New indolylarylsulfones as highly potent and broad spectrum HIV-1 non-nucleoside reverse transcriptase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2014.04.027

New indolylarylsulfone HIV-1 NNRTIs were synthesized to evaluate unexplored substitutions of the benzyl/phenylethyl group linked at the indole-2-carboxamide. Against the NL4-3 HIV-1 WT strain, 17 out 20 compounds were superior to NVP and EFV. Several comp

Full text of DOI:10.1016/j.ejmech.2014.04.027

European Journal of Medicinal Chemistry 80 (2014) 101e111
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
New indolylarylsulfones as highly potent and broad spectrum HIV-1
non-nucleoside reverse transcriptase inhibitors
,
a
a
Valeria Famiglini a, Giuseppe La Regina a , Antonio Coluccia , Sveva Pelliccia ,
Andrea Brancale b, Giovanni Maga c, Emmanuele Crespan c, Roger Badia d,
Bonaventura Clotet d, José A. Esté d, Roberto Cirilli e, Ettore Novellino f, Romano Silvestri a
*
a Istituto Pasteur e Fondazione Cenci Bolognetti, Dipartimento di Chimica e Tecnologie del Farmaco, Sapienza Università di Roma, Piazzale Aldo Moro 5,
I-00185 Roma, Italy
b Welsh School of Pharmacy, Cardiff University, King Edward VII Avenue, Cardiff CF10 3NB, UK
c Institute of Molecular Genetics IGM-CNR, National Research Council, via Abbiategrasso 207, I-27100 Pavia, Italy
d AIDS Research Institute e IrsiCaixa, Hospitals Germans Trias i Pujol, Universitat Autonóma de Barcelona, 08916 Badalona, Spain
e Istituto Superiore di Sanità, Dipartimento del Farmaco, Viale Regina Elena 299, I-00161 Roma, Italy
f Dipartimento di Farmacia, Università di Napoli Federico II, Via Domenico Montesano 49, I-80131, Napoli, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
New indolylarylsulfone HIV-1 NNRTIs were synthesized to evaluate unexplored substitutions of the
benzyl/phenylethyl group linked at the indole-2-carboxamide. Against the NL4-3 HIV-1 WT strain, 17 out
20 compounds were superior to NVP and EFV. Several compounds inhibited the K103N HIV-1 mutant
strain at nanomolar concentration and were superior to EFV. Some derivatives were superior to EFV
against the Y181C and L100I HIV-1 mutant strains. Against the NL4-3 HIV-1 strain, the enantiomers 24
and 25 showed small differences of activity. In contrast, 24 turned out significantly more potent than 25
against the whole panel of mutant HIV-1 strains. The docking studies suggested that the difference in the
observed inhibitory activities of 24 and 25 against the K03N mutation could be due to a kinetic rather
than affinity differences.
Received 22 November 2013
Received in revised form
4 March 2014
Accepted 7 April 2014
Available online 12 April 2014
Keywords:
AIDS
HIV-1
Reverse transcriptase
Nonnucleoside inhibitor
Indolylarylsulfone
Ó 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
mortality maintaining plasma viremia below the detection level in
most patients undergoing treatment for at least six months [4].
The clinical management of human immunodeficiency virus
type 1 (HIV-1) infection and acquired immunodeficiency syndrome
(AIDS) is based on antiretroviral agents [1], while an effective HIV-1
vaccine remains elusive [2]. HIV/AIDS agents may be viewed as
falling into six drug classes: nucleoside (NRTIs) and nucleotide
reverse transcriptase inhibitors (NtRTIs), non-nucleoside reverse
transcriptase inhibitors (NNRTIs), protease inhibitors (PIs), fusion
inhibitors (FIs), entry inhibitors e CCR5 co-receptor antagonists,
and HIV integrase strand transfer inhibitors (INSTIs). These agents
may be formulated either as single drug or multi-drug combination
products [3]. The main treatment available for HIV/AIDS is highly
active antiretroviral therapy (HAART), that includes two or three
(preferably) antiretroviral drugs from different drug classes. Effec-
tive HAART regimens reduce HIV-associated morbidity and
First-generation NNRTIs Nevirapine (NVP), Delavirdine (DLV) or
Efavirenz (EFV) may lead to rapid development of drug resistance;
in particular, the K103N and Y181C are the most prevalent muta-
tions, in clinical HIV-1 isolates [5]. Etravirine (ETV) and Rilpivirine
(RPV) were approved for use in drug combination to manage
treatment-experienced HIV-1 infected people, and naïve and adult
patients, respectively [6]. Despite their effectiveness, the viral
resistance associated to NNRTI drugs and adverse effects continue
to emerge in chronic long-term treatments [7]. In fact there is a
pressing need for new antiretroviral agents.
In the past decade, we have developed indolylarylsulfone (IAS)
HIV-1 NNRTIs [8]. Introduction of two methyl groups at positions 30
and 50 of the 3-phenylsulfonyl moiety led to IAS derivatives with
broader spectrum of activity against mutant HIV-1 strains [9]. The
space surrounding the 2-carboxamide proved to tolerate a wide
variety of substituents (natural or unnatural amino acids, hydrox-
yethyl moiety, Mannich bases) remarkably enhancing potency of
IAS analogues [10e13] (Chart 1).
* Corresponding author.
E-mail addresses: giuseppe.laregina@uniroma1.it (G. La Regina), romano.
silvestri@icloud.com (R. Silvestri).
http://dx.doi.org/10.1016/j.ejmech.2014.04.027
0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.

102
V. Famiglini et al. / European Journal of Medicinal Chemistry 80 (2014) 101e111
The anti-HIV-1 activity of first as well as second generation
(27) with di-tert-butyl dicarbonate in anhydrous dichloromethane
and then with triethylamine to furnish tert-butyl N-((4-
nitrophenyl)methyl)carbamate (28). Compound 28 was reduced
to amino 29 with hydrogen at 35 PSI in the presence of Pd/C as a
catalyst at room temperature for 2 h. Compound 29 was trans-
formed into the corresponding dimethyl derivative 30 by reaction
with dimethylsulfate for 16 h in anhydrous acetone. The subse-
quent treatment of 30 with trifluoroacetic acid in anhydrous
dichloromethane provided salt 31 (Scheme 2).
NNRTIs is hampered by the emergence of NNBS RT mutations.
Recent literature reported that the introduction of an additional
aromatic moiety to the parent compound resulted in NNRTIs with
broad spectrum of activity against the mutant HIV-1 strains [14]. At
the state of the art, only ETV is able to fit well into the NNBS despite
the plasticity of mutated RTs thanks to peculiar binding mode
(horseshoe shape). From docking experiments carried out in the
K103N, L100I, Y181C and Y188L mutated RTs, we observed that IAS
NNRTIs do not adopt the aforementioned horseshoe binding
conformation, but may be considered hybrids between the two
wings and horseshoe conformation [14].
2.2. Separation and absolute configuration assignment of the
enantiomers
Therefore, we focused on the design of inhibitors that would
address this drug design strategy. SAR studies of the pendant
(third) ring at the indole-2-carboxamide was not exhaustively
explored, although two IASs bearing the benzyl or phenylethyl
group showed high antiretroviral activity with IC50 values in the
nanomolar range of concentration [15]. We observed that some
substitutions could mimic the hydroxy group or the carboxamide
function of IASs 2 or 3, respectively. On the basis of these obser-
vations, the current study focused on designing of unexplored
substitutions of the benzyl/phenylethyl group linked at the car-
boxamide nitrogen at position 2 of the indole. We describe the
synthesis and antiretroviral activity of new IAS derivatives 4e23
bearing different substituents of the benzyl/phenylethyl nucleus at
the indole-2-carboxamide (Table 1).
The direct enantioseparation of the racemate 14 was accom-
plished by HPLC on the cellulose derived coated Chiralcel OD chiral
stationary phase (CSP) using the binary mixture n-hexane-ethanol
1:1 as a mobile phase (Chart 2). The optimized analytical enantio-
selective method was easily scaled-up to a semipreparative level.
The absolute configuration assignment was carried out by a two-
step strategy. In the first step, the (S)-enantiomer 25 was synthe-
sized starting from the amine of known stereochemistry (S)-(ꢁ)-
a-
methylbenzylamine. In the second step, the stereochemical course
of reactions was monitored by enantioselective HPLC. The elution
time of (S)-25 was then compared with the enantiomeric peaks of
the racemate 14 under the same enantioselective HPLC conditions
(Chart 2). On the Chiralcel OD CSP, the (R)-enantiomer 24 was
eluted before the (S)-enantiomer 25. The circular dichroism (CD)
spectra of (R)-24 and (S)-25 are shown in Chart 3.
