Structure-activity relationships study of 6-chloro-4-(2-chlorophenyl)-3-(2- hydroxyethyl) quinolin-2(1H)-one derivatives as novel non-nucleoside anti-hepatitis B virus agents

Article abstract of DOI:10.1016/j.ejmech.2010.11.019

A series of novel 6-chloro-4-(2-chlorophenyl)-3-(2-hydroxyethyl) quinolin-2(1H)-one derivatives were synthesized and evaluated for anti-hepatitis B virus (anti-HBV) activities in vitro to explore their structure-activity relationships (SARs). Most of the

Full text of DOI:10.1016/j.ejmech.2010.11.019

European Journal of Medicinal Chemistry 46 (2011) 307e319
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Structureeactivity relationships study of 6-chloro-4-(2-chlorophenyl)-
3-(2-hydroxyethyl) quinolin-2(1H)-one derivatives as novel non-nucleoside
anti-hepatitis B virus agents
,
,
,b
Rui-Hua Guo a b, Quan Zhang a, Yun-Bao Ma a, Jie Luo a, Chang-An Geng a b, Li-Jun Wang a
,
Xue-Mei Zhang a, Jun Zhou a, Zhi-Yong Jiang a, Ji-Jun Chen a
,
*
a State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650204, PR China
b Graduate School of Chinese Academy of Sciences, Beijing 100039, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel 6-chloro-4-(2-chlorophenyl)-3-(2-hydroxyethyl) quinolin-2(1H)-one derivatives were
synthesized and evaluated for anti-hepatitis B virus (anti-HBV) activities in vitro to explore their
structureeactivity relationships (SARs). Most of the synthesized compounds possessed potent anti-HBV
activity, of which the promising compound 44 exhibited significantly inhibitory potency against the
secretion of hepatitis surface antigen (HBsAg) (IC50 ¼ 0.010 mM, SI > 135), hepatitis e antigen (HBeAg)
(IC50 ¼ 0.026 mM, SI > 51) and the replication of HBV DNA (IC50 ¼ 0.045 mM). Preliminary mechanism
study suggested compound 44 could mainly enhance the transcript activity of HBV ENI (enhancer I), EN-
II (enhancer II).
Received 19 July 2010
Received in revised form
6 November 2010
Accepted 11 November 2010
Available online 19 November 2010
Keywords:
Anti-hepatitis B virus activity
Quinolin-2(1H)-one
Crown Copyright Ó 2010 Published by Elsevier Masson SAS. All rights reserved.
Structureeactivity relationships
1. Introduction
As a part of our continuous search for active anti-HBV leads from
natural and synthetic compounds [11e16], a rational screening sug-
Hepatitis B virus, belonging to hepadnavirus (hepatotropic DNA
viruses) family, causes acute and chronic infection of liver [1,2], and
constitutes the world’s ninth leading cause of death [3]. Chronic HBV
infection can lead to cirrhosis of liver, liver failure, and hepatocel-
lular carcinoma (HCC) [4]. An estimated 350e400 million people are
chronically infected by HBV throughout the world with 0.5e1.2
million global deaths per year [5]. Currently only two interferons-
alphas (interferon alpha-2a and PEGylated interferon alpha-2a) and
five nucleoside inhibitors (lamivudine, adefovir dipivoxil, entecavir,
telbivudine, tenofovir disoproxil fumarate) have been approved by
the FDA as single-agent treatments for HBV infection [6,7].
gested that 4-aryl-6-chloroquinolin-2-one (1, Fig. 1) possessed
moderate activity inhibiting the production of HBsAg with an IC50
value of 0.46 mM in HBV-infected HepG 2.2.15 cells. In view of its
novel structural template different from those of all reported anti-
HBV agents, the preliminary structureeactivity relationships of the
relevant compounds were studied leading to the discovery of
6-chloro-4-(2-chlorophenyl)-3-(2-hydroxyethyl)
quinolin-2(1H)-
one (2) with a 5.3-fold increased activity against the secretion of
HBsAg (IC50 ¼ 0.086 mM vs IC50 ¼ 0.46 mM), and with an IC50 value of
0.36 mM on the secretion of HBeAg, which revealed that introduction
of hydroxyethyl to C-3 of 4-aryl-6-chloro-quinoline-2-one was an
important feature in conferring enhanced anti-HBV activity
[12].Therefore, it is of interest to synthesize additional analogues for
investigation of SARs and the development of more potent anti-HBV
agents.
In this paper, we synthesized novel 6-chloro-4-(2-chlorophenyl)-
3-(2-hydroxyethyl) quinolin-2(1H)-one analogues via chemical
modifications on N-1, C-2, and hydroxyethyl moiety and evaluated
their anti-HBV activities. The most active compounds 42e49, 56, and
57 against the secretion of HBsAg andHBeAg were furtherassayed on
their inhibition of HBV DNA replication. Significantly, compound 44
inhibited not only the secretion of HBsAg (IC50 ¼ 0.010 mM, SI > 135),
HBeAg (IC50 ¼ 0.026 mM, SI > 51) but also HBV DNA replication
However, use of a-interferon is limited for its lower success rate
and serious side effects, and nucleoside analogues targeting the viral
DNA polymerase result in development of drug-resistant virus after
long-term treatment [8e10]. Therefore, the current therapies remain
unsatisfactory and development of novel classes of anti-HBV agents
with new structures and mechanisms of action is urgently needed.
Abbreviations: HBV, hepatitis B virus; HBsAg, hepatitis surface antigen; HBeAg,
hepatitis e antigen; HCC, hepatocellular carcinoma; SARs, structureeactivity rela-
tionships; SI, selectivity index; CC50, 50% cytotoxic concentration; IC50, 50% inhib-
itory concentration.
* Corresponding author. Tel./fax: þ86 871 5223265.
E-mail address: chenjj@mail.kib.ac.cn (J.-J. Chen).
0223-5234/$ e see front matter Crown Copyright Ó 2010 Published by Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.11.019

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R.-H. Guo et al. / European Journal of Medicinal Chemistry 46 (2011) 307e319
afforded derivative 14. Compound 2 was refluxed with Lawesson’s
reagent (LR) in toluene to give compound 15(Scheme 3) [14].
To optimize chain length between 6-chloro-4-(2-chlorophenyl)
quinolin-2(1H)-one and ester moieties, compounds 54 and 55 were
prepared by reaction of 2-amino-benzophenone with beta-pro-
piolatone and delta-valerolactone, and then converted to corre-
sponding esters 56 and 57, respectively (Scheme 4).
3. Results and discussion
The synthesized 6-chloro-4-(2-chlorophenyl)-3-(2-hydrox-
yethyl) quinolin-2(1H)-one analogues (2, 8e53, 56, and 57) were
screened for their anti-HBV activities, namely the abilities to inhibit
the secretion of HBsAg and HBeAg in HBV-infected 2.2.15 cells using
lamivudine (3TC, an anti-HBV therapeutic agent) as a positive
control. The potent active compounds 42e49, 56 and 57 were
further evaluated for their activities against HBV DNA replication.
The anti-HBV activity of each compound was expressed as the
concentration of compound that achieved 50% inhibition (IC50) to
the secretion of HBsAg and HBeAg. The cytotoxicity of each
compound was expressed as the concentration of compound
required to kill 50% (CC50) of the HepG 2.2.15 cells. The selectivity
index (SI), a major pharmaceutical parameter that estimates
possible future clinical development, was determined as the ratio of
CC50 to IC50. The results were summarized in Tables 1e3.
Fig. 1. Structures of compounds 1 and 2.
(IC50 ¼ 0.045 mM) in vitro. Preliminarily, the anti-HBV mechanism
study proposed that compound 44 could influence the HBV repli-
cation cycle by enhancing the transcript activity of HBV ENI
(enhancer I), EN-II(enhancer II) on HepG 2 cells.
2. Chemistry
As illustrated in Scheme 1, 6-chloro-4-(2-chlorophenyl)-3-(2-
hydroxyethyl) quinolin-2(1H)-one 2 was conveniently synthesized
from commercially available 2-amino-benzophenone with g-valer-
Compound 2 showed inhibitory potency to the secretion of
HBsAg (IC50 ¼ 0.086 mM) and HBeAg (IC50 ¼ 0.36 mM), but
appeared toxic (CC50 ¼ 0.38 mM), which led to relatively low SI
values (SIHBsAg ¼ 4.4, SIHBeAg ¼ 1.0). The effects of introducing
different patterns of substituents to the N-1 position of compound
2 were studied by replacement of N-1 position with methyl, alkyl,
benzyl, or 2,6-difluorobenzyl groups to yield the derivatives 8e11.
As shown in Table 1, compounds 8e10 enhanced anti-HBV activities
on the secretion of HBsAg and HBeAg, whereas increased the
cytotoxicities, resulting in relatively small SI values compared with
compound 2. Introducing 2,6-difluorobenzyl to the N-1 position of
the 6-chloro-4-(2-chlorophenyl)-3-(2-hydroxyethyl) quinolin-2
(1H)-one core (11) retained potency to the secretion of HBsAg and
led to the decreased cytotoxicity (CC50 > 2.3 mM). However,
compound 11 lost suppressant property on the secretion of HBeAg.
As shown in Table 2, compound 2 was oxidated to aldehyde 12
which retained the activity against secretion of HBsAg and lost the
suppressant property on the secretion of HBeAg. Further oxidation
of aldehyde 12 gave carboxylic acid 13, leading to decreased inhi-
bition to the secretion of HBsAg (IC50 ¼ 0.35 mM) relative to
compound 2 (IC50 ¼ 0.086 mM). Compounds 14e15 lost the
activities against the secretion of HBsAg and HBeAg.
olaction using lithium bis(trimethylsily)amide (LiHMDS) solution in
THF in good yield followed by a tandem amidation/Knoevengel
condensation [17].
The modification of compound 2 performed on N-1 was
summarized in Scheme 2. Treatment of compound 2 with tert-butyl
diphenylchlorosilane (TBDPSCl) in the presence of N-ethyl-
diisopropylamine (DIPEA) in CH2Cl2 afforded compound 3 with
protection of hydroxyl group [18], followed by alkylation with
appropriate alkyl halides in the existence of potassium carbonate in
acetone to produce the N-alkylation derivatives 4e7 [19]. The
remaining O-protecting groups were removed in the presence of
tetra-butylammoniumfluoride (TBAF) in THF to provide the target
compounds 8e11 [20].
Oxidation of compound 2 with Dess-Martin periodinane (DMP)
in CH2Cl2 afforded aldehyde 12 which was treated with NaClO2,
NaH2PO4, and 2-methyl-2-butene in tert-butyl alcohol (t-BuOH)/
H2O (1.5/1) at room temperature to yield carboxylic acid 13 [21].
The presence of free hydroxyl group allowed us to prepare ester
derivatives of compound 2 in order to evaluate the influence of
ester side chain on their anti-HBV activities. Acylation of compound
2 with various carboxylic acids, anhydrides, and acyl chlorides
afforded products 16e53 (Scheme 3).
The subseries of derivatives 16e41 has different patterns of
substitution on hydroxyl group. These compounds generally
exhibited good potency to the secretion of HBsAg, except that
compounds 31, 34, and 36 lost suppressant potency to the secretion
of HBsAg. This result indicated that a range of acyls with different
lipophilic, electronic, and steric characters was tolerated at
hydroxyethyl moiety for their suppressant potency to the secretion
of HBsAg. Compounds 17, 26, 28, 29, 35, 37, 40, and 41 also showed
activity to the secretion of HBeAg. It is worth noting that compound
41 exhibited 2.9-fold and 6.4-fold increased activities against the
secretion of HBsAg (IC50 ¼ 0.030 mM vs IC50 ¼ 0.086 mM) and
HBeAg (IC50 ¼ 0.056 mM vs IC50 ¼ 0.36 mM) comparing with
compound 2, which suggested that cinnamylation of hydroxyl
seemed important for its high potency against the secretion of
HBsAg and HBeAg. Thus, compound 41 was selected as the
benchmark compound for subsequent optimization.
Bioisosteric replacement is a valuable approach in drug design
and can produce compounds with similar biological activity. In order
to convert hydroxyethyl group to aminoethyl moiety, reaction of
compound 2 with methanesulfonyl chloride in the presence of trie-
thylamine in CH2Cl2 did not give methanesulfonylate intermidatebut
O
H
NH
N
O
O
O
Cl
Cl
OH
a
Cl
Cl
2
To extend this result, different patterns of substitutions were
introduced to the phenyl ring of ester moiety. As shown in Table 3,
Scheme 1. Synthesis of compound 2.

