Synthesis and biological evaluation of helioxanthin analogues

Article abstract of DOI:10.1016/j.bmc.2012.11.037

Helioxanthin and analogues have been demonstrated to suppress gene expression of human hepatitis B virus. In the continuous attempt to optimize antiviral activity, various structural motifs were grafted on the helioxanthin scaffold. Many such analogues were synthesized and evaluated for their anti-hepatitis B virus activity. Structure-activity relationships of these helioxanthin derivatives are also discussed. Among these new compounds, 15 exhibits the highest activity against HBV (EC₅₀ = 0.06 μM). This compound can suppress viral surface antigen and DNA expression. Furthermore, viral RNA is also diminished while the core promoter is deactivated upon treatment by 15. A plausible working mechanism is postulated. Our results establish helioxanthin lignans as potent anti-HBV agents with unique mode of action. Since their antiviral mechanism is distinct from current nucleoside/nucleotide drugs, helioxanthin lignans constitute a potentially new class of anti-HBV agents for combination therapy.

Full text of DOI:10.1016/j.bmc.2012.11.037

Bioorganic & Medicinal Chemistry 21 (2013) 2163–2176
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of helioxanthin analogues
b
b
b
b
Damodar Janmanchi a,b, Chih Hsiu Lin a, , Jui-yu Hsieh , Ya-ping Tseng , Tai-an Chen , Hong-jhih Jhuang ,
⇑
Sheau Farn Yeh b,
⇑
a Institute of Chemistry, Academia Sinica, Taipei, Taiwan, ROC
b Institute of Biochemistry and Molecular Biology, National Yang-Ming University, Taipei, Taiwan, ROC
a r t i c l e i n f o
a b s t r a c t
Article history:
Helioxanthin and analogues have been demonstrated to suppress gene expression of human hepatitis B
virus. In the continuous attempt to optimize antiviral activity, various structural motifs were grafted on
the helioxanthin scaffold. Many such analogues were synthesized and evaluated for their anti-hepatitis B
virus activity. Structure–activity relationships of these helioxanthin derivatives are also discussed.
Received 27 August 2012
Revised 20 November 2012
Accepted 21 November 2012
Available online 5 December 2012
Among these new compounds, 15 exhibits the highest activity against HBV (EC50 = 0.06 lM). This com-
pound can suppress viral surface antigen and DNA expression. Furthermore, viral RNA is also diminished
while the core promoter is deactivated upon treatment by 15. A plausible working mechanism is postu-
lated. Our results establish helioxanthin lignans as potent anti-HBV agents with unique mode of action.
Since their antiviral mechanism is distinct from current nucleoside/nucleotide drugs, helioxanthin lign-
ans constitute a potentially new class of anti-HBV agents for combination therapy.
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Helioxanthin derivatives
Hepatitis B virus
Anti-HBV agents
Structure–activity relation
1. Introduction
ly, the polymerase gene encodes for the multifunctional polymer-
ase, and the X gene encodes for the X protein. After translocated
The hepatitis B virus (HBV) is a major world health problem.
According to the world health report released by WHO in 1997,
at least 300 million of are chronically infected with HBV despite
the availability of an effective vaccine.1 The majority of cases occur
in developing country in which nearly 10% of the population are in-
fected. Long term HBV infection can result in acute and fulminant
liver failure. Many chronic patients latter develop liver cirrhosis
and eventually hepatocellular carcinomas. It has been estimated
that HBV-related complications cause 600,000–1.2 million deaths
every year. No therapeutic strategy that can completely eradicate
HBV from the host is hitherto available. Yet, to diminish virus rep-
lication is still crucial for the patients, since it would not only pre-
vent further infection but also attenuated inflammation response
to viral expression.
Knowledge of the viral life cycle and pathogenesis can be an
important guide in the development of effective therapies. The
infection of HBV begins with the internalization viral DNA which
is a circular 3.2 kb genome composed of a partially double-
stranded DNA.2 Its genome encodes for four main genes by a series
of overlapping reading frames. The core gene encodes for core anti-
gen (HBcAg) and the precore gene encodes for the e antigen
(HBeAg). The three envelope genes PreS1, PreS2 and S encode for
the large, middle, and small envelope proteins, respectively. Final-
into the hepatocyte nucleus, the genome is converted into cova-
lently closed circle DNA [cccDNA] which serves as the template
for the transcription of all viral mRNA. During the subsequent rep-
lication, the mRNA not only serves as the template for reverse tran-
scription but also encodes the viral core protein and the HBV
polymerase. In the cytoplasm, the mRNA is translated to make viral
proteins. After encapsulation and maturation, the viral particles are
released from the infected cells.
Interferon (INF) treatment is the first approved therapy for HBV
related liver disease.3 This class of cytokine can bind to receptors
on the hepatocyte membrane and trigger a cascade of intracellular
immune response to combat HBV proliferation.4 However, the inci-
dence of adverse effect in INF treatment, ranging from fatigue to
decrease in platelets, makes it unsuitable for some patients. In
addition, INFs need to be administrated by weekly injection which
diminishes its practicality in area with limited medical resources.
Recently, several nucleoside and nucleotide analogues were
launched to treat chronic HBV infection. This class of compound
suppresses HBV reproduction by inhibiting the viral polymerase.5
Lamivudine6 [(ꢀ)-
L
-20,30-dideoxy-30-thiacytidine] is the first nucle-
otide analogue licensed for chronic hepatitis B infection. Since
then, adefovir7,8 [PMEA, 9-(2-phosphonylmethoxyethyl)adenine],
entecavir9 (BMS-200475, carbocyclic 20-deoxyguanosine), telbivu-
dine10 (synthetic -thymidine nucleoside analogue), and levudine11
L
[L-FMAU, 1-[(2S,3R,4S,5S)-3-fluoro-4-hydroxy-5-(hydroxymethyl)
oxolan-2-yl]-5-methyl-pyri-midine-2,4-dione] were also approved
for clinical use. Unfortunately, drug resistant mutant often emerge
⇑
Corresponding authors.
E-mail addresses: ChihHsiuLina@academiasincia.edu, chemopera@yahoo.com
(C.H. Lin), fyeh@ym.edu.tw (S.F. Yeh).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.11.037

2164
D. Janmanchi et al. / Bioorg. Med. Chem. 21 (2013) 2163–2176
when the target of the drug is polymerase. Indeed, resistant and
cross-resistant strand against lamivudine (3TC) and adefovir ap-
peared after only one to 2 years of treatment.12,13 To overcome
the drug-resistance problem, it is essential to develop non-nucleo-
tide drugs that can block viral replication at a different stage dur-
ing its life cycle. One successful example of this strategy is the
heteroarylpyrimidine Bay 41-4109 reported by Deres and Schrod-
er.14 The authors demonstrated that this drug impedes HBV repli-
cation by inhibiting the assembly of nucleocapsids. Such approach
not only opens unique opportunities in anti-HBV research, but may
also provide new insights to the development of antiviral agent in
general (Fig. 1).
revealed. We also put forward a refined SAR model to guide future
investigation.
2. Results
2.1. Synthesis of helioxanthin analogues
In our effort to explore various anti-HBV agents, we constantly
go back helioxanthin analogues for inspiration. Our strategy is to
identify essential structural motifs that enable a helioxanthin ana-
logue to block HBV production. These key structural elements will
then be combined on the arylnaphthalene scaffold to produce more
potent helioxanthin derivatives. In our earlier studies, we have dis-
covered a few compounds with comparable antiviral activity to the
parent compound.23 The present study is an extension of our
previous effort with similar approach.
There are several different synthetic strategies to arylnaphtha-
lene lignans in the literature, including the dimerization of
dehydro-cinnamate,25,26 Pd-catalyzed benzannulation,27 and
Diels–Alder approach.28 Although each methodology has its merits,
we found the Diels–Alder approach developed by Charlton has the
widest substrate scope and therefore enables us to generate a
Natural products constitute an inexhaustible source of inspira-
tion and challenge for many disciplines in science. For medicinal
chemistry, natural products provide instrumental lead structures
that can be developed into useful pharmacological targets against
pathogenic agents. Arylnaphthalene lignans are a subclass of
botanical lignans. Their basic structural feature is a naphthalene
core with an aryl group at 1 position. A diverse range of medicinal
potentials, including antitumor15–17 and antiviral activities,18 were
discovered for this class of compounds. Helioxanthin is an aryl-
naphthalene lactone lignan isolated from Taiwania cryptomerioides
Hayata19 and Heliopsis scabra Dunal.20 It was found helioxanthin
and its derivatives can inhibit HBV production in various strand
of HBV infected cellular models with sub-micromolar concentra-
tion.21,22 We have also reported helioxanthin can effectively inhibit
HBV gene expression and replication in 3TC-resistance strand of
human hepatoacyte.23 Many helioxanthin congeners were synthe-
sized and two derivative, lactam 5-4-2 and phthalazin dione 8-1,
are even more potent than the parent compound against HBV.24
Furthermore, we have verified that such compounds most likely
target the viral mRNA production machinery instead of the poly-
merase. With this antiviral mechanism, relapse associated with
drug resistance is much less likely. In our previous study, we have
synthesized a wide range of helioxanthin analogues and tested
their anti-HBV activities. A preliminary structure–activity relation
(SAR) model was also proposed.23 In our ongoing venture to dis-
cover new anti-HBV agents and investigate their mechanism, a
new series of nearly 40 helioxanthin derivatives were synthesized
and their anti-HBV activities assessed. Here, we report the
synthetic strategy and the results of anti-HBV testing. A new com-
pound with potency exceeding that of the parent helioxanthin was
library of derivatives in
a few routine steps. The synthesis
(Scheme 1) starts with piperonal. The aldehyde was first protected
as acetal before ortho-metallation was carried out with n-butyl
lithium. The aryl lithium thus generated can react with various
aldehydes to furnish the hydroxyacetal 2. When heated in acetic
acid solution, 2 was converted into bezo[c]furan in situ to react
with maleic anhydride in the key Diels–Alder cycloaddition. The
adduct underwent spontaneous aromatization under the reaction
condition to give 3 which was then reduced to give the lactone
helioxanthin 4 (separated from the regioisomer retro-4). When
we substituted fumaronitrile for maleic anhydride as the dieno-
phile, dinitrile 5 is produced instead (Scheme 2).
By employing various aldehydes after the lithiation step, we
synthesized helioxanthin analogues of which the [1,3]dioxole rings
on the aryl moiety were opened. Lactones (12, 17, 22, 26 and 29)
and their regioisomers (13, 18, and 23) were prepared as described
previously.23 Phenolic hydroxyl groups were integrated as benzyl
protected ether and deprotected after the anhydrides were re-
duced to lactones. Compounds 14 and 19 thus produced are acet-
ylated to give 15 and 20, respectively. 16, 21, and 25 were
F
O
NH2
N
N
NH
Cl
N
MeOOC
HO
HO
N
N
F
N
HO
N
O
P
O
O
N
H
H
O
S
F
Adefovir
Lamivudine
Bay41-4109
O
O
O
NH
NH
NH
O
O
O
O
O
O
O
O
O
O
O
O
5-4-2
O
O
8-1
helioxanthin
Figure 1. Examples of anti-HBV compounds.

D. Janmanchi et al. / Bioorg. Med. Chem. 21 (2013) 2163–2176
2165
O
O
O
O
CHO
O
O
OH
O
O
a
d
b
O
c
O
O
O
O
O
O
O
O
O
O
O
O
O
O
1
2
O
O
e
4
3
CN
CN
O
O
O
O
5
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; (e) fumaronitrile, AcOH, Ac2O, CH2Cl2, 140 °C, 24 h.
produced alone side their regioisomers 14, 19, and 24
(previously unpublished). In hope to improve antiviral activity
through fluorination, compound 27 was synthesized by using
2,2-difluorobenzo[d][1,3]dioxole-5-carbaldehyde as the electro-
philic aldehyde after the lithiation step. In an attempt to identify
the target protein of helioxanthin, we synthesized 30 and 31 with
functionalized long alkyl chain attached to the phenyl group. We
perceive such compounds can be used as probes that may reveal
the action site for this class of agents through various in vivo bio-
conjugative methodologies. We also envision that similar com-
pounds with an anchoring group can be used as stationary phase
in affinity based separation techniques.
In our earlier investigation, lactone 4 was reduced with excess
LiAlH4 to provided diol 32. In the same study, we found that 32
and its acetate derivatives 32(OAc) and 32(OAc)2 are quite active
against HBV. By combining the modification at phenyl moiety with
this diol motif, we wish to generate another series of active ana-
logues. To fulfill this purpose, lactone 14, 19, 24, 26 were first re-
duced to 35, 39, 45, and 50, respectively. The free hydroxyl
groups in these compounds were then esterified with acetic anhy-
dride to produce 36, 37, 40, 41, 46, 47, and 51–53. Compound with
higher hydrophobicity (33, 34, 38, 42–44, 49, and 54) can be syn-
thesized by using hexanoic anhydride in the esterification step.
In these reactions, mixtures of products with different degrees of
esterification are inevitably produced. Fortunately, enough sam-
ples can be purified by flash chromatography for subsequent
testing.
Lactam 5-4-2 reported by Cheng’s group is among the most po-
tent anti-HBV helioxanthin analogues in the literature. A few N-
alkylated derivatives of 5-4-2 were also reported.18 this line, we
devised a convenient and versatile synthetic scheme to this class
of compounds (Scheme 4). Starting from 32, dicarbaldehyde 55
was produced via Swern oxidation. After 55 was treated with 3-
aminopropionitrile and TMSCl, lactam 56 was obtained in good
regioselectivity via an intramolecular Cannizarro reaction.29
Through similar mechanism, 55 also undergoes rapid cyclization
reaction with glycine in boiling acetic acid30 to give the N-acetic
acid lactam 57 and its regioisomer in 5:1 ratio. Although this mix-
ture of free acid isomers cannot be easily separated, desired iso-
mers can be isolated in pure form after the mixture underwent
alkylation with alkyl iodides under basic condition to give 58–60.
Ester 58 was reduced with DIBAL-H to furnish aldehyde 61, which
was further reduced with NaBH4 to provided alcohol 62
(Scheme 4).
2.2. Evaluation of anti-HBV activities
The synthetic analogues were evaluated against human hepa-
titis B virus in human hepatoma cell line, HepA2. Since our prior
studies have revealed that none of the benzylated or anhydride
compounds exhibit any antiviral activity, these compounds were
also skipped in this study. Among 38 new compounds tested,
16 of them were found active. These results are listed in Table 1
together with some published data (4, 14, 19, 32, 32(OAc), and
32(OAc)2) for comparison. Among the active compounds, 15
exhibits the highest EC50 (0.06
more active than the parent helioxanthin 4. Good activities
were also found for compound 27 (EC50 = 0.38 M), 39
(EC50 = 0.67 M), 40 (EC50 = 0.49 M), 41 (EC50 = 0.65 M) and
58 (EC50 = 0.86 M). The activities of these compounds are only
slightly inferior to that of helioxanthin 4. Compound 16
(EC50 = 1.74 M), 33 (EC50 = 1.90 M), 34 (EC50 = 1.90 M), and
42 (EC50 = 1.97 M) are moderately active, about ten times less
active than helioxanthin. Finally, compound 21 (EC50 = 3.94 M),
lM), which is about three times
l
l
l
l
l
l
l
l
l
l
25 (EC50 = 3.40
lM), 36 (EC50 = 3.70
lM), 37 (EC50 = 3.03
lM), 43
(EC50 = 3.33 M), 45 (EC50 = 2.73
l
lM), and 48 (EC50 = 2.77
lM) ex-
hibit detectable activities, yet are considerably less active (>20
times) than helioxanthin. The rest of the compounds are found
to be inactive against HBV activity within the concentration lim-
its. Most compounds in the current study have very low cytotox-
icity. Except for 14 and 59, no detrimental effect on cell viability
was observed for any other compounds. (For CC50 values, see Sup-
plementary data)
2.3. Compound 15 suppressed HBsAg production in HepA2 cells
The suppression of HBsAg production in HepA2 cells is the ma-
jor essay to screen for anti-HBV activity.31 As mentioned before,
compound 15 possesses the lowest EC50 against HBsAg production
in this study, even exceeding that of helioxanthin. Further tests

