First total synthesis of protoapigenone and its analogues as potent cytotoxic agents

Article abstract of DOI:10.1021/jm070363a

Protoapigenone (1), isolated from Thelypteris torresiana, previously showed significant cytotoxic activity against five human cancer cell lines. In a continued structure-activity relationship study, the first total synthesis and modification of 1 were ach

Full text of DOI:10.1021/jm070363a

J. Med. Chem. 2007, 50, 3921-3927
3921
First Total Synthesis of Protoapigenone and Its Analogues as Potent Cytotoxic Agents
An-Shen Lin,†,‡ Kyoko Nakagawa-Goto,‡ Fang-Rong Chang,† Donglei Yu,‡ Susan L. Morris-Natschke,‡ Chin-Chung Wu,†
Shu-Li Chen,† Yang-Chang Wu,*,§,† and Kuo-Hsiung Lee*,‡
Graduate Institute of Natural Products, Kaohsiung Medical UniVersity, Kaohsiung 807, Taiwan, Natural Products Research Laboratories,
School of Pharmacy, UniVersity of North Carolina, Chapel Hill, North Carolina 27599, and National SunYat-Sen UniVersity-Kaohsiung
Medical UniVersity Joint Research Center, Kaohsiung, Taiwan
ReceiVed March 28, 2007
Protoapigenone (1), isolated from Thelypteris torresiana, previously showed significant cytotoxic activity
against five human cancer cell lines. In a continued structure-activity relationship study, the first total
synthesis and modification of 1 were achieved. All synthesized compounds and related intermediates were
evaluated for cytotoxic activity against five human cancer cell lines, HepG2, Hep3B, MDA-MB-231, MCF-
7, and A549. Among them, 24 showed 2.2-14.2-fold greater cytotoxicity than 1 and naphthyl A-ring
analogues remarkably enhanced the activity.
Introduction
bioactivity data, and preliminary structure-activity relationship
(SAR) studies related to 1.
Flavonoids are plant pigments that generally display marvel-
ous colors and are ubiquitous to green plants. Their multiple
bioactivities (leading to the term bioflavonoid) and medicinal
significance have been summarized recently.1,2 In our previous
study, we isolated the unique flavonoid protoapigenone (1) from
Thelypteris torresiana (Gaud.) Alston, which showed potent
cytotoxic activity against HepG2 and Hep3B (liver), MDA-MB-
231 and MCF-7 (breast), and A549 (lung) human cancer cell
lines with IC50 values of 0.94-5.59 µM.3 In our subsequent
study, this compound showed 7.5- and 4.6-fold greater cytotoxic
activity against two human breast cancer cell lines MCF-7 and
MDA-MB-231 compared with the MCF-10A normal human
breast epithelial cell line.4
Chemistry. Our initial attempt to obtain 1 from 2 by
oxidation failed because of oxidation of the 5- and 7-hydroxy
groups. Because of the difficulty of selectively protecting these
two groups on 2 in the presence of the 4′-hydroxyl, diprotected
trihydroxyacetophenones (6 and 7)10 (commercially available)
were selected as starting materials.
