Discovery of 2-pyridone derivatives as potent HIV-1 NNRTIs using molecular hybridization based on crystallographic overlays

Article abstract of DOI:10.1016/j.bmc.2014.01.054

Based on crystallographic overlays of the known inhibitors TMC125 and R221239 complexed in RT, we designed a novel series of 4-phenoxy-6-(phenylamino) pyridin-2(1H)-one derivatives as HIV NNRTIs by molecular hybridization approach. The biological testing results indicated that 2-pyridone scaffold of these inhibitors was indispensable for their anti-HIV-1 activity, and substitution of halogen at the 3-position of the 2-pyridone ring would decrease the anti-HIV activity. Four most potent compounds had anti-HIV-1 III_B activities at low micromolar concentrations (EC₅₀ = 0.15-0.84 μM), comparable to that of nevirapine and delavidine. Some compounds were selected to test their anti-HIV-1 RT inhibitory action and to perform molecular modeling studies to predict the binding mode of these 2-pyridone derivatives.

Full text of DOI:10.1016/j.bmc.2014.01.054

Accepted Manuscript
Discovery of 2-Pyridone Derivatives as Potent HIV-1 NNRTIs Using Molecular
Hybridization Based on Crystallographic Overlays
Wenmin Chen, Yingshan Han, Peng Zhan, Diwakar Rai, Erik De Clercq,
Christophe Pannecouque, Jan Balzarini, Zhongxia Zhou, Huiqing Liu, Xinyong
Liu
PII:
S0968-0896(14)00091-1
DOI:
http://dx.doi.org/10.1016/j.bmc.2014.01.054
BMC 11385
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
7 December 2013
24 January 2014
27 January 2014
Please cite this article as: Chen, W., Han, Y., Zhan, P., Rai, D., De Clercq, E., Pannecouque, C., Balzarini, J., Zhou,
Z., Liu, H., Liu, X., Discovery of 2-Pyridone Derivatives as Potent HIV-1 NNRTIs Using Molecular Hybridization
Based on Crystallographic Overlays, Bioorganic & Medicinal Chemistry (2014), doi: http://dx.doi.org/10.1016/
j.bmc.2014.01.054
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Discovery of 2-Pyridone Derivatives as Potent HIV-1 NNRTIs
Using Molecular Hybridization Based on Crystallographic
Overlays
Wenmin Chen a, Yingshan Han b, Peng Zhan a, Diwakar Rai a, Erik De Clercq c,
Christophe Pannecouque c, Jan Balzarini c, Zhongxia Zhou a, Huiqing Liu a, Xinyong
Liu a, *
aDepartment of Medicinal Chemistry, Key Laboratory of Chemical Biology (Ministry
of Education), School of Pharmaceutical Sciences, Shandong University, No.44, West
Culture Road, 250012, Jinan, Shandong, P.R. China
bMcGill University AIDS Centre, Lady Davis Institute for Medical Research, Jewish
General Hospital, Montreal, Quebec, Canada
cRega Institute for Medical Research, KU Leuven, Minderbroedersstraat 10, B-3000
Leuven, Belgium
Abstract:
Based on crystallographic overlays of the known inhibitors TMC125 and R221239
complexed
in
RT,
we
designed
a
novel
series
of
4-phenoxy-6-(phenylamino)pyridin-2(1H)-one derivatives as HIV NNRTIs by
molecular hybridization approach. The biological testing results indicated that
2-pyridone scaffold of these inhibitors was indispensable for their anti-HIV-1 activity,
and substitution of halogen at the 3-position of the 2-pyridone ring would decrease the
anti-HIV activity. Four most potent compounds had anti-HIV-1 IIIB activities at low
micromolar concentrations (EC50=0.15-0.84 µM), comparable to that of nevirapine
and delavidine. Some compounds were selected to test their anti-HIV-1 RT inhibitory
action and to perform molecular modeling studies to predict the binding mode of
these 2-pyridone derivatives.
Keywords: NNRTIs, HIV, Drug design, 2-Pyridone, Molecular hybridization,
Docking.
*Corresponding author: Tel.: +86-531-88382005; Fax: +86-531-88382731; E-mail address:
xinyongl@sdu.edu.cn (X. Liu).
1

1. Introduction
In the life cycle of human immunodeficiency virus type 1 (HIV-1), reverse
transcriptase (RT) is responsible for the conversion of single-stranded viral RNA into
double-stranded proviral DNA, a prerequisite for integration into host DNA 1. Due to
its important role in the HIV-1 life cycle, RT has been identified as a prime target for
anti-HIV drug discovery. Among currently available RT inhibitors, non-nucleoside
reverse transcriptase inhibitors (NNRTIs) hamper the polymerase activity of HIV RT
through allosterically binding to the NNRTI-binding pocket (NNIBP) to distort the
precise geometry of the DNA polymerase catalytic site 2, 3. Because of their excellent
antiviral potency, high specificity and low toxicity 4, 5, NNRTIs have become the
6
important ingredients of highly active antiretroviral therapy (HAART) . However,
due to the infidelity of HIV-1 RT during replication 7, the emergence of RT mutations
(especially K103N and Y181C) rapidly develop resistance to the first-generation
8
NNRTIs with rigid structures, such as nevirapine (NVP) and efavirenz (EFV) . In an
effort to address the low resistance barrier issue, next generation NNRTIs have been
designed with the structural feature of smaller building blocks connected by flexible
linkers. Representative second generation of NNRTIs with improved drug resistance
9
profiles are the recently marketed diarylpyrimidine analogues (DAPYs) , rilpivirine
(TMC278, RPV) and etravirine (TMC125, ETR) (Fig. 1) 10-12. However, there is still a
need to explore novel NNRTIs to address the current issues of side effects, poor
solubility, cross-resistance, and virologic failure, accompanied by the current NNRTI
therapy 13-17
.
<Fig.1.>
Herein, we decided to employ TMC125 as an appropriate starting point for
development of novel NNRTIs. After extensive literature survey, we focus our
attention
on
a
more
recent
second-generation
congener,
viz.
3-iodo-4-phenoxypyridinone (IOPY) analogue R221239, which serves as a promising
drug candidate due to its excellent profile of resilience to the most common resistance
22
mutations 18-21. The co-crystal structure of R221239 (2BE2) bound in the NNRTI
pocket was overlaid with the published structure of RT-TMC125 complex (3MEC)
23, which demonstrated striking similarities in the geometry of the two bound
inhibitors. It was indicated that TMC125 and R221239 shared some common
2

pharmacophoric features, viz. an aromatic ring (A) to develop π-π stacking
interactions with the residues of W229, Y181 and Y188, a central heterocycle (C)
with hydrogen bond donor and acceptor to participate in hydrogen bondings with the
back bone of the residues K101/K103, and one hydrocarbon-rich region (B) to
involve in hydrophobic contacts with the tolerant region of RT.
<Fig.2.>
Based on above pharmacophoric analysis and the crystallographic overlap
studies, we designed a novel series of 4-phenoxy-6-(phenylamino)pyridin-2(1H)-one
derivatives through combining the privileged structural features of TMC125 and
R221239 using molecular hybridization 24 (Fig. 3): i) Replacing the central pyrimidine
ring of TMC125 with the 2-pyridone ring, which was expected to serve both as
hydrogen bond donor and acceptor. Meanwhile, halogen atoms were introduced to the
3-position of the 2-pyridone ring to evaluate their impacts on antiviral activity; ii)
Fragment switching of the 2-aniline group and the 6-phenoxy group in TMC125 to
the novel 2-pyridone template and modifying the substituents at the two phenyl rings.
The yielded hybrid compounds are expected to maintain pre-selected characteristics
of the original templates. Herein, we described the synthesis and the biological
evaluation of these novel hybrid 2-pyridone derivatives as potent HIV NNRTIs. The
structure and activity relationship (SAR) analysis and molecular modeling studies for
these compounds will also be presented.
<Fig.3.>
2. Results and Discussion
2.1 Chemistry
The synthetic route of 4-phenoxy-6-(phenylamino)pyridin-2(1H)-one derivatives
is outlined in Scheme 1. First, treatment of commercially available 2,6-dichloro
pyridine (1) with 30% aqueous H2O2 solution in CF3COOH provided intermediate
2,6-dichloropyridine-1-oxide (2), which followed by chlorination with POCl3 to
25
afford 2,4,6-trichloro pyridine (3) . Then, 3 was selectively substituted by different
phenols at C-4 position in the presence of K2CO3 to produce
2,6-dichloro-4-phenoxypyridine intermediates 4(a-d) 26, which was subsequently
alcoholyzed by methanol in the presence of sodium methoxide to give intermediate
5(a-d) in good yields (50-78%). Then, intermediate 5(a-d) underwent Pd(OAc)2
catalyzed cross-coupling reaction with corresponding phenylamines to provide
3