2. Chemistry
3. Results and discussion
2.1. Synthetic procedures
3.1. Inhibition of HIV-1 acutely infected cells
Carboxamides 4e11 and 13e23 were synthesized by coupling
reaction of 5-chloro-3-((3,5-dimethylphenyl)sulfonyl)-1H-indole-
2-carboxylic acid (26) [9,10] with the appropriate amine in the
presence of (benzotriazol-1-yloxy)tris(dimethylamino)-phospho-
nium hexafluorophosphate (BOP reagent) and triethylamine in
anhydrous DMF at 25 ꢀC for 12 h. Tiꢀn(II) chloride reduction of the
nitro derivative 11 by heating at 80 C for 3 h in ethyl acetate fur-
nished compound 12 (Scheme 1).
Of the desired structural changes, we initially synthesized new
IAS benzyl derivatives 4e15 (Table 1). Against the NL4-3 HIV-1 WT
strain, compounds 4e15 were all superior to reference drugs NVP
and EFV. Independently on nature and position of the substituent,
derivatives 4e15 inhibited the NL4-3 strain by fifty-percent (EC50
values) in the low-nanomolar (4, 9 and 11) or sub-nanomolar (5, 7,
8, 10, 12 and 14) range of concentrations (MTT method); the latter
group of inhibitors were comparable to the parent unsubstituted
benzyl derivative [15]. Introduction of a fluoro atom the N-benzyl
group provided compounds 7e9 that potently inhibited the NL4ꢁ3
HIV-1 strain (7, 8: EC50 ¼ 0.68 nM; 9: EC50 ¼ 1.1 nM). On the other
hand, introduction of 4-methoxy or 2-nitro group at the benzyl
moiety also provided sub-nanomolar inhibitors of the NL4-3 strain
(5: EC50 ¼ 0.21 nM; 10: EC50 ¼ 0.8 nM). The replacement of the
phenylethyl group with the isomeric 1-phenylethyl moiety gave 14
(EC50 ¼ 0.26 nM) that was more potent than the parent compound.
When tested for their cytotoxicity, 4e11 and 13e15 showed CC50
values >20,000 nM and selectivity indexes (SI ¼ CC50/EC50 ratio)
which were higher than those of NVP and EVF.
(4-Dimethylaminophenyl)methanamine trifluoroacetate (31)
was achieved by treatment of 4-nitrobenzylamine hydrochloride
The introduction of substituents at the phenylethyl moiety led
to NL4-3 inhibitors active in the low nanomolar range, except those
bearing the substituent at the position 4 (compare 16 and 17
(EC50 ¼ 4.0 nM) with 18, and 21 and 22 (EC50 ¼ 6.2 nM) with 23)
suggesting the unfavorable steric effects of this position. As inhib-
itor of the NL4-3 HIV-1 strain, the 2-phenylpropyl derivative 20 was
slightly more potent than the inferior homologue 14; in contrast,
the isomeric derivative 19 inhibited the IIIB HIV-1 strain at higher
concentration.
IAS derivatives 4e23 were evaluated in MT4 cells against
mutant HIV-1 strains harboring K130N, Y181C, Y188L, L100I and
Y181CeK103N single or double amino acid mutations in the RT
(Table 2). Derivatives 5, 7e10, 12, 14, 16 and 20 inhibited the mutant
Chart 1. Structures of IAS compounds 1e23.

V. Famiglini et al. / European Journal of Medicinal Chemistry 80 (2014) 101e111
103
Table 1
Structure and Anti-HIV activity of new IAS derivatives 4e23.a
Compd
R
CC50b (nM)
EC50 ꢂ SD (nM)
HIV-1 NL4-3c
SId
EC50 ꢂ SD (nM)
HIV-1 IIIBe
EC50 (nM)
HIV-2 RODf
4
5
6
7
21221 ꢂ 2077
39,877 ꢂ 13,603
>51,293
2.1 ꢂ 0.6
0.21h
10,105
ndg
nd
189,890
>6255
nd
nd
8.2 ꢂ 6.2
0.68h
nd
nd
>53,085
>78,066
3.6 ꢂ 2.2
>2000i
8
9
>53,085
>53,085
0.68h
>78,066
>48,259
4.3 ꢂ 2.3
>10000i
>10000i
1.1 ꢂ 0.5
5.3 ꢂ 0.49
10
>50,205
0.8 ꢂ 0.4
>62,757
nd
nd
11
12
13
>50,205
2.0 ꢂ 0.60
0.21h
>25,106
8969
nd
nd
nd
nd
nd
nd
6368 ꢂ 919
40,664 ꢂ 6877
12.1 ꢂ 8.1
3361
14j
15
>53,535
>51,974
0.6h
>205,903
>12,375
16 ꢂ 12
>10000i
>2000i
4.2 ꢂ 2.6
28 ꢂ 0.71
16
17
18
44,972 ꢂ 12,055
29,097 ꢂ 8145
32,368 ꢂ 9206
4.0 ꢂ 3.1
4.0 ꢂ 2.9
53.8 ꢂ 32.4
11,243
7274
602
9.4 ꢂ 2.3
11 ꢂ 0
>10,000
>10,000
>10,000
68 ꢂ 46
19j
20j
21
nd
nd
nd
91 ꢂ 9
nd
nd
nd
19,937 ꢂ 5223
>51,974
0.21h
6.2 ꢂ 3.0
94,938
8383
9.1 ꢂ 2.7
>10,000
(continued on next page)

104
V. Famiglini et al. / European Journal of Medicinal Chemistry 80 (2014) 101e111
Table 1 (continued )
Compd
R
CC50b (nM)
EC50 ꢂ SD (nM)
HIV-1 NL4-3c
SId
EC50 ꢂ SD (nM)
HIV-1 IIIBe
EC50 (nM)
HIV-2 RODf
22
23
>51,974
>51,974
6.2 ꢂ 3.4
8383
2270
7.3 ꢂ 0.71
33 ꢂ 4.2
>25,000
>50,000
22.9 ꢂ 14.8
NVP
EFV
AZT
e
e
.
>18,776
>15,839
>30,595
112.4 ꢂ 74.9
15.9 ꢂ 12.7
3.7 ꢂ 3.7
>167
>996
>8269
19.2 ꢂ 0.0
1.5 ꢂ 0.3
nd
>10,000
>10,000
nd
a
Data are mean values of two to three independent experiments each one in triplicate.
CC50: cytotoxic concentration (nM) to induce 50% death of non-infected cells, as evaluated with the MTT method in MT-4 cells.
b
c
EC50 (HIV-1 NL4-3): effective concentration (nM) to inhibit by 50% HIV-1 (NL4ꢁ3 strain) induced cell death, as evaluated with the MTT method in MT-4 cells.
d
e
SI: selectivity index calculated as CC50/EC50 ratio.
EC50 (HIV-1 IIIB): effective concentration (nM) or concentration required to protect CEM cells against the cytopathicity of HIV-1 (IIIB strain) by 50%, as monitored by giant
cell formation.
f
EC50 (HIV-2 ROD): effective concentration (nM) or concentration required to protect CEM cells against the cytopathicity of HIV-2 (ROD strain) by 50%, as monitored by
giant cell formation.
g
nd: no data.
Lowest detectable nM concentration.
Compound precipitation was detected at higher compound concentration.
Data of D,L racemic mixture.
h
i
j
K103N HIV-1 strain in the nanomolar range with EC50s ranging
from 4.2 nM (12) to 37 nM (16). Such compounds were significantly
superior to EFV. The most potent inhibitors 8 and 12 were >546-
fold and >3-fold more potent than EFV and AZT, respectively.
Against the Y181C mutant strain, five compounds, namely 5, 8 and
10e12 were superior to EFV. Against this mutant strain, the most
potent derivative 8 (EC50 ¼ 4.3 nM) went 37-fold higher than EFV
and at the same level of AZT. Compounds 10 and 12 proved to be
2.7- and 1.5-fold superior to EFV as inhibitor of the Y188L HIV-1
strain.
Several new IASs inhibited the mutant L100I HIV-1 strain at
nanomolar concentration, with the exception of compounds 18 and
19. The fluoro derivative 7e9 (EC50 z 5 nM) were uniformly active
against this HIV-1 mutant strain, and went about 4- and 10-fold
more active than NVP and EFV. IAS derivatives 4e23 proved to be
weak inhibitors of the double mutant K103NeY181C HIV-1 strain,
although derivatives 7, 10, 12 and 14 were inhibitory of this mutant
strain in the micromolar range of concentration, and 12 was the
most potent derivative with EC50 ¼ 598 nM.
[16]. Compounds 7e9, 14 and 17 potently inhibited the L100I RT
mutant, and compound 17 proved to highly effective against this
mutant at the same level of ETV. Against the Y181I RT compound 12
that was equipotent to ETV, while the other tested compounds
showed to be weak inhibitors of this HIV-1 muted RT.
