Molecular Modeling, Synthesis, and Anti-HIV Activity of Novel Isoindolinedione Analogues as Potent Non-nucleoside Reverse Transcriptase Inhibitors

Article abstract of DOI:10.1111/cbdd.12620

Different isoindolinedione derivatives bearing imine, amide, thioamide, and sulfonamide linkages have been designed in silico using discovery studio software (BIOVIA, San Diego, CA, USA), synthesized, and evaluated for their anti-HIV activity. SAR studies revealed that the linkages in these molecules did affect their anti-HIV activity and the molecules having sulfonamide linkages were the most potent HIV-RT inhibitors as the S=O bonds of the sulfonamide moiety interacted with Lys103 (NH or carbonyl or both) and Pro236; the NH part of the sulfonamide linkage formed bond with carbonyl of Lys101. blood-brain barrier (BBB) plots were also studied, and it was found that all the designed molecules have potential to cross BBB, a very vital criteria for anti-HIV drugs. In vitro screening was performed using HIV-1 strain IIIB in MT-4 cells using the MTT assay, and it was seen that some of these molecules were effective inhibitors of HIV-1 replication at nanomolar concentration with selectivity indices ranging from 33.75 to 73.33 under in vitro conditions. Some of these molecules have shown good anti-HIV activity at 3-4 nm concentrations. These derivatives have potential to be developed as lead molecules effective against HIV-1. Novel isoindolinedione derivatives as probable NNRTIs have been synthesized and characterized. Some of these molecules have shown good anti-HIV activity at 3-4 nm concentrations. Novel isoindolinedione derivatives as probable NNRTIs have been synthesized and characterized. Some of these molecules have shown good anti-HIV activity at 3 - 4 nM concentrations.

Full text of DOI:10.1111/cbdd.12620

Chem Biol Drug Des 2016; 87: 200–212
Research Article
Molecular Modeling, Synthesis, and Anti-HIV Activity
of Novel Isoindolinedione Analogues as Potent
Non-nucleoside Reverse Transcriptase Inhibitors
grase (IN) of HIV (1–5). To date, the armamentarium of
antiretrovirals against HIV is made up of 36 approved drug
products belonging to six classes: nucleoside and nucleo-
tide reverse transcriptase inhibitors (NRTIs and NtRTIs),
non-nucleoside reverse transcriptase inhibitors (NNRTIs),
protease inhibitors, entry inhibitors, and HIV integrase
strand transfer inhibitors. Highly active antiretroviral therapy
(HAART) based on combination of at least two or different
antiretroviral drugs from different drug classes proved to
improve dramatically the prognosis of HIV-1 infection (6).
Garima Kumari and Ramendra K. Singh*
Nucleic Acids and Antiviral Research Laboratory,
Department of Chemistry, University of Allahabad,
Allahabad, India
*Corresponding author: Ramendra K. Singh,
singhramk@rediffmail.com
Different isoindolinedione derivatives bearing imine,
amide, thioamide, and sulfonamide linkages have been
designed in silico using DISCOVERY STUDIO software (BIO-
VIA, San Diego, CA, USA), synthesized, and evaluated
for their anti-HIV activity. SAR studies revealed that the
linkages in these molecules did affect their anti-HIV
activity and the molecules having sulfonamide linkages
were the most potent HIV-RT inhibitors as the S=O
bonds of the sulfonamide moiety interacted with
Lys103 (NH or carbonyl or both) and Pro236; the NH
part of the sulfonamide linkage formed bond with car-
bonyl of Lys101. blood–brain barrier (BBB) plots were
also studied, and it was found that all the designed
molecules have potential to cross BBB, a very vital cri-
teria for anti-HIV drugs. In vitro screening was per-
formed using HIV-1 strain IIIB in MT-4 cells using the
MTT assay, and it was seen that some of these mole-
cules were effective inhibitors of HIV-1 replication at
nanomolar concentration with selectivity indices rang-
ing from 33.75 to 73.33 under in vitro conditions. Some
of these molecules have shown good anti-HIV activity
at 3–4 nM concentrations. These derivatives have
potential to be developed as lead molecules effective
against HIV-1.Novel isoindolinedione derivatives as
probable NNRTIs have been synthesized and charac-
terized. Some of these molecules have shown good
anti-HIV activity at 3–4 nM concentrations.
Highly active antiretroviral therapy has been proven to be
effective in controlling disease progression and prolonging
the survival of HIV-infected patients. However, the situation
becomes challenging with the development of resistant
viral strains and the emergence of various side-effects (7).
Therefore, new potential drugs that display a broad spec-
trum of activity against clinically relevant HIV-1 mutant
strains are needed.
Non-nucleoside reverse transcriptase inhibitors (NNRTIs) are
non-competitive inhibitors that bind to a hydrophobic
pocket, non-nucleoside binding pocket (NNBP), near the
DNA polymerase active site of the RT enzyme (8–12). The
main attraction of NNRTIs is that these are characterized by
low toxicity and highly favorable pharmacokinetic profile (13).
In spite of various advantages, only four NNRTIs, viz. nevi-
rapine, delavirdine, efavirenz, and etravirine (Figure 1) have
been approved for clinical use by the FDA (14,15). How-
ever, treatment with these NNRTIs results in the rapid
emergence of RT mutants (16). Therefore, there is a need
of developing novel NNRTIs with enhanced therapeutic
value and different resistance mutation profiles to success-
fully employ this class of drugs in HAART.
Key words: anti-HIV activity, blood–brain barrier, isoindoline-
diones, non-nucleoside reverse transcriptase inhibitors
Received 2 February 2015, revised 4 April 2015 and accepted
for publication 6 July 2015
Here, we report a new class of NNRTIs having isoindoline-
dione moiety with imine, amide, thioamide and sulfon-
amide linkers, and different substituents (Figure 1). These
molecules have been designed following Lipinski’s rule of
five and synthesized after successful docking into NNBS
of HIV-RT using DISCOVERY STUDIO 2.5 software (BIOVIA).
The identification of HIV as the causative agent of acquired
immunodeficiency syndrome (AIDS) has led to consider-
able medical advances in order to develop a variety of
drugs targeting mainly the following key enzymes—reverse
transcriptase (RT), protease (PR), and more recently inte-
SAR studies revealed that compounds containing sulfon-
amide linkers were more potent than the analogues having
200
ª 2015 John Wiley & Sons A/S. doi: 10.1111/cbdd.12620