2-methoxypyridine compounds 6(a-f) (62-76%).
Subsequently, the precursor compounds 6(a-f) were converted to
4-phenoxy-6-(phenylamino)pyridin-2(1H)-one compounds 7(a-f) using sodium iodide
and chlorotrimethylsilane under reflux condition with good yields (76%-86%). Finally,
halogenation at the 3-position of 2-pyridone derivatives 7(c-f) with
N-iodosuccinimide (NIS), N-bromosuccinimide (NBS), or N-chlorosuccinimide (NCS)
respectively, afforded corresponding 3-halogenated-2-pyridone compounds 8(c-f) in
25–50% yields 27.
<Scheme 1.>
2.2 Biological Activity
The synthesized compounds were evaluated for their anti-HIV activity in MT-4
cell cultures infected with wild-type HIV-1 strain (IIIB), double mutant HIV-1 strain
RES056 (K103N+Y181C), and HIV-2 strain (ROD), respectively. FDA-approved
drug nevirapine (NVP) and delavidine (DLV) were used as reference drugs for
comparison. The biological results are represented as EC50 values (anti-HIV activity),
CC50 values (cytotoxicity) and SI values (selectivity index, given by the CC50/EC50
ratio) (Tables 1 and Table 2).
In total, 15 compounds were found to exhibit anti-HIV-1 IIIB activity in lower
micromolar range (EC50 = 0.15-9.3 µM) with selectivity index (SI) values ranging
from 5 to > 244. The most potent compound 7d was found to prevent the cytopathic
effect of HIV-1 IIIB with an EC50 value of 0.15 µM, less potent than TMC125 (EC50 =
28, 29
22
1.4 nM)
and R221239 (EC50 = 2.0 nM) , but comparable to NVP (EC50 = 0.18
µM) and superior to DLV (EC50 = 0.91 µM). Additionally, none of these compounds
was active against HIV-1 RES056 strain at subtoxic concentrations.
The contribution of the 2-pyridone scaffold to the biological activities was firstly
assessed. As shown in Table 1, all the synthesized 2-pyridone compounds 7(a-f)
displayed improved anti-HIV-1 IIIB activity and higher SI values as compared to
corresponding 2-methoxy-pyridine analogues 6(a-f), which indicated that the
2-pyridone was an essential scaffold for these 2-pyridone inhibitors to exhibit
anti-HIV-1 activity. Subsequently, to exploit the SAR features of R2, we synthesized
the 2-pyridone analogues 7(a-c) which all bear a 2,4,6-trimethyl group (R1) on the
A-ring and only differ in the R2 substitution on the B-ring. The biological results
4

showed that the 2-pyridone compound 7c (EC50 = 0.37 µM) with a 4-cyano group (R2)
on the B-ring was about 4-fold more active than the corresponding compounds
bearing a 4-methyl group (7b, EC50 = 1.4 µM) or a 4-nitro group (7a, EC50 = 1.4 µM),
which suggested that the 4-cyano group was the optimal substituent on the B-ring, but
more studies are required to confirm this hypothesis (Table 1).
< Table 1.>
Next, keeping the most optimal cyano group as R2 substituent on the B-ring,
different R1 substituents were introduced on the A-ring of these 2-pyridone analogues
(Table 2). Comparing the EC50 and CC50 values of 2-pyridone derivatives 7(c-f), we
found that compound 7d bearing a 4-cyano-2,6-dimethyl-substituent on the A-ring
possessed better activity and a higher SI value than the compounds with a
2,4,6-trimethyl group (7c), 2,6-dimethyl group (7e), or 3,5-dimethyl substitution
pattern (7f) (Table 1). It was noteworthy that introduction of a 3,5-dimethyl group in
the A-ring furnished compound 7f devoid of any anti-HIV-1 activity, whereas this
group was identified as an optimal substituent for IOPY analogues. It appeared that
the meta-position of the A-ring was located in a sterically unfavorable region while
the ortho-/para- positions of the A-ring were favorable to introduce substitutions.
Therefore, the substitution patterns on the A-ring (left wing) of these 2-pyridone
compounds was in agreement with that of DAPYs, but different from that of IOPYs.
Finally, different halogen atoms were introduce into the 3-position of the
2-pyridone compounds 7(c-f) respectively to investigate their influence on antiviral
activity (Table 2). According to the reported literatures 22, 30, the iodine atom at the
3-position of the lead R221239 could develop a electrostatic interaction with the Y188
carbonyl oxygen, which greatly contributed to its binding affinity with RT. Inspired
by this finding, an iodine atom was firstly introduced to the 3-position of the newly
designed 2-pyridone compounds 7(c-f) to explore whether it would contribute to their
biological activity as it did in R221239 22, 31. However, all the 3-iodo substituted
2-pyridone compounds (8c1, 8d1, 8e1) (Table 2) exhibited decreased anti-HIV-1 (IIIB)
activities than the corresponding 3-unsubstituted 2-pyridone compounds (7c, 7d, 7e)
(Table 1). The discrepancy between the SARs of IOPYs and these newly developed
2-pyridone derivatives indicated that the latter may bind to RT in a mode different
from IOPYs. Additionally, the crystallographic overlay studies (Fig. 1) demonstrated
5

that the 3-iodo group of the pyridone moiety in R221239 generally overlapped with
the 5-bromo group of pyrimidine in TMC125. Therefore, the bromo atom was also
introduced into the 3-position of the 2-pyridone, for comparison, as well as the chloro
atom, generating eight compounds (8c2, 8c3, 8d2, 8d3, 8e2, 8e3, 8f2, and 8f3).
Unfortunately, all these 3-bromo/choloro substituted 2-pyridone compounds were not
superior to corresponding unsubstituted analogues 7(c-f) and 3-iodio substituted
analogues (8c1, 8d1, 8e1). These resulted revealed that substituents (X) at the
3-position of the 2-pyridone ring contributed to the antiviral activities in the following
order (of decreasing activity): H > I > Br and Cl.
<Table 2.>
In
summary,
preliminary
SAR
studies
of
these
novel
4-phenoxy-6-(phenylamino)pyridin-2(1H)-one derivatives revealed the important
structural requirements to maintain high potency against HIV-1 virus: i) the optimal
moiety for the left wing (A-ring) was a 4-cyano-2,6-dimethylphenxoy group; ii) the
best group for the right wing (B-ring) was a 4-cyanophenylamino group; iii)
3-unsubstituted-2-pyridone derivatives were superior to 3-halogenated-2-pyridone
analogues.
In addition, all the title compounds were also evaluated for their capability to
inhibit the HIV-2 replication in MT-4 cells, but none was found effective at test
concentration, indicating that newly developed compounds were only specific for
HIV-1 (Supplementary materials).
2.3 Inhibition of HIV-1 RT
To confirm the drug target of these 2-pyridone inhibitors, two compounds (7d
and 7c) with the highest potency were evaluated for their HIV-1 RT inhibitory, using
a Roche RT kit. The colorimetric of the RT assay takes advantage of the ability of RT
to synthesize the DNA, starting from the template/primer hybrid poly(A) ×
oligo(dT)15. The detection and quantification of the synthesized DNA as a parameter
for RT activity follows a sandwich ELISA protocol (as descibled in the Kit):
Biotin-labeled DNA binds to the surface of microplate (MP) modules that have been
precoated with streptavidin. In the next step, an antibody to digoxigenin, conjugated
to peroxidase (anti-DIG-POD), binds to the digoxigenin-labeled DNA. In the final
step, the peroxidase substrate ABTS is added. The peroxidase enzyme catalyzes the
cleavage of the substrate, producing a colored reaction product. The absorbance of the
6

