Synthesis and anti-hepatitis B virus evaluation of novel ethyl 6-hydroxyquinoline-3-carboxylates in vitro

Article abstract of DOI:10.1016/j.bmc.2008.05.029

A series of non-nucleoside ethyl 6-hydroxyquinoline-3-carboxylate derivatives were prepared and evaluated in HepG2.2.15 cells. Most compounds inhibited the expression of viral antigens HBsAg or HBeAg at low concentration. Six compounds, 9f₃, 12b₆, 12f₆, 13b₂, 13b₆, and 13f₆, displayed excellent intracellular inhibitory activity and selectivity towards the replication of HBV DNA. Of these six initial hits, compound 13b₆ was the most active.

Full text of DOI:10.1016/j.bmc.2008.05.029

Bioorganic & Medicinal Chemistry 16 (2008) 6522–6527
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and anti-hepatitis B virus evaluation of novel ethyl
6-hydroxyquinoline-3-carboxylates in vitro
*
Yajing Liu, Yanfang Zhao, Xin Zhai, Xusheng Feng, Jinxin Wang, Ping Gong
Key Lab of New Drugs Design and Discovery of Liaoning Province, School of Pharmaceutical Engineering, Shenyang Pharmaceutical University, 103 Wenhua Road,
Shenhe District, Shenyang 110016, Liaoning, 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 non-nucleoside ethyl 6-hydroxyquinoline-3-carboxylate derivatives were prepared and eval-
uated in HepG2.2.15 cells. Most compounds inhibited the expression of viral antigens HBsAg or HBeAg at
low concentration. Six compounds, 9f3, 12b6, 12f6, 13b2, 13b6, and 13f6, displayed excellent intracellular
inhibitory activity and selectivity towards the replication of HBV DNA. Of these six initial hits, compound
13b6 was the most active.
Received 1 April 2008
Revised 10 May 2008
Accepted 13 May 2008
Available online 17 May 2008
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Hepatitis B virus
Ethyl 6-hydroxyquinoline-3-carboxylates
Synthesis
HepG2.2.15 cells
1. Introduction
Hepatitis B virus (HBV) is a major cause of liver cirrhosis and
hepatocellular carcinoma in humans. Until recently, it was esti-
mated that 400 million people were chronically infected with
HBV worldwide and that HBV was responsible for 750,000 deaths
each year.1 Rates of new HBV infections in developing countries
continue to climb at an alarming rate. Currently, only two interfer-
ons and four nucleoside inhibitors have received FDA approval as
single-agent treatments for HBV infection. Unfortunately, clinical
response rates to interferon-a and peginterferon-a tend to be low
(20–30%).2 On the other hand, the required prolonged regimen of
nucleoside analogues, such as lamivudine, adefovir dipivoxil, ente-
cavir, and telbivudine, almost invariably leads to drug-resistance
problems.3–6 Drugs with novel structures and mechanisms of
action are urgently needed.
Figure 1. Structure of 5-hydroxy-1H-indole-3-carboxylates.
Previously, we described a series of 5-hydroxy-1H-indole-3-car-
boxylate derivatives (I, Fig. 1) with anti-HBV activities in vitro.7,8 In
these studies, the structure–activity relationship (SAR) between
the modification of substituents on the indole cycle and anti-HBV
activity was assessed. In order to explore the SAR surrounding
the indole region of this chemical series, we designed and prepared
some novel ethyl 6-hydroxyquinoline-3-carboxylate derivatives in
which the indole ring was expanded to a quinoline moiety. Other
changes primarily targeted positions 2 and 5 of the quinoline ring.
Aliphatic aminomethyl or N-heteroaromatic methyl functionality
Figure 2. General structure of target compounds.
was introduced at the 5-position, and arylthiomethyl or arylsulfi-
nylmethyl groups were incorporated into the 2-position in order
to investigate the influence of these changes on anti-HBV activity
(see Fig. 2).
2. Chemistry
The synthesis of target compounds 9a–9g was achieved using a
convenient seven-step procedure starting from 3-hydroxy-4-
* Corresponding author. Tel./fax: +86 24 23986429.
E-mail address: gongpinggp@126.com (P. Gong).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.05.029

Y. Liu et al. / Bioorg. Med. Chem. 16 (2008) 6522–6527
6523
Scheme 1. Synthesis of compounds 9a–9g. Reagents and conditions: (a) K2CO3, DMF, 90 °C, 3 h, 90%; (b) fuming HNO3, 40 °C, 5 h, 78%; (c) HCONH2, Zn, absolute alcohol,
reflux, 8 h, 50%; (d) piperidine, isopropanol, reflux, 12–15 h; (e) acetic acid/hydrochloric acid = 2/1 (volume ratio), 80 °C, 5–8 h, 44–59%; (f) aliphatic amine, 37% aqueous
formaldehyde, methanol, HCl, 40 °C, 2–5 h, 68–76%. The overall yield from 10.5% to 15.7%.
be realized through the following two steps: The corresponding
dimethylamine derivatives of 9 were obtained via a Mannich reac-
tion. And then treatment of 9 with imidazole or 2-methylimidazole
in 1,4-dioxane generated five N-heteroaromatic derivatives 12b–
Scheme 2. Synthesis of ethyl 3-oxo-4-((sub)phenylthio)butanoate 6. Reagents and
conditions: KOH, methanol, 25 °C, 8–14 h, 94–98%.
12g in good yields (Scheme 4).
