Synthesis and the biological evaluation of arylnaphthalene lignans as anti-hepatitis B virus agents

Article abstract of DOI:10.1016/j.bmc.2009.12.038

We have previously shown that helioxanthin can suppress human hepatitis B virus gene expression. A series of helioxanthin analogues were synthesized and evaluated for their anti-hepatitis B virus activity. Modifications at the lactone rings and methylenedioxy unit of helioxanthin can modulate the antiviral activity. Among them, compound 32 is the most effective anti-HBV agent. Compound 32 can suppress the secretion of viral surface antigen and e antigen in HepA2 cells with EC₅₀ values of 0.06 and 0.14 μM, respectively. Compound 32 not only inhibited HBV DNA with wild-type and lamivudine-resistant strain but also suppressed HBV mRNA, core protein and viral promoters. In this study, a full account of the preparation, structure-activity relationships of helioxanthin analogues, and the possible mechanism of anti-HBV activity of this class of compounds are presented. This type of compounds possesses unique mode of action differing from existing therapeutic drugs. They are potentially new anti-HBV agents.

Full text of DOI:10.1016/j.bmc.2009.12.038

Bioorganic & Medicinal Chemistry 18 (2010) 1213–1226
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and the biological evaluation of arylnaphthalene lignans as
anti-hepatitis B virus agents
Damodar Janmanchi a,b, , Ya Ping Tseng a, , Kuei-Chen Wang a, Ray Ling Huang c, Chih Hsiu Lin b,
,
*
Sheau Farn Yeh a,
*
a Institute of Biochemistry and Molecular Biology, National Yang-Ming University, Taipei, Taiwan, ROC
b Institute of Chemistry, Academia Sinica, Taipei, Taiwan, ROC
c Department of Biological Science and Technology, Meiho Institute of Technology, Pingtung, Taiwan, ROC
a r t i c l e i n f o
a b s t r a c t
Article history:
We have previously shown that helioxanthin can suppress human hepatitis B virus gene expression. A
series of helioxanthin analogues were synthesized and evaluated for their anti-hepatitis B virus activity.
Modifications at the lactone rings and methylenedioxy unit of helioxanthin can modulate the antiviral
activity. Among them, compound 32 is the most effective anti-HBV agent. Compound 32 can suppress
the secretion of viral surface antigen and e antigen in HepA2 cells with EC50 values of 0.06 and
Received 12 October 2009
Revised 10 December 2009
Accepted 11 December 2009
Available online 21 December 2009
0.14 lM, respectively. Compound 32 not only inhibited HBV DNA with wild-type and lamivudine-resis-
Keywords:
tant strain but also suppressed HBV mRNA, core protein and viral promoters. In this study, a full account
of the preparation, structure–activity relationships of helioxanthin analogues, and the possible mecha-
nism of anti-HBV activity of this class of compounds are presented. This type of compounds possesses
unique mode of action differing from existing therapeutic drugs. They are potentially new anti-HBV
agents.
Helioxanthin derivatives
Hepatitis B virus
Anti-HBV
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
tein and the HBV DNA polymerase. HBV-specific protein, RNA, and
DNA are all involved in the process of viral replication. The com-
Infection of hepatitis B virus (HBV) frequently results in acute
and chronic hepatitis which could lead to liver cirrhosis and hepa-
tocellular carcinoma (HCC). Such infection remains a public health
problem worldwide. More than 400 million people worldwide are
chronically infected with the hepatitis B virus.1 Though immuniza-
tion against HBV has been shown as an effective way to prevent
chronic HBV infection, yet effective drugs to eradicate HBV in
chronic carriers are still urgently needed. The HBV genome is a
3.2 kb partially double-stranded circular DNA with four open read-
ing frames that encode the viral core antigen (HBcAg), the viral
DNA polymerase, the viral surface antigen (HBsAg), and the X pro-
tein.2 The HBV genome is transcribed to produce the 3.5-, 2.4-, 2.1-,
and 0.7-kb viral RNAs. Synthesis of HBV RNA is under the control of
the core, S1, S2 and X promoters, and enhancers, I and II.3 During
the HBV replication, the 3.5 kb mRNA not only serves as the tem-
plate for reverse transcription but also encodes the viral core pro-
plexity of HBV life cycle provides opportunities as well as chal-
lenges in developing effective therapeutic agents for its infection.
Several nucleoside analogues were recently introduced to treat
chronic HBV infection. Notable examples include lamivudine [3TC,
(ꢀ)b-
phonylmethoxyethyl) adenine],5,6 entecavir (BMS-200475, carbo-
cyclic 20-deoxyguanosine),7 telbivudine ( -thymidine),8 and Clevu-
dine -FMAU, 1-[(2S,3R,4S,5S)-3-fluoro-4-hydroxy-5-(hydroxy-
L
-20,30-dideoxy-30-thiacytidine],4 adefovir [PMEA, 9-(2-phos-
L
[L
methyl)oxolan-2-yl]-5-methylpyrimidine-2,4-dione].9 Since these
drugs mainly targeted the viral polymerase,10 resistance and
cross-resistance against lamivudine (3TC) and adefovir have
emerged after only one to two years of treatment.11,12 Unfortu-
nately, prolonged antiviral therapy is required to eradicate HBV
from the chronically infected patients.13 The point mutations that
lead to the emergence of resistance were also identified recently.14
Since each of the viral replication elements could be targeted to
stall HBV production, increasing attention is being focused on find-
ing anti-HBV agents unaffected by resistance problem. Various
non-nucleoside analogues, such as heteroarylpyrimidine Bay 41-
410915 and oxadiazole derivatives,16 have been reported to inhibit
hepatitis B virus replication by depleting the nucleocapsids or
reducing the intracellular virion particles. This approach has
opened a new avenue in anti-HBV research today. Inspired by
these successes, we also attempt to develop non-nucleoside agent
Abbreviations: HE-145, helioxanthin; HBV, hepatitis B virus; HBsAg, HBV surface
antigen; HBeAg, HBV e antigen; HBcAg, HBV core protein; 3TC, lamivudine (ꢀ)b-
L-
20,30-dideoxy-30-thiacytidine; S1P, HBV surface promoter I; S2P, HBV surface
promoter II; CP, HBV core promoter; XP, HBV X promoter; E/XP, HBV enhancer I;
EIA, enzyme immunoassay; ELISA, enzyme-linked immunosorbent assay.
* Corresponding authors. Tel.: +886 2 2826 7117; fax: +886 2 2826 4843 (S.F. Yeh).
E-mail addresses: chl@chem.sinica.edu.tw (C.H. Lin), fyeh@ym.edu.tw (S.F. Yeh).
These authors contributed equally to this work.
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.12.038

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D. Janmanchi et al. / Bioorg. Med. Chem. 18 (2010) 1213–1226
with novel modes of action to overcome the drug resistance
problem.
4 and regio-isomer 5 (retro-helioxanthin). Bromination of 4 with
N-bromosuccinimide in DMF at room temperature afforded bro-
mo-helioxanthin 6 or 7 depending on the equivalence of NBS used.
Further treatment of 6 and 7 with CuCN in DMF gave cyano-helio-
xanthin 8 and 9, respectively. Hydrolysis of 4 in basic methanol
produced sodium salt 10. Attempt to fluorinate 4 with selectfluor
led to an unexpected oxidation reaction and produced the ortho-
quinone 11, instead of the anticipated fluoro-helioxanthin.
Ester 12 was obtained from the esterification of anhydride 3
with catalytic HCl in ethanol. The anhydride 3 was converted to
imide 13 by reacting with ethanolamine. Subsequent acetylation
of 13 with acetic anhydride in pyridine gave 14. Reduction of 3
with LiAlH4 led to the diol 15. Acetylation of 15 gave a mixture
of 16 and 17 that was readily separable by flash chromatography.
Alcohol 16 was oxidized with PDC in dichloromethane to produce
aldehyde 18. Bromination of diol 15 with PBr3 gave 19 that was
conveniently oxidized to dicarbaldehyde 20 in hot DMSO in the
presence of sodium bicarbonate.
For many years, natural products have been the most abundant
and consistently sources in drug discovery. Previously, we reported
that a naturally occurring arylnaphthalene lignan, helioxanthin
(isolated from Taiwanina cryptomerioides Hayata), can effectively
inhibit HBV gene expression and replication in human hepatoma
cells.17,18 In searching for more potent anti-HBV drugs, we synthe-
sized numerous helioxanthin analogues with particular emphasis
on the modification of the naphthalene rim, lactone, and phenyl
ring of parent structure. Herein, we report a full description of
the synthesis of helioxanthin analogues and the biological evalua-
tion of their anti-HBV activities. We also demonstrated the antivi-
ral activity of an active analogue (32) that rivals the parent
compound in its ability to inhibit the levels of HBV DNA, mRNA,
viral proteins, and viral promoters.
2. Results
To obtain the naphtholic diester 21, the dienophile in the key
Diels–Alder reaction was switched from maleic anhydride to di-
methyl acetylenedicarboxylate (DMAD). Hydrolysis in basic meth-
anol removed the less hindered methyl group and produced 22.
Ether 23 was obtained by Williamson’s methylation of 21 with
CH3I. Prolonged alkaline hydrolysis of 23 likewise hydrolyzed the
less hindered methyl ester and produced compound 24.
The anhydrides 25–29 were synthesized in similar manner as 3
following Charlton’s protocol (Scheme 5). The required hydroxy-
acetals (1a–1e) were synthesized from the lithiated 1 and various
known aldehydes. Using maleic anhydride as the dienophile in
Diels–Alder reaction, we obtained anhydrides 25–29. The lactones
30, 33, 36, 39, and 40 and their retro-isomers 31, 34, and 37 were
obtained by reduction with NaBH4. Selective cleavage of the benzyl
protecting group present in lactones 30, 33, and 36 was carried out
with BBr3 to furnish 32, 35, and 38.
2.1. Synthesis of helioxanthin and its structural analogues
The synthetic exploration is outlined in Schemes 1–5. Several
multi-step procedures have been developed for the total synthesis
of helioxanthin. These methodologies include a dimerization ap-
proach,19 inter- and intramolecular Diels–Alder reactions,20–23
and a Pd-catalyzed benzannulation reaction.19 Among the known
procedures for the preparation of helioxanthin 4, we found that
the protocol reported by Charlton and co-workers24 provides the
greatest flexibility necessary for the large scale synthesis of various
analogues. In this strategy, piperonal was first converted to acetal 1
by treating with ethylene glycol and catalytic amount of p-
TsOHꢁH2O in refluxing benzene. The labile acetal was immediately
lithiated with nBuLi at ꢀ78 °C. Another portion of piperonal was
added to this resulting lithium reagent to produce hydroxyl acetal
2. This crude hydroxy acetal was converted into a highly reactive
benzo[c]furan in refluxing AcOH/Ac2O and this intermediate was
trapped in situ by maleic anhydride via Diels–Alder reaction. The
Diels–Alder adduct then underwent spontaneous aromatization
under the reaction condition to furnish anhydride 3. When treated
with sodium borohydride, the anhydride 3 was reduced to lactone
2.2. Evaluation of antiviral activities
Human hepatoma cell line, HepA2, was derived from HepG2
cells by transfecting a tandem repeated full-length HBV DNA.
HepA2 cells continually synthesize and secrete HBsAg and HBeAg
Scheme 1. Reagents and conditions: (a) ethylene glycol, p-toluenesulfonic acid, benzene, reflux; (b) n-BuLi, piperonal, THF, ꢀ78 °C to room temperature; (c) maleic
anhydride, AcOH, Ac2O, CH2Cl2, 140 °C, 24 h; (d) NaBH4, THF, room temperature, 2 h.

D. Janmanchi et al. / Bioorg. Med. Chem. 18 (2010) 1213–1226
1215
Scheme 2. Reagents and conditions: (a) N-bromosuccinimide, DMF, room temperature, 24 h; (b) CuCN, DMF, reflux, 12 h; (c) NaOH, MeOH, reflux, 12 h; (d) selectfluor,
CH3CN, reflux, 24 h.
Scheme 3. Reagents and conditions: (a) EtOH, concd HCl, reflux, 12 h; (b) amino ethanol, Et3N, toluene, reflux, 5 h; (c) Ac2O, pyridine, room temperature, 12 h; (d) LAH, THF,
0 °C, 1 h; (e) PDC, CH2Cl2, room temperature, 1 h; (f) PBr3, toluene, room temperature to 40 °C, 3 h; (g) NaHCO3, DMSO, 100 °C, 10 min.
into the culture medium. This cell line is an excellent assay system
for screening natural or synthetic compounds for anti-HBV activ-
ity.25 We examined helioxanthin and its analogues for their biolog-
ical activities against human hepatitis B virus. Among all 34
compounds tested, helioxanthin 4 and derivative 32 were found
more than one order of magnitude. Analogues 16 (EC50 = 0.42
l
M),
M), and
M) also exhibit antiviral activities against HBV.
10 (EC50 = 0.89
35 (EC50 = 1.89
lM), 17 (EC50 = 1.22 lM), 15 (EC50 = 1.41 l
l
Moderate to low anti-HBV effects were observed for analogue 20
(EC50 = 2.39 M), 38 (EC50 = 3.06 M), and 39 (EC50 = 7.64 M).
l
l
l
to possess the best activity (EC50 = 0.16
lM and 0.14
lM, respec-
All other compounds in this study displayed no detectable anti-
tively), whereas retro-isomer 5 (EC50 = 2.18
lM) is less active by
HBV activity within the same range of concentrations.