Our successful flavonoid synthesis started with a Claisen-
Schmidt condensation carried out individually on 6 and 7 with
4-benzyloxybenzaldehyde in the presence of aqueous potassium
hydroxide to provide 8 (87%) and 9 (79%).11 Chalcone 9 was
further cyclized with a catalytic amount of iodine in DMSO to
give the corresponding 4′-benzyloxyflavonoid 11 (74%).11
Similar cyclization of 8 gave only a low yield of the expected
product. Instead, the use of pyridine as solvent caused an
unexpected cleavage of the MOMa (methoxymethyl) ether from
the 5-position, along with cyclization, to produce 10 (86%). The
benzyl groups of 10 and 11 were removed by treatment with
catalytic 10% palladium carbon under H2 to afford 4′-hydroxy-
flavonoids 12 (86%) and 13 (83%).12 Oxidation of 12 and 13
with [bis(trifluoroacetoxy)iodo]benzene (TAIB) in acetonitrile/
H2O at room-temperature gave the novel flavonoids 14 (22%)
and 15 (33%).13,14 The 5-hydroxy group was unaffected under
these conditions because of strong intermolecular hydrogen
bonding with the carbonyl group on the 4-position. When MeOH
rather than acetonitrile/H2O was used as solvent, trimethox-
yprotoapigenone (16) (33%) was produced from 13. Finally, 1
was obtained by cleavage of the MOM group on 14 using 15%
HCl in i-PrOH (yield 47%),10,12 while the methoxy groups on
15 were not cleaved using 47% HBr/HOAc at reflux as the
demethylation condition (Scheme 1).15,16
Flavonoid 1 has an unusual nonaromatic B-ring with a
hydroxy group on the C-1′ position, which might arise from
oxidation of a 4′-hydroxyphenyl. Apigenin (2), which is the
conceivable biosynthetic precursor of 1, kaempferol (3), and
quercetin (4) are well-known flavonoids bearing a 4′-hydrox-
yphenyl B-ring (Figure 1). These three compounds showed no
antiproliferative activity (IC50 > 20 µg/ mL) against six human
cancer cell lines,5 while 1 displayed significant cytotoxic
activity. This unique difference spurred our interest in the
characteristics of the 1′-hydroxycyclohexa-2′,5′-dien-4′-one B-
ring in this flavonoid skeleton. Although the antitumor mech-
anism of this functional group is still not clear, recent studies
on some quinol derivatives showed that the oxidized species
are more active than their reduced counterparts.6-9
To investigate a new class of anticancer agents based on this
novel plant-derived natural flavonoid, the first total synthesis
of 1 and the preparation of some analogues were accomplished.
All newly synthesized compounds including structurally related
intermediates were assayed for in Vitro cytotoxic activity against
five human cancer cell lines, e.g., HepG2, Hep3B, MDA-MB-
231, MCF-7, and A549. In this paper, we describe the synthesis,
In addition, five commercially available 4′-hydroxyflavonoids
(17, 18, 19, 25, and 28) were oxidized with TAIB using
acetonitrile/H2O or methanol as the solvent to obtain the related
quinol derivatives, 20 (36%) and 21 (28%) from 17, 22 (16%)
and 23 (15%) from 18, 24 (20%) from 19, 26 (16%) and 27
(15%) from 25, and 29 (23%) from 28 (Scheme 2).13,14
Accordingly, the first total synthesis of 1 as well as the synthesis
of twelve A-ring and C-1′ analogues, 14-16, 20-24, 26, 27,
and 29, were accomplished.
* To whom correspondence should be addressed. For K. H. Lee. Phone:
919-962-0066. Fax: 919-966-3893. E-mail: khlee@unc.edu. For Y.-C. Wu.
Phone: 886-7-3121101/ext 2197. Fax: 886-7-3114773. E-mail: yachwu@
knu.edu.tw.
† Kaohsiung Medical University.
‡ University of North Carolina.
§ National SunYat-Sen University-Kaohsiung Medical University Joint
Research Center.
a Abbreviations: TAIB, [bis(trifluoroacetoxy)iodo]benzene; MOM,
methoxymethyl.
10.1021/jm070363a CCC: $37.00 © 2007 American Chemical Society
Published on Web 07/10/2007

3922 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 16
Lin et al.
Figure 1. Chemical structures of 1 and related flavonoids.
