Design, synthesis, and antihepatitis B virus activities of novel 2-pyridone derivatives

Article abstract of DOI:10.1021/jm901237x

A series of novel 2-pyridone derivatives were synthesized and evaluated for their antihepatitis B virus (HBV) activity and cytotoxicity in vitro.Moderate to good activity against HBV DNA replication was observed in these 2-pyridone analogues. The most active compounds were 5d and 6l, with good inhibitory activity against HBV DNA replication (IC₅₀ = 0.206 and 0.12 μM, respectively) and remarkable high selectivity (selectivity indexes of >532 and 467, respectively). A pharmacophore model of the synthesized compounds was proposed by the GASP program. The pharmacophore model consists of three hydrophobic points, four HBApoints, and one HBD point. The 2-pyridone derivatives represent a novel class of HBV inhibitors, which are worth further optimization. 2009 American Chemical Society.

Full text of DOI:10.1021/jm901237x

660 J. Med. Chem. 2010, 53, 660–668
DOI: 10.1021/jm901237x
Design, Synthesis, and Antihepatitis B Virus Activities of Novel 2-Pyridone Derivatives
Zhiliang Lv, Chunquan Sheng,* Tiantian Wang, Yikai Zhang, Jia Liu, Jilu Feng, Hailing Sun, Hanyu Zhong,
Chunjuan Niu, and Ke Li*
Department of Medicinal Chemistry, College of Pharmacy, Second Military Medical University, 325 Guohe Road, Shanghai 200433,
People’s Republic of China
Received August 18, 2009
A series of novel 2-pyridone derivatives were synthesized and evaluated for their antihepatitis B virus
(HBV) activity and cytotoxicity in vitro. Moderate to good activity against HBV DNA replication was
observed in these 2-pyridone analogues. The most active compounds were 5d and 6l, with good
inhibitory activity against HBV DNA replication (IC50 = 0.206 and 0.12 μM, respectively) and
remarkable high selectivity (selectivity indexes of >532 and 467, respectively). A pharmacophore
model of the synthesized compounds was proposed by the GASP program. The pharmacophore model
consists of three hydrophobic points, four HBA points, and one HBD point. The 2-pyridone derivatives
represent a novel class of HBV inhibitors, which are worth further optimization.
Introduction
influenza-like symptoms, depression, and insomnia) are ob-
served.8,9 Lamivudine has been used for the treatment of HBV
infection for a few years. It is orally effective and well toler-
ated.10 However, the emergence of resistance to lamivudine has
been reported.11 Adefovir dipivoxil was approved by the U.S.
Food and Drug Administration in 2002 for the treatment of
chronic hepatitis B infection. Adefovir dipivoxil can reduce
serum HBV DNA levels significantly and can lead to serocon-
version in 20-27% of the patients treated for 12 weeks at a
daily dose of 30 mg or greater.12 However, adefovir dipivoxil
therapy is limited by dose-related side effects such as nephro-
toxicity, lactic acidosis, and severe hepatomegaly with steato-
sis.13 More recently, telbivudine and entecavir were marked
with pharmacological profile. However, their chemical struc-
tures are similar to those of lamivudine and adefovir dipivoxil.
They may act by the same biological mechanism and encounter
the same problems as lamivudine and adefovir dipivoxil.
Therefore, there is a tremendous clinical need to develop novel
classes of antiviral agents for the treatment of HBV infection.
In the screening of an in-house library for anti-HBV activity,
we identified 5-(2-hydroxy-4-methoxybenzoyl)-1-phenylpyri-
din-2(1H)-one (5a) as a modest inhibitor of HBV, with an
IC50 value of 20 μM in inhibiting HBV DNA replication and
low cytotoxicity (CC50=179 μM) in vitro. Because the chemical
scaffold of compound 5a differs from those of all reported anti-
HBV agents, we were interested in synthesizing its derivatives
and analyzing their structure-activity relationships (SARs).
In the present investigation, we designed and synthesized a
new series of 5-(2-hydroxy-4-methoxybenzoyl)pyridin-2-one
derivatives with compound 5a as the starting point. The anti-
HBV activities of the compounds were investigated, and a
pharmacophore model was built to guide further structural
optimization.
Hepatitis B virus (HBVa) is a cause of chronic liver disease
worldwide, affecting more than 5% of the world’s popula-
tion.1 HBV can cause both acute and chronic infections.
Chronic HBV infection leads to liver damage, cirrhosis, and
liver cancer with high mortality. There are approximately
350-400 million who are chronically infected, resulting in
0.5-1.2 million deaths annually.2,3 HBV infection represents
a serious global medical, social, and economic burden.
Important viral-encoded genes include the envelope pro-
tein, hepatitis B surface antigen (HBsAg), the primary serum
marker of viral infection; a structural nucleocapsid, hepatitis
B core antigen; and a soluble nucleocapsid protein, hepatitis B
e antigen (HBeAg), which combinedwithserumlevelsofHBV
DNA are serum markers of active viral replication.2 The
replication of HBV shares unusually similar features with
retroviruses, and eradication of HBV infection turns out to be
difficult because stable, covalently closed circular DNA
(cccDNA), which is relatively resistant to antiviral action
and immune clearance, becomes established in hepatocyte
nuclei and integrated into the host genome.2,4
Seven drugs have been approved by the U.S. Food and Drug
Administration (FDA) for the treatment of HBV infection
either as monotherapy or in combination. These drugs are
categorized as interferons (interferon-R and pegylated interfer-
on R-2a) and nucleotide analogues (lamivudine, telbivudine,5
entecavir,6 adefovir and tenofovir3,7) (Figure 1). Until now, the
major therapeutic option for HBV carriers was R-interferon.
Unfortunately, its use is limited because the success rate is low,
the therapy is expensive, and serious side effects (such as
*To whom correspondence should be addressed. For C.S.: phone/
fax, 86-21-81871233; e-mail, shengcq@hotmail.com. For K.L.: phone/
fax, 86-21-81871237; e-mail, proflike@sina.com.
a Abbreviations: HBV, hepatitis B virus; HBsAg, hepatitis B surface
antigen; HBeAg, hepatitis B e antigen; cccDNA, covalently closed
circular DNA; SARs, structure-activity relationships; SI, selectivity
index; HBA, hydrogen bond acceptor; HBD, hydrogen bond donor;
vdW, van der Waals.
Chemistry
The synthetic route of compound 4 was depicted in
Scheme 1. 3-Iodo-7-methyloxy-4H-chromone (3) was synthe-
sized by a reported procedure.14 Coupling of compound 3
r
pubs.acs.org/jmc
Published on Web 12/15/2009
2009 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2 661
Figure 1. Chemical structures of the anti-HBV agents and lead compound 5a.
Scheme 1. Synthesis of the Key Intermediate 4a
a Reagents and conditions: (a) ether, Na, ethyl formate, 0 °C, 8 h, yield 95%; (b) CH3COOH, conc HCl, 100 °C, 30 min, yield 98%; (c) piperidine, methanol,
reflux, 3 h, yield 99%; (d) dichloromethane, I2, room temp, overnight, yield 97%; (e) PdCl2(Ph3P)2, CuI, K2CO3, THF/H2O, 100 °C, 10 h, yield 80%.
with methyl acrylate by a Heck reaction15,16 afforded the key
intermediate 4. In order to establish a robust synthetic proce-
dure of compound 4, the reaction conditions were optimized.
Various solvents, bases, metal catalysts, and temperatures
were investigated. The mixed solvent (THF/H2O = 9:1) was
found to be the best choice with K2CO3 as the base and
PdCl2(Ph3P)2 as the metal catalyst. The optimal reaction
temperature and time were 100 °C and 10 h, respectively.
In the presence of triethylamine, target compounds 5a-s
were obtained by reacting intermediate 4 with various sub-
stituted phenylamines and alkylamines (molar ratio of 1: 1) via
three sequencing steps in one pot. First, the ester group was
ammonolyzed by amines. Second, the chromone ring was
opened by the attack of the amine group. Last, the 2-pyrinone
ring was formed by a rearrangement process. When intermediate
4 was reacted with excess benzylamines and alkylamines,
compounds 6a-o were obtained. However, reacting com-
pound 4 with excess phenylamines could not afford corre-
sponding Schiff bases. In order to determine the configuration
of the Schiff base, the crystal structure of compound 6c was
determined. Figure 2 shows the ORTEP drawing of compound
6c, indicating that the Schiff base is in cis-configuration.
