Indolylarylsulfones 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

Article abstract of DOI:10.1021/jm801470b

New potent indolylarylsulfone (IAS) HIV-1 NNRTIs were obtained by coupling natural and unnatural amino acids to the 2-carboxamide and introducing different electron-withdrawing substituents at position 4 and 5 of the indole nucleus. The new IASs inhibited

Full text of DOI:10.1021/jm801470b

1922
J. Med. Chem. 2009, 52, 1922–1934
Indolylarylsulfones 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
Francesco Piscitelli,† Antonio Coluccia,| Andrea Brancale,| Giuseppe La Regina,† Anna Sansone,† Cesare Giordano,
Jan Balzarini,∞ Giovanni Maga,‡ Samantha Zanoli,‡ Alberta Samuele,‡ Roberto Cirilli,# Francesco La Torre,#
Antonio Lavecchia,§ Ettore Novellino,§ and Romano Silvestri*,†
Istituto Pasteur-Fondazione Cenci Bolognetti, Dipartimento di Chimica e Tecnologie del Farmaco, Sapienza UniVersita` 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
Chimica Biomolecolare del CNR, Sapienza UniVersita` di Roma, Piazzale Aldo Moro 5, I-00185 Roma, Italy, Dipartimento di Chimica
Farmaceutica e Tossicologica, UniVersita` di Napoli Federico II, Via Domenico Montesano 49, I-80131, Napoli, Italy, Rega Institute for
Medical Research, Katholieke UniVersiteit LeuVen, B-3000 LeuVen, Belgium, Istituto di Genetica Molecolare-Consiglio Nazionale delle
Ricerche, Via Abbiategrasso 207, I-27100 PaVia, Italy, and Dipartimento del Farmaco, Istituto Superiore di Sanità, Viale Regina Elena 299,
I-00161 Roma, Italy
ReceiVed NoVember 21, 2008
New potent indolylarylsulfone (IAS) HIV-1 NNRTIs were obtained by coupling natural and unnatural amino
acids to the 2-carboxamide and introducing different electron-withdrawing substituents at position 4 and 5
of the indole nucleus. The new IASs inhibited the HIV-1 replication in human T-lymphocyte (CEM) cells
at low/subnanomolar concentration and were weakly cytostatic. Against the mutant L100I, K103N, and
Y181C RT HIV-1 strains in CEM cells, sulfones 3, 4, 19, 27, and 31 were comparable to EFV. The new
IASs were inhibitors to Coxsackie B4 virus at low micromolar (2-9 µM) concentrations. Superimposition
of PLANTS docked conformations of IASs 19 and 9 revealed different hydrophobic interactions of the
3,5-dimethylphenyl group, for which a staking interaction with Tyr181 aromatic side chain was observed.
The binding mode of 19 was not affected by the L100I mutation and was consistent with the interactions
reported for the WT strain.
Introduction
combination of three (recommended) or four antiretroviral drugs,
highly active antiretroviral therapy (HAART) produces an
effective and prolonged reduction of morbidity and mortality
in AIDS patients.5 However, HAART does not completely
eradicate the viral infection; thus, the required long-term drug
administrations provoke drug resistance and unwanted side
effects.6
Acquired immunodeficiency syndrome (AIDSa) causes over
5700 deaths every year, and at the same time over 6800 people
become infected with human immunodeficiency virus (HIV).1
Both prevention and treatment of HIV are still inadequate. The
challenge toward new anti-HIV/AIDS drugs is the discovery
of (i) an effective vaccine, (ii) better tolerated drugs, and (iii)
agents endowed with improved properties against both drug
resistance and cross-resistance.2
Drugs currently approved for the treatment of HIV infection
fall into six target classes. In addition to (i) nucleoside (NRTIs)
and nucleotide (NtRTIs) reverse transcriptase inhibitors, (ii) non-
nucleoside reverse transcriptase inhibitors (NNRTIs), (iii) pro-
tease inhibitors (PIs), and (iv) fusion inhibitors (FIs),3 two new
drugs, (v) the entry inhibitor CCR5 co-receptor antagonist
maraviroc and (vi) the integrase inhibitor raltegravir, became
available in 2007.4 These drugs slow the viral infection and
multiplication, affecting the progression of the disease. By
NNRTIs showed low toxicity and favorable pharmacokinetic
properties. The major problem of earlier inhibitors, namely, the
rapid emergence of drug resistance, was overcome by new
generation agents that succeeded in inhibiting both HIV-1 wild
type (WT) and viral strains carrying NNRTI resistance muta-
tions.7 Etravirine was approved in 2008 by the U.S. FDA for
the drug combination treatment of HIV-1 infected people who
experienced drug resistance to other drugs of this class.8
The development of indolylarylsulfones (IASs) NNRTIs was
based on the Merck derivative L-737,126 (1)9 as reference
compound. The potent activity of IAS 2 against the NNRTI-
resistant mutants was correlated to the presence of a 3-(3,5-
dimethylphenyl)sulfonyl moiety.10 Potent IAS derivatives were
obtained by adding to the 2-carboxamide chain of 2 simple
amino acids such as glycine and D,L-alanine to give derivatives
(for example, 3 and 4) endowed with activities against HIV-1
WT and NNRTI-resistant mutants superior to that of the parent
indole 1.11
* To whom correspondence should be addressed. Phone: +39 06 4991
3800. Fax: +39 06 491 491. E-mail: romano.silvestri@uniroma1.it.
†
Dipartimento di Chimica e Tecnologie del Farmaco, Sapienza Universita`
di Roma.
|
Cardiff University.
Consiglio Nazionale delle Ricerche, Roma.
Katholieke Universiteit Leuven.
Consiglio Nazionale delle Ricerche, Pavia.
Istituto Superiore di Sanità, Roma.
∞
‡
On the other hand, the activity against the K103N RT mutant
virus exhibited by imidazole 512 prompted the synthesis of
derivatives bearing either the N-(2-hydroxyethyl)carboxamide
functionality (6)13 or substituted N′-carboxyhydrazide derivatives
(7)14 at position 2 of the indole. DuPont Merck synthesized
analogues of efavirenz (8) containing two halogen atoms at
positions 5 and 6 of the quinazolinone ring which showed
#
§
Universita` di Napoli Federico II.
a Abbreviations, IAS, indolylarylsulfone; HIV, human immunodeficiency
virus; AIDS, acquired immunodeficiency syndrome; RT, reverse tran-
scriptase; NRTI, nucleoside/nucleotide reverse transcriptase inhibitor;
NNRTI, non-nucleoside reverse transcriptase inhibitor; PI, protease inhibitor;
FI, fusion inhibitor; HAART, highly active antiretroviral therapy; WT, wild
type; NVP, nevirapine; EFV, efavirenz.
10.1021/jm801470b CCC: $40.75
2009 American Chemical Society
Published on Web 03/12/2009

Indolylarylsulfones
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7 1923
Chart 1. Structure of Reference Compounds 1-12 and New IASs 13-34
broader spectrum than the monosubstituted counterparts.15
Recently we synthesized IASs derivatives bearing two halogen
atoms at the indole ring. The 5-chloro-4-fluoro- (9) and 4,5-
difluoro- (10) IASs turned out to be potent inhibitors of HIV-1
WT and the NNRTI-resistant Y181C RT and K103N-Y181C
RT HIV-1 strains. In particular, compound 10 was exceptionally
potent against RT WT and RTs carrying the K103N, Y181I,
L100I mutations.16 In 2008, Idenix Pharmaceuticals and Merck
& Co. showed a renewed interest in IASs and replaced the
3-arylsulfonyl group by either an arylphosphonyl17 (11) or a
sulfonamide18 (12) group. Continuing our research project, we
were obtained by reaction of 47-50 with 2-aminoethane-
sulfonamide hydrochloride20 or 2-aminopropanesulfonamide
hydrochloride in the presence of BOP reagent and triethylamine
(Scheme 1).
2-Aminopropanesulfonamide hydrochloride (67) was syn-
thesized by reaction of mesylate 6821 with potassium thioacetate
in DMF to afford 69, which was oxidized with hydrogen
peroxide to give the sulfonic acid 70. Treatment of 70 with
phosgene gave the corresponding sulfonyl chloride 71, which
was transformed into sulfonamide 72 with gaseous ammonia
and then deprotected with hydrogen over carbon to provide 67
(Scheme 2).
planned the synthesis of new IASs 13-34 characterized by (a)
natural and unnatural amino acids at the 2-carboxamide and
(b) different electron-withdrawing substituents at positions 4
and 5 of the indole (Chart 1).
Results and Discussion
The antiretroviral activity (EC50 values) of IAS derivatives 3,
4, and 13-34 was evaluated against the HIV-1 WT in human
T-lymphocyte (CEM) cells, using IASs 2, 7, and 9 as reference
compounds. Compounds 3, 4, and 13-34 proved to be highly
potent against HIV-1 in human T-lymphocyte (CEM) cells and
showed inhibitory potencies in low/subnanomolar range of con-
centrations which were comparable with the previously reported
lead compounds 2,10 7,14 and 9.16 Against HIV-1 WT, the
inhibitory activity was only marginally affected by the substituent
introduced on the indole nucleus, and with only one exception (23),
5-chloro-IASs were the most potent inhibitors (Table 1).
The derivatives were also evaluated for their antiproliferative
activities against murine leukemia cells (L1210) and human
T-lymphocyte cells (Molt4/C8, CEM). Generally, the IASs inhib-
ited the cell proliferation in the micromolar range, that is, at
compound concentrations that are usually 3-4 orders of magnitude
higher than their antivirally active concentrations. 5-Nitro-IASs
bearing an unbranched amino acid unit were often devoid of any
Chemistry
The synthesis of new IASs is depicted in Scheme 1. Reaction
of ethyl 5-chloro- (35), 5-bromo- (36), 5-nitro- (37), or 5-chloro-
4-fluoroindole-2-carboxylate19 (38) with N-(3,5-dimethylphe-
nyl)succinimide in the presence of boron trifluoride diethyl
etherate at room temperature in dichloromethane afforded the
corresponding 3-(3,5-dimethylphenyl)indole-2-carboxylates
39-42. The sulfides 39-42 were oxidized to sulfones 43-46
with 3-chloroperoxybenzoic acid in chloroform at 0 °C.
Subsequent lithium hydroxyde hydrolysis of esters 43-46 at
room temperature afforded the corresponding acids 47-50.
Coupling reaction of 47-50 with an appropriate amino acid in
the presence of BOP reagent and triethylamine in DMF
overnight provided IAS derivatives bearing an amino acid unit
(51-66). Treatment of 51-66 with 28% ammonium hydroxyde
in ethanol at 60 °C or microwave irradiation at 150 W and 130
°C for 10 min provided 3, 4, and 13-36. Sulfonamides 27-34

