Design, Synthesis, and Biological Evaluation of Novel 2-(Pyridin-3-yloxy)acetamide Derivatives as Potential Anti-HIV-1 Agents

Article abstract of DOI:10.1111/cbdd.12657

Through a structure-based molecular hybridization and bioisosterism approach, a series of novel 2-(pyridin-3-yloxy)acetamide derivatives were designed, synthesized, and evaluated for their anti-HIV activities in MT-4 cell cultures. Biological results showed that three compounds (Ia, Ih, and Ij) exhibited moderate inhibitory activities against wild-type (wt) HIV-1 strain (III_B) with EC₅₀ values ranging from 8.18 μm to 41.52 μm. Among them, Ij was the most active analogue possessing an EC₅₀ value of 8.18 μm. To further confirm the binding target, four compounds were selected to implement an HIV-1 RT inhibitory assay. In addition, preliminary structure-activity relationship (SAR) analysis and some predicted physicochemical properties of three active compounds Ia, Ih, and Ij were discussed in detail. Molecular docking studies were also carried out to investigate the binding modes of Ij and the lead compound GW678248 in the binding pocket of RT, which provided beneficial information for further rational design of non-nucleoside reverse transcriptase inhibitors.

Full text of DOI:10.1111/cbdd.12657

Chem Biol Drug Des 2016; 87: 283–289
Research Article
Design, Synthesis, and Biological Evaluation of Novel
2-(Pyridin-3-yloxy)acetamide Derivatives as Potential
Anti-HIV-1 Agents
Boshi Huang1, Xiao Li1, Peng Zhan1,*, Erik De
(1). Up to date, five NNRTI drugs (Figure S1), including the
first generation NNRTIs (nevirapine, delavirdine, efavirenz)
and the second generation NNRTIs (etravirine, rilpivirine),
have been approved by FDA for clinical use, and signifi-
Clercq2, Dirk Daelemans2, Christophe
Pannecouque2 and Xinyong Liu1,*
1Department of Medicinal Chemistry, Key Laboratory of
cantly reduced the morbidity and mortality of AIDS. How-
Chemical Biology (Ministry of Education), School of
Pharmaceutical Sciences, Shandong University, 44 West
Culture Road, Ji’nan 250012, Shandong, China
2Rega Institute for Medical Research, KU Leuven
Minderbroedersstraat 10, Leuven B-3000, Belgium
*Corresponding authors: Peng Zhan and Xinyong Liu,
zhanpeng1982@sdu.edu.cn and xinyongl@sdu.edu.cn
ever, long-term use of NNRTIs inevitably leads to the rapid
emergence of viral drug resistance and serious adverse
effects (2). Therefore, the development of novel NNRTI
drugs with efficient anti-HIV-1 potency against both wild-
type (wt) and mutant strains is still urgently required to
combat the increasing AIDS pandemic (3).
Fortunately, the structural diversity of HIV-1 NNRTIs and
the flexibility of NNRTI binding pocket (NNIBP) in RT gave
a broad space for novel lead discovery, and the pharma-
cophore similarity of NNRTIs also provided valuable clues
to expedite process of lead discovery and optimization (4).
Through a structure-based molecular hybridization and
bioisosterism approach, a series of novel 2-(pyridin-
3-yloxy)acetamide derivatives were designed, synthe-
sized, and evaluated for their anti-HIV activities in
MT-4 cell cultures. Biological results showed that three
compounds (Ia, Ih, and Ij) exhibited moderate inhibitory
activities against wild-type (wt) HIV-1 strain (IIIB) with
EC50 values ranging from 8.18 lM to 41.52 lM. Among
them, Ij was the most active analogue possessing an
EC50 value of 8.18 lM. To further confirm the binding
target, four compounds were selected to implement an
HIV-1 RT inhibitory assay. In addition, preliminary
structure–activity relationship (SAR) analysis and some
predicted physicochemical properties of three active
compounds Ia, Ih, and Ij were discussed in detail.
Molecular docking studies were also carried out to
investigate the binding modes of Ij and the lead
compound GW678248 in the binding pocket of RT,
which provided beneficial information for further
rational design of non-nucleoside reverse transcriptase
inhibitors.