Results and Discussion
In Vitro Virological Examination and Preliminary Struc-
ture-Activity Relationships (SARs). The potential anti-HBV
activity and cytotoxicity of the synthesized 2-pyridones
were tested in HepG2 2.2.15 cells, and adefovir was used
as a reference antiviral drug. The results are summarized
in Table 1. The anti-HBV activity of each compound was

662 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2
Lv et al.
expressed as the concentration of compound that achieved
50% inhibition (IC50) of HBsAg, HBeAg, and HBV DNA
replication. The cytotoxicity of each compound was ex-
pressed as the concentration of compound required to kill
50% (CC50) of the HepG2 2.2.15 cells. The selectivity index
(SI), an important pharmaceutical parameter that estimates
possible future clinical development, was determined as the
ratio of CC50 value to IC50 value. The bioactivity of each
compound was evaluated by the combination of its IC50
values and SI.
Compounds 5a-l have different patterns of substitution
on the phenyl ring attached to the N atom of 2-pyridones. As
shown in Table 1, these compounds generally exhibited
moderate to good anti-HBV potency. Various substitutions
on the phenyl ring are important for the antiviral activity.
4-Ethoxyl (compound 5d) and 3-methyl (compound 5f) analo-
gues showed excellent anti-HBV activity with their IC50
values against HBV DNA replication of 0.206 and 0.8 μM,
respectively, which were more potent than reference drug
adefovir. They also showed higher inhibitory activity against
HBsAg and HBeAg than adefovir. Moreover, compounds
5d and 5f were almost nontoxic against HepG2 2.2.15 cells,
which resulted in their high SI values. When the 3-methyl
group of compound 5f was moved to position 2 (compound
5g) and position 4 (compound 5e), their anti-HBV activity
was decreased by 6- to 14-fold. 2,5-2CH3 derivative
(compound 5h) also showed good inhibitory activity against
HBV DNA replication (IC50 = 0.5 μM). However, it was
very toxic with a CC50 value of 0.5 μM (SI = 1). If the
4-methyl group of compound 5e was replaced by a chlorine
atom (compound 5i), the inhibitory activity against HBV
DNA replication was decreased slightly (IC50 = 6.2 μM).
Figure 2. ORTEP drawing of compound 6c.
Table 1. In Vitro Anti-HBV Activity and Cytotoxicity of the Target Compounds
HBsAg
HBeAg
IC50 (μM)b
DNA replication
IC50 (μM)b
compd
5a
CC50 (μM)a
IC50 (μM)b
SIc
SIc
2.36
SIc
8.95
179
346
176
1.02
d
76
20
5.2
5b
5c
5d
5e
5f
>113.9
>113.9
>109.6
>119.4
29
222
49
1.56
66.54
>1.5
>532
>23.9
198.75
>10.85
1.0
>113.9
>109.6
>119.4
159
d
>2.3
>1.99
>59.7
530
76
d
55
0.206
5
0.8
d
2
0.3
5.48
>6.63
0.04
2.69
d
5g
5h
5i
>119.4
0.5
164
18
13
0.1
38
>1194
0.01
11
0.5
61
1.6
102.5
d
6.2
26.45
>1.2
d
5j
>117.9
177
133
>117.9
>109.3
>110.2
99
>117.9
>109.3
774
0.73
46.3
>132.7
39
98.3
NAe
11.2
99.6
33.3
28.6
20
5k
5l
<1.07
<1.21
0.15
d
<1.07
0.17
11.88
0.15
3.93
5m
5n
5o
5p
5q
5r
15
131
20.55
2.83
>139.3
>132.7
46
67
79
<0.5
1.72
0.82
0.84
3.17
2.55
0.71
0.62
<0.12
0.77
0.41
0.89
<0.17
1.93
1.33
<1
<0.5
2.03
2.34
3.95
696.1
232
244
846.2
276
77
>846.2
58
<0.82
4.0
170.3
100.8
>363.7
31.5
92.6
2.1
4.09
2.3
5s
>97
4.4
<2.5
23.18
1.70
<0.67
3.24
0.55
6a
6b
6c
6d
6e
6f
102
51
40
72
30
0.5
8
10
13
>86.9
13
16.0
<0.12
0.25
3.81
0.15
>86.9
40
64.5
3
10
3.33
2.07
201.4
32
20
496.5
36
>402.8
2
<0.5
16.0
97.5
1
6g
6h
6i
32
0.92
>117.8
201
123
>117.8
71
10
<0.17
5.45
16.4
21.7
4
387
164
96.75
<0.35
3.55
6j
>462.4
33.8
0.12
30
6k
6l
120
56
>121.9
111
29
>121.9
56
<1
1.0
0.50
3.38
d
466.67
3.27
6m
6n
6o
adefovir
98
5
54
19.6
>121.9
20
540
>121.9
98
305
>2.26
0.5
1.89
>121.9
12
0.517
d
1.67
0.20
1.77
40
286
1044.5
a CC50 is 50% cytotoxicity concentration in HepG2 2.2.15 cells. b IC50 is 50% inhibitory concentration. c Selectivity index (SI = CC50/IC50). d No SI
can be obtained. e NA = not active.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2 663
The introduction of a nitro group (compound 5k) or acetyl
group (compound 5l) on position 4 of the phenyl ring led to
the loss of anti-HBV activity.
HBeAg are 31.5, 40, and 4.4 μM, respectively. Introducing a
fluorine atom on the position 2 of the benzyl group (com-
pound 6b, IC50 = 92.6 μM) led to a decrease of antiviral
activity. However, moderate anti-HBV activity was observed
for compound 6e (HBV DNA replication, IC50 = 3 μM)
after the addition of another fluorine atom on the position
4 of the benzyl group of compound 6b. Moreover, the 3-F
derivative (compound 6c) also revealed good activity against
HBV DNA replication, HBsAg, HBeAg (IC50 = 2.1, 13, and
0.5 μM, respectively). The most potent compound in
this subseries is compound 6g (HBV DNA replication,
IC50=1 μM), indicating that the methoxyl at position 4 of
the benzyl group is important for the antiviral activity.
Compared with compounds 6a-g, the dialkyl substituted
derivatives (compounds 6i-o) generally showed lower anti-
viral activity. The loss of antiviral activity was observed for
the propyl, cyclopropyl, and isopropyl derivatives (com-
pounds 6k, 6j, and 6n). The addition of a double bond to
the propyl group afforded compound 6o, which showed
Decreased anti-HBV activity was observed with the re-
placement of the substituted phenyl groups of compounds
5a-l by the alkyl groups (compounds 5m-s). Compound
5m, with a cyclohexyl group attached to the N atom of
2-pyridone, was almost inactive against HBV DNA replica-
tion (IC50 = 99.6 μM). The replacement of the cyclohexyl
group of compound 5m by a propyl group (compounds 5n)
or pentyl group (compounds 5p) yielded 3- to 5-fold im-
provement in anti-HBV activity. However, the introduction
of a 2-(naphthalene-1-yl-amino)ethenyl group at this posi-
tion resulted in the loss of antiviral activity, suggesting that a
steric group larger than the phenyl group was not tolerated at
this position.
Compounds 6a-o have the same substitutions on both the
N atom of 2-pyridone and the imino group. Compound 6a
showed moderate anti-HBV activity. The IC50 values of
compound 6a against HBV DNA replication, HBsAg, and
moderate activity against HBV DNA replication (IC50
=
12 μM). When the propyl group of compound 6k was
extended to the butyl group (compound 6l), the anti-HBV
activity was increased obviously. It is worth noting that
compound 6l is the most potent one among the synthesized
compounds. The IC50 value of compound 6l against HBV
DNA replication is 0.12 μM, which is 4-fold more potent
than adefovir. Moreover, compound 6l also showed high SI
value (SI = 466.67), suggesting that it is a good starting point
for further optimization.
From the biological data, preliminary SARs of the synthe-
sized compounds are as follows. For compounds 5a-s, the
N-aryl derivatives showed better anti-HBV activity than the
N-alkyl derivatives. The substitutions on the phenyl ring
were important for the antiviral activities. An electron with-
drawing group was not favorable at position 4 of the phenyl
ring. The introduction of an electron-donating group (such as
methyl or methoxyl) enhanced the antiviral activity, especially
in the inhibition of HBV DNA replication. The dibenzyl or
dialkyl substituted derivatives (compounds 6a-o) did not
show improved anti-HBV activity, suggesting that the imino
groups are not important for their anti-HBV activities. Be-
cause the present study is a preliminary structural modification
and the target of these compounds remains unknown, the
SARs of 2- pyridones remain to be further studied.
Figure 3. Pharmacophore model of the 2-pyrinone derivatives.
HY1, HY2, and HY3 are hydrophobic points (shown as cyan balls)
located around the centers of the aromatic rings. HBA1, eHBA2,
HBA3, and HBA4 are hydrogen bond (HB) acceptor points (shown
as green balls). HBD1 is a HB donor point (shown as purple balls).