1924 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7
Scheme 1. Synthesis of Compounds 3, 4, and 13-34a
Piscitelli et al.
a Reagents and reaction conditions: (a) BF3 ·Et2O, dichloromethane, room temp to 45 °C, 4 h; (b) 3-chloroperoxybenzoic acid, chloroform, 0 °C, 2 h; (c)
LiOH, aqueous tetrahydrofuran, room temp, 5 h; (d) amino acid methyl or ethyl ester hydrochloride, BOP reagent, triethylamine, dimethylformamide, room
temp, overnight; (e) 28% NH4OH, ethanol, 60 °C, 3 h; (f) 28% NH4OH, closed vessel, 130 °C, 150 W, 10 min; (g) 2-aminoethanesulfonamide hydrochloride
or 2-aminopropane sulfonamide hydrochloride, BOP reagent, triethylamine, dimethylformamide, room temp, overnight.
Scheme 2. Synthesis of Compound 67a
a Reagents and reaction conditions: (a) potassium thioacetate, DMF, room temp, overnight; (b) hydrogen peroxide, acetic acid, room temp, overnight; (c)
phosgene, DMF, dichloromethane, 2 h, overnight, nitrogen atmosphere; (d) gaseous NH3, benzene, 60 °C, 2 h; (e) hydrogen, palladium over carbon, aqueous
methanol, 37% HCl, room temp, 3 h.
significant cytostatic effect at 500 µM (i.e., 14, 21, and 29). On
the contrary, the bromine atom at position 5 of the indole had the
tendency to strengthen the cytotoxicity (Table 2).
The most active compounds 3, 4, 19, 27, and 31 have been
evaluated against mutant HIV-1 strains that harbor the L100I,
K103N, Y181C, Y181I, and Y188L mutations in their RT (Table
3). The compounds showed high antiviral potency against the
mutant L100I and K103N RT HIV-1 strains. Against these mutant
strains, the compounds were always superior to EFV (Table 3,
footnote b) and were comparable with data reported for MK-496522
and TMC-120;23 however, rilpivirine (TMC-278) was overall 10-
fold more potent.23
The compounds lost antiviral potency against the mutant
Y181C RT HIV-1 strain. In this respect, EFV was comparable

Indolylarylsulfones
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7 1925
Table 1. Structure, and Anti-HIV Activitiy of IAS Derivatives 3, 4, and
13-34 and Reference Compounds 2, 7, and 9 in Human T-Lymphocyte
(CEM) Cellsa
Table 2. Inhibitory Effects of Compounds 3, 4, and 13-34 on the
Proliferation of Murine Leukemia Cells (L1210) and Human
T-Lymphocyte Cells (Molt4/C8, CEM)a
IC50 (µM)
compd
L1210
Molt4/C8
CEM
3
220 ( 2
40 ( 5
>500
15 ( 7
17 ( 0
>500
34 ( 1
3.8 ( 1.3
8.7 ( 1.4
44 ( 13
22 ( 11
9 ( 1
92 ( 13
31 ( 15
g500
13
14
15
4
53 ( 3
12 ( 1
10 ( 0
96 ( 4
30 ( 10
26 ( 2
46 ( 4
>500
42 ( 3
48 ( 1
16 ( 4
37 ( 13
42 ( 3
20 ( 7
17 ( 2
408 ( 131
42 ( 3
15 ( 0
11 ( 1
20 ( 3
18 ( 6
g500
31 ( 19
11 ( 1
10 ( 0
66 ( 17
21 ( 3
16 ( 2
36 ( 1
g500
30 ( 2
32 ( 1
18 ( 9
36 ( 11
27 ( 4
14 ( 5
14 ( 1
200 ( 73
31 ( 0
13 ( 3
12 ( 1
15 ( 1
16 ( 1
301 ( 211
g500
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
2
25 ( 12
>500
33 ( 0
36 ( 1
16 ( 11
41 ( 1
25 ( 0
12 ( 2
11 ( 0
244 ( 1
36 ( 3
8.8 ( 0.7
9.3 ( 0.5
11 ( 0
12 ( 1
20 ( 2
432 ( 96
14 ( 6
7
9
>500
46 ( 27
>28 ( 18
a Data are mean values of at least two independent experiments.
Table 3. Anti-HIV-1 Activity of Compounds 3, 4, 19, 27, and 31
against Mutant HIV-1 Strains in CEM Cell Culturesa
IC50 (nM)
compd
L100I
K103N
Y181C
Y181I
Y188L
3
4
19
27
31
NVP
8 ( 7
9 ( 7
29 ( 18
14 ( 6
74 ( 18
96 ( 34
78 ( 23
>1000
>1000
>1000
690 ( 440
660 ( 480
g1000
>1000
>1000
7 ( 2
19 ( 9
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 cythopathicity of HIV by 50%, as monitored by giant cell
formation. c Compound precipitation was detected at higher compound
concentrations.
1.2 ( 1
2.0 ( 8
40 ( 32
36 ( 32
230 ( 190 >1000
390 ( 120 >1000
60 ( 4 2900 ( 1320 11000 ( 4100 >10000 >10000
EFVb 22 ( 14 130 ( 180
160 ( 180 12 ( 14 760 ( 630
a Data are mean values of two to three independent experiments. b The
determinations of EFV showed a high degree of variability.
to the tested compounds against the mutant Y181C RT HIV-1
strain and showed a better inhibition profile against the Y181I
strain. Against the mutant Y188L RT HIV-1 strain, compounds
3 and 4 were equipotent to EFV. These results were in
agreement with the inhibitory effect of the compounds noted
for the corresponding mutant RT enzymes (Table 4).
this mutant. We identified compound 31 containing the 2-ami-
nopropane-1-sulfonamide motif as a potent inhibitor of WT and
L100I RTs.
To evaluate the influence of the chiral center at the amino
acid unit, the racemates 4, 16, and 23 were separated by chiral
HPLC. Against HIV-1 WT RT, the enantiomers 4a and 4b, 16a
and 16b, and 23a and 23b obtained from the corresponding
racemic mixtures 4, 16, and 23, respectively, showed negligible
differences of activity (Table 5). Against the K103N mutation,
the D-enantiomers 4b and 16b were about 5 times more potent
than the corresponding L-enantiomers 4a and 16a, and the
enantiomers 23a and 23b did not show significant difference
of activity.
Compounds 3, 4, 13-34 were evaluated for their antiviral
activity against vesicular stomatitis virus, Coxsackie B4 virus,
and respiratory syncytial virus in HeLa cell cultures (Table 6).
The IASs showed no activity against vesicular stomatitis virus
in HeLa cells. Several compounds (3, 4, 13, 15, 16, 22, 23, 26,
28, 29, and 30-32) were inhibitors to Coxsackie B4 virus at
low micromolar (2-9 µM) concentrations. Seven compounds
(16, 23, 24, 28, and 31-33) inhibited this viral strain at EC50
) 2-5 µM, a concentration that is 25 times lower than required
The compounds were potent inhibitors of the RT of HIV-1
WT, and they were always superior to nevirapine (NVP) and
often comparable to efavirenz (EFV) (Table 4). Against the RT
WT, the sulfonamides 27 and 31 showed inhibitory concentra-
tions comparable to the reference compounds 210 and 916 and
1000 and 200 times superior to NVP and EFV, respectively.
Fifteen compounds inhibited in the submicromolar range the
mutant K103N RT HIV-1, which is the major mutation
emerging in patients treated with EFV, whose viral loads
rebound after an initial response to the drug.7a Against the
K103N RT HIV-1 strain, the most active compounds 3, 4, 19,
23, 24, and 31 were notably more active than NVP and EFV,
albeit less active than the references 2 and 9. Both 5-chloro
and 5-bromo-IASs were potent inhibitors of the L100I RT
mutant, and compound 31 was exceptionally effective against

1926 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7
Piscitelli et al.
Table 4. HIV-1 RT Inhibitory Activity of Compounds 3, 4, and 13-34
against the WT and Mutant Enzymes Carrying Single Amino Acid
Substitutionsa
Table 6. Cytotoxicity and Antiviral Activity of Compounds 3, 4, 13,
15-17, 20, 22-26, and 28-33 against Vesicular Stomatitis Virus,
Coxsackie B4 Virus, and Respiratory Syncytial Virus in HeLa Cell
Culturesa
b
IC50 (nM)
c
EC50 (µM), virus
compd
WT
L100I
K103N
Y181I
MCC b (µM)
Coxsackie B4
3
26
26
34
25
23
28
22
62
27
86
27
34
120
81
33
359
0.4
2
61
ndd
0.4
3
28
36
0.3
10
3
161
199
206
319
112
41
209
270
143
169
204
130
252
296
191
390
25
280
782
417
1042
190
509
362
1522
351
420
474
783
288
321
1088
3387
584
529
787
ndd
1645
2211
2909
2459
2493
1810
4211
6277
5055
8566
5534
7165
4137
15830
11920
>40000
>40000
>40000
>40000
ndd
13
14
15
4
vesicular respiratory
compd
HeLa Vero
HeLa
Vero
stomatitis
syncytial
3
100
100
>100
100
100
20
>100
100
>100
>100
100
>100
>20
>100
>20
>100
>4
>100
20
>100
16
>100
4
g20
4
>100
9
6
2
>100
>20
>100
>20
>20
>4
13
14
15
4
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30c
31
32
33
34
2
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
2
20
>100
20
20
>100
100
100
100
>100
100
100
g20
100
100
100
>100
>4
>4
>100
>4
>4
g20
>100
16
>20
>20
>20
20
>4
>4
20
20
>20
g4
>20
>4
>100
>100
>100
>250
10
g20
>4
>4
11
>100
9
4.5
g20
g20
9
>100
20
12
g20
>100
100
g20
g20
100
100
20
>20
>100
>20
>20
>20
>20
>20
>4
16
>100
>20
16
>20
>20
>20
9
13
388
ndd
4
>4
233
1328
3277
3
3
800
6
1944
8550
26220
>40000
140
20
g20
>4
g4
g20
7
3
3
g20
>4
>100
>100
27
12
125
364
267
60
g100 100
g100
>20
>20
>4
g20
>4
>100
>100
>100
>250
22
>20
>20
12
16
g20
12
>100
>100
100
>250
g250
100
100
20
g20
20
>100
>100
>100
100
g20
g20
g20
100
>100
>100
>100
>250
>250
7
9
ndd
2
ndd
29
NVPe
EFVe
400
80
9000
7000
>20000
>20000
400
ndd
7
9
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 Insoluble in the aqueous medium during
the experiment. d nd: no data. e Data from literature.16
(S)-DHPAd >250
>250
50
ribavirin
>250
a Data are mean values of two independent experiments performed in
triplicate. b MCC: minimum cytotoxic concentration (µM) required to cause
microscopically detectable alteration of normal cell morphology. c EC50:
effective dose (µM) required to reduce virus-induced cytopathogenicity by
50%. d Acyclonucleoside (S)-9-(2,3-dihydroxypropyl)adenine.
Table 5. Inhibitory Activity of Racemates 4, 16, and 23 and
Enantiomers 4a, 4b, 16a, 16b, 23a, and 23b against HIV-1 RT WT and
RTs Carrying Single Amino Acid Substitutionsa
IC50b(nM)
compd
chirality
WT
K103N
the compounds. Several substituted benzimidazole-based struc-
tures have been identified in the past to be endowed with anti-
Coxsackie virus activity in cell culture (i.e., MRL-1237, TBZE-
029, enviradene, and enviroxime) (for an overview see ref 24).
One of these compounds (e.g., TBZE-029) was, like the present
structures, initially discovered as an NNRTI active against HIV-
1. TBZE-029 is active at an EC50 value of 1.2 µg/mL, a
concentration close to the EC50 of the most inhibitory indole
derivatives (i.e., 13, 16, 31, 32). The TBZE-029 compound
affects viral RNA synthesis by targeting the nonstructural protein
2C. Therefore, it would be most interesting to investigate the
most active indole derivatives against this virus-specific protein
to reveal a potential viral target for this novel class of
compounds. Therefore, we believe that this study allowed
disclosure of a new lead class of Coxsackie B4 virus and
respiratory syncytial virus inhibitors. Coxsackie B virus may
be the causative agent of viral polymyositis and rhabdomyolysis,
a pathological condition that may be associated with immun-
odepressed conditions.25 Such agents may provide a starting
point for the development of new effective inhibitors of both
HIV-1 RT and Coxsackie B4 virus.
4
4a
4b
16
16a
16b
23
23a
23b
D, L
L
20
26
17
16
13
477
1005
253
555
1504
324
1690
1340
1417
D
D, L
L
D
16
D, L
L
490
315
147
D
a Data represent mean values of at least three separate experiments.
b Compound dose (IC50, nM) required to inhibit by 50% the RT activity of
the indicated strain.
for ribavirin. The compounds were also evaluated against
Coxsackie B4 virus in Vero cell cultures. The antiviral potency
was in general less pronounced in Vero than in HeLa cell
cultures. Compounds 20, 23, 28, 31, and 32 showed anti-
Coxsackie B4 virus activity in both HeLa and Vero cell cultures.
The inhibitory concentrations of compounds 16 and 32 against
respiratory syncytial virus were ∼4 µM. The activity against
Coxsackie B4 virus seemed to be positively affected by the
presence of the bromine atom at position 5 of the indole and
an amino acid unit bearing a R- or ꢀ-methyl group. It is noted
that the antivirally active compounds have a limited selectivity
index (ratio MIC/EC50) that is several orders of magnitude lower
than the selectivity index for HIV, and it can be questioned
whether the observed antiviral activity is due to a direct anti-
Coxsackie virus effect or caused by the cytotoxic activity of
The new IASs were also evaluated against a wide panel of
other viral strains including parainfluenza virus 3, reovirus 1,
sindbis virus, and Punta Toro virus in Vero cell cultures, herpes
simplex virus-1 (KOS), herpes simplex virus-2 (G), vaccinia
virus, herpes simplex virus-1 TK- (KOS ACV) in HEL cell
cultures, influenza A virus (H1N1 and H3N2 subtypes) and