Benzophenone derivatives are one representative class of
HIV-1 NNRTIs with excellent activities against wt and a
wide range of drug-resistant HIV-1 strains. Among them,
GW678248 (1) (Figure S2) displayed remarkable inhibitory
activities against wt, K103N single mutant and
K103N + Y181C double mutant strains in the nanomolar
range, with EC50 values of 0.52, 1.0, and 1.4 nM accord-
ingly, and its prodrug is undergoing phase II clinical trials
(5,6).
Arylthiotetrazolyl acetanilide analogue 2, which was discov-
ered in the course of a ultra high-throughput screening
(uHTS) campaign, exhibited strong inhibition against wt
and the clinically relevant K103N + Y181C double mutant
HIV-1 RT, in low nanomolar and submicromolar level,
respectively. Subsequent structural modifications led to
the identification of compound 3, which possesses some
different chemical features, such as parazole scaffold
instead of tetrazole scaffold and oxygene instead of sulfur.
Surprisingly, compound 3 showed a dramatically increased
inhibitory activity against K103N + Y181C double mutant
RT (7) (Figure S2).
Key words: anti-HIV activity, bioisosterism, HIV-1 RT,
molecular hybridization, non-nucleoside reverse transcriptase
inhibitors
Received 16 June 2015, revised 15 August 2015 and
accepted for publication 27 August 2015
Due to the unique antiviral potency, high specificity and
low cytotoxicity, HIV-1 non-nucleoside reverse transcrip-
tase inhibitors (NNRTIs) have become indispensable com-
ponents of highly active antiretroviral therapy (HAART), an
efficient and standard treatment regimen for AIDS therapy
In continuation of developing potent NNRTIs as promising
antiretroviral agents,
a series of 2-(pyridin-3-yloxy)ac-
etamide derivatives were designed derived from the privi-
leged structures of lead compounds GW678248 (1) and 3
ª 2015 John Wiley & Sons A/S. doi: 10.1111/cbdd.12657
283

Huang et al.
utilizing the molecular hybridization and bioisosterism prin-
ciples. In the newly designed compounds, a naphthyl or a
para-substituted phenyl was adapted from compound 3,
which was reported to occupy the aromatic-rich sub-
pocket in NNIBP. And a pyridyl ring, as a common surro-
gate of phenyl ring, was used as the central ring based on
bioisosterism. Furthermore, the right oxyacetamide moiety
was employed from the two lead compounds with the aim
of generating indispensable interactions with RT at the RT/
solvent interface. Different substituents, varying in elec-
tronic nature and size, were applied in the right phenyl
wing for further investigating the structure–activity relation-
ship (SAR) features of the newly synthesized compounds
(Figure S3).
with saturated brine and dried over anhydrous Na2SO4, fil-
tered and concentrated under reduced pressure. Recrys-
tallization of the residue by methanol afforded intermediate
I as a white solid, which could be used in the next step
with no further purification. Yield: 55%. mp: 177.0–
177.8 °C. 1H NMR (400 MHz, DMSO-d6, p.p.m.) d: 9.84
(s, 1H, OH), 8.20 (d, J = 4.44 Hz, 1H, pyridine-H), 7.96
(t, J = 6.84 Hz, 2H, Naph-H), 7.59–7.38 (m, 6H, Naph-H,
pyridine-H), 7.32 (dd, J = 8.16 Hz, J = 4.52 Hz, 1H,
pyridine-H), 13C NMR (100 MHz, DMSO-d6, p.p.m.) d:
152.19, 146.82, 140.47, 136.77, 133.60, 131.65, 128.51,
128.33, 127.79, 126.36, 126.24, 126.07, 125.72, 124.17,
123.26. ESI-MS: m/z 220.4 (M-1), C15H11NO (221.08).
2-(p-Tolyl)pyridin-3-ol (II): A mixture of 2-bromopyridin-3-ol
(S) (0.20 g, 1.14 mmol), p-tolylboronic acid (0.19 g,
1.38 mmol), K3PO4 (0.61 g, 2.30 mmol), and Pd(PPh3)4
(0.09 g, 0.08 mmol) was dissolved in a mixed solvent
(dioxane: water = 5 mL: 1 mL). The solution was stirred at
90 °C under a nitrogen atmosphere followed by TLC until
its completion. After evaporation of the excess solvent
under reduced pressure, water was added and the solu-
tion was extracted with CH2Cl2 (3 9 40 mL). The organic
layer was combined, washed with saturated brine, dried
over anhydrous Na2SO4, filtered and concentrated under
reduced pressure. Finally, the residue was further purified
by flash column chromatography and subsequently recrys-
tallized with methanol to give pure intermediate II as a
white solid. Yield: 36%. mp: 193.0–194.0 °C. 1H NMR
(400 MHz, DMSO-d6, p.p.m.) d: 10.08 (s, 1H, OH), 8.14
(d, J = 4.40 Hz, 1H, pyridine-H), 7.93 (d, J = 8.12 Hz, 2H,
Ph-H), 7.31 (d, J = 8.12 Hz, 1H, pyridine-H), 7.23 (d,
J = 8.04 Hz, 2H, Ph-H), 7.16 (dd, J = 8.12 Hz,
J = 4.48 Hz, 1H, pyridine-H), 2.34 (s, 3H, CH3), 13C NMR
(100 MHz, DMSO-d6, p.p.m.) d: 151.86, 144.89, 140.57,
137.47, 135.70, 129.19 (2 9 Ph-C), 128.78 (2 9 Ph-C),
123.89, 123.46, 21.32 (CH3). ESI-MS: m/z 184.2 (M-1),
C12H11NO (185.08).
Herein, we reported the synthesis and antiviral activity
in vitro of these novel 2-(pyridin-3-yloxy)acetamide deriva-
tives. The SARs were well discussed, and the molecular
modeling study and the molecular physicochemical prop-
erty analysis were also carried out to gain further insights
into this series of analogues.
Experimental Section
Chemistry
All melting points (mp) were determined on a micromelting
point apparatus and are uncorrected. Mass spectra were
performed on
a LC Autosampler Device: Standard
G1313A instrument by electrospray ionization. 1H NMR
and 13C NMR spectra were obtained on a Bruker AV-400
€
spectrometer (Bruker BioSpin, Fallanden, Switzerland) in
the indicated solvent DMSO-d6. Chemical shifts were
expressed in d units (p.p.m.), using TMS as an internal
standard, and J values were reported in hertz (Hz). TLC
was performed on Silica Gel GF254. Spots were visualized
by irradiation with UV light (k 254 nm). Flash column chro-
matography was carried out on columns packed with silica
gel 60 (200–300 mesh). Solvents were of reagent grade
and, if needed, were purified and dried by standard meth-
ods. The key reagents were purchased from commercial
suppliers and with no further purification when used.
Rotary evaporators were served in concentration of the
reaction solutions under reduced pressure.
III–V were prepared according to the same procedure
described as II, while amount of the initial materials and
reaction solvents extended 10 times accordingly.
2-(4-(Trifluoromethyl)phenyl)pyridin-3-ol (III): White solid,
yield: 43%. mp: 234.6–235.4 °C. 1H NMR (400 MHz,
DMSO-d6, p.p.m.) d: 10.45 (s, 1H, OH), 8.26 (d,
J = 8.16 Hz, 2H, Ph-H), 8.21 (d, J = 4.28 Hz, 1H,
pyridine-H), 7.80 (d, J = 8.28 Hz, 2H, Ph-H), 7.40
(d, J = 8.12 Hz, 1H, pyridine-H), 7.28 (dd, J = 8.16 Hz,
J = 4.40 Hz, 1H, pyridine-H), 13C NMR (100 MHz, DMSO-
d6, p.p.m.) d: 152.53, 142.92, 142.38, 140.99, 129.86,
128.40 (q, JC–F = 32.00 Hz, 2C, Ph-C), 125.12 (q, JC–
General procedure for the synthesis of
intermediates (I–V)
2-(Naphthalen-1-yl)pyridin-3-ol (I): The starting material 2-
bromopyridin-3-ol (S) (0.20 g, 1.14 mmol), 1-naphthylboric
acid (0.24 g, 1.38 mmol), K2CO3 (0.24 g, 1.72 mmol), and
Pd(PPh3)4 (0.09 g, 0.08 mmol) were added to a mixed sol-
vent (dioxane: water = 5 mL: 1 mL). The mixture was stir-
red at 90 °C under a nitrogen atmosphere until the
completion of the reaction as detected by TLC. After cool-
ing to room temperature, the solid was filtered, washed by
water, and the total filtrate was extracted with EtOAc
(3 9 40 mL). The combined organic phase was washed
= 4.00 Hz, 2C, Ph-C), 124.88 (q, JC–F = 270.00 Hz,
F
2C, Ph-C), 124.84, 124.48. ESI-MS: m/z 240.2 (M + 1),
C12H8F3NO (239.06).
