Total synthesis of moniliformediquinone and calanquinone A as potent inhibitors for breast cancer

Article abstract of DOI:10.1016/j.tet.2011.06.054

The first synthesis of moniliformediquinone has been achieved in which the longest linear sequence is only nine steps. The synthesis proceeds in 23% overall yield from commercially available 2,4,5-trimethoxybenzaldehyde. The key transformations include a

Full text of DOI:10.1016/j.tet.2011.06.054

Tetrahedron 67 (2011) 6166e6172
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Total synthesis of moniliformediquinone and calanquinone A as potent inhibitors
for breast cancer
,
b
Shankar Thangaraj a, Wen-Shing Tsao a, Yi-Wei Luo a, Yean-Jang Lee a , Chia-Fu Chang ,
*
Chun-Cheng Lin b, Biing-Jiun Uang b, Chia-Chun Yu c, Jih-Hwa Guh c, Che-Ming Teng d
a Department of Chemistry, National Changhua University of Education, 1 Ching-Der Rd., Changhua 50058, Taiwan
b Department of Chemistry, National Tsing Hua University, Hsinchu 30013, Taiwan
c School of Pharmacy, College of Medicine, National Taiwan University, Taipei, Taiwan
d Pharmacological Institute, College of Medicine, National Taiwan University, Taipei, Taiwan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 12 May 2011
Received in revised form 17 June 2011
Accepted 21 June 2011
Available online 25 June 2011
The first synthesis of moniliformediquinone has been achieved in which the longest linear sequence is
only nine steps. The synthesis proceeds in 23% overall yield from commercially available 2,4,5-
trimethoxybenzaldehyde. The key transformations include a Pd-catalyzed coupling between a phenyl
triflate and an acetylene, and a TiCl4-mediated cyclization of a benzoquinone intermediate. In addition,
in vitro inhibitory effects of moniliformediquinone, denbinobin, moscatilin, and calanquinone A were
determined to have IC50 values of 0.7, 1.6, 2.5, and 1.5
m
M, respectively.
Ó 2011 Elsevier Ltd. All rights reserved.
Dedicated to my father Ching-Chi Lee on the
occasion of his 92nd birthday
Keywords:
Moniliformediquinone
Calanquinone A
Denbinobin
Moscatilin
TiCl4-mediated
1. Introduction
O
O
O
OMe
cyclization
oxidation
O
Plants of the Dendrobium genus (Orchidaceae) are used in tra-
ditional herbal medicines in Asia, and studies have shown that they
contain a wide variety of medicinal properties.1 In 2001, Chang et al.
collected Dendrobium moniliforme (L.) Sw. in Taiwan and isolated
moniliformediquinone (1) and denbinobin (2) from the stems.1a
The structures of moniliformediquinone and denbinobin were de-
termined to be 1 and 2 by examination of IR, UV, NMR, and
high-resolution mass spectra (Scheme 1). Recently, denbinobins’
biological functions have been intensively studied and found to
involve anticancer, anti-HIV type-1 replication, anti-inflammatory,
and antioxidant effects.2
MeO
MeO
oxidation
OMe
MeO
O
O
OMe
OMe
R1O
3
HO
OR1
denbinobin (2)
moniliformediquinone (1)
Pd-coupling
OTf
OMe
OMe
R2
Pd-coupling
R
+
or Corey-Fuchs
R1O
OR1
MeO
MeO
OMe
OMe
4a R1 = Ac, 4b R1 = Me
6
R = CHO,
7
R = Br, 8 R = I
5a R2 = H, 5b R2 = Br
Perhaps the most direct route to denbinobin (2) would use a
DielseAlder reaction between an appropriate styrene and benzo-
quinone to construct the phenanthraquinone core.3 Unfortunately,
this approach was not successful. Kraus et al.4 have employed
a variety of methodologies to synthesize the denbinobins. For
Scheme 1. Retrosynthetic analysis of moniliformediquinone (1) and denbinobin (2).
instance, the phosphazene base P4-tBu-mediated cyclization of al-
dehydes and toluenes was achieved to give the key phenanthrenes.
A concise synthesis of denbinobin was accomplished by Liou et al.5
via an intramolecular free radical cyclization of
a stilbene
intermediate. However, the starting materials were not readily
available, which complicated synthetic approaches. A synthesis of
* Corresponding author. Tel.: þ886 4 7232105x3511; fax: þ886 4 7211190; e-mail
address: leeyj@cc.ncue.edu.tw (Y.-J. Lee).
0040-4020/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2011.06.054

S. Thangaraj et al. / Tetrahedron 67 (2011) 6166e6172
6167
the related calanquinone has also been described,6 but a synthesis of
moniliformediquinone has not been reported. Consequently, there
is no information available about moniliformediquinone’s behavior
in vitro and in vivo. Since moniliformediquinone is the first natural
product proposed to possess a phenanthradiquinone core, we un-
dertook the synthesis of moniliformediquinone to confirm its
overall structural assignment and to investigate its biological ac-
tivity. We also sought to examine the in vitro/in vivo anti-tumor
activity of denbinobin. In addition, we wanted to examine the
binding affinity of proteineligand complexes by using an automated
docking method7 that has been used for drug lead discovery.
Retrosynthetic analysis (Scheme 1) suggested that the phe-
nanthradiquinone moiety of moniliformediquinone (1) could come
from the free hydroxyl group induced para-oxidation of denbinobin
(2). The phenanthraquinone core structure of 2 was envisioned to
arise from either the sequential intramolecular-cyclization/oxida-
tion or oxidation/cyclization of the corresponding cis-stilbene 3,
which could potentially be attained from a Pd-catalyzed cross
coupling reaction between aryl acetylene 5 and phenyl triflate 4.
Alternatively, the phenanthracenes can be obtained with photo-
cyclization and the oxidation of cis-stilbene 3.3,8 In contrast to
photocyclizing stilbene 3 in the presence of an oxidant, the 9,10-
dihydrophenanthracene moiety can also be formed by direct
intramolecular arylation from a brominated dihydrostilbene de-
rivative.9 Although stilbene 3 has been synthesized by Eling10 and
Kim,11 the synthesis was time-consuming and complicated. To
overcome these technical difficulties and the need for expensive
starting materials we report our study on the synthesis of the key
stilbene 3, which was accessed by a Pd-catalyzed coupling between
the readily available phenyl triflate 4 and substituted benzenes 6e8
(Scheme 1).