samples was determined using a microplate (ELISA) reader and was directly
correlated to the level of RT activity in the sample.
The results showed that the anti-RT activity of 7d was comparable to that of
NVP, but lower than that of ETR. Another compound 7c was less potent than both
ETR and NVP (Table 3). The biological results proved that the newly discovered
2-pyridone derivatives displayed anti-HIV-1 activity by inhibiting RT, and belonged
to HIV-1 NNRTIs.
<Table 3.>
2.4 Molecular modeling analysis
Molecular docking using surflex-dock module of Sybyl-X 1.1 was performed to
predict the binding mode of the representative 2-pyridone inhibitors (7d and 8d1)
with RT and to rationalize the results of SAR studies. Coordinates of the NNIBP were
taken from the crystal structure of the RT/TMC125 complex (PDB code: 3MEC) due
to the high degree of structural similarity between TMC125 and the newly designed
2-pyridone compounds. This program docks a ligand automatically into the binding
site of a receptor, using a protomol-based method and an empirically derived scoring
function. The root-meansquare deviations (RMSD) value for the re-dock result of
bound ligand TMC-125 is 0.4320 Å, indicative of that this docking program is
reliable and accurate.
The highest-scored docking conformation of 7d at the binding site of RT was
displayed in overlap with the crystallized position of TMC125 (blue) by PyMOL
version 1.5 (http://www.pymol.org/). Inspection of the binding conformation of 7d
revealed a fair superposition to that of TMC125 (Fig. 4). The central ring of 7d was
orientated by dual hydrogen bonds between the CONH moiety of the 2-pyridone with
the back bone C=O and NH of K101, respectively. Indeed, according to the biological
results, replacement of the 2-pyridone ring with 2-methoxypyridine decreased the
anti-HIV-1 activity (Table 1), which proves the importance of the central 2-pyridone
ring for interactions with RT. A third hydrogen bond between the NH group of the
4-cyano phenylamino moiety with K101 was observed. Additionally, the benzonitrile
moiety of 7d, which overlapped with the right ring of TMC125, was located in a
hydrophobic channel surrounded by P236, V106 and Y318. The dimethylcyanophenyl
of 7d developed favorable face-to-face π-π interactions with the aromatic rings of
Y181 and Y188. It is noteworthy that the orientation of the dimethylcyanophenyl
group of 7d was slightly different from that of TMC125. This conformational
7

perturbation may reduce the tight contact of the dimethylcyanophenyl group with the
conserved residue W229, which may explain the low sensitivity of compound 7d to
the drug-resistant HIV-1 strain (RES056 strain: K103N+Y181C).
<Fig. 4.>
Introduction of an iodine atom at the 3-position of 7d yielded the less active
compound 8d1. We found that the distance between the iodine atom of 8d1 with the
C=O of Y188 (6.04 Å) was beyond the minimal van der Waals contact distance (3.55
Å), which may explain why the iodine atom of 8d1 did not contribute to the
protein-ligand binding affinity as it did to IOPYs 21, 22. Instead, the steric hindrance of
the iodine atom pushed 8d1 to move towards the bottom of the binding pocket, which
disrupted the dual hydrogen bonds between the CONH moiety of the 2-pyridone with
the back bone of K101 (Fig. 5). The absence of the multiple hydrogen bonds
weakened the binding affinity of 8d1, which may ultimately lead to the 19-fold lower
activity of 7d1 (EC50 = 2.9 µM) than that of 7d (EC50 = 0.15 µM) (Fig. 5).
<Fig. 5.>
The docking studies revealed the binding orientation of these 2-pyridone
derivatives and provided critical insights for further development of these compounds.
3. Conclusion
In
summary,
we
designed
a
series
of
4-phenoxy-6-(phenylamino)pyridin-2(1H)-one derivatives as potent HIV-1 NNRTIs
using molecular hybridization based on crystallographic overlay studies of the lead
compounds TMC125 and R221239. The biological assay showed that 15 compounds
exhibited significant potency against wild-type HIV-1 strain (IIIB) at micromolar
concentrations (EC50 = 0.15-9.3 µM). Among them, compound 7d displayed the
highest activity with an EC50 value of 0.15 µM and SI of 166, providing a good
starting point for further structural optimization. Additionally, the molecular modeling
studies of representative 2-pyridone inhibitors with RT revealed that these inhibitors
adopt a similar binding mode with TMC125. Further efforts to improve the antiviral
activity of these 2-pyridone typed NNRTIs by rational design to improve their drug
resistance profiles and water solubility are currently underway and will be reported in
due course.
8

4.1 Experimental Section
4.1. Chemistry
Mass spectrometry was performed on an API 4000 triple-quadrupole mass
spectrometer (Applied Biosystems/MDS Sciex, Concord, ON, Canada). IR spectra
were recorded with Nicolet-6700 model FT-IR spectrometer (Thermo Scientific,
1
Waltham, MA, USA) using KBr pellets. H-NMR and 13C-NMR spectra were
recorded on a Bruker Avance DRX 600 spectrometer (Bruker, Fällanden, Switzerland),
Bruker AV-400 spectrometer (Bruker BioSpin, Switzerland), or Bruker AVANCE-300
spectrometer (Bruker BioSpin, Switzerland), using solvents as indicated (CDCl3 or
DMSO-d6). Chemical shifts were reported in δ values (ppm) with tetramethylsilane as
the internal reference, and J values were reported in hertz (Hz). Melting points (mp)
were determined on a micromelting point apparatus (Tian Jin Analytical Instrument
Factory, Nankai, Tianjin, China). Flash column chromatography was performed on
columns packed with silica gel 60 (200–300 mesh) (Qingdao waves silica gel
desiccant co., Ltd, Qingdao, China). Thin layer chromatography was performed on
pre-coated HUANGHAI® HSGF254, 0.15-0.2 mm TLC-plates (Yantai Jiangyou
Silica Gel Development Co., Ltd., Yantai, Shandong, China). Solvents (Tianjin Fuyu
Fine Chemical Co., Ltd., Wuqing, Tianjin, China) were of reagent grade and were
purified and dried by standard methods when necessary. The key reactants including
2,6-dichloropyridine, Pd(OAc)2, XantPhos, chlorotrimethylsilane, N-iodosuccinimide
NIS, N-bromosuccinimide NBS and N-chlorosuccinimide NCS, etc. were purchased
from Adamas-beta Co. Ltd (Shanghai, China).
4.1.1. General procedure for synthesis of 2,6-dichloropyridine-1-oxide (2)
A solution of 2,6-dichloropyridine (1) (10.0 g, 0.067 mol) in a mixture of
CF3COOH (80 mL) and 30% aq. H2O2 (25 mL) was heated at reflux for 3h. Then,
saturated Na2S2O4 solution was added to the mixture until hydrogen peroxide was
consumed completely via an inspection with potassium iodide-starch test paper.
Thereafter, the reaction mixture was concentrated under reduced pressure to about 10
mL and then poured on 50 mL water. The unreacted 2,6-dichloropyridine (1) was
formed as a precipitate and was then removed by filtration. The filtrate was basified
by addition of solid Na2CO3, causing the precipitation of a white solid, which was
filtered and dried, giving the crude 2,6-dichloropyridine-1-oxide (2) as a white solid,
yield 55%, mp: 137-138°C.
4.1.2. General procedure for synthesis of 2,4,6-trichloropyridine (3)
9