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D. Janmanchi et al. / Bioorg. Med. Chem. 18 (2010) 1213–1226
Scheme 4. Reagents and conditions: (a) DMAD, AcOH, Ac2O, CH2Cl2, 140 °C, 24 h; (b) KOH/MeOH, 65 °C, 2 h; (c) CH3I, K2CO3, DMF, room temperature, 3 h; (d) KOH/MeOH,
65 °C, 12 h.
Scheme 5. Reagents and conditions: (a) n-BuLi, 3-benzyloxy-4-methoxybenzaldehyde (for 25) or 4-benzyloxy-3-methoxybenzaldehyde (for 26) or 3,4-dibenzyloxybenz-
aldehyde (for 27) 3,4-dimethoxybenzaldehyde (for 28) or p-trifluoromethoxybenzaldehyde (for 29), THF, ꢀ78 °C to room temperature; (b) maleic anhydride, AcOH, Ac2O,
CH2Cl2, 140 °C, 24 h; (c) NaBH4, THF, room temperature, 2 h; (d) BBr3, CH2Cl2, ꢀ78 °C, 2 h.
2.3. Compound 32 suppressed HBsAg and HBeAg production in
HepA2 cells
these synthesized helioxanthin analogues, compound 32 is the
most effective in anti-HBsAg production. Further tests were there-
fore conducted. HepA2 cells were seeded in a 24-well culture plate
to which various concentrations of 32 were added. After 48 h of
incubation, HBsAg or HBeAg in the media were determined by
Natural product, helioxanthin can suppress both HBsAg and
HBeAg production in HepA2 cells.17 As shown in Table 1, among

D. Janmanchi et al. / Bioorg. Med. Chem. 18 (2010) 1213–1226
1217
Table 1
Biological activity of suppression of HBV surface antigen (HBsAg) of helioxanthin or derivatives in HepA2 cells
a
b
a
b
Compound
EC50
CC50
(lM)
Compound
EC50
CC50 (lM)
3
4
5
6
7
>12.5
11.79 2.10
11.26 1.15
>12.5
6.33 1.82
>12.5
>12.5
>12.5
>12.5
7.49 2.14
>12.5
>12.5
>12.5
>12.5
>12.5
6.91 0.22
4.69 1.70
5.31 0.21
21
22
23
24
25
26
27
30
31
32
33
35
36
37
38
39
40
>12.5
>12.5
>12.5
>12.5
>12.5
>12.5
>12.5
>12.5
>12.5
0.14 0.02
>12.5
1.89 0.21
>12.5
>12.5
3.06 0.75
7.64 2.04
>12.5
7.04 1.63
7.01 0.62
8.93 1.19
7.87 0.88
>12.5
11.68 1.37
>12.5
>12.5
>12.5
10.05 1.06
>12.5
>12.5
>12.5
0.16 0.03
2.18 1.21
>12.5
>12.5
>12.5
>12.5
0.89 0.12
>12.5
>12.5
>12.5
8
9
10
11
12
13
14
15
16
17
18
20
>12.5
1.41 0.31
0.42 0.21
1.22 0.87
>12.5
>12.5
7.01 1.19
5.86 1.17
>12.5
2.39 0.53
Note: Cultured HepA2 cells were seeded on 96-well culture plates and treated with various concentration of compounds in DMEM for 2 days. Cultured cells were collected
and the number of cells was examined using MTT assay.
a
Concentration of suppression of HBsAg at 50% of untreated cells.
Concentration of cell growth at 50% of untreated cells.
b
ELISA assay. Compound 32 potently suppressed both the endoge-
nously expressed HBsAg and HBeAg production with an EC50 about
0.06 lM and 0.14 lM, respectively (Fig. 1). The efficacy of 32 is
similar to helioxanthin 4 (Table 1) which was used as a positive
control throughout this study. It is worth pointing out that the sup-
pressive activity of 32 on HBsAg and HBeAg production in HepA2
cells is highly specific and is not due to the cytotoxic effect of this
compound. The treated cells were viable and continued to prolifer-
ate during the 48 h incubation period.
Figure 2. Effects of compound 32 or 3TC treatment on the HBV DNA level in media
of 1.3ES2 and M33 cells. Quantitative real-time PCR was used to detect HBV titer in
the media of wild (1.3ES2) cells and 3TC-resistant mutant (M33) cells. Cultured
cells were seeded on 100 mm culture dishes and treated with various concentra-
2.4. Compound 32 suppressed viral replication of both wt HBV
and lamivudine-resistant HBV
tions of compound 32 (1.0, 3.0 and 5.0 lV and 5.0 lM compound 4 or 14 or 3TC in
We then tested the effect of 32 on HBV DNA in 1.3ES2 cells (sta-
bly transfected with a 1.3-fold wt HBV ayw strain genome26 in
HepG2 cells) and M33 (HepG2 cell line [HBV] containing the lam-
ivudine-resistant HBV with L515M/M539V double mutation in the
DNA polymerase region). 1.3ES2 and M33 cells were both treated
with various concentrations of 32, 14, or 4. After 6 days of treat-
ment, the amount of released viral-associated HBV DNA secreted
into the medium was determined using quantitative real-time
PCR (Fig. 2). Compound 32 reduced HBV DNA in a dose-dependent
manner both in 1.3ES2 and M33 cells. In contrast, lamivudine only
showed inhibitory effect in 1.3ES2 but not in M33 cells. The sup-
serum-free (SF) DMEM for 72 h. Media were collected for real-time PCR analysis
using primer pair designed against HBV DNA. Data were expressed as mean SD
(n = 3).
pression of HBV DNA by the lactone 32 is again structure-specific
since the imide analog 14 showed no detectable suppression activ-
ity in either 1.3ES2 or M33 cells.
2.5. Compound 32 suppressed all HBV transcripts in 1.3ES2 cells
To determine whether the suppression of viral replication by 32
is mediated through interference with the expression of viral tran-
scripts, total cellular RNA was extracted and examined by Northern
blot analysis. 1.3ES2 cells were grown in DMEM with 10% fetal calf
serum till confluence. The medium was then changed to serum-
free DMEM with various concentrations of 32, 4, and 14 for 2 days.
As shown in Figure 3, 32 significantly decreased 3.5-kb (HBeAg
mRNA and pregenomic RNA) and 2.4/2.1-kb (large HBsAg mRNA
and middle/major HBsAg mRNA) HBV transcripts in a dose-depen-
dent manner in 1.3ES2 cells. In contrast, 14 had no such effect on
HBV RNA levels.
2.6. Compound 32 reduced HBV core protein levels in 1.3ES2
cells
Figure 1. Effects of compound 32 on HBsAg and HBeAg production in HepA2 cells.
HepA2 cells were seeded on 24-well plates at a density of 8 ꢂ 104 cells/cm2 in
DMEM with 10% fetal calf serum and allowed to attach overnight. The cells were
then washed twice with phosphate-buffered saline (pH 7.0) and treated with
Since 3.5 kb HBV transcript was decreased after drug treatment,
it was anticipated that this class of compounds would also affect
viral protein expression. To test this prediction, we treated
1.3ES2 cells with 32, 14, and 4 for 2 days and performed Western
various concentrations of compound 32 or 4 (3.0 lV in serum-free DMEM for 48 h.
The amounts of HBsAg and HBeAg in the culture medium were determined by
enzyme immunoassay. Viable cells in each well were determined by the WST-1
assay. Data are expressed as mean SD (n = 3).

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D. Janmanchi et al. / Bioorg. Med. Chem. 18 (2010) 1213–1226
analogues are classified as type B. Anhydride 3, retro-helioxanthin
5, diester 12, and diol derivatives 15–20 are examples of this
group. Type C compounds are those with structural modifications
occur on the phenyl substitution. Compounds 30, 32, 33, 35, 36,
38, 39 and 40 in Scheme 5 are all members of this family. The next
three classes of compounds (D, E, and F type, respectively) are con-
sisted of synthetic derivatives with two types of structural modifi-
cation occur in the same molecule. For example, all compounds in
Scheme 4 are modified both on the naphthyl (type A) and lactone
ring (type B), and these compounds are classified as type D. Several
compounds in Scheme 5 contain alterations both at lactone ring
and phenyl substitution and are likewise assigned to type E. Final-
ly, compounds 7 and 9 are modified both on naphthyl and phenyl
ring. These two compounds are classified as type F. The complete
classification of structural analogues is shown in Table 2.
Figure 3. Effects of compound 32 on steady-state mRNA level of HBV RNAs in
1.3ES2 cells. 1.3ES2 cells were seeded on 100 mm culture dishes and treated with
various concentration of compound 32 (0, 1.5, 3.0 and 9.0 lM) and 3.0 lM
compound 4 or 14 in serum-free DMEM medium for 48 h. Total RNA was extracted
from serum free (SF) and compound 32-treated cells and analyzed by Northern
hybridization with HBV DNA probe as described in Section 5. The mRNA of the
glyceraldehydes-3-phosphate dehydrogenase (GAPDH) gene was used as an
internal marker. Data are representative of three independent experiments.
By correlating the EC50 values in Table 1 and the structural cat-
egories in Table 2, we found that all compounds belonging to type
A, D and F are inactive. The total absence of any antiviral activity in
such a wide array of structurally diverse compounds is somewhat
surprising at first. However, it is immediately clear that all three
types of compounds contain some modification(s) on the naphthyl
ring. When this region is modified, even slightly, the antiviral
activity vanishes altogether. Whether the incorporated substitu-
tion is a hydrogen bonding donor (OH), hydrogen bonding acceptor
(CN), or neutral (Br) group makes no difference. It can be reason-
ably deduced from these observations that the naphthalene rim
in helioxanthin is the most tightly bounded area to its target in
vivo. Furthermore, to complement the property of this aromatic
binding motif, the binding pocket is likely a hydrophobic pocket
mostly consisted of aromatic residues. This postulation can be
tested with affinity labeling or crystallography in the future.
The other three classes of compounds all contain structural
modifications at either the lactone or the phenyl ring. In these 23
synthetic analogues, 10 compounds exhibit antiviral activities.
Curiously, all the active compounds belong to B or C type. None
of the 5 compounds in type E exhibits any antiviral activity. The
most intuitive rationale for this observation is that the in vivo
binding sites of both lactone and phenyl rings are relatively flexi-
ble. Therefore, minor structural alterations at either site can be
made without disrupting the antiviral activity. This loose binding
mode is in sharp contrast to the tight binding of naphthyl rim de-
picted previous. On the other hand, when both the lactone and the
phenyl ring are modified (type E), no antiviral activity was de-
tected. From this observation, we can reasonably deduce that the
loose binding for these two moieties are somewhat cooperative.
Apparently, the overall binding pocket can tolerate certain single
site alteration as in type B and C, but the E type ‘double mutation’
analogues can no longer efficiently bind to their target. Seven of
these compounds (3, 4, 6, 10, 13, 21, and 23) have been tested
against HBV before by Cheng’s group.23 Despite of some numerical
difference, the trend observed in the published results is qualita-
tively consistent with our present data.
blot analysis using anti-HBV core protein antibody to examine if
the drugs could suppress core protein expression in the cells. As
shown in Figure 4, the expression of core protein decreased in a
dose-dependent manner upon treatment by compound 32. On
the other hand, 14 exhibited no effect on HBV core protein
expression.
2.7. Compound 32 selectively suppresses viral promoter activity
for HBV surface antigen, core antigen and enhancer I in HepA2
cells
The observation that 32 effectively reduced the level of viral
transcripts raised the possibility that 32 might also affect the viral
promoter activity. We examined the activities of four viral promot-
ers including large viral surface antigen (S1P), major viral surface
promoter (S2P), viral core protein (CP) and viral X protein (XP).
Furthermore, enhancer I (E/XP) was also subjected to the same test.
In these experiments, luciferase was used as the reporter assay. As
shown in Figure 5, we found that compound 32 and 4 selectively
suppressed S1P, S2P, CP, and E/XP activities but has no effect on
XP activity in HepA2 cells. None of the promoters or enhancer I
activities was suppressed by 14 (Fig. 5).
3. Discussion
3.1. SAR evaluation
Some important structure–activity relationship principles can
be deduced from a meticulous analysis of the data listed in Table
1. To simplify the discussion, we first categorized all the synthetic
analogues into six classes. In the first type of derivatives (denoted
type A), alterations are made on the rim of naphthalene ring. Bro-
minated and cyanated derivatives 6, 8 both belong to this class.
When the lactone moiety of helioxanthin is modified, these
A more detailed structure–activity relationship of compounds
in type B and C emerged upon closer inspection. We first noticed
none of the anhydrides (3, 25–27) and imides (13, 14) derivatives
exhibits any antiviral activity. An obvious rationale for these re-
sults is that these compounds with extra carbonyl groups can not
be fitted into the binding pocket and therefore become inactive.
Especially, the anhydrides and imides both possess the cyclic dicar-
bonyl structural motif and the extra carbonyl groups in these com-
pounds always points at a fixed direction due to the rigidity of the
five member rings. This explanation sufficiently accounts for why
none of the anhydrides and imides derivatives exhibit any activity.
However, this overly-simplified explanation did not take into ac-
count the moderate activities exhibited by the aldehyde derivative
20. This peculiarity will be discussed next.
Figure 4. Effects of compound 32 on HBV core protein levels in 1.3ES2 cells. 1.3ES2
cells were seeded on 100 mm culture dishes and treated with various concentration
of compound 32 (0, 1.5, 3.0 and 9.0 lM) and 3.0 lM compound 4 or 14 in serum-
free (SF) DMEM medium. After 48 h, cell lysates were prepared. Total protein
lysates were resolved on SDS–polyacrylamide gel electrophoresis gels and visual-
ized by immunoblotting using a mouse anti-HBV core antibody. The amount of a-
tubulin present was also probed as internal controls. Data are representative of
three independent experiments.