2-(4-(tert-Butyl)phenyl)pyridin-3-ol (IV): White solid, yield:
1
32%. mp: 176.0–176.6 °C. H NMR (400 MHz, DMSO-d6,
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Chem Biol Drug Des 2016; 87: 283–289

2-(Pyridin-3-yloxy)acetamides as Anti-HIV-1 Agents
p.p.m.) d: 10.07 (s, 1H, OH), 8.14 (d, J = 4.40 Hz, 1H,
pyridine-H), 7.94 (d, J = 8.40 Hz, 2H, Ph-H), 7.44
(d, J = 8.44 Hz, 2H, Ph-H), 7.31 (d, J = 8.12 Hz, 1H,
pyridine-H), 7.16 (dd, J = 8.08 Hz, J = 4.48 Hz, 1H, pyri-
dine-H), 1.32 (s, 9H, t-Bu), 13C NMR (100 MHz, DMSO-
d6, p.p.m.) d: 151.89, 150.60, 144.97, 140.61, 135.70,
129.02 (2 9 Ph-C), 124.92 (2 9 Ph-C), 123.84, 123.47,
34.77, 31.60 (3 9 CH3). ESI-MS: m/z 228.6 (M + 1), C15
H17NO (227.13).
(50% cell culture infectious dose) or culture medium was
added to either the infected or mock-infected wells of the
microtitre tray. Mock-infected cells were used to evaluate
the effect of test compounds on uninfected cells to assess
its cytotoxicity. Exponentially growing MT-4 cells were cen-
trifuged for 5 min at 1000 rpm, 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 microtitre tray wells. Five days after infection, the viabil-
ity of mock- and HIV-infected cells was examined spec-
trophotometrically by the MTT method. The 50% cytotoxic
concentration (CC50) was defined as the concentration of
the test compound that reduced the viability of the mock-
infected MT-4 cells by 50%. The concentration achieving
50% protection from the cytopathic effect of the virus in
infected cells was defined as the 50% effective concentra-
tion (EC50).
2-(4-Methoxyphenyl)pyridin-3-ol (V): Yellow solid, yield:
1
40%. mp: 180.0–180.6 °C. H NMR (400 MHz, DMSO-d6,
p.p.m.) d: 10.07 (s, 1H, OH), 8.12 (d, J = 4.28 Hz, 1H,
pyridine-H), 8.02 (d, J = 8.76 Hz, 2H, Ph-H), 7.29
(d, J = 8.00 Hz, 1H, pyridine-H), 7.14 (dd, J = 8.08 Hz,
J = 4.48 Hz, 1H, pyridine-H), 6.98 (d, J = 8.80 Hz, 2H,
Ph-H), 3.80 (s, 3H, CH3), 13C NMR (100 MHz, DMSO-
d6, p.p.m.) d: 159.44, 151.61, 144.60, 140.50, 130.96,
130.58 (2 9 Ph-C), 123.81, 123.07, 113.60 (2 9 Ph-C),
55.55 (OCH3). ESI-MS: m/z 203.3 (M+2), C12H11NO2
(201.08).