2166
D. Janmanchi et al. / Bioorg. Med. Chem. 21 (2013) 2163–2176
O
O
O
O
O
O
O
O
O
c
+
a
b
O
OH
O
O
O
O
O
O
O
O
O
O
O
R2
R2
R2
R2
R1
R1
R1
1
R1
12: R1 = OCH3, R2 = OBn
13: R1 = OCH3, R2 = OBn
6: R1 = OCH3, R2 = OBn
7: R1 = OBn, R2 = OCH3
2a: R1 = OCH3, R2 = OBn
2b: R1 = OBn, R2 = OCH3
d
d
14: R1 = OCH3, R2 = OH
e
8: R1 = R2 = OBn
9: R1 = R2 = OCH3
16: R1 = OCH3, R2 = OH
2c: R1 = R2 = OBn
15
: R1 = OCH3, R2 = OAc
2d: R1 = R2 = OCH3
10
: R1, R2 = OCF2O
2e: R1, R2 = OCF2O
2f: R1 = O(CH2)8CN, R2 = OBn
17: R1 = OBn, R2 = OCH3
18: R1 = OBn, R2 = OCH3
d
11
: R1 = O(CH2)8CN, R2 = OBn
19: R1 = OH, R2 = OCH3
d
e
21: R1 = OH, R2 = OCH3
20
: R1 = OAc, R2 = OCH3
22: R1 = R2 = OBn
24: R1 = R2 = OH
23: R1 = R2 = OBn
d
d
d
25: R1 = R2 = OH
26
: R1 = R2 = OCH3
: R1, R2 = OCF2O
28
: R1, R2 = OCF2O
27
29
: R1 = O(CH2)8CN, R2 = OBn
30
: R1 = O(CH2)8CN, R2 = OH
e
31
: R1 = O(CH2)8CN, R2 = OAc
Scheme 2. Reagents and conditions: (a) n-BuLi, 3-benzyloxy-4-methoxybenzaldehyde (for 7), 4-benzyloxy-3-methoxybenzaldehyde (for 8), 3,4-dibenzyloxybenzaldehyde
(for 9), 2,2-difluorobenzo[d][1,3]dioxole-5-carbaldehyde (for 10), 2,2-difluorobenzo[d][1,3]dioxole-5-carbaldehyde (for 11), and 9-(2-(benzyloxy)-4-formylphenoxy)non-
anenitrile (for 12), 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; (e) Ac2O, pyridine, room temperature, 12 h.
were therefore conducted on this compound. HepA2 cells were
seeded in a 24-well culture plate to which a range of concentra-
tions of compound 15 were added. After an incubation period of
48 h, HBsAg in the media were determined by ELISA assay. The re-
Table 1
The suppression of HBV surface antigen (HBsAg) by helioxanthin derivatives in HepA2
cells
EC50
(l
M)
EC50 (lM)
sult clearly revealed compound 15 potently suppressed the endog-
enously expressed HBsAg production in a dose-dependent manner
(Fig. 2). In all concentrations tested, the efficacy of 15 is superior to
helioxanthin, which was used as a positive control throughout this
study. It is worth noting that the suppressive activity of 15 on
HBsAg production in HepA2 cells is highly specific and not due
to general cytotoxic effect. The treated cells were viable and con-
tinued to proliferate throughout the incubation period. Structur-
ally, compound 20 is almost identical with 15, yet inactive. This
compound, when used as the negative control, does not show
any suppression activity in HepA2 cells. We can therefore conclude
the suppression of HBsAg by the lactone 15 must be highly struc-
ture-specific.
4
5
14
15
16
19
20
21
25
26
27
30
31
32
32(OAc)
32(OAc)2
33
0.16 0.03
>6.0
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
56
57
58
59
60
62
0.49 0.066
0.65 0.03
1.97 0.21
3.33 0.70
>12.5
2.73 0.76
>12.5
>12.5
2.77 0.35
>12.5
>12.5
>12.5
>12.5
>12.5
>12.5
>6.0
>6.0
0.86 0.0057
>12.5
>6.0
0.14 0.02
0.06 0.01
1.74 0.09
1.89 0.21
>6.0
3.94 0.81
3.4 0.53
>6.0
0.38 0.02
>12.5
>12.5
1.41 0.31
0.42 0.21
1.22 0.87
1.9 0.40
1.9 0.66
>12.5
3.7 0.70
3.03 0.20
>12.5
2.4. Compound 15 suppressed viral replication of HBV
34
35
36
37
38
39
We next examined the effect of 15 on HBV DNA in 1.3ES2 cells
(stably transfected with a 1.3-fold wide-type HBV ayw strain gen-
ome in HepG2 cells32). In this investigation, 1.3ES2 cells were trea-
ted with various concentrations of 15, helioxanthin 4, and 20 for
72 h. After the treatment, the amount of viral-associated HBV
DNA released into the medium was determined using quantitative
real-time PCR (Fig. 3). Like the result on the suppression of HBsAg,
compound 15 reduced HBV DNA in a dose-dependent manner in
>6.0
0.67 0.04
Cultured HepA2 cells were seeded on 96-well culture plates and treated with
various concentrations of compounds in DMEM for 2 days. Cultured cells were
collected and the number of cells was examined using MTT assay.
EC50: concentration of suppression of HBsAg at 50% of untreated cells.

D. Janmanchi et al. / Bioorg. Med. Chem. 21 (2013) 2163–2176
2167
Figure 2. Compound 15 suppressed the HBsAg production in HepA2 cells. HepA2 cells were seeded on 24-well plates at a density of 1 ꢁ 105 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 various concentrations of compound
15 or 4 and 20 in serum-free DMEM for 48 h. The amounts of HBsAg in the culture medium were determined by enzyme immunoassay. Viable cells in each well were
determined by the MTT assay. Data are expressed as mean S.D. (n = 3).
Figure 3. Effect of of compound 15 on the HBV DNA level and its analogs in the
media on 1.3ES2 cells. Quantitative real time PCR was used to detect wild type HBV
titer in the media of 1.3ES2 cells. Cultured cells were seeded on 100 mm culture
dishes and treated with various concentration of compound 15 (0, 1.0, and 5.0
lM)
Figure 4. Effects of compound 15 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
or 20 (5.0 M) and helioxanthin 4 (5.0 M) in serum-free (SF) DMEM for 72 h and
l
l
media collected for real-time PCR analysis using primer pair HBV DNA as template.
Data are expressed as mean S.D. (n = 3).
various concentration of compound 15 (0, 1.0, and 5.0
lM), or 20 (5.0 lM) and 4
(5.0 M) in serum-free DMEM medium for 48 h. Total RNA was extracted from
l
serum free (SF) and compounds-treated cells and analyzed by Northern hybridiza-
tion with HBV DNA probe as described in Section 2.5. The mRNA of the
glyceraldehydes-3-phosphate dehydrogenase (GAPDH) gene was used as an
internal marker.
1.3ES2 cells. In contrast, 20 exhibited no such effect on HBV DNA
replication under any concentration.
2.5. Compound 15 suppressed all HBV transcripts in 1.3ES2 cells
To determine whether the suppression of viral replication by 15
is mediated through interfering 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 15, heliothanxin 4, and 20.
After 48 h. we found compound 15 significantly decreased the
3.5-kb (HBeAg mRNA and pregenomic RNA) and 2.4/2.1-kb (large
HBsAg mRNA and middle/major HBsAg mRNA) HBV RNA levels
in a dose-dependent manner in 1.3ES2 cells (Fig. 4). On the other
hand, 20 had no such effect on HBV RNA levels.
Figure 5. Compound 15 suppressed the HBV core promoter activity in HepA2 cells.
HepA2 cells were transfected with CP (C) of HBV promoter region with luciferase
reported gene. After transfection, HepA2 cells were treated with serum free (SF),
compounds 15 or 4 (0, 0.03, 0.3 and 3.0 lM) in serum-free DMEM for 24 h,
respectively. The transfection efficiency was corrected by cotransfecting b-gal
expression vector and assayed b-gal activity simultaneously. Data were expressed
as mean S.D. (n = 3).
2.6. Compound 15 significantly suppresses viral core promoter
activity in HepA2 cells
Having demonstrated 15 can effectively reduce the level of viral
transcripts, we suspect that 15 might operate through regulating
the viral promoter. Since HBV core promoter region contains many
binding sites for hepatocyte nuclear transcriptional factor, we pro-
ceed to construct plasmids containing the core promoters (CP) fol-
lowed by the luciferase reporter gene to examine the effect of 15
on HBV promoter activity. As shown in Figure 5, we found that
compound 15 suppresses CP activities in HepA2 cells in a dose-
dependent manner. Although this effect is also observed in
helioxanthin 4, 15 exerts much stronger inhibition with 40%
suppression at 0.03
lM. Conversely, with much higher concentra-
tion at 3.0 M, 4 can only attain 30% suppression.
l

