, cytostatic concentration: compound concentration (μM) required
to reduce by 50% the number of viable cells in mock-infected CEM cultures. d Syncytia cell formation was examined microscopically. e Selectivity index
was calculated as CC50/EC50 ratio. f Compound precipitation was detected at higher compound concentrations.
the sulfone group and the amidic nitrogen atom at the side chain
stabilized the compound conformation; (iv) the 3,5-dimethyl-
phenyl moiety formed hydrophobic interactions with an aro-
matic cleft formed by the side chains of Tyr181, Tyr188, Trp229,
and Pro96 residues (Figure 1). Our interest was focused on the
possibility of enlarging the number of interactions at the p51 and
p66 interface cleft.
From analysis of the results for pyrrolidine (12) and
piperidine (16) compounds, it was interesting to observe
H-bond/ionic interactions between the nitrogen atom
(eventually protonated at physiological condition) of the
saturated heterocycle and the Glu138:B carboxylic function.
At the same time, the carbon atoms of the heterocycle moiety
interacted with Val179 and Ile180 residues inside the cleft
and established hydrophobic contacts with the side chains of
Glu138:B and Thr139:B (orange residues in Figure 2). The
phenyl ring of the derivative 24 could be stabilized only by
hydrophobic interactions. In the case of compound 28, the
superior homologue of 12, the ethylene linker forced the
heterocyclic nitrogen distance from Glu138:B carboxylic
function from 3 to >4 Å, thus leading to breaking of the
H-bond/ionic interaction (Figure 3). No major difference
was observed between the binding mode of 36 and its inferior
homologue 24 (data not shown).
A series of molecular dynamics simulations were also per-
formed to study the stability of the interactions reported above.
In particular, we focused our attention on the reference com-
pound 4 and derivative 12. For both inhibitors the binding pose
was stable during the whole simulation time (rmsd 4: 1.13 Å;
rmsd 12: 0.98 Å) and the key H-bond with Lys101 had a
frequency of formation of 90%, suggesting a high stability. The
hydrophobic interactions with Tyr180, Tyr188, and Trp229
were also stable. Some differences were observed in the interac-
tion that compounds 4 and 12 made in the RT p51 and p66
interface cleft. In the case of compound 4 the H-bond between
Glu138:B acid function had a rate of formation of 40% and some
hydrophobic interactions were established between the methyl
groups in the side chain of 4 with Glu138:B and Val179:B. With
derivative 12, the H-bond with Glu138:B was more stable with a
higher rate of formation (74%). Furthermore, in addition to the
hydrophobic contacts with Glu138:B and Val179:B, a stable non-
polar interaction was engaged with Thr139:B. These results are
in accordance with the experimental data and support our
rationale.
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Table 4. HIV-1 RT Inhibitory Activity of Compounds 28-47
and Reference Compounds 4, NVP, and EFV against the WT
and Mutant Enzymes Carrying Single Amino Acid
Substitutionsa
Table 5. Anti-HIV-1 Activity of Compounds 20, 24-26, 28,
36, 40, and 44 and Reference Compounds NVP and EFV
against Mutant HIV-1 Strains in MT-4 Cell Culturesa
EC50 a (nM)
IC50 b (nM)
compd
IIIB
L100I
K103N
Y181C
Y188L
E138K
compd
WT
L1001
K103N
Y181I
20
24
25
26
28
36
40
44
4
3.2 ( 0.8 8.0 ( 5.6 11 ( 3
23 ( 6
46 ( 11 480 ( 90 18 ( 5
59 ( 37 g450 51 ( 41
54 ( 30 470 ( 200 16 ( 0
440 ( 60 14 ( 0
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
4d
80
248
315
594
1547
225
108
421
747
27
660
>40000
>40000
>40000
>40000
>40000
>40000
>40000
>40000
>40000
>40000
>40000
ndc
11 ( 7
16 ( 3
10 ( 7
19 ( 1
21 ( 3
15 ( 1
6.1 ( 2.4 11 ( 0
11 ( 8 15 ( 3
7 ( 5.1 11 ( 5
65
697
280
158
99
1347
2405
6682
1365
>40000
6371
10390
1684
>40000
ndc
84 ( 8
19 ( 1
11 ( 1
80 ( 5
52 ( 6
15 ( 0
380 ( 20 >1000
100 ( 10 >1000
67 ( 22 >1000
260 ( 100
65 ( 3
63
18 ( 1
55
300 ( 140 440 ( 200 >1000
>1000
>1000
380 ( 20
1.6 ( 1.3 9 ( 7
60 ( 4
14 ( 6
96 ( 34 660 ( 480 ndc
711
77
NVP 23 ( 4
>1000
>1000 >1000 150 ( 75
EFVb 1.9 ( 0.3 22 ( 14 130 ( 180 160 ( 180 760 ( 630 9.5 ( 0.3
128
60
ndc
23
a Data represent the average values ( SD of two to three independent
experiments. The cytopathicity assay was based on cell viability mea-
surements determined by the MTT method. The EC50 represents the
compound concentration required to reduce cell viability by 50%. b The
determinations of EFV showed a high degree of variability. c nd: no data.
40
ndc
14
34
ndc
85
ndc
>40000
ndc
ndc
34
23
272
>40000
3700
ethylene spacer. Regardless of the length of the spacer group, the
new IASs were potent inhibitors of HIV-1 WT replication in
CEM cells and showed inhibitory concentrations in the low
nanomolar range. The substituents introduced at positions 4 and
5 of the indole did not show a significant effect on the antiviral
activity against the HIV-1 WT. 5-Bromo and 5-nitro-IASs
bearing the ethylene linker were less cytostatic than the corre-
sponding 5-chloro and 5-chloro-4-fluoro derivatives. Against the
mutant L100I and K103N RT HIV-1 strains, compounds 20,
24-26, 36, and 40 showed antiviral potency superior to that of
NVP and EFV and were comparable to IAS 4.13 Against these
mutant strains, derivatives 20, 24-26, and 40 were equipotent to
ETV. Compounds 12, 40, and 20 proved to inhibit effectively
different HIV clades in PBMC with EC50 values in the higher
picomolar or lower nanomolar range of concentrations. These
IASs may be useful in EFV-based HIV-1 therapies that show the
emergence of the L100I and K103N mutations. Molecular
docking experiments on this novel series of IAS analogues have
also suggested that the H-bond interaction between the nitrogen
atom in the carboxamide chain of IAS and Glu138:B is important
in the binding of these compounds. These results are in
accordance with the experimental data obtained on the WT
and on the mutant strains tested. These compounds are the first
IASs bearing a third cyclic moiety, which may be considered as
hydrids between two-wings and horseshoe-conformation
NNRTIs.27 Such results provide useful information for our
research project looking at an active conformation for future
HIV-1 NNRTIs.