Indolylarylsulfones
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7 1927
Table 7. The rmsd Values for Co-Crystallized Inhibitors by PLANTS,
FlexX, and MOE
rmsd
PDB code
PLANTS 1.0
FlexX 3.0
MOE 2007/09
1vrt
0.23
0.80
0.40
0.85
0.26
0.48
1.18
1.59
2.01
1.28
1.04
0.90
1.53
1.20
5
1.47
4.60
2.10
2rf2
2zd1
1fk0
1s1t
2opq
influenza B virus in MDCK cell cultures, and feline corona virus
(FIPV) and feline herpes virus in CRFK cell cultures. No marked
inhibitory activity was detected (Tables 1S-4S, Supporting
Information).
Molecular Modeling Studies
The binding mode of the IASs was extensively studied by
means of docking experiments into the non-nucleoside binding
site (NNBS) of the RT. The latest available 12a/RT cocrystal
structure (PDB code 2rf2) solved by Merck18 was used as a
new starting point for docking studies because of the high
structural correlation between the cocrystallized inhibitor 12a
and IASs. In our previous studies,13a,14 we used Autodock 3.026
software to generate IASs docking poses. However, Autodock
3.0 software consistently yielded two different clusters of poses,
and the crystallographic conformation was often the lower
scoring pose. To obtain more accurate docking results and also
to validate the generated poses with crystallographic information,
we decided to test the docking softwares PLANTS,27 FlexX,28
and MOE29 docking algorithm.
Figure 1. Binding conformation of 19 in the NNBS of HIV-1 WT
RT.
We selected seven PDB structures of inhibitors cocrystallized
with either HIV-1 WT RT or mutated RTs (2rf2,18 1vrt,30
2zd1,31 1fk0,32 1s1t,33 2opq,34 and 1jkh35). The best scoring
poses predicted by the different algorithms were compared with
the crystallographic conformations obtained from each complex
in terms of the rmsd. Our experiments showed that only
PLANTS was able to correctly dock each molecule with a rmsd
of <1 (Table 7). The other docking software always achieve
bigger rmsd values than PLANTS on the whole test set.
Therefore, we decided to use PLANTS to dock the new IAS
series. Interestingly, the best scoring conformation obtained by
PLANTS clustering algorithm was always superimposed to
Merck’s crystal (Figure 1S, Supporting Information).
Figure 2. Binding conformation of 19 in the NNBS of HIV-1 L100I
RT.
In order to investigate the possible binding mode of the new
IASs, we selected the highly potent compound 19 (IC50 ) 26
nM; EC50 ) 0.70 nM) for docking studies into the NNBS of
the HIV-1 WT RT. From docking, feature interactions of 19
into the NNBS were (i) the H-bond between the indole NH
and the carbonyl oxygen of Lys101 (in stick in the Figure 1),
(ii) the interactions of the N-(3-amino-3-oxopropyl)carboxamide
moiety with the residues Lys101 and Glu138B at the bottom
of the binding pocket (they seemed to be particularly important
for the anti-HIV activity), (iii) the sulfone group allowing 19
to assume a butterfly-like conformation that is common to many
other NNRTIs, (iv) the chlorine atom at position 5 of the indole
occupying a hydrophobic pocket formed by Val106 and Leu234
and forming steric rather than electrostatic interaction, and (v)
the 3,5-dimethylphenyl moiety forming hydrophobic interactions
with an aromatic cleft formed by the side chains of Tyr181,
Tyr188, Trp229, and Pro95 residues (Figure 1).
stacking interaction with the Tyr181 aromatic side chain was
observed (Figure 2S, Supporting Information).
The IASs binding mode was also extensively studied in
mutated RTs. The results obtained by docking the IASs
analogues with PLANTS in L100I mutated RT (PDB codes 1s1t,
2opq) showed that the mutation of leucine to isoleucine does
not affect the binding mode and were consistent with the
interactions reported for the WT strain. Furthermore, all
compounds show a similar binding mode, consistent with the
low EC50 values (Figure 2).
We have also repeated the docking simulations on the K103N
and Y181I mutated RTs, and the results obtained did not show
significant differences with the IASs binding mode observed
with the wild type. However, the lower antiviral activity of the
IASs against these mutants could be the consequence of the
reduced interaction between 19 and the mutated residues. In
particular, for the Y181I RT HIV-1 strain, the limited anti-HIV
efficiency could be caused by the loss of the favorable
protein-ligand π-π interactions, while in the mutant K103N
RT, the loss of the favorable hydrophobic interaction between
the ligand and the side chain of the lysine 103 might be
Worthy of note, a superimposition of PLANTS docked
conformations of 19 and the highly active derivative 916 (IC50
) 3 nM; EC50 ) 1.0 nM) showed different hydrophobic
interactions of the 3,5-dimethylphenyl group, for which a