2168
D. Janmanchi et al. / Bioorg. Med. Chem. 21 (2013) 2163–2176
The third class of compounds are modify both at the [1,3]diox-
3. Discussion
3.1. Refine SAR model
ole and the lactone ring. Based on the structures at the original lac-
tone region, these hybrid compounds can be categorized into two
sub-types, the retro isomer family (16, 21, and 26) and the diol
family (35, 39, 45, and 50). Compound 16 and 39 are the most ac-
tive compounds in the retro family and diol family, respectively.
The phenyl groups in both compounds carry the same substitution
pattern, a para methoxy and a meta hydroxyl. Interestingly, the
same substitution pattern is also found on 14, the most active diox-
ole modified helioxanthin. Compounds with other substitution
patterns are much less active. Based on such resemblance in SAR
trends, we postulate the binding modes of these derivatives are
quite similar to the parent helioxanthin.
Our previous study convincingly established that helioxanthin
analogues with slight modifications at the lactone or [1,3]dioxole
moieties can retain anti-HBV activities. On the contrary, function-
alization around the naphthalene rim always results in inactive
compounds. Under this guideline, all compounds tested in the
present report are modified at the lactone area, the phenyl area,
or both. By carefully correlating compound structures with their
anti-HBV activities, a refined SAR model was developed to account
for the observations. To simplify the discourse, compounds with-
out acetate or hexanoate derivatization will be discussed first.
Those esterified derivatives will be considered separately.
Compounds only modified at the [1,3]dioxole ring (14, 19, 24,
26, 27, and 30) will be discussed first. When the [1,3]dioxole ring
is replaced by a hydroxyl group and a methyl ether, the resulting
compounds 14 and 19 are both active. Yet, despite their structural
similarity, the former possesses potency near that of helioxanthin
4 while the latter is only one tenth as active. Two more [1,3]diox-
ole-ring-opened analogues, dihydroxy 24 and dimethoxy 26, are
reported in this study. Although these two compounds show very
different sizes and polarities at the phenyl moiety, both are weakly
active. The closest structural analogue to 4, the [1,3]dioxole-fluori-
nated 27, has about one quarter activity as 4. Unfortunately, the in-
tended probe compound 30 is inactive within the concentration
tested. These results reveal the delicate nature of the interaction
site with phenyl [1,3]dioxole. This site must be quite size selective
to distinguish the tight binding 14 and 4 from their smaller and lar-
ger congener 24 and 26. Such selectivity definitely attributes to the
lack of activity in 30 and renders the phenyl site unsuitable for tag-
ging labels. Furthermore, the discrepancy between 14 and 19
clearly demonstrated this bind site is not only size selective but
also sensitive to the surface potential of the substrates. Both the
size selectivity and surface potential matching probably attributed
to the slightly diminished activity of 27 compared to 4.
We next examined the derivatives only with their lactone por-
tion modified (retro-4, 5, 32, 56–62). The earlier study has estab-
lished that retro-4 is moderately active. Lactone 4 can be reduced
to acyclic diol 32 yet remain moderately active. More remarkably,
lactam 5-4-2 is more active than 4 despite a non-hydrogen bond-
ing lactone oxygen atom is replaced by a strong hydrogen bond do-
nor N–H. The structural diversity of these active derivatives seems
to suggest the anti-HBV activities might survive even with more
drastic alteration at the lactone site. Unfortunately, the present re-
sults confirm the exact opposite. The activities of these lactone
modified compounds are extremely sensitive to the size and polar-
ity of the groups incorporated. For example, the acyclic dinitrile 5,
unlike acylic 32, is completely inactive. The most unexpected fail-
ure is from the N-alkylated lactam series (56–62). The parent 5-4-2
is so highly potent that we anticipated at least moderate activities
from its derivatives. However, the derivatives are inactive whether
the N-alkyl group carries a neutral nitrile (56), an ionizable carbox-
ylate (57), or a polar hydroxyl (62). The only exception is the
N-methyl acetate 58 which is somewhat less active than 4. The
activities disappear when the length of the alkyl chain increases
beyond five carbons. Since this delicate size effect was also
observed by Cheng’s group, it can therefore be postulated the bind-
ing site of the lactone moiety is a rigid pocket with a hydrophobic
bottom. Hydrophilic end groups in compound 56, 57, and 62 are
therefore excluded from the site. Derivative with nonpolar substi-
tution up to four atoms as in 58 can still fit in and therefore exhib-
its activity. On the other hand, the pocket cannot accommodate
longer alkyl chains and therefore renders 59 and 60 inactive.
We next examine the SAR of acetate and hexanoate deriva-
tives, including 15, 20 and most compounds in Scheme 3. At first
sight, these results seem to contradict our previous SAR model.
We already stated the binding sites of both [1,3]dioxole and lac-
tone must be quite sensitive to the size of the substrates. There-
fore, esterification at either the diol or the phenolic sites should
render most of these compounds inactive because these binding
sites cannot accommodate bulky ester groups. However, in most
instances, the acetylated derivatives exhibit similar or improved
activities when compared with the parent alcohols. Compound
15 is even more active then helioxanthin itself. The striking ef-
fect of acetylation is also manifested when 35 is compared to
acetylated derivative 36 and 37 as the inactive parent diol was
‘activated’ by acetylation. More surprisingly, many hexanoate es-
ters still exhibit weak yet detectable activities. These observa-
tions strongly suggest substitution size is relatively
unimportant in these ester derivatives. Bearing in mind the
unambiguous size effect witnessed in the unesterified com-
pounds, we hereby propose a model to harmonize the discrep-
ancy between in these two series of compounds. We believe,
instead of working directly on the target, the esters act as pro-
drugs that undergo hydrolysis in vivo to regenerate the active
diol or phenol compounds. In such a model, the ester com-
pounds are masked forms of their parent alcohols. The lower
activity found for the hexanoates might reflect their resistance
to hydrolysis. However, this model cannot answer why many es-
ters possess higher activities than the parent alcohols. To ac-
count for this peculiarity, we hypothesize that esterification
might improve the bio-viability of this class of compounds. Since
the free diols are more hydrophilic and less soluble than the es-
ter derivatives, it is likely that esterification makes the com-
pounds more permeable to membranes and less prone to
aggregate; both important factors in optimizing drug candidates.
It is also possible that the acetylation protect the phenolic hy-
droxyl against oxidation and therefore increase its life time
in vivo.
3.2. Anti-HBV mechanism of compound 15
In order to establish the antiviral mechanism of these helioxan-
thin analogues, we conduct further tests with the most active com-
pound, 15, in this study. In these investigations, it is demonstrated
that 15 does not only decrease the HBsAg and HBV DNA level, but
the viral RNA expression is also clearly suppressed. Similar phe-
nomenon is also observed when the infected cells are treated with
helioxanthin or 8-1. Such observation strongly indicates that the
viral life cycle is intercepted by this class of agents before the re-
verse transcription from RNA to DNA takes place. According to this
conception, helioxanthin derivatives must operate through a to-
tally different mechanism from the nucleotide and nucleotide mi-
mic drugs, which inhibit the viral polymerase and therefore only
reduce the viral DNA. On the other hand, helioxanthin analogues
are also distinct from those potent heteroaryldihydropyrimidine

D. Janmanchi et al. / Bioorg. Med. Chem. 21 (2013) 2163–2176
2169
32(OAc): R1 = R2 = OCH2O, R3 = OAc, R4 = OH
32(OAc)2: R1 = R2 = OCH2O, R3 = R4 = OAc
O
c
32: R1, R2 = OCH2O, R3 = R4 = OH
R3
R4
O
a
O
O
33: R1, R2 = OCH2O, R3 = OCOC5H11, R4 = OH
34: R1, R2 = OCH2O, R3 = R4 = OCOC5H11
O
b
O
R2
R2
36; R1 = R2 = OCH3, R3 = OAc, R4 = OH
37
R1
c
R1
35: R1 = R2 = OCH3, R3 = R4 = OH
; R1 = R2 = OCH3, R3 = R4= OAc
4: R1, R2 = OCH2O
b
38: R1 = R2 = OCH3, R3 = OH, R4 = OCOC5H11
40; R1 = OCH3, R3 = OAc, R2 = R4 = OH
41
14: R1 = OCH3, R2 = OH
19
: R1 = OH, R2 = OCH3
c
24: R1 = R2 = OH
39: R1 = OCH3, R2 = R3 = R4 = OH
26: R1 = R2 = OCH3
: R1 = OCH3, R2 = R3 = R4 = OAc
42: R1 = OCH3, R3 = OCOC5H11, R2 = R4 = OH
43: R1 = OCH3, R2 = OH, R3 = R4 = OCOC5H11
b
44
: R1 = OCH3, R2 = R3 = R4 = OCOC5H11
46: R2 = OCH3, R3 = OAc, R1 = R4 = OH
c
45: R2 = OCH3, R1 = R3 = R4 = OH
47: R2 = OCH3, R1 = OH, R3 = R4 = OAc
48: R2 = OCH3, R1 = R3 = R4 = OAc
b
49: R2 = OCH3, R1 = OH, R3 = R4 = OCOC5H11
51: R1 = R2 = OH, R3 = R4 = OAc
c
50
: R1 = R2 = R3 = R4 = OH
52: R1 = OH, R2 = R3 = R4 = OAc
53: R1 = R2 = R3 = R4 = OAc
54: R1 = R2 = OH, R3 = R4 = OCOC5H11
b
Scheme 3. Reagents and conditions: (a) LAH, THF, 0 °C, 1 h; (b) hexanoic anhydride, pyridine, room temperature, 12 h; (c) Ac2O, pyridine, room temperature, 12 h
compounds which interfere with the viral capsid assembly and
therefore should also have little effect on RNA level.
emerge after the administration of nucleotide analogue will still
respond to treatment by 15. This highlights the potential of these
natural product analogues in long-term anti-HBV therapy or com-
bination therapy.
Such comprehensive suppression of viral transcripts and pro-
teins and at every levels of life cycle is the signature of helioxan-
thin and its analogues. Since the 3.5-kb pregenomic RNA does
not only encode the core and polymerase proteins but also serves
as a template for reverse transcription, we can reasonably infer
that compound 15 suppresses viral proliferation by inhibiting the
production of this key RNA transcript at a very upstream event.
Since the viral core promoter plays a critical role in viral gene
expression33–35 we next examined the viral core promoter (CP)
activity using luciferase as the reporter assay (Fig. 5). Indeed, we
found 15 effectively inhibit CP activity with potency comparable
to the most active congener, 5-4-2. Based on these results, we con-
clude the anti-HBV mechanism of 15 is most likely though interfer-
ence with the transcriptional machinery, much like the parent
helioxanthin 4, the phthalazine dione derivative 8-1 and lactam
5-4-2. In earlier studies, hepatocyte nuclear factor HNF-4-1 and
HNF-4-2 are implicated as the target of 8-1. We too believe 15
may down regulate similar transcriptional factor. This series of
studies firmly establishes the family of helioxanthin derivatives
as effective anti-HBV drugs or lead compounds. It is also notewor-
thy that 4, 14, 8-1, and 5-4-2 have all been found active against
lamivudine-resistant strand that contains L515M and M539V dou-
ble mutations in the polymerase. As our results indicate, 15 is
probably a precursor of 14 and it targets viral CP rather than poly-
merase. We are confident that many drug resistant strands that
In conclusion, we have synthesized a new series of helioxanthin
analogues. Skeletal variations include modifications at [1,3]dioxole
ring, lactone ring, and both. Further derivatizations are carried out
with esterification of various alcohols. Compound 15 was revealed
to be even more active than the parent helioxanthin. By correlating
the molecular structures with anti-HBV activities, a refined model
of SAR was proposed. By assuming that the esterified compounds
can serve as the precursors of the free alcohol, the apparent contra-
dictory SAR results from these two classes of derivatives was re-
solved. We believe these SAR insights can provide vital clues in
designing more active lead compounds with good bio-viability.
For example, a hypothetical compound that contains the lactam
motif in 5-4-2 and the opened [1,3]dioxole in 15 can exceed both
predecessors in anti-HBV potency. As expected, 15 does not only
reduce HBV DNA but also decreases RNA like its parent com-
pounds. Since the viral core promoter is deactivated by 15, we infer
this class of structures can interrupt the transcriptional machinery
in hepatocyte and thus nullify HBV reproduction. This mechanism
is distinct from the existing nucleotide analogue therapeutics like
lamivudine and adefovir, which target the viral polymerase. Such
uniqueness mode of action makes 15 a worthy candidate for fur-
ther investigation in combination therapy. For example, a cocktail
of 15, lamivudine, and Bay 41-4109 might eradicate HBV infection

2170
D. Janmanchi et al. / Bioorg. Med. Chem. 21 (2013) 2163–2176
O
O
OH
OH
N
CN
b
O
a
O
O
O
O
O
O
O
O
O
O
O
O
32
55
56
c
O
N
R
O
O
R = CH2CO2H
O
O
57:
d
f
e
58: R = CH2CO2CH3
61: R = CH2CHO
60: R = CH2CO2C8H17
59: R = CH2CO2C5H11
g
h
62: R = CH2CH2OH
Scheme 4. Reagents and conditions: (a) (COCl)2, DMSO, Et3N, CH2Cl2, ꢀ78 °C, 12 h; (b) 3-aminopropionitrile, CH3CN/DMF, TMSCl, room temperature, 12 h; (c) glycine, AcOH,
reflux, 5–10 min; (d) CH3I, K2CO3, DMF, room temperature, 2 h; (e) C5H11I, K2CO3, DMF, room temperature, 2 h; (f) C8H17I, K2CO3, DMF, room temperature, 2 h; room
temperature, 2 h; (g) DIBAL-H, toluene, ꢀ40 °C, 2 h; (h) NaBH4, THF, room temperature, 1 h.
by intercepting viral life cycle at many stages. Alone this direction,
we are currently testing these compounds as novel anti-HBV
agents in HBV transgenic mice.
(4.7 ml) was heated at 140 °C for 24 h. After this period, the reac-
tion mixture was cooled to room temperature, diluted with CH2Cl2
(50 ml), and washed with 5% NaHCO3 solution (3 ꢁ 50 ml). The or-
ganic fraction was dried over MgSO4 and concentrated to furnish
yellow solid compound 6 (1.85 g, 62%). 1H NMR (400 MHz, CDCl3)
d 8.21 (s, 1H), 7.58 (d, 1H, J = 8.5 Hz), 7.40 (d, 1H, J = 8.5 Hz), 6.89
(d, 1H, J = 7.8 Hz), 6.83–6.80 (m, 2H), 6.05 (AB, 2H, J = 18.6 Hz),
5.97 (AB, 2H, J = 12.2 Hz); 13C NMR (100 MHz, CDCl3) d 148.73,
148.50, 147.37, 144.20, 143.04, 136.00, 129.64, 129.07, 124.27,
123.17, 119.07, 116.30, 115.39, 114.52, 111.17, 109.79, 108.06,
107.52, 102.48, 101.48; IR (CH2Cl2, cast) 2227, 1622, 1488, 1444,
4. Synthetic procedures
4.1. General protocols for synthesis
1H and 13C NMR spectra were obtained on Bruker AC 300, AMX
400, AV 400, or DRX 500 MHz spectrometer. Chemical shifts of 1H
were calibrated with the residual proton peak(s) in deuterated sol-
vent as the internal references (CHCl3 at d 7.24 ppm, CH3COCH3 at d
2.49 ppm, and CH3OH at d 4.78 ppm). Chemical shifts of 13C spec-
trum were calibrated accordingly (the central peak of CHCl3 at d
77.00 ppm, carbonyl of CH3COCH3 at 206.00 ppm, and CH3OH at
d 49.15 ppm). Mass spectra were recorded on a JMS-700 double
focusing mass spectrometer (JEOL, Tokyo, Japan). Commercially
available solvents and reagents were used without further purifica-
tion except for THF and CH2Cl2 which are dried over Na/benzophe-
none and CaH2, respectively and distilled before use. All reactions
were carried out under a dry N2 atmosphere and thoroughly mixed
with magnetic stirs. Acetal 1, hydroxyacetal 2, anhydride 3, helio-
xanthin 4, 12–14, 17–19, 22–24, 32, 32(OAc) and 32(OAc)2 were
prepared as published before.
1318, 1298, 1235, 1094, 1039 cmꢀ1
20H10O4N2 m/z (M+) 342.0641, found 342.0644.
; FAB-HRMS Calcd for
C
4.1.2. 10-(3-Benzyloxy-4-methoxy-phenyl)-furo[30,40:6,7]
naphtho[1,2-d][1,3]dioxol-7,9-dione (6), 10-(4-Benzyloxy-3-
methoxy-phenyl)-furo[30,40:6,7]naphtho[1,2-d][1,3]dioxol-7,9-
dione (7), 10-(3,4-Bis-benzyloxy-phenyl)-furo[30,40:6,7]naphtho
[1,2-d][1,3]dioxole-7,9-dione (8), 10-(3,4-Dimethoxyphenyl)-
furo[30,40:6,7]naphtho[1,2-d][1,3]dioxole-7,9-dione (9),
10-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)furo[30,40:6,7]
naphtho[1,2-d][1,3]dioxole-7,9-dione (10) and
9-(2-(Benzyloxy)-4-(7,9-dioxo-7,9-dihydrofuro[30,40:6,7]
naphtho[1,2-d][1,3]dioxol-10-yl)phenoxy)nonanenitrile (11)
Anhydrides 6–11 were synthesized as described for 3. The ace-
tal 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 (3-benzyloxy-4-methoxybenzaldehyde for
4.1.1. 9-(Benzo[d][1,3]dioxol-5-yl)naphtho[1,2-d][1,3]dioxole-
7,8-dicarbonitrile (5)
Mixture of hydroxyacetal 2 (3 g, 8.71 mmol), fumaronitrile
(680 mg, 8.71 mmol), acetic anhydride (12.7 ml), and acetic acid