18
30
170
2395
4719
950
66
>40000
>40000
>40000
68
>40000
>40000
>40000
18500
190
>40000
>40000
>40000
>40000
2493
23
112
9000
ndc
NVPd
EFVd
400
80
7000
>20000
>20000
400
a Data represent mean values of at least three separate experiments.
b Compound concentration (IC50, nM) required to inhibit by 50% the
RT activity of the indicated strain. c nd: no data. d Reference 13.
The IASs binding mode was extensively studied in the mutated
RTs. The mutation of Leu100 to Ile seemed not to affect the
binding mode of IASs 12-43, and all the favorable interactions
observed for the WT RT were still detectable for the L100I
mutation, as it was confirmed by the small difference between
L100I and WT IC50 experimental values: 12, IC50 WT = 12 nM and
IC50 L100I = 26 nM (Figure 1S, Supporting Information).
In the interaction of 12 with the K103N mutated RT, all
binding interactions were retained with the only exception of the
favorable hydrophobic interaction between the indole core and
the carbon atoms of the Lys103 side chain (12, IC50 K103N = 96
nM) (Figure 2S, Supporting Information).
In the case of the mutation of Tyr181 to Cys, the favorable π-
π interaction between the 3-(3,5-dimethylphenyl) moiety and
the phenyl ring of tyrosine completely disappeared. The analysis
was in accordance with the biological data justifying the weak
anti-HIV-1 activity (12, IC50 Y181C = 442 nM). Of particular
note, from the docking studies no H-bond between the carbonyl
oxygen of Glu138:B and the nitrogen atom of the saturated
heterocyclic ring of IAS was observed for K103N and Y181C
mutations.
’ EXPERIMENTAL SECTION
Chemistry. MW-assisted reactions were performed on a Discover
S-Class (CEM) single mode reactor, controlling instrument parameters
with PC-running Synergy 1.4 software. Temperature, irradiation power,
maximum pressure (Pmax), PowerMAX (in situ cooling during the MW
irradiation), ramp and hold times, and open and closed vessel modes
were set as indicated. Reactions in an open vessel were carried out in 100 mL
round-bottom flasks equipped with a Dimroth reflux condenser and
’ CONCLUSIONS
We synthesized new IAS derivatives bearing cyclic substitu-
ents at the indole-2-carboxamide linked through a methylene/
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Table 6. Inhibitory Activity of Compounds 12, 20, and 40 against Different HIV-1 Clades in Peripheral Blood Mononuclear Cells
(PBMC)
EC50 a (nM)
compd
clade A (UG273)
clade B (BaL)
clade C (DJ259)
clade A/E (ID12)
group O (BCF06)
12
20
40
0.1
0.1
2.1
0.1
0.1
2.3
0.1
0.5
3.5
0.3
0.3
2.9
0.2
0.9
3.2
a EC50: 50% effective concentration or compound concentration required to inhibit HIV-1 p24 production in virus-infected PBMC. Data are the mean of
two independent experiments.
Figure 2. Binding conformation of 12 (cyan) and 16 (magenta) into
the NNBS of HIV-1 WT RT. Hydrogen atoms are not shown for clarity.
H-Bonds are reported as dotted yellow lines.
Figure 3. Binding conformation of 12 (cyan) and 28 (pink) into the
NNBS of HIV-1 WT RT. Hydrogen atoms are not shown for clarity.
H-Bonds are reported as dotted yellow lines.
a cylindrical stirring bar (length 20 mm, diameter 6 mm). Reactions in a
closed vessel were performed in capped MW-dedicated vials (10 or 35 mL)
with cylindrical stirring bar (length 8 mm, diameter 3 mm). The
temperature of the reaction was monitored by a built-in infrared (IR)
sensor. After completion of the reaction, the mixture was cooled to 25 ꢀC
via air-jet cooling. Melting points (mp) were determined on a SMP1
apparatus (Stuart Scientific) and are uncorrected. IR spectra were run on
a SpectrumOne FT-ATR spectrophotometer (Perkin Elmer). Band
position and absorption ranges are given in cm-1. Proton nuclear
magnetic resonance (1H NMR) spectra were recorded on a 400 MHz
FT spectrometer (Bruker) in the indicated solvent. Chemical shifts are
expressed in δ units (ppm) from tetramethylsilane. Column chroma-
tography was performed on columns packed with alumina from Merck
(70-230 mesh) or silica gel from Macherey-Nagel (70-230 mesh).
Aluminium oxide thin layer chromatography (TLC) cards from Fluka
(aluminium oxide precoated aluminium cards with fluorescent indicator
visualizable at 254 nm) and silica gel TLC cards from Macherey-Nagel
(silica gel precoated aluminum cards with fluorescent indicator visualiz-
able at 254 nm) were used for TLC. Developed plates were visualized by
a Spectroline ENF 260C/FE UV apparatus. Organic solutions were
dried over anhydrous sodium sulfate. Evaporation of the solvents was
carried out on a B€uchi Rotavapor R-210 equipped with a B€uchi V-850
vacuum controller and B€uchi V-700 (∼5 mbar) and V-710 (∼2 mbar)
vacuum pumps. Elemental analyses of tested compounds were found
within (0.4% of the theoretical values. Purity of tested compounds was
>95%. 5-Chloro-3-[(3,5-dimethylphenyl)sulfonyl]-1H-indole-2-carbo-
xamide (2),11 5-chloro-3-[(3,5-dimethylphenyl)sulfonyl]-4-fluoro-1H-
indole-2-carboxamide (6),14 5-bromo-3-[(3,5dimethylphenyl)sulfon-
yl]-1H-indole-2-carboxamide (48),11 5-chloro-3-[(3,5dimethylphenyl)sulf-
onyl]-1H-indole-2-carboxylic acid (50),13 5-bromo3-[(3,5dimethylphenyl)
sulfonyl]-1H-indole-2-carboxylic acid (51),13 3-[(3,5dimethylphenyl)
sulfonyl]-5-nitro-1H-indole-2-carboxylic acid (52),13 5-chloro-3-[(3,5-
dimethylphenyl)sulfonyl]-4fluoro-1H-indole2-carboxylic acid (53),13 ethyl
5-chloro-3-[(3,5dimethylphenyl)sulfonyl]-1H-indole-2-carboxylate (54),13
ethyl
5-bromo3-[(3,5dimethylphenyl)sulfonyl]-1H-indole2-carboxylate
(55),13 ethyl 3-[(3,5-dimethylphenyl)sulfonyl]-5-nitro-1H-indole-2-carbox-
ylate (56),13 and ethyl 5-chloro-3-[(3,5-dimethylphenyl)sulfonyl]-1H-
indole-2-carboxylate (57)13 were prepared as previously reported.