OR
1. CBr4/PPh3,
Et3N, CH2Cl2
rt, 93%
MeO
MeO
CHO
OMe
MeO
4, Pd(PPh3)4
OMe
OMe
OR
Et3N, dioxane
100 oC
OMe
2. n-BuLi, THF
-78 oC, 97% MeO
OMe
6
5a
10a R = Ac 67%
10b R = Me 85%
MeO
OMe
Pd-C, H2
Pd(OAc)2
NBS or NIS
CH2Cl2
MeOH,
EtOAc
KOH, DMF
MeO
MeO
145 o
C
OMe
RO
11a R = Ac 90%
11b R = Me 94%
OMe
RO
OR
OR
12a R = Ac 90%
O
OMe
12b R = Me 96%
AgO
X
X
MeO
6 N HNO3
MeO
O
OMe
MeO
OMe
OMe
MeO
15 X = Br 62%
16 X = I 56%
17 X = H 64%
13 X = Br 95%
14 X = I 90%
Scheme 2. Preparation of cis-stilbenes, dihydrostilbenes, and phenethylbenzoquinones.
the cation radical occurs to provide spiro 18 instead of the fused
product. In addition, 18 was determined to be the para-spi-
rodienone not the ortho-spirodienone 25. This assignment was
made from the significant NOE observed between H-20 and CH3-2
in the NOESY spectrum of 18. In contrast, no NOE was observed
between CH3-2 and CH3-5, whereas it was observed in the ortho-
spirodienone 25. In the case of 13e16, we attributed our failure to
achieve the desired fused-cyclization product with Pd-catalysis9 or
AIBN/Bu4SnH5 due to the 2- and 4-methoxy groups and the
electron-withdrawing nature of the conjugated ketones. We be-
lieve that these substituents reduced the reactivity of both the Heck
and free radical reactions. In all cases, these reactions led to the
isolation of only the debrominated product and recovered starting
material. Inspired by work from Kraus et al.,4a we investigated the
possibility of using SnCl4 to effect the cyclization (Scheme 3).
2. Results and discussion
The commercial availability of phloroglucinol prompted us to
prepare the substituted phenyl triflate 4 from this compound. It
was anticipated that 11 could smoothly be prepared as shown in
Scheme 2. A CoreyeFuchs reaction converted benzaldehyde 6 to its
corresponding phenylacetylene 5a in 90% yield. In the case of 4 and
5a, a Sonogashira coupling with Pd(PPh3)4 in Et3N and dioxane at
100 ꢀC under N2 pressure gave acetylene 10b in 85% yield. It is
worth noting that with Pd(PPh3)4 2 equiv of 5a were required to
yield the desired cross-coupled product 10b and to avoid forming
the homocoupled diphenylbutadiyne. With 10b in hand, reduction
with Lindlar’s catalyst12 in MeOH/EtOAc under 50 psi H2 generated
the partially reduced cis-stilbene 11b, albeit with low conversion.
As an alternative, we used a Pd-catalyzed semihydrogenation re-
duction13 in DMF/KOH to effect the highly chemo- and stereo-
selective formation of cis-stilbene 11b in 94% yield. At this point, the
key step of the synthesis was the cyclization reaction of 11b;
NOE
H
OMe
OMe
PhI(OCOCF3)2
BF3•OEt2
68%
O
3'
2'
6
3
2
5
1
6
2
5
3
OMe
1
OMe
OMe
4
MeO
O
or VOF3, 82%
OMe
4
MeO
NOE
MeO
OMe
18
MeO
OMe
MeO
OMe
12b
25
O
O
BF3•OEt2
84% (19 : 20 = 1 : 4)
or TiCl4
89% (19 : 20 = 7 : 1)
O
O
+
MeO
MeO
MeO
OMe
O
O
MeO
20
OMe
MeO
17
MeO
19
TMSI, DDQ
(KSO3)2NO, AcONa
moniliformediquinone (1)
denbinobin (2)
19
hy
/I2,3,8 PhI(OCOCF3)2/BF3$OEt2,14 or
DMF, MeOH
91%
CH2Cl2
73%
however, treatment with
15
VOF3 did not successfully form the desired phenanthrene prod-
uct. Instead, the major product was trans-stilbene, which arose
from cis-trans isomerization to form the more stable product.
Consequently, we sought to manipulate the reduction of 11b in
a separate step. We found that 11a and 11b reacted smoothly with
PdeC/H2 to give 90% and 96% yields of 12a and 12b, respectively. In
addition, dihydrostilbenes 13 and 14 and benzoquinones 15e17
were synthesized to optimize the arylation reactions with Pd-
catalysis or AIBN/Bu4SnH (Scheme 2).
Scheme 3. Synthesis of moniliformediquinone and denbinobin.
We evaluated the intramolecular Lewis acid-mediated cycliza-
tion with 17 and SnCl4 under a range of reaction conditions; how-
ever, these conditions only resulted in decomposition and the
isolation of recovered starting material. When 17 was treated with
BF3$OEt2, it was converted to 1,4-phenanthraquinone 19 and spi-
rodiketone 20 in 17% and 67% yield, respectively. It is interesting to
note that the Michael addition to 20 was regioselective. The
regiochemical assignments of 20 were based on characteristic
spectroscopic signatures and X-ray crystallographic analysis.16 To
our delight, 17 underwent a reaction with TiCl4 to provide the de-
sired product 19 in 78% yield and 20 in 11% yield, respectively. We
With 12b in hand, we next followed the procedures of Kita14 and
Wang.15a The substrate underwent an oxidative biaryl coupling
reaction to give spirodienone 18 in 68% yield. The reaction of an
electron-rich aromatic ring with the reagent PhI(OCOCF3)2/
BF3$EtO2 results in the formation of a cation radical intermediate.14
The subsequent nucleophilic attack by the other aromatic ring on

6168
S. Thangaraj et al. / Tetrahedron 67 (2011) 6166e6172
propose that this reaction involves the kinetic chelation of TiCl4 by
the carbonyl group, followed by Michael addition to the electron-
deficient carbon center in benzoquinone 17, based upon the yield
of 19 was decreased at high concentration and temperature. It is
notable that we could not achieve the cyclization of 17 to 19 with
many other Lewis acids, including SnCl2, BCl3, Sc(OTf)3, ZrCl4, AlCl3,
CeCl3, and ZnCl2. At this point, we do not know the detailed
mechanism of the cyclization reaction with BF3$OEt2 and TiCl4. The
selective demethylation3 and subsequent oxidation of 19 with TMSI
and DDQ afforded denbinobin (2) in 73% yield, which was then
oxidized with Fremy’s salt to afford the natural product, mon-
iliformediquinone (1), in 91% yield. HRMS, 1H, and 13C NMR spectra
of the synthesized products 1 and 2 are in agreement with those
reported for the naturally derived materials.1a It is worth noting
that our synthetic strategy can also be employed in the syntheses of
the naturally occurring moscatilin (21) and calanquinone A (22) in
51% and 14% overall yield, respectively, from the corresponding
benzaldehydes (Fig. 1).
3. Conclusion
In summary, the first total synthesis of moniliformediquinone
(1) has been achieved; the longest linear sequence is only nine
steps. The synthesis proceeds in 23% overall yield from commer-
cially available trimethoxybenzaldehyde. Although the rationale for
the intramolecular Lewis acid-mediated cyclization is unclear,
a concise route to three natural products (2, 21, and 22) has been
accomplished in 25%, 51%, and 14% overall yield, respectively.
Moreover, our results demonstrate the usefulness of automated
docking methods for the discovery of drug leads. Consequently, the
rationally optimized and designed denbinobin dimer (Binding en-
ergy: Ki ꢁ9.47 kcal/mol: 113 nM) was identified as a potentially
important inhibitor of the ERRg receptor. Furthermore, 1, 2, 21, and
22 exhibited strong anti-tumor effects in cancer cells. Large-scale
preparations of these compounds are currently underway, and
their biological activities will be investigated so that their efficacy
as anti-tumor agents can be evaluated.