Modeling, Synthesis and Anti-HIV Activity
N
N
O
O
N
R
X
Y
N
N
N
N
CF3
O
N
N
HN
N
NHSO2Me
HN
O
Cl
CH
3O
N
NH
Br
N
X= CH, CO, SO2, CS
Y= N, NH
N
H
O
H
NH2
O
Nevirapine
Efavirenz
Delavirdine
Etravirine Isoindolinedione derivatives
Figure 1: Structural compatibility of approved NNRTIs and isoindolinedione derivatives against HIV-1.
amide and thioamide linkers, which, in turn, were more
potent than the molecules with imine linkers. The influence
of these linkers on anti-HIV activity of various compounds
is reported and discussed. The anti-HIV activity of these
compounds has been evaluated both by computational
and experimental methods. Also a study on blood–brain
barrier (BBB) has also been carried out to see whether
these molecules will be able to cross BBB or not.
microwave (MW) oven at an appropriate power for an
appropriate time (till completion of the reaction as moni-
tored by TLC). After cooling to room temperature, the pro-
duct was dissolved in DCM, filtered, concentrated in
vacuo, and crystallized using hexane.
2-[4-(Benzylidene-amino)-2-methyl-phenyl]-5-
chloro-isoindole-1,3-dione (2)
Yield 97%; Power 600 W, 15 min; Rf 0.66 (DCM); m.p.
140–142 °C; kmax(MeOH) 265 nm; 1H NMR dH (DMSO-
d6)- 8.12 (s, 1H, -CH=N), 7.94–7.68 (m, 3H, ArH),
7.42–7.29 (m,5H, ArH), 7.32- 7.14 (m, 3H, ArH), 2.3 (s,
3H, CH3); 13C NMR d p.p.m.- 178.7 (C=N), 172.6 (C=O),
172.2 (C=O), 150.4 (ArC-N), 149.7 (ArC-N=), 148.4 (ArC-
Cl), 148.2 (ArC-CH3), 146.8, 144.9, 143.9, 142.5, 140.6,
139.8, 139.4, 136.2, 135.5, 134.1, 133.2, 132.8, 129.4
(Aryl Carbons), 17.8 (-CH3); MS (EI+) m/z 374.08 (100%);
Anal. Calcd C22H15ClN2O2: C, 70.50; H, 4.03; N, 7.47
Found C, 70.51; H, 4.02; N, 7.45.
Experimental Procedures
All microwave-irradiated reactions were carried out in a
domestic microwave oven. Silica gel G for TLC was
obtained from E Merck India Ltd (New Mumbai, India). All
reactions were performed in oven-dried flasks using anhy-
drous solvents. Melting points were determined with an
electrothermal apparatus and are uncorrected. UV spectra
were recorded on a Perkin-Elmer spectrophotometer (Perkin
Elmer, Billerica, MA, USA). 1H NMR spectra were recorded
on a Bruker AVANCE 400 MHz Fourier transform spectrom-
eter and using an internal deuterium lock. Chemical shifts
are quoted in parts per million downfield from tetramethylsi-
lane. 13C-NMR spectra were recorded at 100 MHz on Bru-
ker AMX 400 MHz spectrometer in CDCl3. Mass spectra
were recorded on Micromass Quattro II. Elemental analysis
was carried out using Heraeus Vario EL III Carlo Erba 1108
(Elemental Microanalysis Ltd, Okehampton, UK).
5-Chloro-2-{4-[(4-methoxy-benzylidene)-amino]-2-
methyl-phenyl}-isoindole-1,3-dione (3)
Yield 93%; Power 600 W, 10 min; Rf 0.63 (DCM); m.p.
182 °C; kmax(MeOH) 289 nm; 1H NMR dH (DMSO-d6)-
8.28 (s, 1H, -CH=N), 8.24–7.82 (m, 3H, ArH), 7.64–7.22
(m, 3H, ArH), 7.5–6.9 (m, 4H, ArH), 3.76 (s, 3H,
-OCH3),2.24 (s, 3H, CH3); 13C NMR d p.p.m.- 178.4
(C=N), 171.2 (C=O), 170.6 (C=O), 168.7 (ArC-OCH3),
150.8 (ArC-N), 149.4 (ArC-Cl), 146.5 (ArC-CH3), 144.2,
142.4, 141.8, 141.6, 140.9, 138.2, 136.6, 134.8, 132.8,
131.2, 129.2, 128.6, 127.9 (Aryl Carbons), 72.4 (-OCH3),
18.2 (-CH3); MS (EI+) m/z 404.09 (100%); Anal. Calcd
N-(4-Amino-2-methylphenyl)-4-chloro-phthalimide
(1)
For the synthesis of 1, 4-chlorophthalimide (1 mmol) was
ground together with anhydrous potassium carbonate
(0.55 mmol) in a glass mortar. The mixture was then trans-
ferred to a round-bottomed flask; 4-chloro-3-methylaniline
(1.2 mmol) was added and the reaction mixture refluxed
for 3 h. The resulting solid was then washed with 60%
ethanol and recrystallized with glacial acetic acid to yield a
yellow-colored powder. Yield 80%, Rf 0.46 (DCM), m.p.
208–210 °C.
C
23H17ClN2O3: C, 68.23; H, 4.23; N, 6.92 Found C,
68.25; H, 4.21; N, 6.95.
5-Chloro-2-{4-[(4-hydroxy-benzylidene)-amino]-
2-methyl-phenyl}-isoindole-1,3-dione (4)
Yield 95%; Power 800 W, 15 min; Rf 0.36 (DCM:MeOH,
9.8:0.2); m.p. 200 °C; kmax(MeOH) 310 nm; 1H NMR dH
(DMSO-d6)- 8.34 (s, 1H, -CH=N); 8.26–7.9 (m, 3H, ArH),
7.6–7.2 (m, 3H, ArH), 7.4 (d, 2H, ArH), 6.7 (d, 2H, ArH),
5.14 (s, 1H, -OH), 2.34 (s, 3H, CH3); 13C NMR d p.p.
m.- 179.2 (C=N), 172.6 (C=O), 171.8 (C=O), 170.9 (ArC-
General procedure for preparation of imines (2–6)
1
(0.5 mmol) mixed with the respective aldehyde
(0.5 mmol) in 50-ml beaker was irradiated in
a
a
Chem Biol Drug Des 2016; 87: 200–212
201