To assess whether 2-sulfinyl analogues could reduce cytotoxic-
ity and increase potency, 9b1, 9b2, 12f6, etc. were oxidated using
sodium perborate to provide seven target compounds 13b–13f
(Scheme 5).
methoxylbenzaldehyde (1) depicted in Scheme 1. The commer-
cially available compound 1 was treated with benzyl chloride 2
to give the compound 3,9 which was nitrated with a typical nitra-
tion reagent, fuming nitric acid, to provide 2-nitrobenzaldehyde
4.10 Activated zinc powder and catalytic formamide efficiently re-
duced the nitro group prior to the aldehyde group to get 2-amino-
benzaldehyde 5. The thioether 6 was prepared via etherification of
ethyl 4-chloroacetoacetate 11 with the corresponding thiophenol
10 (Scheme 2).8 Treatment of 2-aminobenzaldehyde 5 with 6 in
the presence of piperidine afforded the key intermediate, ethyl
quinoline-3-carboxylate 7,11 which was subsequently deprotected
with a 2:1 mixture of acetic acid and hydrochloric acid to produce
compound 8. The target compounds, 9a–9g, were then synthesized
via a Mannich reaction as previously demonstrated.7,8 When acetic
acid was employed as the solvent, the reaction generated a mixture
of products, including the desired mono-Mannich derivative and
the bis-Mannich byproduct A (Scheme 3).12 An alternative ap-
proach was explored, and the appropriate aliphatic amines were
treated with compound 8 and 37% aqueous formaldehyde in meth-
anol solvent to yield the expected compounds, 9a–9g.
3. Anti-HBV analysis
All of the target compounds were tested in vitro in HepG2.2.15
cells for anti-HBV activity and cytotoxicity. These compounds’
properties are summarized in Table 1, in which they are compared
to those of lamivudine. With the exception of 9c2, 12g6, and 13c2,
all of the compounds exhibited an overall inhibition of virion
including HBV antigen secretion and HBV DNA replication.
Although this series of compounds did not display any obvious
SAR for anti-HBsAg and anti-HBeAg activity, six structural compo-
nents, represented by 2-(fluoro-substituted phenyl)quinoline
derivatives 9b1, 9b2, 9f4, 9f5, 12b7, and 12f6, were required for
high potency (IC50 < 20 lM). Of these compounds, 9f4 and 9b2 were
the most potent inhibitors of HBsAg production (IC50 = 5.8 lM,
SI = 13.7) and HBeAg production (IC50 = 9.3 lM, SI = 5.1),
respectively.
Twelve compounds (9a3, 9b1,9b2, 9f3, 12b6, 12b7, 12f6, 13b1,
13b2, 13b6, 13f2, and 13f6) exhibited more potent inhibition of
HBV DNA replication (10- to 66-fold) than the control drug, lami-
Imidazole or methylimidazole could not be introduced into the
5-position by a direct Mannich reaction with 8. Alternatively, it can
Scheme 3. The Mannich reaction of 8a in acetic acid.

6524
Y. Liu et al. / Bioorg. Med. Chem. 16 (2008) 6522–6527
vudine. Among these, 9f3, 12b6, 12f6, 13b2, 13b6, and 13f6 pos-
sessed selectivity indices from 5.6 (9f3) to 34.1 (13b6), which were
comparable to or higher than that of lamivudine (7.1).
The anti-HBV activities of compounds 9b1, 12b6, 9f3, 12f6, and
13b6, in which an electron-withdrawing fluoride group was intro-
duced at the phenyl moiety of 2-position, were superior to those
with electron-donating methoxy group or no substitution (9d1,
12e6, 12g6, 13e6, and 9a3). However, when the carbon was replaced
with nitrogen at 2-position of phenyl ring, the anti-HBV activities
were eliminated and the cytotoxicities were enhanced evidently
(TC50s of 9c2 and 13c2 were 17.5 lM and 9.4 lM, respectively).
Interestingly, the IC50 values and SIs of 3,4-difluoro derivatives,
such as 12f6, 13f2, and 13f6, were not better than those of monoflu-
orinated derivatives 12b6, 13b2, and 13b6.
Different biological properties were observed when a variety of
basic moieties were introduced via a Mannich reaction at the 5-po-
sition. Heterocyclic basic groups, such as pyrrolidinyl, piperidinyl,
and imidazolyl groups, were preferred at this position. However,
when morpholinyl and 4-methyl piperazinyl were introduced into
5-position of compounds, such as 9f4 and 9f5, the anti-HBV activi-
ties were eliminated.
Scheme 4. Synthesis of compounds 12b–12g. Reagents and conditions: (a) 33%
solution of dimethylamine, 37% aqueous formaldehyde, methanol, 40 °C, 2–5 h, 68–
72%; (b) 1H-imidazole or 2-methyl-1H-imidazole, hydrochloric acid, 1,4-dioxane,
75–80 °C, 2–3 h, 60–73%.
While replacement of the sulfur group with a sulfinyl did not
evidently alter the drugs’ cytotoxicities, it either increased or
maintained the drugs’ anti-HBV activity (compounds 9b1 vs.
13b1, 12b6 vs. 13b6, 9b2 vs. 13b2, and 12f6 vs. 13f6). The most
promising compound, 13b6, which bore an imidazolyl group at
the 5-position and a 4-fluorophenylsulifinyl at the 2-position, pos-
sessed exceptionally high-potency anti-HBV activity with an IC50 of
4.7 lM, a 66-fold improvement over lamivudine (IC50 = 311.2 lM).
4. Conclusion
In summary,
a series of 6-hydroxyquinoline-3-carboxylate
derivatives based on 5-hydroxy-1H-indole-3-carboxylate deriva-
tives (I, Fig. 1) were synthesized in seven to nine steps and were
assayed for their anti-HBV activity and cytotoxicity in vitro. As
hoped, the reported anti-HBV properties of I also extended to
this new series, and quinoline derivatives have shown higher
Scheme 5. Synthesis of compounds 13b–13f. Reagents and conditions: (a) sodium
perborate, sodium tungstate, acetic acid, 40 °C, 3–5 h, 56–74%.