4. Experimental section
4.1. General details
R2
R1
O
MeO
HO
MeO
Melting points were determined on a Mel Temp II melting point
apparatus and are uncorrected. IR spectra were obtained as KBr
pellets with an FTIR spectrometer. 1H NMR and 13C NMR spectra
were obtained using a 300 MHz spectrometer. COSY, HMQC, and
NOE spectra were provided by a 600 MHz spectrometer. Chemical
OMe
O
MeO
NMe2
OMe
HO
O
OH
OMe
21
Tamoxifen (23) R1 = R2 = H
22
4-OH Tamoxifen R1 = H, R2 = OH
GSK5182 (24) R1 = CH2OH, R2 = OH
shifts are reported in parts per million (d, ppm) using CHCl3 (dH
Fig. 1. The structures of moscatilin (21), calanquinone A (22), and tamoxifens.
7.26) as an internal standard. Solvents were freshly distilled prior to
use from phosphorus pentoxide or CaH2; THF was distilled
from sodium diphenyl ketyl. All reactions were carried out under
Tamoxifen (23) has been used for more than 30 years to treat
breast cancer in women and men, but clinical trials have shown
that it has serious side effects, including blood clots, strokes, uter-
ine cancer, and cataracts.17 Zuercher et al.18 have reported that the
tamoxifen analogue GSK5182 (24) can selectively bind to the or-
a
nitrogen atmosphere unless otherwise stated. Silica gel
(230e400 mesh) was used for chromatography. Organic extracts
were dried over anhydrous MgSO4.
4.1.1. 3,5-Dimethoxyphenyl triflate (4)19. A mixture of 3,5-
dimethoxyphenol (1.00 g, 6.5 mmol) and Et3N (10.0 mL) was dis-
solved in CH2Cl2 (20.0 mL) for 5 min at room temperature, and
followed by addition of (CF3SO2)2O (1.1 mL, 6.5 mmol) dropwise at
ꢁ78 ꢀC. The solution was stirred at ꢁ78 ꢀC for 10 min, and then the
solution was warmed to room temperature. After stirring for
20 min the reaction was quenched with water (10.0 mL), the so-
lution was poured into ice water (50.0 mL) and extracted with
CH2Cl2 (3ꢂ50.0 mL); the combined extracts were washed with
saturated aqueous NaHCO3 (5.0 mL). The organic layer was dried
over Na2SO4, and concentrated in vacuo. The residue was purified
by column chromatography (SiO2, hexane/EtOAc 17/1) to yield 3,5-
dimethoxyphenyl triflate (4) (1.73 g, 93%) as a colorless liquid. 1H
phan estrogen-related receptor ERR
g. We sought to probe the ac-
cessibility of the ERR receptors in MDA-MB-231 and MCF breast
g
tumor cells. To this end, the binding affinity of proteineligand
complexes was first examined by an automated docking method7
(Table 1). The results showed that 1 and 2 bind to the receptor
ERRg, thus, these compounds could be potential inhibitors of breast
cancer similar to GSK5812 (24). Accordingly, the in vitro inhibitory
effects of moniliformediquinone (1), denbinobin (2), moscatilin
(21), and calanquinone A (22) were examined with the SRB method,
and IC50 values of 0.7, 1.6, 2.5, and 1.5
mM were obtained, re-
spectively. In addition, Wu and Lee have reported6 that calanqui-
none A (22), which was isolated from Calanthe arissanensis, showed
potent cytotoxicity (EC50<0.5 mg/mL) against prostate (PC-3 and
DU145) and breast (MCF7) cancer cell lines. It also showed an im-
proved drug resistance profile compared with paclitaxel.
NMR (300 MHz, CDCl3):
NMR (75 MHz, CDCl3):
d
3.78 (s, 6H), 6.41 (s, 2H), 6.44 (s, 1H); 13
55.6, 99.9, 100.2, 118.7 (q, J¼318.8 Hz, CF3),
C
d
Table 1
Compound Ki values and the lowest binding energies from AutoDock Lamarckian Genetic Algorithm
Ligand
Protein (PDB entry) binding energy (kcal/mol)/Ki (mM)
Transcription (ERR
g) (2EWP)
Nuclear receptor (ER
a
) (3ERT)
Gene regulation (1KV6)
Mg2þ transport (Tubulin) (2HXF)
Taxol
Morusin
1
2
21
22
GSK5182
4-OH Tamoxifen
Tamoxifen
23.21/NA
164.73/NA
ꢁ4.68/371.1
ꢁ5.98/41.3
ꢁ6.36/21.89
ꢁ4.72/344.4
ꢁ6.52/16.56
ꢁ2.9/7530
189.74/NA
ꢁ4.05/1070
ꢁ6.90/8.77
ꢁ7.11/6.16
ꢁ5.26/140
ꢁ6.75/11.34
2.41/NA
109.13/NA
ꢁ5.55/85.66
ꢁ6.23/27.24
ꢁ6.74/11.46
ꢁ6.58/15.14
ꢁ7.28/4.28
ꢁ4.0/1170
ꢁ4.13/937
ꢁ6.07/35.69
ꢁ7.63/2.57
ꢁ7.48/3.28
ꢁ6.93/8.38
ꢁ7.07/6.52
ꢁ7.58/2.77
ꢁ7.04/6.88
ꢁ7.11/6.09
ꢁ7.40/3.79
ꢁ4.02/1130
ꢁ4.3/703.33
ꢁ1.69/NA
ꢁ3.08/5500
NA: unavailable.

S. Thangaraj et al. / Tetrahedron 67 (2011) 6166e6172
6169
150.6, 161.4, HRMS (EI) calcd for C9H9O5F3S1 (Mþ) 286.0123, found
286.0113. The spectra of synthetic 4 are agreement with those
reported by Subramanian et al.19
After cooling, the reaction mixture was filtered through Celite. The
filtrate was concentrated, and then purified by column chroma-
tography (SiO2, hexane/EtOAc 5/1) to give 10b (0.84 g, 85%) as light
yellow solid: mp 122e124 ꢀC; IR (KBr) nmax: 3003, 2966, 2940,
2842, 2200, 1586, 1362, 1210, 1066, 826 cmꢁ1, 1H NMR (300 MHz,
4.1.2. 1,1-Dibromo-2-(2,4,5-trimethoxyphenyl)ethene (9)20a. A sus-
pension of CBr4 (6.76 g, 20.0 mmol) in CH2Cl2 (15.0 mL) was cooled
to 0 ꢀC, and a solution of PPh3 (10.7 g, 40.0 mmol) in CH2Cl2
(15.0 mL) was added to the reaction mixture. After the mixture was
stirred for 30 min at 0 ꢀC, the color of the solution changed from
pale yellow to orange. Into the reaction mixture was added a solu-
tion of 2,4,5-trimethoxybenzaldehyde (1.96 g, 10.0 mmol) in CH2Cl2
(5.0 mL) and Et3N (11.4 mL, 80.0 mmol), and then the heteroge-
neous mixture was stirred for 8 h at room temperature. The
resulting product was poured into ice water (50.0 mL), followed by
extraction with CH2Cl2 (3ꢂ50.0 mL). After washing with saturated
aqueous NaHCO3 (5.0 mL), the combined organic layers were dried
over Na2SO4 and filtered. The filtrate was reduced in vacuo, and the
crude product was subjected to column chromatography (SiO2,
hexane/EtOAc 9/1) to provide the dibromide 9 (3.25 g, 93%) as
a pale yellow solid. Characterization of 9: mp 65e66 ꢀC; 1H NMR
CDCl3)
d 3.75 (s, 6H), 3.81 (s, 3H), 3.86 (s, 3H), 3.87 (s, 3H), 6.39
(t, J¼2.4 Hz, 1H), 6.46 (s, 1H), 6.67 (d, J¼2.4 Hz, 2H), 6.96 (s, 1H); 13C
NMR (75 MHz, CDCl3)
d 55.3, 55.9, 56.3, 56.7, 85.4, 92.1, 97.1, 101.4,
103.0, 109.1, 115.8, 124.9, 142.8, 150.4, 155.3, 160.4, HRMS (EI) calcd
for C19H20O5 (Mþ) 328.1311, found 328.1312.