HIV-1 RT inhibition assay
The inhibition assay of HIV-1 RTwt was performed by utiliz-
ing the template/primer hybrid poly(A) 9 oligo(dT)15, digox-
igenin- and biotin-labeled nucleotides, an antibody to
digoxigenin which was conjugated to peroxidase (anti-
DIG-POD), and the peroxidase substrate ABTS. The incor-
poration quantities of the digoxigenin- and biotin-labeled
dUTP into DNA represented the activity of HIV-1 RT. The
HIV-RT inhibition assay was implemented using an RT
assay kit (Roche, Mannheim, Germany), and the proce-
dures for assaying RT inhibition were carried out as
described in the kit protocol (10). Concretely, the reaction
mixture consisted of template/primer complex, 20-deoxy-
nucleotide-50-triphosphates (dNTPs), and RT enzyme in
the lysis buffer with or without inhibitors. After incubation
for 1 h at 37 °C, the reaction mixture was transferred to
streptavidine-coated microtitre plate (MTP). The biotin-la-
beled dNTPs that are incorporated in the template due to
the presence of RT were bound to streptavidine. The
unbound dNTPs were washed using wash buffer, and
then, antidigoxigenin–peroxidase (anti-DIG-POD) was
added in MTP. The DIG-labeled dNTPs incorporated in the
template was bound to anti-DIG-POD antibody. The
unbound anti-DIG-POD was also washed elaborately for
five times using wash buffer, and finally, the peroxide sub-
strate (ABST) was added to the MTP. A colored reaction
product emerged during the cleavage of the substrate cat-
alyzed by a peroxide enzyme. The absorbance of the sam-
ple was determined at OD450 using MTP ELISA reader.
The percentage inhibitory activity of RT inhibitors was cal-
culated by comparison with a sample lacking an inhibitor.
The resulting color intensity is directly proportional to RT
activity. The percentage inhibitory values were calculated
by the following formula: %Inhibition = [O.D. value without
inhibitors (with RT) -O.D. value with RT and inhibitors]/
[O.D. value with RT and inhibitors-O.D. value without RT
and inhibitors]. The IC50 value corresponded to the con-
centration of the tested compound required to inhibit the
incorporation of the labeled dUTP into RT by 50%.
General procedure for the synthesis of target
compounds (Ia-o, IIa, IIb, IIIa, IIIb, IVa, IVb, Va, Vb)
At room temperature, key intermediates I–V (0.15 g,
1 eqv.) and anhydrous K2CO3 (2 eqv.) were added to
anhydrous N,N-Dimethylformamide (DMF, 5 mL), followed
by the addition of the appropriate ClCH2CONHAr (1 eqv.).
Then, the reaction mixture was stirred until its completion
at 50 °C. The excess DMF was evaporated under reduced
pressure, and H2O (40 mL) was added. After extraction
with EtOAc (3 9 40 mL), the combined organic phase
was washed with saturated brine, dried over anhydrous
Na2SO4, filtered and concentrated under reduced pres-
sure. Then, the residue was further purified by flash col-
umn chromatography and recrystallized with EtOAc or
anhydrous methanol to afford pure title compounds Ia-o,
IIa, IIb, IIIa, IIIb, IVa, IVb, Va and Vb.
In vitro anti-HIV assay
Evaluation of the antiviral activity and cytotoxicity of the
synthesized compounds was performed using the MTT
assay as previously described (8,9). Stock solutions (109
final concentration) of test compounds were added in
25 lL volumes to two series of triplicate wells to allow
simultaneous evaluation of their effects on mock- and HIV-
infected cells at the beginning of each experiment. Serial
fivefold dilutions of test compounds were made directly in
flat-bottomed 96-well microtitre trays by adding 100 lL
medium to the 25 lL stock solution and transferring 25-lL
of this solution to another well that contained 100 lL med-
ium using a Biomek 3000 robot (Beckman Instruments,
Fullerton, CA, USA). Untreated control HIV- and mock-in-
fected cell samples were included for each sample. HIV-1
wt strain (IIIB), HIV-1 double mutant strain (RES056) or
HIV-2 strain (ROD) stock (50 lL) at 100–300 CCID50
Chem Biol Drug Des 2016; 87: 283–289
285

Huang et al.