1928 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7
Piscitelli et al.
for thin layer chromatography (TLC). Developed plates were
visualized with a Spectroline ENF 260C/F UV apparatus. Organic
solutions were dried over anhydrous sodium sulfate. Evaporation
of the solvents was carried out on a Bu¨chi rotavapor R-210 equipped
with a Bu¨chi V-850 vacuum controller and Bu¨chi V-700 (∼5 mbar)
and V-710 vacuum (∼2 mbar) pumps. Elemental analyses were
found to be within (0.4% of the theoretical values. Purity of tested
compounds was >95%. Ethyl 5-chloroindole-2-carboxylate (35) was
purchased from Sigma-Aldrich. Ethyl 5-nitroindole-2-carboxylate
(37) was purchased from Acros Organics. Ethyl 5-bromoindole-2-
carboxylate (36)10 and ethyl 5-chloro-4-fluoroindole-2-carboxylate
(38)19 were prepared as previously reported.
N-(2-Amino-2-oxoethyl)-5-chloro-3-[(3,5-dimethylphenyl)sul-
fonyl]-1H-indole-2-carboxamide (3). 3 was prepared as previously
reported.11
N-(1-Amino-1-oxopropan-2-yl)-5-chloro-3-[(3,5-dimethylphe-
nyl)sulfonyl]-1H-indole-2-carboxamide (4). 4 was prepared as
previously reported.11
responsible for the reduced activity. In both cases the geometry
of the H-bonds between the carbonyl oxygen of the E138 with
unsubstituted amidic nitrogen of 19 is worst than that in the
wild type (Figure 3S, Supporting Information). Molecular
modeling was also unable to explain the differences of activity
of the enantiomers against the K103N mutant.
Conclusions
Compounds 3, 4, and 13-34 proved to be highly potent
against HIV-1 replication in human T-lymphocyte (CEM) cells
and showed inhibitory potencies in low/subnanomolar range that
were comparable with those of the previously reported lead
compounds 2,10 7,14 and 9.16 Against HIV-1 WT, the inhibitory
activity seemed only marginally affected by the substituent
introduced on the indole nucleus. In general, none of the IASs
proved to be markedly cytostatic. Against the L100I and K103N
RT HIV-1 strains, 3, 4, 19, 27, and 31 were always superior to
EFV and had similar activity to that reported for MK-496522
and TMC-120.23 These IASs were also equipotent to EFV
against the mutant Y181C RT HIV-1 strain. The D-enantiomers
4b and 16b were about 5 times more potent than the corre-
sponding L-enantiomers 4a and 16a against RT. Several
General Procedure for the Synthesis of Derivatives 13-26.
Example. N-(3-Amino-3-oxopropyl)-5-chloro-3-[(3,5-dimethyl-
phenyl)sulfonyl]-1H-indole-2-carboxamide (19). A sample of 28%
ammonium hydroxide (28 mL) was added to a mixture of 59 (0.48
g, 0.001 mol) in ethanol (48 mL). Reaction mixture was stirred at
60 °C for 3 h. After the mixture was cooled, water was added and
the mixture was extracted with ethyl acetate. The organic layer was
washed with brine, dried, and evaporated to furnish a residue which
was purified by silica gel column chromatography (ethyl acetate
compounds were inhibitory to Coxsackie B4 virus at EC50
)
3-5 µM but were much less selective in their anti-Coxsackie
virus activity than observed for HIV. In view that for picor-
naviruses, no single antiviral drug has ever been approved,36
these agents may serve as basis for the development of drugs
endowed with both anti-HIV-1 RT and Coxsackie B4 virus
inhibitory activities.
1
as eluent) to give 19. Yield 40%, mp >290 °C (from ethanol). H
NMR (DMSO-d6): δ 2.30 (s, 6H), 2.42 (t, J ) 7.1 Hz, 2H), 3.52
(t, J ) 6.4 Hz, 2H), 6.90 (broad s, 1H, disappeared on treatment
with D2O), 7.24 (s, 1H), 7.32 (dd, J ) 8.7 and 1.9 Hz, 1H), 7.41
(broad s, 1H, disappeared on treatment with D2O), 7.51 (d, J )
8.7 Hz, 1H), 7.62 (s, 2H), 7.93 (d, J ) 1.7 Hz, 1H), 9.03 (broad s,
1H, disappeared with treatment with D2O), 12.92 ppm (broad s,
In order to investigate the possible binding mode of the new
IASs, we selected the highly potent compound 19. Superimposi-
tion of PLANTS docked conformations of 19 and the highly
active derivative 9 revealed different hydrophobic interactions
of the 3,5-dimethylphenyl group, for which a stacking interaction
with Tyr181 aromatic side chain was observed. The IAS binding
mode was also extensively studied in mutated RTs. In L100I
mutated RT, the mutation of leucine to isoleucine does not affect
the binding mode, and it was consistent with the interactions
reported for the WT strain. The lower antiviral activity against
the mutant K103N and Y181I RT HIV-1 strains could be the
consequence of the reduced interaction between 19 and the
mutated residues. For the Y181I RT HIV-1 strain, the limited
anti-HIV efficiency could also be caused by the loss of the
favorable protein-ligand π-π interactions, while in the K103N
mutant, the loss of the hydrophobic interaction between the
ligand and the side chain of the lysine 103 might be responsible
for the reduced activity.
1H, disappeared on treatment with D2O). IR: ν 1637, 3222 cm-1
Anal. (C20H20ClN3O4S (433.91)) C, H, N, Cl, S.
.
N-(2-Amino-2-oxoethyl)-5-bromo-3-[(3,5-dimethylphenyl)sul-
fonyl]-1H-indole-2-carboxamide (13). 13 was synthesized as for
1
19 but using 52. Yield 57%, mp 274-276 °C (from ethanol). H
NMR (DMSO-d6): δ 2.32 (s, 6H), 4.01 (s, 2H), 7.27 (s, 1H), 7.33
(broad s, 1H, disappeared on treatment with D2O), 7.47-7.49 (m,
2H), 7.52 (d, J ) 8.2 Hz, 1H), 7.73 (s, 2H), 8.17 (s, 1H), 9.39
(broad s, 1H, disappeared on treatment with D2O), 13.08 ppm (s,
1H, disappeared on treatment with D2O). IR: ν 1639, 1693, 3243,
3445 cm-1. Anal. (C19H18BrN3O4S (464.33)) C, H, Br, N, S.
N-(2-Amino-2-oxoethyl)-3-[(3,5-dimethylphenyl)sulfonyl]-5-
nitro-1H-indole-2-carboxamide (14). 14 was synthesized as for
1
19 but using 53. Yield 42%, mp 259-261 °C (from ethanol). H
NMR (DMSO-d6): δ 2.29 (s, 6H), 4.00 (s, 2H), 7.26 (s, 1H), 7.31
(s, 1H, disappeared on treatment with D2O), 7.46 (s, 1H, disappeared
on treatment with D2O), 7.70-7.71 (m, 3H), 8.16 (d, J ) 8,3 Hz,
1H), 8.89 (s, 1H), 9.33 (s, 1H, disappeared on treatment with D2O),
13.42 ppm (s, 1H, disappeared on treatment with D2O). IR: ν 1681,
3291 cm-1. Anal. (C19H18N4O6S (430.43)) C, H, N, S.
Experimental Section
N-(2-Amino-2-oxoethyl)-5-chloro-3-[(3,5-dimethylphenyl)sul-
fonyl]-4-fluoro-1H-indole-2-carboxamide (15). 15 was synthesized
as for 19 but using 54. Yield 95%, mp 255-257 °C (from ethanol).
1H NMR (DMSO-d6): δ 2.32 (m, 6H), 3.92 (s, 2H), 7.26 (s, 1H),
7.28-7.42 (m, 4H), 7.66 (s, 2H), 9.47 (broad s, 1H, disappeared
on treatment with D2O), 13.27 (broad s, 1H, disappeared on
treatment with D2O). IR: ν 1662, 1703, 3280 cm-1. Anal.
(C19H17ClFN3O4S (437.87)) C, H, Cl, F, N, S.
N-(1-Amino-1-oxopropan-2-yl)-5-bromo-3-[(3,5-dimethylphe-
nyl)sulfonyl]-1H-indole-2-carboxamide (16). 16 was synthesized
as for 19 but using 56. Yield 43%, mp 248-251 °C (from ethanol).
1H NMR (DMSO-d6): δ 1.37 (d, J ) 7.1 Hz, 3H), 2.29 (s, 6H),
4.46-4.54 (qn, J ) 7.1 Hz, 1H), 7.26 (s, 2H), 7.46 (dd, J ) 8.7
and 1.7 Hz, 1H), 7.49-7.52 (m, 2H), 7.68 (s, 2H), 8.15 (d, J )
1.7 Hz, 1H), 9.32 (s, 1H, disappeared on treatment with D2O), 13.05
ppm (s, 1H, disappeared on treatment with D2O). IR: ν 1662, 1672,
3254, 3465 cm-1. Anal. (C20H20BrN3O4S (478.36)) C, H, Br, N, S.
Chemistry. Microwave (MW)-assisted reactions were performed
on Discover S-Class (CEM), setting temperature, irradiation power,
maximum pressure (Pmax), PowerMAX (in situ cooling during the
MW irradiation), ramp and hold times, and open and closed vessel
modes as indicated. Melting points (mp) were determined on a
Bu¨chi 510 apparatus and are uncorrected. Infrared spectra (IR) were
obtained using Perkin-Elmer 1310 and SpectrumOne spectropho-
tometers. Band position and absorption ranges are given in cm-1
.
Proton nuclear magnetic resonance (1H NMR) spectra were recorded
on Bruker AM-200 (200 MHz) and Bruker Avance 400 MHz FT
spectrometers in the indicated solvent. Chemical shifts are expressed
in δ units (ppm) from tetramethylsilane. Column chromatographies
were packed with alumina (Merck, 70-230 mesh) and silica gel
(Merck, 70-230 mesh). Aluminum oxide TLC cards (Fluka,
aluminum oxide precoated aluminum cards with fluorescent indica-
tor at 254 nm) and silica gel TLC cards (Fluka, silica gel precoated
aluminum cards with fluorescent indicator at 254 nm) were used