Kumari and Singh
OH), 158.8 (ArC-N=), 152.4 (ArC-N), 150.2 (ArC-Cl), 148.6
(ArC-CH3), 147.9, 146.2, 141.5, 140.8, 139.4, 137.4,
136.8, 135.4, 133.9. 132.6, 130.4, 129.8, 128.6 (Aryl Car-
bons), 19.2 (-CH3); MS (EI+) m/z 390.08 (100%); Anal.
Calcd C22H15ClN2O3: C, 67.61; H, 3.87; N, 7.17 Found
C, 67.62; H, 3.85; N, 7.14.
1H, NH), 8.0–7.7 (m, 3H, ArH), 7.6–7.2 (m, 7H, ArH), 7.5–
7.4 (m, 3H, ArH), 3.4 (s, 2H, ArH), 2.3 (s, 3H, - CH3); 13C
NMR d p.p.m.- 178.2 (-CONH), 173.4 (C=O), 170.6 (C=O),
147.3 (ArC-Cl), 145.2 (ArC-CH2), 144.5 (ArC-N), 142.4
(ArC-NH), 139.8 (ArC-CH3), 138.2, 136.3, 134.8, 134.4,
133.6, 131.9, 129.6, 128.9, 127.5, 124.8, 122.9, 121.6,
121.3, 120.9, 120.4, 119.3, 118.7, 49.4 (CH2-CONH),
17.8 (-CH3); MS (EI+) m/z 454.12 (100%); Anal. Calcd for
5-Chloro-2-{4-[(4-chloro-benzylidene)-amino]-2-
methyl-phenyl}-isoindole-1,3-dione (5)
C27H19ClN2O3: C, 71.29; H, 4.21; N, 6.16 Found: C,
71.28; H, 4.22; N, 6.18.
Yield 97%; Power 400 W, 5 min; Rf 0.9 (DCM); m.p.
157 °C; kmax(MeOH) 276 nm; 1H NMR dH (DMSO-d6)-
8.26 (s, 1H, -CH=N); 8.04–7.8 (m, 3H, ArH), 7.56–7.30
(m, 4H, ArH), 7.4–6.9 (m, 3H, ArH), 2.38 (s, 3H, CH3); 13C
NMR d p.p.m.- 175.8 (C=N), 173.2 (C=O), 171.8 (C=O),
153.5 (ArC-N=), 150.6 (ArC-N), 148.8 (ArC-Cl), 146.2
(ArC-Cl), 141.8 (ArC-CH3), 141.1, 140.4, 138.9, 136.2,
133.5, 131.8, 128.6, 127.4, 124.8, 122.2, 120.6, 119.5,
118.6, (Aryl Carbons), 16.4 (-CH3); MS (EI+) m/z 408.04
(100%); Anal. Calcd C22H14Cl2N2O2: C, 64.56; H, 3.45; N,
6.8 Found C, 64.53; H, 3.43; N, 6.85.
4-Chloro-N-[4-(5-chloro-1,3-dioxo-1,3-dihydro-
isoindol-2-yl)-3-methyl-phenyl]-benzamide (8)
Yield 77%; Rf 0.38 (DCM:MeOH, 9.9:0.1); m.p.
1
173–175 °C; kmax (MeOH) = 258 nm; H NMR dH (DMSO-
d6) 8.24 (s, 1H, NH), 8.16–7.78 (m, 3H, Ar-H), 7.86 (d,
2H, Ar-H), 7.54–7.38 (m, 3H, Ar-H), 7.4 (d, 2H, Ar-H),
2.18 (s, 3H, -CH3); 13C NMR d p.p.m.- 175.2 (CONH),
173.2 (C=O), 171.1 (C=O), 147.8 (ArC-Cl), 146.4 (ArC-Cl),
144.7 (ArC-N), 143.9 (ArC-NH), 141.5 (ArC-CONH), 138.9
(ArC-CH3), 136.5, 134.8, 133.3, 131.5, 129.8, 128.4,
127.8, 124.4, 122.5, 121.8, 120.9, 119.5, 16.9 (-CH3); MS
(EI+) m/z 424.01 (100%); Anal. Calcd for C22H14Cl2N2O3:
C, 62.13; H, 3.32; N, 6.59 Found: C, 62.12; H, 3.33; N,
6.60.
5-Chloro-2-[2-methyl-4-(3-phenyl-allylideneamino)-
phenyl]-isoindole-1,3-dione (6)
Yield 93%; Power 500 W, 8 min; Rf 0.4 (DCM:MeOH,
9.8:0.2); m.p. 95 °C; kmax(MeOH) 293 nm; 1H NMR dH
(DMSO-d6)-8.22–8.1 (m, 3H, ArH), 7.6 (s, 1H, -CH=N),
7. 5–7.18 (m, 3H, ArH), 7.3–7.16 (m, 5H, ArH), 6.8 (d,
1H, =CH), 5.7 (d, 1H, =CH), 2.26 (s, 3H, CH3); 13C
NMR d p.p.m.- 174.2 (C=N), 173.1 (C=O), 172.2 (C=O),
152.8 (ArC-N=), 147.2 (ArC-N), 145.6 (ArC-Cl), 140.8
(ArC-CH3), 141.2, 140.8, 140.4, 138.9, 138.6, 134.6,
133.8, 132.5, 131.6, 129.9, 127.4, 126.3, 124.7, 119.4
(Aryl Carbons), 140.7 (CH-Ar), 118.6 (CH), 17.8 (-CH3);
MS (EI+) m/z 400.10 (100%); Anal. Calcd C24H17ClN2O2:
C, 71.91; H, 4.27; N, 6.99 Found C, 71.89; H, 4.25; N,
6.97.
N-[4-(5-Chloro-1,3-dioxo-1,3-dihydro-isoindol-2-yl)
-3-methyl-phenyl]-benzamide (9)
Yield 80%; Rf 0.37 (DCM:MeOH, 9.9:0.1); m.p.
1
240–241 °C; kmax (MeOH) = 277 nm; H NMR dH (DMSO-
d6)- 7.96 (s, 1H, NH), 7.7–7.6 (m, 3H, Ar-H), 7.7–7.5 (m,
5H, Ar-H), 7.56- 7.4 (m, 3H, Ar-H), 2.48 (s, 3H, -CH3); 13C
NMR d p.p.m.- 174.8 (CONH), 172.8 (C=O), 171.4 (C=O),
147.8 (ArC-Cl), 145.3 (ArC-N), 143.5 (ArC-NH), 140.9
(ArC-CONH), 138.8 (ArC-CH3), 136.7, 135.2, 133.8,
132.4, 130.5, 129.3, 127.6, 125.1, 123.3, 122.8, 121.1,
120.6, 119.8, 17.9 (-CH3); MS (EI+) m/z 390.04 (100%);
Anal. Calcd for C22H15ClN2O3: C, 67.61; H, 3.87; N, 7.17
Found: C, 67.60; H, 3.88; N, 7.18.
General procedure for preparation of amides 7–11
To a stirred solution of 1 (2 mmol) in pyridine (10 mL)
under ice bath was added dropwise the respective acid
chloride (2.5 mmol). The reaction mixture was stirred over-
night, chilled, and added H2O (1 mL). The mixture was
stirred again for 15 min, and NH4OH (1 mL) was added.
The reaction mixture was partitioned between ether and
H2O. The organic layer was then dried over anhydrous
Na2SO4, filtered, concentrated in vacuo, and crystallized
with hexane to give the amides.
N-[4-(5-Chloro-1,3-dioxo-1,3-dihydro-isoindol-2-
yl)-3-methyl-phenyl]-4-methyl-benzamide (10)
Yield 81%; Rf 0.46 (DCM:MeOH, 9.8:0.2); m.p. 236–
238 °C. kmax (MeOH) = 278 nm; 1H NMR dH (DMSO-d6)-
7.95 (s, 1H, NH), 7.87–7.5 (m, 3H, Ar-H), 7.56–7.31 (m,
3H, Ar-H), 7.56- 7.42 (m, 4H, Ar-H), 2.39 (s, 3H, -CH3),
2.3 (3H, s, CH3); 13C NMR d p.p.m.- 165.8 (CONH), 173.1
(C=O), 172.8 (C=O), 151.1 (ArC-CH3), 147.5 (ArC-Cl),
144.9 (ArC-N), 144.1 (ArC-NH), 140.4 (ArC-CONH), 139.4
(ArC-CH3), 135.7, 134.2, 132.9, 131.4, 129.2, 128.1,
126.9, 125.3, 123.9, 122.2, 121.5, 120.8, 30.7 (-CH3),
18.1 (-CH3); MS (EI+) m/z 404.06 (100%); Anal. Calcd for
N-[4-(5-Chloro-1,3-dioxo-1,3-dihydro-isoindol-2-
yl)-3-methyl-phenyl]-2-naph-thalen-2-yl-acetamide
(7)
Yield 73%; Rf 0.57 (DCM:MeOH, 9.9:0.1); m.p. 125 °C;
kmax (MeOH) = 222 nm; 1H NMR dH (DMSO-d6)- 8.1 (s,
C
23H17ClN2O3: C, 68.23; H, 4.23; N, 6.92 Found: C,
68.21; H, 4.20; N, 6.91.
202
Chem Biol Drug Des 2016; 87: 200–212