D. Janmanchi et al. / Bioorg. Med. Chem. 18 (2010) 1213–1226
1219
Figure 5. Compound 32 suppresses the promoter activity of HBV in HepA2 cells. Different HBV promoter region were cloned into promoterless mammalian expression
vector. HepA2 cells were transfected with S1P (A), S2P (B), CP (C), XP (D) and E/XP (E) of HBV promoter region with luciferase reported gene, respectively. After transfection,
HepA2 cells were treated with serum free (SF), compound 32 (0.9 and 3.0 lM) and 3.0 lM compound 4 or 14 in serum-free DMEM for 48 h. The transfection efficiency was
corrected by cotransfecting b-gal expression vector and assayed b-gal activity simultaneously. Data were expressed as mean SD (n = 3). Data are representative of three
independent experiments.
two ester groups in 12 are clearly much bulkier than the original
lactone and therefore unable to fit into the binding site.
Table 2
Classifications of helioxanthin structural analogues
Unlike in type B analogues, the SAR relationship in type C ana-
logues can be explained with structural factors. In these com-
pounds, all the derivatives with the benzyl protecting group still
attached (30, 33, and 36) are inactive. On the contrary, compounds
with substitution sizes close to the [1,3] dioxole in helioxanthin
(32 and 35) are both active. Catechol type derivative 38 and dime-
thoxy 39 only possess very weak activity. These results indicate
that the binding cleft for the phenyl moiety must be quite sensitive
to the size of the substrate.
Classifications
Compounds
Type A derivatives
Type B derivatives
Type C derivatives
Type D derivatives
Type E derivatives
Type F derivatives
6, 8, and 11
3, 5, 10, 12, 13, 14, 15, 16, 17, 18, and 20
30, 32, 33, 35, 36, 38, 39, and 40
21, 22, 23, and 24
25, 26, 27, 31, and 37
7 and 9
For other type B derivatives, the lactone rings are all changed to
acyclic structures. In these seven compounds (10, 12, 15, 16, 17,
18, and 20), two (12 and 18) are inactive. By varying the sizes
and polarities at the acyclic terminals and testing the antiviral
activities in these compounds, we wished to map the environment
in their binding site. However, it is difficult to develop a unified
model to explain our observations solely based on the size and
polarity of the functional groups. However, we found that data be-
come more intelligible if we assume deacetylation can take place
in vivo. For example, if the acetyl groups are readily hydrolyzed
in vivo, both 16 and 17 can be simply viewed as masked forms
of the active 15. The slight discrepancy in their activities can be
due to the difference in their bio-viability (15 is relatively insolu-
ble). On the other hand, hydrolysis of the inactive 18 leads only
to lactol, an unlikely candidate for antiviral activity. Finally, the
This simple space-fitting model is adequate to account for the
general trend of SAR relationship in C type compounds but fails
to explain the observed discrepancy in activity between com-
pounds 32 and 35. As shown in Table 1, compound 32 is 10 times
more active than 35 despite them are virtually identical in molec-
ular sizes. The negative result observed in 40 might provide a clue
to this puzzle. The lack of activity in this fluorinated derivative
strongly suggests that the binding site is not only selective to the
sizes, but also sensitive to the surface potentials of the substrates.
Since the only notable difference between 32 and 35 is the position
of the electropositive H atom, this surface potential sensitive bind-
ing model gives a plausible account why 32 is more active than 35.
The CC50 values in Table 1 refer to the cytotoxicity of these syn-
thetic analogues. Although such toxicity data cannot be linked to
structural features as we demonstrated for the antiviral activities,

1220
D. Janmanchi et al. / Bioorg. Med. Chem. 18 (2010) 1213–1226
we believe it is still worthwhile to map out a few common struc-
tural motifs that might lead to cytotoxicity. Expectedly, com-
pounds with higher antiviral activities (helioxanthin, 17, and 32)
also exhibit cytotoxic effect. Otherwise, compounds with measur-
able toxicity always carry functional groups that are considered
good electrophiles such as anhydrides in compounds 3 and 21–
26, aldehyde in 18 and 20, and the ortho-quinone in 11 (or the
ortho-quinone precursor in 38). These reactive groups can all react
with various biological nucleophiles and cause adverse effect in
vivo. Therefore, it is sensible to avoid these functional groups when
optimizing these potential drugs in the future.
other worthy candidate for further antiviral combination therapy.
For example, a combined dosage of 32 and 3TC might eradicate
HBV infection in a relatively short time before the virus can devel-
op resistance to the latter. Alone this direction, we are currently
testing these compounds as novel anti-HBV agents in HBV trans-
genic mice and the results will be reported in due course.
4. Experimental section
4.1. General methods for synthesis
In order to clarify whether the molecular mechanism of our
most active compound is similar to that of helioxanthin, further
studies were performed with 32. With these investigations, we
found 32 can simultaneously decrease the HBV RNA level and core
antigen protein expression. Furthermore, inhibition of HBV replica-
tion in cell culture was also observed. In short, the treatment of 32
not only reduces the expression of viral transcripts but there was
significant decline in the amount of virus production by the cells.
Such comprehensive suppression at all levels of viral life cycle is
only observed in parent helioxanthin and two other analogues (lac-
tam and piperazine dione derivatives).18,27 These results demon-
strate the likeliness that 32 interferes with viral replication at a
very upstream event like helioxanthin. Based on the wide scope
of its activity, we propose that 32 suppresses the production of
the 3.5-kb pregenomic RNA. Since this key RNA transcript not only
encodes the core and polymerase proteins but also serves as a tem-
plate for reverse transcription, the observed inhibition of RNA tran-
scription and the reduction of core protein level are reasonably
attributed as consequence of the suppression 3.5-kb RNA tran-
scription. Moreover, we also observed the interference of viral core
promoter activity by 32 (Fig. 4). Therefore, the reduction of 3.5 kb
HBV pregenomic RNA production is most like the result of blocked
CP activity. These results foretold our observations that 32 can de-
crease HBV viral particle production by 1.3ES2 cells (Fig. 2). Based
on these observations, we propose the anti-HBV activity of 32 is
most likely the result of interference with the transcriptional
machinery as been observed before in 4 and its lactam ana-
logue.17,18,27 The mechanistic similarity with helioxanthin 4 firmly
establishes this class of natural product as effective anti-HBV drugs
or lead compounds. In addition, the core protein level was also de-
creased after the treatment of 32 (Fig. 4). However, it remains un-
clear whether this reduction in core protein is the cause or the
consequence of the reduction in capsid formation by 32.
In addition to the antiviral activity against simple wild-type
HBV, we also evaluated the activity of 32 against lamivudine-resis-
tant HBV that contained L515M and M539V double mutations in
the polymerase protein. To our delight, we found the lamivu-
dine-resistant M33 mutant strain was still sensitive to treatment
by 32. This result highlighted the potential of this class of natural
product in long-term anti-HBV therapy or combination therapy.
In conclusion, through our synthetic effort and biological test-
ing, a model of structure–activity relationship in this class of com-
pounds is proposed for the first time. Although the analogues
reported here are only comparable to the parent compound 4 at
best, the established structure–activity relationship can be a valu-
able guide in further design nonetheless. For example, C type
derivatization can be a versatile handle for improving drug stabil-
ity and bio-viability. Like the lead compound helioxanthin, com-
pound 32 not only reduces HBV DNA but also decreases HBV
RNA, HBV core protein expression. 32 also inhibits the activity of
HBV viral promoters and enhancer to affect the viral replication
in human hepatoma cells. The mode of action for 32 should be sim-
ilar to that of parent helioxanthin and its lactam analogous17,27
while differ considerably from the existing therapeutic drugs like
3TC. The uniqueness of these new anti-HBV agents make 32 an-
All reactions are performed under 1 atmosphere of dried nitro-
gen and well mixed with magnetic stirring devices. Reagent grade
chemicals and solvents were used in all reactions. Reaction vessels
were dried in oven before use. Diethyl ether and tetrahydrofuran
were distilled over metallic sodium with benzophenone radical an-
ion as the indicator. Dichloromethane were distilled from CaH2.
Flash column chromatography was performed with Silica Gel 60
(0.040–0.063 mm) as the stationary phase. All ratios of reported
mixed eluents are based on volume.
Anhydride 3, helioxanthin 4, and its retro-isomer 5 were syn-
thesized according to the literature procedure.23
4.1.1. 10-Benzo[1,3]dioxol-5-yl-5-bromo-9H-furo[30,40:6,7]naphtho-
[1,2-d][1,3]dioxol-7-one (6)
To a solution of 4 (100 mg, 0.29 mmol) in DMF (5 ml) was added
N-bromosuccinimide (77 mg, 0.43 mmol). The mixture was stirred
for 24 h at room temperature. After the completion of the reaction,
the reaction mixture was diluted with CH2Cl2 and washed with
water (3 ꢂ 25 ml). The organic fraction was dried over MgSO4
and concentrated. After column chromatography (hexane/
CH2Cl2 = 1:1), yellow solid 6 (89 mg, 74%) was isolated. 1H NMR
(500 MHz, CDCl3) d: 8.86 (s, 1H), 7.62 (s, 1H), 6.86 (d, J = 7.6 Hz,
1H), 6.73–6.75 (m, 2H), 6.04 (dd, J = 1.3, 11.2 Hz, 2H), 5.93 (dd,
J = 1.4, 10.0 Hz, 2H), 5.18 (AB, J = 15.2 Hz, 2H). 13C NMR
(100 MHz, CDCl3) d: 170.7, 147.6, 146.6, 141.8, 140.5, 140.5,
129.9, 129.9, 128.9, 127.2, 122.4, 122.3, 122.3, 117.4, 116.2,
109.5, 108.1, 102.0, 101.3, 69.3. IR (CH2Cl2, cast): 1762, 1624,
1446, 1308, 1234, 1134, 1074, 1041, 758. FAB-LRMS m/z (rel inten-
sity): 426 (M+, 4), 419 (11), 417 (1), 391 (68), 389 (5), 341 (2), 340
(1), 279 (12), 267 (8), 209 (9), 194 (3), 167 (18). FAB-HRMS Calcd
for C20H11O6Br m/z: 425.9737 [M]+, found 425.9729.
4.1.2. 5-Bromo-10-(6-bromo-benzo[1,3]dioxol-5-yl)-9H -furo-
[30,40:6,7]naphtha[1,2-d][1,3]dioxol-7-one (7)
To a solution of 4 (100 mg, 0.29 mmol) in DMF (5 ml) was added
N-bromosuccinimide (127 mg, 0.71 mmol) and the reaction was
treated as described for 6. After column chromatography (hex-
ane/CH2Cl2 = 1:1), 7 (100 mg, 70%) was isolated as a yellow pow-
der. 1H NMR (400 MHz, CDCl3) d: 8.89 (s, 1H), 7.62 (s, 1H), 7.10
(s, 1H), 6.71 (s, 1H), 6.07 (dd, J = 1.2, 6.8 Hz, 2H), 5.94 (dd, J = 1.3,
9.6 Hz, 2H), 5.12 (s, 2H). 13C NMR (125 MHz, CDCl3) d: 170.6,
148.7, 147.2, 146.6, 141.7, 140.6, 129.9, 128.7, 128.7, 127.8,
122.4, 122.2, 117.4, 116.3, 114.2, 112.5, 109.8, 102.4, 102.2, 69.1.
IR (KBr): 1762, 1627, 1476, 1421, 1310, 1232, 1133, 1070, 1021.
FAB-LRMS m/z (rel intensity): 503 (M+, 6), 427 (1), 391 (4), 341
(1), 173 (3). FAB-HRMS Calcd for C20H10O6Br2 m/z: 503.8844
[M]+, found 503.8859.
4.1.3. 10-Benzo[1,3]dioxol-5-yl-7-oxo-7,9-dihydro-furo-
[30,40:6,7]naphtho[1,2-d][1,3]dioxole-5-carbonitrile (8)
CuCN (100 mg, 1.12 mmol) was added to a solution of 6 (50 mg,
0.12 mmol) in DMF (5 ml) and the resulting mixture was refluxed
for 12 h. The reaction mixture was quenched with brine (25 ml)
and extracted with ether (25 ml). The extract was dried over