Against the HIV-1 RTs, the enantiomers 24 and 25 showed IC50
values that were in good agreement with the corresponding
cellular data depicted in Table 3 (Table 5). IASs 24 and 25 were
almost equipotent against the HIV-1 WT RT. As inhibitors of the
HIV-1 K103N mutated RT 24 (IC50 ¼ 90 nM) proved to be remark-
ably more potent (104-fold) than 25. A possible explanation of
these results is discussed in the molecular modeling section. Again,
IAS 24 was superior to 25, although to a less extend, against the
HIV-1 Y181C, L100I and V106A mutated RTs.
3.3. Molecular modeling
We evaluated the binding mode of compounds 4e23 in complex
with the HIV-1 WT RT and K103N mutated RT by following our
previously reported methodology [15]. From the molecular docking
results we observed that the proposed binding mode was consis-
tent through the whole training set and also coherent with the one
previously reported for the IAS family [8e11]. For the WT RT we
highlighted some established pharmacophoric interactions: i) an
H-bond between the indole NH and the carbonyl oxygen of the
Lys101; ii) the steric interactions between the chlorine atom in the
pocket formed by Val106 and Leu234; iii) a series of hydrophobic
interactions between the 3,5-dimethylphenyl moiety in the aro-
matic cleft formed by Tyr181, Tyr188, and Trp229 residues; iv) the
hydrophobic interactions between the substituted phenyl ring and
the aliphatic linker with Val179 and Ile 180 and the side chains of
Glu138:B and Thr139:B. Consistent with the proposed binding
mode, compounds 15 and 23 were partly located out of the NNRTI
entrance pocket and exposed to the solvent. It should be noted that
the two enantiomers 24 and 25 showed a very similar binding
mode (Fig. 1).
To evaluate the influence of the asymmetric center at the 1-
phenylethyl moiety, the racemic mixture 14 was separated using
a chiral HPLC to give the pure enantiomers 24 and 25 (Table 3).
Against the NL4-3 HIV-1 strain, the enantiomers 24 and 25 showed
small differences of activity. In contrast, 24 turned out significantly
more potent than 25 against the whole panel of mutant HIV-1
strains: 30-fold vs the K103N, 40-fold vs Y181C, >189-fold vs
Y188L, and >22-fold vs K103NeY181C.
3.2. Inhibition of HIV-1 reverse transcriptase
As inhibitors of the RT of HIV-1 WT, the majority of the IAS
compounds examined were superior to NVP, with the exception of
derivatives 16 and 23 (IC50 values, Table 4). Several IASs were
comparable or superior (5, 9, 10 and 12) to EFV, and compounds 5
and 12 were in the range of concentration of ETV. Eight compounds
inhibited the mutant K103N RT HIV-1 at submicromolar concen-
tration. As inhibitors of the K103N RT compounds 9, 10 and 12 were
equipotent to ETV and remarkably more potent than NVP and EFV.
The K103N mutation often emerges in EFV-treated patients, who
show rebounding of viral load after an initial response to this drug
Interestingly, the molecular docking into the K103N mutated RT
gave apparently similar results to the corresponding ones obtained
using the WT RT. However, when the binding poses of the enan-
tiomers 24 and 25 were carefully examined, a small, yet significant

V. Famiglini et al. / European Journal of Medicinal Chemistry 80 (2014) 101e111
105
Scheme 1. Synthesis of compounds 4e23. Reagents and reaction conditions: (a) amine, BOP reagent, triethylamine, anhydrous DMF, 25 ꢀC, 12 h, 15e80%; (b) SnCl2∙2H2O, AcOEt,
80 ꢀC, 3 h, 9%.
resistance induced by the K103N mutation is related to a kinetic
effect that affects the on/off rate of the binding of the inhibitor
rather than to the breakage of binding contacts between the in-
hibitor and the RT [18]. The docking results obtained for 24 and 25
suggested that the difference in the observed biological activity for
these compounds could be due to a different binding kinetic rather
than affinity: while compound 24 was able to seal the binding
pocket, 25 left the site accessible to water, leading to a negative
effect on the binding kinetic of this inhibitor.
4. Conclusions
We designed and synthesized new indolylarylsulfones as HIV-1
NNRTIs in order to evaluate unexplored substitutions of the benzyl/
phenylethyl group linked at the carboxamide nitrogen at position 2
of the indole. Against the NL4ꢁ3 HIV-1 WT strain, 17 out 20 com-
pounds were superior to the references NVP and EFV. Several
compounds inhibited the K103N HIV-1 mutant strain at nanomolar
range of concentration and were superior to EFV, and some de-
rivatives were superior to EFV against the Y181C and L100I HIV-1
mutant strains. Against the NL4-3 HIV-1 strain, the enantiomers
24 and 25 showed small differences of activity. In contrast, 24
turned out significantly more potent than 25 against the whole
panel of mutant HIV-1 strains. The enzymatic results for 24 and 25
were in well agreement with the cellular data. The docking studies
suggested that the difference in the observed inhibitory activities of
24 and 25 could be due to a kinetic rather than affinity differences.
Compound 24 represents a robust lead compound to develop
NNRTIs with improved activity and selectivity against K103N that is
the most frequently emerging HIV-1 mutation in EFV-treated pa-
tients [16]. These results warrant further exploration of the ary-
lalkyl moiety at the indole-2-carboxamide to obtain new IAS
derivatives that have potential as novel therapeutic agents to treat
AIDS/HIV-1 infection.
Scheme 2. Synthesis of amine 31. Reagents and reaction conditions: (a) (i) Boc2O,
triethylamine, anhydrous CH2Cl2, 0 ꢀC, 10 min; (ii) 25 ꢀC, 2 h, 91%; (b) Pd/C, MeOH, Ar
stream, 35 PSI, 2 h, 100%; (c) (MeO)2SO2, K2CO3, anhydrous acetone, 25 ꢀC, 16 h, 41%;
(d) TFA, CH2Cl2, 25 ꢀC, 30 min 77%.
Chart 2. Chromatograms of the resolution of the racemate 14 and the (S)-enantiomer
purity control. Column Chiralcel OD 250 mm ꢃ 4.6 mm I.D.; detection UV at 280 nm;
n-hexane-ethanol 1:1 (v/v) as eluent; flow rate 1.0 mL minꢁ1; column temp. 25 ꢀC.
5. Experimental protocols
5.1. Chemistry
All reagents and solvents were commercially available and used
without further purification. Organic solutions were dried over
anhydrous sodium sulfate. Evaporation of the solvents was carried
out on a Büchi Rotavapor R-210 equipped with a Büchi V-850
vacuum controller and Büchi V-700 (w5 mbar) and V-710
(w2 mbar) vacuum pumps. Column chromatography was run on
glass columns packed with aluminum oxide 90 standardized
Chart 3. CD Spectra of enantiomers (R)-24 and (S)-25. Solvent ethanol; temp. 20 ꢀC.
difference was observed. The K103N mutation created a small gap
at the entrance channel of the NNRTI binding site and while the
methyl group of the (R)-enantiomer 24 pointed toward this cleft,
the corresponding group of the (S)-enantiomer 25 pointed toward
the bottom of the cleft, leaving the binding pocket more exposed to
the aqueous environment (Fig. 2). It was reported that the drug
(Merck) or silica gel (MachereyeNagel, 63e200 mm) eluting with
the indicated solvent. Aluminum oxide thin layer chromatography
(TLC) cards from Fluka (aluminum oxide precoated aluminum cards
with fluorescent indicator detectable at 254 nm) and silica gel TLC
cards from MachereyeNagel (silica gel precoated aluminum cards
with fluorescent indicator detectable at 254 nm) were used for TLC.

106
V. Famiglini et al. / European Journal of Medicinal Chemistry 80 (2014) 101e111
Table 2
analyses of tested compounds were found within 0.4% of the
theoretical values. Combustion analysis was used as a method of
establishing compound purity. Purity of tested compounds was
ꢄ95%.
Anti-HIV-1 activity of compounds 4e23 against mutant HIV-1 strains.a,b
Compd EC50 ꢂ SD (nM)/FCc
K103N
Y181C
Y188L
L100I
ndd
K103NeY181C
4
385 ꢂ 6.4
183
215 ꢂ 86
103
>21,221
>10,105
>39,177
>186,557
>51,292
>6255
>21221
>10,105
>39,177
>186,557
>51,292
>6255
5.1.1. General procedure for the preparation of derivatives 4e11 and
13e23. Example. 5-Chloro-3-((3,5-dimethylphenyl)sulfonyl)-N-(4-
methylbenzyl)-1H-indole-2-carboxamide (4)
5
21 ꢂ 4.1
21 ꢂ 8.4
nd
nd
100
100
6
103 ꢂ 113 451 ꢂ 328
A
mixture of 26 [9,10] (100 mg, 0.27 mmol), 4-
13
55
tolylmethanamine (100 mg, 0.08 mL, 0.81 mmol), BOP reagent
(120 mg, 0.27 mmol), and triethylamine (80 mg, 0.11 mL,
0.81 mmol) in anhydrous DMF (5 mL) was stirred at 25 ꢀC for 12 h.