Modeling, Synthesis and Anti-HIV Activity
N-[4-(5-Chloro-1,3-dioxo-1,3-dihydro-isoindol-2-
yl)-3-methyl-phenyl]-acetamide (11)
N-[4-(5-Chloro-1,3-dioxo-1,3-dihydro-isoindol-2-
yl)-3-methyl-phenyl]-methane-sulfonamide (14)
Yield 63%; Rf 0.33 (DCM); m.p. 229–230 °C;
Yield 74%; Rf 0.32 (DCM:MeOH, 9.9:0.1); m.p.
1
158–160 °C; kmax (MeOH) = 230 nm; H NMR dH (DMSO-
k
max(MeOH) = 230 nm; 1H NMR dH (DMSO-d6)- 8.22–
d6)- 8.2 (s, 1H, NH), 8.18–7.72 (m, 3H, Ar-H), 7.48–7.39
(m, 3H, Ar-H), 2.38 (s, 3H, CH3), 2.2 (s, 3H, CH3); 13C
NMR d p.p.m.- 178.2 (CONH), 173.5 (C=O), 171.8 (C=O),
147.3 (ArC-Cl), 146.3 (ArC-NH), 144.4 (ArC-N), 139.6
(ArC-CH3), 135.4, 134.6, 133.8, 131.2, 128.6, 124.3,
122.8, 120.6, 27.5 (-CH3-CONH), 17.9 (-CH3); MS (EI+) m/
z 328.06 (100%); Anal. Calcd for C17H13ClN2O3: C, 62.11;
H, 3.99; N, 8.52 Found: C, 62.09; H, 3.98; N, 8.51.
7.78 (m, 3H, CH3), 7.38–6.26 (m, 3H, Ar-H), 3.68 (s,
1H, NH), 2.86 (s, 3H, CH3), 2.64 (s, 3H, CH3); 13C NMR
d p.p.m.- 175.5 (C=O), 173.2 (C=O), 152.6 (ArC-NH),
147.3 (ArC-Cl), 139.6 (ArC-CH3), 138.7 (ArC-N), 133.9,
133.2, 131.7, 128.8, 125.4, 122.7, 117.8, 115.6, 50.5
(CH3-SO2), 17.2 (-CH3).; MS (EI+) m/z 364.03 (100%);
Anal. Calcd for C16H13ClN2O4S: C, 52.68; H, 3.59; N,
7.68; S, 8.79 Found C, 52.66; H, 3.55; N, 7.63; S,
8.80.
General procedure for preparation of
sulfonamides 12–16
N-[4-(5-Chloro-1,3-dioxo-1,3-dihydro-isoindol-2-
yl)-3-methyl-phenyl]-C,C,C-trifluoro-
methanesulfonamide (15)
To a stirred solution of 1 (0.5 mmol) and TEA (1.2 eq) in
DCM (10 mL) was added dropwise a solution of the
respective sulfonyl chloride (0.5 mmol) in DCM (10 mL).
The mixture was stirred at room temperature for 3 h.
The resulting mixture was partitioned between DCM
and water, dried over anhydrous Na2SO4, concentrated
in vacuo, and crystallized with hexane to yield the
sulfonamides.
Yield 69%; Rf 0.55 (DCM:MeOH, 9.8:0.2); m.p. 120–
124 °C; kmax(MeOH) = 232 nm; 1H NMR dH (DMSO-d6)-
8.24–8.02 (m, 3H, ArH), 7.22–6.14 (m, 3H, ArH), 3.88 (s,
1H, NH),2.36 (s, 3H, CH3); 13C NMR d p.p.m.- 175.6
(C=O), 173.4 (C=O), 159.6 (-CF3), 152.4 (ArC-NH), 147.7
(ArC-Cl), 140.2 (ArC-CH3), 138.9 (ArC-N), 133.8, 133.1,
131.5, 128.8, 125.7, 122.4, 117.9, 115.2, 17.4 (-CH3); MS
(EI+) m/z 418 (100%); Anal. Calcd for C16H10ClF3N2O4S:
C, 45.89; H, 2.41; N, 6.69; S, 7.66 Found C, 45.87; H,
2.40; N, 6.64; S, 7.69.
4-Chloro-N-[4-(5-chloro-1,3-dioxo-1,3-dihydro-
isoindol-2-yl)-3-methyl-phenyl]-
benzenesulfonamide (12)
Yield 79%; Rf 0.51 (DCM:MeOH, 9.8:0.2); m.p. 205 °C;
kmax (MeOH) = 234 nm; 1H NMR dH (DMSO-d6)- 8.20–
7.63 (m, 3H, Ar-H), 7.92 (d, 2H, Ar-H), 7.52 (d, 2H, Ar-H),
7.12–6.14 (m, 3H, Ar-H), 4.22 (s, 1H, NH), 2.02 (s, 3H,
CH3); 13C NMR d p.p.m.- 175.3 (C=O), 173.8 (C=O),
150.4 (ArC-NH), 147.9 (ArC-SO2), 147.2 (ArC-Cl), 146.3
(ArC-Cl), 139.4 (ArC-CH3), 138.3 (ArC-N), 134.6, 133.8,
131.6, 128.7, 127.4, 125.9, 124.8, 123.3, 122.4, 120.8,
119.9, 118.5, 17.5 (-CH3); MS (EI+) m/z 460.01 (100%);
Anal. Calcd for C21H14Cl2N2O4S: C, 54.67; H, 3.06; N,
6.07; S, 6.95 Found C, 54.66; H, 3.04; N, 6.08; S, 6.94.
N-[4-(5-Chloro-1,3-dioxo-1,3-dihydro-isoindol-2-yl)
-3-methyl-phenyl]-4-nitro-benzenesulfonamide
(16)
Yield 77%; Rf 0.51 (DCM); m.p. 220–221 °C;
k
max(MeOH) = 235 nm; 1H NMR dH (DMSO-d6)- 8.36
(d, 2H, ArH), 8.12 (d, 2H, ArH), 7.94–7.68 (m, 3H, ArH),
7.34–6.26 (m, 3H, ArH), 4.3 (s, 1H, NH), 2.32 (s, 3H,
CH3); 13C NMR d p.p.m.- 175.4 (C=O), 173.6 (C=O),
162.7 (ArC-NO2), 155.4 (ArC-SO2), 152.2 (ArC-NH),
147.6 (ArC-Cl), 140.2 (ArC-CH3), 138.8 (ArC-N), 134.6,
133.8, 131.6, 128.7, 127.4, 125.9, 124.8, 123.3, 122.4,
120.8, 119.9, 118.5, 17.5 (-CH3); MS (EI+) m/z 471.03
(100%); Anal. Calcd C21H14ClN3O6S: C, 53.45; H, 2.99;
N, 8.91; S, 6.80 Found C, 53.42; H, 2.95; N, 8.90; S,
6.81.
N-[4-(5-Chloro-1,3-dioxo-1,3-dihydro-isoindol-2-
yl)-3-methyl-phenyl]-2,4,6-tri-
isopropylbenzenesulfonamide (13)
Yield 72%; Rf 0.54 (DCM); m.p. 178–181 °C; kmax
1
(MeOH) = 236 nm; H NMR dH (DMSO-d6)- 8.24–8.16 (m,
3H, ArH), 7.24–6.26 (m, 3H, ArH), 4.18 (s, 1H, NH), 3.24
(m, 2H, 3°-CH), 3.16 (m, 1H, 3°-CH), 2.38 (s, 3H, CH3),
1.32 (d, 12H, - CH3), 1.28 (d, 6H, - CH3); 13C NMR d
p.p.m.- 175.2 (C=O), 172.9 (C=O), 161.5 (ArC-CH), 158.2
(ArC-CH), 158.0 (ArC-CH), 152.3 (ArC-NH), 148.1
(ArC-SO2), 147.9 (ArC-Cl), 139.6 (ArC-CH3), 138.9
(ArC-N), 133.9, 133.1, 131.8, 128.5, 125.9, 124.5, 122.7,
120.5, 119.4, 118.4, 17.4 (-CH3); MS (EI+) m/z 552.18
(100%); Anal. Calcd for C30H33ClN2O4S: C, 65.14; H,
6.01; N, 5.06; S, 5.80 Found C, 65.13; H, 6.00; N, 5.05;
S, 5.78.
General procedure for preparation of thioamides
17–21
A mixture of amide 2 (2 mmol), Lawesson’s reagent
(1.1 mmol), and THF (10 mL) was stirred at 5–10 °C for
2 h. The solvent was evaporated under reduced pres-
sure and the residue extracted with ethyl acetate
(3 9 5 mL). The combined organic layers were washed
with 5% NaHCO3, dried over anhydrous MgSO4, and
evaporated to give the crude product which was
recrystallized from ethyl acetate to afford the pure
product.
Chem Biol Drug Des 2016; 87: 200–212
203