D. Janmanchi et al. / Bioorg. Med. Chem. 18 (2010) 1213–1226
1221
MgSO4 and concentrated. After column chromatography (hexane/
4.1.7. 9-Benzo[1,3]dioxol-5-yl-naphtho[1,2-d][1,3]dioxole-7,8-
CH2Cl2 = 1:3), pure 8 (33 mg 76%) was isolated as a yellow solid.
1H NMR (400 MHz, CDCl3) d: 8.80 (s, 1H), 7.65 (s, 1H), 6.88 (dd,
J = 1.1, 7.2 Hz, 1H), 6.75 (s, 1H), 6.73 (d, J = 1.6 Hz, 1H), 6.06 (dd,
J = 1.3, 4.0 Hz, 2H), 6.03 (dd, J = 1.3, 3.8 Hz, 2H), 5.19 (AB,
J = 15.4 Hz, 2H). 13C NMR (100 MHz, CDCl3) d: 169.8, 147.9, 147.6,
146.7, 145.8, 141.2, 131.3, 130.9, 129.2, 124.4, 124.0, 122.2,
121.1, 117.2, 117.1, 109.3, 108.2, 105.7, 102.9, 101.5, 101.4, 69.3.
IR (CH2Cl2, cast): 2212, 1765, 1606, 1496, 1446, 1312, 1237,
1081, 1031 cmꢀ1. FAB-LRMS m/z (rel intensity): 373 (M+, 46), 344
(13), 327 (26), 281 (14), 267 (10), 207 (17), 136 (100). FAB-HRMS
Calcd for C21H11O6N m/z: 373.0586 [M]+, found 373.0589.
dicarbaxylic acid diethyl ester (12)
Compound 3 was dissolved in ethanol (10 ml) and concentrated
HCl (five drops) was added. The mixture was refluxed overnight.
After the reaction was cooled to room temperature, saturated NaH-
CO3 (40 ml) was added and product was extracted with CH2Cl2
(50 ml). The organic fraction was dried over MgSO4 and concen-
trated. After column chromatography (CH2Cl2), pure 12 (100 mg,
83%) was isolated. 1H NMR (500 MHz, CDCl3) d: 8.50 (s, 1H), 7.56
(d, J = 8.5 Hz, 1H), 7.25 (d, J = 8.2 Hz, 1H), 6.80 (s, 1H), 6.77 (d,
J = 1.8 Hz, 2H), 6.01 (d, J = 1.1 Hz, 1H) 5.96 (d, J = 1.1 Hz, 1H), 5.86
(dd, J = 1.2, 10.5 Hz, 2H), 4.36 (q, J = 7.1 Hz, 2H), 4.08 (q,
J = 7.1 Hz, 2H), 1.37 (t, J = 7.1 Hz, 3H), 1.06 (t, J = 7.1 Hz, 3H). 13C
NMR (100 MHz, CDCl3) d: 168.5, 165.6, 147.1, 147.0, 146.4, 142.2,
133.4, 132.4, 132.4, 131.2, 128.8, 124.4, 123.5, 122.5, 120.5,
112.0, 110.9, 107.0, 101.5, 100.9, 61.4, 61.1, 14.2, 13.8. IR (CH2Cl2,
cast): 1737, 1599, 1489, 1446, 1372, 1326, 1234, 1070, 1039,
929 cmꢀ1. FAB-LRMS m/z (rel intensity): 436 (M+, 100), 435 (20),
377 (18), 376 (5), 317 (21), 287 (17), 275 (7), 259 (6), 189 (10),
109 (38). FAB-HRMS Calcd for C24H20O8 m/z: 436.1158 [M+], found
436.1150.
4.1.4. 10-(6-Bromo-benzo[1,3]dioxol-5-yl)-7-oxo-7,9-dihydro-
furo[30,40:6,7]naphtha[1,2-d][1,3]dioxole-5-carbonitrile (9)
CuCN (180 mg, 2 mmol) was added to a solution of 7 (100 mg,
0.2 mmol) in DMF (10 ml) and the reaction was treated as de-
scribed for 8. After column chromatography (hexane/CH2Cl2 = 1:2),
pure 9 (30 mg, 34%) was isolated as a yellow powder. 1H NMR
(400 MHz, CDCl3) d: 8.86 (s, 1H), 7.67 (s, 1H), 7.11 (s, 1H), 6.71
(s, 1H), 6.03–6.09 (m, 4H), 5.14 (s, 2H). 13C NMR (100 MHz, CDCl3)
d: 169.7, 149.0, 147.5, 146.5, 145.8, 141.3, 131.4, 129.7, 129.3,
125.2, 124.2, 117.4, 117.0, 114.1, 112.7, 109.6, 106.0, 103.2,
102.3, 69.1. IR (CH2Cl2, cast): 2219, 1765, 1620, 1475, 1312,
4.1.8. 10-Benzo[1,3]dioxol-5-yl-8-(2-hydroxy-ethyl)-1,3-dioxa-
8-aza-dicyclopenta[a,g]naphthalene-7,9-dione (13)
1234, 1078, 1035 cmꢀ1
. FAB-LRMS m/z (rel intensity): 452
Compound 3 (200 mg, 0.55 mmol), amino ethanol (37 mg,
0.6 mmol), and triethylamine (5 mg, 0.05 mmol) were dissolved
in toluene (10 ml) and water was removed under reflux condition
with a Dean–Stark apparatus. After 5 h, the reaction mixture was
cooling to room temperature and concentrated under reduced
pressure. The residue was triturated with 0.1 N HCl before satu-
rated NaHCO3 was added. The crude mixture was extracted with
dichloromethane. The organic fraction was dried over MgSO4 and
concentrated. After column chromatography (CH2Cl2/ether = 1:5),
pure 13 (140 mg, 64%) was isolated. 1H NMR (500 MHz, CDCl3) d:
8.24 (s, 1H), 7.64 (d, J = 8.5 Hz, 1H), 7.32 (d, J = 8.4 Hz, 1H), 6.87
(d, J = 7.7 Hz, 2H), 6.79–6.82 (m, 2H), 6.06 (s, 1H), 6.01 (s, 1H),
5.92 (d, J = 9.0 Hz, 2H), 3.85–3.83 (m, 4H), 2.31 (br s, 1H). 13C
NMR (50 MHz, CDCl3) d: 170.9, 167.4, 166.8, 147.7, 147.6, 146.8,
144.6, 136.3, 131.2, 129.1, 125.4, 125.3, 124.5, 122.8, 121.4,
113.1, 109.9, 107.4, 101.9, 101.1, 61.4, 37.1. IR (CH2Cl2, cast):
3482, 1758, 1702, 1624, 1492, 1439, 1290, 1230 cmꢀ1. FAB-LRMS
m/z (rel intensity): 406 ([M+H]+, 13), 405 (8), 371 (1). FAB-HRMS
Calcd for C22H16O7N m/z: 406.0927 [M+H]+, found 406.0923.
([M+H]+, 18), 417 (2), 409 (16), 391 (5), 373 (18), 343 (3), 329
(2), 258 (4), 195 (4), 178 (4), 120 (12). FAB-HRMS Calcd for
C21H10O6NBr m/z: 451.9770 [M+H]+, found 451.9777.
4.1.5. 9-Benzo[1,3]dioxol-5-yl-8-hydroxymethyl-naphtho[1,2-
d][1,3]-dioxole-7-carboxylic acid monosodium salt (10)
To a hot methanol solution of 4 (50 mg, 0.14 mmol in 2 mL) was
added a methanol solution of NaOH (37 mg, 0.92 mmol in 2 mL).
The mixture was refluxed overnight. After the completion of the
reaction, the solution was concentrated under reduced pressure
to give the 100 mg of 10. Phosphate buffer of pH 7.0 was added
to neutralize the excess NaOH in the product. 1H NMR (300 MHz,
D2O) d: 7.72 (s, 1H), 7.00 (d, J = 7.6 Hz, 1H), 6.58 (d, J = 8.1 Hz,
1H), 6.41 (d, J = 7.1 Hz, 1H), 6.21 (s, 1H), 6.15 (d, J = 6.8 Hz, 1H),
5.44–5.50 (m, 2H), 5.09 (s, 2H), 4.05 (s, 2H). 13C NMR (125 MHz,
D2O) d: 176.9, 146.2, 144.6, 140.5, 134.6, 134.0, 133.9, 132.9,
129.6, 128.6, 123.3, 123.2, 119.2, 110.9, 110.4, 107.2, 100.8,
100.5, 59.5. IR (CH2Cl2, cast): 3431, 1756, 1631, 1494, 1439,
1266, 1229, 1070, 1030, 919, 801 cmꢀ1
.
4.1.9. Acetic acid 2-(10-Benzo[1,3]dioxol-5-yl-7,9-dioxo-7,9-di-
hydro-1,3-dioxa-8-aza-dicyclopenta[a,g]naphthalene-8-yl)-ethyl
ester (14)
4.1.6. 4-Benzo[1,3]dioxol-5-yl-3H-naphtho[2,3-c]furan-1,5,6-
trione (11)
A solution of Selectfluor [1-(chloromethyl)-4-fluoro-1,4-diazon-
iabicyclo[2.2.2]octane bis (tetrafluoroborate)] (51 mg, 0.14 mmol)
in acetonitrile (1.5 ml) was added to a solution of 4 (50 mg,
0.14 mmol) in the same medium (5 ml). The resulting mixture
was refluxed for 24 h. The solution was then poured into ether
(25 ml), washed with water (25 ml) and saturated NaHCO3
(20 ml). The ether fraction was dried over MgSO4, filtered, and con-
centrated. Pure 11 (29 mg, 60%) was isolated as a dark solid after
column chromatography (hexane/CH2Cl2 = 1:3). 1H NMR
(500 MHz, CDCl3) d: 7.87 (s, 1H), 7.59 (d, J = 10.1 Hz, 1H), 6.88 (d,
J = 8.3 Hz, 2H), 6.60–6.61 (m, 2H), 6.56 (d, J = 10.1 Hz, 1H), 6.02
(s, 2H), 5.11 (br d, J = 14.5 Hz, 2H). 13C NMR (125 MHz, CDCl3) d:
180.1, 179.6, 169.0, 149.7, 148.4, 148.2, 145.2, 141.8, 137.3,
132.8, 130.8, 128.8, 128.5, 126.2, 120.4, 109.1, 107.8, 101.5, 69.8.
IR (CH2Cl2, cast): 1765, 1670, 1617, 1496, 1450, 1301, 1234,
1035, 925; 804 cmꢀ1. FAB-LRMS m/z (rel intensity): 336 (M+, 38),
189 (7), 165 (12). FAB-HRMS Calcd for C19H12O6 m/z: 336.0634
[M]+, found 336.0640.
To a solution of 13 (30 mg, 0.074 mmol) in pyridine (2 ml) was
added acetic anhydride (15 mg, 0.15 mmol) and the resulting mix-
ture was stirred at room temperature for 16 h. 10% HCl (10 ml) was
then added to the reaction and the mixture was extracted with
CH2Cl2 (20 ml). The organic extract was dried over MgSO4 and con-
centrated. After column chromatography (hexane/CH2Cl2 = 1:3),
compound 14 (20 mg, 61%) was isolated as a yellow solid. 1H
NMR (200 MHz, CDCl3) d: 8.24 (s, 1H), 7.63 (d, J = 8.5 Hz, 1H),
7.31 (d, J = 8.5 Hz, 1H), 6.77–6.88 (m, 3H), 6.00–6.05 (m, 2H),
5.92 (d, J = 3.1 Hz, 2H), 4.26 (t, J = 5.3 Hz, 2H), 3.88 (t, J = 5.3 Hz,
2H), 1.98 (s, 3H). 13C NMR (100 MHz, CDCl3) d: 170.8, 167.3,
166.8, 147.7, 147.5, 146.8, 144.6, 136.3, 131.2, 129.0, 125.4,
125.2, 125.1, 124.5, 122.7, 121.4, 113.0, 109.8, 107.4, 101.9,
101.1, 61.3, 37.0, 20.7; IR (CH2Cl2, cast) 1707, 1649, 1544, 1501,
1454, 1378, 1288, 1230, 1035 cmꢀ1. FAB-LRMS m/z (rel intensity):
448 ([M+H]+, 24), 447 (22), 406 (5), 338 (4), 374 (2), 373 (2), 341
(1), 329 (1), 257 (2), 195 (2), 120 (10). FAB-HRMS Calcd for
C24H18O8N m/z: 448.1032 [M+H]+, found 448.1024.