Results and Discussion
The parent compound 1 showed potent cytotoxic activity
against the tested human cancer cell lines. Comparison of 1 to
its synthetic analogues 14-16 showed that 14, with a MOM
ether at C-7, had comparable or reduced activity compared with
1, and trimethoxy 16 was uniformly less active. However, 5,7-
dimethoxy compound 15 showed enhanced cytotoxic activity
with notable IC50 values of 0.54-1.27 µM against four cell lines
(Hep3B, MDA-MB-231, MCF-7, and A549). Remarkably, 1′-
hydroxy analogues 15, 20, 22, and 26 were 1.6 to >48.0 times
Together with 1, newly synthesized flavonoids 14-16, 20-
24, 26, 27, and 29 were examined for in Vitro cytotoxic activities
against several human cancer lines, including HepG2, Hep3B,
MDA-MB-231, MCF-7, and A549. Table 1 lists the IC50 values
obtained with these compounds as well as the related flavonoids
protoapigenin (30) and 5′,6′-dihydro-6′-methoxyprotoapigenone
(31) (Figure 2), as well as doxorubicin, included as a positive
control.
Scheme 1. Total Synthesis of 1 and Its Analoguesa
a Reagents: (i) K2CO3, MOMCl, acetone, (ii) EtOH, 50% KOH/H2O, 4-benzyloxybenzaldehyde; (iii) I2, DMSO or pyridine; (iv) 10% Pd-C/H2; (v)
TAIB (1 equiv), CH3CN/H2O 9/1 (12f14, 13f15) or MeOH (13f16), 25 °C; (vi) 15% HCl/iPrOH (14f1).

Total Synthesis of Protoapigenone
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 16 3923
Scheme 2. Synthesis of Flavonoid and Naphthoflavone Analogues with Quinol B-Ringa
a Reagent: (i) TAIB (1 equiv), CH3CN/H2O 9/1 or MeOH, 25 °C.
Table 1. Cytotoxic Effects of Synthesized 1 and Analogues
IC50 (µM)a
compound
HepG2
Hep3B
MDA-MB-231
MCF-7
A549
1
14
15
16
20
21
22
23
8.11 ( 0.01
15.58 ( 0.08
5.03 ( 0.01
27.01 ( 0.01
6.73 ( 0.01
40.86 ( 0.08
4.96 ( 0.02
26.90 ( 0.01
0.57 ( 0.02
3.55 ( 0.04
9.94 ( 0.10
14.37 ( 0.03
5.56 ( 0.33
18.49 ( 0.47
0.50 ( 0.04
2.27 ( 0.01
2.39 ( 0.00
0.67 ( 0.01
3.78 ( 0.00
1.26 ( 0.02
5.41 ( 0.00
1.30 ( 0.01
2.32 ( 0.04
0.67 ( 0.00
0.30 ( 0.00
1.10 ( 0.01
0.60 ( 0.01
69.44 ( 0.58
5.47 ( 0.08
0.62 ( 0.03
1.43 ( 0.00
1.73 ( 0.00
0.54 ( 0.01
4.70 ( 0.01
0.71 ( 0.00
5.49 ( 0.01
0.67 ( 0.00
1.90 ( 0.02
0.43 ( 0.00
0.39 ( 0.00
1.16 ( 0.01
1.07 ( 0.01
>69.44
3.74 ( 0.02
5.24 ( 0.01
1.21 ( 0.01
4.21 ( 0.04
1.73 ( 0.01
7.99 ( 0.03
3.44 ( 0.04
5.46 ( 0.06
1.70 ( 0.06
0.66 ( 0.00
2.70 ( 0.01
2.96 ( 0.08
13.85 ( 0.01
24.61 ( 0.02
1.27 ( 0.03
>60.98
5.24 ( 0.05
>74.63
5.07 ( 0.03
57.61 ( 0.31
3.10 ( 0.08
1.81 ( 0.00
25.79 ( 0.05
28.33 ( 0.07
65.42 ( 0.60
41.82 ( 0.23
0.36 ( 0.02
24
26
27
29
30b
>69.44
31b
4.09 ( 0.10
0.14 ( 0.01
18.62 ( 0.29
0.74 ( 0.00
doxorubicinc
a Data are expressed as mean ( SD (mean ) 2). b Data from our previous study. c Positive control.
more active than the related 1′-methoxy analogues, 16, 21, 23,
and 27. These findings indicate the importance of a nonsubsti-
tuted OH at the C-1′ position.