General Procedure for the Preparation of Derivatives 12,
14, 16, 18, 20, and 22. Example: 5-Chloro-3-[(3,5-dimethyl-
phenyl)sulfonyl]-N-(pyrrolidin-1-ylmethyl)-1H-indole-2-
carboxamide (12). A mixture of 2 (0.43 g, 0.00119 mol) in tert-butanol
(20 mL) was heated at 100 ꢀC, and pyrrolidine (0.093 g, 0.1 mL, 0.00131
mol) and 37% formaldehyde (0.039 g, 0.1 mL, 0.00131 mol) were added.
After 4 h, pyrrolidine (0.093 g, 0.1 mL, 0.00131 mol) and 37% formaldehyde
(0.039 g, 0.1 mL, 0.00131 mol) were further added, and the reaction mixture
was stirred at the same temperature for additional 3 h. After cooling, n-hexane
(5 mL) was added and the precipitate collected to give 12 (0.07 g, 13%) as
white solid, mp 193-196 ꢀC. 1H NMR (DMSO-d6): δ 1.66-1.69 (m, 4H),
2.30 (s, 6H), 2.63-2.66 (m, 4H), 4.32 (d, J = 5.7 Hz, 2H), 7.25 (s, 1H), 7.32
(dd,J= 6.6 and 2.1 Hz, 1H), 7.52 (d, J= 8.7 Hz, 1H), 7.61 (d, J = 8.9 Hz, 2H),
7.91(d, J= 2.2 Hz, 1H), 9.22 (br s, 1H, disappeared on treatment with D2O),
13.01 ppm (br s, 1H, disappeared on treatment with D2O). IR: ν 1678, 3120
cm-1. Anal. (C22H24ClN3O3S (445.96)) C, H, Cl, N, S.
Preparative and spectroscopic data of derivatives 14, 16, 18, 20 and
22 are reported in Supporting Information.
General Procedure for the Preparation of Derivatives 13,
15, 17, 19, 21, and 23. Example: 5-Bromo-3-[(3,5-dimethyl-
phenyl)sulfonyl]-N-(pyrrolidin-1-ylmethyl)-1H-indole-2-
carboxamide (13). A mixture of 48 (0.1 g, 0.000 24 mol), parafor-
maldehyde (0.072 g, 0.000 24 mol), and pyrrolidine (0.017 g, 0.02 mL,
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ARTICLE
0.000 24 mol) in benzene (15 mL) was refluxed for 3 h using a Dean-
Stark apparatus. After cooling, the precipitate was collected and washed
with benzene to give 13 (0.07 g, 60%) as white solid, mp 183-186 ꢀC. 1H
NMR (DMSO-d6): δ 1.66-1.70 (m, 4H), 2.29 (s, 6H), 2.63-2.67 (m,
4H), 4.32 (d, J = 4.2 Hz, 2H), 7.26 (s, 1H), 7.41-7.49 (m, 2H), 7.71 (s,
2H), 8.07 (d, J = 1.1 Hz, 1H), 9.21 (br s, 1H, disappeared on treatment
with D2O), 13.01 ppm (br s, 1H, disappeared on treatment with D2O). IR:
ν 1646, 3217 cm-1. Anal. (C22H24BrN3O3S (490.41)) C, H, Br, N, S.
Preparative and spectroscopic data of derivatives 15, 17, 19, 21, and
23 are reported in Supporting Information.
Molecular Modeling. All molecular modeling studies were per-
formed on a MacPro dual 2.66 GHz Xeon running Ubuntu 9.10. The RT
structures were downloaded from the PDB (http://www.rcsb.org/).
Hydrogen atoms were added to the protein, using Molecular Operating
Environment (MOE) 2007.0931 and minimized, keeping all the heavy
atoms fixed until a rmsd gradient of 0.05 kcal mol-1 Å-1 was reached.