A mixture of 2,6-dichloropyridine-1-oxide (2) (8.20 g, 0.050 mol) in POCl3 (25
mL) was refluxed for 4h. Then, the solvent was evaporated under reduced pressure.
The residue was poured into crushed ice and extracted with petroleum ether (3×50
mL). The combined organic phase was dried over K2CO3, filtered, and concentrated in
vacuo. The residue was further purified by flash column chromatography using ethyl
acetate/petroleum ether as an eluent to give 2,4,6-trichloropyridine (3) as colorless
needles, yield 85%, mp: 32-33°C.
4.1.3. General procedure for the synthesis of 2,6-dichloro-4-(phenoxy)pyridines
4(a-d)
A mixture of different phenols (5.0 mmol) and K2CO3 (0.83 g, 6.0 mmol) in 15
mL DMF was stirred at room temperature for 15min. Then, 2,4,6-trichloropyridine (3)
(0.91 g, 5.0 mmol) was added and the mixture as heated to 100°C under a nitrogen
atmosphere. The reaction was followed by TLC until its completion. Then, the solvent
was evaporated off and the residue, after addition of water, was extracted with ethyl
acetate. The organic layer was dried over Na2SO4 and concentrated in vacuo. The
residue was recrystallized from ethyl acetate and petroleum ether or purified by
column chromatography using ethyl acetate and petroleum ether as an eluent to give
4(a-d).
4.1.3.1. 2,6-dichloro-4-(mesityloxy)pyridine (4a)
Colorless crystal, recrystallized from ethyl acetate and petroleum ether, yield: 85%,
mp: 129-130°C; 1H-NMR (400 MHz, CDCl3) δ: 2.06 (s, 6H, CH3), 2.31 (s, 3H, CH3),
6.66 (d, J =1.32 Hz, 2H, Py-H), 6.93 (s, 2H, Ph-H); 13C-NMR (100 MHz, CDCl3) δ:
15.99, 20.79, 109.73, 129.94, 130.09, 136.17, 147.14, 151.68, 167.27; MS-ESI: 282.3
[M+H] + , 284.2 [M+H] +.
4.1.3.2. 2,6-dichloro-4-(4-cyano-2,6-dimethylphenoxy)pyridine (4b)
Colorless crystal, purified by column chromatography using ethyl
acetate/petroleum ether (1:20) as an eluent, yield 68%, mp: 168-169°C; 1H-NMR (400
MHz, CDCl3) δ: 2.17 (s, 6H, CH3), 6.65 (d, J = 1.32 Hz, 2H, Py-H), 7.48 (s, 2H,
Ph-H); 13C-NMR (100 MHz, CDCl3) δ: 16.12, 109.58, 110.78, 118.03, 132.58, 133.39,
152.11, 152.62, 165.72; MS-ESI: 293.4 [M+H]+, 295.3 [M+H] +.
4.1.3.3. 2,6-dichloro-4-(2,6-dimethylphenoxy)pyridine (4c)
Colorless crystal, recrystallized from ethyl acetate and petroleum ether, yield: 80%,
mp: 86-87°C; 1H-NMR (400 MHz, CDCl3) δ: 2.11 (s, 6H, CH3), 6.66 (d, J = 1.32 Hz,
2H, Py-H), 7.13-7.14 (m, 3H, Ph-H); 13C-NMR (100 MHz, CDCl3) δ: 16.09, 109.73,
10

126.60, 129.54, 130.44, 149.31, 151.73, 166.99; MS-ESI: 268.3 [M+H]+, 270.4 [M+H]
+
.
4.1.3.4. 2,6-dichloro-4-(3,5-dimethylphenoxy)pyridine (4d)
Colorless crystal, purified by column chromatography using ethyl
acetate/petroleum ether (1:30) as an eluent, yield 84%, mp: 200-201°C; 1H-NMR (400
MHz, CDCl3) δ: 2.34 (s, 6H, CH3), 6.69 (d, J = 1.32 Hz, 2H, Py-H), 6.76 (s, 2H,
Ph-H), 6.94 (s, 1H, Ph-H); 13C-NMR (100 MHz, CDCl3) δ: 21.30, 110.05, 118.33,
128.11, 140.59, 151.39, 153.00, 167.99; MS-ESI: 268.3 [M+H] +, 270.3 [M+H] +.
4.1.4. General procedure for the synthesis of 2-chloro-6-methoxy-4-phenoxy-
pyridines 5(a-d)
Intermediates 4(a-d) (0.01 mol) were dissolved in MeONa/MeOH (prepared
from 1.15 g Na and 20 mL MeOH) and the mixture was heated to reflux for 4h. Next,
the solvent was evaporated off and the residue, after addition of water, was taken up
in CH2Cl2. The combined organic layer was dried over Na2SO4. Evaporation of the
solvent afforded 2-chloro-6-methoxy-4-phenoxy-pyridines 5(a-d).
4.1.4.1. 2-chloro-4-(mesityloxy)-6-methoxypyridine (5a)
1
White solid, yield 78%, mp: 98°C; H-NMR (400 MHz, CDCl3) δ: 2.06 (s, 6H,
CH3), 2.29 (s, 3H, CH3), 3.89 (s, 3H, OCH3), 5.93 (d, J = 1.56 Hz, 1H, Py-H), 6.44 (d,
J = 1.56 Hz, 1H, Py-H), 6.89 (s, 2H, Ph-H); 13C-NMR (100 MHz, CDCl3) δ: 15.99,
20.77, 54.15, 94.02, 105.11, 129.80, 130.27, 135.56, 147.61, 149.53, 165.34, 167.72;
MS-ESI: 278.3, 280.2 [M+H]+ .
4.1.4.2 2-chloro-4-(4-cyano-2,6-dimethyl)-6-methoxyphenoxypyridine (5b)
White solid, yield 72%, MS-ESI: 289.3, 291.4 [M+H] + .
4.1.4.3 2-chloro-4-(2,6-dimethylphenoxy)-6-methoxypyridine (5c)
White solid, yield 72%, MS-ESI: 264.3, 266.4 [M+H] + .
4.1.4.4 2-chloro-6-methoxy-4-(3,5-dimethylphenoxy)- pyridine (5d)
White solid, yield 68%, MS-ESI: 264.2, 266.3 [M+H] + .
4.1.5. General procedure for the synthesis of 2-methoxypyridine compounds 6(a-f).
In a schlenk-type flask, corresponding phenylamine (0.01 mol) and intermediates
5(a-d) (0.01 mol) were dissolved in dry dioxane (25 mL), and then Pd(OAc)2 (67.3
mg, 0.3mmol), XantPhos (347.2 mg, 0.6 mmol) and NaO-tBu (1.44 g, 0.015 mol)
were added. The mixture was heated to 100°C while stirring under a nitrogen
atmosphere. The reaction was followed by TLC until its completion. After cooling,
the solvent was evaporated under reduce pressure. The residue was purified by flash
11