R.-H. Guo et al. / European Journal of Medicinal Chemistry 46 (2011) 307e319
309
R
R
N
H
N
N
O
O
O
c
b
Cl
OTBDPS
Cl
OH
Cl
OR
Cl
Cl
Cl
2
R = H
8 R = CH
9 R = allyl
10 R = benzyl
11 R = 2,6-difluorobenzyl
4 R = CH
5 R = allyl
6 R = benzyl
a
3
R = TBDPS
7 R = 2,6-difluorobenzyl
Scheme 2. Synthesis of compounds 3e11. Reagents and conditions: (a) TBDPSCl, DIPEA, CH2Cl2, r.t.; (b)MeI or R1Br, K2CO3, acetone, r.t.; (c) TBAF, THF, r.t.
compounds 42e49 demonstrated potent activities against the
secretion of HBsAg and HBeAg, and low cytotoxicities, which
indicated that different types of substituents could be present at
positions 2(OCH3), 3(OCH3, F, CF3), 4(OCH3, CF3), and 3,4(methyl-
enedioxyl, dimethoxyl) without substantially affecting potency to
inhibit the secretion of HBsAg and HBeAg. These changes were
typified by the 30-methoxyl substituted compound 44 with high
potency against the secretion of HBsAg (IC50 ¼ 0.010 mM), HBeAg
(IC50 ¼ 0.026 mM), low cytotoxicity (CC50 > 1.3 mM), resulting in
remarkable SI values (SIHBsAg > 135, SIHBsAg > 51). It was observed
that the addition of a nitrogen to 30-position of the phenyl ring, i.e.,
on moving from compound 41 to pyridine 50, retained suppressant
potency to the secretion of HBsAg (IC50 ¼ 0.034 mM) and led to the
loss of activity against the secretion of HBeAg. However, it also
appeared toxic (CC50 ¼ 0.039 mM), resulting in a relatively low SI
value (SIHBsAg ¼ 1.1). Replacement of phenyl moiety with methyl
group afforded compound 55, which showed high potency to the
secretion of HBsAg (IC50 0.025 mM) and HBeAg
¼
(IC50 ¼ 0.022 mM) but high cytotoxicity (CC50 ¼ 0.030 mM).
Addition of a methyl group to 20-position of compound 51 resulted
in compound 53, which demonstrated low cytotoxicity (CC50 ¼ 0.
35 mM) and slightly decreased activities against the secretion of
HBsAg (IC50 ¼ 0.058 mM) and HBeAg (IC50 ¼ 0.062 mM) comparing
with compound 51. The increase of the number of C]C bond
compound 52 showed similar activity and lower cytotoxicity
(CC50 ¼ 0.44 mM) relative to compound 51.
To identify the optimal chain length between the 6-chloro-4-
(2-chlorophenyl)quinolin-2(1H)-one and ester moieties, different
alkyl linkers of variable length were introduced between the
6-chloro-4-(2-chlorophenyl) quinolin-2(1H)-one and ester, prod-
ucing the three derivatives 44, 56, and 57. Compounds 44, 56, and 57
showed similar activities against the secretion of HBsAg (IC50
¼
0.010e0.020 mM) and HBeAg (IC50 ¼ 0.018e0.028 mM) and cyto-
toxicities (CC50 > 1.1 mM), which suggested that alkyl linkers of
length (n ¼ 1, 2, 3) had little effect on activities against the secretion
of HBsAg and HBeAg.
H
N
H
N
O
O
b
COOH
CHO
Cl
Importantly, the most active compounds 42e49, 56, and 57 with
high activities against HBsAg and HBeAg were selected to investigate
inhibition of HBV DNA replication using lamivudine as the reference
drug. Compounds 42, 44, and 46 exhibited anti-HBV activity with
their IC50 values against HBV DNA replication of 0.11, 0.045, 0.41 mM,
respectively. This result suggested that mono-substituted at 30-
position phenyl ring was an important feature in conferring potent
activity against HBV DNA replication. For different chain length
between 6-chloro-4-(2-chlorophenyl)quinolin-2(1H)-one and ester
moieties compounds 44, 56, and 57, the ethylene analogue 44
(IC50 ¼ 0.045 mM) showed about four times more potent than that of
the corresponding methane analogue 56 (IC50 ¼ 0.16 mM), whereas
propylene analogue 57 lost inhibitory activity. These findings
proposed that ethylene linker between 6-chloro-4-(2-chlorophenyl)
quinolin-2(1H)-one and ester moieties was preferred for potent anti-
HBV DNA activity.
Cl
Cl
Cl
12
13
a
H
N
N
O
O
c
Cl
Cl
OH
Cl
Cl
2
14
d
e
The activity for compound 44 (0.02 mM) in the luciferase
reporter assay [22e24] is shown in Fig. 2 (Cells viability were 95.25%
and 80.40% by MTT assay at 48 and 72 h, respectively). Compound
44 could enhance the transcript activity of HBV enhancers ENI up to
12-fold and ENII up to 14-fold, as well as core/pregenomic, X, preS1
promoter, approximately 4-fold more potent compared with mock-
treated control, however, slightly inhibit preS2 promoter activity.
N
S
H
N
Cl
O
O
Cl
Cl
O
R
Cl
15
4. Conclusions
16-53
Scheme 3. Synthesis of compounds 12e53. Reagents and conditions: (a) DMP, CH2Cl2,
r.t.; (b) NaClO2,NaH2PO4, tBuOH, H2O, r.t.; (c) methanesulfonyl chloride, triethylamine,
CH2Cl2, 0ꢀC; (d) LR, toluene,reflux; (e) R2COOH, DMAP, DCC, CH2Cl2; or (R2CO)2O,
DMAP (cat) , pyridine, 95ꢀC; or R2COCl,pyridine, r.t.
In summary, a series of novel 6-chloro-4-(2-chlorophenyl)-3-(2-
hydroxyethyl) quinolin-2(1H)-one analogues based on compound 2
were synthesized and tested for their in vitro anti-HBV activity. Most

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R.-H. Guo et al. / European Journal of Medicinal Chemistry 46 (2011) 307e319
NH
H
N
H
N
O
O
O
Cl
OH
n
a
b
Cl
O
n
OMe
Cl
Cl
Cl
Cl
+
O
O
O
O
54 n = 1
55 n = 3
56 n = 1
57 n = 3
or
Scheme 4. Synthesis of compounds 54e57. Reagents and conditions: (a) LiHMDS, THF, 0ꢀC; (b) 3-Methoxycinnanic acid, DCC, DMAP, CH2Cl2, r.t.
of the compounds exhibited suppressant property on the secretion
of HBsAg. Interestingly, compounds 42, 44, 46, 48, and 56 displayed
optimal profiles inhibiting not only HBsAg and HBeAg secretion but
also HBV DNA replication, however, 3TC showed significantly
activity against HBV DNA replication. Preliminary mechanism study
suggested that compound 44 mainly promote the transcript activity
of HBV enhancers ENI and ENII to exert anti-HBV action.
chemical shifts were given in d with TMS as internal reference;
column chromatography (CC): silica gel (200e300 mesh; Qingdao
Makall Group Co., Ltd; Qingdao; China); All reactions were moni-
tored using thin-layer chromatography (TLC) on silica gel plates.
5.1.1. 6-Chloro-4-(2-chlorophenyl)-3-(2-hydroxyethyl)quinolin-2
(1H)-one (2) [12]
A
100 mL, 2-neck flask was charged with 2-amino-20,5-
dichlorobenzo-phenone (3.76 mmol, 1.0 g) and THF (20 mL). The
resulting solution was cooled to 0 ꢀC and LiHMDS 28.2 mL (1 M in
THF) was added over 15 min. The internal temperature was
5. Experimental
5.1. Chemistry
controlled 0 ꢀC for 10 min, then
g-valerolactone (16.9 mmol,1.7 mg)
was added over 10 min. The reaction solution was allowed to warm
up to room temperature and stirred at room temperature for 2 h.
Then water (10.2 mL) was added over 10 min, and the reaction
mixture was stirred at room temperature for 24 h. The aqueous
layer was extracted with ethyl acetate (100 mL) and the organic
solution was washed with water (80 mL) and brine (3 ꢁ 50 mL) and
dried anhydrous Na2SO4. Removal of the solvents gave a residue
which was purified by column chromatography on silica gel with
CHCl3/CH3OH (98:2) to give white amorphous powder 2, yield 86%;
MS spectra were run on a VG Auto Spec-3000 spectrometer (VG,
Manchester, England); NMR spectra were recorded on Bruker AM
400 (1H/13C, 400 MHz/100 MHz) or DRX-500 (1H/13C, 500 MHz/
125 MHz) spectrometer (Bruker, Bremerhaven, Germany) and
Table 1
Anti-HBV activity, cytotoxicity, and selectivity index of analogues (2, 8e11) in vitro.a
R1
1H NMR (DMSO-d6, 400 MHz)
d
12.16 (1H, br.s), 7.68 (1H, d, J ¼ 1.6,
N
O
5.2 Hz), 7.59e7.49 (3H, m), 7.41e7.36 (2H, m), 6.56 (1H, s), 4.61 (1H,
br.s), 3.43 (2H, t, J ¼ 7.2 Hz), 2.56e2.48 (1H, m), 2.31e2.29 (1H, m);
Cl
OH
13C NMR (DMSO-d6, 100 MHz)
d 161.6, 144.5, 136.4, 134.1, 131.9,
Cl
131.2, 130.7, 130.5, 129.8, 129.7, 127.9, 125.9, 124.3, 120.4, 117.4, 58.8,
32.3; ESIMS: m/z 334 [M þ H]þ, HRESIMS: calc for C17H14NO2Cl2
[M þ H]þ 334.0401, found 334.0409.
Compd
R1
CC50b (mM)
HBsAgc
HBeAgd
5.1.2. 3-[2-(tert-Butyldiphenylsilyloxy)ethyl]-6-chloro-4-(2-
chlorophenyl)quinolin-2(1H)-one (3) [18]
IC50e(mM)
SIf
4.4
IC50 (mM)
SI
Compound 2 (909 mg, 2.7 mmol) was dissolved in 15 mL of DMF,
stirred and cooled to 0 ꢀC in an ice bath. DIPEA and TBDPSCl (1.5 g,
5.4 mmol) were added to the solution and the resulting mixture
was stirred for 12 h at room temperature. The mixture was
quenched with water. The mixture was extracted with EtOAc. The
organic layer was washed with 5% HCl, brine, dried over MgSO4,
filtered and concentrated. Purification by chromatography on silica
gel (elution with CHCl3/MeOH, 70:1) afforded the white amorphous
2
8
H
CH3
0.38
0.034
0.086
0.022
0.36
0.034
1.0
1.0
1.6
9
0.023
0.026
0.010
2.3
0.012
0.012
1.9
10
0.012
2.2
2.2
power, yield 86%; 1H NMR (CDCl3, 400 MHz)
d 13.1 (1H, br.s), 7.57
F
(5H, m), 7.50e7.18 (11H, m), 6.85 (1H, s), 4.10e4.04 (1H, m),
3.98e3.92 (1H, m), 3.07e3.00 (1H, m), 2.79e2.72 (1H, m), 1.04 (9H,
11
>2.3
0.087
27
>2.6
>2.3
e
s); 13C NMR (CDCl3, 125 MHz)
d 164.1, 146.6, 135.9, 135.42(2C),
F
135.40 (2C), 134.4, 133.9 (2C), 133.8, 132.9, 131.1, 130.0, 129.99 (2C),
129.96 (2C), 129.4, 127.8, 127.7, 127.51(2C), 127.49 (2C), 127.1, 125.3,
117.5, 62.1, 32.0, 27.0 (3C), 19.3; ESIMS: m/z 572 [M þ H]þ, HRESIMS:
calc for C33H32NO2SiCl2 [M þ H]þ 572.1579, found 572.1585.
3TCg
e
30
1.1
30
1.0
a
All values are the mean of two independent experiments, standard deviation
values <10% in all cases.
b
CC50: 50% cytotoxic concentration.
HBsAg: HBV surface antigen.
HBeAg: HBV e antigen.
IC50: 50% inhibitory concentration.
c
5.1.3. General procedure for the preparation of intermediates 4e7
[19]
To a solution of the compound 2 in acetone were added
K2CO3 (1.5 equiv mol), and alkyl halides (1.5 equiv mol) at room
d
e
f
SI (selectivity index) ¼ CC50/IC50
.
g
3TC: Lamivudine, an antiviral agent used as positive control.

