Design and anti-HIV activity of arylsulphonamides as non-nucleoside reverse transcriptase inhibitors

Article abstract of DOI:10.1007/s00044-016-1707-7

Design and anti-HIV activity of a series of arylsulphonamide derivatives as possible non-nucleoside reverse transcriptase inhibitors have been discussed. Compounds designed on the basis of Lipinski’s rule of five and having H-bond donor and acceptor sites were synthesized and screened in vitro to assess their human immunodeficiency virus type 1 reverse transcriptase inhibitory activity using TZM-bl cells. In silico studies using Discovery Studio 3.0 software showed that these molecules formed H-bonds and exhibited π–π, π–+ interactions, with amino acids in the non-nucleoside inhibitor binding pocket, and formed more stable complexes (total interaction energy in the range of (?) 47.85–(?) 77.01 kcal/mol) with human immunodeficiency virus type 1 reverse transcriptase in comparison to nevirapine and etravirine, (?) 45.79 and (?) 61.43 kcal/mol, respectively, and thus, lower EC₅₀ values were predicted. The molecule, 4-(4-chloro-benzenesulphonyl amino)-N-(1H-indazole-5-yl)-benzamide showed significant inhibition of human immunodeficiency virus type 1 growth under in vitro conditions with EC₅₀ value in the range of 4.89 × 10^?5 μm. However, its selectivity index value was 2.45 only, which was much lower than nevirapine and etravirine. The reverse transcriptase analysis, based on luciferase reporter assay, of this compound proved its nature as non-nucleoside reverse transcriptase inhibitors. Though the molecules showed low inhibitory action against human immunodeficiency virus type 1 under in vitro conditions, the idea to develop non-nucleoside reverse transcriptase inhibitors was vindicated.

Full text of DOI:10.1007/s00044-016-1707-7

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-016-1707-7
ORIGINAL RESEARCH
Design and anti-HIV activity of arylsulphonamides as
non-nucleoside reverse transcriptase inhibitors
Anuradha Singh1 Madhu Yadav1 Ritika Srivastava1 Nidhi Singh1
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Rajinder Kaur2 Satish K. Gupta2 Ramendra K. Singh1
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Received: 16 February 2016 / Accepted: 1 August 2016
© Springer Science+Business Media New York 2016
Abstract Design and anti-HIV activity of a series of aryl-
sulphonamide derivatives as possible non-nucleoside
reverse transcriptase inhibitors have been discussed. Com-
pounds designed on the basis of Lipinski’s rule of five and
having H-bond donor and acceptor sites were synthesized
and screened in vitro to assess their human immunodefi-
ciency virus type 1 reverse transcriptase inhibitory activity
using TZM-bl cells. In silico studies using Discovery Studio
3.0 software showed that these molecules formed H-bonds
and exhibited π–π, π–+ interactions, with amino acids in the
non-nucleoside inhibitor binding pocket, and formed more
stable complexes (total interaction energy in the range of (−)
47.85–(−) 77.01 kcal/mol) with human immunodeficiency
virus type 1 reverse transcriptase in comparison to nevir-
apine and etravirine, (−) 45.79 and (−) 61.43 kcal/mol,
respectively, and thus, lower EC50 values were predicted.
The molecule, 4-(4-chloro-benzenesulphonyl amino)-N-
(1H-indazole-5-yl)-benzamide showed significant inhibition
of human immunodeficiency virus type 1 growth under in
vitro conditions with EC50 value in the range of 4.89 × 10−5 μm.
However, its selectivity index value was 2.45 only, which
was much lower than nevirapine and etravirine. The reverse
transcriptase analysis, based on luciferase reporter assay, of
this compound proved its nature as non-nucleoside reverse
transcriptase inhibitors. Though the molecules showed low
inhibitory action against human immunodeficiency virus
type 1 under in vitro conditions, the idea to develop non-
nucleoside reverse transcriptase inhibitors was vindicated.
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●
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Keywords Arylsulphonamide HIV NNRTIs TZM-bl
●
cells Luciferase reporter assay
Introduction
Human immunodeficiency virus type 1 reverse transcriptase
(HIV-1 RT) is one of the most attractive targets in the
chemotherapy of AIDS patients and HIV-1 RT inhibitors,
thus, play a vital role in HIV/AIDS effective therapy in
vogue—the highly active antiretroviral therapy (HAART).
It is noteworthy that HIV-1 RT is essential for viral repli-
cation as it is responsible for conversion of its genomic
ssRNA into dsDNA—the provirus, which initiates the viral
replication process (Kumari and Singh, 2013). HIV-1 RT
has a flexible structure and contains two known sites where
drugs can bind: first is the active binding site, characterized
by its catalytic triad, Asp110, Asp185, Asp186, where
deoxynucleoside triphosphates bind in the normal proce-
dure, and the second is an allosteric site or non-nucleoside
inhibitor binding pocket (NNIBP), characterized by
hydrophobic amino acid residues, and located about 10 Å
away from the catalytic or active site (Jonckheere et al.,
2000; Singh et al., 2010).
Electronic supplementary material The online version of this article
(doi: 10.1007/s00044-016-1707-7) contains supplementary material,
which is available to authorized users.
* Ramendra K. Singh
rksinghsrk@gmail.com
The NNIBP is hydrophobic in nature and contains pri-
marily amino acid residues from the loop β5 (Pro95,
Leu100, Lys101, and Lys103), β6 (Val106 and Val108), the
β9–β10 hairpin (Val179, Tyr181, Tyr188, and Gly190), and
the β12–β13 hairpin (Phe227, Trp229, Leu234, His235, and
1
Bioorganic Research Laboratory, Department of Chemistry,
University of Allahabad, Allahabad 211002, India
2
National Institute of Immunology, Aruna Asaf Ali Marg,
New Delhi, India