Indolylarylsulfones
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7 1929
N-(1-Amino-1-oxopropan-2-yl)-3-[(3,5-dimethylphenyl)sulfo-
nyl]-5-nitro-1H-indole-2-carboxamide (17). 17 was synthesized
as for 19 but using 57. Yield 99%, mp 245-250 °C (from ethanol).
1H NMR (DMSO-d6): δ 1.37 (d, J ) 7.0 Hz, 3H), 2.29 (s, 6H),
4.46-4.54 (qn, J ) 7.1 Hz, 1H), 7.27 (s, 1H), 7.50 (s, 1H,
disappeared on treatment with D2O), 7.66 (s, 1H, disappeared on
treatment with D2O), 7.68 (s, 2H), 7.71 (d, J ) 9.1 Hz, 1H), 8.17
(dd, J ) 9.0 and 2.0 Hz, 1H), 8.90 (d, J ) 1.9 Hz, 1H), 9.29 (s,
1H, disappeared on treatment with D2O), 12.95 ppm (s, 1H,
ppm (s, 1H, disappeared on treatment with D2O). IR: ν 1610, 1644,
3299, 3425 cm-1. Anal. (C21H22BrN3O4S (492.39)) C, H, Br, N, S.
N-(4-Amino-4-oxobutan-3-yl)-3-[(3,5-dimethylphenyl)sulfonyl]-
5-nitro-1H-indole-2-carboxamide (25). A mixture of 65 (0.30 g,
0.0006 mol) and 28% ammonium hydroxide (2 mL) was placed
into the MW cavity (closed vessel, Pmax ) 250 PSI). MW
irradiation of 150 W was used, the temperature being ramped from
25 °C to 130 °C, while stirring. Once 130 °C was reached, taking
about 3 min, the reaction mixture was held at this temperature for
10 min. The reaction mixture was neutralized with 1 N HCl and
extracted with ethyl acetate. The organic layer was washed with
brine, dried, and evaporated. The residue was purified by silica gel
column chromatography (ethyl acetate-ethanol 9:1 as eluent) to
disappeared on treatment with D2O). IR: ν 1673, 3275, 3409 cm-1
.
Anal. (C20H20N4O6S (444.46)) C, H, N, S.
N-(1-Amino-1-oxopropan-2-yl)-5-chloro-3-[(3,5-dimethylphe-
nyl)sulfonyl]-4-fluoro-1H-indole-2-carboxamide (18). 18 was
synthesized as for 19 but using 58. Yield 79%, mp 249-250 °C
(from ethanol). 1H NMR (DMSO-d6): δ 1.39 (d, J ) 7.1 Hz, 3H),
2.32 (m, 6H), 4.44 (q, J ) 7.2 Hz, 1H), 7.26 (s, 1H), 7.29 (broad
s, 1H, disappeared on treatment with D2O), 7.35 (broad s, 1H,
disappeared on treatment with D2O), 7.36-7.40 (m, 2H), 7.68 (s,
2H), 9.37 (broad s, 1H, disappeared on treatment with D2O), 13.26
(broad s, 1H, disappeared on treatment with D2O). IR: ν 1655, 1698,
3245 cm-1. Anal. (C20H19ClFN3O4S (451.90)) C, H, Cl, F, N, S.
N-(3-Amino-3-oxopropyl)-5-bromo-3-[(3,5-dimethylphenyl)-
sulfonyl]-1H-indole-2-carboxamide (20). 20 was synthesized as
for 19 but using 60. Yield 99%, mp 258-260 °C (from ethanol).
1H NMR (DMSO-d6): δ 2.32 (s, 6H), 2.47 (t, J ) 7.1 Hz, 2H),
3.58 (t, J ) 7.1 Hz, 2H), 6.98 (broad s, 1H, disappeared on treatment
with D2O), 7.26 (s, 1H), 7.44-7.49 (m, 3H), 7.64 (s, 2H), 8.10 (s,
1H), 9.03 (s, 1H, disappeared on treatment with D2O), 13.07 ppm
(s, 1H, disappeared on treatment with D2O). IR: ν 1635, 1665, 3212,
3273, 3473 cm-1. Anal. (C20H20BrN3O4S (478.36)) C, H, Br, N, S.
N-(3-Amino-3-oxopropyl)-3-[(3,5-dimethylphenyl)sulfonyl]-5-
nitro-1H-indole-2-carboxamide (21). 21 was synthesized as for
1
give 25. Yield 65%, mp 264-268 °C (from ethanol). H NMR
(DMSO-d6): δ 1.25 (d, J ) 6.6 Hz, 3H), 2.29-234 (m, 8H),
4.32-4.44 (m, 1H), 6.94 (broad s, 1H, disappeared on treatment
with D2O), 7.29 (s, 1H), 7.47 (broad s, 1H, disappeared on treatment
with D2O), 7.66 (s, 2H), 7.71 (d, J ) 9.1 Hz, 1H), 8.19 (dd, J )
9.0 and 2.0 Hz, 1H), 8.85 (d, J ) 1.4 Hz, 1H), 9.00 (broad s, 1H,
disappeared with treatment with D2O), 13.46 ppm (broad s, 1H,
disappeared on treatment with D2O). IR: ν 1639, 1657, 3232, 3437
cm-1. Anal. (C21H22N4O6S (458.49)) C, H, N, S.
N-(4-Amino-4-oxobutan-2-yl)-5-chloro-3-[(3,5-dimethylphe-
nyl)sulfonyl]-4-fluoro-1H-indole-2-carboxamide (26). 26 was
synthesized as for 19 but from 66. Yield 22%, mp 217-219 °C
(from ethanol). 1H NMR (DMSO-d6): δ 1.19 (d, J ) 6.7 Hz, 3H),
2.17-2.20 (m, 1H), 2.31 (s, 6H), 2.44-2.49 (m, 1H), 4.29-4.37
(m, 1H), 6.89 (broad s, 1H, disappeared on treatment with D2O),
7.24 (s, 1H), 7.31-7.38 (m, 2H), 7.43 (broad s, 1H, disappeared
on treatment with D2O), 7.66 (s, 2H), 8.96 (broad s, 1H, disappeared
on treatment with D2O), 13.22 (broad s, 1H, disappeared on
treatment with D2O). IR: ν 1625, 1691, 3194 cm-1. Anal.
(C21H21ClFN3O4S (465.93)) C, H, Cl, F, N, S.
1
19 but using 61. Yield 99%, mp 260 °C (from ethanol). H NMR
(DMSO-d6): δ 2.29 (s, 6H), 2.44 (t, J ) 7.1 Hz, 2H), 3.54 (q, J )
6.6 Hz, 2H), 6.69 (broad s, 1H, disappeared on treatment with D2O),
7.25 (s, 1H), 7.45 (broad s, 1H, disappeared on treatment with D2O),
7.64 (s, 2H), 7.68 (d, J ) 9.1 Hz, 1H), 8.16 (dd, J ) 9.1 and 2.3
Hz, 1H), 8.84 (d, J ) 2.2 Hz, 1H), 9.06 (broad s, 1H, disappeared
on treatment with D2O), 13.44 ppm (broad s, 1H, disappeared on
General Procedure for the Synthesis of Derivatives 26-34,
52-54, and 56-66. Example. Methyl 3-[N-[5-chloro-3-[(3,5-
dimethylphenyl)sulfonyl]-1H-indole-2-carboxamido]]pro-
panoate (59). A mixture of 4711 (0.70 g, 0.0019 mol), methyl
ꢀ-alanine hydrochloride (0.52 g, 0.0037 mol), triethylamine (0.57
g, 0.79 mL, 0.0056 mol), and BOP (0.80 g, 0.0019 mol) in DMF
(10 mL) was stirred at room temperature overnight. Water was
added, and the mixture was extracted with ethyl acetate. The organic
layer was washed with brine, dried, and evaporated to dryness. The
residue was purified by silica gel column chromatography (ethyl
acetate as eluent) to give 59. Yield 81%, mp 197-200 °C (from
ethanol). 1H NMR (DMSO-d6): δ 2.29 (s, 6H), 2.65 (t, J ) 6.7 Hz,
2H), 3.58 (t, J ) 6.3 Hz, 2H), 3.61 (s, 3H), 7.24 (s, 1H), 7.31 (dd,
J ) 8.8 and 1.9 Hz, 1H), 7.51 (d, J ) 8.8 Hz, 1H), 7.60 (s, 2H),
7.90 (d, J ) 2.0 Hz, 1H), 9.08 (broad s, 1H, disappeared with
treatment with D2O), 13.00 ppm (broad s, 1H, disappeared on
treatment with D2O). IR: ν 1738, 3204 cm-1. Anal. (C21H21ClN2O5S
(448.92)) C, H, N, Cl, S.
treatment with D2O). IR: ν 1639, 11656, 3190, 3288, 3436 cm-1
Anal. (C20H20N4O6S (444.46)) C, H, N, S.
.
N-(3-Amino-3-oxopropyl)-5-chloro-3-[(3,5-dimethylphenyl)-
sulfonyl]-4-fluoro-1H-indole-2-carboxamide (22). 22 was syn-
thesized as for 19 but using 62. Yield 94%, mp 240-242 °C (from
1
ethanol). H NMR (DMSO-d6): δ 2.32 (m, 6H), 2.43 (t, J ) 7.3
Hz, 2H), 3.49 (t, J ) 7.1 Hz, 2H), 6.88 (broad s, 1H, disappeared
on treatment with D2O), 7.25 (s, 1H), 7.31-7.39 (m, 3H), 7.65 (s,
2H), 9.05 (broad s, 1H, disappeared on treatment with D2O), 13.13
(broad s, 1H, disappeared on treatment with D2O). IR: ν 1649, 1704,
3267, 3496 cm-1. Anal. (C20H19ClFN3O4S (451.90)) C, H, Cl, F,
N, S.
N-(4-Amino-4-oxobutan-3-yl)-5-chloro-3-[(3,5-dimethylphe-
nyl)sulfonyl]-1H-indole-2-carboxamide (23). 23 was synthesized
as for 19 but starting from 63. Yield 43%, mp 237-240 °C (from
N-(4-Amino-4-oxobutan-2-yl)-5-chloro-3-[(3,5-dimethylphe-
nyl)sulfonyl]-4-fluoro-1H-indole-2-carboxamide (26). 26 was
synthesized as for 19 but from 66. Yield 22%, mp 217-219 °C
(from ethanol). 1H NMR (DMSO-d6): δ 1.19 (d, J ) 6.7 Hz, 3H),
2.17-2.20 (m, 1H), 2.31 (s, 6H), 2.44-2.49 (m, 1H), 4.29-4.37
(m, 1H), 6.89 (broad s, 1H, disappeared on treatment with D2O),
7.24 (s, 1H), 7.31-7.38 (m, 2H), 7.43 (broad s, 1H, disappeared
on treatment with D2O), 7.66 (s, 2H), 8.96 (broad s, 1H, disappeared
on treatment with D2O), 13.22 (broad s, 1H, disappeared on
treatment with D2O). IR: ν 1625, 1691, 3194 cm-1. Anal.
(C21H21ClFN3O4S (465.93)) C, H, Cl, F, N, S.
1
ethanol). H NMR (DMSO-d6): δ 1.26 (d, J ) 6.6 Hz, 3H), 2.32
(s, 6H), 2.44 (dd, J ) 15.7 and 7.9 Hz, 1H), 2.67 (dd, J ) 15.8
and 5.0 Hz, 1H), 4.33-4.40 (m, 1H), 7.26 (s, 1H), 7.31 (broad s,
1H disappeared on treatment with D2O), 7.34 (d, J ) 8.7 Hz, 1H),
7.51 (broad s, 1H, disappeared on treatment with D2O), 7.54 (d, J
) 8.6 Hz, 1H), 7.64 (s, 2H), 7.95 (s, 1H), 9.02 (broad s, 1H,
disappeared with treatment with D2O), 12.72 ppm (broad s, 1H,
disappeared on treatment with D2O). IR: ν 1647, 1711, 3204, cm-1
Anal. (C21H22ClN3O4S (447.94)) C, H, N, Cl, S.
.
N-(4-Amino-4-oxobutan-3-yl)-5-bromo-3-(3,5-dimethylphe-
nylsulfonyl)-1H-indole-2-carboxamide (24). 24 was synthesized
as for 19 but using 64. Yield 8%, mp 225-228 °C (from ethanol).
1H NMR (DMSO-d6): δ 1.22 (d, J ) 6.6 Hz, 3H), 2.29-2.32 (m,
7H), 2.45-2.49 (m, 1H), 4.27-4.32 (m, 1H), 6.90 (broad s, 1H,
disappeared on treatment with D2O), 7.26 (s, 1H), 7.41-7.46 (m,
2H), 7.48 (d, J ) 8.7 Hz, 1H), 7.62 (s, 2H), 8.08 (d, J ) 1.5 Hz,
1H), 8.9 (broad s, 1H, disappeared on treatment with D2O), 13.00
5-Chloro-3-[(3,5-dimethylphenyl)sulfonyl]-N-(2-sulfamoylethyl)-
1H-indole-2-carboxamide (27). 27 was synthesized as 59 using
2-aminoethanesulfonamide hydrochloride.20 Yield 21%, mp 245-247
°C (from ethanol). 1H NMR (DMSO-d6): δ 2.32 (s, 6H), 3.27-3.35
(m, 2H), 3.72-3.77 (m, 2H), 7.05 (broad s, 2H, disappeared on
treatment with D2O), 7.26 (s, 1H), 7.35 (dd, J ) 8.8 and 2.1 Hz,
1H), 7.55 (d, J ) 8.8 Hz, 1H), 7.67 (s, 2H), 7.92 (d, J ) 2.1 Hz,
1H), 9.19 (broad s, 1H, disappeared with treatment with D2O), 13.05