R.-H. Guo et al. / European Journal of Medicinal Chemistry 46 (2011) 307e319
311
Table 2
Anti-HBV activity, cytotoxicity, and selectivity index of analogues (12e41) in vitro.a
H
N
H
O
N
N
N
O
O
S
O
R2
Cl
O
R2
Cl
Cl
Cl
Cl
Cl
Cl
Cl
16 - 41
15
14
12, 13
Compd
R2
CC50b (mM)
HBsAgc
HBeAgd
IC50e(mM)
SIf
4.4
1.6
2.8
IC50 (mM)
SI
1.0
<1
<1
e
2
e
0.38
0.12
0.95
>0.28
>2.4
0.027
0.086
0.076
0.35
>0.28
>2.4
0.36
>0.12
>0.95
>0.28
>2.4
12
13
14
15
16
CHO
COOH
e
e
e
e
e
CH3
0.074
<1
>0.027
<1
17
0.095
0.059
0.037
1.6
0.062
1.5
18
0.062
1.7
>0.062
<1
19
20
21
22
23
0.052
0.20
0.035
0.082
0.040
0.15
1.5
2.4
1.9
>0.052
>0.20
>0.078
>0.11
>0.10
<1
<1
<1
<1
<1
0.078
0.11
COOH
CN
<1
0.10
0.075
1.3
1.4
1.4
O
24
25
0.074
0.13
0.032
0.095
>0.074
>0.13
<1
<1
O
O
O
26
27
0.10
0.11
0.057
0.074
1.8
1.4
0.071
1.4
H
N
>0.11
<1
O
28
29
0.046
0.038
0.046
0.027
1.0
1.4
0.046
0.034
1.0
1.1
1.5
30
0.16
0.085
1.9
0.11
(continued on next page)

312
R.-H. Guo et al. / European Journal of Medicinal Chemistry 46 (2011) 307e319
Table 2 (continued )
R2
CC50 (mM)
HBsAgc
HBeAgd
b
Compd
IC50e(mM)
SIf
IC50 (mM)
SI
O
O
31
>1.8
1.5
>1.2
>1.8
e
O
N
C
32
33
34
0.082
0.043
1.6
0.051
0.032
1.2
1.6
>0.082
>0.043
1.6
<1
<1
1.42
NO2
1.4
3.0
1.0
N
35
36
37
0. 17
>1.7
0.055
>1.7
0.055
>1.7
3.0
S
O
e
e
0.098
0.051
1.9
5.7
0.058
1.6
O
N
38
0.96
0.17
>0.96
<1
<1
O
O
39
40
41
0.081
0.046
0.056
0.033
1.5
1.4
>0.081
O
0.035
1.3
0.23
30
0.030
27
7.6
1.1
0.056
30
4.0
1.0
3TC
e
a
All values are the mean of two independent experiments, standard deviation values <10% in all cases.
b
c
d
e
f
CC50: 50% cytotoxic concentration.
HBsAg: HBV surface antigen.
HBeAg: HBV e antigen.
IC50: 50% inhibitory concentration.
SI (selectivity index) ¼ CC50/IC50
.
temperature. The resulting mixture was stirred at room tempera-
ture until the starting material disappeared on the TLC. The reaction
mixture was filtered, concentrated under reduced pressure to
obtain the intermediates (4e7), which were used in the next step
without further purification.
5.1.4.1. 6-Chloro-4-(2-chlorophenyl)-3-(2-hydroxyethyl)-1-methyl-
quinolin-2(1H)-one (8). White amorphous power, two step yield
78% (after chromatography with petroleum ether/acetone, 90:10);
1H NMR (CDCl3, 500 MHz)
d
7.56 (1H, d, J ¼ 9.0 Hz), 7.57 (5H, m),
6.87 (1H, d, J ¼ 2.0 Hz), 3.82e3.76 (3H, m), 3.69e3.67 (2H, m),
2.82e2.80 (1H, m), 2.61e2.57 (1H, m); 13C NMR (CDCl3, 125 MHz)
5.1.4. General procedure for the preparation of derivatives 8e11
[20]
d 163.6, 144.6, 137.2, 134.2, 131.5, 131.0, 130.6, 130.3, 130.2, 130.1,
128.2, 127.4, 126.5, 122.0, 115.7, 62.4, 32.8, 30.4; ESIMS: m/z 348
[M þ H]þ, HRESIMS: calc for C18H16NO2Cl2 [M þ H]þ 348.0558,
found 348.0561.
To a solution of compounds (4e7) in THF at room temperature
was added TBAF (2 equiv mol) in one portion. After stirring at room
temperature for 12 h, the solvent was evaporated under reduced
pressure and the residue was purified by chromatography to give
the products (8e11).
5.1.4.2. 1-Allyl-6-chloro-4-(2-chlorophenyl)-3-(2-hydroxyethyl)qui-
nolin-2(1H)-one (9). White amorphous power, two step yield 61%

R.-H. Guo et al. / European Journal of Medicinal Chemistry 46 (2011) 307e319
313
Table 3
Anti-HBV activity, cytotoxicity, and selectivity index of analogues (41e53, 56 and 57) in vitro.a
H
H
N
N
O
O
O
2'
R
O
3'
4'
Cl
O
Cl
O
R
Cl
Cl
50-52
41-49
H
H
N
N
O
O
4'
3'
OCH3
O
O
Cl
O
Cl
n
2'
Cl
Cl
O
53
56, 57
0
0
0
Compd
n
R2
R3
R4
R
CC50b (mM)
HBsAgc
HBeAgd
HBV DNA
IC50e(mM)
SIf
7.6
>48
13
>135
>2.5
>24
>6.1
>8.1
>58
IC50 (mM)
SI
IC50 (mM)
g
41
42
43
44
45
46
47
48
49
e
e
e
e
e
e
e
e
e
H
H
OCH3
H
H
H
H
H
H
H
F
H
OCH3
H
CF3
H
H
H
H
OCH3
H
e
e
e
e
e
e
e
e
e
0.23
>1.6
0.77
>1.3
>1.2
>1.4
>1.4
e
0.030
0.033
0.061
0.010
0.49
0.058
0. 23
0.15
0.056
0.027
0.024
0.026
0.048
0.022
0.019
0.22
4.0
>58
32
>51
>25
>63
>73
>5.4
>21
NDh
0.11
NAi
0.045
NA
0.41
NA
0.16
NA
H
CF3
OCH2O
OCH3
OCH3
>1.2
0.021
0.057
N
50
e
e
e
e
0.039
0.034
1.1
2.2
<1
ND
51
52
e
e
e
e
e
e
e
e
CH3
0.030
0.44
0.025
0.023
1.2
19
0.022
0.026
1.3
17
ND
ND
53
56
57
3TC
e
1
3
e
e
e
e
e
e
e
e
e
e
e
e
0.35
>1.8
>1.1
30
0.058
0.011
0.020
27
6.1
0.062
0.018
0.028
30
5.6
ND
0.16
NA
>170
>55
1.1
>100
>39
1.0
0.0012
a
All values are the mean of two independent experiments, standard deviation values <10% in all cases.
b
c
d
e
f
CC50: 50% cytotoxic concentration.
HBsAg: HBV surface antigen.
HBeAg: HBV e antigen.
IC50: 50% inhibitory concentration.
SI (selectivity index) ¼ CC50/IC50
.
g
h
i
Concentrations of compounds achieving 50% inhibition of cytoplasmic HBV DNA replication.
Not determined.
Not active.
(after chromatography with CHCl3/MeOH, 70:1); 1H NMR (CDCl3,
2.91e2.85 (1H, m), 2.70e2.61 (1H, m); 13C NMR (CDCl3, 100 MHz)
163.8, 145.1, 136.5, 135.6, 134.2, 132.9, 131.4, 130.5, 130.3, 130.2
(2C), 128.9 (2C), 128.2, 127.5, 127.4, 126.4 (2C), 126.3, 122.2, 116.5,
62.4, 47.0, 32.8; ESIMS: m/z 424 [M þ H]þ, HRESIMS: calc for
C24H20NO2Cl2 [M þ H]þ 424.0871, found 424.0876.
400 MHz)
d
7.56 (1H, dd, J ¼ 7.6, 2.0 Hz), 7.47e7.31 (4H, m),
d
7.26e7.20 (1H, m), 6.87 (1H, d, J ¼ 2.4 Hz), 6.03e5.94 (1H, m),
5.29e4.99 (4H, m), 3.79e3.73 (1H, m), 3.68e3.63 (1H, m),
2.84e2.78 (1H, m), 2.61e2.55 (1H, m); 13C NMR (CDCl3, 100 MHz)
d
163.1, 144.9, 136.4, 134.2, 132.9, 131.3, 131.2, 130.5, 130.3, 130.1,
130.0, 128.1, 127.3, 126.3, 122.0, 117.6, 116.3, 62.2, 45.6, 32.8; ESIMS:
m/z 396 [M þ Na]þ, HRESIMS: calc for C20H18NO2Cl2 [M þ H]þ
374.0714, found 374.0705.
5.1.4.4. 1-(2,6-Difluorobenzyl)-6-chloro-4-(2-chlorophenyl)-3-(2-
hydroxyethyl)quinolin-2(1H)-one (11). White amorphous power,
two step yield 75% (after chromatography with CHCl3/MeOH, 97:3);
1H NMR (CDCl3, 400 MHz)
d
7.57 (1H, dd, J ¼ 8.8, 1.6 Hz), 7.49e7.23
5.1.4.3. 1-Benzyl-6-chloro-4-(2-chlorophenyl)-3-(2-hydroxyethyl)
quinolin-2(1H)-one (10). White amorphous power, two step yield
72% (after chromatography with CHCl3/MeOH, 97:3); 1H NMR
(6H, m), 6.91e6.86 (3H, m), 5.85 (1H, d, J ¼ 16.0 Hz), 5.72 (1H, d,
J ¼ 16.0 Hz), 3.70 (2H, m), 2.89e2.83 (1H, m), 2.65e2.59 (1H, m); 13C
NMR (CDCl3,100 MHz) d 163.5,162.4,160.0,144.9,136.1,134.2,132.9,
(CDCl3, 400 MHz)
d
7.59 (1H, dd, J ¼ 7.2, 2.0 Hz), 7.50e7.41 (2H, m),
131.2,130.5,130.2,130.1,130.0,129.6,128.1,127.3,126.4,122.3,115.5,
111.9, 111.8, 111.6, 62.2, 36.0, 32.8; ESIMS: m/z 482 [M þ Na]þ, HRE-
SIMS: calc for C24H18NO2F2Cl2 [M þ H]þ 460.0682, found 460.0679.
7.38e7.23 (8H, m), 6.88 (1H, d, J ¼ 2.4 Hz), 5.70 (1H, d, J ¼ 15.6 Hz),
5.56 (1H, d, J ¼ 15.6 Hz), 3.85e3.89 (1H, m), 3.74e3.68 (1H, m),