Ligand structures were built with MOE and minimized
using the MMFF94x force field until a rmsd gradient of 0.05 kcal
mol-1 Å-1 was reached. The docking simulations were performed
using PLANTS.29 We set the binding lattice as a sphere of 12 Å
binding site radius from the center of 2rf232 co-crystallized
inhibitor. Also, in this case we used all default settings. The Y181I
mutation was obtained by mutating the specific residue in the 1jkh38
crystal using the rotamer explorer tool in MOE and using the lowest
energy conformation obtained. The calculation of the rmsd values
between the cocrystallized and docked conformation were calculated
by the MOE SVL script.39
General Procedure for the Preparation of Derivatives 24-
26, 29, 30, 32-34, 36-38, 40-42, and 44-46. Example: N-
Benzyl-5-chloro-3-[(3,5-dimethylphenyl)sulfonyl]-1H-in-
dole-2-carboxamide (24). BOP (0.28 g, 0.0016 mol) was added to
a mixture of 50 (0.5 g, 0.0014 mol), benzylamine (0.30 g, 0.03 mL,
0.0028 mol), and triethylamine (0.42 g, 0.58 mL, 0.0042 mol) in DMF
(28 mL). The reaction mixture was stirred at room temperature
overnight, diluted with water and extracted with ethyl acetate. The
organic layer was washed with brine, dried, and filtered. Removal of the
solvent gave a residue that was purified by column chromatography
(silica gel, ethyl acetate as eluent) to give 24 (0.27 g, 43%) as yellow
Molecular dynamics was performed with the AMBER 9 suite.40,41
The minimized structure was solvated in a periodic octahedron simula-
tion box using TIP3P water molecules, providing a minimum of 10 Å of
water between the protein surface and any periodic box edge. Chlorine
ions were added to neutralize the charge of the total system. The water
molecules and chlorine ions were energy-minimized keeping the co-
ordinates of the protein-ligand complex fixed (1000 cycle), and then
the whole system was minimized (5000 cycle). Following minimization,
the entire system was heated to 298 K (5 ps). The production simulation
was conducted at 298 K with constant pressure and periodic boundary
condition. Shake bond length condition was used (ntc = 2). Compounds
were parametrized by Antechamber42,43 using AM1 charges. Trajec-
tories analysis were carried our by ptraj44 program. The images in the
manuscript were created with PyMOL.45
Inhibition of HIV-Induced Cytopathicity. The methodology
for the anti-HIV assays had been described previously.46 Briefly, human
CEM cell cultures (∼3 ꢀ 105 cells mL-1) were infected with ∼100
CCID50 HIV-1(IIIB) or HIV-2 (ROD) per mL and seeded in 200 μL
well microtiter plates, containing appropriate dilutions of the test
compounds. After 4 days of incubation at 37 ꢀC, syncytia cell formation
was examined microscopically in the CEM cell cultures.
The anti-HIV activity of several compounds against wild-type HIV-1
strain IIIB and a variety of RT mutant HIV-1 strains in MT-4 cell
cultures was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) method.47 Briefly, virus stocks
were titrated in MT-4 cells and expressed as the 50% cell culture infective
dose (CCID50). MT-4 cells were suspended in culture medium at 1 ꢀ
105 cells/mL and infected with HIV at a multiplicity of infection of 0.02.
Immediately after viral infection, 100 μL of the cell suspension was
placed in each well of a flat-bottomed microtiter tray containing various
concentrations of the test compounds. The test compounds were
dissolved in DMSO at 50 mM or higher. After 4 days of incubation at
37 ꢀC, the number of viable cells was determined using the MTT
method. The selection and characterization of mutant virus strains have
been performed previously.
Inhibition of Different HIV-1 Clades in PBMC. PBMC from
healthy donors were isolated by density gradient centrifugation and
stimulated with PHA at 2 μg/mL (Sigma) for 3 days at 37 ꢀC. The PHA-
stimulated blasts were washed twice with phosphate-buffered saline and
counted by trypan blue. The cells were then seeded at 0.5 ꢀ 106 cells per
well into a 48-well plate containing varying concentrations of compound
in cell culture medium (RPMI 1640) containing 10% fetal calf serum and
interleukin-2 (25 units/mL, R&D Systems Europe, Abingdon, U.K.).
The virus stocks were diluted in medium and added at a final dose of 250
pg of p24/mL as determined by a viral core antigen (Ag) specific
enzyme-linked immunosorbent assay. Cell supernatant was collected at
days 10-12, and HIV-1 core Ag in the culture supernatant was analyzed
1
solid, mp 259-262 ꢀC (from ethanol). H NMR (CDCl3): δ 2.26
(s, 6H), 4.80 (d, J = 2.9 Hz, 2H), 7.15 (s, 1H), 7025-7.27 (m, 2H),
7.35-7.50 (m, 7H), 8.24-8.25 (m, 1H), 10.17 ( br s, 1H, disappeared
on treatment with D2O), 11.25 ppm (br s, 1H, disappeared on
treatment with D2O). IR:
ν
1638, 3209 cm-1
.
Anal.
(C24H21ClN2O3S (452.95)) C, H, Cl, N, S.
Preparative and spectroscopic data for derivatives 25, 26, 29, 30,
32-34, 36-38, 40-42, and 44-46 are reported in Supporting
Information.
General Procedure for the Preparation of Derivatives 27,
28, 31, 35, 39, 43, and 47. Example: N-Benzyl-5-chloro-
3-[(3,5-dimethylphenyl)sulfonyl]-4-fluoro-1H-indole-2-car-
boxamide (27). A mixture of 57 (0.1 g, 0.00024 mol) and benzyl-
amine (0.078 g, 0.2 mL, 0.00073 mol) in ethanol (0.15 mL) was placed
into the MW cavity (closed vessel mode, Pmax = 250 PSI). MW
irradiation of 150 W was used, the temperature being ramped from
25 ꢀC to 150 ꢀC, while stirring. Once 150 ꢀC was reached, taking about
3 min, the reaction mixture was held there for 15 min. The precipitate
was collected and recrystallized from ethanol to give 27 (0.084 g, 75%)
as white solid, mp 229-231 ꢀC. 1H NMR (DMSO-d6): δ 2.30 (s, 6H),
4.56 (d, J = 5.7 Hz, 2H), 7.25 (s, 1H), 7.28 (t, J = 7.2 Hz, 1H), 7.32-7.39
(m, 4H), 7.48 (d, J = 7.1 Hz, 2H), 7.67 (s, 2H), 9.50 (br s, 1H,
disappeared on treatment with D2O), 13.29 ppm (br s, 1H, disappeared
on treatment with D2O). IR: ν 1638, 3220 cm-1. Anal. (C17H15N3O5S
(470.94)) C, H, Cl, F, N, S.
Preparative and spectroscopic data of derivatives 28, 31, 35, 39, 43,
and 47 are reported in Supporting Information.
3-[(3,5-Dimethylphenyl)sulfonyl]-5-nitro-1H-indole-2-car-
boxamide (49). A mixture of ethyl 3-[(3,5-dimethylphenyl)
sulfonyl]-5-nitro-1H-indole-2-carboxylate13 (0.56 g, 0.0014 mol) was
heated with 30% ammonium hydroxide (25 mL) and ammonium
chloride (40 mg) in a sealed tube at 100 ꢀC overnight. After cooling,
the reaction mixture was poured on ice-water, stirred for 15 min, and
extracted with ethyl acetate. The organic layer was washed with brine,
dried, and filtered. Removal of the solvent gave a residue that was
purified by column chromatography (silica gel, chloroform:ethanol =
95:5) to furnish 49 as white solid (0.31 g, 60%) mp >300 ꢀC (from
ethanol). 1H NMR (DMSO-d6): δ 2.33 (s, 6H), 7.31 (s, 1H), 7.68-7.75
(m, 3H), 8.16-8.24 (m, 1H), 8.34 (br s, disappeared on treatment with
D2O, 1H), 8.50 (br s, disappeared on treatment with D2O, 1H), 8.87-
8.89 (m, 1H), 13.47 ppm (br s, disappeared on treatment with D2O,
1H). IR (Nujol): ν 1653, 3235, 3310 cm-1. Anal. (C17H15N3O5S
(373.39)) C, H, N, S.