1930 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7
Piscitelli et al.
ppm (broad s, 1H, disappeared on treatment with D2O). IR: ν 1641,
3228 cm-1. Anal. (C19H20ClN3O5S2 (469.96)) C, H, N, Cl, S.
5-Bromo-3-[(3,5-dimethylphenyl)sulfonyl]-N-(2-sulfamoylethyl)-
1H-indole-2-carboxamide (28). 28 was synthesized as for 59 but
using 48 and 2-aminoethanesulfonamide hydrochloride.20 Yield
3H), 2.31 (s, 6H), 3.13 (dd, J ) 13.5 and 9.0 Hz, 1H), 3.45 (dd, J
) 13.5 and 3.6 Hz, 1H), 4.43-4.50 (m, 1H), 7.01 (broad s, 2H,
disappeared on treatment with D2O), 7.25 (s, 1H), 7.31-7.41 (m,
2H), 7.65 (s, 2H), 9.16 (broad s, 1H, disappeared with treatment
with D2O), 13.19 ppm (broad s, 1H, disappeared on treatment with
D2O). IR: ν 1629, 3293 cm-1. Anal. (C20H21ClFN3O5S2 (501.98))
C, H, N, Cl, F, S.
Ethyl 2-[N-[5-Chloro-3-[(3,5-dimethylphenyl)sulfonyl]-1H-
indole-2-carboxamido]]acetate (51). 51 was prepared as previously
reported.11
1
32%, mp 260 °C (from ethanol). H NMR (DMSO-d6): δ 2.32 (s,
6H), 3.31 (t, J ) 7.5 Hz, 2H), 3.73 (t, J ) 6.8 Hz, 2H), 7.06 (broad
s, 2H, disappeared on treatment with D2O), 7.26 (s, 1H), 7.45 (dd,
J ) 8.7 and 1.7 Hz, 1H), 7.50 (d, J ) 8.7 Hz, 1H), 7.67 (s, 2H),
8.06 (d, J ) 1.7 Hz, 1H), 9.19 (broad s, 1H, disappeared with
treatment with D2O), 13.08 ppm (broad s, 1H, disappeared on
Ethyl 2-[N-[5-Bromo-3-[(3,5-dimethylphenyl)sulfonyl]-1H-in-
dole-2-carboxamido]]acetate (52). 52 was synthesized as for 59
but using 48 and ethyl glycine hydrochloride. Yield 48%, mp
treatment with D2O). IR:
(C19H20BrN3O5S2 (514.41)) C, H, N, Br, S.
ν
1646, 3226 cm-1
.
Anal.
1
3-[(3,5-Dimethylphenyl)sulfonyl]-5-nitro-N-(2-sulfamoylethyl)-
1H-indole-2-carboxamide (29). 29 was synthesized as for 59 but
using 49 and 2-aminoethanesulfonamide hydrochloride.20 Yield
14%, mp 265-268 °C (from ethanol). 1H NMR (DMSO-d6): δ 1.06
(t, J ) 7.0 Hz, 2H), 2.32 (s, 6H), 3.72-3.76 (m, 2H), 7.06 (broad
s, 2H, disappeared on treatment with D2O), 7.28 (s, 1H), 7.68 (s,
2H), 7.73 (d, J ) 9.1 Hz, 1H), 8.19 (dd, J ) 9.1 and 2.2 Hz, 1H),
8.82 (d, J ) 1.8 Hz, 1H), 9.24 (broad s, 1H, disappeared on
treatment with D2O), 13.48 ppm (broad s, 1H, disappeared on
treatment with D2O). IR: ν 3205, 1637 cm-1. Anal. (C19H20N3O5S2
(514.41)) C, H, N, S.
5-Chloro-3-[(3,5-dimethylphenyl)sulfonyl]-4-fluoro-N-(2-sul-
famoylethyl)-1H-indole-2-carboxamide (30). 30 was synthesized
as for 59 but using 50 and 2-aminoethanesulfonamide hydrochlo-
ride.20 Yield 28%, mp 238-240 °C (from ethanol). 1H NMR
(DMSO-d6): δ 2.32 (s, 6H), 3.30 (t, J ) 7.8 Hz, 2H), 3.69 (t, J )
6.8 Hz, 2H), 7.03 (broad s, 2H, disappeared on treatment with D2O),
7.26 (s, 1H), 7.33-7.41 (m, 2H), 7.65 (s, 2H), 9.25 (broad s, 1H,
disappeared with treatment with D2O), 13.22 ppm (broad s, 1H,
disappeared on treatment with D2O). IR: ν 1646, 3242 cm-1. Anal.
(C19H19ClFN3O5S2 (487.95)) C, H, N, Cl, F, S.
199-201 °C (from ethanol). H NMR (DMSO-d6): δ 1.25 (t, J )
7.1 Hz, 3H), 2.31 (s, 6H), 4.17-4.26 (m, 4H), 7.27 (s, 1H),
7.47-7.53 (m, 2H), 7.70 (s, 2H), 8.18 (d, J ) 1.7 Hz, 1H), 9.48
(broad s, 1H, disappeared on treatment with D2O), 13.13 (broad s,
1H, disappeared on treatment with D2O). IR: ν 1639, 1732, 3213
cm-1. Anal. (C21H21BrN2O5S (493.37)) C, H, Br, N, S.
Ethyl 2-[3-[(3,5-Dimethylphenyl)sulfonyl]-5-nitro-1H-indole-
2-carboxamido]acetate (53). 53 was synthesized as for 59 but using
49 and ethyl glycine hydrochloride. Yield 48%, mp 224-226 °C
1
(from ethanol). H NMR (DMSO-d6): δ 1.24 (t, J ) 7.1 Hz, 3H),
2.29 (s, 6H), 4.21-4.23 (m, 4H), 7.27 (s, 1H), 7.69-7.70 (m, 3H),
8.18 (dd, J ) 9.1 and 2.3 Hz, 1H), 8.91 (d, J ) 2.2 Hz, 1H), 9.43
(broad s, 1H, disappeared on treatment with D2O), 13.88 ppm (broad
s, 1H, disappeared on treatment with D2O). IR (Nujol): ν 1647,
1749, 3215 cm-1. Anal. (C21H21N3O7S (459.47)) C, H, N, S.
Ethyl 2-[5-Chloro-3-[(3,5-dimethylphenyl)sulfonyl]-4-fluoro-
1H-indole-2-carboxamido)acetate (54). 54 was synthesized as for
59 but using 50 and ethyl glycine hydrochloride. Yield 86%, mp
1
221-223 °C (from ethanol). H NMR (DMSO-d6): δ 1.23 (t, J )
7.1 Hz, 3H), 2.30 (m, 6H), 4.12-4.20 (m, 4H), 7.24 (s, 1H),
7.33-7.40 (m, 2H), 7.62 (s, 2H), 9.51 (broad s, 1H, disappeared
on treatment with D2O), 13.24 (broad s, 1H, disappeared on
treatment with D2O). IR: ν 1638, 1745, 3212 cm-1. Anal.
(C21H20ClFN2O5S (466.91)) C, H, Cl, F, N, S.
5-Chloro-3-[(3,5-dimethylphenyl)sulfonyl]-N-(1-sulfamoylpro-
pan-2-yl)-1H-indole-2-carboxamide (31). 31 was synthesized as
for 59 but using 67. Yield 12%, mp 212-214 °C (from ethanol).
1H NMR (DMSO-d6): δ 1.42 (d, J ) 6.6 Hz, 3H), 2.32 (s, 6H),
3.19 (dd, J ) 13.9 and 8.3 Hz, 1H), 3.42 (dd, J ) 13.8 and 4.4 Hz,
1H), 4.48-4.51 (m, 1H), 7.02 (broad s, 2H, disappeared on
treatment with D2O), 7.26 (s, 1H), 7.34 (dd, J ) 8.6 and 1.8 Hz,
1H), 7.54 (d, J ) 8.8 Hz, 1H), 7.66 (s, 2H), 7.91 (d, J ) 1.8 Hz,
1H), 9.09 (broad s, 1H, disappeared with treatment with D2O), 12.98
ppm (broad s, 1H, disappeared on treatment with D2O). IR: ν 1643,
3255 cm-1. Anal. (C20H22ClN3O5S2 (483.99)) C, H, N, Cl, S.
5-Bromo-3-[(3,5-dimethylphenyl)sulfonyl]-N-(1-sulfamoylpro-
pan-3-yl)-1H-indole-2-carboxamide (32). 32 was synthesized as
for 59 but using 48 and 67. Yield 8%, mp 155-159 °C (from
Ethyl 2-[N-[5-Chloro-3-[(3,5-dimethylphenyl)sulfonyl]-1H-
indole-2-carboxamido]]propanoate (55). 55 was prepared as
previously reported.11
Ethyl 2-[N-[5-Bromo-3-[(3,5-dimethylphenyl)sulfonyl]-1H-in-
dole-2-carboxamido]]propanoate (56). 56 was synthesized as for
59 but using 48 and ethyl alanine hydrochloride. Yield 85%, mp
211-214 °C (from ethanol).1H NMR (DMSO-d6): δ 1.47 (d, J )
7.2 Hz, 3H), 2.32 (s, 6H), 3.73 (s, 3H), 4.63 (q, J ) 7.1 Hz, 1H),
7.27 (s, 1H), 7.48 (dd, J ) 8.8 and 1.8 Hz, 1H), 7.52 (d, J ) 8.7
Hz, 1H), 7.67 (s, 2H), 8.16 (d, J ) 2.9 Hz, 1H), 9.49 (broad s, 1H,
disappeared on treatment with D2O), 13.11 (broad s, 1H, disap-
peared on treatment with D2O). IR: ν 1644, 1752, 3216 cm-1. Anal.
(C21H21BrN2O5S (493.37)) C, H, Br, N, S.
1
ethanol). H NMR (DMSO-d6): δ 1.41 (d, J ) 6.7 Hz, 3H), 2.32
(s, 6H), 3.17-3.23 (m, 1H), 3.38-3.43 (m, 1H), 4.46-4.53 (m,
1H), 7.03 (broad s, 2H, disappeared on treatment with D2O), 7.27
(s, 1H), 7.46 (dd, J ) 8.7 and 1.8 Hz, 1H), 7.50 (d, J ) 8.7 Hz,
1H), 7.66 (s, 2H), 8.06 (d, J ) 1.8 Hz, 1H), 9.11 (broad s, 1H,
disappeared with treatment with D2O), 13.00 ppm (broad s, 1H,
disappeared on treatment with D2O). IR: ν 1637, 3257 cm-1. Anal.
(C20H22BrN3O5S2 (528.44)) C, H, N, Br, S.
Ethyl 2-[3-[(3,5-Dimethylphenyl)sulfonyl]-5-nitro-1H-indole-
2-carboxamido)propanoate (57). 57 was synthesized as for 59
but using 49and ethyl alanine hydrochloride. Yield 99%, mp
1
232-235 °C (from ethanol). H NMR (DMSO-d6): δ 1.23 (t, J
) 7.1 Hz, 3H), 1.45 (d, J ) 7.2 Hz, 3H), 2.29 (s, 6H), 4.18 (q,
J ) 7.1 Hz, 2H), 4.56-4.60 (m, 1H), 7.27 (s, 1H), 7.66 (s, 2H),
7.70 (d, J ) 9.1 Hz, 1H), 8.18 (dd, J ) 9.1 and 2.3 Hz, 1H),
8.88 (s, 1H), 9.45 (broad s, 1H, disappeared on treatment with
D2O), 13.82 ppm (broad s, 1H, disappeared on treatment with
D2O). IR (Nujol): ν 1651, 1741, 3194 cm-1. Anal. (C22H23N3O7S
(473.50)) C, H, N, S.
3-[(3,5-Dimethylphenyl)sulfonyl]-5-nitro-N-(1-sulfamoylpro-
pan-2-yl)-1H-indole-2-carboxamide (33). 33 was synthesized as
for 59 but using 49 and 67. Yield 21%, mp 226-229 °C (from
1
ethanol). H NMR (DMSO-d6): δ 1.42 (d, J ) 6.7 Hz, 3H), 2.32
(s, 6H), 3.16-3.22 (m, 1H), 3.37-3.42 (m, 1H), 4.47-4.52 (m,
1H), 7.05 (broad s, 2H, disappeared on treatment with D2O), 7.29
(s, 1H), 7.67 (s, 2H), 7.73 (d, J ) 9.0 Hz, 1H), 8.19 (dd, J ) 9.0
and 2.2 Hz, 1H), 8.81 (d, J ) 1.9 Hz, 1H), 9.15 (broad s, 1H,
disappeared with treatment with D2O), 13.44 ppm (broad s, 1H,
disappeared on treatment with D2O). IR: ν 1655, 3239 cm-1. Anal.
(C20H22N4O7S2 (494.54) C, H, N, S.
Ethyl 2-[5-Chloro-3-[(3,5-dimethylphenyl)sulfonyl]-4-fluoro-
1H-indole-2-carboxamido)propanoate (58). 58 was synthesized
as for 59 but using 50 and ethyl alanine hydrochloride. Yield 90%,
1
mp 198-200 °C (from ethanol). H NMR (DMSO-d6): δ 1.25 (t,
J ) 7.1 Hz, 3H), 1.45 (d, J ) 7.2 Hz, 3H), 2.33 (m, 6H), 4.18 (q,
J ) 7.0 Hz, 2H), 4.56 (q, J ) 7.3 Hz, 1H), 7.26 (s, 1H), 7.35-7.43
(m, 2H), 7.68 (s, 2H), 9.51 (broad s, 1H, disappeared on treatment
with D2O), 13.31 (broad s, 1H, disappeared on treatment with D2O).
IR: ν 1643, 1731, 3222 cm-1. Anal. (C22H22ClFN2O5S (480.94))
C, H, Cl, F, N, S.
5-Chloro-3-[(3,5-dimethylphenyl)sulfonyl]-4-fluoro-N-(1-sul-
famoylpropan-2-yl)-1H-indole-2-carboxamide (34). 34 was syn-
thesized as for 59 but using 50 and 67. Yield 20%, mp 193-197
1
°C (from ethanol). H NMR (DMSO-d6): δ 1.40 (d, J ) 6.5 Hz,