314
R.-H. Guo et al. / European Journal of Medicinal Chemistry 46 (2011) 307e319
dd, J ¼ 7.2, 2.4 Hz), 7.11 (1H, d, J ¼ 2.0 Hz), 4.58 (2H, m), 3.11 (1H, m),
2.95 (1H, m); 13C NMR (CDCl3, 100 MHz)
167.2, 145.6, 141.6, 133.8,
d
132.7, 130.4, 130.2, 130.1, 129.6, 129.1, 127.2, 125.1, 124.0, 122.1, 120.7,
69.2, 27.3; ESIMS: m/z 316 [M þ H]þ, HRESIMS: calc for C17H12NOCl2
[M þ H]þ 316.0295, found 316.0304.
5.1.8. 6-Chloro-4-(2-chlorophenyl)-2,3-dihydrothieno[2,3-b]
quinoline (15) [14]
A solution of compound 2 (50 mg, 0.15 mmol), Lawesson’s
reagent (121 mg, 0.3 mmol) in toluene (5 mL) was heated at
refluxed temperature for 2 h. The reaction mixture was concen-
trated under reduced pressure to give a residue which was purified
by silica gel column chromatography with petroleum ether/acetone
(100:1) to afford compound 15 (39 mg), yield 81%. 1H NMR (CDCl3,
Fig. 2. Normalized fold change in HBV promoter activity. Normalized fold change in
HBV four promoters C: the core promoter, X: X promote, S1: S1 promoter and S2: S2
promoter, two enhancer elements (En I: enhancer I, En II: enhancer II) activity. HepG 2
cells were transfected with a constant amount of pGL3 Vector (expressing firefly
luciferase) as Basic Control, phRL-CMV vector (expressing Renilla luciferase) as internal
control. Firefly and Renilla luciferase activities were assayed using the Dual-LuciferaseÒ
Reporter Assay System. Data represent the average ꢂ standard deviation of triplicate
samples. Normalized fold change in activity between test groups: Fold
activity ¼ compound 44 (0.02 mM) treated group promoter activity/mock-treated
group promoter activity.
500 MHz)
J ¼ 7.3, 1.8 Hz), 7.17 (1H, d, J ¼ 2.2 Hz), 3.48e3.40 (2H, m), 3.25e3.19
(1H, m), 3.13e3.06 (1H, m); 13C NMR (CDCl3,100 MHz)
168.1,146.0,
d
7.90 (1H, d, J ¼ 8.8 Hz), 7.59e7.42 (4H, m), 7.23 (1H, dd,
d
138.9, 134.5, 133.3, 132.9, 131.3, 130.4, 130.3, 130.2, 130.0, 128.9,
127.3, 125.5, 124.2, 32.5, 30.6; ESIMS: m/z 332 [M þ H]þ, HRESIMS:
calc for C17H12NSCl2 [M þ H]þ 332.0067, found 332.0060.
5.1.9. General procedure for the preparation of derivatives 16, 18, 22
and 26
To a solution of compound 2, 4-dimethylaminopyridine (DMAP,
0.2 equiv mol), and an appropriate anhydride (1.2 equiv mol) in
anhydrous pyridine (5e10 mL), was heated overnight at 95 ꢀC until
the starting material was not observed by TLC. The reaction mixture
was diluted with 50 mL of EtOAc and washed three times with
50 mL of 5% HCl and and saturated NaHCO3 (3 ꢁ 30 mL). The
organic layer was dried over anhydrous Na2SO4 and concentrated
under reduced pressure. The residue was chromatographed using
a silica gel column to afford the products.
5.1.5. 2-[6-Chloro-4-(2-chlorophenyl)-2-oxo-1,2-dihydroquinolin-
3-yl]acetaldehyde (12)
To a solution of compound 2 (50 mg, 0.15 mmol) in dry CH2Cl2
(10 mL) was added Dess-Martin periodinane (DMP, 0.3 mmol, 15 wt
% in CH2Cl2) at room temperature. After 2.5 h, the reaction mixture
was quenched with a 1:1 mixture of saturated Na2S2O3 (50 mL) and
NaHCO3 (50 mL). The resulting mixture was extracted with CH2Cl2.
The CH2Cl2 layer was dried over anhydrous Na2SO4 and concen-
trated under reduced pressure, and chromatographed on silica gel
eluting with petroleum ether/acetone (4:1) to give 60% yield of
5.1.9.1. 2-[6-Chloro-4-(2-chlorophenyl)-2-oxo-1,2-dihydroquinolin-
3-yl]ethylacetate (16). White amorphous power, yield 95% (after
chromatography with CHCl3/MeOH, 98:2); 1H NMR (CDCl3,
compound 12 (30 mg). 1H NMR (CDCl3, 500 MHz)
d 9.73 (1H, s), 7.59
(1H, d, J ¼ 8.0 Hz), 7.51e7.41 (4H, m), 7.22 (1H, dd, J ¼ 7.4, 1.5 Hz),
6.92 (1H, d, J ¼ 2.1 Hz), 3.70 (1H, d, J ¼ 17.2 Hz), 3.32 (1H, d,
500 MHz)
d
13.13 (1H, br.s), 7.59 (1H, dd, J ¼ 9.5, 2.0 Hz), 7.46 (4H,
J ¼ 17.2 Hz); 13C NMR (CDCl3, 125 MHz)
d
198.0, 163.4, 148.2, 136.1,
m), 7.25 (1H, m), 6.86 (1H, d, J ¼ 1.8 Hz), 4.30 (2H, t, J ¼ 6.8 Hz), 2.95
133.7, 132.6, 130.9, 130.7, 130.4, 130.2, 128.4, 127.5, 125.70, 125.66,
121.0, 117.8, 43.0; ESIMS: m/z 332 [M þ H]þ, HRESIMS: calc for
C17H12NO2Cl2 [M þ H]þ 332.0245, found 332.0253.
(1H, m), 2.67 (1H, m), 1.97 (3H, s); 13C NMR (CDCl3, 125 MHz)
d
170.8, 164.0, 147.1, 136.0, 134.2, 133.0, 130.7, 130.4, 130.3, 130.1,
129.1,128.1, 127.2, 125.5, 121.1,117.6, 62.3, 28.1, 21.1; FABMS: m/z 376
[M þ H]þ, HRESIMS: calc for C19H16NO3Cl2 [M þ H]þ 376.0507,
found 376.0497.
5.1.6. 2-[6-Chloro-4-(2-chlorophenyl)-2-oxo-1,2-dihydroquinolin-
3-yl]acetic acid (13)
Compound 13 was prepared according to the literature [18] as
5.1.9.2. 2-[6-Chloro-4-(2-chlorophenyl)-2-oxo-1,2-dihydroquinolin-
3-yl]ethylisobutyrate (18). White amorphous power, yield 70%
(after chromatography with CHCl3/MeOH, 98:2); 1H NMR (CDCl3,
white amorphous power, yield 72%. 1H NMR (C5D5N, 400 MHz)
d
8.71 (1H, s), 7.74e7.62 (5H, m), 7.09 (1H, d, J ¼ 2.2 Hz), 4.17 (1H, d,
J ¼ 16.3 Hz), 3.65 (1H, d, J ¼ 16.3 Hz); 13C NMR (C5D5N, 100 MHz)
500 MHz)
d
7.58 (1H, d, J ¼ 7.6 Hz), 7.51e7.43 (4H, m), 7.25 (1H, dd,
d
173.7, 163.2, 146.3, 138.0, 135.1, 133.3, 131.7, 131.4, 130.82, 130.78,
J ¼ 1.7, 6.4 Hz), 6.86 (1H, d, J ¼ 2.7 Hz), 4.31 (2H, t, J ¼ 7.2 Hz),
129.8, 128.5, 127.7, 126.0, 121.7, 118.4, 35.7; EIMS: m/z 347 [M]þ,
HRESIMS: calc for C17H12NO3Cl2 [M þ H]þ 348.0194, found
348.0195.
2.99e2.94 (1H, m), 2.71e2.65 (1H, m), 2.49e2.44 (1H, m), 1.83 (6H,
m); 13C NMR (CDCl3, 100 MHz)
d 176.9, 163.9, 147.0, 136.0, 134.2,
132.9, 130.7, 130.4, 130.3, 130.1, 129.1, 128.0, 127.2, 125.4, 121.1, 117.7,
62.1, 33.8, 28.2, 18.9 (2C); ESIMS: m/z 426 [M þ Na]þ, HRESIMS: calc
for C21H19NO3Cl2Na [M þ Na]þ 426.0627, found 426.0639.
5.1.7. 6-Chloro-4-(2-chlorophenyl)-2,3-dihydrofuro[2,3-b]
quinoline (14)
Methanesulfonyl chloride (372 mg, 3.3 mmol) was added
dropwise to a solution of compound 2 (300 mg, 0.9 mmol) and Et3N
(109 mg, 1.1 mmol) in CH2Cl2 (10 mL) at 0 ꢀC. The reaction was
warmed to room temperature and stirred 4 h. The reaction was
then quenched with 5% HCl and extracted with EtOAc. The
combined extracts were washed with brine, dried with Na2SO4, and
concentrated under reduced pressure. Chromatography of residue
on silica gel (petroleum ether/acetone, 20:1) give 124 mg (44%),
5.1.9.3. 4-{2-[6-Chloro-4-(2-chlorophenyl)-2-oxo-1,2-dihy-
droquinolin-3-yl]ethoxy}-4-oxobutanoic acid (22). White amor-
phous power, yield 68% (after chromatography with CHCl3/MeOH,
98:2), 1H NMR (CDCl3, 400 MHz)
d
7.58 (1H, d, J ¼ 7.6 Hz), 7.57e7.45
(4H, m), 7.23 (1H, d, J ¼ 6.8 Hz), 6.86 (1H, s), 4.22 (1H, t, J ¼ 6.0 Hz),
2.92e2.88 (1H, m), 2.66e2.63 (1H, m), 2.40e2.33 (4H, m); 13C NMR
(CDCl3, 100 MHz)
d 179.0, 172.9, 163.9, 147.6, 135.4, 133.9, 132.8,
130.6, 130.6, 130.5, 130.2, 128.9, 128.5, 127.3, 125.5, 121.2, 117.7, 62.3,
33.4, 33.2, 27.8, 18.9; ESIMS: m/z 470 [M þ Na]þ, HRESIMS: calc for
C22H20NO5Cl2 [M þ H]þ 448.0718, found 448.0731.
yellow amorphous power, 1H NMR (CDCl3, 400 MHz)
J ¼ 8.8 Hz), 7.47 (1H, dd, J ¼ 5.6, 1.6 Hz), 7.40e7.31 (3H, m), 7.72 (1H,
d 7.70 (1H, d,