column chromatography using ethyl acetate/petroleum ether as eluent to give
compounds 6(a-f).
4.1.5.1. 4-(mesityloxy)-6-methoxy-N-(4-nitrophenyl)pyridin-2-amine (6a)
1
Yellow solid, yield 72%, mp: 197-199°C; H-NMR (400 MHz, DMSO-d6) δ:
2.05 (s, 6H, 2CH3), 2.27 (s, 3H, CH3), 3.89 (s, 3H, OCH3), 5.81 (d, J = 1.64 Hz, 1H,
Py-H), 5.86 (d, J = 1.44 Hz, 1H, Py-H), 7.01 (s, 2H, Ph-H), 7.84 (d, J = 9.32 Hz, 2H,
Ph-H), 8.14 (d, J = 9.36 Hz, 2H, Ph-H), 9.80 (s, 1H, NH); 13C-NMR (100 MHz,
DMSO-d6) δ: 16.11, 20.81, 54.30, 88.65, 90.16, 117.18, 125.67, 130.13, 130.45,
135.31, 139.90, 147.90, 148.42, 154.63, 165.01, 167.57; ESI-MS: 380.5 [M+H]+ .
4.1.5.2. 4-(mesityloxy)-6-methoxy-N-(p-tolyl)pyridin-2-amine (6b)
1
White solid, yield 70%, mp: 107-108°C; H-NMR (400 MHz, CDCl3) δ: 2.08 (s,
6H, 2CH3), 2.27 (s, 3H, CH3), 2.30 (s, 3H, CH3), 3.83 (s, 3H, OCH3), 5.48 (d, J =
1.60 Hz, 1H, Py-H), 5.91 (d, J = 1.60 Hz, 1H, Py-H), 6.25 (s, 1H, NH), 6.86 (s, 2H,
Ph-H), 7.09 (d, J = 8.24 Hz, 2H, Ph-H), 7.21 (d, J = 8.36 Hz, 2H, Ph-H); 13C-NMR
(100 MHz, CDCl3) δ: 16.05, 20.75, 53.49, 86.37, 87.13, 120.42, 129.49, 129.63,
130.61, 132.07, 134.88, 148.11, 155.77, 165.21, 168.14; ESI-MS: 349.4 [M+H]+ .
4.1.5.3. 4-((4-(mesityloxy)-6-methoxypyridin-2-yl)amino)benzonitrile (6c)
1
White solid, yield 76%, mp: 198-200°C; H-NMR (400 MHz, CDCl3) δ: 2.09 (s,
6H, 2CH3), 2.29 (s, 3H, CH3), 3.89 (s, 3H, OCH3), 5.70 (d, J = 1.52 Hz, 1H, Py-H),
5.91 (d, J = 1.48 Hz, 1H, Py-H), 6.66 (s, 1H, NH), 6.89 (s, 2H, Ph-H), 7.53(s, 2H,
Ph-H); 13C-NMR (100 MHz, DMSO-d6) δ: 16.04, 20.78, 53.87, 88.63, 89.81, 103.35,
117.73, 119.61, 129.65, 130.51, 133.29, 135.24, 144.95, 147.90, 153.49, 165.22,
168.06; ESI-MS: 360.4, 362.4 [M+H]+ .
4.1.5.4.
4-((2-((4-cyanophenyl)amino)-6-methoxypyridin-4-yl)oxy)-3,5-dimethyl
benzonitrile (6d)
1
White solid, yield 62%, mp: 229-230°C; H-NMR (400 MHz, CDCl3) δ: 2.18 (s,
6H, 2CH3), 3.91 (s, 3H, OCH3), 5.63 (d, J = 1.64 Hz, 1H, Py-H), 5.89 (d, J = 1.64 Hz,
1H, Py-H), 6.86 (s, 1H, NH), 7.41 (s, 2H, Ph-H), 7.55 (d, J = 8.84 Hz, 2H, Ph-H),
7.60 (d, J = 8.92 Hz, 2H, Ph-H); 13C-NMR (100 MHz, CDCl3) δ: 16.10, 54.00, 88.31,
89.50, 103.62, 109.55, 117.96, 118.45, 119.53, 132.92, 133.12, 133.30, 144.78,
153.83, 153.93, 165.30, 166.57; ESI-MS: 371.4 [M+H]+ .
4.1.5.5. 4-((6-methoxy- 4-(2,6-dimethylphenoxy)- pyridin-2-yl)amino)benzonitrile (6e)
12

1
White solid, yield 72%, mp: 159-160°C; H-NMR (400 MHz, CDCl3) δ: 2.13 (s,
6H, 2CH3), 3.89 (s, 3H, OCH3), 5.70 (d, J = 1.64 Hz, 1H, Py-H), 5.90 (d, J = 1.60 Hz,
1H, Py-H), 6.72 (s, 1H, NH), 7.07-7.08 (m, 3H, Ph-H), 7.52-7.53 (m, 4H, Ph-H);
13C-NMR (100 MHz, CDCl3) δ: 16.11, 53.98, 88.53, 89.69, 103.60, 117.88, 119.54,
125.81, 129.09, 130.97, 133.32, 144.79, 150.10, 153.47, 165.13, 167.94; ESI-MS:
346.3 [M+H] +.
4.1.5.6. 4-((6-methoxy-4-(3,5-dimethylphenoxy)- pyridin-2-yl)amino)benzonitrile (6f)
1
White solid, yield 70%, mp: 150-152°C; H-NMR (400 MHz, DMSO-d6) δ: 2.29
(s, 6H, 2CH3), 3.87 (s, 3H, OCH3), 5.87 (d, J = 1.72 Hz, 1H, Py-H), 5.92 (d, J = 1.68
Hz, 1H, Py-H), 6.79 (s, 2H, Ph-H), 6.92 (s, 1H, Ph-H), 7.65 (d, J = 8.84 Hz, 2H,
Ph-H), 7.80 (d, J = 8.80 Hz, 2H, Ph-H), 9.57 (s, 1H, NH); 13C-NMR (100 MHz,
DMSO-d6) δ: 21.24, 54.19, 89.63, 91.65, 101.65, 117.94, 118.83, 120.16, 127.34,
133.54, 140.20, 146.16, 154.27, 164.84, 168.24; ESI-MS: 346.3 [M+H]+ .
4.1.6. General procedure for synthesis of the 2-pyridone compounds 7(a-f)
chlorotrimethylsilane (2.72 g, 0.025 mol) was added to a suspension of 6(a-f)
(0.005 mol) and sodium iodide (3.75 g, 0.025 mol) in acetonitrile. This mixture was
stirred at reflux. Upon completion of the reaction, the solvent was evaporated in
vacuo. The residue was suspended in ethyl acetate, washed by 10% aq. Na2S2O3
solution (10 mL). The precipitate was then filtered and recrystallized from methanol
to give target compounds 7(a-f).
4.1.6.1. 4-(mesityloxy)-6-((4-nitrophenyl)amino)pyridin-2(1H)-one (7a)
1
Yellow solid, yield 85%, decomposed at 260°C; H-NMR (400 MHz, DMSO-d6)
δ: 2.05 (s, 6H, CH3), 2.27 (s, 3H, CH3), 5.66 (s, 1H, Py-H), 5.74 (s, 1H, Py-H), 6.99 (s,
2H, Ph-H), 7.88 (d, J = 9.24 Hz, 2H, Ph-H), 8.10 (d, J = 9.52 Hz, 2H, Ph-H), 9.67 (s,
1H, NH), 10.59 (s, 1H, CONH); 13C-NMR (100 MHz, DMSO-d6) δ: 16.13, 20.80,
88.01, 89.28, 117.08, 125.52, 130.07, 130.49, 135.18, 139.70, 147.95, 148.76, 154.63,
164.57, 167.67; ESI-MS: 366.4 [M+H]+ .
4.1.6.2. 4-(mesityloxy)-6-(p-tolylamino)pyridin-2(1H)-one (7b)
1
White solid, yield 82%, mp: 187-188°C; H-NMR (400 MHz, DMSO-d6) δ: 2.04
(s, 6H, 2CH3), 2.24 (s, 3H, CH3), 2.25 (s, 3H, CH3), 5.12 (s, 1H, Py-H), 5.47 (d, J =
1.64 Hz, 1H, Py-H), 6.95 (s, 2H, Ph-H), 7.06 (d, J = 8.24 Hz, 2H, Ph-H), 7.35 (d, J =
8.12 Hz, 2H, Ph-H), 8.43 (s, 1H, NH), 10.18 (s, 1H, CONH); 13C-NMR (100 MHz,
DMSO-d6) δ: 16.10, 20.79, 20.87, 83.76, 86.32, 120.02, 129.71, 129.89, 130.45,
13