Table 1
Anti-HBV activity and cytotoxicity of target compounds in vitro
Compound
TC50 (lM)a
HBsAg
HBeAg
HBV DNA replication
IC50 (lM) SI
1.3
IC50 (lM)b
SIc
—
6.9
5.6
IC50 (lM)
SI
d
9a3
9b1
9b2
9c2
60.7
120.2
47.3
—
—
—
9.3
—
—
—
5.1
—
45.2
32.8
10.2
17.4
8.5
—
3.7
4.6
17.5
—
—
—
9d1
9f3
9f4
9f5
58.5
135.1
79.7
55.3
201.5
78.5
186.5
132.6
188.8
90.4
166.3
160.3
9.4
194.2
16.5
90.8
—
—
5.8
11.2
42.8
—
—
—
—
36.5
25.1
26.2
—
33.1
—
—
—
13.7
4.9
6.0
—
—
—
—
2.5
6.6
6.1
—
5.9
—
20.5
—
10.9
—
2.9
—
7.3
—
—
24.1
—
—
41.7
30.6
—
5.6
—
—
6.2
2.6
—
14.1
—
4.1
15.5
34.1
—
12b6
12b7
12e6
12f6
12g6
13b1
13b2
13b6
13c2
13e6
13f2
13f6
Lamivudine
—
—
16.2
65.1
16.4
—
4.8
2.9
8.1
—
—
9.4
—
—
—
21.9
10.7
4.7
—
—
21.1
98.1
—
—
—
7.9
1.6
—
—
—
—
5.3
3.1
7.3
7.1
—
—
—
—
—
—
—
—
12.5
311.2
2213.8
a
TC50 is 50% cytotoxic concentration in HepG2.2.15 cells.
IC50 is 50% inhibitory concentration.
Selectivity index (SI: TC50/IC50).
b
c
d
No antiviral activity at the concentration lower than its TC50
.

Y. Liu et al. / Bioorg. Med. Chem. 16 (2008) 6522–6527
6525
anti-HBeAg activities than indole derivatives. Most of these com-
pounds demonstrated potential HBV inhibition, and compound
13b6 was the most potent, highly specific inhibitor of HBV
DNA replication in cell culture.
Inhibition assay data reported in this study showed that the
substituents on the phenyl ring of 2-position might play an impor-
tant role in the tested drugs’ activities. Fluoride could enhance the
inhibition of HBV DNA replication, while 2-pyridyl derivatives
could eliminate the anti-HBV activities completely. The different
Mannich basic groups placed at the 5-position demonstrated sig-
nificant distinctness of anti-HBV effects. Oxidation of the com-
pounds’ sulfide into a sulfinyl group had little influence on anti-
HBV activity and cytotoxicity.
Based on the study, the further structural alterations were
focused on 5-position and the phenyl at 2-position of quinoline
ring. The various halogens, such as chlorine, bromine, and io-
dine, would be introduced into phenyl which also could be re-
placed by S-heteroaromatic. Some N-heterocycles, such as
pyrrolyl, pyrrolinyl, imidazolinyl, etc., would be incorporated
into position 5.
perature for 30 min. Compound 8 (0.01 mol) was added, and the
mixture was heated to 40 °C for 2–5 h. The MeOH was removed
under vacuum, and the residue was poured into water (100 mL).
The resulting mixture was adjusted to pH 7 with 20% hydrochloric
acid, and the solid product was collected by filtration and washed
with water. The crude product was recrystallized from acetone to
obtain compounds 9a–9g.
5.1.3.1. Ethyl 6-hydroxy-7-methoxy-2-((phenylthio)methyl)-5-
((piperidin-1-yl)methyl)quinoline-3-carboxylate (9a3). White
powder (3.5 g, 75%); Mp: 174–176 °C; MS [MH+] (m/z): 467.2; 1H
NMR (DMSO-d6): d 1.38 (t, 3H, J = 7.1 Hz, –CH2CH3), 1.85 (m, 6H,
piperidinyl), 2.67 (m, 4H, piperidinyl), 4.07 (s, 3H, –OCH3), 4.35
(q, 2H, J = 7.1 Hz, –CH2CH3), 4.41 (s, 2H, –CH2N–), 4.79 (s, 2H,
–CH2S–), 7.16 (m, 3H, –PhH), 7.28 (m, 2H, –PhH), 7.21 (s, 1H,
C8–H), 8.67 (s, 1H, C4–H); IR (KBr, cmÀ1): 3421.9 (OH), 1703.4
(C@O).
5.1.3.2. Ethyl
2-((4-fluorophenylthio)methyl)-5-((dimethyla-
mino)methyl)-6-hydroxy-7-methoxyquinoline-3-carboxylate
(9b1). White powder (3.2 g, 72%); Mp: 183–185 °C; MS [MH+] (m/
z): 445.1; 1H NMR (DMSO-d6): d 1.37 (t, 3H, J = 7.2 Hz, –CH2CH3),
2.80 (d, 6H, J = 4.0 Hz, –N(CH3)2), 4.06 (s, 3H, –OCH3), 4.36 (q, 2H,
J = 7.2 Hz, –CH2CH3), 4.72 (s, 2H, –CH2S–), 4.74 (d, 2H, J = 4.0 Hz,
–CH2N–), 7.13 (t, 2H, J = 8.7 Hz, –PhH), 7.41 (q, 2H, J = 5.5 Hz, –
PhH), 7.53 (s, 1H, C8–H), 8.87 (s, 1H, C4–H); IR (KBr, cmÀ1):
3431.2 (OH), 1701.4 (C@O).