R.-H. Guo et al. / European Journal of Medicinal Chemistry 46 (2011) 307e319
315
5.1.9.4. 2-[6-Chloro-4-(2-chlorophenyl)-2-oxo-1,2-dihydroquinolin-
3-yl]ethyl-2-acetoxyacetate (26). White amorphous power, yield
69% (after chromatography with CHCl3/MeOH, 98:2); 1H NMR
(2H, t, J ¼ 6.8 Hz), 1.27e1.23 (8H, m), 0.85 (3H, t, J ¼ 6.8 Hz); 13C
NMR (CDCl3,100 MHz) d 173.6,164.0,147.1,136.0,134.2,133.0,130.7,
130.4, 130.2, 130.1, 129.1, 128.1, 127.2, 125.5, 121.1, 117.7, 62.0, 34.3,
31.6, 29.1, 28.9, 28.2, 24.8, 22.5, 14.0; ESIMS: m/z 460 [M þ H]þ,
HRESIMS: calc for C25H28NO3Cl2 [M þ H]þ 460.1446, found
460.1447.
(CDCl3, 500 MHz)
(4H, m), 7.25 (1H, dd, J ¼ 7.0, 2.0 Hz), 6.87 (1H, s), 4.54 (2H, s), 4.40
(2H, t, J ¼ 7.0 Hz), 2.99e2.94 (1H, m), 2.76e2.70 (1H, m), 2.11 (3H, s);
d
13.00 (1H, br.s), 7.61 (1H, d, J ¼ 8.0 Hz), 7.51e7.46
13C NMR (CDCl3, 125 MHz)
d 170.2, 167.6, 163.7, 147.5, 135.9, 134.0,
132.9, 130.7, 130.5, 130.5, 130.2, 128.5, 128.3, 127.3, 125.6, 121.1, 117.7,
63.1, 60.6, 28.0, 20.4; ESIMS: m/z 434 [M þ H]þ, HRESIMS: calc for
C21H18NO5Cl2 [M þ H]þ 434.0562, found 434.0557.
5.1.10.5. 2-[6-Chloro-4-(2-chlorophenyl)-2-oxo-1,2-dihydroquinolin-
3-yl]ethylcyanoformate (23). White amorphous power, yield 59%
(after chromatography with CHCl3/MeOH, 98:2); 1H NMR (CDCl3,
400 MHz)
6.89 (1H, s), 4.45 (2H, m), 3.40 (2H, s), 2.98 (1H, m), 2.78 (1H, m);
13C NMR (CDCl3, 100 MHz)
163.8, 162.8, 147.6, 135.9, 133.9, 132.8,
130.8, 130.6, 130.5, 130.3, 128.4, 128.2, 127.5, 125.6, 121.0, 117.6, 64.7,
d
7.63 (1H, d, J ¼ 6.8 Hz), 7.53e7.47 (4H, m), 7.28 (1H, m),
5.1.10. General procedure for the preparation of derivatives 17,
19e21, 23e25, 27e53
d
To a solution of compound 2 in acetone, an appropriate carbox-
ylic acid (1.2 equiv mol), and 4-dimethylaminopyridine (DMAP,
0.2 equiv mol) in anhydrous CH2Cl2 was added N’,N’-dicyclohex-
ylcarbodiimide (DCC, 1.2 equiv mol) at 0 ꢀC. The resulting mixture
was stirred at room temperature overnight until the starting
material disappeared on the TLC. The reaction mixture was filtered
and the filtrate was concentrated under reduced pressure. The
residue was taken up in CH2Cl2 (3 ꢁ 50 mL). The CH2Cl2 solution was
washed with saturated NaHCO3 (3 ꢁ 30 mL). The organic layer was
dried over anhydrous Na2SO4, concentrated under reduced pres-
sure, and chromatographed over silica gel to afford the products.
27.8, 24.8; ESIMS: m/z 423 [M
þ
Na]þ, HRESIMS: calc for
C20H15N2O3Cl2 [M þ H]þ 401.0459, found 401.0464.
5.1.10.6. 2-[6-Chloro-4-(2-chlorophenyl)-2-oxo-1,2-dihydroquinolin-
3-yl]ethyl 2-methoxyacetate (24). White amorphous power, yield
90% (after chromatography with CHCl3/MeOH, 100:1); 1H NMR
(CDCl3, 400 MHz)
d
7.58 (1H, dd, J ¼ 7.6, 1.6 Hz), 7.48 (4H, m), 7.26
(1H, dd, J ¼ 7.2, 2.0 Hz), 6.86 (1H, d, J ¼ 2.1 Hz), 4.32 (2H, t,
J ¼ 6.8 Hz), 2.97 (1H, m), 2.69 (1H, m), 2.45 (2H, m), 1.07 (3H, t,
J ¼ 7.6 Hz); 13C NMR (CDCl3, 100 MHz)
d 174.2, 164.0, 147.0, 136.0,
134.1, 133.0, 130.7, 130.4, 130.3, 130.1, 129.1, 128.0, 127.1, 125.4, 121.0,
117.6, 62.1, 28.2, 28.2, 9.0; ESIMS: m/z 428 [M þ Na]þ, HRESIMS: calc
for C20H18NO4Cl2 [M þ H]þ 406.0612, found 406.0616.
5.1.10.1. 2-[6-Chloro-4-(2-chlorophenyl)-2-oxo-1,2-dihydroquinolin-
3-yl]ethylpropionate (17). White amorphous power, yield 90% (after
chromatography with CHCl3/MeOH, 98:2); 1H NMR (CDCl3,
400 MHz)
d
7.58 (1H, dd, J ¼ 7.6, 1.6 Hz), 7.48 (4H, m), 7.26 (1H, dd,
5.1.10.7. 2-[6-Chloro-4-(2-chlorophenyl)-2-oxo-1,2-dihydroquinolin-
3-yl]ethyl 2-ethoxyacetate (25). White amorphous power, yield 70%
(after chromatography with CHCl3/MeOH, 100:1), 1H NMR (CDCl3,
J ¼ 7.2, 2.0 Hz), 6.86 (1H, d, J ¼ 2.1 Hz), 4.32 (2H, t, J ¼ 6.8 Hz), 2.97
(1H, m), 2.69 (1H, m), 2.45 (2H, m), 1.07 (3H, t, J ¼ 7.6 Hz); 13C NMR
(CDCl3, 100 MHz)
d
174.2, 164.0, 147.0, 136.0, 134.1, 133.0, 130.7,
400 MHz)
d
13.11 (1H, br.s), 7.60 (1H, d, J ¼ 7.4 Hz), 7.48 (4H, m), 7.28
130.4, 130.3, 130.1, 129.1, 128.0, 127.1, 125.4, 121.0, 117.6, 62.1, 28.2,
(1H, dd, J ¼ 7.8, 2.0 Hz), 6.87 (1H, s), 4.41 (2H, t, J ¼ 6.6 Hz), 4.01 (2H,
27.4, 9.0; FABMS: m/z 390 [M
þ
H]þ, HRESIMS: calc for
s), 3.53 (2H, m), 2.86 (1H, m), 2.71 (1H, m), 1.20 (3H, t, J ¼ 6.8 Hz);
C20H18NO3Cl2 [M þ H]þ 390.0663, found 390.0657.
13C NMR (CDCl3, 100 MHz)
d 170.3, 163.9, 147.2, 136.0, 134.1, 132.9,
130.7, 130.5, 130.4, 130.2, 128.7, 128.1, 127.3, 125.5, 121.0, 117.6, 68.0,
67.1 62.5, 28.1, 14.9; FABMS: m/z 420 [M þ H]þ; HRESIMS: calc for
C21H19NO4Cl2Na [M þ Na]þ 442.0588, found 442.0594.
5.1.10.2. 2-[6-Chloro-4-(2-chlorophenyl)-2-oxo-1,2-dihydroquinolin-
3-yl]ethylbutyrate (19). White amorphous power, yield 78% (after
chromatography with CHCl3/MeOH, 98:2); 1H NMR (CDCl3,
400 MHz)
d
7.60 (1H, d, J ¼ 7.6 Hz), 7.48 (4H, m), 7.26 (1H, d,
5.1.10.8. 2-[6-Chloro-4-(2-chlorophenyl)-2-oxo-1,2-dihydroquinolin-
3-yl]ethyl-2-acetamidoacetate (27). White amorphous power, yield
70% (after chromatography with CHCl3/MeOH, 97:3); 1H NMR
J ¼ 7.2 Hz), 6.86 (1H, s), 4.27 (2H, t, J ¼ 6.8 Hz), 2.92 (1H, m,), 2.69
(1H, m), 2.22 (2H, t, J ¼ 7.4 Hz), 1.57 (2H, t, J ¼ 7.4 Hz), 0.88 (3H, t,
J ¼ 7.4 Hz); 13C NMR (CDCl3, 100 MHz)
d
173.7, 163.2, 147.0, 135.8,
(CDCl3, 400 MHz)
6.48 (1H, s), 4.37 (2H, m), 3.99 (2H, d, J ¼ 4.0 Hz), 2.92 (1H, m), 2.72
(1H, m), 2.0 (3H, s); 13C NMR (CDCl3, 100 MHz)
170.4, 170.3, 163.6,
d 12.63 (1H, br.s), 7.61e7.23 (6H, m), 6.85 (1H, s),
134.1,132.8, 130.6, 130.4,130.3, 130.1,129.1, 128.0, 127.2, 125.5, 121.0,
117.3, 62.0, 36.1, 28.1, 18.2, 13.5; FABMS: m/z 404 [M þ H] þ, HRE-
SIMS: calc for C21H20NO3Cl2 [M þ H]þ 404.0820, found 404.0826.
d
147.3, 135.8, 134.0, 132.8, 130.6, 130.5, 130.5, 130.2, 128.8, 128.3,
127.4, 125.5, 121.1, 117.5, 63.5, 43.4, 28.0, 22.8; ESIMS: m/z 455
[M þ Na]þ, HRESIMS: calc for C21H19N2O4Cl2 [M þ H]þ 433.0721,
found 433.0721.
5.1.10.3. 2-[6-Chloro-4-(2-chlorophenyl)-2-oxo-1,2-dihydroquinolin-
3-yl]ethylpentanoate (20). White amorphous power, yield 82%
(after chromatography with CHCl3/MeOH, 70:1); 1H NMR (CDCl3,
400 MHz)
d
13.21 (1H, br.s), 7.60 (1H, d, J ¼ 7.6 Hz), 7.51e7.44 (4H,
5.1.10.9. 2-[6-Chloro-4-(2-chlorophenyl)-2-oxo-1,2-dihydroquinolin-
3-yl]ethylcyclopentanecarboxylate (28). White amorphous power,
yield 77% (after chromatography with CHCl3/MeOH, 70:1); 1H NMR
m), 7.27 (1H, dd, J ¼ 7.2, 2.0 Hz), 6.87 (1H, d, J ¼ 2.0 Hz), 4.31 (2H, t,
J ¼ 6.8 Hz), 2.97 (1H, m), 2.67 (1H, m), 2.23 (2H, t, J ¼ 7.6 Hz), 1.53
(2H, m), 1.28 (2H, m), 0.85 (3H, t, J ¼ 7.2 Hz); 13C NMR (CDCl3,
(CDCl3, 400 MHz)
d
13.10 (1H, br.s), 7.60 (1H, dd, J ¼ 7.6, 1.6 Hz),
100 MHz)
d
173.6, 164.0, 147.1, 136.0, 134.2, 133.0, 130.7, 130.4, 130.3,
7.51e7.43 (4H, m), 7.27 (1H, m), 6.86 (1H, d, J ¼ 2.0 Hz), 4.30 (2H, t,
130.1, 129.1, 128.1, 127.2, 125.5, 121.1, 117.7, 62.0, 34.0, 28.2, 26.9,
J ¼ 7.6 Hz) 3.01e2.94 (1H, m), 2.71e2.64 (1H, m),1.83e1.50 (9H, m);
22.2, 13.7; ESIMS: m/z 440 [M
þ
Na]þ, HRESIMS: calc for
13C NMR (CDCl3, 100 MHz)
d 176.6, 163.9, 147.0, 136.0, 134.2, 132.9,
C22H22NO3Cl2 [M þ H]þ 418.0976, found 418.0972.
130.7, 130.4, 130.3, 130.1, 129.2, 128.1, 127.2, 125.5, 121.1, 117.7, 62.1,
43.7, 30.0 (2C), 28.2, 25.8 (2C); ESIMS: m/z 452 [M þ Na]þ, HRE-
SIMS: calc for C23H22NO3Cl2 [M þ H]þ 430.0976, found 430.0973.
5.1.10.4. 2-[6-Chloro-4-(2-chlorophenyl)-2-oxo-1,2-dihydroquinolin-
3-yl]ethyloctanoate (21). White amorphous power, yield 70% (after
chromatography with CHCl3/MeOH, 97:3); 1H NMR (CDCl3,
5.1.10.10. 2-[6-Chloro-4-(2-chlorophenyl)-2-oxo-1,2-dihy-
400 MHz)
d
13.24 (1H, br.s), 7.60 (1H, d, J ¼ 8.0 Hz), 7.51e7.43 (4H,
droquinolin-3-yl]ethyl cyclohexanecarboxylate (29). White amor-
phous power, yield 78% (after chromatography with CHCl3/MeOH,
m), 7.27 (1H, d, J ¼ 7.2 Hz), 6.87 (1H, d, J ¼ 2.0 Hz), 4.32 (2H, t,
J ¼ 6.8 Hz), 2.97 (1H, m), 2.69 (1H, m), 2.22 (2H, t, J ¼ 7.6 Hz), 1.55
70:1); 1H NMR (CDCl3, 400 MHz)
d 13.12 (1H, br.s), 7.60 (1H, d,