The complementary HB donor and acceptor points of the receptor
are also represented as balls with the same color as the acceptor or
donor atom of the template molecules in the direction of lone pair or
hydrogen. The green lines show directions of the HB interactions.
Scheme 2. Synthesis of the Target Compounds 5a-sa
a Reagents and conditions: (a) methanol, substituted aniline, triethylamine, reflux, 8 h; (b) methanol, alkylamine, triethylamine, reflux, 8 h,
yield 35-89%.

664 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2
Scheme 3. Synthesis of the Target Compounds 6a-oa
Lv et al.
a Reagents and conditions: (a) methanol, substituted benzylamine, triethylamine, reflux, overnight; (b) methanol, alkylamine, triethylamine, reflux,
overnight, yield 55-90%.
Pharmacophore Model of 2-Pyridones. The synthesized 2-
pyridones represent a new class of anti-HBV compounds.
However, the target of 2-pyridone derivatives is unknown.
Therefore, the construction a pharmacophore model is a
useful method for further structural optimization. More-
over, the pharmacophore model can also be used in virtual
screening and identifying new lead structures. According to
the chemical diversity and anti-HBV potency, compounds 5f
and 6c were selected as template molecules. Their low-energy
conformations were obtained by energy minimization and
simulated annealing. Then the pharmacophore model was
derived by means of a genetic algorithm similarity program
GASP.17 Several pharmacophore points were identified
(Figure 3), including three hydrophobic, four hydrogen bond
(HB) acceptor, and one HB donor points. Three phenyl
groups (HY1, HY2, and HY3) of compound 5f were identi-
fied as hydrophobic points. HY1 and HY2 are identical
among the compounds. HY3 highlighted the importance of
the substituted phenyl group attached to the N atom of 2-
pyrinones, which was consistent with the SAR results. An-
other phenyl group of compound 6c was not recognized as a
hydrophobic point, indicating that the substituted imino
group was not important for the anti-HBV activity. All four
oxygen atoms of compound 5f were identified as HB accep-
tor points (HBA1-HBA4). Moreover, the hydrogen atoms
of the hydroxyl groups of compounds 5f and 6c were
recognized as a HB donor point (HBD1). However, the
HBA and HBD points are common structural elements
among the synthesized compounds. Their function needs
to be demonstrated by further structural modifications.
and one HBD point. The pharmacophore model can provide
useful information for further structural modification. The
present findings make them attractive candidates for further
optimization as well as the investigation of the mechanism of
action.
Experimental Section
Chemistry. General Methods. Melting points were measured
on an uncorrected X-5 digital melting point apparatus (Gongyi
City Yuhua Instrument Co., Ltd.; China). IR spectra were
recorded on a PE Spectrum One FI-IR spectrometer as KBr
1
pellets. H NMR and 13C NMR spectra were recorded on a
BRUKER AVANCE 300 spectrometer (Bruker Company,
Germany), using TMS as an internal standard and CDCl3 or
DMSO-d6 as solvents. Chemical shifts are given in ppm (δ).
Elemental analyses were performed with a MOD-1106 instru-
ment and were consistent with theoretical values within 0.4%.
The mass spectra were recorded on an Esquire 3000 LC-MS
mass spectrometer. Crystal structure was determined on a
Bruker SMART CCD (Bruker Company, Germany). Silica
gel thin-layer chromatography was performed on precoated
plates GF-254 (Qingdao Haiyang Chemical, China). All sol-
vents and reagents were analytically pure, and no further
purification was needed. All starting materials were commer-
cially available. The purity of final compounds was assessed
on the basis of analytical HPLC, and the results were greater
than 95%.
(E)-Methyl 3-(5-Hydroxy-7-methoxy-4-oxo-4H-chromen-3-
yl)acrylate (4). To a 500 mL round-bottom flask fitted with
mechanical stirrer, a mixed solvent of DMF and H2O (225 mL of
DMF and 25 mL of H2O), 3-iodo-7-methyloxy-4H-chromone
(3) (30.2 g, 0.1 mol), methyl acrylate (11.1 g, 0.15 mol), Pd-
(Ph3P)2Cl2 (0.7 g, 0.001 mol), CuI (1.9 g, 0.01 mol), and K2CO3
(13.8 g, 0.1 mol) were added successively. The mixture was
heated to 70 °C and stirred for 4 h. Then the mixture was filtered.
The filtrate was poured into 800 mL of ice-water and then
extracted with EtOAc (200 mL ꢀ 3). The extract was washed
with saturated NaCl solution (50 mL ꢀ 3), dried over anhydrous
MgSO4, and concentrated to a volume of approximately 300 mL.
Brown crystals (4) were collected after the concentrated solution
was cooled down to 4 °C and maintained overnight. Yield 21 g
(80.5%). 1H NMR (CDCl3, 300 MHz) δ 3.78 (s, 3H), 3.86 (s, 3H),
6.84 (d, 1H, J = 2.4 Hz), 6.99 (dd, 1H, J1 = 2.4 Hz, J2 = 9 Hz),
7.24 (d, 1H, J = 15.9 Hz), 7.37 (d, 1H, J = 15.9 Hz), 8.04 (s, 1H),
8.16 (d, 1H, J = 9 Hz). ESI-MS (m/z): 261.1 [M þ 1]. Anal.
(C14H12O5) C, H, N.
Conclusion
In summary, a series of novel 2-pyridone analogues based
on compound 5a were synthesized and tested for their in vitro
anti-HBV activity, using adefovir as a reference control. Most
of the compounds revealed moderate to good anti-HBV
activity. Compounds 5d, 5f, 5h, 6g, and 6l were active with
IC50 values against HBV DNA replication in the range of
0.12-1.0 μM. The most promising results were observed for
compounds 5d and 6l, with IC50 values against HBV DNA
replication of 0.206 and 0.12 μM, respectively, with high
selectivity (SI of >532 and 467, respectively). A pharmaco-
phore model of the synthesized compounds was constructed,
which consists of three hydrophobic points, four HBA points,
5-(2-Hydroxy-4-methoxybenzoyl)-1-phenylpyridin-2(1H)-one
(5a). A solution of compound 4 (1.04 g, 0.004 mol), aniline

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2 665
(0.41 g, 0.0044 mol), and triethylamine (3 drops) in MeOH (45
mL) was stirred under reflux for 8 h. After the mixture was
cooled to room temperature and the solvent removed, the crude
product was purified by chromatography over silica gel (eluent
petroleum ether/acetic ether, 9:1 to 3:1 v/v) to give compound 5a
as a yellow crystal (Scheme 2). Yield 0.95 g (76%). Mp:
7.81 (dd, 1H, J1 = 9.6H, J2 = 2.4 Hz), 7.86 (d, 1H, J = 2.7 Hz),
12.23 (s, 1H). ESI-MS ( m/z): 326.2 [M þ 1]. Anal. (C20H 17NO4)
C, H, N.
1-(2,5-Dimethylphenyl)-5-(2-hydroxy-4-methoxybenzoyl)py-
ridin-2(1H)-one (5h). The titled compound was prepared from 4
and 2,5-dimethylaniline according to the method of compound
5a. Yield 83%, white crystal. Mp: 128-129.3 °C. 1H NMR
(CDCl3, 300 MHz,): δ 2.16 (s, 3H), 2.37 (s, 3H), 3.86 (s, 3H), 6.45
(dd, 1H, J1 = 9 Hz, J2 = 2.7 Hz), 6.51 (d, 1H, J = 2.7 Hz), 6.72
(d, 1H, J = 9.6 Hz), 7.05 (s, 1H), 7.24 (m, 2H), 7.52 (d, 1H, J = 9
Hz), 7.81 (m, 2H), 12.24 (s, 1H). 13C NMR (CDCl3, 300 MHz): δ
17.15, 20.77, 55.65, 101.49, 107.64, 112.58, 117.44, 120.82,
127.42, 130.44, 131.12, 131.49, 133.20, 137.36, 139.19, 139.54,
163.04, 161.57, 165.81, 166.22, 193.73. ESI-MS (m/z): 350.13
[M þ 1]. Anal. (C21H19NO4) C, H, N.
1
179-179.7 °C. H NMR (CDCl3, 300 MHz): δ 3.86 (s, 3H),
6.46 (dd, 1H, J1 = 9 Hz, J2 = 2.7 Hz), 6.51 (d, 1H, J = 2.7 Hz),
6.70 (d, 1H, J = 9.3 Hz), 7.41 (m, 2H), 7.53 (m, 2H), 7.56 (m,
2H), 7.81 (dd, 1H, J1 = 9.3 Hz, J2 = 2.7 Hz), 7.93 (d, 1H, J =
2.7 Hz), 12.23 (s, 1H). ESI-MS (m/z): 322 [M þ 1]. Anal.
(C19H15NO4) C, H, N.