130.89, 134.95, 138.61, 148.04, 154.03, 164.16, 168.11; ESI-MS: 335.5 [M+H]+ .
4.1.6.3. 4-((4-(mesityloxy)-6-oxo-1,6-dihydropyridin-2-yl)amino)benzonitrile (7c)
1
White solid, yield 82%, mp: 246-247°C; H-NMR (400 MHz, DMSO-d6) δ: 2.07
(s, 6H, CH3), 2.34 (s, 3H, CH3), 5.05 (d, J = 1.56 Hz, 1H, Py-H), 5.94 (d, J = 1.52 Hz,
1H, Py-H), 6.89 (s, 2H, Ph-H), 7.08 (d, J = 8.60 Hz, 2H, Ph-H), 7.51 (d, J = 8.52 Hz,
2H, Ph-H), 9.33 (s, 1H, NH), 11.11 (bs, 1H, CONH); 13C-NMR (100 MHz, DMSO-d6)
δ: 15.94, 20.84, 82.27, 89.68, 105.81, 118.85. 119.48, 129.65, 130.17, 133.63, 135.62,
143.20, 146.86, 147.50, 164.86, 170.94; MS-ESI: 346.3 [M+H]+ .
4.1.6.4.
4-((6-((4-cyanophenyl)amino)-2-oxo-1,2-dihydropyridin-4-yl)oxy)-3,5-
dimethylbenzonitrile (7d)
1
White solid, yield 78%, decomposed at 250-252°C; H-NMR (400 MHz,
DMSO-d6) δ: 2.15 (s, 6H, CH3), 5.61 (d, J = 1.64 Hz, 1H, Py-H), 5.69 (d, J = 1.64 Hz,
1H, Py-H), 7.62 (d, J = 8.72 Hz, 2H, Ph-H), 7.72 (s, 2H, Ph-H), 7.83 (d, J = 8.72 Hz,
2H, Ph-H), 9.31 (s, 1H, NH), 10.56 (bs, 1H, CONH); 13C-NMR (100 MHz, DMSO-d6)
δ: 15.93, 87.35, 88.53, 101.63, 109.07, 117.97, 118.89, 120.21, 133.31, 133.43,
133.52, 146.27, 154.02, 155.07, 164.68, 166.43; MS-ESI: 357.3 [M+H]+ .
4.1.6.5.
4-((4-(2,6-dimethylphenoxy)-6-oxo-1,6-dihydropyridin-2-yl)amino)
benzonitrile (7e)
1
White solid, yield 86%, mp: 182-184°C; H-NMR (300 MHz, DMSO-d6) δ: 2.09
(s, 6H, 2CH3), 5.59 (d, J = 1.5 Hz, 1H, Py-H), 5.70 (d, J = 1.8 Hz, 1H, Py-H),
7.10-7.20 (m, 3H, Ph-H), 7.62 (d, J = 9.0 Hz, 2H, Ph-H), 7.82 (d, J = 9.0 Hz, 2H,
Ph-H), 9.39 (s, 1H, NH); 13C-NMR (75 MHz, DMSO-d6) δ: 15.69, 86.88, 88.14,
100.99, 117.45, 119.76, 125.74, 129.09, 130.42, 132.94, 145.85, 149.64, 154.30,
163.96, 166.98; ESI-MS: 332.5 [M+H]+ .
4.1.6.6.4-((4-(3,5-dimethylphenoxy)-6-oxo-1,6-dihydropyridin-2-yl)amino)benzonitril
e (7f)
1
White solid, yield 76%, mp: 246-248°C; H-NMR (300 MHz, DMSO-d6) δ: 2.29
(s, 6H, 2CH3), 5.67 (d, J = 2.40 Hz, 1H, Py-H), 5.83 (d, J = 2.00 Hz, 1H, Py-H), 6.79
(s, 2H, Ph-H), 6.91 (s, 1H, Ph-H), 7.63 (d, J = 9.0 Hz, 2H, Ph-H), 7.85 (d, J = 9.0 Hz,
2H, Ph-H), 9.41 (s, 1H, NH), 10.56 (s, 1H, CONH); 13C-NMR (75 MHz, DMSO-d6) δ:
20.74, 88.54, 90.08, 100.88, 117.35, 118.41, 119.79, 126.74, 132.93, 139.62, 145.93,
153.79, 154.25, 163.90, 167.87; ESI-MS: 332.5 [M+H]+.
4.1.7.
General
procedure
for
synthesis
of
14

4-phenoxy-6-(phenylamino)pyridin-2(1H)-one derivatives 8(c-f)
Halogenated reagent (N-iodosuccinimide NIS, or N-bromosuccinimide NBS or
N-chlorosuccinimide NCS) (1.2 mmol) was added to a solution of 7(c-f) (1.0 mmol)
in AcOH (4.0 mL) and CF3COOH (2.2 mL). The mixture was stirred at room
temperature until the starting reactant 7(c-f) was completely consumed as detected by
TLC. Then the mixture was poured into ice-water and neutralized with 28% aqueous
NH4OH. The resulting precipitate was separated by filtration, washed with ethyl
acetate, and subsequently purified by flash column chromatography using ethyl
acetate/petroleum ether (1:2) as eluent to give the product of 8(c-f).
4.1.7.1. 4-((5-iodo-4-(mesityloxy)-6-oxo-1,6-dihydropyridin-2-yl)amino)benzonitrile
(8c1)
1
Light yellow solid, yield 35%, decomposed at 254°C; H-NMR (400 MHz,
DMSO-d6) δ: 2.04 (s, 6H, CH3), 2.28 (s, 3H, CH3), 5.43 (s, 1H, Py-H), 7.01 (s, 2H,
Ph-H), 7.61 (d, J = 8.84 Hz, 2H, Ph-H), 7.82 (d, J = 8.72 Hz, 2H, Ph-H), 9.39 (s, 1H,
NH), 11.34 (s, 1H, 1-NH); 13C-NMR (100 MHz, DMSO-d6) δ: 16.11, 20.08, 83.68,
86.29, 119.97, 129.71, 129.90, 130.45, 130.86, 134.96, 138.63, 148.02, 154.02,
164.18, 168.09; MS-ESI: 472.3 [M+H]+.
4.1.7.2. 4-((5-bromo-4-(mesityloxy)-6-oxo-1,6-dihydropyridin-2-yl)amino)benzonitrile
(8c2)
1
White solid, yield 32%, mp: 130-132°C; H-NMR (400 MHz, DMSO-d6) δppm:
2.05 (s, 6H, CH3), 2.28 (s, 3H, CH3), 5.49 (s, 1H, Py-H), 7.01 (s, 2H, Ph-H), 7.60 (d, J
= 8.76 Hz, 2H, Ph-H), 7.80 (d, J = 8.00 Hz, 2H, Ph-H), 9.37 (s, 1H, NH), 11.36 (s,
1H, 1-NH); 13C-NMR (100 MHz, DMSO-d6) δppm: 15.97, 20.81, 101.75, 117.94,
120.18, 130.22, 130.24, 133.40, 135.51, 146.07, 148.27, 160.63, 163.08; MS-ESI:
424.5 [M+H]+ .
4.1.7.3. 4-((5-chloro-4-(mesityloxy)-6-oxo-1,6-dihydropyridin-2-yl)amino)benzonitrile
(8c3)
1
White solid, yield 28%, mp: 144-146°C; H-NMR (400 MHz, DMSO-d6) δppm:
2.05 (s, 6H, CH3), 2.28 (s, 3H, CH3), 5.52 (s, 1H, Py-H), 7.02 (s, 2H, Ph-H), 7.60 (d, J
= 8.88 Hz, 2H, Ph-H), 7.80 (s, 2H, Ph-H), 9.38 (s, 1H, NH), 11.38 (s, 1H, 1-NH);
13C-NMR (100 MHz, DMSO-d6) δppm: 15.94, 20.81, 101.72, 117.87, 120.18, 130.22,
130.24, 133.40, 135.53, 146.09, 148.14, 159.65, 162.02; MS-ESI: 380.4 [M+H]+,
382.4 [M+H]+.
4.1.7.4. 4-((6-((4-cyanophenyl)amino)-3-iodo-2-oxo-1,2-dihydropyridin-4-yl)oxy)-3,5-
15