Kumari and Singh
N-[4-(5-Chloro-1,3-dioxo-1,3-dihydro-isoindol-2-
yl)-3-methyl-phenyl]-2-naphthalen-2-yl-
thioacetamide (17)
(EI+) m/z 420.06 (100%); Anal. Calcd C23H17ClN2O2S: C,
65.63; H, 4.07; N, 6.66; S, 7.62 Found C, 65.61; H, 4.02;
N, 6.64; S, 7.66.
Yield 77%; Rf 0.52 (DCM:MeOH, 9.9:0.1); m.p. 132 °C;
1
kmax (MeOH) = 228 nm; H NMR dH (DMSO-d6)- 8.04–7.8
(m, 3H, ArH), 7.6–7.2 (m, 7H, ArH), 7.1–6.4 (m, 3H, ArH),
4.34 (s, 1H, NH), 2.78 (s, 2H, CH2), 1.96 (s, 3H, CH3); 13C
NMR d p.p.m.- 198.2 (-CSNH), 173.4 (C=O), 170.6 (C=O),
147.3 (ArC-Cl), 145.2 (ArC-CH2), 144.5 (ArC-N), 142.4
(ArC-NH), 139.8 (ArC-CH3), 138.5, 136.9, 134.6, 134.7,
133.8, 131.5, 129.9, 128.4, 127.7, 124.9, 122.5, 121.6,
121.8, 120.9, 120.4, 119.5, 118.4, 62.4 (CH2-CSNH),
17.8 (-CH3); MS (EI+) m/z 470.09 (100%); Anal. Calcd
C27H19ClN2O2S: C, 68.86; H, 4.07; N, 5.95; S, 6.81
Found C, 68.82; H, 4.05; N, 5.96; S, 6.80.
N-[4-(5-Chloro-1,3-dioxo-1,3-dihydro-isoindol-2-
yl)-3-methyl-phenyl]-thioacetamide (21)
Yield 79%; Rf 0.36 (DCM:MeOH, 9.9:0.1); m.p. 172–
174 °C; kmax (MeOH) = 234 nm; 1H NMR dH (DMSO-d6)-
8.24–8.16 (m, 3H, ArH), 7.26–6.28 (m, 3H, ArH), 4.2 (s,
1H, NH), 2.18 (s, 3H, CH3), 0.9 (s, 3H, CH3); 13C NMR d
p.p.m.- 196.2 (CSNH), 173.5 (C=O), 171.8 (C=O), 147.3
(ArC-Cl), 146.3 (ArC-NH), 144.4 (ArC-N), 139.6 (ArC-CH3),
135.8, 134.6, 133.8, 131.4, 128.6, 124.5, 122.8, 120.6,
40.5 (-CH3-CSNH), 17.9 (-CH3); MS (EI+) m/z 344.04
(100%); Anal. Calcd C17H13ClN2O2S: C, 59.21; H, 3.80;
N, 8.12; S, 9.30 Found C, 59.20; H, 3.78; N, 8.11; S,
9.23.
4-Chloro-N-[4-(5-chloro-1,3-dioxo-1,3-dihydro-
isoindol-2-yl)-3-methyl-phenyl]-thiobenzamide (18)
Yield 78%; Rf 0.36 (DCM:MeOH, 9.9:0.1); m.p. 181–
183 °C; kmax (MeOH) = 254 nm; 1H NMR dH (DMSO-d6)-
8.3–8.18 (m, 3H, ArH), 7.5–7.3 (m, 4H, ArH), 7.2–6.14 (m,
3H, ArH), 3.9 (s, 1H, NH), 2.22 (s, 3H, CH3); 13C NMR d
p.p.m.- 197.2 (CSNH), 173.2 (C=O), 171.1 (C=O), 151.5
(ArC-CSNH), 147.8 (ArC-Cl), 146.4 (ArC-Cl), 145.7 (ArC-
N), 144.9 (ArC-NH), 138.9 (ArC-CH3), 136.5, 134.8, 133.3,
131.5, 129.8, 128.4, 127.8, 124.4, 122.5, 121.8, 120.9,
119.5, 16.9 (-CH3); MS (EI+) m/z 440.01 (100%); Anal.
Calcd C22H14Cl2N2O2S: C, 59.87; H, 3.20; N, 6.35; S,
7.27 Found C, 59.82; H, 3.23; N, 6.36; S, 7.23.
Anti-HIV assay
Evaluation of the antiviral activity of the compounds
against HIV-1 strain IIIB in MT-4 cells was performed using
the MTT assay. Stock solutions (10 9 final concentration)
of test compounds were added in 25 lL volumes to two
series of triplicate wells so as to allow simultaneous evalu-
ation of their effects on mock- and HIV-infected cells at
the beginning of each experiment. Serial 5-fold dilutions of
test compounds were made directly in flat-bottomed 96-
well microtiter trays using a Biomek 3000 robot (Beckman
instruments, Fullerton, CA, USA). Untreated control HIV-
and mock-infected cell samples were included for each
sample.
N-[4-(5-Chloro-1,3-dioxo-1,3-dihydro-isoindol-2-
yl)-3-methyl-phenyl]-thiobenz-amide (19)
Yield 74%; Rf 0.34 (DCM:MeOH, 9.9:0.1); m.p. 248–
251 °C; kmax (MeOH) = 272 nm; 1H NMR dH (DMSO-d6)-
8.22–8.16 (m, 3H, ArH), 7.3–7.2 (m, 5H, ArH), 7.1–6.18
(m, 3H, ArH), 4.3 (s, 1H, NH), 2.34 (s, 3H, CH3); 13C NMR
d p.p.m.- 198.8 (CSNH), 172.8 (C=O), 171.4 (C=O), 151.9
(ArC-CSNH), 147.8 (ArC-Cl), 145.3 (ArC-N), 143.5 (ArC-
NH), 138.8 (ArC-CH3), 136.7, 135.2, 133.8, 132.4, 130.5,
129.3, 127.6, 125.1, 123.3, 122.8, 121.1, 120.6, 119.8,
17.9 (-CH3); MS (EI+) m/z 406.04 (100%); Anal. Calcd
HIV-1 (IIIB) stock (50 lL) at 100–300 CCID50 (cell culture
infectious dose) or culture medium was added to either
the HIV-infected or mock-infected wells of the microtiter
tray. Mock-infected cells were used to evaluate the effect
of test compound on uninfected cells in order to assess
the cytotoxicity of the test compound. Exponentially grow-
ing MT-4 cells were centrifuged for 5 min at 1000 r.p.m.,
and the supernatant was discarded. The MT-4 cells were
resuspended at 6 9 105 cells/mL, and 50-lL volumes
were transferred to the microtiter tray wells. Five days after
infection, the viability of mock- and HIV-infected cells was
examined spectrophotometrically by the MTT assay.
C
22H15ClN2O2S: C, 64.94; H, 3.72; N, 6.88; S, 7.88
Found C, 64.90; H, 3.71; N, 6.85; S, 7.84.
N-[4-(5-Chloro-1,3-dioxo-1,3-dihydro-isoindol-2-
yl)-3-methyl-phenyl]-4-methyl-thiobenzamide (20)
Yield 77%; Rf 0.44 (DCM:MeOH, 9.8:0.2); m.p. 251–
253 °C; kmax (MeOH) = 282 nm; 1H NMR dH (DMSO-d6)-
8.1–7.9 (m, 3H, ArH), 7.24–6.44 (m, 3H, ArH), 7.18–6.8
(m, 4H, ArH), 4.0 (s, 1H, NH), 2.4 (s, 3H, CH3), 2.37 (3H,
s, CH3); 13C NMR d p.p.m.-197.8 (CSNH), 173.1 (C=O),
172.8 (C=O), 151.1 (ArC-CH3), 148.9 (ArC-CSNH), 147.5
(ArC-Cl), 144.9 (ArC-N), 144.1 (ArC-NH), 139.4 (ArC-CH3),
135.7, 134.2, 132.9, 131.4, 129.2, 128.1, 126.9, 125.3,
123.9, 122.2, 121.5, 120.8, 30.9 (-CH3), 18.1 (-CH3); MS
The MTT assay is based on the reduction of yellow-col-
ored
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) by mitochondrial dehydrogenase of
metabolically active cells to a blue-purple formazan that
can be measured spectophometrically. The absorbances
were read in an eight-channel computer-controlled pho-
tometer at two wavelengths (540 and 690 nm). All data
were calculated using the median OD (optical density)
value of three wells. The 50% cytotoxic concentration
(CC50) was defined as the concentration of the test
compound that reduced the absorbance (OD540) of the
204
Chem Biol Drug Des 2016; 87: 200–212

