Synthesis, biological evaluation and molecular modeling studies of N-aryl-2-arylthioacetamides as non-nucleoside HIV-1 reverse transcriptase inhibitors

Article abstract of DOI:10.1111/j.1747-0285.2010.01017.x

A series of N-aryl-2-arylthioacetamide derivatives (2-4) designed as non-nucleoside reverse transcriptase inhibitors was synthesized and evaluated for their inhibitory activity against HIV-1 (IIIB) replication in MT-4 cell cultures. The compounds 2-4 were

Full text of DOI:10.1111/j.1747-0285.2010.01017.x

ª 2010 John Wiley & Sons A/S
doi: 10.1111/j.1747-0285.2010.01017.x
Chem Biol Drug Des 2010; 76: 330–339
Research Article
Synthesis, Biological Evaluation and Molecular
Modeling Studies of N-aryl-2-arylthioacetamides
as Non-nucleoside HIV-1 Reverse Transcriptase
Inhibitors
transcriptase inhibitors (NNRTIs) that inhibit the enzyme by an allo-
steric interaction with a site adjacent to the NRTI binding site (the
non-nucleoside inhibitor binding site, namely NNBS). Non-nucleoside
reverse transcriptase inhibitors have gained an increasingly impor-
tant role in the therapy of HIV infection in multidrug regimens and
highly active antiretroviral therapy (HAART), such as nevirapine, del-
avirdine, efavirenz (7–9), 4,5,6,7-tetrahydroimidazo[4,5,1-jk][1,4]ben-
zodiazepin- 2(1H )-one and -thione (TIBO) derivatives (10),
thiocarboxanilides (11) and pyridinones (12). However, the emer-
gence of drug-resistant viral strains (13–15) has limited the thera-
peutic efficiency of these inhibitors. Therefore, there is an urgent
need to develop novel classes of NNRTIs with activity against the
drug-resistant mutants.
Zhu Xiaohe, Qin Yu, Yan Hong*,
Song Xiuqing and Zhong Rugang
College of Life Science and Bio-engineering, Beijing University of
Technology, Pingleyuan Street No. 100, Chaoyang District, Beijing
100124, China
*Corresponding author: Yan Hong, hongyan@bjut.edu.cn
A series of N-aryl-2-arylthioacetamide derivatives
(2–4) designed as non-nucleoside reverse transcrip-
tase inhibitors was synthesized and evaluated for
their inhibitory activity against HIV-1 (IIIB) replica-
tion in MT-4 cell cultures. The compounds 2–4
were performed by the reaction of thiols and 2-
chloro-N-substituted-acetamides and active in the
lower micromolar concentration (1.25–20.83 lM).
The studies of structure–activity relationship sug-
gested that 1H-benzo[d]imidazole ring at arylthio
moiety strongly improved the anti-HIV activity and
consistent with the experimental data. The results
of molecular modeling and docking within the RT
non-nucleoside binding site using AUTODOCK con-
firmed that the 3 series, similar to other non-
nucleoside reverse transcriptase inhibitors such
as N-(5-chloro-2-pyridinyl)-N’-[2-(4-ethoxy-3-fluoro-
2-pyridinyl)ethyl]-thiourea (PETT), was assumed in
a butterfly-like conformation and helped to ratio-
nalize some SARs and the biological activity data.
Recently, a novel class of HIV-1 NNRTIs, N-aryl-2-arylthioaceta-
mides, e.g.: sulfanyltriazoles A (16), sulfanyltetrazoles B (17), sulfa-
nylthiadiazoles C (18) and VRX-480773 (19), has been identified by
the submicromolar activity and significant in vitro activity, especially
the VRX-480773 inhibited viruses from efavirenz-resistant molecular
clones (Figure 1). Studies of crystal structures of the RT complex
with inhibitors suggested that NNRTIs share a common mode of
action and interact with a hydrophobic pocket. Upon binding, N-
aryl-2-arylthioacetamides assumed a typical butterfly-like conforma-
tion, the arylthio moiety and the phenyl ring mimicking the butterfly
wings. Structure–activity relationship (SAR) studies showed that the
arylthio moiety strongly influenced the antiviral activity, leading to
different results depending on steric ⁄ electronic properties of
atoms ⁄ groups. Furthermore, introduction of a set of electron-with-
drawing groups at N-aryl moiety was highly positive for anti-HIV-1
activity. On the basis of these mode, the lead compounds (Figure 1)
had been mainly based on independent variations of the portions
and established the potent derivatives 2–4 (Figure 2) featured by
the following patterns: (i) the substitution of pyridine, 1H-
benzo[d ]imidazole and 1,3,4-thiadiazole rings at arylthio moiety, (ii)
the substitution of the phenyl ring or replacement of the phenyl ring
with a pyrimidyl ring at N-aryl moiety.