Med Chem Res
Pro236) of the p66 palm sub domain and the β15 (Tyr318)
of the p66 thumb sub domain, as well as the β7–β8 con-
necting loop (Gly138) of the p51 fingers sub domain. Most
of the amino acid residues that form the binding pocket are
hydrophobic (Pro59, Leu100, Val106, Val179, Leu234, and
Pro236) and five of them are aromatic residues (Tyr181,
Tyr188, Phe227, Trp229, and Tyr232). Some hydrophilic
residues, like Lys101, Lys103, Ser105, Asp132, and
Glu224 are also present. The binding of non-nucleoside
reverse transcriptase inhibitors (NNRTIs) results in a con-
comitant shift of the β4–β7–β8 sheet and the three catalytic
aspartic acid residues cause conformational changes in
HIV-1 RT. These changes form the basis of inhibitory
action of NNRTIs (Tronchet and Seman, 2003; Zhou et al.,
2006). The NNRTIs include a variety of conformationally
restrained two-ring and three-ring structures with varying
degree of flexibility and some common underlying chem-
istry and interact at allosteric site on HIV-1 RT. NNRTIs
are specific only for HIV-1 RT because of the presence of a
flexible hydrophobic pocket in which NNRTIs can fit
(Martins et al., 2008; Singh et al., 2015).
Most of the NNRTIs adopt a “butterfly” conformation
with a central lipophilic domain (body) flanked by two
hydrophobic (normally a benzene ring and an extended π
system) moieties (wings). The hydrophobic wings interfere
efficiently with functional aromatic amino acid residues
within the binding pocket (Tyr181, Tyr188, Trp229, and
Try318). Apart from stacking interactions, electrostatic
interactions (with Lys101, Lys103, and Gly138), van der
Waals (vdW) interactions (with Leu100, Val106, Val179,
Tyr181, Gly190, Trp229, Leu234, and Trp318), and
hydrogen bonding between NNRTIs and the main chain
peptide bonds contribute to the interaction and binding
efficacies of the drugs with the NNRTI-specific pocket. The
hydrophobic interactions of the side chains of the residues,
Tyr181, Tyr188, and Trp229, with the hydrophobic moi-
eties of the NNRTIs appear to be important for inhibitor
binding. Since most of the NNRTIs contain polar groups,
they have the potential to form hydrogen bonds with sur-
rounding amino acid residues either directly or via water
bridges (Kumari and Singh, 2013; Zhan et al., 2013).
The recently developed second generation NNRTIs
shows a “U” (or “horse-shoe”) conformation, which gives
an increased plasticity to these derivatives and NNIBP and
this appears to be a deciding factor for potency against wild
type (WT) and mutant HIV-1 RTs. The latest approved
NNRTIs, etravirine and rilpivirine and another compound,
dapivirine, under clinical investigation in phase I/II clinical
trials are success stories of such an approach (Wang et al.,
2014).
size, specific chemical structure, and binding mode of the
NNRTI. Limits to the pocket’s flexibility are not fully
understood and this makes accurate predictions of the structure
of RT-NNRTI complexes very challenging with the available
molecular modeling techniques. Molecular modeling analyses
of the RT-NNRTI complexes suggest that potent inhibitors
tend to bind in the NNIBP in low energy conformations. The
analyses suggest that inhibitors with selected conformational
degrees of freedom, such as torsional flexibility around stra-
tegically located chemical bonds, can compensate for the
effects of drug-resistance mutations. Using inhibitor flexibility
in designing drugs to overcome the effects of resistant muta-
tions has broader implications for diseases where drug resis-
tance is a primary concern (Zhan et al., 2016).
Unique antiviral activity, high specificity and low toxicity
of NNRTIs make them attractive ingredient of HAART.
Currently, there are five FDA approved drugs in this class
(Fig. 1), namely nevirapine, efavirenz, delaviridine, etravir-
ine, and rilpivirine, which effectively inhibit WT HIV, but
are less effective against clinically important RT mutant
viruses having mutation at Tyr188, Tyr181, and Lys103
amino acid residues. Therefore, there is an urgent need to
develop novel, highly potent NNRTIs with broad spectrum
of antiviral activity. We, therefore, focused our attention on
the design and synthesis of some possible NNRTIs. The
synthetic pathway was planned in a way to accommodate
well documented pharmacophoric moieties of interest, such
as, amide, aryl and heteroaryl moieties that proved to con-
tribute to anti-HIV (Kumari and Singh, 2012; Kumari et al.,
2011; Singh and Singh, 2011; Esposito et al., 2012).
Experimental section
Design of NNRTIs
We have analyzed some physically significant descriptors
and pharmaceutically relevant properties of compounds,
like molecular weight, H-bond donors, H-bond acceptors,
log P (octanol/water), on the basis of Lipinski’s rule of five,
using Molinspiration and ChemDraw softwares.
Chemistry
Chemicals were obtained from E. Merck India Ltd., India
and Sigma-Aldrich Chemical Company, USA. Melting
points (m.p.) determined on electro thermal apparatus are
uncorrected. Silica gel for TLC was obtained from E. Merck
India Ltd. and spots were identified by iodine and UV lamp
exposure. UV measurements were carried out on Lambda
25 spectrophotometer. 1H nuclear magnetic resonance
(NMR) spectra and 13CMR were recorded on DRX 400
MHz instrument using CDCl3 as solvent and tetra methyl
The overall structure of HIV-1 RT has segmental flexibility
that also varies according to the nature of the bound NNRTI.
The NNIBP is elastic and its conformation depends on the

Med Chem Res
N
N
a
b
N
N
O
N
HN
N
N
NHSO2Me
N
N
N
CF3
O
N
HN
O
N
HN
NH
N
NH
Br
N
N
O
H
N
NH2
H
O
Nevirapine
Delaviridine
Efavirenz
Etravirine
Rilpivirine
c
Cl
Hydrophilic sites
O
O
S
O
N
NH
H
R
N-heteroaryls
Hydrophobic sites
Fig. 1 a First generation NNRTIs and b second generation NNRTIs are FDA approved NNRTIs and c Designed prototype
silane as an internal standard. Mass spectra were obtained
using a Thermofinnigan TRACE-DSQ Electro Spray Ioni-
zation mass spectrometer. Elemental analyses were carried
out on a Perkin-Elmer 240-C analyzer. All solvents were
dried and distilled prior to use.
added drop wise in cold and anhydrous conditions. Reaction
mixture was stirred at 25 °C for 1 h and the solvent removed
in vacuo. The residual oil was dissolved in dry pyridine and
used directly in the next step without further purification.
General procedure for synthesis of title compounds
4-(4-Chloro-benzenesulphonylamino)-benzoic acid (2)
To a stirred solution of 3 (1 mmol) in dry pyridine (3 mL)
maintained in an ice bath, added drop wise amine deriva-
tive (1 mmol) dissolved in dry acetonitrile (4 mL) and
pyridine (1 mL), and stirred the reaction mixture at room
temperature. After completion of the reaction, the solvent
was evaporated under pressure and the residual mass dis-
solved in ethyl acetate (30 mL). This organic fraction was
washed consecutively with 5 % NaHCO3 solution (20 mL),
NaCl (20 mL), H2O (20 mL), dried on anhydrous Na2SO4,
filtered and reduced under vacuum (40–50 °C). The title
compound was purified by recrystallization from ethanol:
water.
To an aqueous solution of 4-amino benzoic acid 1 (3.0 g,
21.8 mmol) maintained at pH 9 with aqueous sodium
bicarbonate solution, 4-chloro benzenesulphonyl chloride
(4.617 g, 21.8 mmol) was added. Contents were stirred at
room temperature until the complete consumption of the
sulphonyl chloride (as indicated by TLC). The medium of
the reaction mixture was changed to pH 1 using hydro-
chloric acid (1 M) and the precipitate obtained was filtered,
washed with water and dried. The resulting solid was
recrystallized from ethanol to get white solid (96 %); m.p.
105 °C, Rf 0.6 (DCM/MeOH, 9.0:1.0); 1H NMR (400 MHz,
Chloroform-d): δ (ppm): 7.92–7.86 (m, 2H, Ar–H), 7.72–
7.66 (m, 2H, Ar–H), 7.64–7.58 (m, 2H, Ar–H), 7.01–6.94
(m, 2H, Ar–H), 5.76 (s, 1H, −NH).
4-(4-Chloro-benzenesulphonylamino)-N-pyridin-2-yl-
benzamide (4)
4-(4-Chloro-benzenesulphonylamino)-benzoyl chloride (3)
White solid (60 %); m.p. 130 °C; Rf 0.48 (DCM/MeOH,
9.5:0.5); 1H NMR (400 MHz, Chloroform-d): δ (ppm): 8.96
(s, 1H, –CONH–), 8.87 (dd, J = 8.1, 1.0 Hz, 1H, C3–H
pyridine), 7.90 (td, J = 8.0, 1.2 Hz, 1H, C4–H pyridine),
To compound 2 (1 mmol) dissolved in dry acetonitrile and a
small amount of pyridine, oxalyl chloride (1.2 mmol) was