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Journal of Medicinal Chemistry
ARTICLE
by a p24 Ag enzyme-linked immunosorbent assay kit (PerkinElmer Life
Sciences).
’ ABBREVIATIONS USED
IAS, indolylarylsulfone; HIV-1, human immunodeficiency
virus type 1; AIDS, acquired immunodeficiency syndrome; RT,
reverse transcriptase; NRTI, nucleoside reverse transcriptase
inhibitor; NNRTI, non-nucleoside reverse transcriptase inhibi-
tor; HAART, highly active antiretroviral therapy; WT,
wilde type; NVP, nevirapine; EFV, efavirenz; ETV, etravir-
ine; CEM cells, human T-lymphocyte cells
Cytostatic Assays. The cytostatic concentration was calculated as
the CC50, the compound concentration required to reduce CEM cell
proliferation by 50% relative to the number of cells in the untreated
controls. CC50 values were estimated from graphic plots of the number
of cells determined by Coulter counter measurements (percentage of
control) as a function of the concentration of the test compounds.
Enzymatic Assay Procedures. Chemicals. [3H]dTTP (40 Ci/
mmol) was from Amersham, and unlabeled dNTPs were from Boeh-
ringer. Whatman was the supplier of the GF/C filters. All other reagents
were of analytical grade and purchased from Merck or Fluka.
Nucleic Acid Substrates. The homopolymer poly(rA) (Pharmacia)
was mixed at weight ratios in nucleotides of 10:1 with the oligomer
oligo(dT)12-18 (Pharmacia) in 20 mM Tris-HCl (pH = 8.0), containing
20 mM KCl and 1 mM EDTA, heated at 65 ꢀC for 5 min, and then slowly
cooled at room temperature.
Expression and Purification of Recombinant HIV-1 RT
Forms. The coexpression vectors pUC12N/p66(His)/p51with the
WT or the mutant forms of HIV-1 RT p66 were kindly provided by Dr.
S. H. Hughes (NCI-Frederick Cancer Research and Development
Center). Proteins were expressed in E. Coli and purified as described.48
HIV-1 RT RNA-Dependent DNA Polymerase Activity As-
say. RNA-dependent DNA polymerase activity was assayed as follows:
a final volume of 25 μL contained reaction buffer (50 mM Tris-HCl pH
7.5, 1 mM DTT, 0.2 mg/mL BSA, 4% glycerol), 10 mM MgCl2, 0.5 μg of
poly(rA)/oligo(dT)10:1 (0.3 μM 30-OH ends), 10 μM [3H]dTTP (1
Ci/mmol), and 2-4 nM RT. Mixtures were incubated at 37 ꢀC for the
indicated time. Then 20 μL aliquots were spotted on glass fiber filters
GF/C which were immediately immersed in 5% ice-cold TCA. Filters
were washed twice in 5% ice-cold TCA and once in ethanol for 5 min,
dried and acid-precipitable radioactivity was quantitated by scintillation
counting.
’ REFERENCES
(1) (a) Barre-Sinoussi, F.; Chermann, J.; Rey, F.; Nugeyre, M.;
Chamaret, S.; Gruest, J.; Dauguet, C.; Axler-Blin, C.; Vezinet-Brun, F.;
Rouzioux, C.; Rozenbaum, W.; Montagnier, L. Isolation of a T-lympho-
tric retrovirus from a patient for a risk of acquired immunodeficiency
syndrome (AIDS). Science 1983, 220, 868–871. (b) Gallo, R. C.; Sarin,
P. S.; Gelmann, E. P.; Robert-Guroff, M.; Richardson, E.; Kalyanaraman,
V. S.; Mann, D.; Sidhu, G. D.; Stahl, R. E.; Zolla.Pazner, S.; Leibowitch,
J.; Popovic, M. Isolation of human T-cell leukemia virus in acquired
immunodeficiency syndrome (AIDS). Science 1983, 220, 865–867.
(2) 2009 AIDS epidemic update.
(3) (a) Mehellou, Y.; De Clercq, E. Twenty-six years of anti-HIV
drug discovery: where do we stand and where do we go? J. Med. Chem.
2010, 53, 521–538. (b) De Clercq, E. Highlights in the discovery of
antiviral drugs: a personal retrospective. J. Med. Chem. 2010,
53, 1438–1450.
(4) Este, J. A.; Cihlar, T. Current status and challenges of antire-
troviral research and therapy. Antiviral Res. 2010, 85, 25–33.
(5) (a) Menꢁendez-Arias, L. Molecular basis of human immunodefi-
ciency virus drug resistance: an update. Antiviral Res. 2010, 85, 210–231.
(b) Paredes, R.; Clotet, B. Clinical management of HIV-1 resistance.
Antiviral Res. 2010, 85, 245–265.
(6) Hawkins, T. Understanding and managing the adverse effects of
antiretroviral therapy. Antiviral Res. 2010, 85, 201–209.
Inhibition Assay. Reactions were performed under the conditions
described for the HIV-1 RT RNA-dependent DNA polymerase activity
assay. Incorporation of radioactive dTTP into poly(rA)/oligo(dT) at
different substrate (nucleic acid or dTTP) concentrations was mon-
itored in the presence of increasing fixed amounts of inhibitor. Data were
then plotted according to Lineweaver-Burke and Dixon. For Ki
determination, an interval of inhibitor concentrations between 0.2Ki
and 5Ki was used.