D. Janmanchi et al. / Bioorg. Med. Chem. 21 (2013) 2163–2176
2171
6, 4-benzyloxy-3-methoxybenzaldehyde for 7, 3,4-dibenzyloxy
benzaldehyde for 8, 3,4-dimethoxybenzaldehyde for 9, 2,2-dif-
luorobenzo[d][1,3]dioxole-5-carbaldehyde for 10, and 9-(2-(ben-
zyloxy)-4-formylphenoxy) nonanenitrile for 11). 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 cor-
responding hydroxyacetals. The crude hydroxyacetals 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, the
anhydrides were filtered as yellow solids and used in the subse-
quent reaction without further purification or characterization.
1460, 1273, 1249, 1198, 1127, 1073 cmꢀ1; FAB-HRMS Calcd for
22H16O7 m/z (M+) 392.0896, found 392.0898.
C
4.1.6. 10-(4-Hydroxy-3-methoxyphenyl)furo[30,40:6,7]
naphtho[1,2-d][1,3]dioxol-9(7H)-one (21)
Compound 21 was synthesized as described for 16 in 72% iso-
lated yield. 1H NMR (400 MHz, CDCl3) d 7.77 (s, 1H), 7.51 (d, 1H,
J = 8.6 Hz), 7.33 (d, 1H, J = 8.6 Hz), 6.94 (d, 1H, J = 8.0 Hz), 6.86 (d,
1H, J = 1.6 Hz), 6.83 (dd, 1H, J = 1.6, 8.0 Hz), 5.85 (s, 2H), 5.76 (s,
1H), 5.34 (s, 2H), 3.84 (s, 3H); 13C NMR (100 MHz, CDCl3) d
169.27, 145.46, 145.40, 144.97, 143.53, 138.31, 137.58, 132.77,
127.14, 122.85, 122.25, 120.77, 120.65, 120.14, 113.55, 113.30,
112.68, 101.38, 67.75, 55.97; FAB-HRMS Calcd for C20H15O6 m/z
([M+H]+) 351.0869, found 351.0870.
4.1.3. 2-Methoxy-5-(7-oxo-7,9-dihydrofuro[30,40:6,7]
naphtho[1,2-d][1,3]dioxol-10-yl) phenyl acetate (15)
4.1.7. 10-(3,4-Dihydroxyphenyl)furo[30,40:6,7]naphtho[1,2-
d][1,3]dioxol-9(7H)-one (25)
Compound 25 was synthesized as described for 16 in 69% iso-
lated yield. 1H NMR (400 MHz, CDCl3) d 7.75 (s, 1H), 7.48 (d, 1H,
J = 8.6 Hz), 7.31 (d, 1H, J = 8.6 Hz), 6.81–6.79 (m, 2H), 6.68 (dd,
1H, J = 1.9, 8.1 Hz), 5.84 (AB, 2H, J = 5.8 Hz), 5.35 (s, 2H); 13C NMR
To a solution of 14 (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 overnight. 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 concentrated.
After column chromatography (hexane/CH2Cl2 = 3:1), 15 (16 mg,
73%) was isolated. 1H NMR (400 MHz, CDCl3) d 8.39 (s, 1H), 7.67
(d, 1H, J = 8.7 Hz), 7.28 (d, 1H, J = 8.7 Hz), 7.18 (dd, 1H, J = 2.1,
8.3 Hz), 7.03–7.00 (m, 2H), 5.92 (s, 2H), 5.21 (AB, 2H, J = 15.2 Hz),
3.89 (s, 3H) 2.30 (s, 3H); 13C NMR (100 MHz, CDCl3) d 171.10,
168.85, 150.91, 146.96, 141.75, 139.72, 139.19, 130.69, 129.09,
128.16, 127.50, 127.01, 125.39. 124.29, 121.31, 121.12, 111.80,
111.65, 101.69, 69.53, 55.89, 20.70; EI-HRMS Calcd for C22H16O7
m/z (M+) 392.0896, found 392.0892.
(100 MHz, CDCl3)
d 170.55, 145.11, 144.09, 143.72, 142.93,
138.72, 137.34, 132.76, 127.67, 122.13, 121.98, 120.64, 120.41,
120.15, 116.74, 114.40, 113.78, 101.55, 68.30; ESI-HRMS Calcd
for C19H12O6Na m/z ([M+Na]+) 359.0532, found 359.0542.
4.1.8. 10-(2,2-Difluorobenzo[d][1,3]dioxol-5-yl)furo[30,40:6,7]
naphtho[1,2-d][1,3]dioxol-7(9H)-one (27) and 10-(2,2-
Difluorobenzo[d][1,3]dioxol-5-yl)furo[30,40:6,7]naphtho[1,2-
d][1,3]dioxol-9(7H)-one (28)
Anhydride 10 (500 mg, 1.1 mmol) was reduced with NaBH4
(130 mg, 3.43 mmol) in dry THF (20 ml) as described before. After
column chromatography (hexane/CH2Cl2 = 2:3), pure 27 (340 mg,
70%) and 28 (100 mg, 20%) were isolated. Compound 27: 1H NMR
(400 MHz, CDCl3) d 8.43 (s, 1H), 7.70 (d, 1H, J = 8.6 Hz), 7.31 (d,
1H, J = 8.6 Hz), 7.12 (d, 1H, J = 8.6 Hz), 7.05–7.03 (m, 2H), 5.92
(AB, 2H, J = 15.2 Hz), 5.16 (AB, 2H, J = 15.2 Hz); 13C NMR
4.1.4. 10-(3-Hydroxy-4-methoxyphenyl)furo[30,40:6,7]
naphtho[1,2-d][1,3]dioxol-9(7H)-one (16)
To a solution of 13 (40 mg, 0.09 mmol) in anhydrous CH2Cl2
(3.0 ml) at ꢀ78 °C was slowly added a solution of BBr3 (1 M in
CH2Cl2, 0.09 ml) and the mixture was stirred for 2 h at the same
temperature. After this time, MeOH (4 ml) was added to the reac-
tion mixture and the resulting solution was warmed back to room
temperature and concentrated in vacuo. The residue was dissolved
in ether (30 ml) and washed with saturated NaHCO3 (20 ml), water
(20 ml), and brine (20 ml). The organic extract was dried over
MgSO4 and concentrated. After column chromatography (hexane/
CH2Cl2 = 1:2), 16 (24 mg, 75%) was isolated. 1H NMR (400 MHz,
(125 MHz, CDCl3)
d 170.73, 147.15, 143.53, 141.54, 139.79,
132.73, 131.72, 130.64, 128.07, 127.71, 125.63, 124.30, 121.18,
121.10, 112.04, 110.57, 109.05, 101.70, 69.18; IR (CH2Cl2, cast)
1762, 1632, 1498, 1459, 1443, 1239, 1151, 1072, 1031 cmꢀ1
FAB-HRMS Calcd for
;
C
20H10O6F2 m/z (M+) 384.0445, found
384.0442. Compound 28: 1H NMR (400 MHz, CDCl3) d 7.83 (s,
1H), 7.54 (d, 1H, J = 8.6 Hz), 7.35 (d, 1H, J = 8.6 Hz), 7.09–7.01 (m,
3H), 5.87 (AB, 2H, J = 11.2 Hz), 5.37 (s, 2H); 13C NMR (100 MHz,
CDCl3) d 169.13, 145.22, 143.56, 143.25, 142.96, 137.43, 136.20,
132.68, 131.75, 131.29, 124.95, 122.49, 121.48, 121.16, 119.78,
113.89, 110.99, 108.22, 101.61, 68.00; IR (CH2Cl2, cast) 1763,
1618, 1499, 1457, 1376, 1314, 1239, 1155, 1023, cmꢀ1; FAB-HRMS
Calcd for C20H10O6F2 m/z (M+) 384.0445, found 384.0442.
CDCl3)
d 7.77 (s, 1H), 7.50 (d, 1H, J = 8.6 Hz), 7.32 (d, 1H,
J = 8.6 Hz), 6.90–6.88 (m, 2H), 6.83 (dd, 1H, J = 2.0, 8.2 Hz), 5.86
(AB, 2H, J = 6.0 Hz), 5.60 (s, 1H), 5.34 (d, 2H, J = 1.0 Hz), 3.94 (s,
3H). 13C NMR (100 MHz, CDCl3) d 169.22, 146.37, 144.99, 144.41,
143.59, 138.10, 137.54, 132.73, 128.57, 122.20, 121.41, 120.85,
120.69, 120.10, 116.09, 113.56, 109.23, 101.45, 67.77, 55.74;
FAB-HRMS Calcd for C20H15O6 m/z ([M+H]+) 351.0869, found
351.0875.
4.1.9. 9-(2-(Benzyloxy)-4-(7-oxo-7,9-dihydrofuro[30,40:6,7]
naphtho[1,2-d][1,3]dioxol-10-yl)phenoxy)nonanenitrile (29)
Compound 11 (500 mg, 1.1 mmol) was reduced with NaBH4
(130 mg, 3.43 mmol) in dry THF (20 ml) as described before. After
workup, mixture of lactone isomers was used in the subsequent
reaction without further purification or characterization.
4.1.5. 2-Methoxy-4-(7-oxo-7,9-dihydrofuro[30,40:6,7]
naphtho[1,2-d][1,3]dioxol-10-yl)phenyl acetate (20)
To a solution of 19 (20 mg, 0.056 mmol) in pyridine (3 ml) was
added acetic anhydride (6 mg, 0.056 mmol) and the reaction mix-
ture was allowed to proceed as described for 15. After column
chromatography (hexane/CH2Cl2 = 1:2), pure 20 (28 mg, 72%) was
isolated. 1H NMR (500 MHz, CDCl3) d 8.43 (s, 1H), 7.70 (d, 1H,
J = 8.7 Hz), 7.30 (d, 1H, J = 8.7 Hz), 7.10 (d, 1H, J = 8.4 Hz), 6.92–
6.90 (m, 2H), 5.90 (AB, 2H, J = 8.5 Hz), 5.21 (AB, 2H, J = 15.2 Hz),
3.80 (s, 3H) 2.34 (s, 3H); 13C NMR (100 MHz, CDCl3) d 171.00,
168.87, 150.67, 147.01, 141.71, 139.64, 139.61, 135.47, 130.70,
128.72, 127.71, 125.50, 122.43, 121.20, 121.13, 113.57, 111.89,
101.62, 69.53, 56.05, 20.73; IR (CH2Cl2, cast) 1761, 1630, 1507,
4.1.10. 9-(2-Hydroxy-4-(7-oxo-7,9-dihydrofuro[30,40:6,7]
naphtho[1,2-d][1,3]dioxol-10-yl)phenoxy)nonanenitrile (30)
To a solution of 29 (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 debenzylation was performed and worked
up as described before. After column chromatography (CH2Cl2/
methanol = 19:1), 30 (24 mg, 77%) was isolated. 1H NMR
(500 MHz, CDCl3) d 8.39 (s, 1H), 7.67 (d, 1H, J = 8.6 Hz), 7.28 (d,