Med Chem Res
7.33 (ddd, J = 7.9, 4.9, 0.9 Hz, 1H, C5-H pyridine), 8.69
(dd, J = 5.0, 1.3 Hz, 1H, C6-H pyridine), 7.72–7.66 (m, 2H,
Ar–H), 7.64–7.58 (m, 2H, Ar–H), 7.54–7.48 (m, 2H, Ar–
H), 7.09–7.03 (m, 2H, Ar–H), 6.17 (s, 1H, –NH); 13C NMR
(CDCl3, 100 MHz): δ = 167.17 (C-1), 151.60 (C-10),
148.28 (C-14), 140.99 (C-23), 139.32 (C-12, C-20), 138.32
(C-21, C-25), 137.24 (C-5), 130.15 (C-22, C-24), 128.44
(C-3, C-7), 122.30 (C-4, C-6), 121.95 (C-2), 117.96 (C-13),
114.40 (C-11). UV (EtOH): λmax 213 nm; MS (EI+, 70
eV): m/z (%): 387 (M+); anal. calcd. for C18H14ClN3O3,S:
C 55.74, H 3.64, Cl 9.14, N 10.83, O 12.381, S 8.27; found;
C 56, H 3, Cl 9, N 10.1, S 8.
4-(4-Chloro-benzenesulphonylamino)-N-(6-ethoxy-
benzothiazol-2-yl)-benzamide (7)
Light brown solid (76 %); m.p. 257 °C; Rf 0.48 (DCM/
MeOH, 9.5:0.5); 1H NMR (400 MHz, Chloroform-d): δ
(ppm): 7.72–7.57 (m, 2H, Ar–H), 7.54–7.47 (m, 2H, Ar–H),
7.09–7.03 (m, 3H, Ar–H), 6.07 (s, 1H, –NH), 4.11 (q, J =
5.9 Hz, 2H, –CH2), 1.34 (d, J = 11.8 Hz, 3H, –CH3); 13C
NMR (CDCl3, 100 MHz): δ = 167.84 (C-1), 156.28 (C-11),
153.50 (C-17), 150.11(C-13), 140.99 (C-29), 139.32 (C-
26), 137.24 (C-5), 133.95 (C-27, C-31), 132.83 (C-14),
130.15 (C-28, C-30), 128.44 (C-3, C-7), 128.19 (C-2),
122.30 (C-4, C-6), 118.42 (C-15), 113.35 (C-16), 106.93
(C-18), 63.77 (C-20), 14.71 (C-21); UV (EtOH): λmax 208
nm, 255 nm; MS (EI, 70 eV): m/z (%): 487.04(M+), 489(41
%); anal. calcd. for: C22H18ClN3O4,S2: C 54.15, H 3.72, Cl
7.27, N 8.61; found; C 54, H 3.8, Cl 7.00, N 8.5, S 13.02.
N-(5-Bromo-pyridin-2-yl)-4-(4-chloro
benzenesulphonylamino)-benzamide (5)
Light brown solid (83 %); m.p. 147 °C; Rf 0.48 (DCM/
MeOH, 9.5:0.5); 1H NMR (400 MHz, Chloroform-d): δ
(ppm): 9.10 (s, 1H, –CONH–), 7.36 (d, J = 8.1 Hz, 1H, C3-
H pyridine), 7.76 (dd, J = 8.0, 1.2 Hz, 1H, C4-H pyridine),
8.49 (d, J = 1.3 Hz, 1H, C6-H pyridine), 7.72–7.66 (m, 2H,
Ar–H), 7.64–7.58 (m, 2H, Ar–H), 7.52–7.45 (m, 2H, Ar–
H), 7.09–7.03 (m, 2H, Ar–H), 6.16 (s, 1H, –NH); 13C NMR
(CDCl3, 100 MHz): δ = 167.17 (C-1), 150.96 (C-21),
149.34 (C-21), 140.99 (C-17), 140.38 (C-23), 139.32 (C-
14), 137.24 (C-5), 133.95 (C-15, C-19), 130.15 (C-16, C-
18), 128.44 (C-3, C-7), 122.30 (C-4, C-6), 121.95 (C-2),
118.47 (C-22), 114.61(C-24); UV (EtOH): λmax 345 nm;
MS (EI+, 70 eV): m/z (%): 447 (M+); anal. calc. for
C18H14BrClN3O3,S: C 46.32, H 2.81, Br 17.12, Cl 7.60,
N 9.00, S 6.87; found; C 46, H 2.75, Cl 7.5, Br 17, N 8.9,
S 6.5.
3-(4-Chloro-benzenesulphonylamino)-N-(6-chloro-
benzothiazol-2-yl)-benzamide (8)
Brown crystals (66 %); m.p. 265 °C; Rf = 0.47 (DCM/
MeOH, 9.9:0.1); 1H NMR (400 MHz, Chloroform-d): δ
(ppm): 8.10 (d, J = 1.5 Hz, 1H, Ar–H), 7.86 (d, J = 7.5 Hz,
1H, Ar–H), 7.72–7.66 (m, 2H, Ar–H), 7.64–7.58 (m, 2H,
Ar–H), 7.54 (dd, J = 7.5, 1.5 Hz, 1H, Ar–H), 7.48 (dt, J =
7.4, 2.1 Hz, 1H, Ar–H), 7.38–7.27 (m, 2H, Ar–H), 7.14 (t,
J = 2.0 Hz, 1H, Ar–H), 6.01 (s, 1H, –NHSO2); 13C NMR
(CDCl3, 100 MHz): δ = 169.01 (C-1), 156.28 (C-11),
151.65 (C-13), 140.99 (C-27), 140.36 (C-4), 139.32 (C-24),
133.95 (C-2), 132.69 (C-25, C-29), 130.15 (C-14), 128.47
(C-26, C-28), 125.83 (C-6), 125.30 (C-7), 124.65 (C-16),
120.21 (C-17), 120.18 (C-5, C-18), 120.06 (C-3), 118.46
(C-15); UV (EtOH): λmax 210 nm; MS (EI, 70 eV): m/z
(%): 476.94(M+); anal. calcd. for: C20H13Cl2N3O3,S2: C
50.21, H 2.74, Cl 14.82, N 8.78; found; C 50, H 2.5, Cl
14.70, N 8.6, S 13.02.
4-(4-Chloro-benzenesulphonylamino)-N-(5-nitro-pyridin-2-
yl)-benzamide (6)
Light yellow solid (56 %); m.p. 138–139 °C; Rf 0.45 (DCM/
MeOH, 9.5:0.5); 1H NMR (400 MHz, Chloroform-d): δ
(ppm): 7.64 (d, J = 8.1 Hz, 1H, C3-H pyridine), 8.49 (dd, J
= 8.0, 1.2 Hz, 1H, C4-H pyridine), 9.40 (d, J = 1.3 Hz, 1H,
C6-H pyridine), 9.31 (s, 1H, –CONH), 7.72–7.66 (m, 2H,
Ar–H), 7.52–7.45 (m, 2H, Ar–H), 7.09–7.03 (m, 2H, Ar–
H), 6.18 (s, 1H, –NH); 13C NMR (CDCl3, 100 MHz): δ =
167.17 (C-1), 153.48 (C-10), 149.59 (C-14), 140.99 (C-26),
139.32 (C-23), 137.24 (C-5), 133.95 (C-24, C-28), 133.34
(C-12), 133.13 (C-13), 130.15 (C-25, C-27), 128.44 (C-3,
C-7), 122.30 (C-4, C-6), 121.94 (C-2), 112 (C-11); UV
(EtOH): λmax 382 nm; MS (EI+, 70 eV): m/z (%): 433
(M+); anal. calcd. for C18H14ClN4O5,S: C 49.83, H 3.25, Cl
8.17, N 12.91, S 7.39; found; C 50, H 3.00, Cl 8.18, N 12.8,
S 7.35.
N-(6-Bromo-benzothiazol-2-yl)3-(4-Chloro-
benzenesulphonylamino)-benzamide (9)
Dark brown solid (69 %); m.p. 279 °C; Rf = 0.49 (DCM/
MeOH, 9.9:0.1); 1H NMR (400 MHz, Chloroform-d): δ
(ppm): δ = 7.53–7.81 ppm (m, 4H), 4.1 (s, 1H, Ar–NH),
6.5–7.4 (m, 4H, Ar), 7.86 (s, 1H, NH), 7.7–8.2 (m, 3H, Ar);
13C NMR (CDCl3, 100 MHz): δ = 169.01 (C-1), 156.28 (C-
22), 152.88 (C-24), 140.99 (C-17), 140.36 (C-4), 139.32
(C-14), 133.95 (C-2, C-15, C-19), 132.26 (C-25), 130.15
(C-16, C-18), 128.47 (C-6), 128.10 (C-27), 125.83 (C-7),
123.09 (C-29), 120.19 (C-3, C-5), 118.86 (C-26), 112.03
(C-28); UV (EtOH) λmax 216 nm; MS (EI, 70 eV): m/z (%):
522.92(M+); anal. calcd. for: C20H13BrN3O3,S2: C 45.95,