Key words: antiviral agents, molecular docking, N-aryl-2-arylthioac-
etamides, non-nucleoside reverse transcriptase inhibitors, structure–
activity relationships
Received 31 January 2010, revised 7 June 2010 and accepted for publi-
cation 16 June 2010
HIV-1 reverse transcriptase (RT) is an essential enzyme converting
the single-stranded viral RNA genome into linear double-stranded
DNA prior to its integration into the host genomic DNA (1). Because
of its important role in the HIV-1 life cycle, RT is one of the most
attractive targets for the development of new antiretroviral agents
(2–4). Two functionally distinct classes of HIV-1 RT inhibitors have
been discovered and used in clinically or clinical trials (5,6): nucleo-
side reverse transcriptase inhibitors (NRTIs) that interact competi-
tively with the catalytic site of the RT, and non-nucleoside reverse
In this study, we described a facile synthesis of N-aryl-2-arylthioac-
etamide derivatives 2–4 and an evaluation for their inhibitory activ-
ity against HIV-1 (IIIB). With the aim to rationalize the biological
results and to predict the activity of novel NNRTIs of 2–4, we
focused on the SAR and the correlation between the inhibitory
activities and the binding free energies, obtained by structural and
330

Studies of N-aryl-2-arylthioacetamides
A
B
Figure 1: Lead N-aryl-2-aryl-
thioacetamide inhibitors of HIV-1
RT.
C
Figure 2: Novel synthesized N-aryl-2-arylthioacetamide derivatives.
molecular modeling studies on the HIV-1 RT NNBS using AUTODOCK
4.0 (Scripps Research Institute, La Jolla, CA, USA).
0 ꢀC. The reaction mixture was stirred at room temperature for an
additional four hours. Then, it was slowly poured into 100 mL of
ice water. The aqueous solution was extracted with CH2Cl2
(2 · 100 mL), the organic phase was washed and dried over
Na2SO4, filtered and the solvent was evaporated to furnish a solid
residue which was purified by crystallization from a mixture of ethyl
acetate ⁄ petroleum ether.
Methods and Materials
Melting points were determined on a XT-4A melting point appa-
ratus and were uncorrected. Infrared (IR) spectra (KBr) were
recorded on a Bruker vertex 70 spectrophotometer. 1H NMR
spectra were obtained using a Bruker ARX-400 MHz spectropho-
tometer, and chemical shift values are expressed in d values
(ppm) relative to tetramethylsilane (TMS) as internal standard.
Coupling constants are given in Hz, and signals are quoted as
follows: s (singlet), d (doublet), t (triplet), m (multiplet) and br s
(broad singlet). All NH and OH protons were exchangeable with
D2O. Electrospray ionization mass spectra (ESI-MS) were mea-
sured on a ZAB-HS & ESQUIRE6000 mass spectrometer. All com-
pounds were routinely checked by TLC and 1H NMR. TLC was
performed by using aluminum-baked silica gel plates (60 HF-254,
Combinol Reagent Corp., Yantai, China). The developed chromato-
grams were visualized under ultraviolet light at 254 nm or by
iodine vapor. All solvents were reagent grade and, when neces-
sary, were purified and dried by standard methods.
2-Chloro-N-phenylacetamide (1a)
Yield 91.3%; m.p. 129.7–130.9 ꢀC; 1H NMR (400 MHz, CDCl3): d
(ppm) 4.17 (s, 2H, CH2), 7.20 (m, 1H, PhCH-p, J = 7.2 Hz), 7.39 (m,
2H, PhCH-m, J = 7.8, 1.9 Hz), 7.54 (m, 2H, PhCH-o, J = 7.5, 1.2 Hz),
8.30 (br s, 1H, NH ).
2-Chloro-N-(2,5-di-fluoro-phenyl)acetamide (1b)
Yield 87.0%; m.p. 116.5–117.6 ꢀC; 1H NMR (400 MHz, CDCl3): d
(ppm) 4.19 (s, 2H, CH2), 6.80 (m, 1H, PhCH-p, J = 5.6 Hz), 7.20 (m,
1H, PhCH-m, J = 6.2 Hz), 7.54 (m, 1H, PhCH-o, J = 2.1 Hz), 9.35 (br
s, 1H, NH ).
2-Chloro-N-(3,5-di-trifluoromethyl-phenyl)acetamide
General procedures for the preparation of 2-chloro-
N-substituted-acetamides (1)
(1c)
1
Yield 94.8%; m.p. 85.4–86.5 ꢀC; H NMR (400 MHz, CDCl3): d (ppm)
Chloroacetyl chloride (33.0 mmol) was added dropwise to a mixture
of the appropriate amine (28.0 mmol) and acetic acid (25 mL) at
4.14 (s, 2H, CH2), 7.68 (s, 1H, PhCH-p), 8.22 (s, 2H, PhCH-o), 9.48
(br s, 1H, NH ).
Chem Biol Drug Des 2010; 76: 330–339
331

Xiaohe et al.
2-Chloro-N-(4,6-dimethoxy-pyrimidin-2-yl)acetamide
(1d)
Yield 74.9%; m.p. 158.3–159.8 ꢀC; 1H NMR (400 MHz, CDCl3): d
(ppm) 3.91 (s, 6H, 2 · OCH3), 4.26 (s, 2H, CH2), 5.76 (s, 1H, Pyrimi-
dine-H5), 9.62 (br s, 1H, NH ).
(m, 1H, Pyridine-H4), 8.50–8.51(d, 1H, Pyridine-H6, J = 3.6 Hz), 10.34
(s, 1H, NH); MS (ESI) m ⁄ z (%): 307.0 [M+H]+, 329.0 [M+Na]+, 345.0
[M+K]+.