The reaction mixture was 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:n-hexane ¼ 1:2 as
eluent) to furnish 4 (70 mg, 56%), mp 240e242 ꢀC (from ethanol).
7
7.8 ꢂ 2.3
220 ꢂ 260
ndc
5.4 ꢂ 1.2
7.9
2144 ꢂ SD
11.5
324
3153
8
4.4 ꢂ 3.3
6.5
4.3 ꢂ 5.7
6.3
nd
nd
5.0 ꢂ 0.9
13,866 ꢂ SD
20,391
7.4
9
16 ꢂ 15
160 ꢂ 57
5.9 ꢂ 0.4
5.4
50,325 ꢂ SD
14.5
145
45,750
10
11
12
13
14e
15
16
17
18
19e
20e
21
22
23
NVP
EFV
AZT
6.0 ꢂ 4.0
7.5
16 ꢂ 14
20
281 ꢂ 181 nd
1868 ꢂ 2410
2335
351
120 ꢂ 60
70 ꢂ 80
>50,206
>25,103
nd
>50,206
>25,103
598 ꢂ 321
2847
1H NMR (DMSO-d6):
2H), 7.17 (d, J ¼ 7.8 Hz, 2H), 7.24 (s, 1H), 7.32e7.35 (m, 3H), 7.53 (d,
J ¼ 8.9 Hz,1H), 7.58 (s, 2H), 7.93 (d, J ¼ 1.8 Hz,1H), 9.39 (t, J ¼ 5.3 Hz,
1H, disappeared on treatment with D2O), 13.05 ppm (br s, 1H,
d
2.26 (s, 6H), 2.29 (s, 3H), 4.53 (d, J ¼ 6.0 Hz,
60
35
4.3 ꢂ 2.1
20
11 ꢂ 9.8
52
491 ꢂ 112 nd
2338
322 ꢂ 161 1028 ꢂ 524 >40,321
nd
>40,321
>3332
27
85
>3332
n .
1651, 2920, 3219 cmꢁ1
33 ꢂ 6.4
720 ꢂ 690
nd
26 ꢂ 24
43
3267 ꢂ SD
disappeared on treatment with D2O). IR:
55
1200
5445
Anal. Calcd. for C25H23ClN2O3S: C, 64.30%; H, 4.96%, N, 6.00%; Cl,
7.59%; S, 6.87%. Found: C, 64.08%; H, 4.90%, N, 5.84%; Cl, 7.41%; S,
6.60%.
180 ꢂ 21
43
>2000f
>476
nd
nd
nd
65 ꢂ 21
>51,794
>12,332
44,972
11,243
29,097
15
37 ꢂ 28
250 ꢂ 28
24 ꢂ 4.2
6.0
9.3
63
180 ꢂ 71
45
870 ꢂ 130
218
31 ꢂ 12
5.1.2. 5-Chloro-3-((3,5-dimethylphenyl)sulfonyl)-N-(4-
methoxybenzyl)-1H-indole-2-carboxamide (5)
7.8
7274
ꢄ2000
1700 ꢂ 490 nd
190 ꢂ 64
3.5
32,368
602
It was synthesized as 4 starting from 26 and (4-methoxyphenyl)
ꢄ37
36
methanamine. Yield 37%, mp 250e254 ꢀC (from ethanol). 1H NMR
530 ꢂ 110 >1000
>1000
>11
380 ꢂ 340 >2079
5.8g
>11
4.2
nd
23
(DMSO-d6):
d
2.26 (s, 6H), 3.74 (s, 3H), 4.53 (d, J ¼ 5.7 Hz, 2H), 6.92
29 ꢂ 11
291 ꢂ 78
>19,937
>94,938
nd
>19,937
>94,938
>51,937
>8383
>51,937
>8383
>51,937
>2268
>3756
>33
(d, J ¼ 8.3 Hz, 2H), 7.24 (s, 1H), 7.32e7.37 (m, 3H), 7.52 (d, J ¼ 8.8 Hz,
1H), 7.57 (s, 2H), 7.93 (s, 1H), 9.37 (t, J ¼ 5.0 Hz, 1H, disappeared on
treatment with D2O), 13.05 ppm (br s, 1H, disappeared on treat-
138
1386
100 ꢂ 69
16
850 ꢂ 7.1
1371
20 ꢂ 15
3.2
ment with D2O). 13C NMR (DMSO-d6):
d
21.33, 43.08, 55.71, 112.06,
260 ꢂ 210 1200 ꢂ 71
nd
nd
31 ꢂ 2.8
42
194
5.0
114.43, 115.53, 119.55, 124.25, 125.29, 125.92, 127.87, 129.63, 130.77,
>50,000
2183
>3756
>33
1800 ꢂ 40
nd
133.54, 135.28, 137.65,139.56, 143.14,159.11,159.82 ppm. IR: n 1643,
79
3209 cmꢁ1. Anal. Calcd. for C25H23ClN2O4S: C, 62.17%; H, 4.80%, N,
5.80%; Cl, 7.34%; S, 6.64%. Found: C, 61.92%; H, 4.77%, N, 5.61%; Cl,
7.18%; S, 6.41%.
>3756
>33
>3756
>33
60 ꢂ 4
1.8
130 ꢂ 180 160 ꢂ 180
760 ꢂ 630 22 ꢂ 14
>317
>20
8.2
10
48
1.4
nd
16 ꢂ 12
6.0 ꢂ 3.4
33 ꢂ 18
16 ꢂ 13
5.1.3. 5-Chloro-N-(4-chlorobenzyl)-3-((3,5-dimethylphenyl)
sulfonyl)-1H-indole-2-carboxamide (6)
4.3
1.6
8.9
1.0
a
Data are mean values of two to three independent experiments each one in
It was synthesized as 4 starting from 26 and (4-chlorophenyl)
triplicate.
methanamine. Yield 74%, mp 260e265 ꢀC (from ethanol). 1H NMR
b
EC50: effective concentration (nM) to inhibit by 50% cell death induced by the
(DMSO-d6):
d
2.26 (s, 6H), 4.57 (d, J ¼ 5.7 Hz, 2H), 7.24 (s, 1H), 7.33
indicated mutant HIV-1 strain, as evaluated with the MTT method in MT-4 cells.
c
FC: fold change obtained as ratio between EC50s of the indicated drug resistant
(d, J ¼ 8.9 Hz, 1H), 7.41e7.48 (m, 4H), 7.53 (d, J ¼ 8.8 Hz, 1H), 7.59 (s,
mutant HIV-1 strain and HIV-1 WT NL4-3 strain.
2H), 7.92 (s, 1H) 9.47 (br s, 1H, disappeared on treatment with D2O),
d
nd: no data.
Data of D,L racemic mixture.
Compound precipitation was detected at higher compound concentration.
FC: fold change obtained as ratio between EC50s of the indicated drug resistant
13.07 ppm (br s, 1H, disappeared on treatment with D2O). IR:
n
e
1650, 3214 cmꢁ1. Anal. Calcd. for C24H20Cl2N2O3S: C, 59.14%; H,
4.14%, N, 5.75%; Cl, 14.55%; S, 6.58%. Found: C, 58.92%; H, 4.09%, N,
5.52%; Cl, 14.38%; S, 6.42%.
f
g
mutant HIV-1 strain and HIV-1 WT IIIB strain.