(7) Ceccherini-Silberstein, F.; Svicher, V.; Sing, T.; Artese, A.;
Santoro, M. M.; Forbici, F.; Bertoli, A.; Alcaro, S.; Palamara, G.;
d’Arminio Monforte, A.; Balzarini, J.; Antinori, A.; Lengauer, T.; Perno,
C. F. Characterization and structural analysis of novel mutations in
human immunodeficiency virus type 1 reverse transcriptase involved in
the regulation of resistance to nonnucleoside inhibitors. J. Virol. 2007,
81, 11507–11519.
(8) Sweeney1, Z. K.; Klumpp, K. Improving non-nucleoside reverse
transcriptase inhibitors for first-line treatment of HIV infection: the
development pipeline and recent clinical data. Curr. Opin. Drug Discovery
Dev. 2008, 11, 454–470.
’ ASSOCIATED CONTENT
(9) (a) Vingerhoets, J.; Azijn, H.; Fransen, E.; De Baere, I.;
Smeulders, L.; Jochmans, D.; Andries, K.; Pauwels, R.; de Bethune,
M.-P. TMC125 displays a high genetic barrier to the development of
resistance: evidence from in vitro selection experiments. J. Virol. 2005,
79, 12773–12782. (b) Haubrich, R.; Gubernick, S.; Yasothan, U.;
Kirkpatrick, P. Etravirine. Nat. Rev. Drug Discovery 2008, 7, 287–288.
(10) (a) Williams, T. A.; Ciccarone, T. M.; Saari, W. S.; Wai, J. S.;
Greenlee, W. J.; Balani, S. K., Goldman, M. E.; Theoharides, A. D.
Indoles as Inhibitors of HIV Reverse Transcriptase. Eur. Pat. Appl. EP 0
530 907 A1, 1992. (b) Williams, T. M.; Ciccarone, T. M.; MacTough,
S. C.; Rooney, C. S.; Balani, S. K.; Condra, J. H.; Emini, E. A.; Goldman,
M. E.; Greenlee, W. J.; Kauffman, L. R.; O’Brien, J. A.; Sardana, V. V.;
Schleif, W. A.; Theoharides, A. D.; Anderson, P. S. 5-Chloro-
3-(phenylsulfonyl)indole-2-carboxyamide: a novel non-nucleoside in-
hibitor of the HIV-1 reverse transcriptase. J. Med. Chem. 1993, 36, 1291–
1294.
S
Supporting Information. Additional synthesis and char-
b
acterization information. This material is available free of charge
via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*Phone: þ39 06 4991 3800. Fax: þ39 06 4969 3268. E-mail:
romano.silvestri@uniroma1.it.
’ ACKNOWLEDGMENT
The authors are indebted to Istituto Pasteur, Fondazione
Cenci Bolognetti (Grant 2009) and FILAS (Finanziaria Laziale
di Sviluppo, Grant 2010) for financial support. The authors also
thank I. Nikolova and A. Galabov, Bulgarian Academy of
Sciences, Sofia, Bulgaria, for the biological evaluation against
Coxsackie B4 virus.
(11) Silvestri, R.; De Martino, G.; La Regina, G.; Artico, M.; Massa,
S.; Vargiu, L.; Mura, M.; Loi, A. G.; Marceddu, T.; La Colla, P. Novel
indolyl aryl sulfones active against HIV-1 carrying NNRTI resistance
mutations: synthesis and SAR studies. J. Med. Chem. 2003, 46, 2482–
2493.
1596
dx.doi.org/10.1021/jm101614j |J. Med. Chem. 2011, 54, 1587–1598

Journal of Medicinal Chemistry
ARTICLE
(12) Silvestri, R.; Artico, M.; De Martino, G.; La Regina, G.; Loddo,
R.; La Colla, M.; La Colla, P. Simple, short peptide derivatives of a
sulfonylindolecarboxamide (L-737,126) active in vitro against HIV-1
wild-type and variants carrying non-nucleoside reverse transcriptase
inhibitor resistance mutations. J. Med. Chem. 2004, 47, 3892–3896.
(13) Piscitelli, F.; Coluccia, A.; Brancale, A.; La Regina, G.; Sansone,
A.; Giordano, C.; Balzarini, J.; Maga, G.; Zanoli, S.; Samuele, A.; Cirilli,
R.; La Torre, F.; Lavecchia, A.; Novellino, E.; Silvestri, R. Indolylar-
ylsulfones bearing natural and unnatural amino acids. Discovery of
potent inhibitors of HIV-1 non-nucleoside wild type and resistant
mutant strains reverse transcriptase and coxsackie B4 virus. J. Med.
Chem. 2009, 52, 1922–1934.
(14) La Regina, G.; Coluccia, A.; Piscitelli, F.; Bergamini, A.; Sinistro,
A.; Cavazza, A.; Maga, G.; Samuele, A.; Zanoli, S.; Novellino, E.; Artico,
M.; Silvestri, R. Indolyl aryl sulfones as HIV-1 non-nucleoside reverse
transcriptase inhibitors: role of two halogen atoms at the indole ring in
developing new analogues with improved antiviral activity. J. Med. Chem.
2007, 50, 5034–5038.
(15) Ludovici, D. W.; Kavash, R. W.; Kukla, M. J.; Ho, C. Y.; Ye, H.;
De Corte, B L.; Andries, K.; de Bꢁethune, M.-P.; Azijn, H.; Pauwels, R.;
Moereels, H. E. L.; Heeres, J.; Koymans, L. M. H.; de Jonge, M. R.; Van
Aken, K. J. A.; Daeyaert, F. F. D.; Lewi, P. J.; Das, K.; Arnold, E.; Janssen,
P. A. J. Evolution of anti-HIV drug candidates part 2: diaryltriazine
(DATA) analogues. Bioorg. Med. Chem. Lett. 2001, 11, 2229–2234.
(16) (a) Klunder, J. M.; Hoermann, M.; Cywin, C. L.; David, E.;
Brickwood, J. R.; Schwartz, R.; Barringer, K. J.; Pauletti, D.; Shih, C.-K.;
Erickson, D. A.; Sorge, C. L.; Joseph, D. P.; Hattox, S. E.; Adams, J.;
Grob, P. M. Novel nonnuceloside inhibitors of HIV-1 reverse tran-
scriptase. 7. 8-Arylethyldipyridodiazepinones as potent broad-spectrum
inhibitors of wild-type and mutant enzymes. J. Med. Chem. 1998,
41, 2960–2971. (b) Cywin, C. L.; Klunder, J. M.; Hoermann, M.;
Brickwood, J.; David, E.; Grob, P. M.; Schwartz, R.; Pauletti, D.;
Barringer, K. J.; Shih, C.-K.; Sorge, C. L.; Erickson, D. A.; Hattox,
S. E. Novel nonnuceloside inhibitors of HIV-1 reverse transcriptase. 7.
8-Aryloxymethyl and 8-arylthiomethydipyridodiazepinones. J. Med.