2-(1H-benzo[d]imidazol-2-ylthio)-N-phenylacetamide
(3a)
General procedures for the preparation of N-aryl-
2-arylthioacetamide derivatives (2–4)
Yield 86.2%; m.p. 199.1–200.5 ꢀC; IR (KBr, cm)1): 3436 (NH), 1677
(C = O); 1H NMR (400 MHz, CDCl3): d (ppm) 4.28 (s, 2H, -CH2),
7.04–7.08 (t, 1H, PhCH-p, J = 7.6Hz), 7.11–7.15 (m, 2H, PhCH-m),
The corresponding thiol (11.7 mmol) was added to a solution of
NaOH (0.56 g, 14.1 mmol) in water (5 mL) while stirring at room
temperature. A solution of respective 2-chloro-N-substituted-acetam-
ide 1a–d (9.8 mmol) in ethanol (20 mL) and KI (0.5 g, 3.0 mmol)
was added dropwise. Stirring was continued for 1 h, the solvent
was removed under reduced pressure, and the residue was treated
with water (50 mL) and CH2Cl2 (30 mL). The phases were separated
and the aqueous layer was extracted with CH2Cl2 (2 · 30 mL). The
combined organic phases were dried and concentrated to give a
solid, then recrystallized from a mixture of CH2Cl2 ⁄ petroleum ether.
7.29–7.33 (t, 2H, PhCH-o, J = 8.0 Hz), 7.41–7.51 (d, 2H, 2-Ph-H4,5
,
J = 5.6 Hz), 7.57–7.60 (d, 2H, 2-Ph-H3,6, J = 8.0 Hz), 10.51 (s, 1H,
Imidazole-H), 12.66 (s, 1H, NH); MS (ESI) m ⁄ z (%): 281.9 [M–H]).
2-(1H-benzo[d]imidazol-2-ylthio)-N-(2,5-di-fluoro-
phenyl)acetamide (3b)
Yield 86.8%; m.p. 193.0–194.8 ꢀC; IR (KBr, cm)1): 3432 (NH), 1679
(C = O); 1H NMR (400 MHz, CDCl3): d (ppm) 4.13 (s, 2H, -CH2),
6.79–6.85 (m, 1H, Ph-H4), 7.17–7.25 (m, 3H, 2-Ph-H4,5, Ph-H3), 7.56
(d, 2H, 2-Ph-H3,6, J = 4.7 Hz), 8.23–8.28 (m, 1H, Ph-H6), 11.60
(s, 1H, Imidazole-H), 11.84 (s, 1H, NH); MS (ESI) m ⁄ z (%): 317.9
[M–H]).
N-phenyl-2-(pyridin-2-ylthio)acetamide (2a)
Yield 87.5%; m.p. 61.3–62.9 ꢀC; IR(KBr, cm)1): 3436 (NH), 1684
(C = O); 1H NMR (400 MHz, CDCl3): d (ppm) 3.90 (s, 2H, -CH2),
7.05–7.09 (t, 1H, PhCH-p, J = 7.4 Hz), 7.14–7.17 (q, 1H, Pyridine-H5,
J = 5.2 Hz), 7.27–7.33 (m, 3H, PhCH-m, Pyridine-H3), 7.49–7.50 (d,
2H, PhCH-o, J = 8.0 Hz), 7.58–7.62 (m, 1H, Pyridine-H4, J = 7.6 Hz),
8.55–8.56 (t, 1H, Pyridine-H6, J = 4.0 Hz), 10.13 (s, 1H, NH); MS
(ESI) m ⁄ z (%): 266.9 [M+Na]+.
2-(1H-benzo[d]imidazol-2-ylthio)-N-(3,5-
bis(trifluoromethyl)phenyl)acetamide (3c)
Yield 89.2%; m.p. 202.4–203.8 ꢀC; IR (KBr, cm)1): 3432 (NH), 1680
(C = O); 1H NMR (400 MHz, CDCl3): d (ppm) 4.34 (s, 2H, -CH2),
7.11–7.15 (m, 2H, 2-Ph-H4,5), 7.42–7.47 (t, 2H, 2-Ph-H3,6, J = 18.2,
1.4Hz), 7.79 (s, 1H, Ph-H4), 8.25 (s, 2H, Ph-H2,6), 11.14 (s, 1H, Imid-
azole-H), 12.69 (s, 1H, NH); MS (ESI) m ⁄ z (%): 418.0 [M–H]).
N-(2,5-di-fluoro-phenyl)-2-(pyridin-2-ylthio)acetamide
(2b)
Yield 50.6%; m.p. 112.5–113.8 ꢀC; IR(KBr, cm)1): 3435 (NH), 1694
1
(C = O); H NMR (400 MHz, CDCl3): d (ppm) 3.90(s, 2H, -CH2), 6.65–
2-(5-amino-1,3,4-thiadiazol-2-ylthio)-N-pheny-
lacetamide (4a)
6.69 (m, 1H, Ph-H4), 6.93–6.99 (m, 1H, Ph-H3), 7.13–7.16 (m, 1H, Ph-
H6), 7.29–7.32 (d, 1H, Pyridine-H5, J = 8.4 Hz), 7.57–7.61 (m, 1H,
Pyridine-H3), 8.21–8.26 (m, 1H, Pyridine-H4), 8.54–8.55 (t, 1H, Pyri-
dine-H6, J = 2.4 Hz), 10.85 (s, 1H, NH); MS (ESI) m ⁄ z (%): 302.9
[M+Na]+.