5.1.4. 5-Chloro-3-((3,5-dimethylphenyl)sulfonyl)-N-(2-
fluorobenzyl)-1H-indole-2-carboxamide (7)
Developed plates were visualized by a Spectroline ENF 260C/FE UV
apparatus. Flash chromatography was carried out on Interchim
Spot II Flash, using Merck SuperVarioFlash D26 cartridges packed
It was synthesized as 4 starting from 26 and (2-fluorophenyl)
methanamine. Yield 22%, mp 267e270 ꢀC (from ethanol). 1H NMR
with Merck Geduran 60 (40e63
m
m) silica gel. Melting points (mp)
(DMSO-d6):
d
2.28 (s, 6H), 4.63 (d, J ¼ 5.4 Hz, 2H), 7.21e7.27 (m, 3H),
were determined on a SMP1 apparatus (Stuart Scientific) and are
uncorrected. IR spectra were run on a SpectrumOne FT-ATR spec-
trophotometer (Perkin Elmer). Band position and absorption ranges
are given in cmꢁ1. Proton (1H NMR) and carbon (13C NMR) nuclear
magnetic resonance spectra were recorded on a 400 MHz FT
spectrometer (Bruker) in the indicated solvent. Chemical shifts are
7.34e7.42 (m, 2H), 7.54e7.60 (m, 4H), 7.95 (s, 1H), 9.47 (t, J ¼ 4.9 Hz,
1H, disappeared on treatment with D2O), 13.11 ppm (br s, 1H, dis-
appeared on treatment with D2O). 13C NMR (DMSO-d6):
d 21.18,
37.23, 115.44, 115.54, 115.75, 119.35, 124.07, 124.84, 124.89, 125.04,
125.23, 125.43, 127.63, 129.73, 129.86, 130.43, 130.49, 135.08, 139.37,
143.05, 158.94, 160.07 ppm. IR:
24H20ClFN2O3S: C, 61.21%; H, 4.28%, N, 5.95%; Cl, 7.53%; F, 4.03%; S,
n
1644, 3213 cmꢁ1. Anal. Calcd. for
expressed in
d
units (ppm) from tetramethylsilane. Elemental
C

V. Famiglini et al. / European Journal of Medicinal Chemistry 80 (2014) 101e111
107
Table 3
Anti-HIV-1 activity of racemate 14 and its enantiomers 24 and 25 against mutant HIV-1 strains.a,b
Compd
(Chirality)
EC50 ꢂ SD (nM)/FCc
WT
K103N
Y181C
Y188L
ndf
L100I
K103NeY181C
14d
(R,S)
24
(R)
25
0.6e
33 ꢂ 6.4
55
720 ꢂ 690
1200
26 ꢂ 24
43
3267 ꢂ 2110
5445
2.1 ꢂ 1.9
6.3 ꢂ 4.2
4.3 ꢂ 3.2
86 ꢂ 43
193 ꢂ 64
92
>36,404
>5778
nd
1670 ꢂ 1177
2.0
41
795
128 ꢂ 107
20
3469 ꢂ 1735
550
nd
>36,404
>5778
(S)
a
Data are mean values of two to three independent experiments each one in triplicate.
b
c
d
e
f
EC50: effective concentration (nM) to inhibit by 50% cell death induced by the indicated mutant HIV-1 strain, as evaluated with the MTT method in MT-4 cells.
FC: fold change obtained as ratio between EC50s of the indicated drug resistant mutant HIV-1 strain and HIV-1 WT NL4-3 strain.
Data of D,L racemic mixture.
Lowest detectable nM concentration.
nd: no data.
6.81%. Found: C, 60.92%; H, 4.22%, N, 5.77%; Cl, 7.29%; F, 3.83%; S,
6.65%.
7.32 (d, J ¼ 10. Hz, 1H), 7.52 (d, J ¼ 11.9 Hz, 1H), 7.60 (s, 2H), 7.7
(d, J ¼ 10.3 Hz, 2H), 7.9 (s, 1H), 8.21 (d, J ¼ 10.6 Hz, 2H), 9.58 (br
s, 1H, disappeared on treatment with D2O), 11.68 ppm (br s, 1H,
disappeared on treatment with D2O). IR:
n .
1647, 3225 cmꢁ1
5.1.5. 5-Chloro-3-((3,5-dimethylphenyl)sulfonyl)-N-(3-
fluorobenzyl)-1H-indole-2-carboxamide (8)
Anal. Calcd. for C24H20ClN3O5S: C, 57.89%; H, 4.05%, N, 8.44%; Cl,
7.12%; S, 6.44%. Found: C, 57.65%; H, 3.97%, N, 8.32%; Cl, 6.94%; S,
6.28%.
It was synthesized as 4 starting from 26 and (3-fluorophenyl)
methanamine. Yield 22%, mp 260e264 ꢀC (from ethanol). 1H NMR
(DMSO-d6):
d
2.28 (s, 6H), 4.62 (d, J ¼ 5.8 Hz, 2H), 7.10e7.15 (m,
1H), 7.25e7.45 (m, 5H), 7.55 (d, J ¼ 8.7 Hz, 1H), 7.63 (s, 2H),
7.94 (d, J ¼ 1.7 Hz, 1H), 9.51 (t, J ¼ 5.8 Hz, 1H, disappeared on
treatment with D2O), 13.1 ppm (br s, 1H, disappeared on treat-
5.1.9. 5-Chloro-N-(4-dimethylaminobenzyl)-3-((3,5-
dimethylphenyl)sulfonyl)-1H-indole-2-carboxamide (13)
It was synthesized as
dimethylaminophenyl)methanamine trifluoroacetate (31). Yield
77%, mp 230 ꢀC dec (from ethanol). 1H NMR (DMSO-d6):
2.25 (s,
6H), 2.87 (s, 6H), 4.45 (d, J ¼ 5.9 Hz, 2H), 6.72 (d, J ¼ 8.6 Hz, 2H), 7.25
(d, J ¼ 8.8 Hz, 3H), 7.33 (d, J ¼ 9.1 Hz, 1H), 7.51e7.55 (m, 3H), 7.94 (s,
1H), 9.3 (br s, 1H, disappeared on treatment with D2O), 13.03 (br s,
4
starting from 26 and (4-
ment with D2O). IR:
24H20ClFN2O3S: C, 61.21%; H, 4.28%, N, 5.95%; Cl, 7.53%; F, 4.03%;
n . Anal. Calcd. for
1649, 3206 cmꢁ1
C
d
S, 6.81%. Found: C, 61.03%; H, 4.25%, N, 5.82%; Cl, 7.30%; F, 3.88%;
S, 6.55%.
5.1.6. 5-Chloro-3-((3,5-dimethylphenyl)sulfonyl)-N-(4-
fluorobenzyl)-1H-indole-2-carboxamide (9)
1H, disappeared on treatment with D2O). IR:
n 1649, 3192,
3282 cmꢁ1. Anal. Calcd. for C26H26ClN3O3S: C, 62.96%; H, 5.28%, N,
8.47%; Cl, 7.15%; S, 6.46%. Found: C, 62.81%; H, 5.22%, N, 8.29%; Cl,
6.96%; S, 6.28%.
It was synthesized as 4 starting from 26 and (4-fluorophenyl)
methanamine. Yield 22%, mp 275e277 ꢀC (from ethanol). 1H NMR
(DMSO-d6):
d
2.28 (s, 6H), 4.58 (d, J ¼ 5.8 Hz, 2H), 7.19e7.25 (m, 3H),
7.35 (dd, J ¼ 2.0 and 8.7 Hz, 1H), 7.48e7.56 (m, 3H), 7.61 (s, 2H), 7.94
(d, J ¼ 2.0 Hz, 1H), 9.47 (t, J ¼ 5.7 Hz, 1H, disappeared on treatment
with D2O), 13.1 ppm (br s, 1H, disappeared on treatment with D2O).
5.1.10. 5-Chloro-3-((3,5-dimethylphenyl)sulfonyl)-N-(1-
phenylethyl)-1H-indole-2-carboxamide (14)
IR: n
1648, 3210 cmꢁ1. Anal. Calcd. for C24H20ClFN2O3S: C, 61.21%; H,
It was synthesized as
phenylethanamine. Yield 57%, mp 193e197 ꢀC (from ethanol). 1H
NMR (DMSO-d6):
4
starting from 26 and 1-
4.28%, N, 5.95%; Cl, 7.53%; F, 4.03%; S, 6.81%. Found: C, 60.98%; H,
4.21%, N, 5.70%; Cl, 7.32%; F, 3.80%; S, 6.66%.
d
1.52 (d, J ¼ 6.7 Hz, 3H), 2.27 (s, 6H), 5.19e5.22
(m,1H), 7.25e7.39 (m, 5H), 7.47 (d, J ¼ 7.4 Hz, 2H), 7.53 (d, J ¼ 8.8 Hz,
1H), 7.57 (s, 2H), 7.94 (s, 1H), 9.42 (d, J ¼ 7.8 Hz, 1H, disappeared on
treatment with D2O), 13.02 ppm (s, 1H, disappeared on treatment
5.1.7. 5-Chloro-3-((3,5-dimethylphenyl)sulfonyl)-N-(2-
nitrobenzyl)-1H-indole-2-carboxamide (10)
with D2O). 13C NMR (DMSO-d6):
d
21.20, 22.82, 49.58, 111.77, 115.38,
It was synthesized as 4 starting from 26 and (2-nitrophenyl)
119.42, 124.03, 125.20, 125.83, 126.69, 127.50, 127.76, 128.88, 133.35,
methanamine. Yield 75%, mp 180 ꢀC dec. (from ethanol). 1H NMR
135.17, 137.47, 139.46, 143.01, 143.82, 158.81 ppm. IR:
n 1647, 3214,
(DMSO-d6):
d
2.28 (s, 6H), 4.88 (d, J ¼ 4.7 Hz, 2H), 7.25 (s, 1H), 7.35
3273 cmꢁ1. Anal. Calcd. for C25H23ClN2O3S: C, 64.30%; H, 4.96%, N,
6.00%; Cl, 7.59%; S, 6.87%. Found: C, 64.11%; H, 4.91%, N, 5.78%; Cl,
7.42%; S, 6.70%.