316
R.-H. Guo et al. / European Journal of Medicinal Chemistry 46 (2011) 307e319
J ¼ 8.0 Hz), 7.50e7.44 (4H, m), 7.27 (1H, m), 6.86 (1H, d, J ¼ 2.0 Hz),
4.28 (2H, t, J ¼ 6.8 Hz), 2.97 (1H, m), 2.67 (1H, m), 2.21 (1H, m)
1.83e1.70 (5H, m), 1.39e1.12 (5H, m); 13C NMR (CDCl3, 100 MHz)
117.0, 63.7, 27.8; ESIMS: m/z 505 [M þ H]þ, HRESIMS: calc for
C24H17N2O5Cl2 [M þ H]þ 483.0514, found 483.0526.
d
175.9, 163.9, 146.0, 136.0, 134.2, 132.9, 130.7, 130.4, 130.2, 130.1,
5.1.10.16. 2-[6-Chloro-4-(2-chlorophenyl)-2-oxo-1,2-dihy-
129.2, 128.0, 127.2, 125.5, 121.1, 117.6, 62.0, 43.1, 28.9 (2C), 28.1, 25.7,
droquinolin-3-yl]ethylnicotinate (35). White amorphous power,
25.4 (2C); ESIMS: m/z 466 [M
þ
Na]þ, HRESIMS: calc for
yield 65% (after chromatography with CHCl3/MeOH, 25:1); 1H NMR
C24H24NO3Cl2 [M þ H]þ 444.1133, found 444.1135.
(CDCl3, 400 MHz)
d
13.19 (1H, br.s), 9.12 (1H, d, J ¼ 1.4 Hz) 8.70 (1H,
dd, J ¼ 4.8, 1.7 Hz), 8.21 (1H, m), 7.58 (1H, d, J ¼ 7.2 Hz), 7.52e7.40
(3H, m), 7.40e7.25 (2H, m), 7.19 (1H, dd, J ¼ 7.6, 1.5 Hz), 6.8 (1H, s),
4.60 (2H, m), 3.12e3.07 (1H, m), 2.89e2.84 (1H, m); 13C NMR (CDCl3,
5.1.10.11. 2-[6-Chloro-4-(2-chlorophenyl)-2-oxo-1,2-dihy-
droquinolin-3-yl]ethyl-3-cyclohexylpropanoate (30). White amor-
phous power, yield 80% (after chromatography with CHCl3/MeOH,
100 MHz) d 164.5, 162.7, 150.2, 147.0, 135.7, 135.5, 133.8, 132.7, 130.6,
70:1); 1H NMR (CDCl3, 500 MHz)
d
7.60 (1H, d, J ¼ 8.0 Hz), 7.52e7.42
130.39, 130.35, 130.3, 130.1, 128.8, 128.0, 127.2, 125.4, 123.2, 120.8,
118.2, 117.0, 63.6, 27.8; ESIMS: m/z 461 [M þ Na]þ, HRESIMS: calc for
C23H16N2O3Cl2Na [M þ Na]þ 461.0435, found 461.0445.
(4H, m), 7.24 (1H, m), 6.89 (1H, s), 4.23 (2H, t, J ¼ 6.9 Hz), 2.91 (1H,
m), 2.64 (1H, m), 2.39 (2H, t, J ¼ 7.8 Hz), 2.21 (2H, t, J ¼ 7.8 Hz),
1.74e1.55 (9H, m), 1.43 (2H, m); 13C NMR (CDCl3, 100 MHz)
d 174.7,
169.1,158.9,143.0,130.6, 129.1,128.1,125.9,125.7,125.4,124.1,123.9,
122.5, 120.9, 116.6, 112.8, 57.2, 32.4, 28.2, 28.1, 27.4, 27.0, 23.2, 21.7,
21.42, 21.40; ESIMS: m/z 494 [M þ Na]þ, HRESIMS: calc for
C26H27NO3Cl2Na [M þ Na]þ 494.1265, found 494.1272.
5.1.10.17. 2-[6-Chloro-4-(2-chlorophenyl)-2-oxo-1,2-dihy-
droquinolin-3-yl]ethylthiophene-2-carboxylate (36). White amor-
phous power, yield 70% (after chromatography with CHCl3/MeOH,
98:2); 1H NMR (CDCl3, 400 MHz)
d
13.11 (1H, br.s), 7.73e7.23 (8H,
m), 7.02 (1H, m), 6.85 (1H, s), 4.77 (2H, m), 3.30 (1H, m), 3.00 (1H,
m); 13C NMR (CDCl3, 100 MHz)
163.8, 162.0, 147.0, 136.0, 134.1,
5.1.10.12. 2-[6-Chloro-4-(2-chlorophenyl)-2-oxo-1,2-dihy-
d
droquinolin-3-yl]ethyl-4,7,7-trimethyl-3-oxo-2-oxa-bicyclo[2.2.1]
heptane-1-carboxylate (31). White amorphous power, yield 65%
(after chromatography with CHCl3/MeOH, 98:2); 1H NMR (CDCl3,
133.9, 133.4, 132.8, 132.2, 130.8, 130.4, 130.3, 130.2, 129.1, 128.0,
127.5, 127.3, 125.5, 121.1, 117.5, 63.0, 28.3; ESIMS: m/z 466
[M þ Na]þ, HRESIMS: calc for C22H15NO3Cl2SNa [M þ Na]þ
466.0047, found 466.0054.
400 MHz)
d 12.9 (1H, br.s), 7.60e7.35 (6H, m), 6.86 (1H, m),
4.57e4.51 (1H, m), 4.41e4.35 (1H, m), 3.03e2.97 (1H, m), 2.76e2.70
(1H, m), 2.41e2.34 (1H, m), 2.00e1.82 (2H, m), 1.67e1.58 (1H, m),
1.07 (3H, s), 0.97 (3H, s), 0.82 (3H, s); 13C NMR (CDCl3, 100 MHz)
5.1.10.18. 2-[6-Chloro-4-(2-chlorophenyl)-2-oxo-1,2-dihy-
droquinolin-3-yl]ethylfuran-2-carboxylate (37). White amorphous
power, yield 56% (after chromatography with CHCl3/MeOH, 98:2);
d
178.2, 167.4, 163.7, 147.5, 135.9, 133.9, 132.6, 131.2, 130.9, 130.5,
130.4, 130.0, 129.9, 128.3, 127.6, 121.1, 117.5, 91.0, 63.4, 54.7, 54.2,
1H NMR (CDCl3, 400 MHz)
d 13.18 (1H, br.s), 7.58e7.25 (7H, m), 7.02
30.5, 28.8, 28.1, 16.60, 16.57, 9.6; ESIMS: m/z 536 [M þ Na]þ, HRE-
(1H, m), 7.12 (1H, d, J ¼ 3.6 Hz), 6.86 (1H, d, J ¼ 2.0 Hz), 6.45 (1H, m),
SIMS: calc for C27H25NO5Cl2Na [M
536.0997.
þ
Na]þ 536.1007, found
4.58 (2H, m), 3.10 (1H, m), 2.79 (1H, m); 13C NMR (CDCl3, 100 MHz)
d
163.9, 158.4, 147.2, 146.2, 144.7, 136.1, 134.1, 132.8, 130.7, 130.5,
130.3, 130.1, 128.8, 128.0, 27.1, 125.5, 121.1, 17.9, 117.7, 111.8, 62.7,
28.3; ESIMS: m/z 450 [M þ Na]þ, HRESIMS: calc for C22H16NO4Cl2
[M þ H]þ 428.0456, found 428.0462.
5.1.10.13. 2-[6-Chloro-4-(2-chlorophenyl)-2-oxo-1,2-dihy-
droquinolin-3-yl]ethyl-1-acetylpiperidine-4-carboxylate (32). White
amorphous power, yield 67% (after chromatography with CHCl3/
MeOH, 98:2); 1H NMR (CDCl3, 500 MHz)
7.50e7.43 (4H, m), 7.24 (1H, m), 6.84 (1H, d, J ¼ 2.0 Hz), 4.30 (2H,
m), 3.71 (1H, m), 3.05 (1H, t, J ¼ 10.4 Hz), 2.91 (1H, m), 2.71 (2H, m),
2.48e2.42 (1H, m), 2.05 (3H, s), 1.57 (2H, m); 13C NMR (CDCl3,
d
7.59 (1H, d, J ¼ 7.5 Hz),
5.1.10.19. 2-[6-Chloro-4-(2-chlorophenyl)-2-oxo-1,2-dihy-
droquinolin-3-yl]ethyl-2-(1,3-dioxoisoindolin-2-yl)acetate
(38). White amorphous power, yield 80% (after chromatography
with CHCl3/MeOH, 100:1); 1H NMR (CDCl3, 500 MHz)
d 11.81 (1H,
125 MHz)
d
173.9, 168.9, 163.7, 147.1, 136.0, 134.1, 132.9, 130.5, 130.4,
br.s), 7.79 (2H, m), 7.69 (2H, m), 7.57 (1H, m), 7.47 (2H, m), 7.41e7.32
(2H, m), 7.22e7.19 (1H, m), 6.80 (1H, d, J ¼ 2.6 Hz), 4.35e4.31 (4H,
m), 2.92e2.85 (1H, m), 2.68e2.62 (1H, m); 13C NMR (CDCl3,
130.2, 128.9, 128.1, 127.3, 125.5, 121.0, 117.6, 62.5, 45.6, 40.8, 30.9,
28.2, 28.0 (2C), 27.7, 21.4; ESIMS: m/z 487 [M þ H]þ, HRESIMS: calc
for C25H25N2O4Cl2 [M þ H]þ 487.1191, found 487.1181.
125 MHz)
d 167.5, 167.1, 162.9, 147.1, 135.8, 134.2, 134.0, 132.8, 131.8,
130.5, 130.4, 130.2, 128.6, 128.0, 127.4, 125.5, 123.5, 121.0, 117.2, 63.6,
5.1.10.14. 2-[6-Chloro-4-(2-chlorophenyl)-2-oxo-1,2-dihy-
38.8, 27.9; ESIMS: m/z 543 [M
þ
Na]þ, HRESIMS: calc for
droquinolin-3-yl]ethyl benzoate (33). White amorphous power,
C27H18N2O5Cl2Na [M þ Na]þ 543.0490, found 543.0490.
yield 69% (after chromatography with CHCl3/MeOH, 98:2); 1H NMR
(CDCl3, 400 MHz)
(7H, m), 7.18 (1H, d, J ¼ 7.5 Hz), 6.86 (1H, s), 4.57 (2H, t, J ¼ 6.8 Hz),
3.13 (1H, m), 2.83 (1H, m); 13C NMR (CDCl3, 100 MHz)
166.4, 164.0,
147.2,136.0,134.1,132.84,132.80,130.7,130.4,130.3,130.1,129.6,129.
2, 129.2 (2C), 128.2 (2C), 128.1, 127.3, 125.4, 121.2, 117.8, 58.8, 32.3;
ESIMS: m/z 460 [M þ Na] þ, HRESIMS: calc for C24H17NO3Cl2Na
[M þ Na]þ 460.0483, found 460.0482.
d
7.96 (2H, m), 7.58 (1H, d, J ¼ 6.4 Hz), 7.49e7.28
5.1.10.20. 2-[6-Chloro-4-(2-chlorophenyl)-2-oxo-1,2-dihy-
droquinolin-3-yl]ethyl-2-phenoxyacetate (39). White amorphous
power, yield 68% (after chromatography with petroleum ether/
d
acetone, 80:20); 1H NMR (CDCl3, 500 MHz)
d 13.15 (1H, br.s), 7.60
(1H, d, J ¼ 7.9 Hz), 7.51e7.43 (4H, m), 7.30e7.18 (3H, m), 6.94e6.80
(4H, m), 4.60e4.43 (4H, m), 3.00 (1H, m), 2.74 (1H, m); 13C NMR
(CDCl3,125 MHz) d 168.8,163.9,157.7,147.3,136.0,134.1,132.9,130.7,
130.5, 130.4, 130.2, 129.5 (2C), 128.6, 128.1, 127.3, 125.5, 121.7, 121.0,
117.7, 114.5 (2C), 65.4, 63.1, 28.0; ESIMS: m/z 490 [M þ Na]þ, HRE-
SIMS: calc for C25H19NO4Cl2Na [M þ Na]þ 490.0588, found 490.0595.
5.1.10.15. 2-[6-Chloro-4-(2-chlorophenyl)-2-oxo-1,2-dihy-
droquinolin-3-yl]ethyl4-nitrobenzoate
power, yield 68% (after chromatography with CHCl3/MeOH, 98:2);
1H NMR (CDCl3, 400 MHz)
12.89 (1H, br.s), 8.12 (4H, m), 7.60 (1H,
(34). White amorphous
d
5.1.10.21. 2-[6-Chloro-4-(2-chlorophenyl)-2-oxo-1,2-dihy-
d, J ¼ 6.4 Hz), 7.51e7.36 (5H, m), 6.85 (1H, s), 4.58 (2H, t, J ¼ 7.2 Hz),
droquinolin-3-yl]ethyl 3-phenoxypropanoate (40). White amor-
phous power, yield 58% (after chromatography with CHCl3/MeOH,
3.14e3.09 (1H, m), 2.90e2.85 (1H, m); 13C NMR (CDCl3, 100 MHz)
d
164.5, 162.7, 150.3, 147.0, 135.7, 135.5, 133.8, 132.6, 130.6 (2C),
100:1); 1H NMR (CDCl3, 400 MHz)
d
13.16 (1H, br.s), 7.58 (1H, dd,
130.4, 130.4, 130.3, 130.1, 128.8, 128.0, 127.2, 125.4, 123.2 (2C), 120.9,
J ¼ 8.8, 2.0 Hz), 7.48e7.41 (4H, m), 7.27e7.20 (3H, m), 6.93e6.80