Yield 76.2%; m.p. 174.4–175.6 ꢀC; IR (KBr, cm)1): 3300 (NH), 1665
1
(C = O), 1629 (C = N); H NMR (400 MHz, CDCl3): d (ppm) 3.99 (s,
2H, -CH2), 7.05–7.09 (t, 1H, PhCH-p, J = 7.4Hz), 7.30–7.34 (m, 4H,
Ph-H), 7.56–7.58 (d, 2H, -NH2, J = 7.6 Hz), 10.25 (s, 1H, NH); MS
(ESI) m ⁄ z (%):288.9 [M+Na]+, 304.9 [M+K]+.
N-(3,5-bis(trifluoromethyl)phenyl)-2-(pyridin-2-
ylthio)acetamide (2c)
2-(5-amino-1,3,4-thiadiazol-2-ylthio)-N-(2,5-di-
fluorophenyl)acetamide (4b)
Yield 57.4%; m.p. 110.6–111.7 ꢀC; IR(KBr, cm)1): 3431 (NH), 1669
(C = O); 1H NMR (400 MHz, CDCl3): d (ppm) 3.88 (s, 2H, -CH2),
7.21–7.26 (q, 1H, Pyridine-H5, J = 5.2Hz), 7.35–7.37 (d, 1H, Pyridine-
H3, J = 8.0 Hz), 7.55(s, 1H, Ph-H4), 7.62–7.66 (m, 1H, Pyridine-H4),
7.98 (s, 2H, Ph-H2,6), 8.56–8.57 (d, 1H, Pyridine-H6, J = 5.2 Hz),
10.94(s, 1H, NH); MS (ESI) m ⁄ z (%): 381.0 [M+H]+, 403.0 [M+Na]+.
Yield 81.1%; m.p. 179.3–180.2 ꢀC; IR (KBr, cm)1): 3306 (NH), 1672
1
(C = O), 1629 (C = N); H NMR (400 MHz, CDCl3): d (ppm) 4.11 (s,
2H, -CH2), 6.69 (s, 2H, -NH2), 6.85–6.91 (m, 1H, Ph-H4), 7.19–7.26
(m, 1H, Ph-H3), 8.13–8.18 (m, 1H, Ph-H6), 9.81 (s, 1H, NH); MS (ESI)
m ⁄ z (%): 301.0 [M+H]+, 324.9 [M+Na]+, 340.9 [M+K]+.
N-(4,6-di-methoxy-pyrimidin-2-yl)-2-(pyridin-2-
ylthio)acetamide (2d)
2-(5-amino-1,3,4-thiadiazol-2-ylthio)-N-(3,5-
bis(trifluoromethyl)phenyl)acetamide (4c)
Yield 82.2%; m.p. 184.2–185.9 ꢀC; IR(KBr, cm)1): 3489 (NH), 1698
Yield 87.4%; m.p. 117.6–118.5 ꢀC; IR(KBr, cm)1): 3433 (NH), 1678
1
1
(C = O); H NMR (400 MHz, CDCl3): d (ppm) 3.92 (s, 6H, 2 · OCH3),
(C = O), 1615 (C = N); H NMR (400 MHz, CDCl3): d (ppm) 4.10 (s,
4.16 (s, 2H, -CH2), 5.75 (s, 1H, ArH), 7.08–7.11 (q, 1H, Pyridine-H5,
J = 5.2 Hz), 7.30–7.32 (d, 1H, Pyridine-H3, J = 8.0 Hz), 7.55–7.59
2H, -CH2), 6.72 (s, 2H, -NH2), 7.73 (s, 1H, Ph-H4), 8.29 (s, 2H, Ph-H2,6),
10.26 (s, 1H, NH); MS (ESI) m ⁄ z (%): 424.9 [M+Na]+, 440.9 [M+K]+.
332
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Studies of N-aryl-2-arylthioacetamides
2-(5-amino-1,3,4-thiadiazol-2-ylthio)-N-(4,6-di-
methoxy-pyrimidin-2-yl)acetamide (4d)
the Brookhaven Protein Data Bank (PDB entry code 1dtt). After the
removal of the inhibitor from the complex, polar hydrogen atoms
and the Kollman-united charges were added to the macromolecule.
Yield 89.3%; m.p. 187.1–188.5 ꢀC; IR (KBr, cm)1): 3462 (N-H), 1667
1
(C = O), 1630 (C = N); H NMR (400 MHz, CDCl3): d (ppm) 3.88 (s,
6H, 2 · OCH3), 4.38 (s, 2H, -CH2), 5.93 (s, 1H, ArH), 7.29 (s, 2H,
NH2), 10.69 (s, 1H, NH); MS (ESI) m ⁄ z (%): 329.0 [M+H]+, 350.9
[M+Na]+, 366.9 [M+K]+.