Among the quinol derivatives, changing the C-5 substituent
from hydrogen to methoxy (20 vs 22, 21 vs 23) did not affect
potency, while variation in the C-7 functional group did exert
an influence. For example, compound 24 with a C-7 methoxy
group showed the highest potency against all five cancer cell
lines with IC50 values of 0.43-3.10 µM. Amazingly, this
compound also exhibited significantly enhanced activity against
the HepG2 human liver cancer cell line with an IC50 value of
0.57 µM and consequently was 14.2 times more active than 1,
while most analogues showed comparatively weak activity
against this cell line. Compound 22, which lacks a C-7 methoxy

3924 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 16
Lin et al.
Figure 2. Structures of 1-related cytotoxic flavonoids.
Figure 3. SAR in newly synthesized cytotoxic flavonoids.
group, was generally less active than 24, but was about 2-fold
more active than 1 against all cell lines. In particular, growth
inhibition of Hep3B (IC50 ) 1.30 µM) and MDA-MB-231 (IC50
) 0.67 µM) was significantly increased.
Naphthoflavones 26, 27, and 29, which have an additional
conjugated aromatic ring attached to the A-ring, showed better
activity compared with 1 against the three cell lines Hep3B,
MDA-MB-231, and MCF-7. Particularly, analogue 26 was more
potent than doxorubicin against the Hep3B cell line with an
IC50 value of 0.30 µM. In addition, this compound was 7.7 times
more potent than 1 against A549 cell growth with an IC50 value
of 1.81 µM.
Our prior report stated that the 4′-oxocyclohexa-2′,5′-dienyl
moiety as found in 1 is critically important for biological activity
in flavonoids.3 Accordingly, we found in this study that the
natural product 30, which instead contains a 4′-hydroxy-
cyclohexa-2′,5′-dienyl moiety, was active against only the
HepG2 cell line. However, corresponding synthetic 1′-hydrox-
yquinol analogues 20, 22, and 24, with different combi-
nations of hydrogen, hydroxy, and methoxy substituents
rather than two hydroxy groups on the A-ring, showed
significant cytotoxic activity. In addition, we previously found
that 31, which has only a single double bond in the B-ring,
exhibited lower cytotoxic activity; thus, a symmetrical
double bond structure in the B-ring might also be important.3
The summary of this preliminary SAR study is shown in
Figure 3.
In conclusion, among all screened compounds, the 1′-
hydroxycyclohexa-2′,5′-dien-4′-one B-ring is critically important
in the bioflavonoid skeleton, but substituent changes on the
A-ring can affect selectivity or potency against different kinds
of cancer cell lines. A methoxy group on the C-7 position can
enhance cytotoxic selectivity toward the human HepG2 liver
cancer cell line, and an additional conjugated aromatic ring on
the A-ring increases cytotoxicity against all five tested human
cancer cell lines. These prototype analogues of 1 have demon-
strated valuable growth inhibition of selected human cancer cell
lines. In addition, our preliminary data (unpublished) indicate
that treatment of MDA-MB-231 with 1 leads to cell cycle arrest
at the G2/M phase and apoptosis; the precise mechanisms
responsible for these effects are currently under investigation.
We are synthesizing additional candidate structures that will
address specific cancer cell lines.
Experimental Section
Materials and Methods. Melting points were measured with a
Fisher-John melting apparatus without correction. 1H NMR spectra
were measured on a 300 MHz Varian Gemini 2000 spectrometer.