Indolylarylsulfones
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7 1931
Methyl 3-[N-[5-Bromo-3-[(3,5-dimethylphenyl)sulfonyl]-1H-
indole-2-carboxamido]]propanoate (60). 60was synthesized as for
59 but using 48 and methyl ꢀ-alanine hydrochloride. Yield 95%,
disappeared on treatment with D2O). IR: ν 1643, 1731, 3218 cm-1
.
Anal. (C23H24ClFN2O5S (494.96)) C, H, Cl, F, N, S.
Ethyl 5-Chloro-3-[(3,5-dimethylphenyl)thio]-1H-indole-2-car-
1
boxylate (39). 39 was prepared as previously reported.10
mp 208-211 °C (from ethanol). H NMR (DMSO-d6): δ 2.31 (s,
6H), 2.68 (t, J ) 6.7 Hz, 2H), 3.58-3.63 (m, 5H), 7.25 (s, 1H),
7.44-7.50 (m, 2H), 7.62 (s, 2H), 8.08 (s, 1H), 9.12 (broad s, 1H,
disappeared on treatment with D2O), 13.03 (broad s, 1H, disap-
peared on treatment with D2O). IR: ν 1644, 1736, 3291 cm-1. Anal.
(C21H21BrN2O5S (493.37)) C, H, Br, N, S.
Methyl 3-[3-[(3,5-Dimethylphenyl)sulfonyl]-5-nitro-1H-indole-
2-carboxamido)propanoate (61). 61 was synthesized as for 59
but using 49 methyl ꢀ-alanine hydrochloride. Yield 99%, mp
224-226 °C (from ethanol). 1H NMR (DMSO-d6): δ 2.30 (s, 6H),
2.66 (t, J ) 6.6 Hz, 2H), 3.56-4.62 (m, 5H), 7.26 (s, 1H), 7.62 (s,
2H), 7.68 (d, J ) 9.0 Hz, 1H), 8.16 (dd, J ) 9.0 and 2.12 Hz, 1H),
8.81 (d, J ) 2.0 Hz, 1H), 9.11 (broad s, 1H, disappeared on
treatment with D2O), 14.57 ppm (broad s, 1H, disappeared on
treatment with D2O). IR: ν 1656, 1732, 3223, 3322 cm-1. Anal.
((C21H21N3O7S (459.47)) C, H, N, S.
Methyl 3-[5-Chloro-3-[(3,5-dimethylphenyl)sulfonyl]-4-fluoro-
1H-indole-2-carboxamido)propanoate (62). 62 was synthesized
as for 59 but using 50 and methyl ꢀ-alanine hydrochloride. Yield
73%, mp 205-268 °C (from ethanol). 1H NMR (DMSO-d6): δ 2.34
(s, 6H), 2.68 (d, J ) 6.6 Hz, 2H), 3.57-3.63 (m, 5H), 7.27 (s,
1H), 7.33-7.41 (m, 2H), 7.66 (s, 2H), 9.18 (broad s, 1H,
disappeared on treatment with D2O), 13.24 (broad s, 1H, disap-
peared on treatment with D2O). IR: ν 1639, 1734, 3221 cm-1. Anal.
(C21H20ClFN2O5S (466.91)) C, H, Cl, F, N, S.
Ethyl 5-Bromo-3-[(3,5-dimethylphenyl)thio]-1H-indole-2-car-
boxylate (40). 40 was prepared as previously reported.10
Ethyl 3-[(3,5-Dimethylphenyl)thio]-5-nitro-1H-indole-2-car-
boxylate (41). Boron trifluoride diethyletherate (0.14 g, 0.12 mL,
0.001 mol) was added to a mixture of ethyl 5-nitro-1H-indole-2-
carboxylate (0.72 g, 0.0031 mol) and 1-(3,5-dimethylphenylthi-
o)pyrrolidine-2,5-dione (0.77 g, 0.0033 mol) in anhydrous dichlo-
romethane (20 mL) while cooling over an ice bath. After 2 h at
room temperature, boron trifluoride diethyletherate (0.28 g, 0.24
mL, 0.002 mol) was added and the mixture was refluxed for 2 h.
After the mixture was cooled, dichloromethane was added and the
organic layer was washed with brine, dried, and evaporated. The
residue was purified by silica gel column chromatography
(dichloromethane-petroleum ether 1:1 as eluent) to give 41. Yield
53%, mp 212-213 °C (from ethanol). 1H NMR (DMSO-d6): δ 1.29
(t, J ) 7.1 Hz, 3H), 2.16 (s, 6H), 4.36 (q, J ) 7.1 Hz, 2H), 6.82
(s, 3H), 7.69 (d, J ) 9.0 Hz, 1H), 8.17 (dd, J ) 9.0 and 2.1 Hz,
1H), 8.25 (d, J ) 2.1 Hz, 1H), 12.99 ppm (broad s, 1H, disappeared
on treatment with D2O). IR (Nujol): ν 1660, 3270 cm-1. Anal.
(C19H18N2O4S (370.42)) C, H, N, S.
Ethyl 5-Chloro-3-[(3,5-dimethylphenyl)thio]-4-fluoro-1H-in-
dole-2-carboxylate (42). 42 was prepared as previously reported.16
Ethyl 5-Chloro-3-[(3,5-dimethylphenyl)sulfonyl]-1H-indole-
2-carboxylate (43). 43 was prepared as previously reported.10
Ethyl 5-Bromo-3-[(3,5-dimethylphenyl)sulfonyl]-1H-indole-
2-carboxylate (44). 44 was prepared as previously reported.10
Ethyl 3-[(3,5-Dimethylphenyl)sulfonyl]-5-nitro-1H-indole-2-
carboxylate (45). 3-Chloroperoxybenzoic acid (0.69 g, 0.004 mol)
was added to an ice-cooled solution of 41 (0.50 g, 0.001 35 mol)
in chloroform (22 mL). Reaction mixture was stirred at room
temperature for 2 h. Water was added, and the mixture was extracted
with ethyl acetate. The organic layer was washed with brine, dried,
evaporated. The residue was purified by alumina chromatography
(ethyl acetate as eluent) to give 45. Yield 53%, mp 255-256 °C
Ethyl 3-[N-[5-Chloro-3-[(3,5-dimethylphenyl)sulfonyl]-1H-
indole-2- carboxamido]butanoate (63). 63 was synthesized as for
59 but using ethyl 3-aminobutanoate. Yield 86%, mp 179-181 °C
1
(from ethanol). H NMR (DMSO-d6): δ 1.19 (t, J ) 7.1 Hz, 3H),
1.28 (d, J ) 6.7 Hz, 3H), 2.32 (s, 6H), 2.54 (dd, J ) 15.7 and 7.4
Hz, 1H), 2.67 (dd, J ) 15.6 and 6.0 Hz, 1H), 4.10 (q, J ) 7.1 Hz,
2H), 4.36-4.42 (m, 1H), 7.27 (s, 1H), 7.35 (dd, J ) 8.8 and 2.0
Hz, 1H), 7.54 (d, J ) 8.7 Hz, 1H), 7.64 (s, 2H), 7.94 (d, J ) 1.4
Hz, 1H), 9.03 (broad s, 1H, disappeared with treatment with D2O),
13.00 ppm (broad s, 1H, disappeared on treatment with D2O). IR:
ν 1643, 1732, 3212, 3285 cm-1. Anal. (C23H25ClN2O5S (476.97))
C, H, N, Cl, S.
1
(from ethanol). H NMR (DMSO-d6): δ 1.34 (t, J ) 7.1 Hz, 3H),
2.37 (s, 6H), 4.43 (q, J ) 7.1 Hz, 2H), 7.34 (s, 1H), 7.72 (s, 2H),
7.83 (d, J ) 9.2 Hz, 1H), 8.29 (dd, J ) 9.2 and 1.9 Hz, 1H), 9.21
(d, J ) 1.9 Hz, 1H), 13.72 ppm (broad s, 1H, disappeared on
treatment with D2O, 1H). IR (Nujol): ν 1680, 3375 cm-1. Anal.
(C19H18N2O6S (402.42)) C, H, N, S.
Ethyl 3-[N-[5-Bromo-3-[(3,5-dimethylphenyl)sulfonyl]-1H-in-
dole-2-carboxamido]]butanoate (64). 64 was synthesized as for
59 but using 48 and ethyl 3-aminobutanoate. Yield 99%, mp
1
181-185 °C (from ethanol). H NMR (DMSO-d6): δ 1.19 (t, J )
7.1 Hz, 3H), 1.28 (d, J ) 6.6 Hz, 3H), 2.30 (s, 6H), 2.49-2.53 (m,
1H), 2.69-2.74 (m, 1H), 4.01 (q, J ) 7.1 Hz, 2H), 4.36-4.41 (m,
1H), 7.28 (s, 1H), 7.46 (dd, J ) 8.8 and 1.8 Hz, 1H), 7.49 (d, J )
8.7 Hz, 1H), 7.64 (s, 2H), 8.09 (d, J ) 1.7 Hz, 1H), 9.02 (broad s,
1H, disappeared on treatment with D2O), 13.02 ppm (s, 1H,
Ethyl 5-Chloro-3-[(3,5-dimethylphenyl)sulfonyl]-4-fluoro-1H-
indole-2-carboxylate (46). 46 was prepared as previously re-
ported.16
5-Chloro-3-[(3,5-dimethylphenyl)sulfonyl]-1H-indole-2-car-
boxylic Acid (47). 47 was prepared as previously reported.11
5-Bromo-3-[(3,5-dimethylphenyl)sulfonyl]-1H-indole-2-car-
boxylic Acid (48). Lithium hydroxide monohydrate (1.79 g, 0.0428
mol) was added to a mixture of 4410 (6.07 g, 0.0139 mol) in
tetrahydrofuran (16 mL) and water (16 mL). After the mixture was
stirred at room temperature for 5 h, lithium hydroxide monohydrate
(1.79 g, 0.0428 mol) was added and the mixture was maintained at
room temperature for 3 days. Water and 1 N HCl were added (pH
∼ 2), and the mixture was extracted with ethyl acetate. The organic
layer was washed with brine, dried, filtered, and evaporated. The
crude product was crystallized from ethanol to give 48. Yield 96%,
disappeared on treatment with D2O). IR: ν 1643, 1730, 3193 cm-1
.
Anal. (C23H25BrN2O5S (521.42)) C, H, Br, N, S.
Ethyl 3-[3-[(3,5-Dimethylphenyl)sulfonyl]-5-nitro-1H-indole-
2-carboxamido)butanoate (65). 65 was synthesized as for 59 but
using 49 and ethyl 3-aminobutanoate. Yield 82%, mp 278-279
1
°C (from ethanol). H NMR (DMSO-d6): δ 1.20 (t, J ) 7.5 Hz,
3H), 1.29 (d, J ) 6.6 Hz, 3H), 2.32 (s, 6H), 2.53-2.57 (m, 1H),
2.68-2.76 (m, 1H), 4.12 (q, J ) 7.1 Hz, 2H), 4.36-4.41 (m, 1H),
7.29 (s, 1H), 7.65 (s, 2H), 7.72 (d, J ) 9.1 Hz, 1H), 8.18 (dd, J )
8.3 and 2.3 Hz, 1H), 8.85 (d, J ) 2.5 Hz, 1H), 9.05 (broad s, 1H,
disappeared on treatment with D2O), 13.46 ppm (s, 1H, disappeared
on treatment with D2O). IR: ν 1644, 1721, 3178 cm-1. Anal.
(C23H25N3O7S (487.53)). C, H, N, S.
1
mp 280-284 °C (from ethanol). H NMR (DMSO-d6): δ 2.30 (s,
6H), 7.21 (s, 1H), 7.46-7.53 (m, 2H), 7.70 (s, 2H), 8.37 (s, 1H),
13.06 (broad s, 1H, disappeared on treatment with D2O), 14.02
(broad s, 1H, disappeared on treatment with D2O). IR: ν 1708, 3169,
3386 cm-1. Anal. (C17H14BrNO4S (408.27)) C, H, Br, N, S.
3-[(3,5-Dimethylphenyl)sulfonyl]-5-nitro-1H-indole-2-carbo-
xylic Acid (49). 49 was synthesized as for 48 but using 45. Yield
83%, mp 266-275 °C (from ethanol). 1H NMR (DMSO-d6): δ 2.27
(s, 6H), 7.22 (s, 1H), 7.64 (s, 2H), 7.69 (d, J ) 9.0 Hz, 1H), 8.18
(dd, J ) 9.0 and 2.2 Hz, 1H), 9.11 (d, J ) 2.17 Hz, 1H), 13.9
(broad s, 1H, disappeared on treatment with D2O), 14.15 ppm (broad
Ethyl 3-[5-Chloro-3-[(3,5-dimethylphenyl)sulfonyl]-4-fluoro-
1H-indole-2-carboxamido)butanoate (66). 66 was synthesized as
for 59 but using 50 and ethyl 3-aminobutanoate. Yield 88%, mp
1
172-174 °C (from ethanol). H NMR (DMSO-d6): δ 1.19 (t, J )
7.1 Hz, 3H), 1.24 (d, J ) 6.6 Hz, 3H), 2.31 (s, 6H), 2.41-2.47 (m,
1H), 2.71-2.77 (m, 1H), 4.09 (q, J ) 7.1 Hz, 2H), 4.31-4.39 (m,
1H), 7.25 (s, 1H), 7.31-7.39 (m, 2H), 7.64 (s, 2H), 9.05 (broad s,
1H, disappeared on treatment with D2O), 13.16 ppm (s, 1H,