dimethylbenzonitrile (8d1)
1
Light brown solid, yield 42%, decomposed at 235-236°C; H-NMR (400 MHz,
DMSO-d6) δ: 2.15 (s, 6H, CH3), 5.39 (s, 1H, Py-H), 7.60 (d, J = 8.76 Hz, 1H, Py-H),
7.76-7.80 (m, 4H, Ph-H), 9.30 (s, 1H, NH), 11.40 (bs, 1H, CONH); 13C-NMR (100
MHz, DMSO-d6) δppm: 15.90, 101.93, 109.43, 118.08, 118.83, 120.13, 133.06,
133.43, 133.66, 145.87, 154.48, 163.49, 179.79; MS-ESI: 483.3 [M+H]+.
4.1.7.5. 4-((3-bromo-6-((4-nitrophenyl)amino)-2-oxo-1,2-dihydropyridin-4-yl)oxy)-3,5
-dimethylbenzonitrile (8d2)
White solid, yield 28%, decomposed at 242-243°C; 1H-NMR (400 MHz,
DMSO-d6) δppm: 2.14 (s, 6H, CH3), 5.44 (s, 1H, Py-H), 7.63-7.98 (m, 5H, Ph-H),
9.39 (s, 1H, NH), 11.70 (bs, 1H, CONH); ESI-MS: 435.4 [M+H]+.
4.1.7.6.
4-((3-chloro-6-((4-nitrophenyl)amino)-2-oxo-1,2-dihydropyridin-4-yl)oxy)-
3,5-dimethylbenzonitrile (8d3)
1
White solid, yield 34%, decomposed at 290°C; H-NMR (400 MHz, DMSO-d6)
δppm: 2.15 (s, 6H, CH3), 5.48 (s, 1H, Py-H), 7.62 (d, J = 8.56 Hz, 2H, Ph-H), 7.80 (s,
4H, Ph-H), 9.37 (s, 1H, NH), 11.56 (bs, 1H, CONH); 13C-NMR (100 MHz, DMSO-d6)
δppm: 15.76, 88.90, 93.66, 101.93, 109.43, 117.92, 118.80, 120.15, 133.08, 133.46,
133.73, 145.87, 154.05, 159.88, 160.87; ESI-MS: 391.3 [M+H]+.
4.1.7.7.
4-((4-(2,6-dimethylphenoxy)-5-iodo-6-oxo-1,6-dihydropyridin-2-yl)amino)
benzonitrile (8e1)
1
White solid, yield 36%, mp: 244-245°C; H-NMR (300 MHz, DMSO-d6) δ: 2.09
(s, 6H, 2CH3), 5.43 (s, 1H, Py-H), 7.13-7.23 (m, 3H, Ph-H), 7.62 (d, J = 8.7 Hz, 2H,
Ph-H), 7.82 (d, J = 9.0 Hz, 2H, Ph-H), 9.43 (s, 1H, NH), 11.35 (s, 1H, CONH);
13C-NMR (75 MHz, DMSO-d6) δ: 15.67, 87.99, 101.20, 117.53, 119.71, 125.96,
129.19, 130.18, 132.91, 145.56, 150.24, 150.24, 162.79, 164.90; ESI-MS: 458.3
[M+H]+.
4.1.7.8. 4-((5-bromo-4-(2,6-dimethylphenoxy)-6-oxo-1,6-dihydropyridin-2-yl)amino)
benzonitrile (8e2)
1
White solid, yield 42%, mp: 240-241°C; H-NMR (400 MHz, DMSO-d6) δppm:
2.10 (s, 6H, 2CH3), 5.49 (s, 1H, Py-H), 7.15-7.24 (m, 3H, Ph-H), 7.62 (d, J = 8.48 Hz,
2H, Ph-H), 7.82 (d, J = 8.20 Hz, 2H, Ph-H), 9.43 (s, 1H, NH), 11.46 (s, 1H, CONH);
13C-NMR (100 MHz, DMSO-d6) δppm: 16.05, 82.52, 89.00, 101.78, 117.97, 120.18,
126.54, 129.75, 130.69, 133.41, 146.04, 150.46, 153.08, 160.68, 162.87; ESI-MS:
16

410.3 [M+H]+ .
4.1.7.9. 4-((5-chloro-4-(2,6-dimethylphenoxy)-6-oxo-1,6-dihydropyridin-2-yl)amino)
benzonitrile (8e3)
1
White solid, yield 40%, mp: 260-261°C; H-NMR (400 MHz, DMSO-d6) δppm:
2.09 (s, 6H, 2CH3), 5.48 (s, 1H, Py-H), 7.15-7.24 (m, 3H, Ph-H), 7.62 (d, J = 8.80 Hz,
2H, Ph-H), 7.81 (dd, J = 8.58 Hz, 2.92 Hz, 2H, Ph-H), 9.42 (s, 1H, NH), 11.43 (s, 1H,
CONH); 13C-NMR (100 MHz, DMSO-d6) δppm: 16.08, 88.72, 93.77, 101.76, 117.97,
120.19, 126.52, 129.73, 130.82, 133.41, 146.06, 150.55, 153.08, 160.76, 164.30;
ESI-MS: 366.3 [M+H]+.
4.1.7.10. 4-((4-(3,5-dimethylphenoxy)-5-iodo-6-oxo-1,6-dihydropyridin-2-yl)amino)
benzonitrile (8f1)
1
White solid, yield 36%, mp: 182-184°C; H-NMR (400 MHz, DMSO-d6) δ: 2.30
(s, 6H, 2CH3), 5.67 (s, 1H, Py-H), 6.80 (s, 2H, Ph-H), 6.94 (s, 1H, Ph-H), 7.62 (d, J =
8.80 Hz, 2H, Ph-H), 7.81 (d, J = 7.92 Hz, 2H, Ph-H), 9.44 (s, 1H, NH), 11.39 (s, 1H,
CONH); 13C-NMR (75 MHz, DMSO-d6) δ: 21.24, 107.74, 118.04, 118.70, 120.22,
127.44, 133.46, 140.32, 146.05, 154.52, 163.28, 167.08; ESI-MS: 458.4 [M+H]+.
4.1.7.11. 4-((5-bromo-4-(3,5-dimethylphenoxy)-6-oxo-1,6-dihydropyridin-2-yl)amino)
benzonitrile (8f2)
1
White solid, yield 32%, mp: 182-184°C; H-NMR (300 MHz, DMSO-d6) δppm:
2.30 (s, 6H, 2CH3), 5.72 (s, 1H, Py-H), 6.95 (s, 1H, Ph-H), 6.82 (s, 2H, Ph-H), 6.05 (s,
1H, Py-H), 7.63 (d, J = 11.6 Hz, 2H, Ph-H), 7.82 (d, J = 11.6 Hz, 2H, Ph-H), 9.45 (s,
1H, NH), 11.46 (s, 1H, CONH); 13C-NMR (75 MHz, DMSO- d6) δppm: 20.73, 90.83,
101.30, 117.47, 118.18, 119.69, 127.04, 132.95, 139.85, 145.52, 153.80, 160.17,
163.78; ESI-MS: 410.3 [M+H]+, 412.3 [M+H]+ .
4.1.7.12. 4-((5-chloro-4-(3,5-dimethylphenoxy)-6-oxo-1,6-dihydropyridin-2-yl)amino)
benzonitrile (8f3)
1
White solid, yield 42%, mp: 200-202°C; H-NMR (400 MHz, DMSO-d6) δppm:
2.30 (s, 6H, 2CH3), 5.75 (s, 1H, Py-H), 6.83 (s, 2H, Ph-H), 6.94 (s, 1H, Ph-H), 7.62 (d,
J = 8.80 Hz, 2H, Ph-H), 7.79 (s, 2H, Ph-H), 9.42 (s, 1H, NH), 11.42 (s, 1H, CONH);
13C-NMR (100 MHz, DMSO-d6) δppm: 21.23, 101.80, 117.92, 118.62, 120.17,
127.55, 133.45, 140.35, 146.05, 154.27, 159.72, 163.08; ESI-MS: 366.3 [M+H]+,
368.2 [M+H]+.
17