Modeling, Synthesis and Anti-HIV Activity
mock-infected control sample by 50%. The concentration
achieved 50% protection from the cytopathic effect of the
virus in infected cells was defined as the 50% effective
concentration (EC50).
Input Site Sphere parameters specify a sphere around the
center of the binding site, where CDOCKER experiment
was to be performed. The center of the Input Site Sphere
was used for initial ligand placement. Ten replicas for each
ligand were generated and randomly distributed around
the center of the allosteric site. Each of these was sub-
jected to MD-based simulated annealing and final refine-
ment by minimization, leading to 10 minimized docked
poses.
Molecular modeling
The computational studies began with the X-ray crystal
structure of HIV-RT complexed with a ligand (PDB: ID
3E01). All computations were performed using DISCOVERY
STUDIO 2.5.
The docked complexes were further refined by the Ligand
Minimization protocol, during which the side-chain atoms
within the Input Site Sphere were free to move. A Smart
Minimizer algorithm with 1000 steps and rms. gradient of
Receptor setup
ꢀ
ꢁ
The target protein (PDB: ID 3E01) was taken, the ligand
was extracted, hydrogens added, and their positions were
optimized using the all-atom CHARMm forcefield (17,18)
and the Adopted Basis set Newton Raphson (ABNR)
method available in DS 2.5 protocol until the root-mean-
0.5 kcal/mol/A was used for ligand minimization.
The final step in docking was the scoring of the refined
docked poses. This was performed using the Score
Ligand Poses protocol of DS 2.5. The Ludi Energy Estimate
(21–23) was used for the refined poses. The ligand pose
which corresponded to the highest Ludi score (24) was
taken as the best-docked pose.
ꢀ
ꢁ
square (rms.) gradient was <0.05 kcal/mol/A. The mini-
mized protein was defined as the receptor using the bind-
ing site module of DS 2.5. The binding site was defined
from the volume of ligand method which was modified to
accommodate all the important interacting residues in the
allosteric site of HIV-RT. The Input Site Sphere was
Visualization was performed using PYMOL software
(www.pymol.org).
ꢀ
ꢁ
defined over the binding site, with a radius of 5 A from the
center of the binding site. The protein, thus characterized,
was taken as the target receptor for the docking proce-
dure.
Validation of the docking technology
The docking methodology was tested on nevirapine, a
well-known marketed NNRTI, which was docked into the
allosteric site of HIV-1 RT after extracting the ligand from
the crystal structure. The Ludi_3 score was found to be
718, and the corresponding Kd value was 0.06 lM. This is
in good agreement with the observed value of 0.05 lM.
Ligand setup
Using the built-and-edit module of DS 2.5, various
ligands were built; all-atom CHARMm forcefield parame-
terization was assigned and then minimized using the
ABNR method. A conformational search of the ligand
was carried out using a stimulated annealing molecular
dynamics (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 transformation
obtained at the end of each cycle was further subjected
to local energy minimization, using the ABNR method.
The 30 energy-minimized structures were then superim-
posed, and the lowest energy conformation occurring in
the major cluster was taken to be the most probable
conformation.
Calculation of BBB
For plotting BBB, ADMET property of DS 2.5 was used.
The minimized ligand was input, and BBB plot was
obtained.
Calculation of H-acceptors, H-donors, and TPSA
values
H-acceptors, H-donors, and TPSA were calculated using
MOLINSPIRATION software (www.molinspiration.com).
Docking and scoring
Results and Discussion
Docking of the HIV-RT with the ligands was carried out
with CDOCKER protocol of DS 2.5. CDOCKER (19,20) is a
grid-based, MD-simulated, annealing-based algorithm that
uses CHARMm. The receptor (protein) was held rigid while
the ligands were flexible during the refinement. The basic
strategy involves the generation of several initial ligand ori-
entations in the allosteric site of the target protein followed
by MD-based simulated annealing, and final refinement by
minimization.
The synthesis of new imine, amide, and sulfonamide
derivatives (2–16) was achieved from the key intermediate,
N-(4-amino-2-methylphenyl)-4-chloro-phthalimine (1), which
was synthesized from 4-chlorophthalimine and 4-chloro-3-
methylaniline (Figure 2). Compound 1 was treated with the
appropriate aldehydes under microwave irradiation to get
the respective imines (2–6). Further, 1 on treatment with
acid chlorides in the presence of pyridine at 0–5 °C gave
Chem Biol Drug Des 2016; 87: 200–212
205