Molecular modeling and analysis of the docked
results
For the docking, a grid spacing of 0.375 ꢀ and 61 · 61 · 61 num-
ber of points were used. Given the known location of the NNRTI-
binding site, the cubic grid box was centered in the catalytic active
region and encompassed the binding site, and the grid center was
designated at dimensions (x, y and z): )1.121, )34.649 and 23.964.
Docked conformations were generated using the LGA with an initial
population size of 150 structures. Further parameters were set to
their default values. For docking assessment, the same docking pro-
tocol was used on the reference drug PETT. AUTODOCK successfully
reproduced the bound conformation with a RMSD value of only
1.31 ꢀ. The first-ranked docked conformation (Best Docked confor-
mation) and the lowest-energy conformation of the most populated
cluster (Best Cluster conformation) were selected as the binding
conformation. Model analyses were performed using the ACCELRYS
DS VISUALIZER 2.0 software (Accelrys, Inc., San Diego, CA, USA).
In vitro assays of anti-HIV activity
The inhibitory activity of the test compounds on HIV-1 IIIB replication
in MT-4 was based on the quantitative detection of HIV-1 p24 core
antigen in the culture supernatant, which was determined with
enzyme-linked immunosorbent assay (ELISA) by Vironostika HIV-1
Antigen MicroeLisa Assay (Organon Teknika, Dublin, Ireland), accord-
ing to the manufacturer's protocol. In brief, 1 · 104 MT-4 cells in
96-well plates were infected with HIV-1 IIIB (100 TCID50) in the
presence or absence of a test compound at graded concentrations,
followed by incubation at 37 ꢀC overnight. Then, the culture super-
natants were removed, and fresh media containing no test
compounds were added. After 2 days of cultivation, the medium
was removed and the cells were fixed with 100 lL of phosphate-
buffered saline (PBS) containing 1% formaldehyde and 0.2% glutar-
aldehyde for 5 min at room temperature. The cells were then
washed three times with PBS and incubated for 1 h at 37 ꢀC with
100 lL of an indicator HRP. The reaction was terminated by addition
of 1N H2SO4. Absorbance at 450 nm was recorded in an ELISA
reader. Recombinant protein p24 was included for establishing stan-
dard dose–response curves. Each sample was tested in triplicate.
Results and Discussion
Chemistry
The N-aryl-2-arylthioacetamide derivatives were synthesized using
the conventional strategy as depicted in Scheme 1. The starting
compound, 2-chloro-N-substituted-acetamides (1), was readily syn-
thesized by the reaction of chloroacetyl chloride and anilines or
pyrimidinylamines in acetic acid at ambient temperature in 75–95%
yields (21). By alkylation of the corresponding sodium thiolate with
1, we prepared N-aryl-2-arylthioacetamide derivatives 2–4. At the
alkylation reactions, the literature procedure (22) was carried out
for several hours in ethanol. Our more versatile procedure used KI
as the catalyst, which afforded the N-aryl-2-arylthioacetamides in
good yields and with short reaction times. The 2–4 were isolated
in 51–89% yields (Table 1) as colorless powders, which were gener-
ally purified by recrystallization with CH2Cl2 and petroleum ether.
The structures of 2–4 were determined by the IR, NMR and mass
spectra. The 1a–d were confirmed in particular by the presence of
a proton of NH as a singlet signal at 8.30–9.62 ppm in 1H NMR
spectra. The IR spectrum of the N-aryl-2-arylthioacetamide deriva-
tives (2–4) contained a characteristic absorption band of the CONH
group at 1665–1700 and 3300–3460 ⁄ cm. The 1H NMR spectra of
2–4, containing the thiomethylene group, revealed the SCH2 pro-
tons at 3.88–4.38 ppm. In addition, the ESI–MS spectra of 2–4
exhibited the anticipated molecular ion peaks, and the fragmenta-
tion ions were consistent with their structures.
Molecular modeling study
The automated docking studies were performed with AUTODOCK 4.0
(20). This automated ligand-docking program uses the Lamarckian
genetic algorithm (LGA) to explore the full range of ligand confor-
mational flexibility with partial flexibility of the receptor. Lamarckian
genetic algorithm is a hybrid of a genetic algorithm and a local
search algorithm. This algorithm first builds a population of individ-
uals (genes), each gene being a different random conformation of
the docked compound. The local search algorithm then performs
energy minimizations on a user-specified proportion of the popula-
tion of individuals. If the energy of the new individual is lower than
that of the old, the new one is automatically accepted as the next
step in docking.
Preparation of the receptor and ligands
molecules
The three-dimensional structures of ligands were constructed using
standard bond lengths and bond angles of the GAUSSVIEW 3.09 soft-
ware (Gaussian, Inc., Wallingford, CT, USA). Geometry optimizations
were carried out with the semi-empirical AM1 method, and then
output files were minimized by using density functional (DFT)
method by applying the B3LYP (Becke, Lee, Yang and Parr) correla-
tion functional in the second optimization. Gasteiger partial charges
were assigned using the AUTODOCK Tools. The crystal structure of
HIV-1 RT receptor in complex with PETT (N-(5-chloro-2-pyridinyl)-N'-
[2-(4-ethoxy-3-fluoro-2-pyridinyl)ethyl]-thiourea) was retrieved from
Biological activity
The inhibitory activity of N-aryl-2-arylthioacetamide derivatives 2–4
was evaluated against HIV-1 (IIIB) replication in MT-4 cell culture
using ELISA (23,24). The compound Zidovudine (azidothymidine, AZT)
was used as a reference drug. The results, expressed as IC50 val-
ues, are summarized in Table 1. Although the tested compounds
Chem Biol Drug Des 2010; 76: 330–339
333

Xiaohe et al.