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D. Janmanchi et al. / Bioorg. Med. Chem. 18 (2010) 1213–1226
4.1.10. (9-Benzo[1,3]dioxol-5-yl-7–dihydroxmethyl-
4.1.13. Acetic acid 9-benzo[1,3]dioxol-5-yl-8-formyl-
naphtho[1,2-d][1,3]dioxol-8-yl)-methanol (15)
naphtho[1,2-d][1,3]dioxol-7-yl methyl ester (18)
A solution of lithium aluminum hydride (22 mg, 0.58 mmol) in
THF (95 ml) was cooled to 0 °C in an ice bath and a THF solution of
3 (100 mg, 0.29 mmol in 5 mL) was added. The mixture was stirred
for 1 h at the same temperature. Water and a few drops of concen-
trated sulfuric acid were then slowly added. The mixture was then
extracted with CH2Cl2. The organic fraction was dried over MgSO4
and concentrated under reduced pressure to give almost pure 15.
After column chromatography (CH2Cl2), 15 (90 mg, 89%) was iso-
lated as a white solid. 1H NMR (400 MHz, CDCl3) d: 7.76 (s, 1H),
7.40 (d, J = 8.5 Hz, 1H), 7.16 (d, J = 8.5 Hz, 1H), 6.83 (d, J = 7.8 Hz,
1H), 6.76 (d, J = 1.3 Hz, 1H), 6.72 (dd, J = 1.5, 7.8 Hz, 1H), 6.04 (d,
J = 1.2 Hz, 1H), 6.00 (d, J = 1.2 Hz, 1H), 5.78 (dd, J = 1.4, 5.4 Hz,
2H), 4.89 (s, 2H), 4.59 (AB, J = 11.9 Hz, 2H), 2.11 (br s, 2H). 13C
NMR (100 MHz, CDCl3) d: 146.9, 146.8, 144.9, 142.2, 135.8, 135.5,
135.1, 133.7, 129.6, 129.5, 123.1, 122.1, 119.7, 111.1, 110.5,
107.4, 100.9, 65.3, 59.9. IR (CH2Cl2, cast): 3326, 1485, 1443, 1322,
1269, 1234, 1042, 932 cmꢀ1. FAB-LRMS m/z (rel intensity): 352
(M+, 42), 335 (13), 109 (33); FAB-HRMS Calcd for C20H16O6 m/z:
352.0947 [M]+, found 352.0943. Anal. Calcd for C20H16O6: C,
68.18; H, 4.58. Found: C, 67.29; H, 4.72.
Compound 16 (10 mg, 0.025 mmol) was dissolved in CH2Cl2
(5 ml) and pyridinium dichromate was added (43 mg, 0.11 mmol).
The reaction mixture was stirred for 1 h at room temperature. The
reaction mixture was then filtered and washed thoroughly with
CH2Cl2 (10 ml). The organic solution was dried over MgSO4 and
concentrated. After column chromatography (hexane/CH2Cl2 =
1:3), pure 18 (7 mg, 70%) was isolated. 1H NMR (400 MHz, CDCl3)
d: 9.87 (s, 1H), 7.80 (s, 1H), 7.45 (d, J = 8.5 Hz, 1H), 7.28 (d,
J = 8.5 Hz, 1H), 6.82–6.85 (m, 2H), 6.76 (dd, J = 1.6, 7.8 Hz, 1H),
6.07 (d, J = 1.2 Hz, 1H), 6.02 (d, J = 1.2 Hz, 1H), 5.86 (dd, J = 1.2,
10.3 Hz, 2H), 5.56 (s, 2H), 2.15 (s, 3H). 13C NMR (100 MHz, CDCl3)
d: 194.0, 170.7, 147.6, 147.1, 145.6, 143.5, 143.0, 130.9, 130.9,
130.1, 129.6, 128.4, 124.3, 122.4, 118.9, 113.4, 110.9, 107.5,
101.5, 101.3, 64.7, 21.1; IR (CH2Cl2, cast) 1730, 1680, 1585, 1446,
1372, 1234, 1039, 925 cmꢀ1. FAB-LRMS m/z (rel intensity): 392
(M+, 47), 391 (19), 333 (55), 332 (11), 289 (12), 255 (43), 239
(24), 185 (42), 173 (100), 105 (69). FAB-HRMS Calcd for C22H16O7
m/z: 392.0896 [M]+, found 392.0895. Anal. Calcd for C24H20O8: C,
66.05; H, 4.62. Found: C, 66.20; H, 4.86.
4.1.14. 9-Benzo[1,3]dioxol-5-yl-7,8-bis-bromomethyl-
naphtho[1,2-d][1,3]dioxole (19)
4.1.11. Acetic acid 9-benzo[1,3]dioxol-5-yl-8-hydroxmethyl-
naphtho[1,2-d][1,3]dioxol-7-yl methyester (16)
To a stirred solution of 15 (200 mg, 0.57 mmol) in dry toluene
(10 ml) was slowly added a solution of phosphorous tribromide
(218 mg, 0.80 mmol) in dry toluene (2 ml). After the addition
was completed (30 min), the mixture was stirred for 1 h at room
temperature and then an additional 2 h at 40 °C. The mixture
was then poured into ice and extracted with ether (50 ml). The
combined organic extracts was washed with brine (50 ml) and
dried over MgSO4. Crude 19 was obtained (200 mg, 74%) after the
solvent was removed in vacuo. This crude product was used in
the next step directly without further purification.
To a solution of 15 (20 mg, 0.056 mmol) in pyridine (3 ml) was
added acetic anhydride (6 mg, 0.056 mmol) and the resulting mix-
ture was stirred at room temperature for 16 h. After the comple-
tion of the reaction, 10% HCl (5 ml) was added and the mixture
was extracted with ether (10 ml). The ether extract was washed
with saturated NaHCO3 (5 ml), dried over MgSO4, and concen-
trated. After column chromatography (hexane/CH2Cl2 = 3:1), 16
(16 mg, 73%) was isolated as a colorless solid. 1H NMR (400 MHz,
CDCl3) d: 7.79 (s, 1H), 7.39 (d, J = 8.6 Hz, 1H), 7.15 (d, J = 8.5 Hz,
1H), 6.81 (d, J = 7.8 Hz, 1H), 6.76 (d, J = 1.4 Hz, 1H), 6.71 (dd,
J = 1.6, 7.8 Hz, 1H), 6.02 (d, J = 1.2 Hz, 1H), 5.98 (d, J = 1.2 Hz, 1H),
5.76 (dd, J = 1.4, 5.6 Hz, 2H), 5.39 (s, 2H), 4.51 (AB, J = 11.8 Hz,
2H), 2.10 (s, 3H), 1.91 (br s, 1H). 13C NMR (100 MHz, CDCl3) d:
170.7, 146.9, 146.9, 145.0, 141.9, 135.7, 135.4, 132.9, 130.2,
129.5, 123.1, 123.1, 122.3, 119.9, 111.2, 110.5, 107.4, 101.0, 65.1,
59.2, 21.7. IR (CH2Cl2, cast): 3439, 1737, 1599, 1453, 1375, 1234,
1042, 748 cmꢀ1. FAB-LRMS m/z (rel intensity): 394 (M+, 100), 377
(12), 335 (23), 275 (8), 189 (7), 109 (19). FAB-HRMS Calcd for
C22H18O7 m/z: 394.1053 [M]+, found 394.1057. Anal. Calcd for
C22H18O7: C, 67.00; H, 4.60. Found: C, 66.53; H, 4.72.
4.1.15. 9-Benzo[1,3]dioxol-5-yl-naphtho[1,2-d][1,3]dioxole-7,8-
dicarbaldehyde (20)
Compound 19 (120 mg, 0.025 mmol) and NaHCO3 (56 mg,
0.66 mmol) were dissolved in DMSO (10 ml) and the solution
was heated to 100 °C. The reaction was kept at same temperature
for 10 min. After cooled to room temperature, the reaction was
quenched with cold brine (100 ml) and the aqueous mixture was
extracted ether (50 ml ꢂ 2) and the combined organic layers was
washed with saturated NaHCO3 (25 ml) and brine (25 ml). The
ether extract was then dried over MgSO4 and concentrated. After
column chromatography (hexane/CH2Cl2 = 1:3), pure 20 (53 mg,
60%) was isolated. 1H NMR (400 MHz, CDCl3) d: 10.50 (s, 1H),
9.96 (s, 1H), 8.33 (s, 1H), 7.64 (d, J = 8.5, 1H), 7.34 (d, J = 8.5, 1H),
6.86 (d, J = 7.8 Hz, 1H), 6.83 (d, J = 1.4 Hz, 1H), 6.77 (dd, J = 1.6,
7.9 Hz, 1H), 6.08 (d, J = 1.1 Hz, 1H), 6.03 (d, J = 1.1 Hz, 1H), 5.93
(d, J = 1.2 Hz, 1H), 5.90 (d, J = 1.2 Hz, 1H) 13C NMR (100 MHz,
CDCl3) d: 193.6, 192.3, 147.9, 147.8, 147.2, 143.4, 142.4, 132.3,
131.4, 130.7, 129.9, 129.3, 125.3, 124.2, 120.5, 113.8, 110.8,
107.6, 101.9, 101.4. IR (CH2Cl2, cast): 1762, 1684, 1496, 1446,
1234, 1042, 925, 804 cmꢀ1. FAB-LRMS m/z (rel intensity): 349
([M+H]+, 25), 271 (22), 257 (13), 173 (12); FAB-HRMS Calcd for
C20H13O6 m/z: 349.0712 [M+H]+, found 349.0714.
4.1.12. Acetic acid 8-acetoxymethyl-9-benzo[1,3]dioxol-5-yl-
naphtho[1,2-d][1,3]dioxol-7-yl methy ester (17)
To a solution of compound 15 (30 mg, 0.085 mmol) in pyridine
(5 ml) was added acetic anhydride (18 mg, 0.17 mmol) and the
reaction was treated as described for 16. After column chromatog-
raphy (hexane/CH2Cl2 = 1:4), pure 17 (20 mg, 70%) was isolated as
a colorless solid. 1H NMR (400 MHz, CDCl3) d: 7.81 (s, 1H), 7.42 (d,
J = 8.5 Hz, 1H), 7.18 (d, J = 8.5 Hz, 1H), 6.79 (d, J = 7.8 Hz, 1H), 6.72
(d, J = 1.5 Hz, 1H), 6.67 (dd, J = 1.65, 7.8 Hz, 1H), 6.03 (d, J = 1.4 Hz,
1H), 5.99 (d, J = 1.4 Hz, 1H), 5.78 (dd, J = 1.4, 5.5 Hz, 2H), 5.27 (s,
2H), 4.99 (s, 2H), 2.09 (s, 3H), 2.00 (s, 3H). 13C NMR (100 MHz,
CDCl3) d: 170.7, 170.5, 146.9, 146.8, 145.2, 141.9, 137.4, 132.4,
130.5, 130.2, 130.1, 129.7, 123.1, 122.3, 119.7, 111.5, 110.4,
107.4, 101.1, 100.9, 64.8, 61.3, 21.0, 20.9. IR (CH2Cl2, cast): 1720,
4.1.16. 9-Benzo[1,3]dioxol-5-yl-6-hydroxy-naphtho[1,2-
d][1,3]dioxole-7,8-dicarbaxylic acid dimethyl ester (21)
A mixture of hydroxy acetal 1a (1.5 g, 4.35 mmol), dimethyl
acetylenedicarboxylate (625 mg, 4.39 mmol), acetic acid (0.62 ml),
and dichloromethane (2 ml) was heated at 140 °C for 1 h. The mix-
ture was cooled to room temperature, diluted with CH2Cl2 (30 ml),
and washed with 5% NaHCO3 solutions (3 ꢂ 30 ml). The organic
solution was dried over MgSO4 and concentrated. After column
1624, 1485, 1443, 1262, 1204, 1148, 1070, 1031, 929, 801 cmꢀ1
.
FAB-LRMS m/z (rel intensity): 436 (M+, 100), 391 (55), 363 (11),
333 (94), 261 (3), 176 (2). FAB-HRMS Calcd for C24H20O8 m/z:
436.1158 [M]+, found 436.1150. Anal. Calcd for C24H20O8: C,
66.05; H, 4.62. Found: C, 66.20; H, 4.86.