5. Experimental
5.1. Chemistry
Melting points were measured with a Büchi Melting Point B-
540 apparatus (Büchi Labortechnik, Flawil, Switzerland) and are
uncorrected. Mass spectra (MS) were taken in ESI mode on Agilent
1100 LC–MS (Agilent, Palo Alto, CA, USA). 1H NMR spectra were
performed using Bruker 300 MHz and 600 MHz spectrometers
(Bruker Bioscience, Billerica, MA, USA) with TMS as an internal
standard. Compounds 3, 4, and thioether 6 were synthesized in
accordance with literature procedures.9,10
5.1.3.3. Ethyl 2-((4-fluorophenylthio)methyl)-6-hydroxy-7-
methoxy-5-((pyrrolidin-1-yl)methyl)quinoline-3-carboxylate
(9b2). White powder (3.5 g, 74%); Mp: 179–181 °C; MS [MH+] (m/
z): 471.0; 1H NMR (DMSO-d6): d 1.43 (t, 3H, J = 6.9 Hz, –CH2CH3),
1.96 (s, 4H, –pyrrolidinyl), 2.90 (s, 4H, –pyrrolidinyl), 4.03 (s, 3H,
–OCH3), 4.39 (s, 2H, –CH2N–), 4.41 (q, 2H, J = 6.9 Hz, –CH2CH3),
4.76 (s, 2H, –CH2S–), 7.12 (t, 2H, J = 8.6 Hz, –PhH), 7.39 (q, 2H,
J = 5.4 Hz, –PhH), 7.32 (s, 1H, C8–H), 8.65 (s, 1H, C4–H); IR (KBr,
cmÀ1): 3419.5 (OH), 1708.1 (C@O).
5.1.1. 2-Amino-5-benzyloxy-4-methoxybenzaldehyde (5)
A mixture of 4 (28.7 g, 0.1 mol), formamide (2 mL) and absolute
ethanol (60 mL) was heated with vigorous stirring until the solu-
tion boiled gently. Heating was discontinued, and 5 g portions of
32.5 g (0.5 mol) of activated zinc dust were added with sufficient
frequency to sustain boiling while the reaction refluxed for 8 h.
The hot mixture was filtered, and the filtrate was cooled to room
temperature. The yellow crystals were filtered, washed with etha-
nol, and dried to yield 5 (12.8 g, 50%). Mp: 182–184 °C; MS [MH+]
(m/z): 258.1.
5.1.3.4. Ethyl 6-hydroxy-7-methoxy-2-((pyridin-2-ylthio)methyl)-
5-((pyrrolidin-1-yl)methyl)quinoline-3-carboxylate
(9c2). White
powder (3.2 g, 70%); Mp: 142–143 °C; MS [MH+] (m/z): 454.1; 1H
NMR (DMSO-d6): d 1.43 (t, 3H, J = 7.1 Hz, –CH2CH3), 1.89 (s, 4H, –pyr-
rolidinyl), 2.81 (s, 4H, –pyrrolidinyl), 3.92 (s, 3H, –OCH3), 4.11 (s, 2H,
–CH2N–), 4.40 (q, 2H, J = 7.1 Hz, –CH2CH3), 4.72 (s, 2H, –CH2S–), 7.51
(m, 3H, –pyridyl+C8–H), 7.65 (m, 2H, –pyridyl), 8.66 (s, 1H, C4-H); IR
(KBr, cmÀ1): 3430.5 (OH), 1704.6 (C@O).
5.1.2. General procedure for the synthesis of ethyl 6-hydroxy-7-
methoxy-2-((arylthio)methyl)quinoline-3-carboxylate
derivatives (8a–8g)
5.1.3.5. Ethyl 2-((2-methoxyphenylthio)methyl)-5-((dimethyl-
amino)methyl)-6-hydroxy-7-methoxyquinoline-3-carboxylate
(9d1). White powder (3.1 g, 68%); Mp: 171–172 °C; MS [MH+] (m/
z): 457.2; 1H NMR (DMSO-d6): d 1.42 (t, 3H, J = 7.1 Hz, –CH2CH3),
2.91 (s, 6H, –N(CH3)2), 3.80 (s, 3H, –OCH3), 4.03 (s, 3H, –OCH3),
4.31 (s, 2H, –CH2N–), 4.42 (q, 2H, J = 7.1 Hz, –CH2CH3), 4.77 (s,
2H, –CH2S–), 6.81 (m, 2H, –PhH), 7.15 (m, 1H, –PhH), 7.27 (s, 1H,
C8–H), 7.46 (m, 1H, –PhH), 8.62 (s, 1H, C4–H); IR (KBr, cmÀ1):
3427.1 (OH), 1700.8 (C@O).
A mixture of o-aminobenzaldehyde 5 (12.8 g, 0.05 mol), ethyl 3-
oxo-4-(arylthio)butanoate
6
(0.05 mol), piperidine (0.5 mL,
0.005 mol) and isopropanol (10 mL) was stirred at reflux for 12–
15 h and thencooled toroomtemperature. The solventwas removed
under vacuum, and 20 mL of water was added. The aqueous solution
was extracted with CH2Cl2, and the organic phase was washed with
saturated sodium chloride solution and water. The organic phase
was dried (Na2SO4) and evaporated to yield the crude compounds
7 as red oil. A 2:1 mixture of acetic acid and HCl acid (25 mL) was
added directly to the red oil 7, and the resulting solution was heated
at 80 °C for 5–8 h. The reaction mixture was cooled to room temper-
ature, and the isolated solids were filtered and dried to give com-
pounds 8a–8g as buff to yellow powder.