Kumari and Singh
O
O
K2CO3
H2N
N
H2N
Cl
CH3
HN
+
Cl
Cl
CH3
O
O
1
pyridine, 0 o
C
TEA, RT
RCHO MW
R'COCl
R''SO2Cl
O
N
O
O
O
C
O
S
H
C
H
N
R'
R
NH
N
N
R''
N
Cl
Cl
Cl
O
CH3
CH3
O
O
CH3
O
2–6
7–11
12–16
R''
p-Cl-C6H4-
R'
R
C10H7-CH2-
7
C6H5-
12
2
3
p-Cl-C6H4-
C6H5-
1,3,5-(C(CH3)2)3-C6H2-
CH3-
13
14
8
9
p-CH3O-C6H4-
p-HO-C6H4-
p-Cl-C6H4-
4
5
6
CF3-
15
16
p-CH3-C6H4-
CH3-
10
11
p-NO2-C6H4-
C6H5-CH=CH-
Figure 2: Synthesis of imine (2–6), amide (7–11), and sulfonamide (12–16) derivatives of soindolinedione.
the respective amides (7–11) and when treated with
appropriate sulfonyl chloride in the presence of TEA and
DCM at room temperature, gave the respective sulfon-
amides (12–16).
The rationale behind synthesizing isoindolinediones as
molecules with anti-HIV property culminated from docking
studies, which predicted about the possible binding con-
formation of the designed molecules and their interaction
with HIV-1 RT. Docking of the molecules 2–21 using DS
2.5 into the allosteric site of RT (PDB code 3E01) gener-
ated a number of possible binding conformations with the
corresponding Ludi_3 values. The cluster analysis revealed
a predominant orientation of the ligand within the binding
pocket of allosteric site and the conformation with the
highest Ludi_3 value, Table 1, for each molecule was
chosen for further analysis. These linkages were involved
The thioamides (17–21) were synthesized by treatment of
the amide derivatives (2–6) with Lawesson’s reagent in the
presence of THF at 5–10 °C (Figure 3). The desired prod-
ucts were obtained in good yield and were characterized
by various spectroscopic techniques. The spectral data of
all the synthesized compounds are in full agreement with
the proposed structures.
O
O
O
C
S
C
Lawesson’s reagent, 5–10 o
C
R'
R'
NH
N
NH
N
Cl
Cl
CH3
CH3
O
O
17–21
R'
C10H7-CH2-
17
p-Cl-C H -
18
6
4
19 C6H5-
p-CH3-C6H4-
CH3-
20
21
Figure 3: Synthesis of thioamide derivatives
(17–21) of isoindolinedione.
206
Chem Biol Drug Des 2016; 87: 200–212

Modeling, Synthesis and Anti-HIV Activity
Table 1: Anti-HIV activity of isoindoline-
dione derivatives 2–21
a
b
H-
H-
Ludi_3 EC50
(lM)
CC50
Molecule
acceptors donors TPSA logP value
(lM)
SIc
2
4
5
5
4
4
5
5
5
5
5
6
6
6
6
6
4
4
4
4
4
–
0
0
1
0
0
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
–
51.44 5.50
60.67 5.44 1025
71.69 5.17 915
51.44 6.12 1031
51.44 5.45 937
774
0.287 0.317
0.138 0.309
0.263 0.534
0.119 0.327
0.235 0.538
0.020 0.420
0.022 0.256
0.161 0.638
0.017 0.616
0.033 0.334
0.003 0.220
0.005 0.180
0.008 0.269
0.004 0.234
0.003 0.200
0.097 0.415
0.180 0.517
0.362 1.222
0.083 0.409
0.099 0.164
0.050 >10
1.10
2.24
2.03
2.75
3
4
5
6
2.29
7
68.17 5.44 1044
68.17 5.06 1010
21.00
11.64
3.99
8
9
10
68.17 4.50
870
68.17 4.99 1004
36.23
10.12
73.33
36.00
33.75
58.50
66.67
4.28
2.87
3.39
4.93
1.66
11
12
68.17 2.60
85.25 4.66
85.25 7.48
85.25 1.92
85.25 4.09
85.25 3.74
808
776
13
14
–
870
874
917
15
16
17
18
51.10 6.29 1088
51.10 5.91
51.10 5.35
51.10 5.83
51.10 3.45
810
979
990
898
718
19
20
21
Nevirapine
–
–
>200
aConcentration required to reduce HIV-1 (IIIB)-induced cytopathic effect by 50% in MT-4
cells.
bConcentration required to reduce MT-4 cell viability by 50%.
cSelectivity index: ratio CC50/EC50.
in H-bonding along with the carbonyl part of the isoindo-
linedione moiety.
linkage formed an H-bond with Tyr318 and the C=O part
of its phthalimine moiety formed another H-bond with
Val106. In the case of compound 9, no H-bonding
occurred with -CO-NH- moiety but 3 H-bonds were
formed between C=O of the phthalimine moiety and
Lys103 and Pro236. The compound 10 formed no
H-bonds and interacted through п-п interaction with
Tyr188 and p-+ interaction with Lys223.
The best interactions were obtained in the case of sulfon-
amides followed by the amides, thioamides, and imines,
the imines being the least interactive among all the deriva-
tives, which is also the order of their anti-HIV activity.
In the case of compound 12, three H-bonds were formed
between the C=O portion of the isoindolinedione moiety
and Lys103 and Pro236 (Figure 4A,B). In the case of 14
and 15, the S=O bonds of the sulfonamide moiety inter-
acted with Lys103 (NH or carbonyl or both) and Pro236;
the NH part of the sulfonamide linkage formed bond with
carbonyl of Lys101 and thus, formed 6 H-bonds each. In
the case of compound 16, an additional H-bond was
formed between its nitro function on aromatic ring and
carbonyl of Val106. It also interacted with His235 (NH
and carbonyl), and thus, formed seven H-bonds
(Figure 4A). The compound 13 did not dock, probably
due to its large size and thus was unable to fit in the
binding pocket.
The thioamides 17, 18, and 21 formed one H-bond
each with Lys101, Lys103, and Pro236 residues of the
binding site, respectively. In the case of compound 17,
the H-bond was formed between the NH of the
thioamide and Lys101. Compound 18 interacted with
Lys103 of the binding site with its phthalimine moiety.
Compound 21 formed H-bond between the NH of the
amide linkage along with Pro236 in the NNBS. Both the
remaining thioamides 19 and 20 interacted with п-п
interaction and п-+ interaction with Tyr188 and Lys223,
respectively, present in the NNBS. In the case of 20, an
additional p-p interaction with Phe227 was present (Fig-
ure 5).
The amides formed one to four H-bonds with various
amino acids (Lys103, Val106, Tyr188, Tyr229, His235,
Pro236, and Tyr318) present in NNBS on HIV-1 RT. The
compounds 7, 8, and 11 formed H-bonds with their NH
portion (of amide linkage) and Tyr188, His235, or Tyr318.
In the case of compound 7, the C=O portion of amide
On comparing the interactions of the amides with thioa-
mides, it is clear that the amides interacted in a better way
as compared to thioamides (Figure 6). Docking studies
also revealed that imines mostly interacted with p-p inter-
actions with various amino acid residues present on the
allosteric site of HIV-1 RT (Figure 6).
Chem Biol Drug Des 2016; 87: 200–212
207

Kumari and Singh
A
B
Figure 4: Compounds 12 and 16 having
sulfonamide linkages within HIV-1 RT (wild
type) (A) Full view (B) Magnified view (only
surrounding amino acid residues shown in
green, hydrogen bonds shown in yellow).
A
B
Compound 2 formed p-p interactions with Tyr188 and
Tyr318. In the case of compound 3, p-p interactions
occurred with Tyr188 and Tyr318 and p-+ interaction with
Lys223. Compound 5 showed p- p interaction with Tyr188
and p-+ interactions with Lys223 and the compounds 4
and 6 interacted with one H-bond. In the case of com-
pound 4, one H-bond was formed between phenolic –OH
substituent and Lys223, whereas in the case of compound
6, one H-bond was formed between the N functionality of
the imine and Lys103 of HIV-1 RT.
The ligands had docked energies much lower than that of
nevirapine which was used as a reference. However, the
isoindolinedione molecules were less active against HIV-1.
SAR studies revealed that the nature of the linkers played
a vital role in displaying anti-HIV activity and the com-
pounds 12–16 bearing sulfonamide linkage were found to
be most potent. Further, the nature of R group also
affected the anti-HIV activity. The substituents with ben-
zene ring having atoms capable of H-bond formation have
shown better activity. Thus, among sulfonamides, 12, 15,
and 16 have shown good anti-HIV activity. All sulfon-
amides contained H-bond acceptors that directly inter-
acted with Lys103 backbone of HIV-1 RT, which was
The corresponding docked energies are given in Table 2.
The results indicate that the ligands as a whole moved into
a more stable position with much lower docked energy.
208
Chem Biol Drug Des 2016; 87: 200–212