Scheme 1: General prepara-
tion of N-aryl-2-arylthioacetamides
(2–4). Reagents and conditions:
(i) chloroacetyl chloride, AcOH,
0 ꢀC fi rt, 4 h; (ii) thiol, NaOH,
EtOH, KI, rt, 1 h.
Table 1: Preparation of 2a–d, 3a–c and 4a–d and anti-HIV-1 activity valuesa
H
H
N
N
H
N
N
N
X
N
X
X
S
S
H2N
S
R
S
R
N
R
N
H
O
X
O
X
O
X
4
3
2
Compound
X
R
Mp (/C)
Yield (%)
IC50 (lM)b
2a
2b
2c
2d
3a
3b
3c
4a
4b
4c
4d
CH
CH
CH
N
CH
CH
CH
CH
CH
CH
N
H
61.3–62.9
87.5
50.6
57.4
87.4
86.2
86.8
89.2
76.2
81.1
82.2
89.3
>100
>100
>100
>100
5.07 € 0.21
1.25 € 0.08
2.20 € 0.32
20.83 € 1.17
16.10 € 0.24
14.93 € 0.84
7.50 € 1.06
2,5-di-F
3,5-di-CF3
4,6-di-OCH3
H
2,5-di-F
3,5-di-CF3
H
2,5-di-F
3,5-di-CF3
4,6-di-OCH3
112.5–113.8
110.6–111.7
117.6–118.5
199.1–200.5
193.0–194.8
202.4–203.8
174.4–175.6
179.3–180.2
184.2–185.9
187.1–188.5
aZidovudine was used as a reference compound; IC50 for zidovudine was 0.016 lM.
bCompound concentration required to decrease the viability of mock-infected cells by 50%; Data represent mean values for three separate experiments,
variation among triplicate samples was <15%.
exhibited less anti-HIV-1 activities than that of AZT
(IC50 = 0.016 lM) as the control, the majority of them were active
in the lower micromolar concentration (1.25–20.83 lM), appreciably
revealing their promising potential inhibitory activities.
romethyl groups on the N-phenyl ring improved antiviral potency,
whereas their unsubstituted N-phenyl series led to less potent
derivatives in both 1H-benzo[d]imidazole and 5-amino-1,3,4-thiadia-
zole series (compare 3b,c with 3a, and 4b,c with 4a). It is
appeared to confirm our assumption that the analogs bearing an
electron-withdrawing group might be more potent than the congen-
ers with an electron-donating group. Interestingly, the 2,5-difluoro
derivative 3b was two times more potent than 3,5-ditrifluoromethyl
compound 3c, and the same replacement of 4b was led to a
decrease in potency with respect to the 3,5-ditrifluoromethyl com-
pound 4c. The activity trend suggested that the steric ⁄ electronic
properties of the N-phenyl substituents influenced the antiretroviral
activity more than how their positions do.
It has been observed that the electronic properties of the arylthio
substituents significantly affected the activity compared to the N-
aryl substituents. Among them, analogs of 3 series showed IC50
values <5.07 lM. Compound 3b was the most active one with the
highest potency (IC50 = 1.25 lM) lower than the reference drug of
AZT, which indicates that the 1H-benzo[d]imidazole is an acceptable
isosteric replacement for the triazole in the lead compound A.
Interestingly, the activities of 1H-benzo[d]imidazoles 3a–c were
turned out to be the most potent N-aryl-2-arylthioacetamides com-
pared to the corresponding pyridines 2a–d and 5-amino-1,3,4-thi-
adiazoles 4a–d (3a versus 2a and 4a; 3b versus 2b and 4b; 3c
versus 2c and 4c).
Successively, the type of rings on the N-aryl also affected the antiv-
iral potency of 2–4. 4d with heteroaryl ring was indicated the
highest anti-HIV activity in the 4 series, probably because the 4,6-
dimethoxy-pyrimidyl ring might be more favorable to improve a
putative p-stacking interaction between the electron-deficient aryl
ring of the ligand and the electron-rich benzene ring of RT (25).
The electronic proprieties of groups on the N-aryl affected the an-
tiviral potency of 2–4. The introduction of fluorine atoms or tr–uo-
334
Chem Biol Drug Des 2010; 76: 330–339

Studies of N-aryl-2-arylthioacetamides
Molecular docking studies
structure as a means of testing program performance. A superposi-
tion of the most highly scored conformation of the ligand onto its
crystallographic geometry yielded a RMSD (root mean square devia-
tion) of 1.31 ꢀ, which was the best docked conformation by <2 ꢀ
(34), thus revealing that AUTODOCK was successful in reproducing
the binding mode of PETT into the active site of HIV-1 RT.