4.1.6. cis-1-(3,5-Dimethoxy)-2-(2,4,5-trimethoxy)stilbene (11b)21. Acc-
ording to Hua’s procedure, into a sealable tube were introduced 10b
(0.50 g, 1.5 mmol), KOH (0.13 g, 2.3 mmol), Pd(OAc)2 (6.8 mg, 2 mol %),
and DMF (5.0 mL) under a N2 atmosphere. The tube was capped and
the mixture was heated at 145 ꢀC with stirring for 3 h. After cooling,
the crude reaction mixture was diluted with CH2Cl2 (20.0 mL) and
cyclohexane (10.0 mL), the mixture was filtered and the filtrate
evaporated in vacuo. The residue was subjected to flash chromatog-
raphy (SiO2, hexane/EtOAc 5/1) to provide the cis-stilbene 11b (0.46 g,
94%) as light yellow solid: mp 93e94 ꢀC; 1H NMR (300 MHz, CDCl3)
(300 MHz, CDCl3)
d
3.78 (s, 3H), 3.82 (s, 3H), 3.87 (s, 3H), 6.44 (s,
55.9, 56.3,
1H), 7.36 (s, 1H), 7.56 (s, 1H); 13C NMR (75 MHz, CDCl3)
d
d
3.45 (s, 3H), 3.64 (s, 6H), 3.80 (s, 3H), 3.86 (s, 3H), 6.27 (t, J¼2.4 Hz,
56.4, 87.3, 96.5, 111.9, 115.4, 131.8, 142.3, 150.1, 151.6. The spectra of
1H), 6.44 (d, J¼2.4 Hz, 2H), 6.47 (d, J¼12.3 Hz, 1H), 6.48 (s, 1H), 6.65 (d,
synthetic 9 are in agreement with those reported by Konno et al.20a
J¼12.3 Hz, 1H), 6.77 (s, 1H); 13C NMR (75 MHz, CDCl3)
d 55.2, 56.0,
56.1, 56.5, 97.2, 99.4, 106.7, 113.4, 117.2, 125.3, 128.7, 139.5, 142.4, 149.2,
151.7, 160.5, HRMS (EI) calcd for C19H22O5 (Mþ) 330.1467, found
330.1462. The spectra of synthetic 11b are in agreement with those
reported by Kim et al.21
4.1.3. 2,4,5-Trimethoxyphenylacetylene (5a)20. A solution of 1,1-
dibromo-2-(2,4,5-trimethoxyphenyl)ethane (9) (0.98 g, 2.8 mmol)
in THF (15.0 mL) was cooled at ꢁ78 ꢀC for 30 min, and n-BuLi
(3.6 mL, 1.6 M in hexane) was added with dropwise at ꢁ78 ꢀC. The
mixture was stirred at ꢁ78 ꢀC for 30 min, and allowed to warm to
room temperature. Then the resulting solution was quenched with
saturated aqueous NH4Cl (5.0 mL), followed by extraction with
EtOAc (3ꢂ50.0 mL). The organic layers were dried and concen-
trated, and the residue was subjected to column chromatography
(SiO2, hexane/EtOAc 9/1) to obtain 5a (0.52 g, 97%) as a white solid:
4.1.7. cis-1-(3,5-Diacetoxy)-2-(2,4,5-trimethoxy)stilbene (11a). This
compound was prepared from 10a (0.59 g, 1.5 mmol), KOH (0.13 g,
2.3 mmol), and Pd(OAc)2 (6.8 mg, 2 mol %) in DMF (5.0 mL)
according to the preceding procedure. Compound 11a was isolated
in 90% yield as a colorless oil; 1H NMR (300 MHz, CDCl3)
d 2.08 (s,
6H), 3.41 (s, 3H), 3.66 (s, 3H), 3.75 (s, 3H), 6.32 (d, J¼12.3 Hz, 1H),
mp 115e117 ꢀC; 1H NMR (300 MHz, CDCl3)
3H), 3.84 (s, 3H), 3.86 (s, 3H), 6.44 (s, 1H), 6.90 (s, 1H); 13C NMR
(75 MHz, CDCl3) 55.9, 56.3, 56.6, 79.8, 80.2, 96.8, 101.8, 116.3,
d 3.22 (s, 1H), 3.78 (s,
6.35 (s, 1H), 6.27 (t, J¼1.5 Hz, 1H), 6.57 (s, 1H), 6.59 (d, J¼12.3 Hz,
1H), 6.75 (d, J¼1.5 Hz, 2H); 13C NMR (75 MHz, CDCl3)
d 20.0, 55.2,
d
55.3, 55.6, 97.7, 113.4, 114.4, 115.8, 119.1, 125.9, 126.8, 140.2, 142.8,
142.6, 150.7, 156.0. The spectra of synthetic 5a are in agreement
150.3, 151.3, 152.1, 168.5, 13C NMR (75 MHz, (CD3)2CO)
d 21.0, 55.8,
with those reported by Konno and Brooks.20
55.9, 56.3, 97.1, 112.6, 114.0, 116.3, 119.2, 126.5, 126.6, 140.1, 142.5,
149.3, 150.8, 151.7, 168.9, HRMS (EI) calcd for C21H22O7 (Mþ)
386.1366, found 386.1370.
4.1.4. 3,5-Diacetoxy-1-[2-(2,4,5-trimethoxyphenyl)ethynyl]benzene
(10a). To
a mixture of 3,5-diacetoxyphenyl triflate (1.04 g,
3.0 mmol), acetylene 5a (0.58 g, 3.0 mmol), and Pd(PPh3)4 (0.18 g,
5 mol %) in anhydrous dioxane (30.0 mL) was added Et3N (5.0 mL)
at room temperature under nitrogen; the resulting mixture was
heated to reflux at 110 ꢀC for 6 h. After cooling, the reaction mixture
was filtered through Celite. The filtrate was concentrated, and then
purified by column chromatography (SiO2, hexane/EtOAc 5/1) to
afford 10a (0.70 g, 67%) as light yellow solid: mp 118e120 ꢀC; IR
(KBr) nmax: 3073, 3012, 2976, 2938, 2848, 2206, 1767, 1602, 1523,
4.1.8. 3,5-Diacetoxy-1-[2-(2,4,5-trimethoxyphenyl)ethyl]benzene
(12a). The mixture of 10a (0.13 g, 0.3 mmol) and palladium on
carbon (10%, 0.074 g) in MeOH (3.0 mL) and EtOAc (3.0 mL) was
placed with shaking in Parr-Shaker equipment under 50 psi of H2.