Molecular simulations
The molecules (Ij and the lead compound GW678248) for
docking were optimized for 2000-generations until the
R2
H
N
N
O
X
N
c
OH
O
R1
ꢀ
maximum derivative of energy became 0.005 kcal/(mol*A),
a
b
using the Tripos force field. Charges were computed and
added according to Gasteiger–Huckel parameters. The
published 3D crystal structures of wt RT complexes (PDB
code: 3DLE) were retrieved from the Protein Data Bank
and were used for the docking studies by means of Sur-
flex-Dock module of SYBYL-X 1.1. The protein was prepared
using the Biopolymer application accompanying SYBYL
according to SYBYL-X 1.1 manual: The bound ligand was
extracted from the complexes, water molecules were
removed, hydrogen atoms were automatically added, and
charges and atom types were assigned according to
AMBER99. After the protomol was generated, which
referred to a computational representation of the intended
binding site to which putative ligands are aligned, the opti-
mized compounds Ij and GW678248 were docked into
NNIBP, with the relevant parameters set as defaults. The
original ligand of the co-ordinates (3DLE) was used as ref-
erence molecule to calculate the RMSD values. The dock-
ing scores related to binding affinities were calculated
based on hydrophobic, polar, and repulsive interactions as
well as entropic effects and solvation. Top-scoring poses
of compounds were shown by the software of PYMOL ver-
sion 0.99 (http://www.pymol.org/). Only the key residues
for interactions with the inhibitors were labeled and shown
in sticks. The potential hydrogen bonds were presented by
dashed lines in red.
I
Ia-o
N
OH
Br
S
H
N
N
N
OH
O
c
O
R1
R3
II-V
R3
IIIb
Vb
IIa, IIb, IIIa,
IVa, IVb, Va,
Scheme 1: Reagents and conditions: (a) 1-naphthylboric acid,
K2CO3, Pd(PPh3)4, dioxane: water = 5:1, N2 atmosphere, 90 °C;
(b) p-substituted phenylboronic acids, K3PO4, Pd(PPh3)4, dioxane:
water = 5:1, N2 atmosphere, 90 °C; (c) ClCH2CONHAr, K2CO3,
N,N-Dimethylformamide (DMF), 50 °C.
results are summarized in Table S1, with nevirapine (NVP),
efavirenz (EFV), etravirine (ETR), and zidovudine (AZT) as
reference drugs.
As listed in Table S1, these newly synthesized compounds
were not as efficient as we expected and were inferior to
all the reference drugs NVP (EC50 = 0.25 lM), EFV
(EC50 = 0.0050 lM), ETR (EC50 = 0.0041 lM), and AZT
(EC50 = 0.0058 lM). Among them, three compounds (viz.
Ia, Ih, and Ij) showed moderate activity against HIV-1
strain (IIIB) with EC50 values of 41.5 lM, 10.8 lM, and
8.18 lM, respectively. Of the active compounds, Ij was the
most potent analogue with an SI value of 2. However,
none of the target compounds was effective against the
frequently encountered K103N + Y181C double mutant
HIV-1 strain and HIV-2 strain.
Results and Discussion
Chemistry
Newly designed 2-(pyridin-3-yloxy)acetamide derivatives
were expeditiously synthesized via a very short and effi-
cient route. As shown in Scheme 1, commercially available
2-bromopyridin-3-ol (S) was chosen as the starting mate-
rial, which first reacted with 1-naphthylboric acid or the
p-substituted phenylboronic acids to afford the key inter-
mediates I–V, using K2CO3 (in preparing I) or K3PO4 (in
preparing II–V) as a base by Suzuki reaction (11). Finally,
alkylation at the OH group of intermediates I–V yielded 23
title compounds Ia-o, IIa, IIb, IIIa, IIIb, IVa, IVb, Va, and
Vb (12). These new compounds were featured by physico-
chemical and spectral means, and both analytical and
spectral data of these target compounds were totally con-
sistent with their molecular structures.
Further, preliminary SARs were derived from the results
described in Table S1. Compared to II–Va with the same
SO2NH2 substituent at 4-position of the right phenyl ring,
compound Ia kept the moderate activity. On the whole,
the potency of subseries I was superior to that of sub-
series II–V, indicating a naphthyl substituent was favorable
for the anti-HIV-1 activity. What remains to be explained is
chloro-pyridine derivative Ih (EC50 = 10.8 lM) showed
increased anti-HIV-1 activity compared to none-substituent
derivative Ii (EC50 >20.2 lM) and other mono-substituent
derivatives (Ia–Ig) on the right phenyl ring. Moreover,
in the bis-substituent subseries (Ij–Io), Ij with 2-nitro,
4-methyl substituent of the anilide moiety displayed better
potency than its counterpart Ik (EC50 >62.0 lM) and the
rest ones. These results were similar with those of aryla-
zolylthioacetanilide NNRTIs.