Modeling, Synthesis and Anti-HIV Activity
A
B
Figure 5: Comparison of interactions of an
amide derivative (7) with a thioamide
derivative (17) with HIV-1 RT (wild type) (A)
full view (B) magnified view (only surrounding
amino acid residues shown in green,
hydrogen bonds shown in yellow).
A
B
similar to interactions shown by delavirdine and many
‘next-generation inhibitors’. These molecules interacted
with the backbone of Lys103 and filled the space adjacent
to the loop with functionality capable of maintaining sub-
stantial hydrophobic contacts with Pro236.
linkages lacked both C=O and N-H dipoles and hence
exhibited less number of H-acceptors. Similarly, in the
thioamides 17–21, the C=S dipole is weaker as compared
to C=O dipole. In the case of compounds 7–11 with amide
linkages, the presence of a C=O dipole and a small N-C
dipole allowed them to act as H-bond acceptors and the
presence of N-H dipole to function as H-bond donors.
Likewise, the compounds 12–16 with sulfonamide linkage
also acted as potent H-bond acceptors and H-bond
donors because of two S=O and one S-N dipoles. As
these molecules can both donate and accept H-bonds,
they can ionize at an appropriate pH to further enhance
their solubility. Thus, these molecules internalize better in
cells. Moreover, in all cases, the substituted aromatic
derivatives were more potent than the unsubstituted or
aliphatic derivatives.
The amides were also potent but cytotoxic. The most
potent among the amides was compound 10 having a
methyl substituent on the benzene ring attached with
amide linkage. The thioamide derivatives, 17–21, were less
potent than the amide derivatives and were also much
more cytotoxic. The least potent were the compounds 2–6
with imine linkages associated with high cytotoxicity.
The structural features of these molecules also speak
about their activity. The compounds, 2–6, with imine
Chem Biol Drug Des 2016; 87: 200–212
209

Kumari and Singh
Figure 6: Compound 5 having an imine
linkage within HIV-1 RT (wild type) (A) full
view (B) magnified view (only surrounding
amino acid residues shown in green, pi-pi
and pi-+ interactions shown in orange).
A
B
Table 2: DISCOVERY STUDIO 2.5 docking results
Table 3: ADMET_PSA_2D:ADMET_AlogP98 values
of the
compounds
Docked energy (kcal/mol)
Compound
ADMET_PSA_2D:ADMET_AlogP98
Potential energy
2
49.277:5.086
70.092:5.564
70.092:5.564
49.277:6.47
49.277:6.166
68.065:5.902
68.065:5.658
68.065:4.994
68.065:5.48
68.065:3.35
85.365:5.361
85.365:8.279
85.365:3.12
85.365:5.256
128.186:4.591
50.764:6.836
50.764:6.557
50.764:5.893
50.764:4.229
50.764:4.229
55.985:2.293
39.014:3.717
Ligand
van der Waals
Electrostatic
(Total)
3
2
À53.19
À51.04
À49.08
À46.26
À51.39
À54.64
À42.20
À43.21
À47.36
À43.19
À45.75
À8.34
À9.44
À61.53
À60.48
À76.42
À54.80
À60.45
À66.37
À58.40
À48.55
À64.51
À56.23
À52.13
4
3
5
4
À27.34
À8.54
6
5
7
6
À9.06
8
7
À11.73
À16.20
À5.34
9
10
8
9
10
11
12
À17.15
À13.03
À6.38
11
12
13
14
13
14
DID NOT DOCK
À9.81
15
16
À40.92
À46.99
À50.52
À55.65
À46.39
À47.26
À46.81
À42.01
À38.53
À50.73
À63.74
À60.52
À60.91
À50.28
À62.41
À60.07
À48.88
À45.99
15
16
À16.75
À1.00
17
18
17
18
À5.26
19
20
À3.89
19
20
À15.15
À13.26
À6.87
21
Nevirapine
Delavirdine
21
Nevirapine
À7.46
values ranging from 33.75 to 73.33 with minimal cytotoxic-
ity. Similarly, amides too have shown activity but with con-
siderable cytotoxicity and thus have lower SI values. The
compounds with imine and thioamide linkages, however,
did show very little activity because of their high cytotoxicity.
The newly synthesized compounds 2–21 were evaluated
for their anti-HIV activity in MT-4 cells of HIV-1 (III B). The
cytotoxicity of the compounds was also measured in MT-4
cells of HIV-1 (III B). The results obtained from the
cell-based assays are summarized in Table 1 and are
compared with nevirapine as reference drug.
The BBB graph was plotted for all the molecules using DIS-
COVERY STUDIO 2.5, and it was found that in all the cases,
the compounds had fallen within the 99% confidence
eclipse indicating that the compounds may be able to
cross the BBB which is one of the major drawbacks in
case of anti-HIV compounds (Tables 3 and 4). ADME plot
As shown in Table 1, several of the new compounds were
active against HIV-1 at micromolar or nanomolar concentra-
tions with high selectivity indices. The compounds 12–16
with sulfonamide linkages have shown remarkably high SI
210
Chem Biol Drug Des 2016; 87: 200–212

Modeling, Synthesis and Anti-HIV Activity
Table 4: Blood–brain barrier plots of compounds 2, 11, 14, 21, and known drugs nevirapine and delavirdine
Compound
Graph
2
11
14
21
Nevirapine
Delavirdine
is 2D plot calculated using FPSA_2D and AlogP98 proper-
ties. Blood–brain barrier (BBB) plot was drawn for all the
compounds. Hence, it may be presumed that all the com-
pounds may penetrate the BBB and may be effective in
treating HIV/AIDS.
activity at 3–4 nM concentrations. These results are well in
accordance with computational predictions.
Acknowledgments
In conclusion, novel isoindolinedione derivatives as proba-
ble NNRTIs have been synthesized and characterized.
Some of these molecules have shown good anti-HIV
Authors are sincerely grateful to Prof. Erick De Clercq for
anti-HIV screening of the molecules and to Prof. KVR
Chary for providing the NMR spectra of the synthesized
Chem Biol Drug Des 2016; 87: 200–212
211

Kumari and Singh
compounds. The corresponding author thanks DBT-ICMR
for financial support as a special drive for developing
microbicides against HIV. Garima Kumari would like to
thank CSIR-UGC for providing JRF and SRF.
12. Sluis-Cremer N., Temiz N.A., Bahar I. (2004) Confor-
mational changes in HIV-1 reverse transcriptase
induced by non nucleoside reverse transcriptase inhibi-
tor binding. Curr HIV Res;10:323–324.
13. Das K., Lewi P.J., Hughes S.H., Arnold E. (2005) Crys-
tallography and the design of anti-AIDS drugs: confor-
mational flexibility and positional adaptability are
important in the design of non-nucleoside HIV-1
reverse transcriptase inhibitors. Prog Biophys Mol
Biol;88:209–231.
Conflict of Interest
The authors declare no conflict of interest.
14. De Clercq E. (2004) Non-nucleoside reverse transcrip-
tase inhibitors (NNRTIs): past, present, and future.
Chem Biodivers;1:44–64.
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