5-(2-Hydroxy-4-methoxybenzoyl)-1-(4-methoxyphenyl)pyridin-
2(1H)-one (5b). The titled compound was prepared from 4 and
4-methoxyaniline according to the method of compound 5a. Yield
1
85%, yellow crystal. Mp: 152.2-152.6 °C. H NMR (CDCl3,
1-(4-Chlorophenyl)-5-(2-hydroxy-4-methoxybenzoyl)pyridin-
2(1H)-one (5i). The titled compound was prepared from 4 and 4-
chloroaniline according to the method of compound 5a. Yield
65%, light-yellow crystal. Mp: 173.8-175.6 °C. 1H NMR
(CDCl3, 300 MHz): δ 3.87 (s, 1H), 6.46 (dd, 1H, J1 = 9 Hz,
J2 = 2.7 Hz), 6.52 (d, 1H, J = 2.7 Hz), 6.70 (d, 1H, J = 9.6 Hz),
7.36 (d, 2H, J = 6.6 Hz), 7.49 (m, 3H), 7.78 (dd, 1H, J1 = 9.6 Hz,
J2 = 2.4 Hz), 7.90 (d, 1H, J = 2.4 Hz), 12.20 (s, 1H). 13C NMR
(CDCl3, 300 MHz): δ 55.70, 101.54, 107.80, 112.51, 117.96,
120.88, 127.80, 129.80, 133.20, 135.22, 138.46, 139.61, 142.32,
161.54, 165.94, 166.39, 193.62. ESI-MS (m/z): 356.68 [M þ 1].
Anal. (C19H14ClNO4) C, H, N.
300 MHz): δ 3.86 (s, 3H), 3.87 (s, 3H), 6.45 (dd, 1H, J1 = 9 Hz,
J2 = 2.7 Hz), 6.52 (d, 1H, J = 2.7 Hz), 6.69 (d, 1H, J = 9.6 Hz),
7.00 (m, 2H), 7.32 (m, 2H), 7.53 (d, 1H, J = 9 Hz), 7.77 (dd, 1H,
J1 = 9.6 Hz, J2 = 2.7 Hz), 7.93 (d, 1H, J = 2.7 Hz), 12.24 (s, 1H).
ESI-MS (m/z): 352.3 [M þ 1]. Anal. (C20H17NO5) C, H, N.
5-(2-Hydroxy-4-methoxybenzoyl)-1-(2-methoxyphenyl)pyridin-
2(1H)-one (5c). The titled compound was prepared from 4 and 2-
methoxyaniline according to the method of compound 5a. Yield
84%, white needle crystal. Mp: 161.0-162.9 °C. 1H NMR
(CDCl3, 300 MHz): δ 3.85 (s, 3H), 3.87 (s, 3H), 6.44 (dd, 1H,
J1 = 9 Hz, J2 = 2.7 Hz), 6.51 (d, 1H, J = 2.7 Hz), 6.72 (m, 1H),
7.06 (m, 2H), 7.31 (dd, 2H, J1 = 8.1 Hz, J2 = 1.8 Hz), 7.42 (m,
1H), 7.59 (d, 1H, J = 9 Hz), 7.81 (m, 2H), 12.27 (s, 1H). ESI-MS
(m/z): 352.3 [M þ 1]. Anal. (C20H17NO5) C, H, N.
1-(4-Fluorophenyl)-5-(2-hydroxy-4-methoxybenzoyl)pyridin-
2(1H)-one (5j). The titled compound was prepared from 4 and
4-fluoroaniline according to the method of compound 5a. Yield
1
1-(4-Ethoxyphenyl)-5-(2-hydroxy-4-methoxybenzoyl)pyridin-
2(1H)-one (5d). The titled compound was prepared from 4 and
4-ethoxyaniline according to the method of compound 5a. Yield
80%, white crystal. Mp: 178.5-178.8 °C. 1H NMR (CDCl3, 300
MHz): δ 1.32 (t, 3H, J = 7.5 Hz), 3.67 (q, 3H), 6.45 (dd, 1H,
J1 = 9 Hz, J2 = 2.7 Hz), 6.52 (d, 1H, J = 2.7 Hz), 6.69 (d, 1H,
J = 9.6 Hz), 7.00 (t, 2H), 7.32 (t, 2H), 7.53 (d, 1H, J = 9 Hz),
7.77 (dd, 1H, J1 = 9.6 Hz, J2 = 2.7 Hz), 7.93 (d, 1H, J = 2.7 Hz),
12.24 (s, 1H). ESI-MS ( m/z): 366.3 [M þ 1]. Anal. (C21H 19NO5)
C, H, N.
78%, yellow crystal. Mp: 178.8-180.4 °C. H NMR (CDCl3,
300 MHz): δ 3.87 (s, 3H), 6.46 (dd, 1H, J 1 = 9 Hz, J2 = 2.7 Hz),
6.52 (d, 1H, J = 2.4 Hz), 6.71 (d, 1H, J = 9.6 Hz), 7.19 (m, 2H),
7.39 (m, 2H), 7.52 (d, 1H, J = 9 Hz), 7.78 (dd, 1H, J1 = 9.6H,
J2 = 2.4 Hz), 7.92 (d, 1H, J = 2.7 Hz), 12.21 (s, 1H). ESI-MS
( m/z): 340.8 [M þ 1]. Anal. (C19H 14FNO4) C, H, N.
5-(2-Hydroxy-4-methoxybenzoyl)-1-(4-nitrophenyl)pyridin-
2(1H)-one (5k). The titled compound was prepared from 4 and
4-nitroaniline according to the method of compound 5a. Yield
35%, yellow solid. Mp: 158.6-161.1 °C. 1H NMR (CDCl3, 300
MHz): δ 3.88 (s, 1H), 6.47 (dd, 1H, J1 = 9 Hz, J2 = 2.4 Hz), 6.62
(d, 1H, J = 2.4 Hz), 6.73 (d, 1H, J = 9.3 Hz), 7.51 (d, 1H, J = 9
Hz), 7.65 (m, 2H), 7.80 (dd, 1H, J1 = 9.6 Hz, J2 = 2.7 Hz), 7.91
(d, 1H, J = 2.4 Hz), 8.40 (m, 2H). ESI-MS (m/z): 367.68 [M þ 1].
Anal. (C19H14N2O6) C, H, N.
5-(2-Hydroxy-4-methoxybenzoyl)-1-p-tolylpyridin-2(1H)-one
(5e). The titled compound was prepared from 4 and 4-methyla-
niline according to the method of compound 5a. Yield 79%,
yellow crystal. Mp: 189.1-192.0 °C. 1H NMR (CDCl3, 300
MHz): δ 2.42 (s, 3H), 3.86 (s, 3H), 6.45 (dd, 1H, J1 = 9 Hz, J2 =
2.7 Hz), 6.51 (d, 1H, J = 2.4 Hz), 6.70 (d, 1H, 9.6 Hz), 7.27 (m,
3H), 7.53 (d, 1H, J = 9 Hz), 7.79 (dd, 1H, J1 = 9.6H, J2 = 2.4
Hz), 7.93 (d, 1H, J = 2.7 Hz), 12.24 (s, 1H). ESI-MS (m/z): 336.3
[M þ 1]. Anal. (C20H17NO4) C, H, N.
1-(4-Acetylphenyl)-5-(2-hydroxy-4-methoxybenzoyl)pyridin-
2(1H)-one (5l). The titled compound was prepared from 4 and
4-acetylaniline according to the method of compound 5a. Yield
39%, yellow crystal. Mp: 196.4-196.7 °C. 1H NMR (DMSO-d6,
300 MHz): δ 2.62 (s, 3H), 3.77 (s, 3H), 6.49 (m, 2H), 6.59 (d, 1H,
J = 9.6 Hz), 7.53 (d, 1H, J = 8.4 Hz), 7.66 (d, 2H, J = 6.6 Hz),
7.84 (dd, 1H, J1 = 9.6 Hz, J2 = 2.7 Hz), 8.02 (d, 1H, J = 2.4 Hz),
8.08 (d, 2H, J = 6.6 Hz), 11.17 (s, 1H). ESI-MS (m/z): 364 [M þ
1]. Anal. (C21H17NO5) C, H, N.
5-(2-Hydroxy-4-methoxybenzoyl)-1-m-tolylpyridin-2(1H)-one
(5f). The titled compound was prepared from 4 and 3-methylani-
line according to the method of compound 5a. Yield 81%,
yellow crystal. Mp: 123.8-125.8 °C. IR (KBr) ν (cm-1):
3070.34, 3033.05, 2975.80, 2947.44, 1684.65, 1633.84, 1579.98,
1
1273.79. H NMR (CDCl3, 300 MHz): δ 2.43 (s, 3H), 3.87 (s,
1-Cyclohexyl-5-(2-hydroxy-4-methoxybenzoyl)pyridin-2(1H)-
one (5m). The titled compound was prepared from 4 and
cyclopropanamine according to the method of compound 5a.