Chem. 1998, 41, 2972–2984.
(17) Zhao, Z.; Wolkenberg, S. E.; Lu, M.; Munshi, V.; Moyer, G.;
Feng, M.; Carella, A. V.; Ecto, L. T.; Gabryelski, L. J.; Lai, M.-T.; Prasad,
S. G.; Yan, Y.; McGaughey, G. B.; Miller, M. D.; Lindsley, C. W.;
Hartman, G. D.; Vacca, J. P.; Williams, T. M. Novel indole-3-sulfona-
mides as potent HIV non-nucleoside reverse transcriptase inhibitors
(NNRTIs). Bioorg. Med. Chem. Lett. 2008, 18, 554–559.
(18) (a) Ragno, R.; Artico, M.; De Martino, G.; La Regina, G.;
Coluccia, A.; Di Pasquali, A.; Silvestri, R. Docking and 3-D QSAR studies on
indolyl aryl sulfones (IASs). Binding mode exploration at the HIV-1 reverse
transcriptase non-nucleoside binding site and design of highly active N-(2-
hydroxyethyl)carboxyamide and N-(2-hydroxyethyl)carboxyhydrazide de-
rivatives. J. Med. Chem. 2005, 48, 213–223. (b) De Martino, G.; La Regina,
G.; Ragno, R.; Coluccia, A.; Bergamini, A.; Ciaprini, C.; Sinistro, A.; Maga,
G.; Crespan, E.; Artico, M.; Silvestri., R. Indolyl aryl sulfones (IASs) as HIV-
1 non-nucleoside reverse transcriptase inhibitors. Synthesis, biological
evaluation and binding mode studies of new derivatives at indole-2-
carboxamide. Antiviral Chem. Chemother. 2006, 17, 59–77.
(19) Ragno, R.; Coluccia, A.; La Regina, G.; De Martino, G.;
Piscitelli, F.; Lavecchia, A.; Novellino, E.; Bergamini, A.; Ciaprini, C.;
Sinistro, A.; Maga, G.; Crespan, E.; Artico, M.; Silvestri, R. Design,
molecular modeling, synthesis and anti-HIV-1 activity of new indolyl
aryl sulfones. Novel derivatives of the indole-2-carboxamide. J. Med.
Chem. 2006, 49, 3172–3184.
(20) Polinsky, A. Lead-likenss and Drug-likeness. In The Practice of
Medicinal Chemistry, 3rd ed.; Wermuth C. G. Elsevier Ltd.: Oxford, U.K.,
2008; pp 244-254.
(23) Corbett, J. W.; Rodgers, J. D. Discovery of second generation of
quinazoline non-nucleoside reverse transcriptase inhibitors of HIV-1.
Prog. Med. Chem. 2002, 40, 63–105.
(24) ALOGPS and ALOGpS values were calculated at the Web site
www.vcclab.org using ALOGPS version 2.1: Tetko, I. V.; Gasteiger, J.;
Todeschini, R.; Mauri, A.; Livingstone, D.; Ertl, P.; Palyulin, V. A.;
Radchenko, E. V.; Zefirov, N. S.; Makarenko, A. S.; Tanchuk, V. Y.;
Prokopenko, V. V. Virtual computational chemistry laboratory-design
and description. J. Comp. Aid Mol. Des. 2005, 19, 453–463.
(25) VCCLAB, Virtual Computational Chemistry Laboratory.
http://www.vcclab.org.
(26) (a) Silvestri, R.; La Regina, G.; De Martino, G.; Artico, A.;
Befani, O.; Palumbo, M.; Agostinelli, E.; Turini, P. Simple, potent and
selective pyrrole inhibitors of monoamine oxidase type A and type B. J.
Med. Chem. 2003, 46, 917–920. (b) Silvestri, R.; Artico, M.; La Regina,
G.; De Martino, G.; La Colla, M.; Loddo, R.; La Colla, P. Anti-HIV-1
activity of pyrryl aryl sulfone (PAS) derivatives. Synthesis and SAR
studies of novel esters and amides at the position 2 of the pyrrole
nucleus. Farmaco 2004, 59, 201–210. (c) Silvestri, R.; Marfꢀe, G.; Artico,
M.; La Regina, G.; De Martino, G.; Lavecchia, A.; Novellino, E.;
Morgante, E.; Di Stefano, C.; Catalano, G.; Filomeni, G.; Abruzzese,
E.; Ciriolo, M. R.; Russo, M. A.; Amadori, S.; Cirilli, R.; La Torre, F.;
Sinibaldi Salimei, P. Pyrrolo[1,2-b][1,2,5]benzothiadiazepines (PBTDs):
a new class of agents endowed with high apoptotic activity in chronic
myelogenous leukemia K562 cells and in cells from patients at onset and
imatinib-resistant. J. Med. Chem. 2006, 49, 5840–5844.
(27) La Regina, G.; Coluccia, A.; Silvestri, R. Looking for an active
conformation of the future HIV-1 non-nucleoside reverse transcriptase
inhibitors. Antiviral Chem. Chemother. 2010, 20, 231–237.
(28) Goodsell, D. S.; Morris, G. M.; Olson, A. J. Automated docking
of flexible ligands: applications of AutoDock. J. Mol. Recognit. 1996,
9, 1–5.
(29) Korb, O.; St€utzle, T.; Exner, T. E. PLANTS: Application
of Ant Colony Optimization to Structure-Based Drug Design.
In Ant Colony Optimization and Swarm Intelligence, Proceedings
of the 5th International Workshop, ANTS; Dorigo, M., Gambardella,
L. M., Birattari, M., Martinoli, A., Poli, R., St€utzle, T., Eds.; Lecture
Notes in Computer Science, Series 4150; Springer: Berlin, 2006;
pp 247-258.