In attempt to further elucidate the high HIV-1 inhibitory potencies
of the 2–4 at a molecular level, the computer-simulated automated
docking studies were performed using the widely distributed molec-
ular docking software, AutoDock, which has been successfully used
to predict protein recognition and binding (26–28). The HIV-1
RT ⁄ NNRTI complexes of X-ray crystallography had identified that
the NNRTIs show a common configuration resembling a butterfly-
like shape where the wings generally contain aromatic rings that
have p–p interactions with aromatic amino acid residues (29–33).
All test compounds 2–4 were docked into the NNBS using the
recently published HIV-1 RT protein crystal structure of PETT (PDB
entry code 1dtt) as template (29). Prior to automate docking of the
reported inhibitors, PETT itself was docked into the HIV-1 RT crystal
Two different interactional modes of 3b and 4d with the RT recep-
tor were generated from docking. In the most frequently occurring
and most favorable result, 3b was found to bind the NNBS in an
orientation very similar to that of the co-crystallized inhibitor PETT,
and compound 4d displayed a different binding mode. Figure 3
shows the predicted binding mode of 3b and 4d to the active site
of HIV-1 RT. A schematic depiction of the interaction between both
A
B
Figure 3: Stereographic views of the binding mode of compounds (A) 3b and (B) 4d in the HIV-1 RT NNBS. The ligands are represented
as bold sticks, while the amino acid residues lining the RT NNBS are shown as thin sticks. For clarity, only interacting polar hydrogen atoms
are displayed. Hydrogen bonds are depicted as dashed lines.
Chem Biol Drug Des 2010; 76: 330–339
335

Xiaohe et al.
inhibitors and the RT NNBS residues is illustrated in Figure 4. It
can be observed from Figure 3A that the NH function of the 1H-
benzo[d]-imidazole of 3b makes a hydrogen bond with the main
chain carbonyl oxygen of Glu138 (NHÆÆÆO = C, d = 2.08 ꢀ), whereas
compound 4d is involved in two hydrogen bonds with the enzyme
backbone: one involves the amino group of thiadiazole and the car-
bonyl oxygen of Glu138 (NHÆÆÆO = C, d = 2.20 ꢀ) and the other
occurs between the nitrogen of thiadiazole and the main chain NH
group of Lys101 (NÆÆÆHN, d = 1.82 ꢀ).
the biological data, which shows that 3b and 4d are more potent
than 2.
As shown in Figure 4A, the thiomethyl linker of the RT ⁄ 3b complex
is embedded within a hydrophobic pocket made up by the side
chains of Val106 and Val179, while the 1H-benzo[d]-imidazole moi-
ety fills the hydrophobic bottom of the NNBS made up by the aro-
matic rings of Tyr181, Tyr188 and Trp229 as well as by Glu138. In
particular, it is involved in p–p interactions with Tyr181, Tyr188 and
interacts with a tilted T contact with the aromatic indole nucleus of
Trp229. Furthermore, the favorable steric interactions of the N-phe-
nyl ring with a hydrophobic pocket are defined by the side chains
of the Leu100, Lys103, His235 and Tyr318. The fluorine atom estab-
lishes van der Waals contacts with the Pro236 main chain atoms,
and the imidic oxygen is involved in a network of polar interactions
with the Leu100 main chains, thus providing further stabilization to
the complex.
In comparing the docking simulations of 2a–d with that of 3b and
4d in the NNBS, we did not observe any H-bonding interaction
between the nitrogen of pyridine and the enzyme. The lack of this
hydrogen bond prevents the ligand from assuming a bioactive U-
shaped conformation within the NNBS, and this is consistent with
A
Interestingly, the conformation of compound 4d in Figure 4B lies
along the RT NNBS in opposite direction to that of 3b. The pyrimi-
dine ring establishes the p–p interactions with the Tyr181 and
Tyr188 side chain, whereas the thiadiazole group is involved in van
der Waals interactions with Val179, Lys101 and Gly190. Notably,
the model shows the formation of two potential intermolecular
hydrogen bonds, which is one more than that of 3b.
This additional hydrogen bond might explain not only the same
level of antiviral activity of 3b through a stable interaction with RT
but also its reverse binding mode to the NNBS. In fact, if their
binding modes were similar, the thiadiazole group should be located
at the bottom of the RT pocket, as above said for the 1H-benzo[d]-
imidazole ring of 3b. As a consequence, this moiety would not be
suitably situated to form the hydrogen bond with Lys101. The
reverse binding mode of 4d cannot approximate the butterfly-like
structure commonly observed with other NNRTIs (29), but it is com-
pensated by the number of hydrophobic contacts and more hydro-
gen bonds in which 4d is engaged.
B
Inspection of the RT ⁄ 3b complex revealed that the N-phenyl ring is
hosted in a subpocket, made up by the side chains of Leu100,
Lys103, His235 and Tyr318. Notably, these residues are involved
through prominent van der Waals and p–p stacking interactions,
with the p-electron systems located in the butterfly wings of RT
NNBS conformation (30,32). We hypothesized that an efficient occu-
pancy of this region by strategically designed aromatic substituents
should yield more potent anti-HIV-1 agents with higher affinity for
the RT binding pocket. Actually, the analog 3b and 3c were four-
fold and twofold more active than 3a, respectively, suggesting that
there is a wide sterically allowed usable space in the regions host-
ing the N-phenyl and the (hetero)aryl moieties. These observations
could provide the basis for further structural modifications of them.