1932 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7
Piscitelli et al.
s, 1H, disappeared on treatment with D2O). IR (Nujol): ν 1608,
the center of the 2zd131 cocrystallized inhibitor. Also in this case
we used all default settings. The Y181I mutation was obtained by
mutating the specific residue in the 1jkh29 crystal using the rotamer
explorer tool in MOE and using the lowest energy conformation
obtained. The rmsd values between the cocrystallized and docked
conformation were calculated using the MOE SVL script.40 The
images in the manuscript were created with Zodiac39 and PyMOL.41
Antiviral Activity Assays. The compounds were evaluated
against the following viruses: herpes simplex virus type 1 (HSV-
1) strain KOS, thymidine kinase-deficient (TK-) HSV-1 KOS strain
resistant to ACV (ACVr), herpes simplex virus type 2 (HSV-2)
strain, vaccinia virus Lederle strain, respiratory syncytial virus
(RSV) strain Long, vesicular stomatitis virus (VSV), Coxsackie
B4, parainfluenza 3 reovirus-1, sindbis, reovirus-1, Punta Toro,
human immunodeficiency virus type 1 strain IIIB, and human
immunodeficiency virus type 2 strain ROD. The antiviral, other
than anti-HIV, assays were based on inhibition of virus-induced
cytopathicity in human embryonic lung (HEL) fibroblasts, African
green monkey kidney cells (Vero), and human cervix carcinoma
cells (HeLa). Briefly, confluent cell cultures in microtiter 96-well
plates were inoculated with 100 CCID50 of virus (1 CCID50 being
the virus dose to infect 50% of the cell cultures). After a 1-2 h
adsorption period, residual virus was removed and the cell cultures
were incubated in the presence of varying concentrations of the
test compounds. Viral cytopathicity was recorded as soon as it
reached completion in the control virus-infected cell cultures that
were not treated with the test compounds. Antiviral activity was
expressed as the EC50 or concentration required to reduce virus-
induced cytopathogenicity by 50%.
1730, 3191 cm-1. Anal. (C17H14N2O6S (402.43)) C, H, N, S.
5-Chloro-3-[(3,5-dimethylphenyl)sulfonyl]-4-fluoro-1H-indole-
2-carboxylic Acid (50). 50 was synthesized as for 48 but starting
from 46.16 Yield 99%, mp 255-257 °C (from ethanol). H NMR
1
(DMSO-d6): δ 2.35 (s, 6H), 7.30 (s, 1H), 7.37-7.46 (m, 2H), 7.72
(s, 2H), 13.41 (broad s, 1H, disappeared on treatment with D2O),
13.90 ppm (broad s, 1H, disappeared on treatment with D2O). IR
(Nujol): ν 1668, 3236 cm-1. Anal. (C17H13ClFNO4S (381.81)) C,
H, N, S.
Cbz-Ala-ψ[CH2SAc] (69). Potassium thioacetate (3.03 g, 26.5
mmol) was added in one portion to a solution of mesylate 6821
(1.51 g, 5.26 mmol) in DMF (26 mL), and stirring was maintained
at room temperature overnight. The mixture was diluted with water
(50 mL) and extracted with ethyl acetate. The pooled organic phases
were washed with water, 5% sodium hydrogen sulfate, and brine.
After removal of the solvent, the crude product was triturated with
n-hexane to give 69 as a pale-yellow solid (yield 60%). 1H NMR:
δ 1.20 (d, J ) 6.6 Hz, 3H), 2.40 (s, 3H), 3.02 (d, J ) 6.2 Hz, 2H),
3.99-4.05 (m, 1H), 5.05 (s, 2H), 6.99 ppm (broad s, 1H,
disappeared on treatment with D2O), 7.39-7.41 (m, 5H). IR: ν
1715, 3435 cm-1. Anal. (C13H17NO3S (267.34)) C, H, N, S.
Cbz-Ala-ψ[CH2SO2]-Cl (71). A solution of 69 (0.82 g, 3.06
mmol) in acetic acid (5 mL) was treated with a solution hydrogen
peroxide [30% w/w in water (3 mL)] and acetic acid (7 mL). After
the mixture was stirred overnight at room temperature, the excess
peroxide was destroyed by addition of 10% Pd/C (0.050 g). The
mixture was filtered on Celite, concentrated, and coevaporated with
toluene. To the crude sulfonic acid, 70 was suspended in anhydrous
dichloromethane (20 mL), and a solution of phosgene in toluene
(20% m/m, 2.5 mL) and dry DMF (0.4 mL) was added under
nitrogen atmosphere. After 1 h an additional 1 mL of phosgene
solution was added and the mixture was stirred for 2 h under the
same conditions. After removal of the solvent, the crude product
waspurifiedbysilicagelflashchromatography(chloroform-methanol
97:3) to give 71 as a white solid (yield 70%). 1H NMR: δ 1.50 (d,
J ) 6.7 Hz, 3H), 3.86-3.89 (m, 1H), 4.15-4.18 (m, 1H),
4.33-4.37 (m, 1H), 5.12 (s, 2H), 7.01 (broad s, 1H, disappeared
on treatment with D2O), 7.34-7.37 ppm (m, 5H). IR: ν 1719, 3439
cm-1. Anal. (C11H14ClNO4S (291.75)) C, H, N, S.
Cbz-Ala-ψ[CH2SO2]-NH2 (67). Gaseous ammonia was bubbled
for 30 min through an ice-cooled solution of 71 (0.52 g, 1.8 mmol)
in benzene (15 mL). The mixture was heated at 60 °C for 2 h.
After the mixture was cooled, the precipitate was collected, washed
with benzene, and dried to afford 72 as a white solid (yield 73%).
1H NMR: δ 1.24 (d, J ) 6.6 Hz, 3H), 3.13-3.18 (m, 2H),
4.04-4.09 (m, 1H), 5.08 (s, 2H), 6.95 (broad s, 2H, disappeared
on treatment with D2O), 7.34-7.39 ppm (m, 5H). IR: ν 1728, 3452
cm-1. Anal. (C11H16N2O4S (272.32)) C, H, N, S. The sulfonilamide
hydrochloride 67 was obtained by treating 71 with hydrogen in
methanol/water/37% HCl (10:10:1, 20 mL) in the presence of 10%
Pd/C for 3 h at room temperature. The solution was filtered on
Celite and the solvent removed under reduced pressure. The crude
salt was triturated with diethyl ether and used without further
purification in the next coupling step. Anal. (C3H11ClN2O2S
(174.65)) C, H, N, S.
Inhibition of HIV-Induced Cytopathicity in CEM Cells. The
methodology for the anti-HIV assays had been described previ-
ously.42 Briefly, human CEM cell cultures (∼3 × 105 cells/mL)
were infected with ∼100 CCID50 HIV-1(IIIB) or HIV-2 (ROD)
per milliliter 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.
Cytostatic and Cytotoxicity Assays. The cytostatic activity of
the test compounds was determined by seeding the murine leukemia
L1210 or human lymphocyte Molt4/C8 or CEM cells at 5 × 104
to 7.5 × 104 cells/200 µL in 96-well microtiter plates in the absence
or presence of serial dilutions of the test compounds. After 2 days
(L1210) or 3 days (Molt4/C8 or CEM) of incubation at 37 °C, cell
numbers were counted using a Coulter counter. The cytostatic
concentration was calculated as the CC50, or the compound
concentration required to reduce 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 (percentage of
control) as a function of the concentration of the test compounds.
The cytotoxic activity of the test compounds was determined by
administering serial dilutions of the test compounds on confluent
monolayer cultures of human cervix carcinoma HeLa or monkey
kidney Vero cells in 96-well microtiter plates. Cytotoxicity of the
test compounds was expressed as the minimum cytotoxic concen-
tration (MCC) or the compound concentration that caused a
microscopically detectable alteration of cell morphology after 3 days
of incubation.
Molecular Modeling. All molecular modeling studies were
performed on a MacPro dual 2.66 GHz Xeon running Ubuntu 8.
The RT structures were downloaded from the PDB Data Bank
(http://www.rcsb.org/). Hydrogen atoms were added to the protein,
using Molecular Operating Environment (MOE) 2007.09,29 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 FlexX28 with the MOE interface
using default settings and getting 20 poses for compound with
SurfleX37 with the Sybyl8.038 interface, also in this case using
default settings, and getting 10 poses for compound and PLANTS27
with the Zodiac39 interface. In the Zodiac GUI for PLANTS we
set the binding lattice as a sphere of 12 Å binding site radius from
Enzymatic Assay Procedures. Chemicals. [3H]dTTP (40 Ci/
mmol) was from Amersham and unlabeled dNTP from Boehringer.
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) (Phar-
macia) was mixed at weight ratios in nucleotides of 10:1 to 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
wild-type or the mutant forms of HIV-1 RT p66 were kindly
provided by Dr. S. H. Hughes (NCIsFrederick Cancer Research

Indolylarylsulfones
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7 1933
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O’Brien, J. A.; Pettibone, D. J. 2-Heterocyclic indole-3-sulfones as
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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.;
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and Development Center). Proteins were expressed in E. coli and
purified as described.43
HIV-1 RT RNA-Dependent DNA Polymerase Activity
Assay. 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 3′-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 and dried, and acid-
precipitable radioactivity was quantitated by scintillation counting.
Inhibition Assays. Reactions were performed under the condi-
tions 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) concentra-
tions was monitored 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 concentra-
tions between 0.2 Ki and 5 Ki was used.
Acknowledgment. We thank the financial support of the
Istituto Pasteur-Fondazione Cenci Bolognetti, the Italian MUR
(PRIN 2006), and the Concerted Actions of the K. U. Leuven
(GOA 05/19). A.S. was supported by a fellowship from FILAS.
Supporting Information Available: Additional cytotoxicity,
inhibitory, and antiviral data and elemental analysis results. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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