2172
D. Janmanchi et al. / Bioorg. Med. Chem. 21 (2013) 2163–2176
1H, J = 8.6 Hz), 6.90 (d, 1H, J = 2.0 Hz), 6.88 (d, 1H, J = 8.2 Hz), 6.78
(dd, 1H, J = 2.0, 8.2 Hz), 5.92 (AB, 2H, J = 5.5 Hz), 5.71 (s, 1H), 5.18
(AB, 2H, J = 15.1 Hz), 4.12–4.07 (m, 2H), 2.34 (t, 2H, J = 7.0 Hz),
1.87–1.84 (m, 2H), 1.68–1.65 (m 2H), 1.50–1.46 (m, 4H), 1.41–
1.37 (m, 4H); 13C NMR (100 MHz, CDCl3) d 171.23, 146.86,
145.77, 145.17, 141.80, 139.71, 130.78, 129.85, 129.21, 127.25,
125.40, 121.47, 121.08, 120.70, 119.75, 115.45, 111.73, 110.81,
101.53, 69.62, 68.83, 29.25, 29.08, 28.68, 28.54, 25.95, 25.28,
17.14; FAB-HRMS Calcd for C28H27O6N m/z (M+) 473.1838, found
473.1835.
4.1.14. (9-(4-Hydroxy-3-methoxyphenyl)naphtho[1,2-d][1,3]
dioxole-7,8-diyl)dimethanol (45)
Yield 91%, 1H NMR (500 MHz, CDCl3) d 7.77 (s, 1H), 7.41 (d, 1H,
J = 8.6 Hz), 7.17 (d, 1H, J = 8.6 Hz), 6.93 (d, 1H, J = 8.0 Hz), 6.80 (d,
1H, J = 1.6 Hz), 6.77 (dd, 1H, J = 1.6, 8.0 Hz), 5.75 (s, 2H), 5.64 (br
s, 1H), 4.90 (s, 2H), 4.60 (s, 2H), 3.84 (s, 3H). 13C NMR (100 MHz,
CD3OD) d 148.17, 146.89, 146.08, 143.35, 137.41, 137.19, 136.55,
132.78, 131.50, 128.91, 124.10, 123.37, 121.03, 115.44, 115.31,
111.87, 102.11, 64.26, 59.91, 56.58; IR (CH2Cl2, cast) 3442, 1643,
1630, 1509, 1449, 1417, 1376, 1106 cmꢀ1; FAB-HRMS Calcd for
C
20H18O6 m/z ([M+H]+) 354.1103, found 354.1106.
4.1.11. 2-((8-Cyanooctyl)oxy)-5-(7-oxo-7,9-dihydrofuro
[30,40:6,7]naphtho[1,2-d][1,3]dioxol-10-yl)phenyl acetate (31)
To a solution of 30 (20 mg, 0.056 mmol) in pyridine (3 ml)
was added acetic anhydride (6 mg, 0.056 mmol) and the reaction
mixture was allowed to proceed as described for 15. After col-
umn chromatography (hexane/CH2Cl2 = 1:2), 31 (28 mg, 72%)
was isolated. 1H NMR (400 MHz, CDCl3) d 8.40 (s, 1H), 7.68 (d,
1H, J = 8.6 Hz), 7.29 (d, 1H, J = 8.6 Hz), 7.15 (dd, 1H, J = 2.1,
8.4 Hz), 7.02 (d, 1H, J = 2.1 Hz), 6.99 (d, 1H, J = 8.4 Hz), 5.92 (s,
2H), 5.21 (AB, 2H, J = 15.2 Hz), 4.04–4.02 (m, 2H), 2.33 (t, 2H,
J = 7.1 Hz), 2.29 (s, 3H), 1.80–1.79 (m, 2H), 1.67–1.64 (m 2H),
1.49–1.44 (m, 4H), 1.37–1.36 (m, 4H); 13C NMR (100 MHz,
CDCl3) d 171.12, 168.77 150.41, 146.97, 141.77, 139.74, 139.45,
130.73, 128.98, 128.26, 127.49, 126.99, 125.41, 124.13, 121.35,
121.14, 119.76, 112.53, 111.81, 101.68, 69.55, 68.57, 29.16,
29.04, 28.75, 28.58, 25.85, 25.33, 20.62, 17.14; IR (CH2Cl2, cast)
2858, 2245, 1767, 1631, 1511, 1461, 1370, 1271, 1072,
1015 cmꢀ1; FAB-HRMS Calcd for C30H29O7N m/z (M+) 515.1944,
found 515.1948.
4.1.15. 4-(7,8-bis(Hydroxymethyl)naphtho[1,2-d][1,3]dioxol-9-
yl)benzene-1,2-diol (50)
Yield 89%, 1H NMR (400 MHz, CD3OD) d 7.84 (s, 1H), 7.44 (d,
1H, J = 8.6 Hz), 7.16 (d, 1H, J = 8.6 Hz), 6.78 (d, 1H, J = 8.0 Hz),
6.71 (d, 1H, J = 2.0 Hz), 6.60 (dd, 1H, J = 2.0, 8.0 Hz), 5.73 (AB,
2H, J = 9.6 Hz), 5.48 (s, 1H), 4.91 (s, 2H), 4.55 (AB, 2H,
J = 11.4 Hz); 13C NMR (100 MHz, CD3OD)
d 145.88, 145.53,
145.24, 143.23, 137.18, 137.09, 136.33, 132.77, 131.31, 128.72,
123.13, 122.78, 120.89, 118.58, 115.30, 111.65, 101.93, 64.18,
59.78; IR (CH2Cl2, cast) 3390, 1504, 1449, 1322, 1272, 1218,
1070, 1047 cmꢀ1
;
FAB-HRMS Calcd for C19H16O6 m/z ([M]+)
340.0947, found 340.0942.
General procedure for the preparation of 33–54: To a solution of
above appropriate alcohol in pyridine was added acetic anhydride
or hexanoic anhydride and the resulting mixture was stirred at
room temperature for 12 h. For the synthesis of monoesters,
1 equiv of anhydride is used while 2–3 equiv are required for mul-
tiple esterification. After the completion of the reaction, 10% HCl
was added and the mixture was extracted with ether. The ether ex-
tract was washed with saturated NaHCO3, dried over MgSO4, and
concentrated. After column chromatography, the desired products
were isolated.
General procedure for the preparation of 32,1 35, 39, 45 and 50:
As described for 32, a solution of lithium aluminum hydride in THF
was cooled to 0 °C in an ice bath and a THF solution of lactone (4,
15, 20, 23 and 26) was added. The mixture was stirred for 1 h at the
same temperature. Water and a few drops of concentrated sulfuric
acid were then slowly added at 0 °C. The mixture was then
extracted with CH2Cl2. The organic fraction was dried over
MgSO4 and concentrated under reduced pressure to give desired
product.
4.1.16. (9-(Benzo[d][1,3]dioxol-5-yl)-8-(hydroxymethyl)
naphtho[1,2-d][1,3]dioxol-7-yl)methyl hexanoate (33)
Yield 80%, 1H NMR (500 MHz, CDCl3) d 7.80 (s, 1H), 7.40 (d, 1H,
J = 8.5 Hz), 7.16 (d, 1H, J = 8.5 Hz), 6.82 (d, 1H, J = 7.8 Hz), 6.77 (d,
1H, J = 1.2 Hz), 6.73 (dd, 1H, J = 1.2, 7.8 Hz), 6.03 (s, 1H), 5.99 (s,
1H), 5.77 (AB, 2H, J = 7.0 Hz), 5.41, (s, 2H), 4.54 (AB, 2H,
J = 11.9 Hz), 2.35 (t, 2H, J = 7.6 Hz), 1.66–1.60 (m, 2H), 1.29–1.26
(m, 4H), 0.85 (t, 3H, J = 6.8 Hz); 13C NMR (125 MHz, CDCl3) d
173.52, 146.89, 146.83, 144.98, 141.88, 135.59, 135.47, 132.95,
130.39, 130.11, 129.48, 123.07, 122.28, 119.81, 111.16, 110.49,
107.42, 100.98, 64.90, 59.21, 34.37, 31.24, 24.57, 22.26, 13.86; IR
(CH2Cl2, cast) 3455, 1731, 1635, 1602, 1488, 1444, 1373, 1323,
1272, 1234, 1171, 1069, 1043, 933, 799 cmꢀ1; FAB-HRMS calcd
for C26H26O7 m/z (M)+ 450.1679, found 450.1685.
4.1.12. (9-(3,4-Dimethoxyphenyl)naphtho[1,2-d][1,3]dioxole-
7,8-diyl)dimethanol (35)
Yield 91%, 1H NMR (400 MHz, CDCl3) d 7.71(s, 1H), 7.37 (d, 1H,
J = 8.5 Hz), 7.14 (d, 1H, J = 8.5 Hz), 6.86 (d, 1H, J = 7.9 Hz), 6.82–6.80
(m, 2H), 5.72 (AB, 2H, J = 7.2 Hz), 4.82 (AB, 2H, J = 12.2 Hz), 4.53
(AB, 2H, J = 11.8 Hz), 3.91 (s, 3H), 3.78 (s, 3H); 13C NMR
(100 MHz, CDCl3)
d 148.11, 147.88, 144.79, 141.91, 135.70,
135.06, 132.01, 129.57, 129.28, 122.12, 122.04, 119.61, 113.46,
110.99, 110.07, 100.86, 65.13, 59.86, 55.86, 55.76; IR (CH2Cl2, cast)
3406, 1725, 1508, 1450, 1262, 1070, 1024, 797, 734 cmꢀ1; FAB-
HRMS Calcd for C21H20O6 m/z (M+) 368.1260, found 368.1256.
4.1.17. (9-(Benzo[d][1,3]dioxol-5-yl)naphtho[1,2-d][1,3]dioxole-
7,8-diyl)bis(methylene)dihexanoate (34)
4.1.13. (9-(3-Hydroxy-4-methoxyphenyl)naphtho[1,2-d][1,3]
dioxole-7,8-diyl)dimethanol (39)
Yield 80%, 1H NMR (500 MHz, CDCl3) d 7.81 (s, 1H), 7.42 (d,
1H, J = 8.5 Hz), 7.18 (d, 1H, J = 8.5 Hz), 6.78 (d, 1H, J = 7.9 Hz),
6.71 (d, 1H, J = 1.4 Hz), 6.67 (dd, 1H, J = 1.4, 7.9 Hz), 6.02 (S,
1H), 5.98 (S, 1H), 5.78 (AB, 2H, J = 6.9 Hz), 5.27, (s, 2H), 4.97 (s,
2H), 2.34 (t, 2H, J = 7.5 Hz), 2.24 (t, 2H, J = 7.5 Hz), 1.64–1.62
(m, 2H), 1.58–1.55 (m, 2H), 1.29–1.23 (m, 8H), 0.86 (t, 6H,
J = 6.8 Hz); 13C NMR (100 MHz, CDCl3) d 173.53, 173.29, 146.92,
146.83, 145.10, 141.96, 137.28, 132.43, 130.67, 130.51, 129.87,
129.76, 123.08, 122.32, 119.72, 111.47, 110.44, 107.34, 101.05,
100.98, 64.49, 61.09, 34.29, 34.25, 31.28, 24.65, 24.59, 22.29,
13.87; FAB-HRMS calcd for C32H36O8 m/z (M)+ 548.2410, found
548.2413.
Yield 92%, 1H NMR (400 MHz, CDCl3) d 7.75 (s, 1H), 7.38 (d, 1H,
J = 8.5 Hz), 7.15 (d, 1H, J = 8.5 Hz), 6.86 (d, 1H, J = 8.2 Hz), 6.82 (d,
1H, J = 2.0 Hz), 6.75 (dd, 1H, J = 2.0, 8.2 Hz), 5.75 (s, 2H), 5.70 (br
s, 1H), 4.88 (AB, 2H, J = 12.2 Hz), 4.57 (AB, 2H, J = 12.0 Hz), 3.94
(s, 3H); 13C NMR (100 MHz, CDCl3) d 145.90, 144.83, 144.67,
141.99, 135.65, 135.12, 132.71, 129.63, 129.41, 122.10, 121.54,
119.61, 116.28, 111.06, 109.67, 100.92, 65.32, 60.00, 55.92; IR
(CH2Cl2, cast) 3385, 1506, 1450, 1323, 1249, 1227, 1138, 1070,
1025 cmꢀ1; FAB-HRMS Calcd for C20H18O6 m/z (M+) 354.1103,
found 354.1105.