The solvent used were CDCl3 or pyridine-d5. Mass spectra were
measured on a PECIEX API 3000 instrument with turbo ion spray
source, Agilent-1100, LC/MSD-Trap, or Shimadzu LCMS-IT-TOF
with ESI interface. Thin-layer chromatography (TLC) and prepara-
tive TLC were performed on precoated silica GF plates purchased
from Merck, Inc. Biotage Flash+ or Isco Companion systems were
used for flash chromatography. Silica gel (200-400 mesh) from
Aldrich, Inc. was used for column chromatography. Flavonoids were
obtained from Indofine Chemical Company, Inc. All other chemicals
were obtained from Aldrich, Inc.
Cell Viability Assays. Human breast (MCF-7 and MDA-MB-
231), liver (HepG2 and Hep3B), and lung (A549) cancer cell lines
were obtained from American Type Culture Collection. All cell
lines were propagated in RPMI-1640 medium supplement with 10%
(v/v) FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin at
37 °C in a humidified atmosphere of 5% CO2 and 95% air. Cell
viability was measured by the MTT colorimetric method. Cells were
seeded at densities of 5000-10000 cells/well in 96-well tissue
culture plates. On day two, cells were treated with test compounds
for another 72 h. After drug treatment, attached cells were incubated
with MTT (0.5 mg/mL, 1 h) and subsequently solubilized in DMSO.
The absorbency at 550 nm was then measured using a microplate
reader. The IC50 is the concentration of agent that reduced the cell
viability by 50% under the experimental conditions.3,17

Total Synthesis of Protoapigenone
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 16 3925
Protoapigenone (1). Compound 14 (30 mg, 0.09 mmol) was
dissolved in 15% HCl/iPrOH (1:5, 5 mL), and the reaction mixture
was stirred at the room temperature for 140 min. The solvent was
evaporated and the residue chromatographed using silica gel
(isocratic elution, 33% MeOH/67% CHCl3) to give 1 (12.0 mg,
47%) as a yellow solid: ESI-MS (m/z, %): 285 (M- - 1, 100);
1H NMR (pyridine-d5) δ 13.38 (1H, br s, OH-5), 7.24 (2H, d, J )
10.2 Hz, H-2′, H-6′), 7.07 (1H, s, H-3), 6.72 (1H, d, J ) 2.4 Hz,
H-8), 6.60 (1H, d, J ) 2.4 Hz, H-6), 6.54 (2H, d, J ) 10.2 Hz,
H-3′, H-5′); 13C NMR (pyridine-d5) δ 185.2 (C-4′), 182.9 (C-4),
167.8 (C-2), 166.3 (C-7), 163.1 (C-5), 158.7 (C-9), 148.4 (C-2′,
C-6′), 129.5 (C-3′, C-5′), 107.4 (C-3), 105.1 (C-10), 100.2 (C-6),
95.0 (C-8), 69.7 (C-1′).
1-[2-Hydroxy-4,6-bis(methoxymethoxy)phenyl]ethanone (6).
2′,4′,6′-Trihydroxyacetophenone monohydrate (5) (2.0 g, 11.9
mmol) and K2CO3 (11.5 g) were dissolved in anhydrous acetone
(80 mL). Chloromethyl methyl ether (MOMCl, 2.4 g) was added
dropwise to the stirring solution, and then the suspension was
refluxed for 90 min. After cooling, the solid was filtered off, and
the crude product was evaporated and purified by column chro-
matography on silica gel (isocratic elution, 90% n-hexane/10%
EtOAc) to afford 6 (1.5 g, 50%) as a white solid: 1H NMR (CDCl3)
δ 13.72 (1H, br s, OH-2), 6.25 (1H, d, J ) 2.4 Hz, H-5), 6.23 (1H,
d, J ) 2.4 Hz, H-3), 5.24, 5.16 (2H each, s, CH2-MOM), 3.51,
3.46 (3H each, s, OMe-MOM), 2.64 (1H, s, COCH3); 13C NMR
(pyridine-d5) δ 203.2 (CO), 166.8 (C-2), 163.4 (C-4), 160.3(C-6),
106.9 (C-1), 97.1 (C-3), 94.4 (C-5), 93.9 (CH2-MOM, overlapping),
56.6, 56.4 (OMe-MOM), 33.0 (COCH3).