Binding affinities
The binding affinity was evaluated by the binding free energies
(DGb), inhibition constants (Ki) and hydrogen bonding values. The
compounds 2–4 which revealed the highest binding affinities, that
is, the lowest binding free energies, within HIV-1 RT NNBS and the
Figure 4: Schematic diagrams showing the intermolecular inter-
actions between the inhibitors (A) 3b, (B) 4d, and the surrounding
residues of HIV-1 RT NNBS. NNBS, non-nucleoside binding site.
336
Chem Biol Drug Des 2010; 76: 330–339

Studies of N-aryl-2-arylthioacetamides
Table 2: The best docking results based on the binding free energies (DGb) and inhibition constants (Ki) of 2–4
Hydrogen bonds between atoms of compounds and amino acids
RMSDc (ꢀ)
a
Compound
DGb (kcal ⁄ mol)
Kib
Atom of compound
Amino acid
Distance (ꢀ)
Angle (ꢀ)
2a
2b
2c
2d
3a
3b
3c
4a
4b
4c
)4.76
)5.73
)5.57
)5.27
)6.55
)7.00
)6.68
)6.23
)5.81
)6.01
3.26E–4
6.26E–5
8.22E–5
1.37E–4
1.59E–5
7.35E–6
1.27E–5
2.69E–5
5.50E–5
3.96E–5
–
–
–
–
–
–
–
–
–
–
–
2.03
2.08
1.77
–
–
–
–
7.32
4.63
8.49
5.69
7.85
8.76
8.75
7.09
5.67
6.51
–
–
N1–H
N1–H
N1–H
–
CO of Glu 138
CO of Glu 138
CO of Lys 101
–
162.1
154.5
169.7
–
–
–
–
–
C5–NH
C5–NH
N1
C5–NH
N1
CO of Gly 99
CO of Glu 138
HN of Lys 101
CO of Glu 138
HN of Lys 101
CO of Lys 101
2.15
2.20
1.78
2.20
1.82
2.16
142.4
159.4
145.3
135.3
160.5
124.6
4d
)6.50
)6.07
7.83E–5
3.57E–5
3.80
1.31
PETTd
N8–H
aBinding free energy.
bInhibition constant.
cRoot mean square deviation.
dThe native co-crystallized bound ligand (PETT) of HIV-1 RT (PDB code: 1dtt).
conformation as a determinant requisite for antiretroviral activity.
These compounds showed significant anti-HIV-1 activity in the
micromolar concentration range (1.25–20.83 lM). Compounds 3a–c
turned out to be the most potent derivatives compared to 2a–d
and 4a–d, by the replacement of the pyridine or thiadiazole ring of
the arylthio moiety with 1H-benzo[d]imidazole. In particular, intro-
duction of fluorine atoms or trifluoromethyl groups on the N-phenyl
ring of 3 series was endowed with high activity and selectivity. The
most potent 3b displayed an IC50 value of 1.25 lM lower than that
of the lead compound A.
Molecular modeling and docking studies were employed to under-
stand the interactions between these inhibitors and the reverse
transcriptase. The highest potency of 3b would be ascribable to
the stabilization by the hydrogen bond between the NH of the 1H-
benzo[d]-imidazole and the Glu138 main chain carbonyl and van der
Waals contacts between the N-phenyl ring and the Leu100, Lys103,
His235 and Tyr318 side chains, which bind to reverse transcriptase
assuming a 'butterfly-like' orientation. Finally, considering the bind-
ing affinities and conformations of 3 series, further SAR studies,
keeping constant the N-phenyl and the (hetero)aryl substitution pat-
terns emerged as the best in the present work, will be the object
of the following paper.
Figure 5: Correlation between the DGb and IC50 of 3a–c and
4a–d.
hydrogen bond interactions into the target macromolecule, are rep-
resented in Table 2. The 3 series exhibited the lowest free energy
between )7.00 and )6.55 kcal ⁄ mol and 3b with the least binding
free energy ()7.00 kcal ⁄ mol) by fitting well into the groove of the
binding site. The overall correlation between the inhibitory activities
(IC50, lM) of the N-aryl-2-arylthioacetamids 2–4 and the binding
affinities (lower binding free energy) predicted by AUTODOCK was
fairly good for some compounds. Especially, the correlation between
the binding free energy (DGb) and IC50 (lM) values for compounds
3a–c and 4a–d revealed a correlation coefficient (R2) of 0.720 as
shown in Figure 5.
Acknowledgments
This work was financially supported by Key Projects in the National
Science & Technology Pillar Program during the Eleventh Five-Year
Plan Period (No. 2008ZX10001-015) and Funding Project for Aca-
demic Human Resources Development in Institutions of Higher
Learning under the Jurisdiction of Beijing Municipality. We thank
Department of Viro-Pharmacology, Beijing University of Technology,
for their assistances in the experiments.
Conclusions
N-aryl-2-arylthioacetamides 2–4 were designed and synthesized as
a new class of NNRTIs, taking into account the 'butterfly-like'
Chem Biol Drug Des 2010; 76: 330–339
337

Xiaohe et al.
15. Ilina T., Parniak M.A. (2008) Inhibitors of HIV-I reverse transcrip-
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