(30) FlexX version 2.2; BioSolveIT GmbH: Sankt Augustin, Ger-
many; http://www.biosolveit.de/FlexX/.
(31) Molecular Operating Environment (MOE) version 2009.10;
Chemical Computing Group Inc.: Montreal, Canada; http://www.
chemcomp.com/.
(32) Zhao, Z.; Wolkenberg, S. E.; Lu, M.; Munshi, V.; Moyer, G.;
Feng, M.; Carella, A. V.; Ecto, L. T.; Gabryelski, L. J.; Lai, M.-T.; Prasad,
S. G.; Yan, Y.; McGaughey, G. B.; Miller, M. D.; Lindsley, C. W.;
Hartman, G. D.; Vacca, J. P.; Williams, T. M. Novel indole-3-sulfona-
mides as potent HIV non-nucleoside reverse transcriptase inhibitors
(NNRTIs). Bioorg. Med. Chem. Lett. 2008, 18, 554–559.
(33) Kohlstaedt, L. A.; Wang, J.; Friedman, J. M.; Rice, P. A.; Steitz,
T. A. Crystal structure at 3.5 Å resolution of HIV-1 reverse transcriptase
complexed with an inhibitor. Science 1992, 256, 1783–1790.
(34) Das, K.; Bauman, J. D.; Clark, A. D., Jr.; Frenkel, Y. V.; Lewi,
P. J.; Shatkin, A. J.; Hughes, S. H.; Arnold, E. High-resolution structures
of HIV-1 reverse transcriptase/TMC278 complexes: strategic flexibility
explains potency against resistance mutations. Proc. Nat. Acad. Sci. U.S.A.
2008, 105, 1466–1471.
(35) Ren, J.; Milton, J.; Weaver, K. L.; Short, S. A.; Stuart, D. I.;
Stammers, D. K. Structural basis for the resilience of efavirenz (DMP-
266) to drug resistance mutations in HIV-1 reverse transcriptase.
Structure 2000, 8, 1089–1094.
(21) Veber, D. F.; Johnson, S. R.; Cheng, H.-C.; Smith, B. R.; Ward,
K. W.; Kopple, K. D. Molecular properties influence the oral bioavil-
ability of drug candidate. J. Med. Chem. 2002, 45, 2615–2623.
(22) The recombinant HIV-1 RT carrying the Y181I mutation was
comparable to the Y181C substitution in terms of drug resistance, from
an enzymological point of view. See ref 17.
(36) Ren, J.; Nichols, C. E.; Chamberlain, P. P.; Weaver, K. L.; Short,
S. A.; Stammers, D. K. Crystal structures of HIV-1 reverse transcriptases
mutated at codons 100, 106 and 108 and mechanisms of resistance to
non-nucleoside inhibitors. J. Mol. Biol. 2004, 336, 569–578.
(37) Ren, J.; Nichols, C. E.; Chamberlain, P. P.; Weaver, K. L.; Short,
S. A.; Chan, J. H.; Kleim, J. P.; Stammers, D. K. Relationship of potency
1597
dx.doi.org/10.1021/jm101614j |J. Med. Chem. 2011, 54, 1587–1598

Journal of Medicinal Chemistry
ARTICLE
and resilience to drug resistant mutations for GW420867X revealed by
crystal structures of inhibitor complexes for wild-type, Leu100Ile,
Lys101Glu, and Tyr188Cys mutant HIV-1 reverse transcriptases. J.
Med. Chem. 2007, 50, 2301–2309.
(38) Ren, J.; Nichols, C.; Bird, L.; Chamberlain, P.; Weaver, K.;
Short, S.; Stuart, D. I.; Stammers, D. K. Structural mechanisms of drug
resistance for mutations at codons 181 and 188 in HIV-1 reverse
transcriptase and the improved resilience of second generation non-
nucleoside inhibitors. J. Mol. Biol. 2001, 312, 795–805.
(39) Code “scoring.svl” was obtained from SLV Exchange Web site
http://svl.chemcomp.com (Chemical Computing Group, Inc., Mon-
treal, Canada).
(40) Case, D. A; Cheatham, T. E., III; Darden, T.; Gohlke, H.; Luo,
R.; Merz, K. M., Jr.; Onufriev, A.; Simmerling, C.; Wang, B.; Woods, R.
The Amber biomolecular simulation programs. J. Comput. Chem. 2005,
26, 1668–1688.
(41) Meagher, K. L.; Redman, L. T.; Carlson, H. A. Development of
polyphosphate parameters for use with the AMBER force field.
J. Comput. Chem. 2003, 24, 1016–1026.
(42) Wang, J.; Wang, W.; Kollman, P. A.; Case, D. A. Automatic
atom type and bond type perception in molecular mechanical calcula-
tions. J. Mol. Graph. Model. 2006, 25, 247–260.
(43) Wang, J.; Wolf, R. M.; Caldwell, J. W.; Kollman, P. A.; Case,
D. A. Development and testing of a general AMBER force field.
J. Comput. Chem. 2004, 25, 1157–1174.
(44) AmberTools version 1.4; http://ambermd.org/#AmberTools.
(45) PyMOL version 1.2r1; DeLano Scientific LLC: San Carlos, CA
http://www.pymol.org/.
(46) Van Nhien, A. N.; Tomassi, C.; Len, C.; Marco-Contelles, J. L.;
Balzarini, J.; Pannecouque, C.; De Clercq, E.; Postel, D. First synthesis
and evaluation of the inhibitory effects of aza analogues of TSAO on
HIV-1 replication. J. Med. Chem. 2005, 48, 4276–4284.
(47) Pannecouque, C.; Daelemans, D.; De Clercq, E. Tetrazolum-
based colometric assay for the detection of HIV replication inhibitors:
revised 20 years latter. Nat. Protoc. 2008, 3, 427–434.
(48) Maga, G.; Amacker, M.; Ruel, N.; Hubsher, U.; Spadari, S.
Resistance to nevirapine of HIV-1 reverse transcriptase mutants: loss of
stabilizing interactions and thermodynamic or steric barriers are induced
by different single amino acid substitutions. J. Mol. Biol. 1997,
274, 738–747.
1598
dx.doi.org/10.1021/jm101614j |J. Med. Chem. 2011, 54, 1587–1598