5.1.3.6. Ethyl 2-((3,4-difluorophenylthio)methyl)-6-hydroxy-7-
methoxy-5-((piperidin-1-yl)methyl)quinoline-3-carboxylate
(9f3). Yellow powder (3.6 g, 72%); Mp: 168–170 °C; MS [MH]+ (m/
z): 503.2; 1H NMR (DMSO-d6): d 1.35 (t, 3H, J = 7.1 Hz, –CH2CH3),
1.53 (s, 6H, –piperidinyl), 2.64 (s, 4H, –piperidinyl), 3.95 (s, 3H, –
OCH3), 4.06 (s, 2H, –CH2N–), 4.32 (q, 2H, J = 7.1 Hz, –CH2CH3),
4.70 (s, 2H, –CH2S–), 7.14–7.18 (m, 2H, –PhH), 7.40–7.45 (m, 2H,
–PhH+C8–H) 8.67 (s, 1H, C4–H); IR (KBr, cmÀ1): 3415.0 (OH),
1708.1 (C=O).
5.1.3. General procedure for the synthesis of compounds 9a–9g
A solution of aliphatic amine (0.025 mol), 37% HCHO (3 mL,
0.03 mol) and 36% HCl (0.1 mL) in MeOH was stirred at room tem-

6526
Y. Liu et al. / Bioorg. Med. Chem. 16 (2008) 6522–6527
5.1.3.7. Ethyl
2-((3,4-difluorophenylthio)methyl)-6-hydroxy-
lyl-H), 7.04 (s, 1H, –imidazolyl-H), 7.33 (s, 1H, C8–H), 7.58 (m, 3H,
–PhH), 7.67 (s, 1H, –imidazolyl-H), 8.52 (s, 1H, C4–H); IR (KBr,
cmÀ1): 3427.4 (OH), 1703.5 (C@O).
7-methoxy-5-(morpholinomethyl)quinoline-3-carboxylate
(9f4). White powder (4.1 g, 81%); Mp: 191–193 °C; MS [MH+] (m/z):
505.2;1H NMR (DMSO-d6): d 1.35 (t, 3H, J = 7.1 Hz, –CH2CH3), 3.35 (s,
4H, –morpholino), 3.54 (s, 4H, –morpholino), 3.95 (s, 3H, –OCH3),
4.38 (s, 2H, –CH2N–), 4.41 (q, 2H, J = 7.1 Hz, –CH2CH3), 4.65 (s, 2H,
–CH2S–), 7.12–7.35 (m, 3H, –PhH), 7.29 (s, 1H, C8–H), 8.75 (s, 1H,
C4–H); IR (KBr, cmÀ1): 3420.1 (OH), 1708.4 (C@O).
5.1.4.5. Ethyl 5-((1H-imidazole-1-yl)methyl)-2-((3,4-dimethoxy
phenylthio)methyl)-6-hydroxy-7-methoxyquinoline-3-carbox-
ylate (12g6). White powder (3.1 g, 61%); Mp: 179–181 °C; MS
[MH+] (m/z): 510.2; 1H NMR (DMSO-d6): d 1.35 (t, 3H, J = 7.1 Hz, –
CH2CH3), 3.55 (s, 3H, –OCH3), 3.68 (s, 3H, –OCH3), 4.01 (s, 3H, –OCH3),
4.29 (q, 2H, J = 7.1 Hz, –CH2CH3), 4.60 (s, 2H, –CH2S–), 5.62 (s, 2H, –
CH2N–), 6.82 (m, 3H, –PhH), 7.05 (s, 1H, –imidazolyl-H), 7.16 (s,
1H, –imidazolyl-H), 7.37 (m, 1H, C8-H), 7.76 (s, 1H, –imidazolyl-H),
8.80 (s, 1H, C4-H); IR (KBr, cmÀ1): 3409.1 (OH), 1698.3 (C@O).
5.1.3.8. Ethyl 2-((3,4-difluorophenylthio)methyl)-6-hydroxy-7-
methoxy-5-((4-methylpiperazin-1-yl)methyl)quinoline-
3-carboxylate (9f5). Pale yellow powder (3.7 g, 71%); Mp: 186–
188°C; MS [MH+] (m/z): 518.2; 1H NMR (DMSO-d6): d 1.41 (t, 3H,
J = 7.0 Hz, –CH2CH3), 2.36 (s, 3H, –NCH3), 2.78 (m, 8H, –piperazi-
nyl), 4.01 (s, 3H, –OCH3), 4.35 (s, 2H, –CH2N–), 4.46 (q, 2H,
J = 7.0 Hz, –CH2CH3), 4.68 (s, 2H, –CH2S–), 7.15–7.36 (m, 3H, –
PhH), 7.33 (s, 1H, C8–H), 8.71 (s, 1H, C4–H); IR (KBr, cmÀ1):
3427.5 (OH), 1702.3 (C@O).
5.1.5. General procedure for the synthesis of the target
compounds 13b–13f
A solution of compound 9 or 12 (0.01 mol) and sodium perbo-
rate (1.7 g, 0.011 mol) in acetic acid (40 mL) was heated at 40 °C
for 3–5 h. The solvent was evaporated under vacuum, and the res-
idue was poured into water (100 mL). The resulting mixture was
adjusted to pH 8 with 20% NaOH, and the precipitate was filtered,
washed with acetone, dried, and recrystallized from ethanol to ob-
tain the target compounds 13b–13f.