D. Janmanchi et al. / Bioorg. Med. Chem. 18 (2010) 1213–1226
1223
chromatography (CH2Cl2), yellow solid 21 (940 mg, 51%) was iso-
lated. 1H NMR (300 MHz, CDCl3) d: 12.58 (s, 1H), 8.12 (d,
J = 8.9 Hz, 1H), 7.18 (d, J = 8.8 Hz, 1H), 6.69–6.77 (m, 3H). 5.99
(dd, J = 1.3, 8.3 Hz, 2H), 5.83 (dd, J = 1.2, 6.3 Hz, 2H), 3.90 (s, 3H),
3.55 (s, 3H). 13C NMR (100 MHz, CDCl3) d: 170.4, 168.8, 161.7,
148.6, 146.9, 146.5, 142.5, 131.2, 130.9, 124.5, 123.9, 121.6,
120.9, 119.6, 111.2, 110.6, 107.0, 101.6, 100.9, 100.1, 52.8, 51.9.
IR (CH2Cl2, cast): 3489, 1735, 1684, 1492, 1444, 1246, 1037, 932,
812 cmꢀ1. FAB-LRMS m/z (rel intensity): 424 (M+, 18), 393 (10),
392 (4), 371 (2), 178 (1). FAB-HRMS Calcd for C22H16O9 m/z:
424.0794 [M]+, found 424.0800.
110.7, 107.1, 101.8, 100.9, 64.4, 52.3. IR (CH2Cl2, cast): 3425,
1812, 1765, 1578, 1492, 1442, 1365, 1223, 1039, 929 cmꢀ1. FAB-
LRMS m/z (rel intensity): 424 (M+, 18), 393 (35), 391 (22), 338
(9), 176 (14). FAB-HRMS Calcd for m/z: C22H16O9 m/z 424.0794
[M]+, found 424.0784.
4.1.20. 10-(3-Benzyloxy-4-methoxy-phenyl)-furo[30,40:6,7]-
naphtho[1,2-d][1,3]dioxol-7,9-dione (25)
As described previously in the synthesis of 3,21 acetal 1 (7 g,
36.05 mmol) was dissolved in dry THF (100 ml) under nitrogen
and the solution was cooled to ꢀ78 °C. n-BuLi (2.5 M in hexanes,
14.4 ml, 34.84 mmol) was added over 5 min. The mixture was stir-
red for another 15 min, the addition of 3-benzyloxy-4-methoxy-
benzaldehyde (8.8 g, 36.32 mmol) in dry THF (20 ml) then
followed. After stirred for 30 min, the solution was gradually
warmed to room temperature and stirred for another 2.5 h. The
reaction was quenched with water (100 ml) and the resulting mix-
ture was extracted with ether (3 ꢂ 100 ml). The organic fraction
was dried over MgSO4 and concentrated to give crude hydroxyac-
etal 1a. This crude hydroxy acetal was used in the following Diels–
Alder reaction without further purified or characterized.
4.1.17. 9-Benzo[1,3]dioxol-5-yl-6-hydroxy-naphtho[1,2-
d][1,3]dioxole-7,8-dicarbaxylic acid-8-methyl ester (22)
To a solution of KOH (560 mg, 10 mmol) in MeOH (10 ml) was
added 21 (20 mg, 0.047 mmol) and the mixture was refluxed for
2 h. After the completion of the reaction, 10% HCl (10 ml) was
added to neutralize the reaction mixture before the mixture was
extracted with CH2Cl2. The combined organic extracts was dried
over MgSO4 and concentrated. After column chromatography
(CH2Cl2/MeOH = 20:1), pure 22 (11 mg 58%) was isolated as a
white solid. 1H NMR (400 MHz, CDCl3) d: 8.12 (d, J = 8.8 Hz, 1H),
7.19 (d, J = 8.8 Hz, 1H), 6.78 (s, 1H), 6.76 (d, J = 0.8 Hz, 2H), 6.00
(d, J = 1.3 Hz, 1H), 5.97 (d, J = 1.3 Hz, 1H), 5.83 (dd, J = 1.4, 8.4 Hz,
2H), 3.89 (s, 3H). 13C NMR (100 MHz, CDCl3) d: 172.5, 168.9,
162.8, 151.8, 148.9, 146.9, 146.5, 142.6, 131.1, 124.6, 123.9,
121.9, 120.9, 119.9, 111.2, 110.6, 108.4, 107.1, 101.7, 100.9, 52.1.
IR (CH2Cl2, film): 3439, 1734, 1564, 1380, 1225, 1034, 941,
805 cmꢀ1. FAB-LRMS m/z (rel intensity): 410 (M+, 10), 393 (5),
377 (3), 338 (4), 176 (7). FAB-HRMS Calcd for C21H14O9 m/z:
410.0638 [M]+, found 410.0632.
Mixture of hydroxy acetal 1a (14.8 g, 33.90 mmol), maleic
anhydride (3.32 g, 33.95 mmol), acetic anhydride (12.7 ml), and
acetic acid (4.7 ml) was heated at 140 °C for 24 h. After this period,
the reaction mixture was cooled to room temperature, diluted
with CH2Cl2 (100 ml), and washed with 5% NaHCO3 solution
(3 ꢂ 100 ml). The organic fraction was dried over MgSO4 and con-
centrated to furnish yellow solid anhydride 25 (8 g, 52%). This
anhydride was used in the following reaction without further puri-
fication. 1H NMR (400 MHz, CDCl3) d: 8.40 (s, 1H), 7.71 (d,
J = 8.5 Hz, 1H), 7.26–7.41 (m, 6H), 6.93–6.98 (m, 2H), 6.90 (d,
J = 1.2 Hz, 1H), 5.84 (d, J = 1.2 Hz, 1H), 5.67 (d, J = 1.1 Hz, 1H),
5.12 (AB, J = 12.3 Hz, 2H), 3.96 (s, 3H). 13C NMR (100 MHz, CDCl3)
d: 163.0, 161.6, 150.2, 148.5, 147.3, 144.7, 139.6, 137.2, 131.8,
128.4, 127.9, 127.8, 127.3, 126.5, 125.9, 123.2, 122.8, 122.6,
121.9, 115.6, 114.3, 110.7, 102.1, 71.2, 55.9. IR (CH2Cl2, cast):
1833, 1773, 1592, 1510, 1453, 1258, 1223, 1138, 900 cmꢀ1. FAB-
LRMS m/z (rel intensity): 454 (M+, 7), 443 (1), 388 (1), 371 (2),
329 (2). FAB-HRMS Calcd for C27H18O7 m/z: 454.1053 [M]+, found
454.1048.
4.1.18. 9-Benzo[1,3]dioxol-5-yl-6-methoxy-naphtho[1,2-
d][1,3]dioxole-7,8-dicarbaxylic acid dimethyl ester (23)
To a solution of 21 (100 mg, 0.23 mmol) in DMF (5 ml) was
added CH3I (67 mg, 0.47 mmol) and K2CO3 (65 mg, 0.47 mmol).
The mixture was stirred for 3 h at room temperature. After the
completion of the reaction, the mixture was diluted with water
and extracted with CH2Cl2. The organic fraction was dried over
MgSO4 and concentrated. After column chromatography (CH2Cl2),
pure 23 (100 mg, 97%) was isolated. 1H NMR (400 MHz, CDCl3) d:
7.85 (d, J = 8.8 Hz, 1H), 7.25 (d, J = 8.8 Hz, 1H), 6.76 (d, J = 8.1 Hz,
2H), 6.70 (dd, J = 1.42, 7.9 Hz, 1H), 5.99 (dd, J = 1.3, 9.6 Hz, 2H),
5.85 (dd, J = 1.3, 9.6 Hz, 2H), 4.03 (s, 3H), 3.90 (s, 3H), 3.55 (s,
3H). 13C NMR (100 MHz, CDCl3) d: 168.2, 166.8, 155.9, 147.0,
146.9, 146.5, 142.9, 131.6, 131.4, 130.2, 124.7, 123.1, 121.1,
118.1, 117.7, 111.9, 110.7, 107.1, 101.6, 100.9, 63.8, 52.6, 52.2. IR
(CH2Cl2, cast): 1735, 1624, 1492, 1440, 1368, 1218, 1038, 932,
812 cmꢀ1. FAB-LRMS m/z (rel intensity): 438 (M+, 85), 407 (47),
377 (3), 349 (3). FAB-HRMS Calcd for C23H18O9 m/z: 438.0951
[M]+, found 438.0945.
4.1.21. 10-(4-Benzyloxy-3-methoxy-phenyl)-furo[30,40:6,7]-
naphtho[1,2-d][1,3]dioxol-7,9-dione (26), 10-(3,4-bis-benzyl-
oxy-phenyl)-furo[30,40:6,7]naphtho[1,2-d][1,3]dioxole-7,9-dione
(27), 10-(3,4-dimethoxyphenyl)-furo[30,40:6,7]naphtho-
[1,2-d][1,3]dioxole-7,9-dione (28) and 10-(4-trifluoromethoxy-
phenyl)-furo[30,40:6,7]naphtho[1,2-d][1,3]dioxole-7,9-dione
(29)
Anhydrides 26–29 were synthesized as described for 25. The
acetal 1 was dissolved in dry THF under nitrogen and cooled to
ꢀ78 °C. n-BuLi (2.5 M in hexanes) was added drop wise over
5 min. The mixture was stirred for another 15 min before the
addition of appropriate aldehydes (4-benzyloxy-3-methoxybenzal-
dehyde for 26, 3, 4-dibenzyloxy benzaldehyde for 27, 3,4-dime-
thoxybenzaldehyde for 28, 4-trifluoromethoxybenzaldehyde for
29). After stirred for 30 min, the solution was gradually warmed
to room temperature and was stirred for another 2.5 h. The
reaction was quenched with water and the resulting mixture was
extracted with ether. The ether extract was dried over MgSO4
and concentrated to give corresponding hydroxyl acetals. The
crude hydroxyl acetals were mixed with maleic anhydride, acetic
anhydride, and acetic acid in CH2Cl2 and the mixture was heated
to 140 °C for 24 h. After workup, 50% of 26 and 48% of 27 were ob-
tained as yellow solids in pure form. Anhydrides 28 and 29 thus
obtained were impure but used in the subsequent reaction without
further purification or characterization. Compound 26. 1H NMR
4.1.19. 9-Benzo[1,3]dioxol-5-yl-6-methoxy-naphtho[1,2-
d][1,3]dioxol-7,8-dicarbaxylic acid 8-methyl ester (24)
Compound 23 (90 mg, 0.20 mmol) was dissolved in hot MeOH
(5 ml) and a hot MeOH solution of KOH (1.16 g, 20 mmol in
5 mL) was added. The mixture was stirred overnight at 65 °C.
10% HCl (75 ml) was then added and the product was extracted
with CH2Cl2 (50 ml). The organic extract was dried over MgSO4
and concentrated. After column chromatography (hexane/
CH2Cl2 = 1:2), pure 24 (50 mg, 57%) was isolated as a white solid.
1H NMR (300 MHz, CDCl3) d: 7.81 (d, J = 8.7 Hz, 1H), 7.30 (d,
J = 8.9 Hz, 1H), 6.70–6.79 (m, 3H), 6.00 (dd, J = 1.2, 10.3 Hz, 2H),
5.88 (dd, J = 1.3, 8.2 Hz, 2H), 4.12 (s, 3H), 3.61 (s, 3H). 13C NMR
(100 MHz, CDCl3) d: 168.2, 167.6, 157.0, 147.7, 147.2, 146.6,
143.1, 132.4, 130.7, 123.8, 123.3, 121.8, 118.3, 115.1, 112.1,

1224
D. Janmanchi et al. / Bioorg. Med. Chem. 18 (2010) 1213–1226
(400 MHz, CDCl3) d: 8.41 (s, 1H), 7.73 (d, J = 8.6 Hz, 1H), 7.48 (d,
J = 7.2 Hz, 2H), 7.29–7.44 (m, 4H), 6.97 (d, J = 8.1 Hz, 1H), 6.89 (s,
1H), 6.87 (d, J = 8.2 Hz, 1H), 5.92 (s, 2H), 5.21 (AB, J = 12.1 Hz,
2H), 3.85 (s, 3H). 13C NMR (100 MHz, CDCl3) d: 163.0, 161.7,
148.8, 148.7, 148.6, 144.7, 139.7, 136.9, 131.92, 128.6, 128.0,
127.9, 127.5, 127.2, 126.0, 123.3, 122.9, 121.9, 114.3, 113.4,
112.8, 102.2, 71.0, 56.2; IR (CH2Cl2, cast) 1822, 1768, 1512, 1458,
1418, 1371, 1262, 1219, 1136, 898 cmꢀ1. EI-LRMS m/z (rel inten-
sity): 454 (M+, 100), 426 (<1), 422 (<1), 364 (16), 363 (51), 335
(6), 317 (3), 289 (4), 261 (5), 233 (4), 205 (3), 176 (3), 163 (5),
150 (2). EI-HRMS Calcd for C27H18O7 m/z: 454.1053 [M]+, found
454.1060. 27. 1H NMR (400 MHz, CDCl3) d: 8.40 (s, 1H), 7.71 (d,
J = 8.5 Hz, 1H), 7.49 (d, J = 7.3 Hz, 2H), 7.26–7.44 (m, 9H), 7.02 (d,
J = 8.2 Hz, 1H), 6.96 (d, J = 1.8 Hz, 1H), 6.91 (dd, J = 1.9, 8.1 Hz,
1H), 5.84 (s, 1H), 5.72 (s, 1H), 5.22 (d, J = 4.9 Hz, 2H), 5.12 (AB,
J = 11.9 Hz, 2H). 13C NMR (100 MHz, CDCl3) d: 163.0, 161.6, 149.6,
148.5, 148.0, 144.7, 139.6, 137.3, 137.1, 131.8, 128.5, 128.4,
128.0, 127.8, 127.8, 127.5, 127.4, 127.3, 125.9, 123.3, 122.8,
121.9, 116.7, 114.3, 113.6, 102.2, 71.5, 71.1. IR (CH2Cl2, cast):
1833, 1773, 1620, 1492, 1443, 1223, 1039, 900, 744 cmꢀ1. FAB-
LRMS m/z (rel intensity): 529 (M+, 9), 422 (1), 329 (3), 176 (4).
FAB-HRMS Calcd for C33H22O7 m/z: 530.1366 [M]+, found 530.1364.
tion mixture and the resulting solution was warmed back to room
temperature and concentrated in vacuo. The residue was dissolved
in ether (15 ml) and washed with saturated NaHCO3 (10 ml), water
(10 ml), and brine (10 ml). The organic extract was dried over
MgSO4 and concentrated. After column chromatography (hexane/
CH2Cl2 = 1:2), pure 32 (11 mg, 73%) was isolated as a yellow solid.
1H NMR (400 MHz, CDCl3) d: 8.39 (s, 1H), 7.67 (d, J = 8.7 Hz, 1H),
7.28 (d, J = 8.6 Hz, 1H), 6.89–6.91 (m, 2H), 6.80 (dd, J = 2.0, 8.2 Hz,
1H), 5.91 (dd, J = 1.4, 4.5 Hz, 2H), 5.69 (s, 1H), 5.17 (AB,
J = 15.1 Hz, 2H), 3.95 (s, 3H). 13C NMR (100 MHz, CDCl3) d: 171.2,
146.9, 146.3, 145.1, 141.8, 139.7, 130.8, 129.9, 129.2, 127.3,
125.4, 121.5, 121.1, 120.7, 115.5, 111.7, 110.0, 101.5, 69.6, 55.9.
IR (CH2Cl2, cast): 3510, 1751, 1631, 1510, 1453, 1269, 1067,
1017, 804 cmꢀ1. FAB-LRMS m/z (rel intensity): 350 (M+, 9), 338
(8), 326 (3), 279 (11), 255 (31), 253 (14), 239 (14), 219 (11), 185
(20), 173 (54), 149 (100), 113 (18). FAB-HRMS Calcd for C20H14O6
m/z: 350.0790 [M]+, found 350.0781. Anal. Calcd for C27H20O6: C,
73.63; H, 4.58. Found: C, 73.02; H, 4.68.
4.1.24. 10-(4-Benzyloxy-3-methoxy-phenyl)-9H-furo[30,40:6,7]-
naphtho[1,2-d][1,3]dioxol-7-one (33) and 10-(4-benzyloxy-3-
methoxy-phenyl)-7H-furo[30,40:6,7]naphtho[1,2-d][1,3]dioxol-
9-one (34)
4.1.22. 10-(3-Benzyloxy-4-methoxy-phenyl)-9H-furo[30,4’:6,7]-
naphtho[1,2-d][1,3]dioxol-7-one (30) and 10-(3-Benzyloxy-4-
methoxy-phenyl)-7H-furo[30,40:6,7]naphtho[1,2-d][1,3]dioxol-
9-one (31)
Anhydride 26 (500 mg, 1.1 mmol) was reduced with NaBH4
(130 mg, 3.43 mmol) in dry THF (20 ml) as described for 30. After
column chromatography (hexane/CH2Cl2 = 2:3), pure 33 (340 mg,
70%) and 34 (100 mg, 20%) were isolated as yellow solids. Com-
pound 33. 1H NMR (400 MHz, CDCl3) d: 8.40 (s, 1H), 7.69 (d,
J =8.7 Hz, 1H), 7.48 (d, J = 7.3 Hz, 2H), 7.28–7.40 (m, 4H), 6.95
(d, J = 8.0 Hz, 1H,), 6.85 (d, J = 1.8 Hz, 1H), 6.82 (dd, J = 1.9, 8.0 Hz,
1H), 5.90 (dd, J = 1.2, 5.0 Hz, 2H), 5.19 (AB, J = 15.2 Hz, 2H), 5.20
(s, 2H), 3.85 (s, 3H). 13C NMR (100 MHz, CDCl3) d: 171.1, 148.9,
148.0, 146.8, 141.7, 139.6, 136.9, 130.7, 129.8, 129.2, 128.5,
127.9, 127.4, 127.2, 125.4, 121.4, 212.1, 113.1, 111.7, 101.5, 71.0,
69.6, 56.1. IR (CH2Cl2, cast): 1759, 1631, 1514, 1457, 1269, 1134,
Anhydride 25 (200 mg, 0.44 mmol) and NaBH4 (60 mg,
1.58 mmol) were dissolved in dry THF (10 ml) and the solution
was stirred for 1 h at room temperature. After this period, 10%
HCl was added cautiously to the reaction mixture and the reac-
tion was stirred for another 30 min. The mixed solution was ex-
tracted with ether. The organic fraction was dried over MgSO4
and concentrated. After column chromatography (hexane/
CH2Cl2 = 1:2), pure 30 (120 mg, 63%) and 31 (30 mg, 16%) were
isolated as yellow solids. Compound 30. 1H NMR (300 MHz,
CDCl3) d: 8.33 (s, 1H), 7.64 (d, J = 8.5 Hz, 1H), 7.27–7.40 (m, 6H),
6.94 (d, J = 8.2 Hz, 1H), 6.86 (dd, J = 8.2, 1.8 Hz, 1H), 6.81 (d,
J = 1.8 Hz, 1H), 5.83 (d, J = 1.0 Hz, 1H), 5.68 (d, J = 1.2 Hz, 1H),
5.16 (AB, J = 12.8 Hz, 2H), 4.90 (AB, J = 15.2 Hz, 2H), 3.96 (s, 3H).
13C NMR (100 MHz, CDCl3) d: 171.1, 149.5, 147.2, 146.8, 145.8,
141.7, 139.7, 137.0, 130.7, 129.1, 128.6, 127.9, 127.1, 125.3,
121.9, 121.3, 121.1, 115.5, 111.7, 111.2, 101.4, 70.8, 69.4 55.9.
IR (CH2Cl2, cast): 1758, 1627, 1510, 1457, 1258, 1134, 1070,
1021 cmꢀ1. FAB-LRMS m/z (rel intensity): 440 (M+, 53), 439 (6),
413 (2), 350 (13). FAB-HRMS Calcd for C27H20O6 m/z: 440.1260
[M]+, found 440.1268. Anal. Calcd for C27H20O6: C, 73.63; H,
4.58. Found: C, 73.02; H, 4.68. Compound 31. 1H NMR
(300 MHz, CDCl3) d: 7.76 (s, 1H), 7.49 (d, J = 8.7 Hz, 1H), 7.41 (d,
J = 6.8 Hz, 2H), 7.25–7.34 (m, 4H), 6.94 (d, J = 1.6 Hz, 2H), 6.92
(d, J = 1.3 Hz, 1H), 5.76 (d, J = 1.2 Hz, 1H), 5.60 (d, J = 1.2 Hz, 1H),
5.33 (s, 2H), 5.10 (AB, J = 12.2 Hz, 2H), 3.94 (s, 3H). 13C NMR
(100 MHz, CDCl3) d: 169.1, 149.5, 147.0, 144.9, 143.5, 138.1,
137.6, 137.4, 132.7, 128.3, 127.7, 127.6, 127.3, 122.8, 122.1,
120.7, 120.4, 120.0, 116.0, 113.5, 110.4, 101.3, 71.1, 67.70, 55.8.
IR (CH2Cl2, cast): 1761, 1609, 1510, 1453, 1312, 1255, 1124,
1021, 755 cmꢀ1. FAB-LRMS m/z (rel intensity): 441 ([M+H]+, 9),
440 (8), 391 (7), 350 (1). FAB-HRMS Calcd for C27H21O6 m/z:
441.1338 [M+H]+, found 441.1332.
1070, 1017, 737 cmꢀ1
. FAB-LRMS m/z (rel intensity): 441
([M+H]+, 9), 440 (11), 439 (2), 371 (13), 349 (4), 257 (4). FAB-HRMS
Calcd for C27H20O6 m/z: 440.1260 [M]+, found 440.1257. 34. 1H
NMR (300 MHz, CDCl3) d: 7.78 (s, 1H), 7.48–7.52 (m, 3H), 7.30–
7.7.39 (m, 4H), 6.96 (d, J = 8.2 Hz, 1H), 6.89 (d, J = 1.8 Hz, 1H),
6.85 (dd, J = 1.9, 8.1 Hz, 1H), 5.83 (s, 2H), 5.35 (d, J = 0.8 Hz, 2H),
5.20 (AB, J = 12.0 Hz, 2H), 3.83 (s, 3H). 13C NMR (100 MHz, CDCl3)
d: 169.2, 148.4, 148.2, 145.0, 143.6, 138.2, 137.6, 137.3, 132.8,
128.5, 128.4, 127.8, 127.5, 122.2, 122.2, 120.8, 120.7, 120.2,
113.9, 113.6, 112.5, 101.4, 71.0, 67.8, 56.1. IR (CH2Cl2, cast):
1762, 1609, 1510, 1453, 1414, 1312, 1124, 1021 cmꢀ1. FAB-LRMS
m/z (rel intensity): 441 ([M+H]+, 11), 440 (12), 439 (3), 391 (4),
349 (4), 341 (<1). FAB-HRMS Calcd for C27H20O6 m/z: 440.1260
[M]+, found 440.1253.
4.1.25. 10-(4-Hydroxy-3-methoxy-phenyl)-9H-furo[30,40:6,7]-
naphtho[1,2-d][1,3]dioxol-7-one (35)
To a solution of 33 (50 mg, 0.11 mmol) in anhydrous CH2Cl2
(5 ml) at ꢀ78 °C was slowly added a solution of BBr3 (1 M in
CH2Cl2, 0.12 ml). The reaction mixture was allowed to proceed as
described for 32. After column chromatography (hexane/
CH2Cl2 = 1:2), pure 35 (28 mg, 72%) was isolated as a yellow solid.
1H NMR (400 MHz, CDCl3 ) 8.40 (s, 1H), 7.69 (d, J = 8.6 Hz, 1H),
d:
7.29 (d, J = 8.6 Hz, 1H), 6.97 (d, J = 8.3 Hz, 1H), 6.84 (dd, J = 1.8,
8.0 Hz, 1H), 6.81 (d, J = 1.7 Hz, 1H), 5.91 (dd, J = 1.2, 6.6 Hz, 2H),
5.72 (s, 1H), 5.19 (AB, J = 15.1 Hz, 2H), 3.86 (s, 3H). 13C NMR
(100 MHz, CDCl3) d: 171.2, 146.9, 146.1, 145.5, 141.8, 139.7,
130.8, 129.4, 128.7, 127.3, 125.5, 121.9, 121.6, 121.1, 114.0,
111.9, 111.8, 101.5, 69.6, 56.0. IR (CH2Cl2, cast): 3446, 1756,
1631, 1514, 1460, 1265, 1074, 1017 cmꢀ1. FAB-LRMS m/z (rel
intensity): 350 (M+, 24), 326 (13), 281 (39), 255 (32), 221 (88),
4.1.23. 10-(3-Hydroxy-4-methoxy-phenyl)-9H-furo[30,40:6,7]-
naphtho[1,2d][1,3]dioxol-7-one (32)
To a solution of 30 (20 mg, 0.045 mmol) in anhydrous CH2Cl2
(3 ml) at ꢀ78 °C was slowly added a solution of BBr3 (1 M in
CH2Cl2, 0.045 ml) and the mixture was stirred for 2 h at the same
temperature. After this time, MeOH (2 ml) was added to the reac-