Med Chem Res
H 2.51, Br 15.28, Cl 6.78, N 8.04 S 12.27; found; C 45, H
2.3, Br 15.21,Cl 6.73, N 8.0, S 12.02.
(EtOH): λmax 247 nm; MS (EI, 70 eV): m/z (%): 456 (M+),
458 (37 %); anal. calcd. for: C20H13ClN4O5,S: C 52.57, H
2.87, Cl 7.76, N 12.26, S 7.02; found; C 52.5, H 2.55, Cl
7.28, N 12.8, S 7.3.
4-(4-Chloro-benzenesulphonylamino)-N-(1H-indazole-5-
yl)-benzamide (10)
4-(4-Chloro-benzenesulphonylamino)-N-napthalen-2-yl-
benzamide (13)
Dark brown (70 %); m.p. 238 °C; Rf = 0.47 (DCM/MeOH,
9.5:0.5); 1H NMR (400 MHz, Chloroform-d) δ 9.19 (s, 1H,
–CONH–), 9.04 (s, 1H, –NH, indazole), 8.32 (d, J = 1.5 Hz,
1H, C-3 indazole), 7.83 (dd, J = 7.4, 1.6 Hz, 1H, C-6
indazole), 7.72–7.66 (m, 2H, Ar–H), 7.64–7.53 (m, 6H, Ar–
H), 7.09–7.03 (m, 2H, Ar–H), 6.17 (s, 1H); 13C NMR
(CDCl3, 100 MHz): δ = 165.98 (C-1), 140.99 (C-26),
139.32 (C-23), 137.24 (C-5), 135.40 (C-18), 133.95(C-24,
C-28) , 132.08 (C-12), 130.15 (C-15, C-25, C-28), 128.44
(C-3, C-7), 122.30 (C-16, C-13), 121.39 (C-4, C-6), 115.21
(C-2), 110.31 (C-14, C-17); UV (EtOH): λmax 245 nm; MS
(EI, 70 eV): m/z (%): 426 (M+); anal. calcd. for:
C20H15ClN4O3,S: C 56.27, H 3.54, Cl 8.31, N 13.12, S
7.51; found; C 55.5, H 3.55, Cl 8.28, N 13.8, S 7.33.
Brown solid (70 %); m.p. 246 °C; Rf 0.35 (DCM/MeOH,
9.9:0.1); 1H NMR (400 MHz, Chloroform-d): δ (ppm): 9.28
(s, 1H, –CONH–), 7.74–7.54 (m, 7H, Ar–H), 7.40–7.31 (m,
2H, Ar–H), 7.09–7.03 (m, 2H, Ar–H), 6.19 (s, 1H, –NH);
13C NMR (CDCl3, 100 MHz): δ = 165.98 (C-1), 140.99 (C-
27), 139.32 (C-24), 137.24 (C-5), 134.69 (C-19), 133.95
(C-25, C-29), 133.61 (C-11), 130.15 (C-26, C-28), 129.44
(C-13), 129.20 (C-14), 128.44 (C-3, C-7), 128.07 (C-15),
126.45 (C-18), 126.12 (C-16, C-17), 122.30 (C-4, C-6),
121.39 (C-2), 121.05 (C-12); UV (EtOH): λmax 250 nm;
MS (EI, 70 eV): m/z (%): 436 (M+); anal. calcd. for:
C23H17ClN2O3S: C 63.23, H 3.92, Cl 8.11, N 6.41; found;
C 63.20, H 3.85, Cl 8, N 6.5, S 7.21.
N-[(4-Benzoimidazole-1-carbonyl)-phenyl]-4-chloro-
benzenesulphonamide (11)
Molecular modeling analysis
Creamish white crystals (74 %); m.p. 188 °C; Rf 0.46
(DCM/MeOH, 9.5:0.5); H NMR (400 MHz, Chloroform-
1
The structure retrieved from the Protein Data Bank (www.
rcsb.org) with entry code (PDB: ID 3MEC), has been uti-
lized for modeling. The protein was prepared, docked,
scored, and the molecular dynamics (MD) simulation car-
ried out using standard procedures. All computational stu-
dies were done using Discovery Studio 3.0 (DS 3.0,
Accelrys Ltd., UK).
d) δ 8.74 (s, 1H, C-2 benzimidazole), 7.72–7.65 (m, 4H,
benzimidazole), 7.64–7.58 (m, 2H, Ar–H), 7.57–7.51 (m,
2H, Ar–H), 7.15–7.08 (m, 2H), 6.21 (s, 1H, –NHSO2); 13C
NMR (CDCl3, 100 MHz): δ = 166.41 (C-18), 142.62 (C-
20), 140.99 (C-4), 139.32 (C-1), 137.31 (C-23), 136 (C-22,
C-12), 133.95(C-2, C-6), 131.43 (C-15), 130.15 (C-3, C-5),
128.40 (C-14, C-16), 124.30 (C-27), 121.75 (C-26), 119.95
(C-1), 114.82 (C-13,C-17); UV (EtOH): λmax 235 nm; MS
(EI, 70 eV): m/z (%): 411 (M+), 413 (37 %); anal. calcd. for:
C20H14ClN3O3,S: C 58.32, H 3.43, Cl 8.61, N 10.20, S
7.79; found; C 58.0, H 3.44, Cl 8.5, N 10.8, S 7.6.
Receptor set up
The target protein (PDB: ID 3MEC) was taken, ligand
extracted, missing hydrogens added, and their positions
optimized using the all-atom CHARMm forcefield and the
Adopted Basis set Newton–Raphson (ABNR) method
available in DS 3.0 protocol until the root mean square (r.m.
s.) gradient was less than 0.05 kcal/mol/Å (Brooks et al.,
1983). The minimized protein was defined as the “receptor”
using the binding site module of DS 3.0. The binding site
was defined from the volume of ligand method, which was
modified to accommodate all important interacting residues
in the active site of HIV-1 RT. The Input Site Sphere was
defined over the binding site, with a radius of 5 Å from the
center of the binding site. The protein thus characterized
was taken as the target receptor for the docking procedure.
Minimization was performed to relax these newly added
hydrogen atoms by fixing all other non-hydrogen atoms.
The minimized structure of the protein was used in docking
simulations.
4-Chloro N-[4-(4-nitro-benzoimidazole-1-carbonyl)-
phenyl]-benzenesulphonamid (12)
Cream solid (68 %); m.p. 196 °C; Rf 0.47 (DCM/MeOH,
9.5:0.5); 1H NMR (400 MHz, Chloroform-d) δ 8.79 (s, 1H,
C-2 benzimidazole), 8.59 (dd, J = 7.5, 1.5 Hz, 1H, C-7
benzimidazole), 7.94 (dd, J = 7.5, 1.5 Hz, 1H, C-5 benzi-
midazole), 7.52 (t, J = 7.5 Hz, 1H, C-6 benzimidazole),
7.72–7.66 (m, 2H, Ar–H), 7.64–7.57 (m, 4H, Ar–H), 6.97–
6.91 (m, 2H, Ar–H), 5.97 (s, 1H, –NHSO2); 13C NMR
(CDCl3, 100 MHz): δ = 166.41 (C-17), 142.32 (C-24),
141.49 (C-19), 140.99 (C-4), 139.32 (C-1), 137.35 (C-22),
136.40 (C-11), 136.15 (C-2, C-6), 133.95 (C-14), 131.43
(C-3, C-5), 130.15 (C-13, C-15), 128.40 (C-26), 126.72 (C-
25), 123.45 (C-12), 121.75 (C-16), 116.03 (C-27); UV

Med Chem Res
Cl
O
HO
Cl
CI
O
O
c
a
O
S
O
O
S
b
N
H
Cl
O
S
O
NH
O
2
N
NH
R
H
Cl
1
2
3
4-13
R
N
O
+
Br
N
O-
S
N
N
N
O
4
8
5
7
6
Cl
S
H
Br
S
N
N
N
N
N
N
9
10
11
N
-
O
N
+
N
O
12
13
Scheme 1 Reagents and conditions: a water, sodium bicarbonate (1 mol), room temperature, 4 h, HCl (1 mol); b CH3CN and pyridine (3:1), oxalyl
chloride, room temperature, 1 h; c appropriate amine, pyridine, triethyl amine, overnight stirring
Ligand set up
(bond lengths and bond angles fixed and only the rotatable
bonds were allowed to rotate freely) to generate docked
poses parallel to the shape of the binding site. Dreiding
forcefield and grid-based calculated interaction energy,
were used to refine these poses by rigid body minimization
(Hamdouchi et al., 2005). The receptor protein was fixed
during docking. The docked poses were further minimized
by using all-atom CHARMm forcefield and smart mini-
mizer algorithm until the r.m.s gradient for potential energy
was less than 0.001 kcal mol−1 Å−1 and evaluated with a set
of scoring functions. The description of ligand scoring
(-PLP1, -PLP2, -PMF, Lig_Internal_Energy, Binding
Energy, and Dock Score) have already been discussed in
“Result and discussion” section. The atoms of ligand and
the side chains of the residues of the receptor within 5 Å
from the center of the binding site were kept flexible during
minimization. The LUDI III score was used to score the
refined poses. The ligand pose, which corresponded to the
highest LUDI III score was taken as the best docked pose.
Furthermore, determination of binding energy to assess the
binding affinity of ligands for receptor was calculated by
employing highest stable ligand-receptor complex through
the protocol “Calculate Binding Energies” within DS 3.0
using the default settings (Bohm, 1994a, b, 1998; Wang
et al., 2003; Prathipati and Saxena, 2006).
In order to propose new inhibitors, build-and-edit module
of DS 3.0 was used. A series of desired arylsulphonamides,
Scheme 1, having substitution at C1 positions of para amino
benzoic acid nucleus through amide linkage with different
heterocyclic amines was built, all-atom CHARMm force-
field was assigned and then minimized using the ABNR
method. A conformational search of inhibitors was carried
out using a simulated annealing MD approach. The ligand
was heated to a temperature of 700 K and then annealed to
200 K. Thirty such cycles were carried out. The con-
formation obtained at the end of each cycle was further
subjected to local energy minimization, using Smart Mini-
mizer algorithm with 2000 steps and r.m.s. gradient of
0.001 kcal/mol/Å with the help of ABNR method as
described above. The 30 energy-minimized structures were
then superimposed and the lowest energy conformation
occurring in the major cluster was taken to be the most
probable conformation.
Docking and scoring
Molecular docking is a computational method used to pre-
dict the binding of the ligand to the receptor binding site by
varying position and conformation of the ligand keeping the
receptor rigid. Binding mode of inhibitors can be explored
by using the LigandFit docking protocol (Venkatachalam
et al., 2003). LigandFit algorithm performed a shape com-
parison filter with a Monte Carlo conformational search
Validation of the docking methodology
Docking was first tested on known inhibitor—etravirine,
which was docked in the allosteric binding site of HIV-1