5.1.4. General procedure for the synthesis of compounds12b–12g
Either 1H-imidazole or 2-methyl-1H-imidazole (0.05 mol), 36%
HCl (4.3 mL, 0.05 mol) and dimethylamino analogues
(0.01 mol) were dissolved in 1,4-dioxane (30 mL) and stirred at
75–80 °C for 2–3 h. After cooling to room temperature, the result-
ing mixture was poured into water (100 mL), and the precipitate
was collected by filtration and washed with water and acetone.
The crude product was recrystallized from ethanol/water to obtain
the desired compounds 12b–12g.
9
5.1.5.1. Ethyl 2-((4-fluorophenylsulfinyl)methyl)-5-((dimethyl-
amino)methyl)-6-hydroxy-7-methoxyquinoline-3-carboxylate
(13b1). Yellow powder (3.4 g, 74%); Mp: 171–173 °C; MS [MH+]
(m/z): 461.3; 1H NMR (DMSO-d6):
d 1.36 (t, 3H, J = 7.0 Hz,
–CH2CH3), 2.79 (s, 6H, –N(CH3)2), 3.97 (s, 3H, –OCH3), 4.32 (q,
2H, J = 7.0 Hz, –CH2CH3), 4.69 (d, 1H, J = 12.7 Hz, –CH2SO–), 4.75
(s, 2H, –CH2N–), 4.76 (d, 1H, J = 12.7 Hz, –CH2SO–), 7.21 (t, 2H,
J = 8.8 Hz, –PhH), 7.40 (q, 2H, J = 5.5 Hz, –PhH), 7.49 (s, 1H, C8–H),
8.82 (s, 1H, C4–H); IR (KBr, cmÀ1): 3429.6 (OH), 1712.5 (C@O).
5.1.4.1. Ethyl 5-((1H-imidazole-1-yl)methyl)-2-((4-fluorophen-
ylthio)methyl)-6-hydroxy-7-methoxyquinoline-3-carboxylate
(12b6). Pale yellow powder (3.1 g, 66%); Mp: 153–155 °C; MS
[MH+] (m/z): 468.1; 1H NMR (DMSO-d6): d 1.34 (t, 3H, J = 7.0 Hz,
–CH2CH3), 4.01 (s, 3H, –OCH3), 4.30 (q, 2H, J = 7.0 Hz, –CH2CH3),
4.66 (s, 2H, –CH2S–), 5.60 (s, 2H, –CH2N–), 6.81 (s, 1H, –imidazo-
lyl-H), 7.02 (s, 1H, –imidazolyl-H), 7.09 (t, 2H, J = 8.7 Hz, –PhH),
7.38 (m, 3H, –PhH+C8–H), 7.68 (s, 1H, –imidazolyl-H), 8.79 (s, 1H,
C4-H); IR (KBr, cmÀ1): 3416.8 (OH), 1704.1 (C@O).
5.1.5.2. Ethyl 2-((4-fluorophenylsulfinyl)methyl)-6-hydroxy-7-
methoxy-5-((pyrrolidin-1-yl)methyl)-quinoline-3-carboxylate
(13b2). Pale yellow powder (2.7 g, 56%); Mp: 163–165 °C; MS
[MH+] (m/z): 487.1; 1H NMR (DMSO-d6): d 1.34 (t, 3H, J = 7.1 Hz,
–CH2CH3), 1.93 (s, 4H, –pyrrolidinyl), 2.77 (s, 4H, –pyrrolidinyl),
4.01 (s, 3H, –OCH3), 4.37 (q, 2H, J = 7.1 Hz, –CH2CH3), 4.72 (d, 1H,
J = 12.6 Hz, –CH2SO–), 4.77 (s, 2H, –CH2N–), 4.93 (d, 1H,
J = 12.6 Hz, –CH2SO–), 7.31 (t, 2H, J = 8.7 Hz, –PhH), 7.51 (q, 2H,
J = 5.5 Hz, –PhH), 7.35 (s, 1H, C8–H), 8.69 (s, 1H, C4–H); IR (KBr,
cmÀ1): 3422.1 (OH), 1708.6 (C@O).
5.1.4.2. Ethyl 2-((4-fluorophenylthio)methyl)-6-hydroxy-7-
methoxy-5-(2-methyl-1H-imidazol-1-yl)quinoline-3-carboxy-
late (12b7). Yellow powder (3.4 g, 71%); Mp: 167–169 °C; MS [MH+]
(m/z): 482.0; 1H NMR (DMSO-d6): d 1.34 (t, 3H, J = 7.0 Hz, –CH2CH3),
2.44 (s, 3H, –CCH3), 4.04 (s, 3H, –OCH3), 4.31 (q, 2H, J = 7.0 Hz,
–CH2CH3), 4.69 (s, 2H, –CH2S–), 5.47 (s, 2H, –CH2N–), 6.64
(s, 1H, –imidazolyl-H), 6.70 (s, 1H, –imidazolyl-H), 7.11 (t, 2H,
J = 8.7 Hz, –PhH), 7.28 (s, 1H, C8–H), 7.40 (q, 2H, J = 5.4 Hz, –PhH),
8.68 (s, 1H, C4–H); IR (KBr, cmÀ1): 3417.5 (OH), 1703.7 (C@O).