Yield 80%, white crystal. Mp: 199.9-201.8 °C. 1H NMR
(CDCl3, 300 MHz): δ 1.24 (m, 1H), 1.48 (m, 4H), 1.75 (m,
1H), 1.97 (m, 4H), 3.88 (s, 3H), 4.9 (m, 1H), 6.46 (dd, 1H, J1 = 9
Hz, J2 = 2.7 Hz), 6.53 (d, 1H, J = 2.7 Hz), 6.59 (d, 1H, J = 9.6
Hz), 7.45 (d, 1H, J = 9 Hz), 7.66 (dd, 1H, J1 = 9.6 Hz, J2 = 2.7
Hz), 7.92 (d, 1H, J = 2.7 Hz), 12.24 (s, 1H). ESI-MS (m/z): 328.1
[M þ 1]. Anal. (C19H21NO4) C, H, N.
3H), 6.45 (dd, 1H, J1 = 9 Hz, J2 = 2.7 Hz), 6.52 (d, 1H, J = 2.4
Hz), 6.71 (d,1H, J = 9.6 Hz), 7.19 (m, 2H), 7.28 (m,1H), 7.39 (m,
1H), 7.53 (d,1H, J = 9 Hz), 7.78 (dd, 1H, J1 = 9.6H, J2 = 2.4
Hz), 7.93 (d, 1H, J = 2.7 Hz), 12.24 (s,1H). 13C NMR (CDCl3,
300 MHz): δ 21.30, 55.66, 101.48, 107.66, 112.58, 117.50, 120.78,
123.38, 126.99, 129.38, 129.94, 133.27, 139.67, 139.78, 140.06,
142.91, 161.80, 165.84, 166.24, 193.80. ESI-MS ( m/z): 336.3
[M þ 1]. Anal. (C20H 17NO4) C, H, N.
5-(2-Hydroxy-4-methoxybenzoyl)-1-o-tolylpyridin-2(1H)-one
(5g). The titled compound was prepared from 4 and 2-methyla-
niline according to the method of compound 5a. Yield 83%,
5-(2-Hydroxy-4-methoxybenzoyl)-1-propylpyridin-2(1H)-one
(5n). The titled compound was prepared from 4 and propyla-
mine according to the method of compound 5a. Yield 78%,
yellow crystal. Mp: 128.5-129.5 °C. 1H NMR (CDCl3, 300
MHz): δ 0.98 (t, 3H, J=7.5 Hz), 1.80 (q, 2H, J=7.5 Hz), 3.88 (s,
1H), 3.95 (t, 2H, J=7.5 Hz), 6.46 (dd, 1H, J1=9 Hz, J2=2.7 Hz),
1
light-yellow crystal. Mp: 146.2-147.0 °C. H NMR (CDCl3,
300 MHz): δ 2.22 (s, 3H), 3.86 (s, 3H), 6.45 (dd, 1H, J1 = 9 Hz,
J2 = 2.7 Hz), 6.51 (d, 1H, J = 2.4 Hz), 6.74 (d, 1H, J = 9.6 Hz),
7.25 (m, 1H), 7.38 (m, 3H), 7.39 (m, 1H), 7.52 (d,1H, J = 9 Hz),

666 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2
Lv et al.
6.53 (d, 1H, J = 2.7 Hz), 6.59 (d, 1H, J = 9.6 Hz), 7.48 (d, 1H,
J = 9 Hz), 7.69 (dd, 1H, J1 = 9.6 Hz, J2 = 2.7 Hz), 7.86 (d, 1H,
J = 2.7 Hz), 12.23 (s, 1H). ESI-MS ( m/z): 288.23[M þ 1]. Anal.
(C16H 17NO4) C, H, N.
After the mixture was cooled to room temperature and the
solvent removed, the crude product was purified by chroma-
tography over silica gel (eluent petroleum ether/acetic ether, 6:1
to 2:1 v/v) to give compound 6a (Scheme 3). Yield: 68%, yellow
1
1-Butyl-5-(2-hydroxy-4-methoxybenzoyl)pyridin-2(1H)-one
(5o). The titled compound was prepared from 4 and butylamine
according to the method of compound 5a. Yield 87%, white
crystal. Mp: 96.8-97.2 °C. 1H NMR (CDCl3, 300 MHz): δ 0.95
(t, 3H, J = 7.2 Hz), 1.37 (m, 2H), 1.75 (m, 2H), 3.88 (s, 1H), 3.98
(t, 2H, J = 7.2 Hz), 6.46 (dd, 1H, J1 = 9 Hz, J2 = 2.7 Hz), 6.53
(d, 1H, J = 2.7 Hz), 6.59 (d, 1H, J = 9.6 Hz), 7.48 (d, 1H, J = 9
Hz), 7.69 (dd, 1H, J1 = 9.6 Hz, J2 = 2.7 Hz), 7.87 (d, 1H, J = 2.7
Hz), 12.24 (s, 1H). ESI-MS (m/z): 302.3 [M þ 1]. Anal.
(C17H19NO4) C, H, N.
crystal. Mp: 173.1-174.3 °C. H NMR (CDCl3, 300 MHz): δ
3.80 (s, 3H), 4.58 (s, 2H), 5.12 (s, 2H), 6.21 (dd, 1H, J1 = 9 Hz,
J2 = 2.7 Hz), 6.42 (d, 1H, J = 2.7 Hz), 6.70 (d, 1H, J = 9.6 Hz),
6.84 (d, 1H, J = 9 Hz), 7.16 (m, 4H), 7.24 (m, 2H), 7.30 (m, 3H),
7.34 (m, 3H). ESI-MS (m/z): 425.15 [M þ 1]. Anal. (C27H24-
N2O3) C, H, N.
(E)-1-(3-Fluorobenzyl)-5-((2-fluorobenzylimino)(2-hydroxy-
4-methoxyphenyl)methyl)pyridin-2(1H)-one (6b). The titled
compound was prepared from 4 and 2-fluorobenzylamine accord-
ing to the method of compound 6a. Yield 63%, yellow crystal.
Mp: 97.7-99.7 °C. 1H NMR (CDCl3, 300 MHz): δ 3.81 (s, 3H),
4.87 (m, 2H), 4.98 (m, 2H), 6.27 (dd, 1H, J1 = 9 Hz, J2 = 2.7 Hz),
6.46 (d, 1H, J = 2.7 Hz), 6.74 (dd, 1H, J1 = 9.6 Hz, J2 = 2.4 Hz),
6.87 (m, 4H), 7.18 (m, 3H), 7.30 (m, 4H), 15.62 (s, 1H). ESI-MS
(m/z): 461.16 [M þ 1]. Anal. (C27H22F2N2O3) C, H, N.
5-(2-Hydroxy-4-methoxybenzoyl)-1-pentylpyridin-2(1H)-one
(5p). The titled compound was prepared from 4 and pentylamine
according to the method of compound 5a. Yield 88%, white
crystal. Mp: 74-74.6 °C. IR (KBr) ν (cm-1): 3095.73, 3079.71,
3021.74, 2953.41, 2934.19, 2859.94, 1671.19, 1627.07, 1615.70,
1
(E)-1-(3-Fluorobenzyl)-5-((3-fluorobenzylimino)(2-hydroxy-
4-methoxyphenyl)methyl)pyridine-2(1H)-one (6c). The titled
compound was prepared from 4 and 3-fluorobenzylamine ac-
cording to the method of compound 6a. Yield 83%, yellow
crystal. Single crystals for X-ray diffraction were obtained by
slow crystallization of a solution in petroleum ether/AcOEt. Mp:
142.3-144.3 °C. IR (KBr) ν (cm-1): 3061.26, 2934.59, 2847.51,
1665.10, 1592.58, 1226.49, 1019.84, 831.76, 781.16. 1H NMR
(CDCl3, 300 MHz): δ 3.81 (s, 3H), 4.60 (m, 2H), 5.03 (m, 2H),
6.26 (dd, 1H, J1 = 9 Hz, J2 = 2.7 Hz), 6.45 (d, 1H, J = 2.7 Hz),
6.72 (dd, 1H, J1 = 9.6 Hz, J2 = 2.4 Hz), 6.86 (m, 4H), 7.19
(m, 3H), 7.28 (m, 4H), 15.62 (s, 1H), 13C NMR (300 MHz,
CDCl3): δ 51.7, 54.0, 55.43, 101.78, 106.45, 111.86, 113.89,
114.14, 114.42, 114.96, 115.36, 115.64, 121.66, 122.53, 123.63,
130.29, 130.40, 130.77, 132.11, 136.65, 138.96, 161.39, 164.14,
169.57. IR (KBr) ν (cm-1): 3061.26, 2934.59, 2847.51, 1665.10,
1592.58, 1226.49, 1019.84, 831.76, 781.16. ESI-MS (m/z): 461.47
[M þ 1]. Anal. (C27H22F2N2O3) C, H, N.