Med Chem Res
RT, after extracting the ligand from the crystal structure and
then the molecules under study were docked with HIV-1 RT
(Table 2). Results were in good agreement with the
observed values of etravirine. The root mean square
deviation (RMSD) of the best docked poses of the mole-
cules reported herein was less than 2 Å, which was sup-
posed to be an appropriate condition for developing
NNRTIs. Two structures overlapped very well with posi-
tional RMSD of 1.2. The docked pose having the highest
LUDI III score gave the least RMSD with respect to the
crystal conformation for both the ligands. The analysis of
the results showed a sound validation of the docking and
scoring methodology used in this study and the ligand as a
whole moved into a more stable position with a much lower
docked energy.
production stage are stored in the simulation trajectory,
from which structural and energetics properties can be
calculated and subsequently analyzed (Carlsson et al.,
2008).
MD with desmond
The bound and free state of compound 10 was prepared
similarly for MD using the Desmond 3.1 MD package. To
maintain an electrical neutrality of unit cell, sodium and
chloride ions were added to reach a final concentration of
0.15 M Na+ and the system was solvated with TIP3P water
molecules after reorientation to minimize the volume in an
orthorhombic box.
The Amber force fields were applied to these periodic
systems. The default Desmond minimization and equili-
bration procedure was followed, except for the maximum
number of steps for SD and total minimization were
increased to 1000 and 5000 steps, respectively. Simulations
were kept at constant pressure (1 atom) and temperature
(335 K) maintained with a Berendsen barostat and ther-
mostat, respectively. SHAKE was applied to all systems
allowing a 2-fs time-step. Long-range interactions were
treated with the Particle Mesh Ewald method for periodic
boundaries using a nonbonded cutoff of 8.0 Å and the
nonbonded list was updated frequently using the default
settings (Miller et al., 2014).
MD simulation
We performed MD simulation of the bound and free state
for compound 10 using the DS 3.0 software in CHARMm
force field to study the relationship between biological
activity and energetic properties. First, the compound was
solvated in an orthorhombic solvent model with explicit
spherical boundary with harmonic restraint solvation con-
dition, which was used as input for standard dynamics
cascade protocol.
The Standard Dynamics Cascade includes two stages of
energy minimization followed by three stages of dynamics,
including heating, equilibration, and production. The
structures were firstly energy relaxed with 1000 steps of
steepest-descent energy minimization followed by another
1000 steps of conjugate gradient energy minimization. The
RMS gradients 0.1 and 0.001 kcal/mol/Å were used for the
steepest descent (SD) and conjugate gradient algorithms,
respectively. An initial minimization stage, typically using
the robust SD algorithm to resolve any initial poor contacts
within the system without creating large distortions in the
overall structure and a second minimization stage, typically
using the ABNR method was adopted. A MD simulation
heating stage is then employed to add thermal energy to the
system to reach a target temperature. The heating dynamics
started from 50 K and gradually increased to reach the target
temperature of 300 K in 50,000 steps with a time step of 1
fs. Then the structures were forced to a 1 ns equilibration
MD run followed by a 5 ns production MD run. The pur-
pose of the equilibration stage is to ensure that the energy in
the system is distributed appropriately among all degrees of
freedom. This allows the system to achieve thermal equi-
libration at the target temperature. A NPT stimulation
condition (using a leap-frog Verlet integration algorithm)
with 2 fs time step and 310 K temperature was used in the
MD run. The total energy of the stimulated systems turned
out to be stable after 5 ns MD stimulation. The results of the
Biological evaluation
Anti-HIV screening using TZM-bl cells
TZM-bl cells (4 × 104/well) were seeded in 24-well plate
and cultured overnight at 37 °C in humidified atmosphere of
carbondioxide (5 %). Different vials containing HIV-1
NL4.3 at a concentration equivalent to 0.05 multiplicity of
infection with respect to seeded TZM-bl cells were treated
with various compounds along with appropriate vehicle
controls for 1 h at 37 °C. Subsequently, pre-treated virus
was added to TZM-bl cells growing in 24-well plate and
incubated for 4 h at 37 °C in humidified atmosphere of
carbon dioxide (5 %). After incubation, the culture super-
natant was removed and the cells were washed with cold 50
mm PBS followed by addition of fresh culture medium with
or without compounds/formulations. Cells were further
cultured for 48 h in humidified atmosphere of carbondioxide
(5 %). Varying concentrations of known anti-HIV drug,
such as AZT, was used as positive reference control
whereas negative control comprised of untreated cells. After
incubation, the cells were washed twice with PBS and 1X
Promega cell culture lysis buffer (Promega Corporation,
USA) and left for 30 min at 4 °C and lysates were

Med Chem Res
centrifuged at 12,000 rpm for 10 min at 4 °C. The super-
natant was checked for the luciferase activity using the
appropriate substrate (Promega Corporation, USA). The
result expressed as percentage inhibition was calculated by
taking the read out (luciferase activity) in experimental
group (i.e., in the presence of compounds) divided by the
read out in infected cells in the absence of test compound
multiplied by hundred. Percent inhibition was calculated by
subtracting the above value from hundred.
the template was bound to anti-DIG-POD antibody. The
unbound anti-DIG-POD was washed and the peroxide
substrate (ABST) was added to the MTP. A colored reaction
product was produced during the cleavage of the substrate
catalyzed by a peroxide enzyme. The OD of the sample was
determined as OD at 405 nm using microtiter plate ELISA
reader. The resulting color intensity is directly proportional
to the actual RT activity. The percentage inhibitory activity
of RT inhibitors was calculated by comparing to a sample
that did not contain an inhibitor.
MTT cell cytotoxicity assay
The percentage inhibition was calculated by formula as
given:
The cytotoxicity of compounds on TZM-bl cells was
assayed by MTT assay (Mosmann, 1983). The MTT assay
is based on reduction of the yellow colored 3-(4, 5-dime-
thylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT)
by mitochondrial dehydrogenases of metabolically active
cells (live cells) to a blue colored formazan, which can be
measured spectroscopically. Briefly, 3 × 103 TZM-bl cells
were seeded in 96 well cell culture plates (Greiner Bio-One,
GmbH, Frickenhausen, Germany) and incubated at 37 °C
in a humidified atmosphere of carbon dioxide (5 %). After
24 h, cells were treated with varying concentrations of the
compounds ranging from 10–400 μg/mL, for 48 h. Negative
control included cells treated with solvent/medium. After
incubation, cell viability was assessed by adding 20 μL
MTT (5 mg/mL in 50 mM PBS) per well and incubated at
37 °C for 3 h followed by addition of MTT solvent (100 μL/
well; 20 % SDS and 50 % dimethyl formamide in 50 mm
PBS). The absorbance (OD) was read at 570 nm with
reference filter at 690 nm (690 nm reading was used as
blank). Experiments were performed in duplicate and the
percent viability was calculated from the equation:
% Inhibition ¼ 100 ꢀ ½OD 405 nm with inhibitor=
OD 405 nm without inhibitor ´ 100ꢁ
Results and discussion
Designing of NNRTIs
We have examined 55 arylsulphonamide derivatives to
develop some potent and selective inhibitors of HIV-1 RT
by using in silico structure-based approach. On the basis of
preliminary in silico investigations, we selected ten pro-
mising molecules for further studies. All compounds have
amide and sulphonamide linkages. The presence of C=O
dipole, N–C dipole, S=O dipoles, and S–N dipole allowed
these molecules to function as H-bond acceptors as well as
H-bond donors. Since these molecules have both H-donor
and H-acceptor sites, they can ionize at an appropriate pH to
further enhance their solubility. Thus, these molecules have
potential to internalize better in cells. Some physically
significant descriptors and pharmaceutically relevant prop-
erties of designed arylsulphonamides, like molecular
weight, H-bond donors, H-bond acceptors, logP (octanol/
water) have been analyzed on the basis of Lipinski’s rule of
five and the results are summarized in Table 1. Molecules
showing more than one type of violations are supposed to
have problems with bioavailability, hence rejected. The
intermediate TPSA values of these compounds in compar-
ison with nevirapine and etravirine were expected to help in
better cellular internalization.
% Viability
n
o
ðOD drug treated culturesÞꢀðOD untreated virus control culturesÞ
¼
´ 100%
ðOD uninfected culturesÞꢀðOD untreated virus control culturesÞ
HIV-reverse transcriptase (RT) inhibitory activity
The ability of the compounds to inhibit HIV-RT activity
was analyzed using the commercially available ELISA kit
(Roche Diagnostics GmbH, Roche Applied Science, Man-
nheim, Germany) according to the instructions of the
manufacturer. In brief, the reaction mixture containing
template/primer complex, 2′- deoxynucleoside-5′-tripho-
sphates (dNTPs) and RT enzyme in the lysis buffer were
incubated for 1 h at 37 °C and subsequently, the reaction
mixture was transferred to streptavidine-coated microtitre
plate (MTP). The biotin labeled dNTPs that are incorpo-
rated in the template due to activity of RT bind to strepta-
vidine. The unbound dNTPs were washed using wash
buffer and anti-digoxigenin-peroxidase (DIG-POD) was
added onto MTP. The DIG-labeled dNTPs incorporated in
Chemistry
Arylsulphonamide derivatives have been synthesized via 4-
(4chloro benzenesulphonylamino)-benzoyl chloride starting
from p-amino benzoic acid as shown in Scheme 1. For
synthesis of 4-(4-chloro-benzenesulphonylamino)-benzoic
acid, green protocols were applied. To an aqueous solution
of 4-amino benzoic acid 1, maintained at pH 9 with aqueous
sodium bicarbonate solution, 4-chloro benzenesulphonyl
chloride was added drop wise under cold conditions. In this