After shaking for 8 h at ambient temperature, the reaction mixture
was filtered and evaporated to give the crude product, which was
subjected to column chromatography (SiO2, hexane/EtOAc 5/1) to
obtain 12a (0.12 g, 90%) as a colorless oil; 1H NMR (300 MHz, CDCl3)
1357, 1210, 1185, 1066, 895, 863 cmꢁ1
,
1H NMR (300 MHz, CDCl3)
d
2.24 (s, 6H), 2.81 (br s, 4H), 3.74 (s, 3H), 3.75 (s, 3H), 3.83 (s, 3H),
6.49 (s, 1H), 6.56 (s, 1H), 6.71 (s, 1H), 6.77 (s, 2H); 13C NMR (75 MHz,
CDCl3) 21.0, 31.3, 36.3, 56.1, 56.1, 56.5, 97.6, 112.7, 114.1, 119.0,
d
2.20 (s, 6H), 3.77 (s, 3H), 3.81 (s, 3H), 3.82 (s, 3H), 6.42 (s, 1H), 6.81
(t, J¼2.1 Hz, 1H), 6.89 (s, 1H), 7.10 (d, J¼2.1 Hz, 2H); 13C NMR
d
(75 MHz, CDCl3)
d
20.7, 55.7, 56.1, 56.4, 87.6, 90.2, 96.8, 102.2, 115.1,
120.8, 142.6, 144.7, 147.8, 150.7, 151.4, 169.0, HRMS (EI) calcd for
115.5, 121.6, 125.4, 142.6, 150.5, 150.5, 155.4, 168.6; 13C NMR
(75 MHz, (CD3)2CO) 20.0, 55.4, 55.9, 56.0, 88.2, 89.8, 97.7, 102.0,
C21H24O7 (Mþ) 388.1522, found 388.1529.
d
115.8, 116.5, 121.6, 125.4, 143.2, 151.4, 151.7, 156.0, 168.5, HRMS (EI)
4.1.9. 1-(3,5-Dimethoxyphenyl)-2-(2,4,5-trimethoxyphenyl)ethane
(12b). Compound 11b (1.00 g, 3.0 mmol) was dissolved in MeOH
(10.0 mL) and EtOAc (10.0 mL). Palladium on carbon (10%, 0.35 g)
was added and the reaction was placed with shaking in Parr-Shaker
equipment under 50 psi of H2. After shaking for 8 h at ambient
temperature, the reaction mixture was filtered and evaporated to
give the crude product, which was subjected to column chroma-
tography (SiO2, hexane/EtOAc 5/1) to provide 12b (0.96 g, 96%) as
white solid: mp 84.1e86.1 ꢀC; IR (KBr) nmax: 3013, 2972, 2938, 2836,
calcd for C21H20O7 (Mþ) 384.1209, found 384.1213.
4.1.5. 1-(3,5-Dimethoxyphenyl)-2-(2,4,5-trimethoxyphenyl) acety-
lene (10b). To a mixture of 3,5-dimethoxyphenyl triflate (0.86 g,
3.0 mmol), acetylene 5a (1.15 g, 6.0 mmol), Pd(PPh3)2Cl2 (0.21 g,
10 mol %), and CuI (0.11 g, 20 mol %) in anhydrous dioxane (30.0 mL)
was immediately added Et3N (5.0 mL) at room temperature under
nitrogen, and the mixture was heated to reflux at 100 ꢀC for 8 h.

6170
S. Thangaraj et al. / Tetrahedron 67 (2011) 6166e6172
1603, 1523, 1462, 1155, 1046, 852 cmꢁ1, 1H NMR (300 MHz, CDCl3)
2.78e2.80 (m, 4H), 3.75 (s, 6H), 3.77 (s, 3H), 3.78 (s, 3H), 3.86 (s,
3H), 6.29 (t, J¼2.4 Hz,1H), 6.34 (d, J¼2.4 Hz, 2H), 6.51 (s,1H), 6.61 (s,
1H); 13C NMR (75 MHz, CDCl3)
31.7, 36.9, 55.2, 56.2, 56.4, 56.6,
97.7, 97.9, 106.6, 114.3, 121.7, 142.7, 144.7, 147.8, 151.5, 160.6, HRMS
(EI) calcd for C19H24O5 (Mþ) 332.1624, found 332.1626.
182.1, 187.2, HRMS (EI) calcd for C17H17BrO5 (Mþ) 380.0259, found
380.0264.
d
d
4.1.14. 5-Methoxy-2-(2-iodo-3,5-dimethoxyphenethyl)-1,4-benzoqui-
none (16). According to the previous procedure, compound 16 was
isolated in 56% yield as an orange solid: mp 119e120 ꢀC; 1H NMR
(300 MHz, CDCl3)
d
2.72 (t, J¼7.5 Hz, 2H), 2.95 (t, J¼7.5 Hz, 2H), 3.78
4.1.10. 1-(2-Bromo-3,5-dimethoxyphenyl)-2-(2,4,5-trimethoxyphenyl)
ethane (13). NBS (29 mg, 1.6ꢂ10ꢁ1 mmol) was added in one portion
to a well-stirred solution of 12b (50 mg, 1.5ꢂ10ꢁ1 mmol) in CH2Cl2
(5.0 mL) at room temperature, and the mixture was stirred for 8 h.
The resulting solution was quenched by adding saturated aqueous
Na2SO3 (2.0 mL), and extracted with CH2Cl2 (3ꢂ5.0 mL). After re-
moval of solvent, the residue was subjected to column chroma-
tography (SiO2, hexane/ether 5/1) to give 13 (58 mg, 95%) as a light
yellow solid: mp 100e102 ꢀC; IR (KBr) nmax: 3002, 2967, 2939, 2849,
(s, 3H), 3.82 (s, 3H), 3.84 (s, 3H), 5.94 (s, 1H), 6.29 (d, J¼2.7 Hz, 1H),
6.42 (d, J¼2.7 Hz, 1H), 6.48 (s, 1H); 13C NMR (75 MHz, CDCl3)
d 29.6,
39.6, 55.4, 56.2, 56.4, 81.6, 96.9, 106.6, 107.7, 131.0, 145.0, 148.9, 158.5,
158.9, 161.0, 182.1, 187.2, HRMS (EI) calcd for C17H17IO5 (Mþ)
428.0121, found 428.0126.