5.1.5.3. Ethyl 5-((1H-imidazol-1-yl)methyl)-2-((4-fluorophenyl-
sulfinyl)methyl)-6-hydroxy-7-methoxyquinoline-3-carboxylate
(13b6). Yellow powder (3.4 g, 70%); Mp: 193–195 °C; MS [MH+]
(m/z): 484.1; 1H NMR (DMSO-d6):
d 1.35 (t, 3H, J = 7.1 Hz,
5.1.4.3. Ethyl 5-((1H-imidazole-1-yl)methyl)-2-((3-methoxy-
phenylthio)methyl)-6-hydroxy-7-methoxyquinoline-3-car
boxylate (12e6). White powder (3.5 g, 73%); Mp: 173–175 °C;
MS [MH+] (m/z): 480.1; 1H NMR (DMSO-d6): d 1.32 (t, 3H,
J = 7.0 Hz, –CH2CH3), 3.65 (s, 3H, –OCH3), 3.86 (s, 3H, –OCH3),
4.23 (q, 2H, J = 7.0 Hz, –CH2CH3), 4.61 (s, 2H, –CH2S–), 5.43 (s,
2H, –CH2N–), 6.74 (s, 1H, –imidazolyl-H), 6.95 (s, 1H, –imidazo-
lyl-H), 7.10 (m, 4H, –PhH+C8–H), 7.38 (t, 1H, J = 7.5 Hz, –PhH),
7.60 (s, 1H, –imidazolyl-H), 8.40 (s, 1H, C4–H); IR (KBr, cmÀ1):
3418.5 (OH), 1710.2 (C@O).
–CH2CH3), 4.06 (s, 3H, –OCH3), 4.32 (q, 2H, J = 7.1 Hz, –CH2CH3),
4.73 (d, 1H, J = 12.4 Hz, –CH2SO–), 4.92 (d, 1H, J = 12.4 Hz,
–CH2SO–), 5.88 (s, 2H, –CH2N–), 7.37 (t, 2H, J = 8.7 Hz, –PhH),
7.48 (s, 1H, –imidazolyl), 7.57 (m, 3H, –imidazolyl+–PhH), 7.64
(s, 1H, C8–H), 8.83 (s, 1H, –imidazolyl), 9.13 (s, 1H, C4–H); IR
(KBr, cmÀ1): 3438.2 (OH), 1721.5 (C@O).
5.1.5.4. Ethyl 6-hydroxy-7-methoxy-2-((pyridin-2-ylsulfinyl)-
methyl)-5-((pyrrolidin-1-yl)methyl)quinoline-3-carboxylate
(13c2). Yellow powder (3.0 g, 64%); Mp: 146–148 °C; MS [MH+]
(m/z): 470.2; 1H NMR (DMSO-d6):
d 1.32 (t, 3H, J = 7.1 Hz,
5.1.4.4. Ethyl 5-((1H-imidazole-1-yl)methyl)-2-((3,4-difluoro-
phenylthio)methyl)-6-hydroxy-7-methoxyquinoline-3-carbox-
ylate (12f6). White powder (2.9 g, 60%); Mp: 158–160 °C; MS
[MH+] (m/z): 486.3; 1H NMR (DMSO-d6): d 1.33 (t, 3H, J = 7.0 Hz,
–CH2CH3), 3.89 (s, 3H, –OCH3), 4.25 (q, 2H, J = 7.0 Hz, –CH2CH3),
4.67 (s, 2H, –CH2S–), 5.57 (s, 2H, –CH2N–), 6.75 (s, 1H, –imidazo-
–CH2CH3), 1.91 (s, 4H, –pyrrolidinyl), 2.83 (s, 4H, –pyrrolidinyl),
4.01 (s, 3H, –OCH3), 4.28 (s, 2H, –CH2N–), 4.37 (q, 2H, J = 7.1 Hz,
–CH2CH3), 4.74 (d, 1H, J = 12.7 Hz, –CH2SO–), 4.82 (d, 1H,
J = 12.7 Hz, –CH2SO–), 7.29 (s, 1H, C8–H), 7.52 (m, 2H, –pyridyl),
7.74 (m, 2H, –pyridyl), 8.58 (s, 1H, C4–H); IR (KBr, cmÀ1): 3429.2
(OH), 1713.2 (C@O).

Y. Liu et al. / Bioorg. Med. Chem. 16 (2008) 6522–6527
6527
5.1.5.5. Ethyl 5-((1H-imidazol-1-yl)methyl)-2-((3-methoxy-
phenylsulfinyl)methyl)-6-hydroxy-7-methoxyquinoline-3-car-
boxylate (13e6). Yellow powder (3.5 g, 67%); Mp: 176–178 °C; MS
[MH+] (m/z): 496.2; 1H NMR (DMSO-d6): d 1.32 (t, 3H, J = 7.0 Hz, –
CH2CH3), 3.65 (s, 3H, –OCH3), 3.86 (s, 3H, –OCH3), 4.25 (q, 2H,
J = 7.0 Hz, –CH2CH3), 4.61 (d, 1H, J = 12.2 Hz, –CH2SO–), 4.73 (d,
1H, J = 12.2 Hz, –CH2SO–), 5.42 (s, 2H, –CH2N–), 6.75 (s, 1H, –imid-
azolyl), 6.95 (s, 1H, –imidazolyl), 7.05 (m, 4H, –PhH+C8–H), 7.41 (t,
1H, J = 7.5 Hz, –PhH), 7.60 (s, 1H, –imidazolyl), 8.39 (s, 1H, C4–H);
IR (KBr, cmÀ1): 3420.1 (OH), 1718.4 (C@O).
HepG2.2.15 cells by semiquantitative enzyme immunoassay
(EIA) methods using commercial kits (HBsAg, Abbott Laborato-
ries; HBeAg, Diasorin, Inc.) as previously described.8 Intracellular
HBV DNA levels were measured by quantitative Southern blot
hybridization.