R.-H. Guo et al. / European Journal of Medicinal Chemistry 46 (2011) 307e319
317
(4H, m), 4.44e4.33 (2H, m), 4.17 (2H, t, J ¼ 6.4 Hz), 2.99 (1H, m),
2.73 (3H, m); 13C NMR (CDCl3, 100 MHz)
170.9, 164.0, 158.4, 147.2,
136.0, 134.2, 132.9, 130.7, 130.5, 130.4, 130.2, 129.6 (2C), 129.0, 128.1,
127.3, 125.5, 121.1, 120.98, 117.7, 115.4 (2C), 63.2, 62.6, 34.6, 28.1;
ESIMS: m/z 504 [M þ Na]þ, HRESIMS: calc for C26H21NO4Cl2Na
[M þ Na]þ 504.0745, found 504.0758.
5.1.10.27. (E)-2-[6-Chloro-4-(2-chlorophenyl)-2-oxo-1,2-dihy-
droquinolin-3-yl]ethyl-3-[3-(trifluoro methyl) phenyl]acrylate
(46). White amorphous power, yield 91% (after chromatography
with CHCl3/MeOH, 70:1); 1H NMR (CDCl3, 400 MHz)
13.19 (1H,
d
d
br.s), 7.69e7.42 (10H, m), 7.25 (1H, dd, J ¼ 7.2, 1.6 Hz), 6.86 (1H, d,
J ¼ 2.0 Hz), 4.42 (1H, d, J ¼ 16.0 Hz), 4.46 (2H, t, J ¼ 6.8 Hz), 3.07e3.00
(1H, m), 2.83e2.76 (1H, m); 13C NMR (CDCl3,100 MHz)
d 166.2,163.9,
5.1.10.22. 2-[6-Chloro-4-(2-chlorophenyl)-2-oxo-1,2-dihy-
147.3,142.8,136.0,135.1,134.1,133.0,130.9,130.7,130.5,130.4,130.2,
129.4,129.0,128.2,127.3,126.6,126.6,125.5,124.5,124.5,121.1,120.0,
117.7, 62.6, 28.2; ESIMS: m/z 554 [M þ Na]þ, HRESIMS: calc for
C27H19NO3Cl2F3 [M þ H]þ 532.0694, found 532.0693.
droquinolin-3-yl]ethylcinnamate (41). White amorphous power,
yield 50% (after chromatography with petroleum ether/acetone,
90:10); 1H NMR (CDCl3, 400 MHz)
d
7.63e7.26 (12H, m), 6.86 (1H, d,
J ¼ 2.0 H z), 6.36 (1H, d, J ¼ 16 Hz), 4.46 (2H, t, J ¼ 6.4 Hz), 3.04 (1H,
m), 2.77 (1H, m); 13C NMR (CDCl3, 100 MHz)
166.7, 163.8, 147.1,
d
5.1.10.28. (E)-2-[6-Chloro-4-(2-chlorophenyl)-2-oxo-1,2-dihy-
144.7, 135.9, 134.3, 134.1, 132.9, 130.8, 130.5, 130.3, 130.2, 130.17,
129.1, 128.8 (2C), 128.1, 127.9 (2C), 127.2, 125.5, 121.4, 118.0, 117.6,
62.3, 28.2; FABMS: m/z 464 [M þ H] þ, HRESIMS: calc for
C26H19NO3Cl2Na [M þ Na]þ 486.0639, found 486.0636.
droquinolin-3-yl]ethyl-3-[4-(trifluoro
(47). White amorphous power, yield 34% (after chromatography
with CHCl3/MeOH, 70:1); 1H NMR (CDCl3, 400 MHz)
13.03 (1H,
methyl)
phenyl]acrylate
d
br.s), 7.63e7.42 (10H, m), 7.24 (1H, dd, J ¼ 7.5, 1.6 Hz), 6.86 (1H, d,
J ¼ 2.0 Hz), 6.42 (1H, d, J ¼ 16.1 Hz), 4.45 (2H, t, J ¼ 6.7 Hz), 3.06e3.00
5.1.10.23. (E)-2-[6-Chloro-4-(2-chlorophenyl)-2-oxo-1,2-dihy-
(1H, m), 2.82e2.75 (1H, m); 13C NMR (CDCl3,100 MHz)
d 166.2,163.8,
droquinolin-3-yl]ethyl-3-(3-fluorophenyl)
amorphous power, yield 76% (after chromatography with CHCl3/
MeOH, 70:1); 1H NMR (CDCl3, 500 MHz)
13.23 (1H, br.s),
acrylate
(42). White
147.4,142.8,137.7,135.8,134.1,132.9,130.7,130.6,130.4,130.3,129.0,
128.4, 128.1 (2C), 127.3, 125.9, 125.8 (2C), 125.7, 125.6, 121.2, 120.6,
117.7, 62.7, 28.1; ESIMS: m/z 554 [M þ Na]þ, HRESIMS: calc for
C27H18NO3Cl2F3Na [M þ Na]þ 554.0513, found 554.0529.
d
7.60e7.44 (6H, m), 7.33e7.03 (5H, m), 6.86 (1H, s), 6.50 (1H, d,
J ¼ 16.0 Hz), 4.46 (2H, t, J ¼ 6.9 Hz), 3.07e3.02 (1H, m), 2.81e2.76
(1H, m); 13C NMR (CDCl3,125 MHz)
d
166.3,164.0,162.0,147.1,143.2,
5.1.10.29. (E)-2-[6-Chloro-4-(2-chlorophenyl)-2-oxo-1,2-dihy-
136.6, 136.0, 134.2, 133.0, 130.7, 130.4, 130.4, 130.2, 129.2, 128.1,
127.2, 125.5, 123.9, 121.1, 119.5, 117.6, 117.2, 117.0, 114.2, 62.6, 28.2;
ESIMS: m/z 504 [M þ Na]þ, HRESIMS: calc for C26H19NO3Cl2F
[M þ H]þ 482.0726, found 482.0716.
droquinolin-3-yl]ethyl-3-(benzo[d][1,3] dioxol-5-yl)acrylate (48). White
amorphous power, yield 60% (after chromatography with CHCl3/
MeOH, 98:2); 1H NMR (CDCl3, 500 MHz)
d 7.41 (2H, m), 7.32e7.23 (5H,
m), 7.15 (1H, d, J ¼ 8.8 Hz), 7.07 (1H, d, J ¼ 7.5, 1.5 Hz), 6.63 (2H, m),
5.99 (1H, d, J ¼ 15.9 Hz), 5.82 (2H, s), 3.17 (2H, m), 2.77e2.72 (1H, m),
5.1.10.24. (E)-2-[6-Chloro-4-(2-chlorophenyl)-2-oxo-1,2-dihy-
droquinolin-3-yl]ethyl-3-(2-methoxyphenyl) acrylate (43). White
amorphous power, yield 60% (after chromatography with CHCl3/
2.53e2.47 (1H, m); 13C NMR (CDCl3, 100 MHz)
d 167.7, 163.0, 150.1,
148.7, 147.4, 145.1, 136.2, 134.4, 133.1, 131.1, 130.7, 130.6, 130.4, 129.0,
128.3, 127.6, 125.9, 124.8, 121.4, 117.4, 115.9, 108.8, 106.7, 106.6, 101.9,
MeOH,100:1); 1H NMR (CDCl3, 400 MHz)
d
13.16 (1H, br.s), 7.91 (1H,
62.6, 28.6; ESIMS: m/z 530 [M
þ
Na]þ, HRESIMS: calc for
d, J ¼ 16.0 Hz),7.60e7.26 (8H, m), 6.94e6.87 (3H, m), 6.46 (1H, d,
C27H19NO5Cl2Na [M þ Na]þ 530.0537, found 530.0543.
J ¼ 16.0 Hz), 4.47 (2H, m), 3.84 (3H, s), 3.08e3.03 (1H, m), 2.78e2.78
(1H, m); 13C NMR (CDCl3, 100 MHz)
d
167.2,163.9, 158.2, 147.2, 140.1,
5.1.10.30. (E)-2-[6-Chloro-4-(2-chlorophenyl)-2-oxo-1,2-dihy-
droquinolin-3-yl]ethyl-3-(3,4-dimethoxy phenyl)acrylate (49). White
amorphous power, yield 60% (after chromatography with CHCl3/
135.9, 134.2, 132.9, 131.4, 130.8, 130.4, 130.3, 130.1, 129.2, 128.8,
128.1, 127.2, 125.4, 123.3, 121.2, 120.6, 118.6, 117.8, 111.0, 62.2, 55.4,
28.3; ESIMS: m/z 516 [M þ Na]þ, HRESIMS: calc for C27H21NO4Cl2Na
[M þ Na]þ 516.0745, found 516.0759.
MeOH, 98:2); 1H NMR (CDCl3, 400 MHz)
d 13.18 (1H, br.s), 7.59e6.80
(11H, m), 6.24 (1H, d, J ¼ 16.0 Hz), 4.43 (2H, t, J ¼ 6.8 Hz), 3.88 (3H, s),
3.86 (3H, s), 3.05e2.99 (1H, m), 2.80e2.74 (1H, m); 13C NMR (CDCl3,
5.1.10.25. (E)-2-[6-Chloro-4-(2-chlorophenyl)-2-oxo-1,2-dihy-
droquinolin-3-yl]ethyl-3-(3-methoxyphenyl) acrylate (44). White
amorphous power, yield 70% (after chromatography with CHCl3/
100 MHz) d 166.9, 163.9, 151.0, 149.1, 147.1, 144.8, 144.6, 136.0, 134.1,
132.9, 130.7, 130.4, 130.3, 130.2,129.2, 128.0, 127.2, 125.4, 122.5,121.1,
117.7, 115.7, 115.4, 110.9, 62.2, 55.9, 51.6, 28.2; ESIMS: m/z 546
[M þ Na]þ, HRESIMS: calc for C28H23NO5Cl2Na [M þ Na]þ 546.0850,
found 546.0855.
MeOH, 95:5); 1H NMR (CDCl3, 500 MHz)
d 7.60e7.42 (6H, m), 7.27
(3H, m), 7.05 (1H, d, J ¼ 7.5 Hz), 6.97 (1H, s), 6.90 (1H, dd, J ¼ 7.5,
2.5 Hz), 6.86 (1H, d, J ¼ 2.0 Hz), 6.35 (1H, d, J ¼ 16.0 Hz), 4.50 (2H, t,
J ¼ 5.2 Hz), 3.80 (3H, s), 3.03 (1H, m), 2.77 (1H, m); 13C NMR (CDCl3,
5.1.10.31. (E)-2-[6-Chloro-4-(2-chlorophenyl)-2-oxo-1,2-dihy-
droquinolin-3-yl]ethyl-3-(pyridin-3-yl) acrylate (50). White amor-
phous power, yield 70% (after chromatography with CHCl3/MeOH,
125 MHz) d 166.6, 163.8, 159.8, 147.2, 144.6, 135.9, 135.7, 134.1, 132.9,
130.7, 130.5,130.4,130.2,129.8, 129.1, 128.2,127.2,125.5,121.2, 120.6,
118.3, 117.7, 116.1, 112.8, 62.4, 55.2, 28.2; ESIMS: m/z 516 [M þ Na]þ,
HRESIMS: calc for C27H22NO4Cl2 [Mþ H]þ 494.0925, found 494.0932.
25:1); 1H NMR (CDCl3, 400 MHz)
d 13.19 (1H, br.s), 8.85 (1H, s), 8.58
(1H, d, J ¼ 4.4 Hz), 7.73 (1H, d, J ¼ 6.8 Hz), 7.60e7.26 (8H, m), 6.85
(1H, d, J ¼ 1.6 Hz), 6.36 (1H, d, J ¼ 16.0 Hz), 4.46 (2H, t, J ¼ 6.7 Hz),
5.1.10.26. (E)-2-[6-Chloro-4-(2-chlorophenyl)-2-oxo-1,2-dihy-
droquinolin-3-yl]ethyl-3-(4-methoxyphenyl) acrylate (45). White
amorphous power, yield 60% (after chromatography with CHCl3/
3.03 (1H, m), 2.75 (1H, m); 13C NMR (CDCl3,100 MHz)
d 166.0,164.0,
150.9, 149.5, 147.1, 140.9, 136.0, 134.1, 134.1, 132.9, 130.7, 130.4, 130.3,
130.2, 130.1, 129.1, 128.1, 127.2, 125.5, 123.7, 121.0, 120.2, 117.6, 62.8,
28.2; ESIMS: m/z 465 [M þ H]þ, HRESIMS: calc for C25H19N2O3Cl2
[M þ H]þ 465.0772, found 465.0767.
MeOH, 70:1); 1H NMR (CDCl3, 500 MHz)
d 13.28 (1H, br.s),
7.60e7.38 (8H, m), 7.28 (1H, d, J ¼ 8.0 Hz), 6.86 (3H, m), 6.24 (1H, d,
J ¼ 16.0 Hz), 4.45 (2H, m), 3.81 (3H, s), 3.08e3.03 (1H, m), 2.80e2.74
(1H, m); 13C NMR (CDCl3, 125 MHz)
d
167.1, 164.0, 161.3, 147.0, 144.3,
5.1.10.32. (E)-2-[6-Chloro-4-(2-chlorophenyl)-2-oxo-1,2-dihy-
droquinolin-3-yl]ethyl-but-2-enoate (51). White amorphous power,
yield 60% (after chromatography with CHCl3/MeOH, 98:2); 1H
136.0, 134.2, 132.9, 130.8, 130.4, 130.3, 130.1, 129.6 (2C), 129.3, 128.0,
127.2, 127.0, 125.4, 121.1, 117.7, 115.5, 114.2 (2C), 62.3, 55.3, 28.2;
ESIMS: m/z 516 [M þ Na]þ, HRESIMS: calc for C27H21NO4Cl2Na
[M þ Na]þ 516.0745, found 516.0754.
NMR (CDCl3, 500 MHz)
7.25 (1H, dd, J ¼ 9.0, 1.5 Hz), 6.94e6.87 (2H, m), 5.77 (1H, m), 4.38
d
7.59 (1H, d, J ¼ 7.9 Hz), 7.51e7.42 (4H, m),