4.1.15. 50,70-Dimethoxy-spiro(2,5-dimethoxy-cyclohexa-2,5-dien-4-
one-1,10-indan) (18). Method A: To a solution of 12b (0.10 g,
3.0ꢂ10ꢁ1 mmol) and (CF3CO)2O (1.0
mL) in anhydrous CH2Cl2
1592, 1524, 1468, 1363, 1335, 1210, 1205, 1083, 860, 816 cmꢁ1
NMR (300 MHz, CDCl3)
,
1H
(3.0 mL) was added VOF3 (82 mg, 6.6ꢂ10ꢁ1 mmol) at 0 ꢀC under N2,
and then a solution of CF3CO2H (0.2 mL) in CH2Cl2 (2.0 mL) was
added dropwise slowly. After stirring at 0 ꢀC for 4 h, the solution
was diluted with CH2Cl2 (5.0 mL) and washed with cold 1 M
aqueous citric acid (2ꢂ2.0 mL), 3 M NH4OH (3ꢂ3.0 mL), and brine
(2ꢂ4.0 mL). The resulting mixture was dried over Na2SO4 and fil-
tered; the filtrate was concentrated in vacuo, and then the residue
was subjected to column chromatography (SiO2, hexane/EtOAc 4/1)
to give 18 (78 mg, 82%) as an orange solid: mp 188e190 ꢀC; IR (KBr)
nmax: 3073, 2976, 2940, 2842, 1675, 1636, 1597, 1457, 1369, 1195,
d
2.83 (t, J¼4.5 Hz, 2H), 2.94 (t, J¼4.5 Hz,
2H), 3.73 (s, 3H), 3.78 (s, 3H), 3.79 (s, 3H), 3.84 (s, 3H), 3.86 (s, 3H),
6.32 (d, J¼2.4 Hz, 1H), 6.33 (d, J¼2.4 Hz, 1H), 6.51 (s, 1H), 6.63 (s,
1H); 13C NMR (75 MHz, CDCl3)
d 30.0, 37.3, 55.4, 56.1, 56.2, 56.4,
56.6, 97.4, 97.8, 104.7, 106.8, 114.3, 121.4, 142.7, 143.4, 147.8, 151.6,
156.6, 159.3, HRMS (EI) calcd for C19H23BrO5 (Mþ) 410.0729, found
410.0723.
4.1.11. 1-(2-Iodo-3,5-dimethoxyphenyl)-2-(2,4,5-trimethoxyphenyl)
ethane (14). This compound was prepared as a white solid from
12b (50 mg, 1.5ꢂ10ꢁ1 mmol), NIS (37 mg, 1.6ꢂ10ꢁ1 mmol), and one
drop CF3CO2H in CH3CN (5.0 mL) at 50 ꢀC according to the pre-
ceding 13 procedure. Compound 14 was isolated in 90% yield as
a solid: mp 94e96 ꢀC; IR (KBr) nmax: 3003, 2995, 2937, 2848, 1590,
1081, 876, 842, 826 cmꢁ1, 1H NMR (600 MHz, CDCl3)
d 2.11e2.15 (m,
1H), 2.55e2.60 (m, 1H), 3.06e3.09 (m, 2H), 3.56 (s, 3H), 3.58 (s, 3H),
3.63 (s, 3H), 3.76 (s, 3H), 5.48 (s, 1H), 5.65 (s, 1H), 6.20 (d, J¼1.2 Hz,
1H), 6.40 (d, J¼1.2 Hz, 1H); 13C NMR (150 MHz, CDCl3):
d 31.9, 39.5,
53.6, 54.9, 55.1, 55.4, 56.0, 97.0, 100.8, 101.5, 115.5, 123.2, 146.8,
148.8, 156.6, 161.6, 178.7, 183.3, HRMS (EI) calcd for C18H20O5 (Mþ)
316.1311, found 316.1318.
1523, 1464, 1319, 1205, 1157, 1080, 1034, 938, 850, 816 cmꢁ1
,
1H
NMR (300 MHz, CDCl3)
2H), 3.73 (s, 3H), 3.77 (s, 3H), 3.79 (s, 3H), 3.82 (s, 3H), 3.88 (s, 3H),
d
2.81 (t, J¼4.5 Hz, 2H), 2.96 (t, J¼4.5 Hz,
Method B: To a solution of 12b (0.10 g, 3.0ꢂ10ꢁ1 mmol) in an-
hydrous CH2Cl2 (1.0 mL) was added a solution of PhI(OCOCF3)2
6.27 (d, J¼2.7 Hz, 1H), 6.37 (d, J¼2.7 Hz, 1H), 6.51 (s, 1H), 6.67 (s,
1H); 13C NMR (75 MHz, CDCl3)
d
30.3, 41.8, 55.4, 56.2, 56.4, 56.4,
(0.13 g, 3.0ꢂ10ꢁ1 mmol) and BF3$Et2O (55
m
L, 6.2ꢂ10ꢁ1 mmol) in
56.6, 81.7, 96.5, 97.8, 106.7, 114.3, 121.3, 142.7, 146.8, 147.8, 151.6,
158.7, 160.7, HRMS (EI) calcd for C19H23IO5 (Mþ) 458.0590, found
458.0590.
CH2Cl2 (1.0 mL) dropwise at ꢁ40 ꢀC under N2. The reaction mixture
was then stirred at ꢁ40 ꢀC for 1 h and then evaporated in vacuo.
After removal of solvent, the residue was subjected to column
chromatography (SiO2, hexane/EtOAc 4/1) to give 18 (65 mg, 68%)
as an orange solid: mp 188e190 ꢀC.
4.1.12. 5-Methoxy-2-(3,5-dimethoxyphenethyl)-1,4-benzoquinone
(17). To a mixture of 12b (0.41 g, 1.2 mmol) and AgO (0.59 g,
4.8 mmol) in dioxane (25 mL) was added 6 N HNO3 (1.4 mL)
dropwise at room temperature. The suspension was stirred for
20 min, and the reaction was quenched with saturated aqueous
NaHCO3 (10.0 mL). After extraction with EtOAc (3ꢂ20.0 mL), the
organic layers were combined and concentrated and the residue
was subjected to column chromatography (SiO2, hexane/EtOAc 4/
1) to give 17 (0.24 g, 64%) as orange solid: mp 149.5e150.4 ꢀC; IR
(KBr) nmax: 3073, 2982, 2942, 2840, 1675, 1659, 1600, 1472, 1206,
4.1.16. 9,10-Dihydro-3,5,7-trimethoxy-1,4-phenanthrenedione (19)3.
To a solution of 17 (0.23 g, 7.6ꢂ10ꢁ1 mmol) in CH2Cl2 (10.0 mL) was
added TiCl4 (0.10 mL, 9.1ꢂ10ꢁ1 mmol) dropwise at 0 ꢀC. The reaction
was monitored by TLC and quenched after ca. 4 h by treatment with
saturated aqueous NaHCO3 (5.0 mL). The solvent was removed and
the crude product was subjected to flash chromatography (SiO2,
hexane/EtOAc 3/1) to afford 19 (0.18 g, 78%) as a red solid and 20
(25 mg, 11%). Compound 19: mp 194e195 ꢀC; 1H NMR (300 MHz,
1161, 981, 859 cmꢁ1
,
1H NMR (300 MHz, CDCl3)
d
2.71e2.72 (m,
CDCl3)
d
2.56e2.66 (m, 4H), 3.76 (s, 3H), 3.81 (s, 3H), 3.82 (s, 3H), 5.84
20.4, 28.8, 55.4,
4H), 3.74 (s, 6H), 3.79 (s, 3H), 5.90 (s, 1H), 6.28 (t, J¼2.4 Hz, 1H),
(s, 1H), 6.34 (br s, 2H); 13C NMR (75 MHz, CDCl3):
d
6.30 (d, J¼2.4 Hz, 2H), 6.41 (s, 1H); 13C NMR (75 MHz, CDCl3)
55.8, 56.2, 97.6, 105.3, 105.8, 112.1, 139.0, 140.6, 142.5, 158.5, 159.9,
162.2, 180.0, 185.8, HRMS (EI) calcd for C17H16O5 (Mþ) 300.0998,
found 300.1005.
d
30.6, 34.3, 55.2, 56.2, 98.2, 106.4, 107.7, 130.9, 142.6, 149.2, 158.5,
160.8, 182.1, 187.2, HRMS (EI) calcd for C17H18O5 (Mþ) 302.1154,
found 302.1146.