5.2.2. Cytotoxicity assay
The cytotoxicities of compounds 9a–13f to HepG2.2.15 cells
were assessed by MTT assay.15 Briefly, HepG2.2.15 cells were
treated as described above. Untreated control cultures were also
maintained on each 96-well plate. Toxicity was determined by
measuring neutral red dye uptake, as determined from the cells’
5.1.5.6. Ethyl 2-((3,4-difluorophenylsulfinyl)methyl)-6-hydro-
xy-7-methoxy-5-((pyrrolidin-1-yl)methyl)quinoline-3-carboxy-
late (13f2). Yellow powder (3.2 g, 63%); Mp: 181–183°C; MS [MH+]
(m/z): 505.2; 1H NMR (DMSO-d6): d 1.33 (t, 3H, J = 7.1 Hz, –
CH2CH3), 1.93 (s, 4H, –pyrrolidinyl), 2.77 (s, 4H, –pyrrolidinyl),
3.94 (s, 3H, –OCH3), 4.27 (q, 2H, J = 7.1 Hz, –CH2CH3), 4.65 (d, 1H,
J = 12.7 Hz, –CH2SO–), 4.76 (s, 2H, –CH2N–), 4.79 (d, 1H,
J = 12.7 Hz, –CH2SO–), 7.35 (s, 1H, C8–H), 7.56 (m, 3H, –PhH),
8.42 (s, 1H, C4-H); IR (KBr, cmÀ1): 3417.1 (OH), 1709.3 (C@O).
510 nm absorbance relative to untreated cells,
treatment.
9 days of
Acknowledgments
This work was supported by a grant from the National Natural
Science Foundation of China (No. 30672519) and a grant from the
Doctor Foundation of Liaoning Province, China (No. 20061027).
5.1.5.7. Ethyl 5-((1H-imidazol-1-yl)methyl)-2-((3,4-difluoro-
phenylsulfinyl)methyl)-6-hydroxy-7-methoxyquinoline-3-car
boxylate (13f6). Pale yellow powder (3.1 g, 62%); Mp: 183–
185 °C; MS [MH+] (m/z): 502.1; 1H NMR (DMSO-d6): d 1.33 (t,
3H, J = 7.0 Hz, –CH2CH3), 3.87 (s, 3H, –OCH3), 4.25 (q, 2H,
J = 7.0 Hz, –CH2CH3), 4.65 (d, 1H, J = 12.3 Hz, –CH2SO–), 4.83
(d, 1H, J = 12.3 Hz, –CH2SO–), 5.45 (s, 2H, –CH2N–), 6.75 (s, 1H,
–imidazolyl), 7.04 (s, 1H, –imidazolyl), 7.33 (s, 1H, C8–H), 7.55
(m, 4H, –PhH+–imidazolyl), 8.44 (s, 1H, C4–H); IR (KBr, cmÀ1):
3421.6 (OH), 1709.3 (C@O).
References and notes
1. Custer, B.; Sullivan, S. D.; Hazlet, T. K.; Iloeje, U.; Veenstra, D. L.; Kowdley, K. V. J.
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Hepatology 1988, 8, 1651.
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2552.
5.2. Pharmacology
9. Sumiaki, K.; Hirofumi, T.; Tetsuya, K.; Kiyoharu, N.; Manabu, N. Tetrahedron
2004, 60, 4901.
5.2.1. In vitro anti-HBV activity assay
10. Patteux, C.; Foucout, L.; Bohn, P.; Dupas, G.; Leprince, J.; Tonon, M. C.; Dehouck,
B.; Marsais, F.; Papamicael, C.; Levacher, V. Org. Biomol. Chem. 2006, 4, 817.
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12. The spectral data for compound A: White powder; Mp: 184–186; MS [MH+]
The anti-HBV activities of compounds 9a–13f were evaluated
in HepG2.2.15 cells by previously reported methods.13,14 This as-
say included measurement of the drugs’ abilities to inhibit the
production of both HBsAg and HBeAg and to inhibit HBV DNA
replication. Briefly, confluent cell cultures in 96-well flat-bot-
tomed tissue culture plates were treated with various doses of
the test compounds or lamivudine (purchased by Glaxo & Wel-
come Co.) in RPMI 1640 medium supplemented with 2% fetal
bovine serum. Medium was changed daily with fresh test
compounds and positive control for 8 days. Extracellular HBV
surface- and e-antigen secretion levels were evaluated for
(m/z): 564.0; 1H NMR (DMSO-d6):
d 1.28–1.46 (m, 10H, –CH2CH3+–
piperidinyl), 1.54–2.14 (m, 6H, –piperidinyl), 2.16 (t, 2H, –piperidinyl), 2.36
(d, 2H, –piperidinyl), 2.50–2.55 (m, 3H, –piperidinyl), 2.82 (dd, J = 5.3, 12.7 Hz,
1H, –CHCH2–), 3.26 (dd, J = 8.7, 12.7 Hz, 1H, –CHCH2–), 3.98 (s, 3H, –OCH3),
4.07 (s, 2H, –CH2N–), 4.30 (q, J = 7.1 Hz, 2H, –CH2CH3), 5.78 (dd, J = 5.3, 8.7 Hz,
1H, –CHS–), 7.23–7.26 (m, 2H, C8-H+–PhH), 7.29–7.40 (m, 2H, –PhH), 7.42 (d,
2H, –PhH), 8.71 (s, 1H, C4–H). IR (KBr, cmÀ1) 3437 (OH), 1710 (C@O).
13. Korba, B. E.; Milman, G. Antiviral Res. 1991, 15, 217.
14. Sells, M. A.; Zelent, A. Z.; Shvartsan, M.; Acs, G. Biochem. Biophys. Res. Commun.
1988, 156, 1144.
15. Mosmann, T. J. Immunol. Methods 1983, 65, 55.