D. Janmanchi et al. / Bioorg. Med. Chem. 21 (2013) 2163–2176
2173
4.1.18. (9-(3,4-Dimethoxyphenyl)-8-(hydroxymethyl)
4.1.23. (9-(3-Hydroxy-4-methoxyphenyl)-8-(hydroxymethyl)
naphtho[1,2-d][1,3]dioxol-7-yl)methyl acetate (36)
naphtho[1,2-d][1,3]dioxol-7-yl)methyl hexanoate (42)
Yield 71%, 1H NMR (500 MHz, CDCl3) d 7.76 (s, 1H), 7.40 (d, 1H,
J = 8.5 Hz), 7.17–7.13 (m, 2H), 6.96–6.99 (m, 2H), 5.77 (AB, 2H,
J = 6.0 Hz), 4.88 (s, 2H), 4.59 (AB, 2H, J = 12.0 Hz), 3.88(s, 3H),
2.55 (t, 2H, J = 7.5 Hz), 1.75–1.71 (m, 2H), 1.38–1.33 (m, 4H), 0.89
(t, 3H, J = 7.0 Hz); 13C NMR (125 MHz, CDCl3) d 172.05, 150.36,
144.89, 141.95, 138.79, 135.79, 135.27, 134.78, 131.89, 129.61,
129.47, 127.84, 124.84, 122.07, 119.49, 111.24, 111.09, 101.05,
65.29, 59.90, 55.86, 34.06, 31.19, 24.70, 22.31, 13.92; IR (CH2Cl2,
cast) 3393, 1760, 1646, 1505, 1452, 1375, 1322, 1268,
1070 cmꢀ1; FAB-HRMS calcd for C26H28O7 m/z (M)+ 452.1835,
found 452.1842.
Yield 72%, 1H NMR (500 MHz, CDCl3) d 7.82 (s, 1H), 7.42 (d,
1H, J = 8.5 Hz), 7.17 (d, 1H, J = 8.5 Hz), 6.89 (d, 1H, J = 8.1 Hz),
6.85 (dd, 1H, J = 1.6, 8.1 Hz), 6.82 (d, 1H, J = 1.6 Hz), 5.74 (AB,
2H, J = 11.0 Hz), 5.42 (s, 2H), 4.53 (AB, 2H, J = 11.7 Hz), 3.94 (s,
3H), 3.82 (s, 3H), 2.11 (s, 3H); 13C NMR (100 MHz, CDCl3) d
170.74, 148.26, 148.00, 145.03, 141.97, 135.96, 135.38, 131.84,
130.24, 130.13, 129.52, 122.32, 122.02, 119.88, 113.41, 111.19,
110.12, 101.98, 65.17, 59.31, 55.94, 55.82, 21.17; IR (CH2Cl2,
cast) 3497, 1736, 1508, 1451, 1372, 1324, 1230, 1247,
1072 cmꢀ1; FAB-HRMS Calcd for C23H22O7 m/z (M+) 410.1366,
found 410.1362.
4.1.19. (9-(3,4-Dimethoxyphenyl)naphtho[1,2-d][1,3]dioxole-
7,8-diyl)bis(methylene) diacetate (37)
4.1.24. (9-(3-Hydroxy-4-methoxyphenyl)naphtho[1,2-d][1,3]
dioxole-7,8-diyl)bis(methylene) dihexanoate (43)
Yield 75%, 1H NMR (500 MHz, CDCl3) d 7.82 (s, 1H), 7.43 (d, 1H,
J = 8.5 Hz), 7.19 (d, 1H, J = 8.5 Hz), 6.86 (d, 1H, J = 7.8 Hz), 6.77–6.79
(m, 2H), 5.74 (AB, 2H, J = 11.5 Hz), 5.28 (s, 2H), 4.99 (s, 2H), 3.93 (s,
3H), 3.80 (s, 3H), 2.10 (s, 3H), 2.00 (s, 3H); 13C NMR (100 MHz,
CDCl3) d 170.72, 170.36, 148.36, 147.91, 145.17, 142.05, 137.63,
131.30, 130.41, 130.24, 130.05, 129.76, 122.34, 122.04, 119.78,
113.41, 111.52, 110.05, 101.05, 64.81, 61.44, 55.88, 55.79, 21.05,
20.91; IR (CH2Cl2, cast) 1738, 1509, 1452, 1373, 1324, 1232,
Yield 60%, 1H NMR (400 MHz, CDCl3) d 7.80 (s, 1H), 7.40 (d, 1H,
J = 8.5 Hz), 7.17–7.14 (m, 2H), 6.99–6.97 (m, 2H), 5.76 (AB, 2H,
J = 5.5 Hz), 5.41 (s, 2H), 4.53 (AB, 2H, J = 11.9 Hz), 3.88 (s, 3H),
2.54 (t, 2H, J = 7.5 Hz), 2.35 (t, 2H, J = 7.5 Hz), 1.75–1.70 (m, 2H),
1.65–1.59 (m, 2H), 1.40–1.33 (m, 4H), 1.31–1.23 (m, 4H), 0.89 (t,
3H, J = 7.0 Hz), 0.85 (t, 3H, J = 7.0 Hz); 13C NMR (125 MHz, CDCl3)
d 173.53, 171.97, 150.41, 145.02, 141.94, 138.80, 135.55, 134.92,
131.72, 130.44, 130.17, 129.46, 127.83, 124.86, 122.20, 119.69,
111.20, 111.15, 101.09, 64.93, 59.18, 55.85, 34.39, 34.05, 31.26,
31.20, 24.70, 24.59, 22.31, 22.28, 13.92, 13.87; IR (CH2Cl2, cast)
3361, 1736, 1633, 1457, 1376, 1234, 1168, 1073 cmꢀ1; FAB-HRMS
calcd for C32H38O8 m/z (M)+ 550.2567, found 550.2575.
1138, 1075 cmꢀ1
;
FAB-HRMS Calcd for C25H24O8 m/z (M+)
452.1471, found 452.1464.
4.1.20. (9-(3,4-Dimethoxyphenyl)-8-(hydroxymethyl)
naphtho[1,2-d][1,3]dioxol-7-yl)methyl hexanoate (38)
Yield 75%, 1H NMR (500 MHz, CDCl3) d 7.82 (s, 1H), 7.42 (d, 1H,
J = 8.5 Hz), 7.17 (d, 1H, J = 8.5 Hz), 6.90 (d, 1H, J = 8.0 Hz), 6.85 (dd,
1H, J = 1.6, 8.0 Hz), 6.82 (d, 1H, J = 1.6 Hz), 5.74 (AB, 2H, J = 11.1 Hz),
5.42 (s, 2H), 4.53 (AB, 2H, J = 11.1 Hz), 3.94 (s, 3H), 3.82 (s, 3H), 2.35
(t, 2H, J = 7.5 Hz), 1.65–1.61 (m, 2H), 1.29–1.25 (m, 4H), 0.85 (t, 3H,
J = 6.8 Hz); 13C NMR (100 MHz, CDCl3) d 173.55, 148.25, 148.00,
145.00, 142.50, 135.93, 135.44, 131.86, 130.41, 130.06, 129.54,
122.32, 122.05, 119.90, 113.44, 111.17, 110.12, 100.97, 64.98,
59.32, 55.94, 55.83, 34.40, 31.27, 24.60, 22.29, 13.88; IR (CH2Cl2,
cast) 3413, 1711, 1634, 1507, 1451, 1378, 1322, 1262,
1137 cmꢀ1; FAB-HRMS Calcd for C27H30O7 m/z (M+) 466.1992,
found 466.2001.
4.1.25. (9-(3-(Hexanoyloxy)-4-methoxyphenyl)naphtho[1,2-
d][1,3]dioxole-7,8-diyl)bis (methylene)dihexanoate (44)
Yield 22%, 1H NMR (400 MHz, CDCl3) d 7.80 (s, 1H), 7.40 (d, 1H,
J = 8.5 Hz), 7.17 (d, 1H, J = 8.5 Hz), 7.07 (dd, 1H, J = 2.1, 8.3 Hz),
6.91–6.93 (m, 2H), 5.76 (AB, 2H, J = 6.0 Hz), 5.27 (d, 2H,
J = 2.8 Hz), 5.00 (AB, 2H, J = 12.0 Hz), 3.86 (s, 3H), 2.53 (t, 2H,
J = 7.4 Hz), 2.33 (t, 2H, J = 7.4 Hz), 2.23 (t, 2H, J = 7.4 Hz), 1.74–
1.71 (m, 2H), 1.65–1.54 (m, 4H), 1.40–1.33 (m, 4H), 1.31–1.23
(m, 8H), 0.91–0.84 (m, 9H); 13C NMR (100 MHz, CDCl3) d 173.53,
173.18, 171.65, 150.55, 145.17, 142.03, 138.94, 136.60, 131.20,
130.61, 130.47, 129.97, 129.71, 127.64, 124.94, 122.19, 119.65,
111.46, 111.02, 101.20, 64.35, 61.07, 55.81, 34.30, 34.21, 34.02,
31.32, 31.21, 24.73, 24.60, 22.33, 22.30, 13.93, 13.89; IR (CH2Cl2,
cast) 1736, 1505, 1452, 1374, 1321, 1271, 1162, 1075,
1048 cmꢀ1; FAB-HRMS calcd for C38H48O9 m/z (M)+ 648.3298,
found 648.3293.
4.1.21. (9-(3-Hydroxy-4-methoxyphenyl)-8-(hydroxymethyl)
naphtho[1,2-d][1,3]dioxol-7-yl)methyl acetate (40)
Yield 72%, 1H NMR (400 MHz, CDCl3) d 7.75 (s, 1H), 7.39 (d, 1H,
J = 8.5 Hz), 7.16–7.13 (m, 2H), 6.99–6.97 (m, 2H), 5.76 (AB, 2H,
J = 5.9 Hz), 4.87 (s, 2H), 4.58 (AB, 2H, J = 12.0 Hz), 3.89 (s, 3H),
2.28 (s, 3H); 13C NMR (100 MHz, CDCl3) d 169.14, 150.31, 144.89,
141.92, 138.69, 135.80, 135.25, 134.70, 131.92, 129.60, 129.51,
128.00, 124.80, 122.09, 119.48, 111.24, 111.10, 101.04, 65.26,
59.88, 55.89, 20.75; IR (CH2Cl2, cast) 3403, 1762, 1505, 1451,
4.1.26. (9-(4-Hydroxy-3-methoxyphenyl)-8-(hydroxymethyl)
naphtho[1,2-d][1,3]dioxol-7-yl)methyl acetate (46)
Yield 72%, 1H NMR (400 MHz, CDCl3) d 7.79 (s, 1H), 7.42 (d, 1H,
J = 8.5 Hz), 7.17 (d, 1H, J = 8.5 Hz), 7.04 (d, 1H, J = 8.0 Hz), 6.90 (d,
1H, J = 1.8 Hz), 6.87 (dd, 1H, J = 1.8, 8.0 Hz), 5.75 (s, 2H), 4.90 (s,
2H), 4.61 (s, 2H), 3.84 (s, 3H), 2.34 (s, 3H); 13C NMR (125 MHz,
CDCl3) d 169.00, 150.19, 139.10, 135.44, 135.25, 135.15, 133.92,
130.12, 129.69, 129.62, 122.19, 121.74, 119.35, 118.10, 114.47,
113.49, 111.19, 100.97, 65.27, 59.97, 56.01, 20.75; IR (CH2Cl2, cast)
3405, 1762, 1505, 1451, 1370, 1322, 1268, 1210, 1071 cmꢀ1; FAB-
HRMS Calcd for C22H20O7 m/z ([M]+) 396.1209, found 396.1217.
1371, 1322, 1269, 1211, 1070 cmꢀ1
22H20O7 m/z (M+) 396.1209, found 396.1203.
; FAB-HRMS Calcd for
C
4.1.22. (9-(3-Acetoxy-4-methoxyphenyl)naphtho[1,2-d][1,3]
dioxole-7,8-diyl)bis(methylene) diacetate (41)
Yield 25%, 1H NMR (400 MHz, CDCl3) d 7.81 (s, 1H), 7.41 (d, 1H,
J = 8.6 Hz), 7.18 (d, 1H, J = 8.6 Hz), 7.09 (dd, 1H, J = 2.1, 8.3 Hz),
6.94–6.96 (m, 2H), 5.77 (AB, 2H, J = 5.8 Hz), 5.27 (s, 2H), 5.01 (AB,
2H, J = 12.0), 3.88 (s, 3H), 2.27 (s, 3H), 2.09 (s, 3H), 1.99 (s, 3H);
13C NMR (100 MHz, CDCl3) d 170.73, 170.44, 168.77, 150.51,
145.24, 142.01, 138.84, 136.61, 131.16, 130.50, 130.26, 130.17,
129.69, 127.72, 124.91, 122.25, 119.66, 111.55, 111.08, 101.20,
64.84, 61.23, 55.85, 21.04, 20.86, 20.70; FAB-HRMS Calcd for
4.1.27. (9-(4-Hydroxy-3-methoxyphenyl)naphtho[1,2-d][1,3]
dioxole-7,8-diyl)bis(methylene) diacetate (47)
Yield 48%, 1H NMR (500 MHz, CDCl3) d 7.83 (s, 1H), 7.42 (d, 1H,
J = 8.5 Hz), 7.17 (d, 1H, J = 8.5 Hz), 7.04 (d, 1H, J = 8.0 Hz), 6.92 (d,
1H, J = 1.5 Hz), 6.87 (dd, 1H, J = 1.5, 8.0 Hz), 5.74 (s, 2H), 5.42 (s,
2H), 4.54 (s, 2H), 3.77 (s, 3H), 2.34 (s, 3H), 2.11 (s, 3H); 13C NMR
C
26H24O9 m/z (M+) 480.1420, found 480.1418.
(125 MHz, CDCl3) d 170.73, 168.98, 150.15, 145.05, 141.86,