D. Janmanchi et al. / Bioorg. Med. Chem. 18 (2010) 1213–1226
1225
207 (56), 173 (100), 147 (75), 109 (94). FAB-HRMS Calcd for
4.1.29. 10-(4-Trifluoromethoxy-phenyl)-9H-furo[30,40:6,7]-
naphtho[1,2-d][1,3]dioxol-7-one (40)
C20H14O6 m/z: 350.0790 [M]+, found 350.0783.
Anhydride 29 (1 g, 2.48 mmol) was reduced with NaBH4
(330 mg, 8.70 mmol) in dry THF (20 ml) as described for 30. After
column chromatography (hexane/CH2Cl2 = 3:2), pure 40 (610 mg,
63%) was isolated as a yellow solid. 1H NMR (400 MHz, CDCl3) d:
8.44 (s, 1H), 7.71 (d, J = 8.6 Hz, 1H), 7.37 (d, J = 8.7 Hz, 2H), 7.25–
7.32 (m, 3H), 5.89 (s, 2H), 5.16 (s, 2H). 13C NMR (100 MHz, CDCl3)
4.1.26. 10-(3,4-Bis-benzyloxy-phenyl)-9H-furo[30,40:6,7]naph-
tho[1,2-d][1,3]dioxol-7-one (36) and 10-(3,4-Bis-benzyloxy-
phenyl)-7H-furo[30,40:6,7]naphtho[1,2-d][1,3]dioxol-9-one (37)
Anhydride 27 (1 g, 1.88 mmol) was reduced with NaBH4 (24 mg,
6.35 mmol) in dry THF (20 ml) as described for 30 and 31. After
column chromatography (hexane/CH2Cl2 = 3:1), pure 36 (610 mg,
63%) and 37 (140 mg, 14%) were isolated as yellow solids. Com-
pound 36. 1H NMR (400 MHz, CDCl3) d: 8.33 (s, 1H), 7.63 (d,
J = 8.5 Hz, 1H), 7.50 (d, J = 7.0 Hz, 2H), 7.26–7.40 (m, 9H), 7.00 (d,
J = 8.0 Hz, 1H), 6.87 (s, 1H), 6.83 (d, J = 7.8 Hz, 1H), 5.81 (s, 1H),
5.71 (s, 1H), 5.22 (s, 2H), 5.17 (AB, J = 12.6 Hz, 2H), 4.93 (AB,
J = 15.2 Hz, 2H). 13C NMR (100 MHz, CDCl3) d: 171.1, 148.9, 147.9,
146.8, 141.7, 139.7, 137.2, 130.7, 129.9, 129.1, 128.5, 127.9,
127.6, 127.4, 127.2, 125.4, 122.2, 121.4, 121.1, 116.4, 114.1,
111.7, 101.4, 71.2, 71.1, 69.4. IR (CH2Cl2, cast): 1760, 1627, 1509,
1458, 1266, 1129, 1070, 1015, 739 cmꢀ1. FAB-LRMS m/z (rel inten-
sity): 516 (M+, 39), 515 (5), 460 (<1), 426 (9), 391 (7), 371 (2), 335
(5), 181 (7). FAB-HRMS Calcd for C33H24O6 m/z: 516.1573 [M]+,
found 516.1575. 37. 1H NMR (500 MHz, CDCl3) d: 7.77 (s, 1H),
7.50–7.48 (m, 3H), 7.41 (d, J = 7.2 Hz, 2H), 7.29–7.36 (m, 7H),
7.01 (d, J = 8.2 Hz, 1H), 6.97 (d, J = 1.8 Hz, 1H), 6.90 (dd, J = 1.8,
8.2 Hz, 1H), 5.76 (s, 1H), 5.66 (s, 1H), 5.35 (s, 2H), 5.20 (AB,
J = 11.8 Hz, 2H), 5.11 (AB, J = 12.2 Hz, 2H). 13C NMR (100 MHz,
CDCl3) d: 169.1, 149.0, 147.8, 144.9, 143.5, 137.9, 137.5, 137.4,
132.7, 128.6, 128.3, 128.2, 127.8, 127.6, 127.5, 127.4, 127.3,
123.1, 122.2, 120.7, 120.0, 117.2, 113.5, 113.4, 101.3, 71.5, 71.1,
67.7. IR (CH2Cl2, cast): 1759, 1609, 1505, 1455, 1312, 1208, 1122,
1018, 735 cmꢀ1. FAB-LRMS m/z (rel intensity): 516 (M+, 69), 515
(9), 426 (8), 425 (6), 371 (6), 369 (5), 181 (9). FAB-HRMS Calcd
for C33H24O6 m/z: 516.1573 [M]+, found 516.1575.
d: 170.8, 149.1, 148.1, 147.1, 141.6, 139.6, 135.4, 130.7, 130.5,
127.9, 125.6, 121.1, 120.3, 111.9, 101.6, 69.3. IR (CH2Cl2, cast):
1765, 1631, 1458, 1259, 1215, 1677, 1071, 1020 cmꢀ1. FAB-LRMS
m/z (rel intensity): 387 (M+, 100), 386 (11), 359 (6), 315 (5), 299
(3), 289 (2), 189 (3), 176 (3). FAB-HRMS Calcd for C20H11O5F3 m/
z: 388.0559 [M]+, found 388.0558.
5. Materials and methods for cell culture
5.1. Reagents
Hepatitis B surface antigen (HBsAg) and e antigen (HBeAg) en-
zyme immunoassay (EIA) kits were purchased from Bio-Rad (Her-
cules, CA, USA). Fetal calf serum was obtained from Hyclone
(Logan, UT, USA). Dulbecco’s modified Eagle’s medium (DMEM)
was obtained from Gibco/BRL (Gaithersbung, MD, USA). [a-
32P]
dCTP was obtained from PerkinElmer Life Sciences (MA, USA).
Sea Kem LE-agarose was purchased from FMC Bioproducts (Rock-
land, MA, USA).
5.2. Cell culture
Human hepatoblastoma HepA2 cell line is derived from HepG2
cells by transfecting with two tandem repeats of the HBV genome
and continually secretes HBsAg and HBeAg into the culture med-
ium.28 The 1.3ES2 cell line is a clonal derivative of HepG2 cells in
which the 1.3-copies of the entire HBV genome was stably inte-
grated in the host genome.26 These cell lines were used to assess
the antiviral activity of helioxanthin and its analogues. The lamivu-
dine-resistant cell line M33 is also a hepatoblastoma HepG2 cell
line stably transfected with 1.3-copies of entire HBV genome,
which contains the L515M/M539V double mutation.29 This stable
cell line secretes lamivudine-resistant HBV particles containing
viral DNA and DNA polymerase activity. Stock cultures of human
hepatoma cells HepA2, 1.3ES2 and M33 were maintained in DMEM
supplemented with 10% fetal calf serum and antibiotics (100 IU/ml
each of penicillin and streptomycin) in a humidified atmosphere
containing 5% CO2 and 95% air at 37 °C. The cultures were passaged
by trypsinization every 4 days. For bioassays, cells were plated
either in 24-well plates at a density of 8 ꢂ 104 cells/well or in
100-mm culture dishes at a density of 1.5 ꢂ 106 cells/dish in
DMEM medium containing 10% fetal calf serum.
4.1.27. 10-(3,4-Dihydroxy-phenyl)-9H-furo[30,40:6,7]naphtho-
[1,2-d][1,3]dioxol-7-one (38)
To a solution of 36 (50 mg, 0.096 mmol) in anhydrous CH2Cl2
(5 ml) at ꢀ78 °C was added slowly a solution of BBr3 (1 M in
CH2Cl2, 0.22 ml) and the reaction was allowed to proceed as de-
scribed for 32. After column chromatography (CH2Cl2/metha-
nol = 19:1), pure 36 (24 mg, 77%) was isolated. 1H NMR
(300 MHz, CD3COCD3) d: 8.42 (s, 1H), 7.88 (d, J = 8.6 Hz, 1H), 7.41
(d, J = 8.6 Hz, 1H), 6.91 (d, J = 2.2 Hz, 1H), 6.89 (s, 1H), 6.77 (dd,
J = 2.1, 8.0 Hz, 1H), 5.97 (d, J = 3.6 Hz, 2H), 5.61 (s, 2H), 5.24 (AB,
J = 15.0 Hz, 2H). 13C NMR (100 MHz, CD3COCD3) d: 171.0, 147.4,
145.7, 145.2, 142.5, 140.7, 131.7, 130.5, 129.2, 126.9, 126.1,
122.1, 122.0, 121.5, 117.1, 115.4, 112.2, 102.1, 69.9. IR (CH2Cl2,
cast): 3354, 1737, 1624, 1510, 1453, 1269, 1070, 1017, 804 cmꢀ1
.
FAB-LRMS m/z (rel intensity): 336 (M+, 7), 176 (2). FAB-HRMS
Calcd for C19H12O6 m/z: 336.0634 [M]+, found 336.0628.
4.1.28. 10-(3,4-Dimethoxy-phenyl)-9H-furo[30,40:6,7]naphtho-
[1,2-d][1,3]dioxol-7-one (39)
5.3. Quantification of HBsAg and HBeAg
Anhydride 28 (1 g, 2.64 mmol) was reduced with NaBH4
(350 mg, 9.24 mmol) in dry THF (20 ml) as described for 30. After
column chromatography (hexane/CH2Cl2 = 3:1), pure 39 (630 mg,
65%) was isolated. 1H NMR (400 MHz, CDCl3) d: 8.41 (s, 1H), 7.69
(d, J = 8.6 Hz, 1H), 7.30 (d, J = 8.6 Hz, 1H), 6.90–6.92 (m, 2H), 6.83
(d, J = 1.8 Hz, 1H), 5.91 (d, J = 0.6 Hz, 2H), 5.20 (AB, J = 15.1 Hz,
2H), 3.95 (s, 3H), 3.85 (s, 3H). 13C NMR (100 MHz, CDCl3) d:
170.9, 148.8, 148.4, 146.8, 141.6, 139.6, 130.7, 129.2, 127.8,
127.7, 127.1, 125.3, 121.4, 121.1, 121.0, 112.7, 111.6, 110.6,
101.4, 69.4, 55.9, 55.8. IR (CH2Cl2, cast): 1758, 1627, 1514, 1457,
1258, 1134, 1070, 1024 cmꢀ1. FAB-LRMS m/z (rel intensity): 364
(M+, 39), 326 (10), 281 (12), 243 (13), 191(18), 109 (100). FAB-
HRMS Calcd for C21H16H6 m/z: 364.0947 [M]+, found 364.0942.
Cells were seeded either in 96-well plates at a density of 3 ꢂ 104
cells/well or in 24-well plates at a density of 8 ꢂ 104 cells/well in
DMEM medium containing 10% fetal calf serum. After 24 h of incu-
bation, the cells were washed twice with phosphate-buffered sal-
ine (PBS), pH 7.0, and treated with various concentrations of
drugs in serum-free DMEM for 48 h. The HBsAg and HBeAg in
the culture medium were measured by the appropriate enzyme
immunoassay (EIA) kits (Bio-Rad, CA, USA). The viability of cells
was determined by a WST-1 cell proliferation assay. For the
WST-1 assay, WST-1 (Roche Diagnostics, Mannheim, Germany)
was added to each well and incubated for 0.5 h. The amount
of formazan dye formed can be correlated to the number of