(d, J ¼ 8.9 Hz, 1H), 7.54e7.59 (m, 4H), 7.75e7.83 (m, 2H), 7.94 (s,
1H), 8.12 (d, J ¼ 7.9 Hz, 1H) 9.57 (br s, 1H, disappeared on treat-
ment with D2O), 13.12 ppm (br s, 1H, disappeared on treatment
with D2O). 13C NMR (DMSO-d6):
d
21.17, 112.15, 115.43, 119.37,
124.11, 125.20, 125.69, 127.73, 129.12, 130.57, 133.48, 133.53, 134.37,
5.1.11. 5-Chloro-N-(3,5-dimethylbenzyl)-3-((3,5-dimethylphenyl)
135.13, 137.08, 139.40, 142.89, 148.32, 160.37 ppm. IR:
n 1644,
sulfonyl)-1H-indole-2-carboxamide (15)
3214 cmꢁ1. Anal. Calcd. for C24H20ClN3O5S: C, 57.89%; H, 4.05%, N,
8.44%; Cl, 7.12%; S, 6.44%. Found: C, 57.63%; H, 4.00%, N, 8.29%; Cl,
6.98%; S, 6.20%.
It was synthesized as
dimethylphenyl)methanamine. Yield 15%, mp 256e260 ꢀC (from
ethanol). 1H NMR (DMSO-d6):
4 starting from 26 and (3,5-
d
2.26 (s, 12H), 4.5 (d, J ¼ 5.3 Hz, 2H),
6.91 (s, 1H), 7.05 (s, 2H), 7.24 (s, 1H), 7.33 (d, J ¼ 9.0 Hz, 1H), 7.53 (d,
J ¼ 9.2 Hz, 1H), 7.59 (s, 2H), 7.93 (s, 1H), 9.36 (br s, 1H, disappeared
on treatment with D2O), 13.05 ppm (br s, 1H, disappeared on
5.1.8. 5-Chloro-3-((3,5-dimethylphenyl)sulfonyl)-N-(4-
nitrobenzyl)-1H-indole-2-carboxamide (11)
It was synthesized as 4 starting from 26 and (4-nitrophenyl)
treatment with D2O). IR:
26H25ClN2O3S: C, 64.92%; H, 5.24%, N, 5.82%; Cl, 7.37%; S, 6.67%.
Found: C, 64.78%; H, 5.19%, N, 5.69%; Cl, 7.12%; S, 6.40%.
n
1637, 3220, 3289 cmꢁ1. Anal. Calcd. for
methanamine. Yield 30%, mp 150 ꢀC dec (from ethanol). 1H NMR
C
(DMSO-d6):
d
2.25 (s, 6H), 4.69 (d, J ¼ 8.1 Hz, 2H), 7.22 (s, 1H),

108
V. Famiglini et al. / European Journal of Medicinal Chemistry 80 (2014) 101e111
Table 4
5.1.13. 5-Chloro-N-(3-chlorophenethyl)-3-((3,5-dimethylphenyl)
sulfonyl)-1H-indole-2-carboxamide (17)
Anti-HIV-1 activity of compounds 4e12, 14e18 and 21e23 against the WT RT and
mutant RTs carrying single amino acid substitutionsa.
It was synthesized as 4 starting from 26 and 2-(3-chlorophenyl)
Compd
IC50 (nM)b
WT
ethanamine. Yield 25%, mp 212 ꢀC (from aqueous DMF). 1H NMR
K103N
Y181Ic
L100I
(DMSO-d6):
d 2.30 (s, 6H), 2.89e2.99 (m, 2H), 3.57e3.64 (m, 2H),
7.26e7.37 (m, 5H), 7.42 (s, 1H), 7.52e7.61 (m, 3H), 7.92 (s, 1H), 9.12
(t, J ¼ 11.5 Hz, 1H, disappeared on treatment with D2O), 13.04 ppm
4
5
6
7
8
9
10
11
12
14
16
16
17
18
21
22
23
NVP
EFV
ETV
50
3.7
57
180
70
26
23
50
7.7
40
>20,000
>20,000
7500
100
>20,000
>20,000
>20,000
>20,000
>20,000
>20,000
>20,000
>20,000
201
>20,000
>20,000
>20,000
>20,000
>20,000
>20,000
>20,000
>20,000
>20,000
400
ndd
nd
nd
70
40
30
nd
nd
nd
(br s, 1H, disappeared on treatment with D2O). IR:
n 1646, 3190,
3292 cmꢁ1. Anal. Calcd. for C25H22Cl2N2O3S: C, 59.88%; H, 4.42%, N,
5.59%; Cl, 14.14%; S, 6.39%. Found: C, 59.64%; H, 4.39%, N, 5.44%; Cl,
13.96%; S, 6.22%.
160
24
23
218
34
540
90
5.1.14. 5-Chloro-N-(4-chlorophenethyl)-3-((3,5-dimethylphenyl)
sulfonyl)-1H-indole-2-carboxamide (18)
7000
131
83
>20,000
3140
5940
60
12,400
>20,000
>20,000
7000
>20,000
20
5900
270
10
960
200
1300
16,250
9000
nd
It was synthesized as 4 starting from 26 and 2-(4-chlorophenyl)
89
ethanamine. Yield 80%, mp 220e225 ꢀC (from ethanol). 1H NMR
288
190
2000
400
80
(DMSO-d6):
d 2.30 (s, 6H), 2.87e2.94 (m, 2H), 3.55e3.64 (m, 2H),
7.25e7.35 (m, 6H), 7.51e7.61 (m, 3H), 7.93 (d, J ¼ 2.0 Hz, 1H), 9.08
(br s, 1H, disappeared on treatment with D2O), 13.05 ppm (br s, 1H,
disappeared on treatment with D2O). IR:
n .
1649, 3197, 3286 cmꢁ1
10
164
12
Anal. Calcd. for C25H22Cl2N2O3S: C, 59.88%; H, 4.42%, N, 5.59%; Cl,
14.14%; S, 6.39%. Found: C, 59.71%; H, 4.40%, N, 5.40%; Cl, 13.84%; S,
6.12%.
a
Data represent mean values of at least three separate experiments.
Compound concentration (IC50, nM) required to inhibit by 50% the RT activity of
b
the indicated strain.
c
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
[17].
5.1.15. 5-Chloro-3-((3,5-dimethylphenyl)sulfonyl)-N-(1-
phenylpropan-2-yl)-1H-indole-2-carboxamide (19)
d
Data of D,L racemic mixture.
It was synthesized as 4 starting from 26 and 1-phenylpropan-2-
amine. Yield 33%, mp 214e216 ꢀC (from ethanol). 1H NMR (CDCl3):
d
1.36 (d, J ¼ 6.5 Hz, 3H), 2.33 (s, 6H), 2.83e2.88 (m, 1H), 3.04e3.09
(m,1H), 4.45e4.52 (m,1H), 7.17 (s,1H), 7.21e7.24 (m,1H), 7.28e7.34
(m, 5H), 7.38e7.40 (m, 1H), 7.48 (s, 2H), 8.21 (s, 1H), 9.63 (d,
J ¼ 8.4 Hz,1H, disappeared on treatment with D2O),10.32 ppm (br s,
Table 5
Anti-HIV-1 activity of racemate 14 and its enantiomers 24 and 25 against the WT RT
and mutant RTs carrying single amino acid substitutions.a
n .
1642, 3233 cmꢁ1
Compd
IC50 (nM)b
1H, disappeared on treatment with D2O). IR:
Anal. Calcd. for C26H25ClN2O3S: C, 64.92%; H, 5.24%, N, 5.82%; Cl,
7.37%; S, 6.67%. Found: C, 64.81%; H, 5.15%, N, 5.66%; Cl, 7.20%; S,
6.54%.
(Chirality)
WT
40
K103N
Y181I or Y181Cc
8228c
L100I
70
V106A
50
14d
(R,S)
24
(R)
25
540
90
39
50
2531c
50
80
65
28
9400
>20000c
(S)
NVP
EFV
ETV
400
80
10
7000
>20,000
20
>20,000
400
9000
120
12
nd
nd
10
164
a
Data represent mean values of at least three separate experiments.