Hz, H-3′, H-5′), 6.64 (1H, d, J ) 2.1 Hz, H-8), 6.55 (1H, s, H-3),
6.46 (1H, d, J ) 2.1 Hz, H-6), 5.23 (2H, s, CH2-OMOM), 5.13
(2H, s, CH2-OBz), 3.50 (3H, s, OMe); 13C NMR (CDCl3) δ 182.4
(C-4), 163.9 (C-7), 162.8 (C-2), 161.9 (C-5), 161.7 (C-9), 157.4
(C-4′), 136.1 (C-1-OBz), 128.7 (C-2, C-6), 128.2 (C-4-OBz), 128.0
(C-2-OBz, C-6-OBz), 127.4 (C-3-OBz, C-5-OBz), 123.6 (C-1′),
115.3 (C-3, C-5), 106.1 (C-10), 104.3 (C-3), 100.0 (C-6), 94.2 (C-
8), 94.2 (CH2-MOM), 70.1 (CH2-OBz), 56.3 (OMe).
2-[4-(Benzyloxy)phenyl]-5,7-dimethoxychromen-4-one (11).
Compound 9 (1.6 g, 4.0 mmol) was treated analogously as 8 to
give 11 (1.1 g, 74%) as a yellow solid: 1H NMR (pyridine-d5) δ
7.95 (2H, d, J ) 9.0 Hz, H-2′, H-6′), 7.32-7.58 (5H, m, H-OBz),
7.23 (2H, d, J ) 9.0 Hz, H-3′, H-5′), 6.96 (1H, s, H-3), 6.83 (1H,
d, J ) 2.4 Hz, H-8), 6.57 (1H, d, J ) 2.4 Hz, H-6), 5.19 (2H, s,
CH2-OBz), 3.85 (6H, s, OMe); 13C NMR (CDCl3) δ 176.6 (C-4),
164.3 (C-7), 161.6 (C-2), 161.3 (C-5), 160.5 (C-4′), 160.2 (C-9),
137.2 (C-1-OBz), 129.0 (C-3-OBz, C-5-OBz), 128.5 (C-4-OBz),
128.1 (C-2-OBz, C-6-OBz), 128.1 (C-2, C-6), 124.5 (C-1′), 115.6
(C-3, C-5), 108.2 (C-10), 109.8 (C-3), 96.7 (C-6), 93.7 (C-8), 70.3
(CH2-OBz), 56.2, 55.9 (OMe).
General Procedure for Benzyl Group Removal. Synthesis of
Compounds 12 and 13. A mixture of 10 (200 mg, 0.5 mmol), dry
Pd/C (10%, 106 mg), and EtOAc/ MeOH (1:1, 15 mL) was stirred
at 25 °C under an atmosphere of hydrogen for 3 h. The mixture
was filtered through a layer of silica gel on cotton, washed with
EtOAc, concentrated under vacuum, and purified by column
chromatography on silica gel (isocratic elution, EtOAc only) to give
12 (134 mg) as a yellow solid.