1578.74, 1384.13. H NMR (CDCl3, 300 MHz): δ 0.95 (t, 3H,
J = 7.2 Hz), 1.27 (m, 4H), 1.65 (m, 2H), 3.83 (t, 2H, J = 7.2 Hz),
3.88 (s, 1H), 6.46 (dd, 1H, J1 = 9 Hz, J2 = 2.7 Hz), 6.53 (d, 1H,
J = 2.7 Hz), 6.59 (d, 1H, J = 9.6 Hz), 7.48 (d, 1H, J = 9 Hz),
7.69 (dd, 1H, J1 = 9.6 Hz, J2 = 2.7 Hz), 7.87 (d, 1H, J = 2.7 Hz),
12.24 (s, 1H). 13C NMR (CDCl3, 300 MHz): δ 13.87, 22.25,
28.73, 28.79, 50.61, 55.66, 101.48, 107.55, 112.64, 117.38, 119.92,
133.30, 138.86, 142.54, 161.99, 165.81, 166.17, 193.92. ESI-MS
(m/z): 316.2 [M þ 1]. Anal. (C18H21NO4) C, H, N.
5-(2-Hydroxy-4-methoxybenzoyl)-1-isopropylpyridin-2(1H)-
one (5q). The titled compound was prepared from 4 and
isopropylamine according to the method of compound 5a.
Yield 79%, yellow crystal. Mp: 143.8-144.8 °C. 1H NMR
(CDCl3, 300 MHz): δ 1.41 (d, 6H, J = 6.9 Hz), 5.28 (q, 1H, J =
6.9 Hz), 6.46 (dd, 1H, J1 = 9 Hz, J2 = 2.7 Hz), 6.53 (d, 1H, J =
2.7 Hz), 6.63 (d, 1H, J = 9.6 Hz), 7.46 (d, 1H, J = 9 Hz), 7.67
(dd, 1H, J1 = 9.6 Hz, J2 = 2.7 Hz), 7.93 (d, 1H, J = 2.7 Hz),
12.24 (s, 1H). ESI-MS (m/z): 288.2 [M þ 1]. Anal. (C16H17NO4)
C, H, N.
(E)-1-(4-Fluorobenzyl)-5-((4-fluorobenzylimino)(2-hydroxy-
4-methoxyphenyl)methyl)pyridin-2(1H)-one (6d). The titled
compound was prepared from 4 and 4-fluorobenzylamine ac-
cording to the method of compound 6a. Yield 70%, yellow
crystal. Mp: 154.0-155.3 °C. IR (KBr) ν (cm-1): 3101.82,
3067.29, 3047.88, 2954.01, 2940.03, 2871.62, 1665.49, 1307.58,
1508.73, 1382.74, 1223.25. 1H NMR (CDCl3, 300 MHz): δ 3.79
(s, 3H), 4.57 (m, 2H), 5.15 (m, 2H), 6.23 (dd, 1H, J1 = 9 Hz),
J2 = 2.4 Hz), 6.43 (d, 1H, J = 2.7 Hz), 6.71 (d, 1H, J = 9 Hz),
6.83 (d, 1H, J = 9 Hz), 7.02 (m, 3H), 7.12 (m, 4H), 7.24 (m, 3H).
13C NMR (CDCl3, 300 MHz): δ 51.57, 53.61, 55.38, 76.58, 77.01,
77.43, 101.80, 106.31, 110.79, 112.77, 115.51, 115.79, 115.90,
116.19, 121.56, 128.54, 128.64, 130.06, 130.17, 131.52, 132.01,
134.23, 136.53, 138.86, 161.42, 164.12, 167.29, 169.25. ESI-MS
(m/z): 461.16 [M þ 1]. Anal. (C27H22F2N2O3) C, H, N.
1-Allyl-5-(2-hydroxy-4-methoxybenzoyl)pyridin-2(1H)-one (5r).
The titled compound was prepared from 4 and 2-propen-1-
ylamine according to the method of compound 5a. Yield 89%,
1
Yellow crystal. Mp: 131.7-132.6 °C. H NMR (CDCl3, 300
MHz): δ 3.87 (s, 1H), 4.62 (m, 2H), 5.25 (m, 2H), 5.92 (m, 1H),
6.56 (dd, 1H, J1 = 9 Hz, J2 = 2.7 Hz), 6.52 (d, 1H, J = 2.7 Hz),
6.62 (d, 1H, J = 9.6 Hz), 7.46 (d, 1H, J = 9 Hz), 7.70 (dd, 1H,
J1 = 9.6 Hz, J2 = 2.7 Hz), 7.86 (d, 1H, J = 2.7 Hz), 12.24 (s,
1H). 13C NMR (CDCl3, 300 MHz): δ 30.91, 51.62, 55.68, 101.45,
107.63, 112.56, 117.68, 119.78, 119.97, 131.65, 133.31, 139.10,
142.02, 161.78, 165.85, 166.22, 193.84. IR (KBr) ν (cm-1):
3085.15, 3071.62, 2987.67, 2971.99, 2940.53, 2845.23, 1667.92,
1616.20, 1585.08, 1536.00, 1507.79, 1736.73, 1271.19,1111.47,
840.54, 606.96. ESI-MS (m/z): 286.1 [M þ 1]. Anal. (C16H15-
NO4) C, H, N.
(E)-1-(3,4-Difluorobenzyl)-5-((2,4-difluorobenzylimino)(2-hy-
droxy-4-methoxyphenyl)methyl)pyridin-2(1H)-one (6e). The
titled compound was prepared from 4 and 2,4-difluorobenzyla-
mine according to the method of compound 6a. Yield 55%,
yellow crystal. Mp: 102.5-103.8 °C. 1H NMR (CDCl3, 300
MHz): δ 3.80 (s, 3H), 4.54 (m, 2H), 5.05 (m, 2H), 6.29 (dd, 1H,
J1=9 Hz, J2=2.7 Hz), 6.49 (d, 1H, J=2.7 Hz), 6.78 (d, 1H, J=
9 Hz), 6.95 (m, 2H), 7.12 (m, 2H), 7.25 (m, 3H), 15.44 (s, 1H).
ESI-MS (m/z): 497.14 [M þ 1]. Anal. (C27H20F4N2O3) C, H, N.
(E )-1-(3,4-Difluorobenzyl)-5-((3,4-difluorobenzylimino)(2-hy-
droxy-4-methoxyphenyl)methyl)pyridin-2(1H)-one (6f ). The
titled compound was prepared from 4 and 3,4-difluorobenzyla-
mine according to the method of compound 6a. Yield 75%,
yellow crystal. Mp: 129-130.8 °C. IR (KBr) ν (cm-1): 3445.28,
3049.49, 3005.44, 2968.00, 2934.85, 2913.94, 2838.74, 1667.72,
1597.44, 1520.12, 1457.75. 1H NMR (CDCl3, 300 MHz): δ 3.82
(s, 3H), 4.55 (m, 2H), 5.09 (m, 2H), 6.28 (dd, 1H, J1 = 9 Hz, J2 =
2.7 Hz), 6.47 (d, 1H, J = 2.7 Hz), 6.76 (d, 1H, J = 9 Hz), 6.89
(E)-5-(2-Hydroxy-4-methoxybenzoyl)-1-(2-(naphthalen-1-yl-
amino)vinyl)pyridin-2(1H)-one (5s). The titled compound was
prepared from 4 and (E)-N1-(naphthalen-1-yl)ethene-1,2-dia-
mine according to the method of compound 5a. Yield 88%,
yellow crystal. Mp: 203.1-205.1 °C. IR (KBr) ν (cm-1):
3440.21, 3049.82, 2922.37, 2854.85, 1667.34, 1583.46, 1532.23,
1383.58, 1350.42, 1235.41. 1H NMR (DMSO-d6, 300 MHz): δ
3.66 (s, 3H), 5.50 (dd, 1H, J1 = 9 Hz, J2 = 2.4 Hz), 638 (d, 1H,
J = 2.4 Hz), 6.56 (d, 1H, J = 9.3 Hz), 7.04 (d, 1H, J = 9 Hz),
7.13 (d, 1H, J = 8.7 Hz), 7.24 (t, 1H, J = 8.1 Hz), 7.41 (m, 2H),
7.77 (m, 2H), 8.00 (d, 1H, J = 2.4 Hz), 8.10 (d, 1H, J = 7.8 Hz),
11.36 (s, 1H). ESI-MS (m/z): 415.65 [M þ 1]. Anal. (C25H20-
N2O4) C, H, N.