Anti-HIV activity in MT-4 cells
All the target compounds were screened for their in vitro
anti-HIV activity and cytotoxicity in human T-lymphocyte
(MT-4) cell cultures infected with the wt HIV-1 (IIIB) and
HIV-2 strain (ROD), respectively. Four compounds were
also evaluated for their potency against K103N+Y181C
double mutant HIV-1 strain (RES056). The biological
Regarding the cytotoxicity, it can be observed that Ih
possessed the lowest cytotoxicity (CC50 = 179 lM) and
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2-(Pyridin-3-yloxy)acetamides as Anti-HIV-1 Agents
highest safe profile (SI = 17) in the subseries I. The
cytotoxicities of subseries II–V were lower than those of
subseries I (by comparing II–Va with Ia, or II–Vb with Ib).
In addition, in the I–V subseries, it is noteworthy that
compounds bearing a para-SO2NH2 substituent of the
anilide moiety were always found to be less toxic than
the para-CONH2 substituent derivatives. Besides, in the
subseries I, the introduction of COMe group in the para-
position of the anilide moiety gave compounds Id and Il
with pronounced cytotoxicity (CC50 = 8.23 lM, 5.51 lM,
respectively). As a common sense, the MT-4 cell line is
one kind of HTLV-transformed leukemic cells, and thus,
we should pay more attention to the molecules with signifi-
cantly increased cytotoxicities (lower CC50 values) to seek
drug candidates with potential leukemic/tumoral inhibition
serendipitously.
As shown in Figure S4, the detailed binding pattern analy-
sis can be described as follows: (i) The pyridine ring of
compound Ij fits into a hydrophobic subpocket, which is
surrounded by the aromatic side chains of Tyr181, Tyr188,
Phe227, and Trp229. Particularly, it is parallel to the
side-chain phenyl group of Tyr188, generating face-to-face
p–p interactions. And the naphthyl group, which located in
the middle of NNIBP, is also parallel to the side-chain phe-
nyl group of Tyr181. Those features are distinct from those
for GW678248: The 3-chloro-5-cyanobenzene motif is
located in the aromatic-rich subpocket, while the center
phenyl ring is far away from these aromatic residues. (ii)
The anilide moiety of Ij is positioned under the residue
His235 and is oriented directly toward the RT/solvent inter-
face. In addition, the oxygen of NO2 forms a hydrogen
bond with the backbone NH of Lys103, which was very
important in the arylazolylthioacetanilide NNRTIs (14–16).
(iii) Moreover, it is found that the NH of Ij can form double
hydrogen bonds with the backbone carbonyl oxygen of
His235 and side-chain hydroxyl oxygen of Tyr318. By con-
trast, one hydrogen bond is formed between the SO2NH2
substituent of GW678248 and the backbone carbonyl of
Lys104. The triple hydrogen bonds between Ij and NNIBP
would favor the binding affinity and may be responsible for
its moderate anti-HIV-1 potency.
It must be pointed that crystallizations were observed for
all the compounds when the anti-HIV screening per-
formed. As physicochemical properties such as water sol-
ubility can significantly influence the activities of bioactive
molecules (13), the undesired anti-HIV results maybe were
related to low solubility.
In brief, although the present molecular modification has
not generated very promising leads with improved antiviral
potency utilizing this design strategy, the SARs analysis
above provided precious clues for further rational design of
novel NNRTIs.
In silico calculation of physicochemical properties
Furthermore, some physicochemical properties of the
active HIV-1 inhibitors Ia, Ih, Ij and the lead compound
GW678248 (as control) were calculated using the free
online MOLINSPIRATION software (http://www.molinspiration.-
com/). The predicted results were presented in Table S3.
The physicochemical studies suggested that the three
compounds and GW678248 conformed the Lipinski’s rule
of five well, but the former ones could not show the excel-
lent antiviral potency as GW678248. There would be other
factors, which might be responsible for their reduced inhi-
bitory activities.