318
R.-H. Guo et al. / European Journal of Medicinal Chemistry 46 (2011) 307e319
(2H, m), 3.02e3.97 (1H, m), 2.74e2.68 (1H, m), 1.80 (3H, dd, J ¼ 7.1,
1.4 Hz); 13C NMR (CDCl3, 125 MHz)
166.3, 163.9, 147.1, 144.7,136.0,
134.1, 132.9, 130.8, 130.4, 130.3, 130.1, 129.1, 128.1, 127.2, 125.5,
122.6, 121.3, 117.7, 62.0, 28.2, 17.9; ESIMS: m/z 402 [M þ H]þ,
HRESIMS: calc for C21H18NO3Cl2 [M þ H]þ 402.0663, found
402.0673.
5.2. Cell cultures [25]
d
HepG 2.2.15 cells were cultured at 37 ꢀC in a humidified 5% CO2/
air atmosphere (Forma, Series II, Thermo, USA) in DMEM (Dulbec-
co’s modifiedeagle’s medium) supplemented with 10% (vol/vol)
fetal bovine serum, and 5 mM
L-glutamine, 50 U/mL penicillin,
50 g/mL streptomycin. Cells were subcultured once a week, and
m
5.1.10.33. (2E,4E)-2-[6-Chloro-4-(2-chlorophenyl)-2-oxo-1,2-dihy-
droquinolin-3-yl]ethyl-hexa-2,4-dienoate (52). White amorphous
power, yield 65% (after chromatography with CHCl3/MeOH, 98:2);
fresh medium was added every other day.
5.2.1. Determination of HBV replication [26]
1H NMR (CDCl3, 500 MHz)
d
7.61 (1H, d, J ¼ 7.5 Hz), 7.48e7.44 (4H,
HepG 2.2.15 cells were seeded in twenty-four well culture plates
at a density of 5 ꢁ 105 cells per well. Every 2 days, medium was
changed. After 6 days, compounds were added to the cell cultures,
and fresh medium was fed every other day for another 6 days. Cells
were collected and total DNA was isolated by using TIANamp
Gemomic DNA Kit (TIANGEN, Biotech Co., Ltd, China) following the
manufacturer’s instructions. For detection of HBV DNA, a real-time
m), 7.29e7.18 (2H, m), 6.90 (1H, d, J ¼ 2.0 Hz), 6.20e6.09 (2H, m),
6.72 (1H, d, J ¼ 15.5 Hz), 4.41 (2H, m), 3.05e3.00 (1H, m), 2.76e2.72
(1H, m), 1.85 (3H, d, J ¼ 6.0 Hz); 13C NMR (CDCl3, 125 MHz)
d 167.0,
163.6, 147.4, 145.0, 139.3, 135.7, 134.0, 132.9, 130.8, 130.5, 130.3,
130.1, 129.7, 129.1, 128.3, 127.2, 125.5, 121.3, 118.8, 117.7, 62.0, 28.3,
18.6; ESIMS: m/z 428 [M þ H]þ, HRESIMS: calc for C23H20NO3Cl2
[M þ H]þ 428.0820, found 428.0822.
PCR assay was used. Briefly, 10
mL of DNA sample was amplified in
a 25
mL mixture containing 2ꢁSYBR Green PCR Master Mix (Applied
5.1.10.34. (E)-2-[6-Chloro-4-(2-chlorophenyl)-2-oxo-1,2-dihy-
droquinolin-3-yl]ethyl-2-methylbut-2-enoate (53). White amor-
phous power, yield 80% (after chromatography with CHCl3/MeOH,
Biosystems, USA) and 2 primers specific for HBV: a forward primer
(HBV-t1: 50 CAA GGA ACC TCT ATG TAT CCC TCC 30) and reverse
primer (HBV-t2:50 TCC GTC CGA AGG TTT GGT AC 30) covering the
50ebase pair insertion from 541 bp to 591 bp. Amplification and
detection were performed in the Mastercycler ep realplex System
(eppendorf, Masteraycler ep realplex, Germen) with incubation at
95 ꢀC for 2 min and subsequently, 40 three-step cycles (20 s at
95 ꢀC; 15 s at 58 ꢀC; 20 min at 72 ꢀC) were performed. The standard
was prepared on serial dilutions of a known amount of the cloned
HBV plasmid pCP10, carrying two head-to-tail copies of the HBV
genome as positive control was kindly provided by professor J.
Chen (No. 302 PLA Hospital, Beijing, China). The specificity of 2
primers (HBV-t1 and HBV-t2) was confirmed in every PCR run via
dissociation curve analysis.
70:1); 1H NMR (CDCl3, 500 MHz)
d 13.07 (1H, br.s), 7.57 (1H, d,
J ¼ 9.0 Hz), 7.51e7.39 (4H, m), 7.24 (1H, dd, J ¼ 9.5, 2.0 Hz), 6.84 (1H,
d, J ¼ 2.0 Hz), 6.75 (1H, m), 4.34 (2H, m), 3.48 (3H, d, J ¼ 9.5 Hz) 3.00
(1H, m), 2.70 (1H, m), 1.75 (3H, s); 13C NMR (CDCl3, 125 MHz)
d
167.9, 163.7, 147.1, 137.2, 135.9, 134.1, 132.9, 130.6, 130.4, 130.3,
130.2, 129.2, 128.6, 128.2, 127.2, 125.4, 121.2, 117.7, 62.3, 28.1, 14.1,
12.0; EIMS: m/z 415 [M]þ, HRESIMS: calc for C22H20NO3Cl2 [M þ H]þ
416.0820, found 416.0809.
5.1.11. General procedure for the preparation of compounds 54 and
55
Compounds 54 and 55 were prepared in a similar manner as
described for the synthesis of compound 2. These were used in the
next step without further purification.
5.2.2. Analysis of secreted HBV antigens [27,28]
Medium from HepG 2.2.15 cells was collected, centrifuged at
6000.0 g to remove cellular debris, transferred to clean tubes. The
levels of HBsAg and HBeAg were determined utilizing commer-
cially available quantitative ELISA test kits (Autobio diagnostics Co.,
Ltd, China.) and measured with a microplate reader (model 680,
Bio-Rad, USA).
5.1.12. General procedure for the preparation of compounds 56 and
57
Compounds 54 and 55 were prepared in a similar manner as
described for the synthesis of compound 17.
5.1.12.1. (E)-[6-Chloro-4-(2-chlorophenyl)-2-oxo-1,2-dihy-
Acknowledgments
droquinolin-3-yl]methyl-3-(3-methoxyphenyl) acrylate (56). White
amorphous power, yield 70% (after chromatography with CHCl3/
MeOH,100:1); 1H NMR (CDCl3, 500 MHz)
d 13.09 (1H, br.s), 7.58 (2H,
The work was supported by grants from National Natural
Science Foundation of China for Distinguished Young Scholars (No.
81025023), Xibu Zhiguang Joint-Scholarship of Chinese Academy of
Sciences, the External Cooperation Program of Chinese Academy of
Sciences (No. GJHI200818), and CASeCroucher Fund (CAS-CF07/
08.SC03). The authors are grateful to the staff of the analytical
group of the State Key Laboratory of Phytochemistry and Plant
Resources in West China, Kunming Institute of Botany, ChineseA-
cademy of Sciences, for measurements of all spectra.
m), 7.49e7.38 (4H, m), 7.27 (2H, m), 7.06 (1H, d, J ¼ 7.5 Hz), 6.98 (1H,
s), 6.94 (2H, m), 6.34 (1H, d, J ¼ 16.0 Hz), 5.25 (1H, d, J ¼ 11.6 Hz),
4.97 (1H, d, J ¼ 11.6 Hz), 3.80 (3H, s); 13C NMR (CDCl3, 125 MHz)
d
166.4, 163.4, 159.8, 149.7, 144.9, 136.8, 135.6, 133.2, 132.9, 131.5,
130.6, 130.4, 130.1, 129.8, 128.3, 127.2, 126.6, 126.0, 122.5, 120.74,
120.69, 117.9, 116.2, 112.8, 58.8, 55.2; EIMS: m/z 479 [M]þ, HRESIMS:
calc for C26H20NO4Cl2 [M þ H]þ 480.0769, found 480.0771
5.1.12.2. (E)-3-[6-Chloro-4-(2-chlorophenyl)-2-oxo-1,2-dihy-
droquinolin-3-yl]propyl-3-(3-methoxyphenyl)acrylate (57). White
amorphous power, yield 70% (after chromatography with CHCl3/
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