Med Chem Res
Table 1 Arylsulphonamide derivatives 4–13 in the eyes of Lipinski’s
rule of five
hydrogen bond, nonbonded “π–π” and “π–+” interactions as
they help stabilize and strengthen the receptor-ligand
complex. The “π–π” and “π–+” interactions are non-cova-
lent interactions, which have pivotal role in protein-ligand
recognition and are termed as major forces that stabilize the
association. According to Gallivan and Dougherty, “π–+”
interaction energies are considered of the same order of
magnitude as hydrogen bonds or salt bridges and play an
important role in molecular recognition and interaction with
ligands. The distance for “π–+” interactions in the present
studies was observed well below the limit of 6 Å as sug-
gested by Gallivan and Dougherty (1999) for optimum
receptor-ligand recognition.
Compound MWa
H–Ab H–Dc LogPd TPSAe Violations
4
387.848
6
6
9
7
6
6
7
6
9
5
5
7
2
2
2
2
2
2
3
1
1
2
1
3
3.348
4.109
88.15
88.15
0
0
0
1
1
2
0
0
0
1
1
1
5
466.744
432.845
487.99
6
3.259 133.98
7
5.214
5.459
5.59
97.39
88.15
88.15
8
478.382
522.833
426.885
411.874
456.867
436.922
9
10
11
12
13
3.936 103.95
4.414 81.06
4.324 126.89
5.455
1.380
75.26
63.58
Nevirapine 266.306
Etravirine 435.28
Structure-activity relationship (SAR)
5.027 120.65
a
Docking of compounds 4–13 using DS 3.0 into the active
site of target protein (PDB: ID 3MEC) generated a number
of possible binding conformations with the corresponding
Ludi_3 values. The cluster analysis revealed a predominant
orientation of the ligands within the binding pocket of
active site and the conformation with the highest Ludi_3
value for each molecule was chosen for further analysis.
Considering the magnitude of interacting forces for recep-
tor-ligand interaction, the entire set of molecules was rig-
orously docked into the allosteric site of HIV-1 RT, using
the same protocol, and analyzed through the above-men-
tioned parameters, and the results are presented in Tables 2
and 3.
Visual inspection of the minimized complexes of inhi-
bitors with HIV-1 RT showed the optimal electronic and
hydrophobic interactions between the enzyme and the
ligands. These derivatives, like other second generation
non-nucleoside inhibitors, adopted a common “Horse-shoe”
conformation. Nevertheless, there are important differences
in their conformations and specific positioning within the
NNIBP. Compounds 6 and 10 adopted a “Horse-shoe”
conformation (Fig. 2), whereas compounds 4, 5, 7, 8, 9, 11,
12, and 13 showed a “Sea-horse” conformation (Figs. 3, 4),
which closely resembled “Horse-shoe” conformation.
To explore the SAR profile, the role of substituents at 5-
position of pyridine ring of compounds 4, 5, 6 was ana-
lyzed. Three types of derivatives, bearing a hydrogen (4),
bromine (5), and nitro group (6) were studied. The com-
pound 4 bearing a hydrogen at 5-position formed four H-
bonds: the first H-bond between the –SO– group of sul-
phonamide moiety and Lys103 with a distance of 2.7 Å, the
second H-bond between –NH of the sulphonamide moiety
with Tyr318 and other H-bonds formed between benzamide
moiety and Gly190 and Tyr188 present in the NNIBP with
interatomic distance of 3.0 Å each. The compound 5, having
Br atom in place of hydrogen, formed no H-bonds but
showed one π interaction with Tyr181 with an interatomic
Molecular weight
b
Number of H-bond acceptors
Number of H-bond donors
c
d
e
Octanol-water partition coefficient
Total polar surface area
step, we selected water as solvent instead of organic sol-
vents because the reaction of aminobenzoic acids and sul-
phonyl chloride in organic solvents in the presence of
organic amine bases gave poor yield in our earlier experi-
ment, and this was also corroborated by cited literature
(Deng and Mani, 2006) . Contents were stirred at room
temperature and on completion of the reaction, the medium
of the reaction mixture was adjusted to pH 2 using hydro-
chloric acid (1 M) and the precipitate obtained was filtered,
washed with water and dried. The resulting solid was
recrystallized from ethanol.
To a solution of compound 2 in dry acetonitrile con-
taining pyridine as a catalyst, was added oxalyl chloride
drop wise in cold and anhydrous conditions. The reaction
mixture was stirred for 1 h, extra oxalyl chloride removed
under pressure and the resultant solution was added drop
wise to a acetonitrile:pyridine (1:1) solution of different
heterocyclic amines in cold and anhydrous condition to get
the desired compounds, (Scheme 1), which were purified by
recrystallization from ethanol:water in good yields (Rai and
Singh, 2011, Parang et al., 1998).
Molecular modeling analysis
Recently, molecular docking has emerged as a powerful
tool in drug designing, predicting the best mode by which a
given compound fits well into a binding site of a macro-
molecular target (Chen et al., 2006). We have docked the
molecules within the NNIBP of HIV-1RT and docking
results have been discussed using the parameters, like