2174
D. Janmanchi et al. / Bioorg. Med. Chem. 21 (2013) 2163–2176
139.04, 137.98, 135.34, 135.04, 130.32, 129.44, 122.32, 122.19,
121.70, 119.48, 114.46, 111.26, 101.00, 65.06, 59.21, 56.00, 21.16,
20.75; IR (CH2Cl2, cast) 3477, 1738, 1640, 1501, 1453, 1369,
1216, 1116, 1044, 797 cmꢀ1; FAB-HRMS Calcd for C24H22O8 m/z
([M]+) 438.1315, found 438.1306.
129.61, 127.70, 125.32, 122.27, 122.21, 119.30, 111.67, 101.25,
64.78, 60.99, 21.03, 20.81, 20.73, 20.67; FAB-HRMS Calcd for
C
27H24O10 m/z ([M]+) 508.1369, found 508.1378.
4.1.33. (9-(3,4-Dihydroxyphenyl)naphtho[1,2-d][1,3]dioxole-
7,8-diyl)bis(methylene) dihexanoate (54)
4.1.28. (9-(4-Acetoxy-3-methoxyphenyl)naphtho[1,2-d][1,3]
dioxole-7,8-diyl)bis(methylene) diacetate (48)
Yield 61%, 1H NMR (400 MHz, CDCl3) d 7.78 (s, 1H), 7.40 (d, 1H,
J = 8.6 Hz), 7.19 (m, 2H), 7.16 (d, 1H, J = 8.6 Hz), 7.13–7.12 (m, 1H),
5.77 (AB, 2H, J = 6.0 Hz), 4.88 (AB, 2H, J = 12.2 Hz), 4.58 (s, 2H), 2.56
(t, 2H, J = 7.5 Hz), 2.49 (t, 2H, J = 7.5 Hz), 1.78–1.67 (m, 4H), 1.42–
1.32 (m, 8H), 0.92 (t, 3H, J = 7.1 Hz), 0.88 (t, 3H, J = 7.1 Hz); 13C
NMR (100 MHz, CDCl3) d 171.33, 171.20, 144.93, 141.33, 141.10,
137.81, 135.62, 135.36, 134.12, 130.93, 129.77, 129.56, 127.84,
125.34, 122.26, 122.10, 119.02, 111.21, 101.12, 65.16, 59.80,
34.08, 31.28, 31.23, 24.64, 24.55, 22.34, 22.29, 13.90, 13.86; IR
(CH2Cl2, cast) 3420, 1766, 1632, 1503, 1456, 1316, 1270, 1152,
1111, 1023 cmꢀ1; ESI-HRMS Calcd for C31H36O8Na m/z ([M+Na]+)
559.2308, found 559.2313.
Yield 30%, 1H NMR (500 MHz, CDCl3) d 7.83 (s, 1H), 7.42 (d, 1H,
J = 8.5 Hz), 7.19 (d, 1H, J = 8.5 Hz), 7.03 (d, 1H, J = 8.0 Hz), 6.87 (d,
1H, J = 1.7 Hz), 6.83 (dd, 1H, J = 1.7, 8.0 Hz), 5.75 (s, 2H), 5.27 (s,
2H), 5.02 (s, 2H), 3.76 (s, 3H), 2.33 (s, 3H), 2.09 (s, 3H), 1.99 (s,
3H); 13C NMR (100 MHz, CDCl3) d 170.69, 170.22, 168.82, 150.13,
145.22, 141.93, 139.23, 137.41, 136.98, 130.31, 130.24, 130.2,
129.66, 122.32, 122.18, 121.68, 119.40, 114.41, 111.61, 101.10,
64.76, 61.18, 55.95, 21.02, 20.85, 20.73; IR (CH2Cl2, cast) 1738,
1598, 1503, 1452, 1370, 1232, 1120, 1074 1026 cmꢀ1; FAB-HRMS
Calcd for C26H24O9 m/z ([M]+) 480.1420, found 480.1416.
4.1.29. (9-(4-Hydroxy-3-methoxyphenyl)naphtho[1,2-d][1,3]
dioxole-7,8-diyl)bis(methylene) dihexanoate (49)
4.1.34. 9-(Benzo[d][1,3]dioxol-5-yl)naphtho[1,2-d][1,3]dioxole-
7,8-dicarbaldehyde (55)
Yield 63%, 1H NMR (400 MHz, CDCl3) d 7.83 (s, 1H), 7.42 (d, 1H,
J = 8.6 Hz), 7.17 (d, 1H, J = 8.6 Hz), 7.03 (d, 1H, J = 8.0 Hz), 6.91 (d,
1H, J = 1.8 Hz), 6.88 (dd, 1H, J = 1.8, 8.0 Hz), 5.74 (s, 2H), 5.42 (s,
2H), 4.54 (s, 2H), 3.76 (s, 3H), 2.60 (t, 2H, J = 7.5 Hz), 2.35 (t, 2H,
J = 7.5 Hz), 1.79 (m, 2H), 1.64 (m, 2H), 1.40 (m, 4H), 1.30–1.27
(m, 4H), 0.93 (t, 3H, J = 7.0 Hz), 0.86 (t, 3H, J = 7.0 Hz); 13C NMR
To a dichloromethane (2.0 ml) solution of oxalyl chloride
(0.3 ml, 5 equiv) was added dropwise DMSO (0.5 ml, 10 equiv in
1.0 ml CH2Cl2) at ꢀ78 °C, and the resulting mixture was stirred
for 15 min. A dichloromethane solution of 32 (250 mg in 5.0 ml)
was added dropwise at ꢀ78 °C. After the mixture was stirred for
12 h, triethylamine (1 ml, 20 equiv) was added dropwise, and the
reaction mixture was then slowly warmed to room temperature
and stirred for 1 h. The mixture was quenched with water
(50 ml) and extracted with dichloromethane (50 ml ꢁ 2). The com-
bined organic layer was washed with brine, dried (MgSO4), filtered,
and concentrated in vacuo. 230 mg (93%) of dicarbaldehyde was
obtained as almost pure. This was used directly without further
purification.
(100 MHz, CDCl3)
d 173.55, 171.93, 150.20, 141.89, 140.19,
139.14, 137.89, 135.37, 135.12, 130.46, 130.25, 129.46, 122.29,
122.19, 121.74, 119.48, 114.44, 111.23, 101.02, 64.88, 59.23,
55.97, 34.39, 34.04, 31.26, 31.23, 24.75, 24.59, 22.36, 22.29,
13.97, 13.88; IR (CH2Cl2, cast) 3453, 1733, 1641, 1503, 1454,
1273, 1166, 749 cmꢀ1
; ESI-HRMS Calcd for C32H38O8Na m/z
([M+Na]+) 573.2464, found 573.2455.
4.1.30. (9-(3,4-Dihydroxyphenyl)naphtho[1,2-d][1,3]dioxole-
7,8-diyl)bis(methylene) diacetate (51)
4.1.35. 3-(10-(Benzo[d][1,3]dioxol-5-yl)-7-oxo-7H-[1,3]
dioxolo[40,50:3,4]benzo[1,2-f]isoindol-8(9H)-yl)propanenitrile
(56)
Yield 43%, 1H NMR (400 MHz, CDCl3) d 7.79 (s, 1H), 7.40 (d, 1H,
J = 8.5 Hz), 7.20 (m, 2H), 7.16 (d, 1H, J = 8.5 Hz), 7.14 (br s, 1H), 5.76
(AB, 2H, J = 5.7 Hz), 4.88 (AB, 2H, J = 12.2 Hz), 4.59 (s, 2H), 2.32 (s,
3H), 2.25 (s, 3H); 13C NMR (100 MHz, CDCl3) d 168.36, 168.25,
144.95, 141.80, 141.28, 141.03, 137.94, 135.60, 135.31, 134.05,
129.81, 129.55, 127.98, 125.31, 122.21, 122.13, 119.04, 111.23,
101.08, 65.17, 59.81, 20.72; IR (CH2Cl2, cast) 3423, 1766, 1637,
1503, 1451, 1369, 1267, 1209, 1174, 1105 cmꢀ1; FAB-HRMS Calcd
for C23H20O8 m/z ([M]+) 424.1158, found 424.1147.
Compound 55 (50 mg, 0.14 mmol) and 3-aminopropionitrile
(10 mg, 0.14 mmol) were placed in a round-bottomed flask. Mix-
ture of CH3CN/DMF (2:1/3 ml) was used as reaction medium;
TMSCl (13 mg, 0.12 mmol) was added and the mixture was stirred
for 12 h at room temperature. The mixture was extracted with
ethyl acetate. The combined organic layer was evaporated in vac-
uum. The isolation of the product 20 mg (32%) was accomplished
by flash column chromatography (EA/Hexane = 1/1). 1H NMR
(500 MHz, CDCl3) d 8.28 (s, 1H), 7.63 (d, 1H, J = 8.7 Hz), 7.25 (d,
1H, J = 8.7 Hz), 6.86 (d, 1H, J = 7.8 Hz), 6.79 (d, J = 1.4 Hz), 6.78
(dd, 1H, J = 1.4, 7.8 Hz), 6.03 (AB, 2H, J = 15.2 Hz), 5.90 (AB, 2H,
J = 9.6 Hz), 4.40 (AB, 2H, J = 16.5 Hz), 3.85 (t, 2H, J = 6.5 Hz), 2.74
(t, 2H, J = 6.5 Hz); 13C NMR (100 MHz, CDCl3) d 168.70, 147.29,
147.23, 146.01, 141.69, 135.73, 131.15, 130.46, 129.74, 127.29,
124.74, 124.56, 122.40, 120.75, 117.96, 111.29, 109.77, 107.93,
101.34, 101.18, 50.54, 39.28, 17.21; IR (CH2Cl2, cast) 2896, 1691,
1630, 1492, 1441, 1272, 1235, 1062, 1035 cmꢀ1; FAB-HRMS Calcd
for C23H16O5N2 m/z (M+) 400.1059, found 400.1052.
4.1.31. (9-(3-Acetoxy-4-hydroxyphenyl)naphtho[1,2-d][1,3]
dioxole-7,8-diyl)bis(methylene) diacetate (52)
Yield 22%, 1H NMR (500 MHz, CDCl3) d 7.83 (s, 1H), 7.41 (d, 1H,
J = 8.5 Hz), 7.21 (m, 2H), 7.17–7.16 (m, 2H), 5.77 (AB, 2H,
J = 6.1 Hz), 5.42 (s, 2H), 4.54 (s, 2H), 2.32 (s, 3H), 2.25 (s, 3H),
2.11 (s, 3H); 13C NMR (125 MHz, CDCl3) d 170.73, 168.31, 168.23,
145.11, 141.79, 141.33, 141.03, 137.77, 135.31, 134.23, 130.54,
130.34, 129.38, 127.96, 125.34, 122.27, 122.19, 119.26, 111.30,
101.13, 65.01, 59.05, 21.15, 20.72; FAB-HRMS Calcd for C25H22O9
m/z ([M]+) 466.1264, found 466.1269.
4.1.36. 2-(10-(Benzo[d][1,3]dioxol-5-yl)-7-oxo-7H-[1,3]
4.1.32. 4-(7,8-Bis(acetoxymethyl)naphtho[1,2-d][1,3]dioxol-9-
yl)-1,2-phenylene diacetate (53)
dioxolo[40,50:3,4]benzo[1,2-f]isoindol-8(9H)-yl)acetic acid (57)
To a solution of 55 (48 mg, 0.14 mmol) in acetic acid (3 ml) was
added glycine (10 mg, 0.14 mmol) and the mixture was boiled for
5–10 min. After this period, acetic acid was removed under re-
duced pressure. Inseparable mixture of 57 and the retro-isomer
57a was obtained (34 mg, 61%, 57:57a = 5:1). The reported spec-
trum was extracted from the mixture spectrum based on peak
intensity. Compound 57: 1H NMR (400 MHz, CD3OD) d 8.25 (s,
Yield 12%, 1H NMR (500 MHz, CDCl3) d 7.83 (s, 1H), 7.42 (d, 1H,
J = 8.5 Hz), 7.21–7.17 (m, 2H), 7.15 (dd, 1H, J = 1.8, 8.2 Hz), 7.12 (d,
1H, J = 1.8 Hz), 5.77 (s, 2H), 5.27 (s, 2H), 5.02 (AB, 2H, J = 12.1 Hz),
2.30 (s, 3H), 2.24 (s, 3H), 2.09 (s, 3H), 1.99 (s, 3H); 13C NMR
(125 MHz, CDCl3)
d 170.71, 170.35, 168.10, 167.94, 145.32,
141.90, 141.53, 141.21, 137.19, 135.94, 130.58, 130.39, 130.15,