(E)-1-Benzyl-5-((benzylimino)(2-hydroxy-4-methoxyphenyl)-
methyl)pyridin-2(1H)-one (6a). A solution of compound 4 (1.04
g, 0.004 mol), benzylamine (1.07 g, 0.01 mol), and triethylamine
(6 drops) in MeOH (45 mL) was stirred under reflux overnight.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2 667
(m, 2H), 7.03 (m, 2H), 7.16 (m, 3H), 15.44 (s, 1H). ESI-MS (m/z):
497.14 [M þ 1]. Anal. (C27H20F4N2O3) C, H, N.
2838.84, 1667.75, 1614.25, 1592.11, 1541.96, 1528.65, 1384.16,
1224.30. 1H NMR (CDCl3, 300 MHz): δ 0.926(m, 6H), 1.40 (m,
4), 1.63 (m, 2H), 1.75 (m, 2H), 3.41 (t, 2H, J = 6.9 Hz), 3.81 (s,
3H), 3.99 (t, 2H, J = 6.9 Hz), 6.20 (dd, 1H, J1 = 9.3 Hz, J2 = 2.4
Hz), 6.40 (d, 1H, J = 2.4 Hz), 6.67 (d, 1H, J = 9 Hz), 6.85 (d, 1H,
J = 9 Hz), 7.21 (m, 2H), 16.24 (s, 1H). 13C NMR (CDCl3, 300
MHz): δ 13.68, 19.80, 20.30, 31.22, 32.75, 49.16, 50.00, 55.29,
102.15, 105.99, 111.05, 112.23, 121.28, 131.84, 137.30, 138.68,
161.46, 164.42, 168.24, 170.38. ESI-MS (m/z): 357.21 [M þ 1].
Anal. (C21H28N2O3) C, H, N.
(E)-5-((2-Hydroxy-4-methoxyphenyl)(pentylimino)methyl)-1-
pentylpyridin-2(1H)-one (6m). The titled compound was pre-
pared from 4 and pentylamine according to the method of
compound 6a. Yield 90%, yellow crystal. Mp: 95.8-97.4 °C.
IR (KBr) ν (cm-1): 3446.24, 3005.10, 2952.81, 2927.19, 2858.28,
1669.93, 1615.19, 1525.84, 1229.60. 1H NMR (CDCl3, 300
MHz): δ 0.89 (m, 6H), 1.34 (m, 8H), 1.67 (m, 2H), 1.78 (m,
2H), 3.39 (m, 2H), 3.80 (s, 3H), 3.97 (m, 2H), 6.19 (dd, 1H, J1 =
9 Hz, J2 = 2.7 Hz), 6.39 (d, 1H, J = 2.7 Hz), 6.66 (d, 1H, J = 9.6
Hz), 6.85 (d, 1H, J = 9 Hz), 7.22 (m, 2H), 16.26 (s, 1H). 13C
NMR (CDCl3, 300 MHz): δ 13.90, 22.28, 28.75, 28.87,
29.32, 30.42, 49.50, 52.28, 55.31, 76.58, 77.00, 77.43, 102.17,
106.02, 111.06, 112.25, 121.31, 131.86, 137.33, 138.69, 161.47,
164.45, 168.23, 170.40. ESI-MS (m/z): 385.65 [M þ 1]. Anal.
(C23H32N2O3) C, H, N.
(E)-5-((2-Hydroxy-4-methoxyphenyl)(isopropylimino)methyl)-
1-isopropylpyridin-2(1H)-one (6n). The titled compound was
prepared from 4 and isopropylamine according to the method
of compound 6a. Yield 71%, light-yellow crystal. Mp:
152.8-155.6 °C. 1H NMR (CDCl3, 300 MHz): δ 1.261 (d, 6H,
J = 6.3 Hz), 1.37 (d, 6H, J = 6.9 Hz), 3.67 (m, 1H), 3.81 (s, 3H),
5.34 (m, 1H), 6.21 (dd, 1H, J1 = 9 Hz, J2 = 2.7 Hz), 6.42 (d, 1H,
J = 2.7 Hz), 6.67 (d, 1H, J = 9.3 Hz), 6.81 (d, 1H, J = 9H), 7.17
(dd, 1H, J1 = 9.3 Hz, J2 = 2.4 Hz), 7.26 (d, 1H, J = 2.4 Hz),
16.17 (s, 1H). 13C NMR (CDCl3, 300 MHz): δ 21.96, 24.21,
46.63, 50.58, 55.32, 102.06, 105.95, 111.75, 112.40, 120.97,
131.87, 132.29, 137.91, 161.16, 164.16, 166.58, 169.34. ESI-MS
(m/z): 328.1 [M þ 1]. Anal. (C19H24N2O3) C, H, N.
(E)-5-((2-Hydroxy-4-methoxyphenyl)(4-methoxybenzylimino)-
methyl)-1-(4-methoxybenzyl)pyridin-2(1H)-one (6g). The titled
compound was prepared from 4 and 4-methoxybenzylamine
according to the method of compound 6a. Yield 67%, yellow
crystal. Mp: 128.5-129.9 °C. IR (KBr) ν (cm-1): 3444.64,
3063.50, 3030.28, 2999.59, 2951.76, 2930.54, 2833.12, 1666.27,
1611.78, 1514.40, 1257.94, 1227.13, 1174.57. 1H NMR (CDCl3,
300 MHz): δ 3.80 (t, 9H), 4.51 (s, 2H), 5.07 (s, 2H), 6.20 (dd, 1H,
J1 = 9 Hz, J2 = 2.7 Hz), 6.42 (d, 1H, J = 2.9 Hz), 6.72 (m, 1H),
6.85 (m, 5H), 7.03 (m, 2H), 7.16 (m, 2H), 7.22 (m, 2H), 15.49 (s,
1H). ESI-MS (m/z): 485.2 [M þ 1]. Anal. (C29H28N2O5) C, H, N.
(E)-5-((2-Hydroxy-4-methoxyphenyl)(4-methylbenzylimino)-
methyl)-1-(4-methylbenzyl)pyridin-2(1H)-one (6h). The titled
compound was prepared from 4 and 4-methylbenzylamine
according to the method of compound 6a. Yield 66%, yellow
needle crystal. Mp: 158.8-159.5 °C. IR (KBr) ν (cm-1): 3446.44,
3454.69, 3049.93, 3001.00, 2981.32, 2922.59, 2854.95, 1659.67,
1595.70, 1384.37. 1H NMR (CDCl3, 300 MHz): δ 2.34 (s, 6H),
3.80 (s, 3H), 4.54 (m, 2H), 5.09 (m, 2H), 6.20 (dd, 1H, J1 = 9 Hz,
J2 = 2.7 Hz), 6.41 (d, 1H, J = 2.7 Hz), 6.69 (d, 1H, J = 9.9 Hz),
6.82 (d, 1H, J = 9 Hz), 7.04 (m, 2H), 7.10 (m, 2H), 7.15 (m, 4H),
7.18 (m, 2H). 13C NMR (CDCl3, 300 MHz): δ 21.07, 21.14,
51.93, 53.68, 55.35, 76.58, 77.00, 77.43, 101.89, 106.18, 111.41,
112.64, 120.11, 121.27, 127.03, 128.34, 128.57, 129.46, 129.77,
132.05, 132.59, 135.39, 136.90, 138.28, 138.84, 161.61, 164.20,
138.50, 169.02. ESI-MS (m/z): 453.21 [M þ 1]. Anal. (C29H28-
N2O3) C, H, N.
(E)-1-Cyclohexyl-5-((cyclohexylimino)(2-hydroxy-4-methoxy-
phenyl)methyl)pyridin-2(1H)-one (6i). The titled compound was
prepared from 4 and cyclohexylamine according to the method
of compound 6a. Yield 79%, yellow crystal. Mp: 182-184.2 °C.
1H NMR (CDCl3, 300 MHz): δ 1.20 (m, 2H), 1.25 (m, 2H), 1.32
(m, 4H), 1.75 (m, 4H), 1.93 (m, 4H), 3.35 (m, 1H), 3.80 (s, 3H),
4.94 (m, 1H), 6.18 (dd, 1H, J1 = 9 Hz, J2 = 2.7 Hz), 6.39 (d, 1H,
J = 2.7 Hz), 6.67 (d, 1H, J = 9.3 Hz), 6.80 (d, 1H, J = 9 Hz),
7.15 (dd, 1H, J1 = 9 Hz, J2 = 2.7 Hz), 7.28 (d, 1H, J = 2.7 Hz).