4.1.17. 50,70-Dimethoxy-spiro(4-methoxy-3-cyclohexen-2,5-dione-
4.1.13. 5-Methoxy-2-(2-bromo-3,5-dimethoxyphenethyl)-1,4-
benzoquinone (15). This compound was prepared from 13 (0.19 g,
4.6ꢂ10ꢁ1 mmol) and AgO (0.22 g, 1.8 mmol) in dioxane (10 mL) at
room temperature according to the preceding procedure. Com-
pound 15 was isolated in 62% yield as an orange solid: mp
1,10-indan) (20). Into a solution of 17 (0.23 g, 7.6ꢂ10ꢁ1 mmol) in
CH2Cl2 (10.0 mL) was introduced BF3$Et2O (70
m
L, 7.6ꢂ10ꢁ1 mmol)
dropwise at 0 ꢀC, and the reaction was monitored by TLC until 17
was consumed (ca. 2 h). The reaction mixture was then evaporated
in vacuo, and the residue subjected to column chromatography
(SiO2, hexane/EtOAc 3/1) to provide 19 (39 mg, 17%) and 20 (0.15 g,
67%) as an orange solid. Compound 20: mp 145e146 ꢀC; IR (KBr)
nmax: 3013, 2982, 2942, 2842, 1714, 1659, 1603, 1462, 1361, 1222,
95e96 ꢀC; 1H NMR (300 MHz, CDCl3)
d
2.60 (t, J¼7.5 Hz, 2H), 2.80 (t,
J¼7.5 Hz, 2H), 3.63 (s, 3H), 3.68 (s, 3H), 3.71 (s, 3H), 5.79 (s, 1H), 6.22
(s, 2H), 6.29 (s, 1H); 13C NMR (75 MHz, CDCl3)
29.4, 35.1, 55.5, 56.1,
56.2, 97.8, 104.7, 106.7, 107.7, 131.0, 141.6, 148.9, 156.8, 158.5, 159.6,
d
1151, 1083, 839 cmꢁ1 1H NMR (300 MHz, CDCl3)
, d 1.95e2.02 (m,

S. Thangaraj et al. / Tetrahedron 67 (2011) 6166e6172
6171
1H), 2.40e2.49 (m, 1H), 2.81 (d, J¼16.5 Hz, 1H), 2.87e2.95 (m, 1H),
3.04e3.36 (m, 1H), 3.33 (d, J¼16.5 Hz, 1H), 3.65 (s, 3H), 3.75 (s,
3H), 3.79 (s, 3H), 5.96 (s, 1H), 6.24 (d, J¼1.8 Hz, 1H), 6.37 (d,
to make 12. Compound 21 was isolated in 51% yield over five steps as
a white solid: mp 84e86 ꢀC (lit.22 84 ꢀC); 1H NMR (300 MHz, CDCl3)
d
2.80 (br s, 4H), 3.78 (s, 3H), 3.82 (s, 6H), 5.39 (br s, OH), 5.50 (br s,
J¼1.8 Hz, 1H); 13C NMR (75 MHz, CDCl3):
d
31.7, 38.6, 47.3, 54.9,
OH), 6.34 (s, 2H), 6.59 (d, J¼1.8 Hz, 1H), 6.66 (dd, J¼8.1,1.8 Hz, 1H),
55.4, 56.2, 59.0, 97.2, 101.1, 111.7, 123.4, 147.0, 156.2, 161.8, 162.5,
192.2, 199.7, HRMS (EI) calcd for C17H18O5 (Mþ) 302.1154, found
302.1147.
6.82 (d, J¼8.1 Hz, 1H); 13C NMR (75 MHz, CDCl3):
d 37.9, 38.4, 55.8,
56.2, 105.1, 111.2, 114.1, 121.0, 132.7, 132.8, 133.6, 143.7, 146.2, 146.8,
HRMS (EI) calcd for C17H20O5 (Mþ) 304.1311, found 304.1319. Melting
point and the spectra of synthetic 21 are in agreement with those
reported for the natural product.22
4.1.18. 5-Hydroxy-3,7-dimethoxy-1,4-phenanthrenedione, denbino-
bin (2). A solution of 19 (0.10 g, 3.3ꢂ10ꢁ1 mmol) in CH2Cl2 (12.0 mL)
wasstirred atroomtemperature, followedbyadditionof timethylsilyl
4.1.21. 5-Hydroxy-3,6,7-trimethoxy-1,4-phenanthrenedione, calanq-
uinone A (22). According to the developed preparation of 1, com-
pound 22 was prepared from 2,4,5-trimethoxybenzaldehyde in 14%
yield over eight steps as a yellow solid: mp 154e155 ꢀC; 1H NMR
iodide (TMSI, 95
m
L, 6.6ꢂ10ꢁ1 mmol) dropwise. The resulting mixture
was stirred at room temperature for 5 h, and quenched by treatment
with MeOH (4.0 mL). After the reaction mixture was concentrated in
vacuo, the residue was subjected to flash chromatography (SiO2,
hexane/EtOAc 3/1) to give 9,10-dihydro-5-hydroxy-3,7-dimethoxy-
1,4-phenanthrenedione(95mg, 92%)asablack solid:mp162e164 ꢀC;
IR (KBr) nmax: 3369, 3065, 3020, 2966, 2920, 2849, 1636, 1612, 1553,
1440,1305,1264,1127,1066, 843, 826 cmꢁ1,1HNMR(300 MHz,CDCl3)
(300 MHz, CDCl3)
6.85 (s, 1H), 8.04 (d, J¼8.7 Hz, 1H), 8.10 (d, J¼8.7 Hz, 1H), 10.73 (br s,
1H); 13C NMR (75 MHz, CDCl3):
55.9, 56.9, 60.8, 101.1, 107.2, 118.5,
d 3.96 (s, 3H), 4.02 (s, 3H), 4.03 (s, 3H), 6.15 (s,1H),
d
121.7, 128.1, 132.8, 134.8, 136.9, 140.1, 148.1, 155.0, 161.5, 184.4, 186.0,
HRMS (EI) calcd for C17H14O6 (Mþ) 314.0790, found 314.0788. The
spectra of synthetic 22 are in agreement with those reported for the
natural product.6
d
2.66 (br s, 4H), 3.80 (s, 3H), 3.88 (s, 3H), 5.99 (s,1H), 6.40 (d, J¼2.7 Hz,
1H), 6.45 (d, J¼2.7 Hz, 1H), 8.85 (br s, 1H); 13C NMR (75 MHz, CDCl3):
d
21.4, 28.8, 55.3, 56.6, 102.8, 106.7, 108.4, 110.2, 137.0, 141.9, 144.6,
156.6, 158.8, 162.2, 185.3, 185.6, HRMS (EI) calcd for C16H12O5 (Mþ)
284.0685, found 284.0681, HRMS (EI) calcd for C16H14O5 (Mþ)
286.0841, found 286.0833. The spectra are in agreement with those
reported for the synthetic product.3
4.2. Computational methods
4.2.1. Preparation of ligand and protein complexes input structur-
es. First, protein structures were extracted from the Protein Data-
bank (pdb) file (containing only non-hydrogen atoms). The
protein’s pdb files were edited with AutoDockTools-1.5.1 (on
a Linux system) to protonate polar hydrogen atoms, add Kollman
charges, and saved as a pdbqt file. Furthermore, ligand structures
were optimized by Chem 3D Ultra version 8.0 (Run MOPAC mini-
mize energy to minimum rms Gradient 0.001) to get the structure
(pdb file). Alternatively, the X-ray structure of morusin was
extracted from Cambridge Chemical Database (pdb file). The ligand
file was input into AutoDockTools-1.5.1 program, detected the root
of molecule, and output as a pdbqt file as well.