Inhibition of HIV-1 RT (wt)
To further confirm the action mechanism of the newly syn-
thesized compounds, four selected derivatives (Ia, Ih, Ij,
and Im) were tested in recombinant HIV-1 RT inhibitory
assays which use poly (A) 9 oligo (dT)15 as template/pri-
mer (RT kit; Roche). As shown in Table S2, compound Ij
still exhibited the most potent inhibitory activity against
HIV-1 RT with an IC50 value of 38.3 lM among the four
compounds, while was much inferior to that of NVP
(IC50 = 2.32 lM) and ETR (IC50 = 0.03 lM). Even though
there were some differences between the cell screening
test and RT inhibitory assay, the results indicated that the
newly designed compounds could act as typical HIV-1
NNRTIs, but their abilities of binding with RT were insuffi-
cient which needed to be improved by further modifica-
tions.
Conclusions
Briefly, based on the structures of previously reported lead
compounds GW678248 and parazole oxyacetamide
derivative 3, we designed and synthesized a series of
novel anti-HIV-1 agents by employing a structure-based
molecular hybridization and bioisosterism approach. Antivi-
ral screening results showed that three compounds (Ia, Ih,
and Ij) displayed moderate potency against wt HIV-1 strain
(IIIB) with EC50 values ranging from 8.18 to 41.52 lM.
Among them, Ij was the most active analogue possessing
an EC50 value of 8.18 lM. Preliminary SAR analysis was
discussed in detail. Four compounds were selected to
implement an HIV-1 RT inhibitory assay to confirm the
binding target. Some predicted physicochemical properties
of the three active compounds Ia, Ih, and Ij were studied.
Molecular docking studies were also carried out to
Molecular modeling analysis
Molecular modeling was carried out to further investigate
the possible binding mode of the target compounds in
NNIBP of RT. The representative compound Ij and the
lead compound GW678248 were docked into the NNIBP
(PDB code: 3DLE) by Surflex-Dock module of SYBYL-X 1.1
software, and the docking results were shown by PYMOL
0.99. Default parameters were used as described in the
SYBYL-X 1.1 manual unless otherwise specified.
Chem Biol Drug Des 2016; 87: 283–289
287

Huang et al.
investigate the binding modes of Ij and GW678248 in the
binding pocket of RT, which provided beneficial informa-
tion for further rational design of NNRTIs.
activity of GW678248, a novel benzophenone nonnu-
cleoside reverse transcriptase inhibitor. Antimicrob
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Acknowledgments
The financial support from the Key Project of NSFC for Inter-
national Cooperation (No. 81420108027, 30910103908),
National Natural Science Foundation of China (NSFC No.
81102320, 81273354), Research Fund for the Doctoral
Program of Higher Education of China (No. 20110
131130005), Natural Science Foundation of Shandong
Province (ZR2009CM016) and KU Leuven (GOA 10/014) is
gratefully acknowledged. We thank K. Erven, K. Uytter-
sprot and C. Heens for technical assistance with the anti-
HIV assays.
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Chem Biol Drug Des 2016; 87: 283–289

2-(Pyridin-3-yloxy)acetamides as Anti-HIV-1 Agents
(PDB code: 3DLE). Hydrogen bonds are indicated with
dashed lines in red.
Supporting Information
Additional Supporting Information may be found in the
online version of this article:
Table S1. In vitro anti-HIV-1 activity and cytotoxicity of tar-
get compounds (Ia-o, IIa, IIb, IIIa, IIIb, IVa, IVb, Va, Vb).
Figure S1. Structures of FDA-approved NNRTIs.
Table S2. Inhibitory activity of compounds Ia, Ih, Ij, Im,
NVP and ETR against HIV-1 RT.
Figure S2. Structures of lead compounds GW678248 (1),
2 and 3.
Table S3. Prediction of physicochemical properties of Ia,
Ih, Ij and GW678248.
Figure S3. Design of 2-(pyridin-3-yloxy)acetamide deriva-
tives.
Appendix S1. Physical properties and spectrum data of
target compounds.
Figure S4. The predicted binding modes of compound Ij
(yellow) and lead compound GW678248 (green) in NNIBP
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