Compound concentration (IC50, nM) required to inhibit by 50% the RT activity of
b
the indicated strain.
c
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
[17].
d
Data of D,L racemic mixture.
5.1.12. 5-Chloro-N-(2-chlorophenethyl)-3-((3,5-dimethylphenyl)
sulfonyl)-1H-indole-2-carboxamide (16)
It was synthesized as 4 starting from 26 and 2-(2-chlorophenyl)
ethanamine. Yield 33%, mp 217 ꢀC (from aqueous DMF). 1H NMR
(DMSO-d6):
d 2.31 (s, 6H), 3.01e3.09 (m, 2H), 3.57e3.64 (m, 2H),
7.27e7.37 (m, 4H), 7.44e7.56 (m, 3H), 7.63 (s, 2H), 7.95 (d, J ¼ 3.6 Hz,
1H), 9.17 (t, J ¼ 4.7 Hz, 1H, disappeared on treatment with D2O),
13.08 ppm (br s, 1H, disappeared on treatment with D2O). IR:
n
Fig. 1. Binding mode of derivatives 24 (cyan) and 25 (magenta) into the NNBS of the
WT RT. Residues involved in the binding are reported as stick. Residues of B chain are
reported in orange stick. (For interpretation of the references to colour in this figure
legend, the reader is referred to the web version of this article.)
1639, 3211, 3271 cmꢁ1. Anal. Calcd. for C25H22Cl2N2O3S: C, 59.88%;
H, 4.42%, N, 5.59%; Cl,14.14%; S, 6.39%. Found: C, 59.70%; H, 4.36%, N,
5.44%; Cl, 13.88%; S, 6.23%.

V. Famiglini et al. / European Journal of Medicinal Chemistry 80 (2014) 101e111
109
Fig. 2. Binding mode of derivatives 24 (cyan) and 25 (magenta) into the NNBS of the K103N RT. The surface (gray) representation clearly shows that the methyl group of 24 well
closes the entrance channel, while for 25 the methyl group points to the bottom of the cleft. (For interpretation of the references to colour in this figure legend, the reader is referred
to the web version of this article.)
5.1.16. 5-Chloro-3-((3,5-dimethylphenyl)sulfonyl)-N-(2-
phenylpropyl)-1H-indole-2-carboxamide (20)
5.82%; Cl, 7.37%; S, 6.67%. Found: C, 64.80%; H, 5.19%, N, 5.70%; Cl,
7.20%; S, 6.44%.
It was synthesized as 4 starting from 26 and 2-phenylpropan-1-
amine. Yield 33%, mp 209e212 ꢀC (from ethanol). 1H NMR (DMSO-
5.1.20. N-(4-Aminobenzyl)-5-chloro-3-((3,5-dimethylphenyl)
sulfonyl)-1H-indole-2-carboxamide (12)
d6):
d
1.31 (d, J ¼ 6.7 Hz, 3H), 2.30 (s, 6H), 3.04e3.09 (m, 1H), 3.53 (t,
J ¼ 5.9 Hz, 2H), 7.22e7.32 (m, 7H), 7.52 (d, J ¼ 9.0 Hz, 1H), 7.57 (s,
2H), 7.90 (s, 1H), 9.07 (t, J ¼ 5.1 Hz, 1H, disappeared on treatment
with D2O), 12.98 ppm (br s, 1H, disappeared on treatment with
To a solution of 11 (100 mg, 0.24 mmol) in ethyl acetate (14 mL)
was added tin(II) chloride dihydrate (160 mg, 0.72 mmol), and the
reaction mixture was heated at reflux for 3 h. After cooling, the
reaction mixture was adjusted to pH 8 with a saturated solution of
sodium hydrogencarbonate, filtered and extracted with ethyl ac-
etate. Organic layer was dried and filtered. Removal of the solvent
gave a residue that was purified by column chromatography (silica
gel, ethyl acetate:n-hexane ¼ 7:3 as eluent) to furnish 12 (10 mg,
D2O). IR:
n
1641, 3230 cmꢁ1. Anal. Calcd. for C26H25ClN2O3S: C,
64.92%; H, 5.24%, N, 5.82%; Cl, 7.37%; S, 6.67%. Found: C, 64.72%; H,
5.19%, N, 5.68%; Cl, 7.21%; S, 6.49%.
5.1.17. 5-Chloro-3-((3,5-dimethylphenyl)sulfonyl)-N-(2-
methylphenethyl)-1H-indole-2-carboxamide (21)
9%), mp 185 ꢀC dec (from ethanol). 1H NMR (DMSO-d6):
d 2.26 (s,
It was synthesized as 4 starting from 26 and 2-(2-tolyl)ethan-
6H), 4.38 (s, 2H), 5.03 (s, 2H, disappeared on treatment with D2O),
6.52e6.56 (m, 2H), 7.06e7.10 (m, 2H), 7.23e7.25 (m, 1H), 7.31e7.36
(m, 1H), 7.50e7.61 (m, 3H), 7.93e7.95 (m, 1H), 9.26 (br s, 1H,
disappeared on treatment with D2O), 13.03 ppm (br s, 1H, dis-
amine. Yield 25%, mp 214e216 ꢀC (from ethanol). 1H NMR (DMSO-
d6):
d 2.31 (s, 6H), 2.36 (s, 3H), 2.88e2.95 (m, 2H), 3.47e3.59 (m,
2H), 7.11e7.17 (m, 3H), 7.24e7.27 (m, 2H), 7.32e7.38 (m, 1H), 7.52e
7.56 (m, 1H), 7.63 (s, 2H), 7.95 (d, J ¼ 2.1 Hz, 1H), 9.14 (br s, 1H,
disappeared on treatment with D2O), 13.03 ppm (br s, 1H, dis-
appeared on treatment with D2O). IR:
n .
1667, 2980, 3225 cmꢁ1
Anal. Calcd. for C24H22ClN3O3S: C, 61.60%; H, 4.74%, N, 8.98%; Cl,
7.58%; S, 6.85%. Found: C, 61.37%; H, 4.68%, N, 8.74%; Cl, 7.39%; S,
6.61%.
appeared on treatment with D2O). IR:
n .
1651, 3226, 3282 cmꢁ1
Anal. Calcd. for C26H25ClN2O3S: C, 64.92%; H, 5.24%, N, 5.82%; Cl,
7.37%; S, 6.67%. Found: C, 64.77%; H, 5.21%, N, 5.68%; Cl, 7.09%; S,
6.44%.
5.1.21. tert-Butyl 4-nitrobenzylcarbamate (28)
A
mixture of (4-nitrophenyl)methanamine hydrochloride
5.1.18. 5-Chloro-3-((3,5-dimethylphenyl)sulfonyl)-N-(3-
methylphenethyl)-1H-indole-2-carboxamide (22)
(500 mg, 2.65 mmol) and di-tert-butyl dicarbonate (634 mg,
2.91 mmol) in anhydrous dichloromethane (10.8 mL) was cooled to
0 ꢀC in ice-water bath. Triethylamine (691 mg, 0.96 mL, 6.39 mmol)
was added dropwise and the resulting mixture was stirred at room
temperature for 2 h. The reaction mixture was diluted with water
and extracted with dichlorometane. The organic layer was washed
with brine, dried and filtered. Removal of the solvent gave com-
It was synthesized as 4 starting from 26 and 2-(3-tolyl)ethan-
amine. Yield 31%, mp 202e204 ꢀC (from ethanol). 1H NMR (DMSO-
d6):
d
2.27 (s, 3H), 2.30 (s, 6H), 2.87 (t, J ¼ 7.0 Hz, 2H), 3.55e3.59 (m,
2H), 7.03 (d, J ¼ 7.8 Hz, 1H), 7.09e7.19 (m, 3H), 7.26 (s, 1H), 7.33 (d,
J ¼ 8.6 Hz, 1H), 7.52 (d, J ¼ 8.7 Hz, 1H), 7.60 (s, 2H), 7.92 (s, 1H), 9.08
(br s, 1H, disappeared on treatment with D2O), 13.01 ppm (br s, 1H,
pound 28 (610 mg, 91%) as a slurry. 1H NMR (DMSO-d6):
9H), 4.23 (d, J ¼ 5.7 Hz, 2H), 7.46e7.56 (m, 3H), 8.18 ppm (d,
J ¼ 8.4 Hz, 2H). IR:
1675, 2982, 3350 cmꢁ1
d 1.37 (s,
disappeared on treatment with D2O). IR:
n .
1648, 3211, 3289 cmꢁ1
Anal. Calcd. for C26H25ClN2O3S: C, 64.92%; H, 5.24%, N, 5.82%; Cl,
7.37%; S, 6.67%. Found: C, 64.70%; H, 5.16%, N, 5.59%; Cl, 7.20%; S,
6.45%.
n
.