A mixture of the preceding synthetic hydroxyphenanthrenedione
(95 mg, 3.3ꢂ10ꢁ1 mmol) and DDQ (60 mg, 2.6ꢂ10ꢁ1 mmol) in an-
hydrous toluene (5.0 mL) was heated to reflux at 120 ꢀC under N2, and
the reaction was monitored by TLC and quenched after ca. 12 h. After
cooling, the resulting mixture was filtered and the solvent was re-
moved, followed purification by column chromatography (SiO2,
hexane/EtOAc 5/1) to achieve 2 (75 mg, 80%) as gray solid: mp
215e217 ꢀC; IR (KBr) nmax: 3446, 3066, 2966, 2943, 2841, 1675, 1627,
1586, 1510, 1422, 1330, 1240, 1170, 1083, 852 cmꢁ1,1H NMR (300 MHz,
CDCl3)
6.91 (d, J¼2.7 Hz, 1H), 8.04 (d, J¼8.7 Hz, 1H), 8.10 (d, J¼8.7 Hz, 1H),
11.01 (br s, 1H); 13C NMR (75 MHz, CDCl3):
55.5, 56.9, 101.7, 107.3,
d
3.92 (s, 3H), 3.96 (s, 3H), 6.14 (s, 1H), 6.80 (d, J¼2.7 Hz, 1H),
d
108.6, 117.1, 122.6, 128.5, 132.3, 137.3, 139.9, 156.3, 160.7, 161.2, 184.3,
186.4, HRMS (EI) calcd for C16H12O5 (Mþ) 284.0685, found 284.0681.
The spectra of synthetic 2 are in agreement with those reported for
the natural product.1a,5
4.2.2. Grid and docking studies. To predict the energetically favor-
able positions of ligand molecules in the interior structure of the
3
ꢀ
protein target, a rectangular grid box of 21.75ꢂ21.75ꢂ21.75 A with
ꢀ
grid points separated by 0.333 A, it was centered on the midpoint of
the ligand binding pocket. It was essential to modify the AutoGrid
program to enable docking in the presence of metals and hetero-
atoms. Subsequently, automated docking studies were performed
with Lamarckian Genetic Algorithm to provide the binding energies
and conformations of protein-ligand complexes.
4.1.19. 2,6-Dimethoxy-1,4,5,8-phenanthrenetetrone, moniliformediqui-
none (1). To a solution of 2 (0.10 g, 3.5ꢂ10ꢁ1 mmol) in MeOH and
DMF (10.0 mL; v/v 1/1) was added a solution of freshly prepared
(KSO3)2NO (0.33 g, 1.2 mmol) in water (6.0 mL) and 1 M aqueous
NaOAc solution (0.18 mL) at room temperature. After being stirred
for 12 h at room temperature, the reaction mixture was extracted
with ether (3ꢂ10.0 mL). The combined organic phase was washed
with water (2.0 mL) and brine (2.0 mL), dried over anhydrous
Na2SO4, and concentrated in vacuo. The residue was subjected to
flash chromatography (SiO2, hexane/EtOAc 5/1) to provide 1 (95 mg,
91%) as yellow solid: mp 258e260 ꢀC; IR (KBr) nmax: 3082, 2986,
2946, 2843, 1688, 1652, 1610, 1458, 1354, 1232, 1139, 1085, 872,
4.3. Sulforhodamine B (SRB) assays
Cells were seeded in 96-well plates in medium with 5% fetal
bovine serum (FBS). After 24 h, cells were fixed with 10% tri-
chloroacetic acid (TCA) to represent cell population at the time of
compound addition (T0). After additional incubation of dimethyl
sulfoxide (DMSO) or test compound for 48 h, cells were fixed with
10% TCA and sulforhodamine (SRB) at 0.4% (w/v) in 1% acetic acid
was added to stain cells. Unbound SRB was washed out by 1% acetic
acid and SRB-bound cells were solubilized with 10 mM Trizma base.
The absorbance was read at a wavelength of 515 nm. Using the
following absorbance measurements, such as time zero (T0), control
growth (C), and cell growth in the presence of the compound (Tx),
the percentage growth was calculated at each of the compound
concentrations levels. Percentage growth inhibition was calculated
as: 100ꢁ[(TxꢁT0)/(CꢁT0)]ꢂ100. Growth inhibition of 50% (IC50) is
determined at the compound concentration which results in 50%
822 cmꢁ1, 1H NMR (300 MHz, CDCl3)
d 3.92 (s, 3H), 3.95 (s, 3H), 6.17
(s, 1H), 6.32 (s, 1H), 8.36 (d, J¼8.1 Hz, 1H), 8.41 (d, J¼8.1 Hz, 1H); 13C
NMR (75 MHz, CDCl3):
d 56.6, 56.8, 108.2, 111.7, 129.6, 130.6, 133.1,
134.5, 135.0, 137.2, 158.9, 163.0, 178.5, 179.2, 182.4, 182.5, HRMS (EI)
calcd for C16H10O6 (Mþ) 298.0477, found 298.0482. The spectra of
synthetic 1 are in agreement with those reported for the natural
product.1a
4.1.20. 1-(4-Hydroxy-3-methoxyphenyl)-2-(4-hydroxy-2,5-dimethox-
yphenyl)ethane, moscatilin (21). The moscatilin (21) was prepared
from 4-hydroxy-3-methoxybenzaldehyde, using the procedure used

6172
S. Thangaraj et al. / Tetrahedron 67 (2011) 6166e6172
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reduction of total protein increase in control cells during the
compound incubation.
Acknowledgements
The research was supported by the National Science Council
(Research Grants NSC98-2119-M-018-002-MY3 and NSC99-2811-M-
018-011), National Tsing Hua University (NSC99-2323-B-007-001),
and the Department of Health, Taiwan (DOH99-TD-C-111-004). We
thank Professor T. Ross Kelly for valuable proofreading and the re-
vised manuscript. We also thank Dr. Fang-Rong Chang for helpful
discussions.
7. Tseng, T.-H.; Chuang, S.-K.; Hu, C.-C.; Chang, C.-F.; Huang, Y.-C.; Lin, C.-W.; Lee,
Y.-J. Tetrahedron 2010, 66, 1335e1340.
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.tet.2011.06.054.
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