ESI-MS (m/z): 409 [M þ 1]. Anal. (C25H32N2O3) C, H, N.
(E)-1-Cyclopropyl-5-((cyclopropylimino)(2-hydroxy-4-methoxy-
phenyl)methyl)pyridin-2(1H)-one (6j). The titled compound was
prepared from 4 and cycloprpylamine according to the method
of compound 6a. Yield 75%, yellow crystal. Mp: 178.4-180.0 °C.
IR (KBr) ν (cm-1): 3446.29, 3421.37, 3079.42, 2938.77, 1663.68,
1588.65, 1287.39. 1H NMR (CDCl3, 300 MHz): δ 0.94 (m, 2H),
0.97 (m, 4H), 1.17 (m, 2H), 2.86 (m, 1H), 3.41 (m, 1H), 3.80
(s, 3H), 6.29 (dd, 1H, J1 = 9 Hz, J2 = 2.7 Hz), 6.44 (d, 1H, J =
2.7 Hz), 6.65 (d, 1H, J = 9.3 Hz), 6.87 (d, 1H, J = 9 Hz), 7.07
(m, 3H), 7.27 (m, 1H), 7.33 (m, 1H), 14.79 (s, 1H). 13C NMR
(CDCl3, 300 MHz): δ 6.96, 10.27, 32.59, 33.53, 55.33, 101.63,
105.71, 112.29, 113.41, 120.83, 131.18, 136.97, 139.06, 162.88,
163.02, 164.48, 166.46. ESI-MS (m/z): 325.15 [M þ 1]. Anal.
(C19H20N2O3) C, H, N.
(E)-1-Allyl-5-((allylimino)(2-hydroxy-4-methoxyphenyl)methyl)-
pyridin-2(1H)-one (6o). The titled compound was prepared from
4 and 2-propen-1-ylamine according to the method of com-
pound 6a. Yield 73%, yellow crystal. Mp: 139.2-141.6 °C. IR
(KBr) ν (cm-1): 3445.94, 3421.28, 3061.74, 2923.71, 2851.53,
1665.02, 1613.79, 1592.52, 1522.85, 1383.92. 1H NMR (CDCl 3,
300 MHz): δ 3.82 (s, 3H), 4.08 (s, 2H), 4.62 (s, 2H), 5.17 (m, 1H),
5.20 (m, 3H), 5.98 (m, 2H), 6.24 (dd, 1H, J1 = 9 Hz, J2 = 2.4
Hz), 6.44 (d, 1H, J = 2.7 Hz), 6.90 (d, 1H, J = 9.6 Hz), 6.72 (d,
1H, J = 9 Hz), 7.24 (m, 2H), 16.93 (s, 1H). ESI-MS (m/z): 325.16
[M þ 1]. Anal. (C19H 20N2O3) C, H, N.
In Vitro Cytotoxicity Study of Target Compounds. Cytotoxi-
city of all synthesized compounds was assessed by MTT assays
as previously described.18 The compounds, dissolved in DMSO,
were serially diluted in cell culture media and then added onto
cell monolayers. HepG2 2.2.15 cells were maintained in 96-well
tissue culture plates for 48 h before being treated with com-
pounds. The cells were subsequently incubated at 37 °C for
9 days. Cells with media alone were used as the blank control,
while adefovir was used as positive controls. MTT reagents were
added at a concentration of 5 g/L at 4 h before the cells were
harvested and lysed with 10% sodium dodecyl sulfate (SDS) and
50% N,N-dimethylformamide (DMF), pH 7.2. The OD values
of cell lysates were read at a wavelength of 570 nm, and the
percentages of cell death were determined.19
General Method for the HBsAg, HBeAg, and HBV DNA
Inhibition Assays. HBV antigen and HBV DNA inhibitory
efficacies of these compounds were evaluated in cultured HepG2
2.2.15 cells. Nine days after treatment with different dosages of
the compounds, cell culture supernatants were collected, and
HBV antigen activities were tested by enzyme linked immuno-
sorbent assay (ELISA). Levels of HBV-DNA in the culture
(E)-5-((2-Hydroxy-4-methoxyphenyl)(propylimino)methyl)-1-
propylpyridin-2(1H)-one (6k). The titled compound was pre-
pared from 4 and propylamine according to the method of
compound 6a. Yield 71%, yellow crystal. Mp: 129.5-131 °C. 1H
NMR (CDCl3, 300 MHz): δ 0.99 (m, 6H), 1.69 (m, 2H), 1.82 (m,
2H), 3.38 (t, 2H, J = 0.72 Hz), 3,81 (s, 3H), 3.95 (t, 2H, J=0.72),
6.20 (dd, 1H, J1 = 9 Hz, J2 = 2.4 Hz), 6.39 (d, 1H, J = 2.4 Hz),
6.67 (d, 1H, J = 9.3 Hz), 6.85 (d, 1H, J = 9 Hz), 7.21 (m, 2H).
13C NMR (300 MHz CDCl3) δ 11.00, 11.75, 22.41, 23.99, 51.17,
51.84, 55.31, 102.15, 106.05, 111.02, 112.17, 121.31, 131.87,
137.40, 138.74, 161.50, 164.47, 168.30, 170.49. ESI-MS (m/z):
329.1 [M þ 1]. Anal. (C19H24N2O3) C, H, N.
(E)-1-Butyl-5-((butylimino)(2-hydroxy-4-methoxyphenyl)me-
thyl)pyridin-2(1H)-one (6l). The titled compound was prepared
from 4 and butylamine according to the method of compound
6a. Yield 73%, yellow crystal. Mp: 85.6-86.6 °C. IR (KBr) ν
(cm-1): 3447.08, 3065.86, 3000.35, 2954.68, 2932.74, 2871.64,

668 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2
Lv et al.
media were quantified by fluorescent PCR as previously
described.20 PCR primers used were as follows: forward, 50-
TGTCCTGGTTATCGCTGG-30; reverse, 50-CAAACGGG-
CAACATA-CCTT-30. Probe used was 50-(FAM)-TGTGTCT-
GCGGCGTTTTATCAT-(TAMRA)-30. PCR reactions were
run on a MJ Research PTC-200, and data were analyzed by
OpticonMonitor, version 2.01, software. The antiviral activities
of the compounds are summarized in Table 1.
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Computational Details. GASP17 and SYBYL molecular mod-
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model. The three-dimensional structures of compounds 5f and
6c were built from the SYBYL fragment library. Energy mini-
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˚
vergence criterion of 0.001 kcal/(mol A). Charges were calcu-
3
€
lated by the Gasteiger-Huckel method. Simulated annealing
was then performed. The system was heated to 1000 K for 1.0 ps
and then annealed to 250 K for 1.5 ps. The annealing function
was exponential; 50 such cycles of annealing were run, and the
resulting 50 conformers were optimized using methods de-
scribed above. The lowest energy conformation was selected.
The GASP program uses a genetic algorithm for determining
the correspondence between functional groups in the super-
imposed ligands and the alignment of these groups in a common
geometry for receptor binding. A population of chromosomes is
randomly constructed with each chromosome representing a
possible alignment. Torsion angles for the rotatable bonds are
adjusted when searching for molecule alignment. The fitness
score of a given alignment is a weighted sum of three terms: the
number and similarity of the overlaid elements, the common
volume of the molecules, and the internal van der Waals (vdW)
energy of each molecule. The calculation terminates when the
fitness of the population does not further improve by a specified
value or when the preset number of genetic operations is
completed. The GASP settings were as follows. The GASP
parameters were set to the following default values: population
size 100; selection pressure 1.1; maximum number of operations
100 000; operation increment 6500; fitness increment 0.01; point
cross weight 95.0; allele mutate weight 95.0; full mutation weight
0.0; full cross weight 0.0; internal vdW energy coefficient 1; HB
weight coefficient 750; vdW contact cutoff 0.8.
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Acknowledgment. We thank Mei Zhou of Tri-I Biotech,
Inc. (Shanghai, People’sRepublic ofChina), for her assistance
in pharmacophore modeling.
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C.-L.; Shi, L.-P.; Ren, Y.-D.; Zuo, J.-P.; Lu, W. Identification of 1-
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(20) Li, Y.-F.; Wang, G.-F.; He, P.-L.; Huang, W.-G.; Zhu, F.-H.; Gao,
H.-Y.; Tang, W.; Luo, Y.; Feng, C.-L.; Shi, L.-P.; Ren, Y.-D.; Lu,
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63144), 2003.
Supporting Information Available: HPLC analysis data of
compounds 4, 5a-s, and 6a-o. This material is available free of
charge via the Internet at http://pubs.acs.org.
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