5.1.22. tert-Butyl 4-aminobenzylcarbamate (29)
5.1.19. 5-Chloro-3-((3,5-dimethylphenyl)sulfonyl)-N-(4-
methylphenethyl)-1H-indole-2-carboxamide (23)
To a mixture of 28 (590 mg, 2.33 mmol) in methanol (20.6 mL)
was added Pd/C (62 mg; 10% pp). The reaction mixture was hy-
drogenated under 35 PSI pressure at room temperature and for 2 h.
The reaction mixture was filtered and evaporated. Residue was
purified by silica gel column chromatography (ethyl acetate:n-
hexane ¼ 1:1 as eluent) to furnish 29 (520 mg; 100%) as an oil. 1H
It was synthesized as 4 starting from 26 and 2-(4-tolyl)ethan-
amine. Yield 43%, mp 206e207 ꢀC (from ethanol). 1H NMR (DMSO-
d6):
d 2.26 (s, 3H), 2.30 (s, 6H), 2.84e2.87 (m, 2H), 3.52e3.58 (m,
2H), 7.11 (d, J ¼ 6.8 Hz, 2H), 7.20 (d, J ¼ 7.4 Hz, 2H), 7.26 (s, 1H), 7.33
(d, J ¼ 7.9 Hz, 1H), 7.52 (d, J ¼ 8.5 Hz, 1H), 7.60 (s, 2H), 7.92 (s, 1H)
9.08 (br s, 1H, disappeared on treatment with D2O),13.02 ppm (br s,
NMR (DMSO-d6):
d
1.37 (s, 9H), 3.91 (d, J ¼ 6.0 Hz, 2H), 4.90 (s, 2H,
disappeared on treatment with D2O), 6.47 (d, J ¼ 8.1 Hz, 2H), 6.87
(d, J ¼ 8.0, 2H), 7.13 ppm (s, 1H, disappeared on treatment with
1H, disappeared on treatment with D2O). IR:
n 1642, 3208,
3299 cmꢁ1. Anal. Calcd. for C26H25ClN2O3S: C, 64.92%; H, 5.24%, N,
D2O). IR:
n
1695, 2974 cm-1.

110
V. Famiglini et al. / European Journal of Medicinal Chemistry 80 (2014) 101e111
5.1.23. tert-Butyl (4-dimethylaminophenyl)carbamate (30)
5.2.2. Enzymatic assay procedures
A mixture of 29 (230 mg; 1.03 mmol), anhydrous potassium
carbonate (355 mg, 2.6 mmol) and dimethylsulfate (129 mg, 0.1 mL;
1.03 mmol) in anhydrous acetone (25.5 mL) was stirred at room
temperature for 16 h. The reaction mixture was diluted with water
and extracted with ethyl acetate. Organic layer was washed with
brine, dried and filtered. Removal of the solvent gave a residue that
was purified by flash chromatography (silica gel, ethyl acetate/n-
hexane as eluent) to furnish 28 (100 mg, 41%) as a slurry. 1H NMR
5.2.2.1. Chemicals. [3H]dTTP (40 Ci/mmol) was from Amersham
and unlabeled dNTP’s from Boehringer. Whatman was the supplier
of the GF/C filters. All other reagents were of analytical grade and
purchased from Merck or Fluka. The homopolymer poly(rA)
(Pharmacia) was mixed at weight ratios in nucleotides of 10:1, to
the oligomer oligo(dT)12e18 (Pharmacia) in 20 mM TriseHCl (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. The coex-
pression 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 (NCI-Frederick Cancer Research and Development Center).
Proteins were expressed in E. coli and purified as described [21].
RNA-dependent DNA polymerase activity was assayed as follows: a
(DMSO-d6):
d
1.37 (s, 9H), 2.84 (s, 6H), 3.97 (d, J ¼ 5.7 Hz, 2H), 6.65
(d, J ¼ 8.4 Hz, 2H), 7.04 (d, J ¼ 8.2 Hz, 2H), 7.20 ppm (s, 1H, dis-
appeared on treatment with D2O).
5.1.24. 4-Dimethylaminophenylmethanamine trifluoroacetate (31)
A mixture of 30 (100 mg, 0.39 mmol), dichloromethane (0.4 mL)
and trifluoroacetic acid (0.4 mL) was stirred at room temperature
for 30 min. The reaction mixture was diluted with diethyl ether and
the solvent was evaporated. The obtained product was used
without further purification.
final volume of 25
pH 7.5,1 mM DTT, 0.2 mg/mL BSA, 4% glycerol),10 mM MgCl2, 0.5
of poly(rA)/oligo(dT)10:1 (0.3 M [3H]-dTTP
M 30-OH ends), 10
(1 Ci/mmol) and 2e4 nM RT. Reactions were incubated at 37 ꢀC
for the indicated time. 20 L-Aliquots were then spotted on glass
mL contained reaction buffer (50 mM TriseHCl
mg
m
m
m
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. 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 monitored in the presence of increasing fixed
amounts of inhibitor. Data were then plotted according to Line-
weavereBurke and Dixon. For Ki determination, an interval of in-
hibitor concentrations between 0.2 Ki and 5 Ki was used.
5.1.25. Separation of racemate 14 into the enantiomers 24 and 25
5.1.25.1. HPLC. HPLC enantioseparations were performed by using
the stainless-steel Chiralcel OD (250 mm ꢃ 4.6 mm i.d. and
250 ꢃ 10 mm i.d.) (Chiral Technologies Europe, Illkirch, France)
columns. All chemicals solvents for HPLC were purchased from
Aldrich (Italy) and used without further purification. The analytical
HPLC apparatus consisted of a PerkineElmer (Norwalk, CT, USA)
200 lc pump equipped with a Rheodyne (Cotati, CA, USA) injector, a
20 ml sample loop, an HPLC Dionex CC-100 oven (Sunnyvale, CA,
USA) and a Jasco (Jasco, Tokyo, Japan) Model CD 2095 Plus UV/CD
detector. For semipreparative separations a PerkineElmer 200 LC
pump equipped with a Rheodyne injector, a 1 mL sample loop, a
PerkineElmer LC 101 oven and Waters 484 detector (Waters Cor-
poration, Milford, MA, USA) were used. The signal was acquired and
processed by Clarity software (DataApex, Prague, The Czech
Republic).
5.3. Molecular modeling
All molecular modeling studies were performed on a MacPro
dual 2.66 GHz Xeon running Ubuntu 12.04 LTS. The RT structures
were downloaded from the PDB (WT RT: 2rf2 [17]; K103N mutated
RT: 1fk0 [22]). Hydrogen atoms were added to the protein using
Molecular Operating Environment (MOE) 2007.09 [23] and mini-
mized, keeping all the heavy atoms fixed until a rmsd gradient of
5.1.25.2. Circular dichroism. The circular dichroism (CD) spectra
were measured by using a Jasco Model J-700 spectropolarimeter.
The optical path and temperature were set at 0.1 mm and 20 ꢀC,
respectively. The spectra are average computed over three instru-
mental scans and the intensities are presented in terms of ellipticity
values (mdeg).
ꢀꢁ1
0.05 kcal molꢁ1
A
was reached. Ligand structures were built with
MOE and minimized using the MMFF94ꢃ force field until a rmsd
ꢀꢁ1
gradient of 0.05 kcal molꢁ1
A
was reached. The docking simula-
tions were performed using PLANTS 1.1 [24]. The images in the
manuscript were generated by using PyMOL 1.2 [25] and MOE
softwares.
5.2. Biological assays
Acknowledgments
5.2.1. Inhibition of HIV-induced cytopathicity
This research project was supported by grant 2012 of Istituto
Pasteur-Fondazione Cenci Bolognetti and the Spanish MINECO
SAF2010-21617-C02 and BFU 2012-31569. Authors thank Prof. Jan
Balzarini, Rega Institute for Medical Research, Katholieke Uni-
versiteit Leuven, B-3000 Leuven, Belgium, for the evaluation of
some compounds against HIV-1 IIIB and HIV-2 ROD strains.
Biological activity of the compounds was tested in the lymphoid
MT-4 cell line (received from the NIH AIDS Reagent Program)
against the WT HIV-1 NL4-3 strain and the different mutant HIV-1
strains, as described before [19]. Briefly, MT-4 cells were infected
with the appropriate HIV-1 strain (or mock-infected to determine
cytotoxicity) in the presence of different drug concentrations. At
day five post-infection, a tetrazolium-based colorimetric method
(MTT method) was used to evaluate the number of viable cells. The
methodology for the anti-HIV assays in CEM cells had been
described previously [20]. Briefly, human CEM cell cultures
(w3 ꢃ 105 cells/mLꢁ1) were infected with w100 CCID50 HIV-1 IIIB
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