Med Chem Res
Table 2 DS 3.0 docking results showing the scoring of ligands and stability of HIV-1 RT-ligand complexes
c
g
pred
h
Name
I.Ea
DSb
-PLP1c
-PLP2
-PMFd
LIGe
Ludi_2f
ΔG
Ludi_3f
Predicted EC50
4
−77.01
−58.35
−47.85
−76.07
−66.50
−72.37
−69.20
−64.79
−73.68
−61.29
−45.79
−61.43
59.49
50.06
61.25
57.89
59.92
59.91
49.79
50.17
54.40
48.21
40.69
55.05
107.3
99.68
77.55
119.6
109.74
106.18
105.49
82.52
94.4
80.66
100.4
87.35
70.66
68.57
69.6
−6.04
−4.44
−8.23
−8.31
−2.65
−2.63
−4.50
−6.47
−6.33
−6.03
−5.35
−6.46
484
313
394
506
528
532
372
423
409
527
393
501
−6.86
−4.43
−5.58
−7.17
−7.48
−7.54
−5.27
−6.0
752
450
566
779
851
865
543
589
583
847
557
766
0.003 × 10−5
1.9 × 10−5
5
76.64
6
122.71
113.59
109.06
108.53
84.34
0.21 × 10−5
0.001 × 10−5
0.0003 × 10−5
0.0002 × 10−5
0.37 × 10−5
0.12 × 10−5
0.14 × 10−5
0.0003 × 10−5
0.27 × 10−5
0.002 × 10−5
7
8
9
10
96.28
59.09
51.67
57.67
103.6
80.9
11
97.94
12
102.25
109.7
96.09
103.95
90.29
100.08
−5.80
−7.47
−5.57
−7.10
13
14 Nevirapine
94.81
15 Etravirine
107.62
a
Total interaction energy (kcal/mol)
Dock score
b
c
d
e
f
Piecewise linear potential
Potential of mean force
Ligand internal_energy
Ludi_2 and Ludi_3, empirical scoring functions derived from the Ludi algorithm
Predicted binding free energy(kcal/mol)
g
h
Predicted 50 % effective concentration required to inhibit HIV-1 replication (μM)
distance of 5.9 Å. The nitro group introduced at 5-position,
in compound 6, formed one H-bond with Lys101 and
showed π-cation interaction with Trp229 with interatomic
distance of 5.9 Å. The compound 4 showed highest stabi-
lization energy, i.e., (−) 77.01 kcal/mol whereas compounds
5 and 6 showed (−) 58.35 and (−) 47.85 kcal/mol, respec-
tively. Thus, it may be assumed that at 5-position on pyr-
idine ring smaller groups are preferred for better orientation
of the molecules in the hydrophobic pocket.
Compound 10 contains indazole group as heterocyclic
amine part, which in the protein-ligand complex is sur-
rounded by the β5–β6 loop (Pro95, Leu100, Lys101, and
Lys103). The indazole ring showed π-cation interaction
with Lys101 and N atom of indazole nucleus formed one
hydrogen bond with Gly99 (3 Å). Central benzene ring of
compound 10 showed π interaction with Tyr181. The sta-
bilization energy of the protein-ligand complex in the case
of compound 10 was (−) 69.20 kcal/mol.
In compounds 7, 8, and 9 pyridine ring is replaced by
benzothiazole group where compound 7 has an ethoxy,
compound 8 a chloro, and compound 9 a bromo group at 4-
position. All three compounds have shown similar orien-
tation within NNIBP. However, heterocyclic core of the
molecules despite being flanked by the aromatic amino acid
residues did not show π interaction. Sulphonamide linkage
formed one H-bond with Lys103 in all three compounds.
The compound 7 showed highest stabilization energy, i.e.,
(−) 76.07 kcal/mol whereas compounds 8 and 9 showed (−)
66.50 and (−) 72.37 kcal/mol, respectively. So it may be
assumed that a larger group in benzothiazole ring better
stabilizes the protein-ligand complex.
So, it was clear from the SAR analysis that more stable
protein-ligand complexes were formed with compounds 4
and 7, and this behavior can be explained on the basis of
different orientations adopted by the molecules as a whole
inside the NNIBP and this fact also indicated about the
degree of flexibility of NNIBP.
Compounds 11 and 12 contain hydrogen and nitro group,
respectively, at 4-position of benzimidazole nucleus as
heterocyclic part, which during complexation with HIV-RT
is surrounded by the β5–β6 loop. In both compounds ben-
zimidazole nucleus showed π-cation interaction with
Lys103 (6.5 Å and 6.0 Å). In compound 11, –NH– part of
amide linkage formed one hydrogen bond with Tyr181 (2.8
Å). The stabilization energy of compounds 11 and 12 was
(−) 64.79 and (−) 73.68 kcal/mol, respectively. Thus, nitro
group at 4-position of benzimidazole nucleus better stabi-
lized the RT-ligand complex.
In compound 13, –SO– group of sulphonamide moiety
formed one H-bond with Val189 (2.9 Å), –NH– group of
amide linkage formed another H-bond with H is235 (2.9 Å)
and benzene ring of arylsulphonamide unit showed
π-interaction with Tyr181 (5.0 Å). The stabilization energy
of the protein-ligand complex in this case was (−) 61.29
kcal/mol, very close to that of etravirine [(−) 61.43
kcal/mol].

Med Chem Res

Med Chem Res
4
5
7
8
Fig. 2 Interaction of compound 4, 5, 7, and 8 with HIV-RT showing “Sea-Horse” conformation
In all molecules, H-bonds were formed between –SO–
and –NH– of the sulphonamide moiety and different amino
acid residues, like Lys101, Lys 103 and Tyr188, Tyr318,
respectively, present in the NNIBP of HIV-1 RT. In addi-
tion to this, in the case of compounds 4, 11, and 13, H-
bonds were formed between carbonyl group of benzamide
moiety and different amino acid residues, like Tyr181,
Gly190, and His235 present in the NNIBP.
hydrophobic amino acid Lys103, present in the NNIBP,
through favorable π-stacking and aliphatic interactions,
whereas the benzene ring of arylsulphonamide and hetero-
cyclic amine cores acted as wing I and II, respectively.
The SAR data, as shown in Table 3 revealed that the
arylsulphonamide core played an important role in the sta-
bilization of these compounds within HIV-1 RT. These
compounds interacted with residues Lys101, Lys103,
Gly99, Gly190, and Tyr318 through H-bonds and with
residues Lys103, Trp229, Tyr181, Tyr188, and Tyr318
The proposed molecules interacted with the aromatic
amino acids Tyr181, Tyr188, Tyr318, and Trp229, and the

Med Chem Res
9
11
12
13
Fig. 3 Interaction of compound 9, 11, 12, and 13 with HIV-RT showing “Sea-Horse” conformation
through π-interactions, whereas in the case of nevirapine, H-
bonding was absent. The ligands interacted through π-π
interactions with Tyr181 and Trp188. In the case of etra-
virine Lys101 was involved in H-bonding and Tyr181 in π–
π interactions. SAR studies on all inhibitors revealed that
sulphonamide linkage played a crucial role in the stabili-
zation of protein-ligand complex. As discussed previously,
the sulphonamides containing H-bond acceptors directly
interacted with Lys103 backbone of HIV-1 RT, which was
similar to interactions shown by many “next generation
inhibitors”. In the case of compounds 4, 6, 7, 8, 9, and 11,
the sulphonamide linkage directly interacted with Lys103
backbone of HIV-1 RT. Superimposition of all minimized
frames showed that the proposed inhibitors did bind to the
HIV-1 RT inside the NNIBP in a common mode as pro-
posed for second generation NNRTI—etravirine.
Scoring
Ligand scoring is a method to rapidly estimate the binding
affinity of a ligand and is based on ligand pose geometry
docked into a target receptor structure. To define binding
affinity of ligands with receptor in a better way, hybrid title
analogs and reference etravirine and nevirapine (for

Med Chem Res
6
10
Fig. 4 Interaction of compound 6 and 10 with HIV-RT showing “Horse-shoe” conformation
Fig. 5 Molecular dynamics trajectory plots correlating RMSD deviation from the compound 10 heavy atoms (1) coordinates, 10-HIV-RT Cα
atoms (2) and side chain (3) over a simulation time of 50 ns
comparison) were rigorously analyzed through scoring
functions, such as PLP1, PLP2, PMF, Lig_Internal_Energy,
binding energy, dock score, and ultimately Ludi3.
Piecewise Linear Potential is a fast and simple docking
function that has been shown to explain well the protein-
ligand binding affinities. PLP scores are measured in arbi-
trary units and reported as negative values. Higher PLP
scores indicate stronger receptor-ligand binding. Two
versions of the PLP function available are: PLP1 and PLP2
(Gehlhaar et al., 1995). In the PLP1 function, each non-
hydrogen ligand or non-hydrogen receptor atom is assigned
a PLP atom type. Hydrogen atoms are excluded from
consideration. In PLP2 function, PLP atom typing remains
the same as in PLP1. In addition, an atomic radius is
assigned to each atom except for hydrogen. It is worthwhile
to mention that majority of title compounds showed