4.2. Biological activity evaluation
4.2.1 In vitro anti-HIV assay
The target compounds were evaluated for their activity against wild-type HIV-1
(strain HIV-IIIB), double RT mutant strain HIV-1 IIIB RES056 (RT K103N + Y181C)
and HIV-2 (strain ROD) in MT-4 cells using the MTT method as previously described
32. Briefly, stock solutions (10×final concentration) of test compounds were added in
25 µL 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 five-fold dilutions of test compounds were made directly in flat-bottomed
96-well microtiter trays by adding 100 µL medium to the 25 µL stock solution and
transferring 25 µL of this solution to another well that contained 100 µL medium
using a Biomek 3000 robot (Beckman instruments, Fullerton, CA). Untreated control
HIV- and mock-infected cell samples were included for each sample.
33
34
HIV-1(IIIB) or HIV-2 (ROD) stock (50 µL) at 100-300 CCID50 (50% cell
culture infectious dose-50%) or culture medium was added to either the infected or
mock-infected wells of the microtiter plate. 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 growing MT-4 cells were centrifuged for 5 min at
200 g and the supernatant was discarded. The MT-4 cells were resuspended at 6 × 105
cells/mL, and 50-µL 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. 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
against the cytopathic effect of the virus in infected cells was defined as the 50%
effective concentration (EC50).
4.2.2 HIV-1 RT inhibition assay
A RT assay kit produced by Roche was used for the RT inhibition assay. All the
reagents for performing the RT reaction are contained in the kit and the ELSIA
35,
procedures for RT inhibition assay was performed as described in the kit protocol.
36
Briefly, the reaction mixture containing HIV-1 reverse transcriptase (RT) enzyme,
reconstituted template and viral nucleotides [digoxigenin (DIG)-dUTP, biotin-dUTP
and dTTP] in the incubation buffer with or without inhibitors was incubated for 1 h at
18

37 °C. Then, the reaction mixture was transferred to a streptavidine-coated microtitre
plate (MTP) and incubated for another 1 h at 37 °C. The biotin-labeled dNTPs that
was incorporated in cDNA chain in the presence of RT were bound to streptavidine.
The unbound dNTPs were removed by rinsing using washing buffer followed by the
addition of anti-DIG-POD working solution into the MTPs. After incubation for 1 h at
37 °C, the DIG-labeled dNTPs incorporated in cDNA were bound to the
anti-DIG-POD antibody. The unbound anti-DIG-PODs were removed and the
peroxide substrate (ABST) solution was added to the MTPs. Incubation of the
reaction mixture at 25 °C until a green color was sufficiently developed for detection.
The absorbance of the sample was determined at O.D. 405 nm using microtiter plate
ELISA reader. The percentage inhibitory activity of RT inhibitors was calculated by
formula as given below:
%Inhibition =[O.D. value with RT but without inhibitors- O.D. value with RT
and inhibitors]/ [O.D. value with RT and inhibitors- O.D. value without RT and
inhibitors]35
The IC50 values corresponded to the concentrations of the inhibitors required to
inhibit biotin-dUTP incorporation by 50%.
4.3 Molecular Simulation
The molecules (7d, 8d1 and TMC125) for docking was optimized for
2000-generations until the maximum derivative of energy became 0.005kcal/(mol*Å),
using the Tripos force field. Charges were computed and added according to
Gasteiger–Huckel parameters. The published three-dimensional crystal structures of
RT complexes with TMC125 (PDB code: 3MEC) were retrieved from the Protein
Data Bank and were used for the docking studies by means of surflex-docking module
of Sybyl-X 1.1. The protein was prepared by using the Biopolymer application
accompanying Sybyl: The bound ligand was extracted from the complexes, water
molecules were removed, hydrogen atoms were added, side chain amides and side
chains bumps were fixed, and charges and atom types were assigned according to
AMBER99. After the protomol was generated, the optimized compounds 7d, 8d1 and
TMC125 were surflex-docked into the binding pocket of NNRTIs, with the relevant
parameters set as defaults. The original ligand (TMC-125) of the coordinates (3MEC)
was used as reference molecule to calculate the RMSD values. The docking scores
related to binding affinities were calculated based on hydrophobic, polar, and
19

repulsive interactions as well as entropic effects and solvation. Top-scoring poses of
7d were shown in overlap with the bound ligand (TMC-125) and the conformation of
8d1 in the binding site of RT respectively, by the software of PyMOL version 1.5
(http://www.pymol.org/). The secondary structure of RT are shown in cartoons, and
only the key residues for interactions with the inhibitors were shown in sticks and
labeled. The potential hydrogen-bondings were presented by dashed lines.
Conflict of interest
None.
Acknowledgments
We thank K. Erven, K. Uyttersprot and C. Heens for technical assistance with the
anti-HIV assays. The financial support from the National Natural Science Foundation
of China (NSFC No. 81273354, No. 81102320, No. 30873133, No. 30772629, No.
30371686), Key Project of NSFC for International Cooperation (No. 30910103908),
Research Fund for the Doctoral Program of Higher Education of China (No.
20110131130005), Shandong Provincial Natural Science Foundation (No.
ZR2009CM016) and KU Leuven (GOA 10/014) is gratefully acknowledged.
20

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22

Figure Captions
Fig. 1. Structures and anti-HIV-1 activities of DAPY and IOPY analogues 22, 29
.
Fig. 2. Crystallographic overlays of TMC125 (purple) and R221239 (pink) with RT
(performed by SYBYL-X
http://www.pymol.org/).
1.1, shown by PyMOL version 1.5;
Fig. 3. The structure of novel 2-pyridone-typed NNRTIs designed by molecular
hybridization of TMC-125 and R221239.
Fig. 4. The structural overlap of the most potent compound 7d (blue) and TMC125
(orange) (Proposed hydrogen bonds are indicated with dashed lines in red. The
docking result was performed by SYBYL-X 1.1, shown by PyMOL).
Fig. 5. The structural overlap of compound 7d (blue) and 3-iodo-2-pyridone
compound 8d1 (yellow) (Proposed hydrogen bonds are indicated with dashed lines in
red. The docking result was performed by SYBYL-X 1.1, shown by PyMOL).
Scheme 1. Synthetic route of target compounds 6, 7 and 8. Reagents and conditions:
(a) 30% H2O2, CF3COOH, reflux; (b) POCl3, reflux; (c) K2CO3, DMF, N2, 100°C; (d)
MeONa, MeOH, reflux; (e) Pd(OAc)2, XantPhos, NaO-tBu, N2, 1,4-dioxane, 100°C;
(f) TMSCl, NaI, CH3CN, reflux; (g) NIS/NBS/NCS, AcOH, CF3COOH, rt.
Table 1. In vitro anti-HIV-1 activity of 2-methoxypyridine derivatives 6(a-f)and
2-pyridone derivatives 7(a-f) .
Table 2. In vitro anti-HIV-1 activity of the 2-pyridone compounds with different
substitutions (X) on the 3-position of the 2-pyridone ring.
Table 3. Inhibitory activity of representative 2-pyridone compounds against HIV-1
RT.
23

Fig. 1. Structures and anti-HIV-1 activities of DAPY and IOPY analogues [22, 23].
24

Fig. 2. Crystallographic overlay of TMC125 (purple) and R221239 (pink) with RT
(performed by SYBYL-X 1.1, shown by PyMOL).
25

Fig. 3. Molecular hybridization of TMC-125 and R221239 to create the novel
2-pyridone-typed NNRTIs
26

Fig. 4. The structural overlap of the most potent compound 7d (blue) and TMC125
(orange) (Proposed hydrogen bonds are indicated with dashed lines in red. The
docking result was performed by SYBYL-X 1.1, shown by PyMOL).
27

Fig. 5. The structural overlap of compound 7d (blue) and 3-iodo-2-pyridone
compound 8d1 (yellow) (Proposed hydrogen bonds are indicated with dashed lines in
red. The docking result was performed by SYBYL-X 1.1, shown by PyMOL).
28

Scheme 1. Synthetic route of target compounds 6, 7 and 8. Reagents and conditions:
(a) 30% H2O2, CF3COOH, reflux; (b) POCl3, reflux; (c) K2CO3, DMF, N2, 100°C; (d)
MeONa, MeOH, reflux; (e) Pd(OAc)2, XantPhos, NaO-tBu, N2, 1,4-dioxane, 100°C;
(f) TMSCl, NaI, CH3CN, reflux; (g) NIS/NBS/NCS, AcOH, CF3COOH, rt.
29