1226
D. Janmanchi et al. / Bioorg. Med. Chem. 18 (2010) 1213–1226
metabolically active cells and is quantitatively determined using a
scanning multi-well spectrophotometer (ELISA reader) at absor-
bance 450 nm.
promoter from pXP-CAT were inserted into the MluI-HindIII site
of the pGL3-Basic vector (Promega, Madison, WI). The pS1P-Luc,
pS2P-Luc, pCP-Luc, pE/XP-Luc plasmids were generous gifts from
Dr. Chungming Chang (National Health Research Institutes,
Taiwan).
5.4. Quantitative detection of HBV DNA by real-time light cycler
PCR
Acknowledgments
We have used dilutions of known amounts of HBV-DNA from
plasmid as a control. The standard curve showed a good linear
range when 103–107 copies of plasmid DNA were used as tem-
plates (data not shown). Cells were seeded in 100 mm culture
dishes at a density of 5 ꢂ 106 cells/well, then treated with various
concentrations of drug in serum-free DMEM for 72 h. For quantifi-
cation of HBV DNA, viral DNA was extracted from culture media
using High Pure Viral Nucleic Acid Kit (Roche, Mannheim, Ger-
many) according to the manufacturer’s instructions. The PCR prim-
ers used were purchased from Tib-Molbiol (Berlin, Germany). The
oligonucleotide sequences of primers were: HBV Forward: 50-CA
GGTCTGTGCCAAG-30 (the accession number of GenBank: V01460,
nt 1168–1182) HBV Reverse: 50-TGCGGGATAGGACAA-30 (nt
1359–1345). The PCR cycling program consisted of an initial dena-
turing step at 95 °C for 10 min, followed by 45 amplification cycles
at 95 °C for 12 s, 54 °C for 20 s.
The authors thank Drs. Kak-Shan Shia and Shu-Jen Chen for the
advice and comments on the manuscript; Dr. Chungming Chang for
providing pS1P-Luc, pS2P-Luc, pCP-Luc, pE/XP-Luc plasmids. This
work was supported by grants: NSC 97-3112-B-010-003 from Na-
tional Science Council and Ministry of Education, Aim for the Top
University Plan, Republic of China.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2009.12.038.
References and notes
1. Chang, M. H.; Chen, C. J.; Lai, M. S.; Hsu, H. M.; Wu, T. C.; Kong, M. S.; Liang, D.
C.; Shau, W. Y.; Chen, D. S. N. Eng. J. Med. 1997, 336, 1855.
2. Robinson, W. S. Annu. Rev. Microbiol. 1977, 31, 357.
3. Yuh, C. H.; Ting, L. P. J. Virol. 1990, 64, 4281.
4. Dienstag, J. L.; Perrillo, R. P.; Schiff, E. R.; Bartholomew, M.; Vicary, C.; Rubin, M.
N. Eng. J. Med. 1995, 333, 1657.
5.5. RNA isolation and Northern blot analysis
Total RNA was extracted from the cells using the phenol and
guanidium isothiocyanate method.30 The RNA (20
lg) was dena-
5. Walsh, K. M.; Woodall, T.; Lamy, P.; Wight, D. G.; Bloor, S.; Alexander, G. J. Gut
2001, 49, 436.
tured by 2.2 M formaldehyde, separated on a denaturing formalde-
hyde 1.2% agarose gel, and transferred to a nylon membrane
(Hybond-XL, Amersham). The membrane was hybridized with a
32P-radiolabelled full-length HBV probe. The relative amount of to-
tal RNA applied was normalized to that of glyceraldehyde-3-phos-
phate dehydrogenase.
6. Franco, J.; Saeian, K. J. Vasc. Interv. Radiol. 2002, 13, S191.
7. Langley, D. R.; Walsh, A. W.; Baldick, C. J.; Eggers, B. J.; Rose, R. E.; Levine, S. M.;
Kapur, A. J.; Colonno, R. J.; Tenney, D. J. J. Virol. 2007, 81, 3992.
8. Ruiz-Sancho, A.; Sheldon, J.; Soriano, V. Expert Opin. Biol. Ther. 2007, 7, 751.
9. Lee, H. S.; Chung, Y. H.; Lee, K. S.; Byun, K. S.; Paik, S. W.; Han, J. Y.; Yoo, K.; Yoo,
H. W.; Lee, J. H.; Yoo, B. C. Hepatology 2006, 43, 982.
10. Zoulim, F. J. Clin. Virol. 2001, 21, 243.
11. Yeh, C. T.; Chien, R. N.; Chu, C. M.; Liaw, Y. F. Hepatology 2000, 31, 1318.
12. Locarnini, S.; Mason, W. S. J. Hepatol. 2006, 44, 422.
13. Mohanty, S. R.; Kupfer, S. S.; Khiani, V. Nat. Clin. Pract. Gastroenterol. Hepatol.
2006, 3, 446.
14. Lee, Y. S.; Suh, D. J.; Lim, Y. S.; Jung, S. W.; Kim, K. M.; Lee, H. C.; Chung, Y. H.;
Yoo, W.; Kim, S. O. Hepatology 2006, 43, 1385.
15. Deres, K.; Schroder, C. H.; Paessens, A.; Goldmann, S.; Hacker, H. J.; Weber, O.;
Kramer, T.; Niewohner, U.; Pleiss, U.; Stoltefuss, J.; Graef, E.; Koletzki, D.;
Masantschek, R. N.; Reimann, A.; Jaeger, R.; Gross, R.; Beckermann, B.;
Schlemmer, K. H.; Haebich, D.; Rubsamen-Waigmann, H. Science 2003, 299,
893.
5.6. Transient transfections and luciferase assay
HepA2 cells were transfected with various plasmids by using
lipofectamine 2000 transfection reagent (Invitrogen). The transfec-
ted cells were changed to a serum-free DMEM with drug for two
days. To prepare total cell lysate from transfected cells for lucifer-
ase activity measurements, medium was aspirated from the cell
culture and the cells were gently rinsed with PBS. Cells were
scrapped from the plates and collected through centrifugation.
The supernatant was collected for protein and luciferase activity
measurements immediately following lysate preparation. Protein
concentrations of the resultant cell lysates were measured by the
Bradford method. Lysates prepared from transfected cells were
analyzed for luciferase activity using a luminometer and the Pro-
mega Luciferase Assay System as described by the manufacturer
(Promega). Luciferase activities were normalized to the amount
of protein in each lysate. For all transient transfections with pro-
moter-luciferase reporter constructs, the level of luciferase activity
was determined without drug treatment to be set to one. The
transfection efficiency was normalized using the activity of b-
galactosidase as an internal control. The values are means plus
and minus the standard error of the mean of at least three indepen-
dent experiments.
16. Tan, T. M.; Chen, Y.; Kong, K. H.; Bai, J.; Li, Y.; Lim, S. G.; Ang, T. H.; Lam, Y.
Antiviral Res. 2006, 71, 7.
17. Tseng, Y. P.; Kuo, Y. H.; Hu, C. P.; Jeng, K. S.; Janmanchi, D.; Lin, C. H.; Chou, C. K.;
Yeh, S. F. Antiviral Res. 2008, 77, 206.
18. Li, Y.; Fu, L.; Yeo, H.; Zhu, J. L.; Chou, C. K.; Kou, Y. H.; Yeh, S. F.; Gullen, E.;
Austin, D.; Cheng, Y. C. Antiviral Chem. Chemother. 2005, 16, 193.
19. Mizufune, H.; Nakamura, M.; Mitsudera, H. Tetrahedron Lett. 2001, 42,
437.
20. Holmes, T. L.; Stevenso, R. J. Chem. Soc. C-Org. 1971, 2091.
21. Stevenson, R.; Weber, J. V. J. Nat. Prod. 1989, 52, 367.
22. Charlton, J. L.; Oleschuk, C. J.; Chee, G. L. J. Org. Chem. 1996, 61, 3452.
23. Yeo, H.; Li, Y.; Fu, L.; Zhu, J. L.; Gullen, E. A.; Dutschman, G. E.; Lee, Y.; Chung, R.;
Huang, E. S.; Austin, D. J.; Cheng, Y. C. J. Med. Chem. 2005, 48, 534.
24. Cow, C.; Leung, C.; Charlton, J. L. Can. J. Chem.-Revue Canadienne De Chim. 2000,
78, 553.
25. Yeh, S. F.; Hong, C. Y.; Huang, Y. L.; Liu, T. Y.; Choo, K. B.; Chou, C. K. Antiviral
Res. 1993, 20, 185.
26. Chou, Y. C.; Jeng, K. S.; Chen, M. L.; Liu, H. H.; Liu, T. L.; Chen, Y. L.; Liu, Y. C.; Hu,
C. P.; Chang, C. J. Virol. 2005, 79, 1813.
27. Ying, C.; Li, Y.; Leung, C. H.; Robek, M. D.; Cheng, Y. C. Proc. Natl. Acad. Sci. U.S.A.
2007, 104, 8526.
28. Chang, C. M.; Jeng, K. S.; Hu, C. P.; Lo, S. J.; Su, T. S.; Ting, L. P.; Chou, C. K.; Han, S.
H.; Pfaff, E.; Salfeld, J., et al EMBO J. 1987, 6, 675.
5.7. Plasmids
29. Huang, R. L.; Huang, Q.; Chen, C. F.; Chang, T. T.; Chou, C. J. Chin. Pharm. J. 2003,
55, 371.
30. Gruffat, D.; Piot, C.; Durand, D.; Bauchart, D. Anal. Biochem. 1996, 242, 77.
31. Mullis, K.; Faloona, F.; Scharf, S.; Saiki, R.; Horn, G.; Erlich, H. Cold Spring Harb.
Symp. Quant. Biol. 1986, 51, 263.
The plasmids were constructed by standard DNA cloning proce-
dures and polymerase chain reaction (PCR) methods.31 The HBV se-
quence used in this study is of ayw subtype.32 The accession
number of GeneBank: V01460, Eco RI site as nucleotide 1. To
generate pXP-Luc, the Xbal-HindIII fragments containing the X
32. Galibert, F.; Mandart, E.; Fitoussi, F.; Tiollais, P.; Charnay, P. Nature 1979, 281,
646.