Med Chem Res
Table 4 Standard dynamics cascade summary of compound 10 in free state and with HIV-RT docked complexes
Sl. no.
1
Step
Summary
Compound 10 in free state
Compound 10 HIV-RT complexes
Minimization 1
Initial potential energy (kcal/mol)
Potential energy (kcal/mol)
vdW energy (kcal/mol)
32.10076
32.10134
2.74492
−8340.58952
−10539.55789
−4621.70880
−8522.25643
0.86388
Electrostatic energy (kcal/mol)
Final RMS gradient (kcal/(mol × Å)
Initial potential energy (kcal/mol)
Potential energy (kcal/mol)
vdW energy (kcal/mol)
−4.19950
8.66859
2
3
Minimization 2
32.10076
32.08020
2.63992
−10,539.55866
−12,770.44898
−4487.25436
−10,893.78873
0.39797
Electrostatic energy (kcal/mol)
Final RMS gradient (kcal/(mol × Å)
Initial potential energy (kcal/mol)
Total energy (kcal/mol)
−4.18604
0.86311
Heating
32.08025
109.71697
64.94987
44.76710
357.58459
4.54424
−12,770.44309
2700.20118
−5060.07977
7760.28095
286.75339
Potential energy (kcal/mol)
Kinetic energy (kcal/mol)
Temperature (K)
vdW energy (kcal/mol)
−3840.98991
−10,666.67554
20.66100
Electrostatic energy (kcal/mol)
Final RMS gradient (kcal/(mol × Å)
Initial potential energy (kcal/mol)
Total energy (kcal/mol)
−4.36391
21.59726
64.94987
110.51382
68.93120
41.58262
332.14803
9.53231
4
Equilibration
−5060.07977
2995.97563
−5158.54680
8154.52243
301.32117
Potential energy (kcal/mol)
Kinetic energy (kcal/mol)
Temperature (K)
vdW energy (kcal/mol)
−3815.51532
−11,001.36621
20.75912
Electrostatic energy (kcal/mol)
Final RMS gradient (kcal/(mol × Å)
Initial potential energy (kcal/mol)
Total energy (kcal/mol)
−3.93326
24.43701
68.93120
110.32260
72.40481
37.91779
302.87458
3.91600
5
Production
−5158.54680
2985.11516
−5204.98398
8190.09914
302.63578
Potential energy (kcal/mol)
Kinetic energy (kcal/mol)
Temperature (K)
vdW energy (kcal/mol)
−3826.92446
−11,266.66037
20.95726
Electrostatic energy (kcal/mol)
Final RMS gradient (kcal/(mol × Å)
−4.95205
22.27851
comparable PLP scores to the reference ligands and com-
pounds 4 (107.3), 8 (109.06), 9 (108.5), and 13 (109.7)
exhibited PLP1 score very close to etravirine (107.62).
The PMF scoring functions were developed on the basis
of statistical analysis of the 3D structure of protein-ligand
complexes and were found to correlate well with protein-
ligand binding free energies. The scores are calculated by
summing pair wise interaction terms over all interatomic
pairs of the receptor-ligand complex (Muegge and Martin,
1999). In a comparison test, it was found that all title
compounds showed significant PMF scores with reference
to standard ligand. The internal nonbonded ligand energy is
calculated for each new conformation that is generated. The
unsuitable conformations with high internal ligand energies
(typically resulting from internal close contacts) are dis-
carded. The internal ligand energy consists of a vdW term
and an optional electrostatic term. The nonbonded vdW
energy is computed using a standard 9–6 (unsoftened)
potential using force field parameters consistent with the
force field employed. Efficient ligand is classified with high
negative ligand internal energy. All title hybrid analogs
demonstrated significantly high negative values as com-
pared to that of the reference.
All the scoring data for the title hybrid compounds along
with reference are presented in Table 2. Candidate ligand
poses were evaluated and prioritized according to the Dock

Med Chem Res
Score function. It was clear from Table 2 that the reference
ligand etravirine exhibited dock score of 55.05 and the
hybrid compounds 4–13 showed dock score ranging from
48.21 to 59.91, which suggested that all molecules have
similar mode of binding and significant binding affinity
toward NNIBP receptor domain as that of the reference
ligand. Further, the docking energy as shown in Table 2
reflected the stability of RT-ligand complexes, which
showed that majority of the ligands moved into a stable
position comparable to nevirapine and etravirine. Binding
energy of the title molecules as well as of reference mole-
cules was analyzed from their corresponding docked con-
formations in receptor. Results illustrated that binding
energy of compounds 6 (−5.58), 10 (−5.27), 11 (−6.0), and
12 (−5.80) little bit similar to that of reference nevirapine
(−5.57), whereas in the case of compounds 4 (−6.86), 7
(−7.17), 8 (−7.48), 9 (−7.54), and 13 (−7.47), the binding
energy was similar to that of reference etravirine (−7.10).
The binding free energy (ΔG) and Predicted EC50 for each
derivative were predicted using the Ludi_2 and Ludi_3
scoring functions, respectively (Lagos et al., 2008; Kumar
et al., 2010). The present study on all these compounds
proved that higher binding energy (ΔG) was associated with
higher affinity, which corresponded to lower EC50 value as
shown in Table 2.
Molecular dynamic analysis
Further, the stability of the compound 10-RT complex
obtained from the docking calculations was assessed via
MD simulations. The RMSD values for the protein Cα
atoms, side chain as well as heavy atoms were calculated by
aligning the MD production phase trajectories to their initial
structures. The RMSD plots show that 10-RT complex was
stable during the production phase of the MD simulations
(Fig. 5). The summary of standard dynamic cascade as
presented in Table 6 further supported that 10-RT complex
demonstrated higher stability as compared to that of the
compound 10 in the unbound state.
Table 5 Cytotoxicity and anti-HIV activity of compounds 4–13
b
c
Compound % Inhibition
EC50 (μM)
CC50 (μM) SId
(at 50 μM)a
4
−30
−52
−36
−30
−50
−39
59
–
–
–
–
–
–
–
–
–
–
–
–
–
5
–
On the basis of extensive docking experiments using the
DS 3.0software, ten promising compounds, 4–13, out of 55
initially taken for this study, were finally synthesized and
screened for their anti-HIV activity.
6
–
7
–
8
–
9
–
10
0.00489 ×
10−5
0.0120 ×
10−5
2.45
Biological evaluation
Anti-HIV activity
11
12
13
−33
−52
−49
–
–
–
–
–
–
–
–
–
Nevirapine 99
0.00050 ×
10−5
>10
>200
All compounds were evaluated for their biological activity
against WT HIV-1 strain III-B tusing TZM-b1 cells and The
results expressed as EC50, CC50, and selectivity index (SI)
values in Table 4. Etravirine and nevirapine were used as
reference drugs. The result of anti-HIV assay demonstrated
that the molecules did inhibit HIV expression, albeit, to a
very low degree. The compound 10, nevertheless, exhibited
high EC50 value in the range of 4.89 × 10−5 nm but,
a
Data represent the mean of two and three independent assays for
EC50 and CC50, respectively
b
EC50 is the 50 % effective concentration, which is the concentration
needed to inhibit 50 % virus replication in vitro
c CC50 is the 50 % cytotoxic concentration, which is the concentration
required to cause 50 % death of uninfected cells
d
SI ratio CC50/EC5
Table 6 HIV-RT assay of
compound 10
Sample
Inhibition of RT activity (%)
Time (in min)
0
0
0
0
0
5
0
10
0
15
0
20
0
25
0
30
1
35
1.1
40
1.7
9.12
12.1
45
45
0
10 (12.5 µM)
10 (25 µM)
4
0
8.41
9.46
7.44
9.5
8.37
12.23
45.26
9.49
10 (50 µM)
26
57
14.16
40
10.61
48.4
12.89
45.62
9.28
13.29
45.81
11.75
43.42
Nevirapine (1 µM)
44.85

Med Chem Res
Compliance with ethical standards
unfortunately, a low SI value—2.45, in comparison to
etravirine and nevirapine lowered its usefulness. Analysis of
the anti-HIV screening results (Table 4) revealed that all
molecules 4, 5, 6, 7, 10, 11, and 12 showed very low level
of in vitro anti-HIV activity in contrast to the predicted
EC50 values (Table 2) on the basis of docking studies with
HIV-1 RT.
Conflict of interest The authors declare that they have no conflict of
interest.
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On the basis of extensive docking experiments, ten pro-
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Acknowledgments Financial assistance in the form of WOS-A
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DST, New Delhi is sincerely acknowledged. SK Gupta would like to
acknowledge JC Bose Fellowship by Science and Engineering
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