D. Janmanchi et al. / Bioorg. Med. Chem. 21 (2013) 2163–2176
2175
1H), 7.72 (d, 1H, J = 8.7 Hz), 7.29 (d, 1H, J = 8.7 Hz), 6.89–6.83 (m,
3H), 6.00 (AB, 2H, J = 9.8 Hz), 5.89 (AB, 2H, J = 11.5 Hz), 4.43 (AB,
2H, J = 16.9 Hz), 4.17 (s, 2H); 13C NMR (100 MHz, CDCl3) d 170.59,
148.82, 148.76, 147.44, 143.04, 138.25, 132.95, 132.12, 131.04,
129.51, 125.91, 125.06, 124.01, 122.06, 112.18, 111.15, 108.87,
102.66, 102.58, 71.49, 51.90; IR (CH2Cl2, cast) 3419, 1736, 1631,
1487, 1439, 1373, 1320, 1273, 1232, 1039 cmꢀ1; 57+57a: ESI-
The mixture was stirred 2 h at same temperature. After completion
of the reaction, the reaction mixture was washed with water
(20 ml), extracted with dichloromethane (20 ml) and dried over
MgSO4. After evaporation of solvents, 30 mg (81%) of 61 obtained
and was used further without purification and characterization.
4.1.40. 10-(Benzo[d][1,3]dioxol-5-yl)-8-(2-hydroxyethyl)-8,9-
dihydro-7H-[1,3]dioxolo[40,50:3,4]benzo[1,2-f]isoindol-7-one
(62)
HRMS calcd for
404.0776.
C
22H14NO7 m/z ([MꢀH]ꢀ) 404.0770, found
Compound 61 (30 mg, 0.077 mmol) and NaBH4 (10 mg,
0.26 mmol) were stirred in dry THF (3 ml) for 1 h at room temper-
ature. After this period, 10% HCl was added cautiously to the reac-
tion mixture and the reaction was stirred for another 30 min. The
mixed solution was extracted with ether. The organic fraction
was dried over MgSO4 and concentrated. After column chromatog-
raphy, 23 mg (76%) of 62 isolated. 1H NMR (400 MHz, CDCl3) d 8.26
(s, 1H), 7.62 (d, 1H, J = 8.6 Hz), 7.23 (d, 1H, J = 8.6 Hz), 6.86 (d, 1H,
J = 7.7 Hz), 6.79–6.77 (m, 2H), 6.03 (AB, 2H, J = 11.7 Hz), 5.89 (AB,
2H, J = 6.7 Hz), 4.35 (AB, 2H, J = 16.8 Hz), 3.88 (t, 2H, J = 4.9 Hz),
3.72 (t, 2H, J = 4.9 Hz); 13C NMR (100 MHz, CDCl3) d 169.63,
147.22, 147.13, 145.82, 141.64, 136.23, 131.40, 130.49, 129.38,
128.02, 124.65, 124.26, 122.48, 120.59, 111.11, 109.82, 107.86,
101.28, 101.14, 61.67, 51.33, 46.52; IR (CH2Cl2, cast) 3353, 1677,
1619, 1492, 1438, 1374, 1294, 1230, 1036 cmꢀ1; FAB-HRMS Calcd
for C22H18O6N m/z ([M+H]+) 392.1134, found 392.1133.
4.1.37. Methyl 2-(10-(benzo[d][1,3]dioxol-5-yl)-7-oxo-7H-
[1,3]dioxolo[40,50:3,4]benzo[1,2-f]isoindol-8(9H)-yl)acetate (58)
To a DMF solution of 57 (25 mg, 0.06 mmol 2.0 ml) was added
K2CO3 (17 mg, 0.12 mmol) and CH3I (18 mg, 0.13 mmol). The mix-
ture was stirred 2 h. After completion of the reaction, the reaction
mixture was washed with water (10 ml ꢁ 2) and extracted with
dichloromethane (20 ml). After purified by column chromatogra-
phy (DCM/Hexane = 2/1), 22 mg (81%) of 58 and 4 mg (15%) of
58a were obtained. Compound 58: 1H NMR (500 MHz, CDCl3) d
8.31 (s, 1H), 7.63 (d, 1H, J = 8.6 Hz), 7.23 (d, 1H, J = 8.6 Hz), 6.85
(d, 1H, J = 7.8 Hz), 6.81 (d, 1H, J = 1.5 Hz), 6.79 (dd, 1H, J = 1.5,
7.8 Hz), 6.02 (AB, 2H, J = 17.4 Hz), 5.89 (AB, 2H, J = 8.2 Hz), 4.36
(s, 2H), 4.35 (AB, 2H, J = 16.1 Hz), 3.72 (s, 3H); 13C NMR
(100 MHz, CDCl3)
d 169.37, 168.64, 147.19, 147.11, 145.89,
141.63, 136.20, 131.39, 130.48, 129.48, 127.28, 124.73, 122.52,
120.74, 111.13, 109.86, 107.83, 101.29, 101.13, 52.32, 50.18,
43.83; IR (CH2Cl2 cast) 1744, 1692, 1635, 1486, 1459, 1434, 1273,
1234, 1061, 1064, 927, 803 cmꢀ1; ESI-HRMS Calcd for C23H17NO7-
Na m/z ([M+Na])+ 442.0903, found 442.0902.
5. Materials and methods for cell culture
5.1. Reagents
4.1.38. Pentyl 2-(10-(benzo[d][1,3]dioxol-5-yl)-7-oxo-7H-
[1,3]dioxolo[40,50:3,4]benzo[1,2-f]isoindol-8(9H)-yl)acetate (59)
and octyl 2-(10-(benzo[d][1,3]dioxol-5-yl)-7-oxo-7H-
Hepatitis B surface antigen (HBsAg) enzyme immunoassay (EIA)
kits were purchased from Bio-Rad (Hercules, 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). MTT (3-[4, 5-dimethylthiazol-2-yl]-2,5-
diphenyl-tetrazolium bromide), was purchased from Sigma Chem-
ical Co. (St. Louis. MO, USA).
[1,3]dioxolo[40,50:3,4]benzo[1,2-f]isoindol-8(9H)-yl)acetate (60)
To a solution of 57 in DMF was added K2CO3 and C5H11I (for the
synthesis of 59) and C8H17I (for 60) and the reactions were treated
as described for 58. After column chromatography 59 (68%) and
59a (13%); 60 (66%) and 60a (14%) were isolated, respectively.
Compound 59: 1H NMR (400 MHz, CDCl3) d 8.31 (s, 1H), 7.63 (d,
1H, J = 8.6 Hz), 7.24 (d, 1H, J = 8.6 Hz), 6.85 (d, 1H, J = 7.8 Hz),
6.80 (d, 1H, J = 1.4 Hz), 6.78 (dd, 1H, J = 1.4, 7.8 Hz), 6.02 (AB, 2H,
J = 14.0 Hz), 5.89 (AB, 2H, J = 6.6 Hz), 4.35 (s, 2H), 4.35 (AB, 2H,
J = 16.2 Hz), 4.11 (t, 2H, J = 6.6 Hz), 1.60–1.57 (m, 2H), 1.36–1.30
(m, 2H), 0.89 (t, 3H, J = 7.4 Hz); 13C NMR (100 MHz, CDCl3) d
168.98, 168.63, 147.18, 147.09, 145.86, 141.63, 136.25, 131.42,
130.47, 129.44, 127.36, 124.71, 122.51, 120.73, 111.11, 109.85,
107.82, 101.28, 101.12, 65.32, 50.17, 43.92, 30.51, 19.05, 13.63;
IR (CH2Cl2, cast) 1740, 1696, 1633, 1490, 1460, 1373, 1273, 1234,
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.36 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. Stock cul-
tures of human hepatoma cells HepA2 and 1.3ES2 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 1 ꢁ 105 cells/
well or 96-well plates at a density at of 3 ꢁ 104 cells/well in 100-
mm culture dishes at a density of 1.5 ꢁ 106 cells/dish in DMEM
medium containing 10% fetal calf serum.
;
1038 cmꢀ1 ESI-HRMS Calcd for C26H23NO7Na m/z ([M+Na])+
484.1372, found 484.1368. 60: 1H NMR (400 MHz, CDCl3) d 8.31
(s, 1H), 7.64 (d, 1H, J = 8.6 Hz), 7.24 (d, 1H, J = 8.6 Hz), 6.85 (d,
1H, J = 7.8 Hz), 6.81 (d, J = 1.3 Hz), 6.79 (dd, 1H, J = 1.6, 7.8 Hz),
6.02 (AB, 2H, J = 14.4 Hz), 5.89 (AB, 2H, J = 6.6 Hz), 4.35 (s, 2H),
4.35 (AB, 2H, J = 16.2 Hz), 4.10 (t, 2H, J = 6.7 Hz), 1.61–1.58 (m,
2H), 1.25–1.22 (m, 10H), 0.84 (t, 3H, J = 6.9 Hz); 13C NMR
(400 MHz, CDCl3)
d 168.97, 168.64, 147.19, 147.10, 145.87,
141.64, 136.26, 131.44, 130.49, 129.45, 127.37, 124.72, 122.52,
120.74, 111.11, 109.86, 107.83, 101.28, 101.12, 65.63, 50.18,
43.95, 31.72, 29.11, 28.49, 25.83, 22.59, 14.04; IR (CH2Cl2, cast)
1681, 1635, 1443, 1413, 1378, 122, 1039, 764 cmꢀ1; ESI-HRMS
Calcd for C30H31NO7Na m/z ([M+Na])+ 540.1998, found 540.1985.
5.3. Quantification of HBsAg
Cells were seeded either in 96-well plates at a density of 3 ꢁ 104
cells/well or in 24-well plates at a density of 1 ꢁ 105 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 amount of HBsAg
4.1.39. 2-(10-(Benzo[d][1,3]dioxol-5-yl)-7-oxo-7H-[1,3]dioxolo
[40,50:3,4]benzo[1,2-f]isoindol-8(9H)-yl)acetaldehyde (61)
To a solution of 58 (40 mg, 0.095 mmol) in dry toluene (3 ml)
was added dropwise DIBALH (1 M in toluene, 0.16 ml) at ꢀ40 °C.

2176
D. Janmanchi et al. / Bioorg. Med. Chem. 21 (2013) 2163–2176
production in the culture medium was determined by enzyme
immunoassay (EIA) kits (Bio-Rad, CA, USA). The viability of cells
was determined by a MTT cell proliferation assay.37
Acknowledgments
The authors thank Dr. Chungming Chang for providing the pCP-
Luc plasmid. This work was supported by Grants from Academia
Sinica, National Science Council (NSC 98-2320-B-010-024-MY3),
and Ministry of Education, Republic of China (Aim for the Top Uni-
versity Plan).
5.4. Quantitative detection of HBV DNA by real-time light cycler
PCR
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 ꢁ 106 cells/well before 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-CAG-
GTCTGTGCCAAG-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 denaturing
step at 95 °C for 10 min, followed by 45 amplification cycles at
95 °C for 12 s, 54 °C for 20 s.
A. Supplementary data
Supplementary data associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/j.bmc.
2012.11.037.
References and notes
1. World Health Organization. Hepatitis B. World Health Organization Fact Sheet
204 (Revised August 2008). (http://www.who.int/mediacentre/factsheets/
fs204/en/index.html).
2. Robinson, W. S. Annu. Rev. Microbiol. 1977, 31, 357.
3. Lok; Mc Mahon Hepatology 2007, 45, 2.
4. Pang, K. R.; Wu, J. J.; Huang, D. B.; Tyring, S. K.; Baron, S. Methods Mol. Med.
2005, 116, 1.
5. Zoulim, F. J. Clin. Virol. 2001, 21, 243.
6. Dienstag, J. L.; Perril, N. Engl. J. Med. 1995, 333, 1657.
7. Walsh, K. M.; Woodlo, R. P.; Schiff, E. R.; Bartholomew, M.; Vicary, C.; Rubin,
Mall, T.; Lamy, P.; Wight, D. G.; Bloor, S.; Alexander, G. J. Gut 2001, 49, 436.
8. Franco, J.; Saeian, K. J. Vasc. Interv. Radiol. 2002, 13, S191.
9. 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.
10. Ruiz-Sancho, A.; Sheldon, J.; Soriano, V. Expert. Opin. Biol. Ther. 2007, 7, 751.
11. 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.
5.5. RNA isolation and Northern blot analysis
Total RNA was extracted from the cells using the phenol and
guanidium isothiocyanate method.38 The RNA (20 mg) was dena-
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.
12. Yeh, C. T.; Chien, R. N.; Chu, C. M.; Liaw, Y. F. Hepatology 2000, 31, 1318.
13. Locarnini, S.; Mason, W. S. J. Hepatol. 2006, 44, 422.
14. 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.; Helga, R. W. Science 2003, 299, 893.
15. Sanghyun, L.; Hyun, S. B.; Yong, P. K.; Bak-Kwang, K.; Seon, H. C.; Kazuo, O.;
Hyun, S. Phytother. Res. 2005, 19, 103.
5.6. Transient transfections and luciferase assay
16. Kuo, Y. H.; Wu, M. D.; Hung, C. C.; Huang, R. L.; Yang Kuo, L. M.; Shen, Y. C.; Ong,
C. W. Bioorg. Med. Chem. 2005, 13, 1555.
17. Cheng, Y. C.; Ying, C. X.; Leung, C. H.; Li, Y. J. Clin. Virol. 2005, 34, S147.
18. 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, 2, 534.
19. He, K.; Zeng, L.; Shi, G.; Zhao, G. X.; Kozlowski, J. F.; McLaughlin, J. L. J. Nat. Prod.
1997, 60, 38.
20. Burden, R. S.; Crombie, L.; Whiting, D. A. Tetrahedron Lett. 1968, 1035.
21. 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.
22. 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.
23. Janmanchi, D.; Tseng, Y. P.; Wang, K. C.; Huang, R. L.; Lin, C. H.; Yeh, S. F. Bioorg.
Med. Chem. 2010, 18, 1213–1226. Ying, C. X.
24. Li, Y.; Leung, C. H.; Robek, M. D.; Cheng, Y. C. Proc. Natl. Acad. Sci. U.S.A. 2007,
104, 8526.
25. Holmes, T. L.; Stevenso, R. J. Chem. Soc. C-Org. 1971, 2091.
26. Stevenson, R.; Weber, J. V. J. Nat. Prod. 1989, 52, 367.
27. Mizufune, H.; Nakamura, M.; Mitsudera, H. Tetrahedron Lett. 2001, 42, 437.
28. Charlton, J. L.; Oleschuk, C. J.; Chee, G. L. J. Org. Chem. 1996, 61, 3452.
29. Wan, J.; Wu, B.; Pan, Y. Tetrahedron 2007, 63, 9338.
30. Grigg, R.; Gunaratne, H. Q. N.; Sridharan, V. J. Chem. Soc., Chem. Commun. 1985,
1183.
31. Yeh, S. F.; Hong, C.Y.; Huang, Y. L.; Liu, T. Y.; Choo, K. B.; Chou, C. K. Antiviral Res.
1993, 20, 185–193.
HepA2 cells were transfected with pCP-Luc plasmid using lipo-
fectamine 2000 transfection reagent (Invitrogen). The transfected
cells were changed to a serum-free DMEM with drug for 48 h. To
prepare total cell lysate from transfected cells for luciferase activity
measurements, the 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 superna-
tant was collected for protein and luciferase activity measurements
immediately following lysate preparation. Protein concentrations
of the resultant cell lysates were measured by the Bradford meth-
od. Lysates prepared from transfected cells were analyzed for lucif-
erase activity using a luminometer and the Promega Luciferase
Assay System as described by the manufacturer (Promega). Lucifer-
ase activities were normalized to the amount of protein in each ly-
sate. For all transient transfections with promoter–luciferase
reporter construct, 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 independent experiments.
32. 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. 1813, 2005, 79.
´
33. Gilbert, S.; Galarneau, L.; Lamontagne, A.; Roy, S.; Belanger, L. J. Virol. 2000, 74,
5032.
34. Li, J.; Ou, J. H. J. Virol. 2001, 75, 8400.
35. Yu, X.; Mertz, J. E. J. Virol. 2003, 77, 2489.
36. 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. EMBO J. 1987, 6, 675.
37. Denizot, F.; Lang, R. J. Immunol. Methods 1986, 89(2), 271.
38. Gruffat, D.; Piot, C.; Durand, D.; Bauchart, D. Anal. Biochem. 1996, 242, 77.
39. Galibert, F.; Mandart, E.; Fitoussi, F.; Tiollais, P.; Charnay, P. Nature 1979, 281,
646.
5.7. Plasmid
The HBV sequence used in this study is of ayw subtype.39 The
accession number of GeneBank: V01460, Eco RI site as nucleotide
1. The pCP-Luc, plasmid was generous gift from Dr. Chungming
Chang (National Health Research Institutes, Taiwan).