3-[4-(Benzyloxy)phenyl]-1-[2-hydroxy-4,6-bis(methoxymethox-
y)phenyl]prop-2-en-1-one (8). Compound 6 (1.8 g, 7.1 mmol) and
4-benzyloxybenzaldehyde (3.0 g, 14.3 mmol) were dissolved in 50%
EtOH, KOH/H2O solution (20 mL). The reaction was stirred at rt
for 30 h, and then the solvent was evaporated under reduced
pressure. The mixture was chromatographed on silica gel (isocratic
elution, n-hexane/EtOAc, 6:1) to afford 8 (2.8 g, 87%) as a white
solid: 1H NMR (CDCl3) δ 13.95 (1H, br s, OH-2′), 7.84 (1H, d, J
) 15.6 Hz, H-â), 7.78 (1H, d, J ) 15.6 Hz, H-R), 7.56 (2H, d, J
) 8.7 Hz, H-2, H-6), 7.32-7.46 (5H, m, H-OBz), 7.01 (2H, d, J
) 8.7 Hz, H-3, H-5), 6.32 (1H, d, J ) 2.4 Hz, H-5′), 6.25 (1H, d,
J ) 2.4 Hz, H-3′), 5.29, 5.19 (2H each, s, CH2-MOM), 5.11 (2H,
s, CH2-OBz), 3.54, 3.49 (3H each, s, OMe-MOM); 13C NMR
(CDCl3) δ 192.8 (CO), 167.3 (C-4′), 163.3 (C-2′), 160.6 (C-6′),
159.8 (C-4), 142.6 (C-â), 136.4 (C-1-OBz), 130.1 (C-2, C-6), 128.6
(C-3-OBz, C-5-OBz), 128.4 (C-1), 128.1 (C-R), 127.4 (C-2-OBz,
C-6-OBz), 125.0 (C-4-OBz), 115.3 (C-3, C-5), 107.5 (C-1′), 97.5
(C-5′), 95.1 (C-3′), 94.7, 94.0 (CH2-MOM), 70.1 (CH2-OBz), 56.8,
56.4 (OMe-MOM).
3-[4-(Benzyloxy)phenyl]-1-(2-hydroxy-4,6-dimethoxyphenyl)-
prop-2-en-1-one (9). Compound 7 (1.0 g, 5.1 mmol) was reacted
with 4-benzyloxybenzaldehyde (2.2 g, 10.2 mmol) as described
above for 6 to afford 9 (1.6 g, 79%) as a white solid: 1H NMR
(CDCl3) δ 13.87 (1H, br s, OH-2′), 7.82 (1H, d, J ) 15.6 Hz, H-â),
7.77 (1H, d, J ) 15.6 Hz, H-R), 7.56 (2H, d, J ) 9.0 Hz, H-2,
H-6), 7.32-7.46 (5H, m, H-OBz), 7.00 (2H, d, J ) 9.0 Hz, H-3,
H-5), 6.11 (1H, d, J ) 2.4 Hz, H-5′), 5.96 (1H, d, J ) 2.4 Hz,
H-3′), 5.11 (2H, s, CH2-OBz), 3.92, 3.83 (3H each, s, OMe); 13C
NMR (CDCl3) δ 192.6 (CO), 168.4 (C-4′), 166.0 (C-2′), 162.4 (C-
6′), 160.5 (C-4), 142.4 (C-â), 136.5 (C-1-OBz), 130.1 (C-2, C-6),
128.7 (C-3-OBz, C-5-OBz), 128.5 (C-1), 128.1 (C-R), 127.5 (C-
2-OBz, C-6-OBz), 125.2 (C-4-OBz), 115.2 (C-3, C-5), 106.3 (C-
1′), 93.8 (C-5′), 91.2 (C-3′), 70.1 (CH2-OBz), 55.8, 55.6 (OMe).
2-[4-(Benzyloxy)phenyl]-5-hydroxy-7-(methoxymethoxy)-
chromen-4-one (10). Compound 8 (500 mg, 1.1 mmol) was
dissolved anhydrous pyridine (5 mL), and a catalytic amount of
iodine (28.4 mg, 0.1 mmol) was added. The solution mixture was
stirred and refluxed. After 3 h, 10% Na2S2O3 (30 mL) was added
to remove iodine. The mixture was extracted with EtOAc (50 mL)
and then purified by column chromatography on silica gel (isocratic
elution, EtOAc only) to give 10 (380 mg, 86%) as a yellow solid:
1H NMR (CDCl3) δ 12.77 (1H, br s, OH-5), 7.81 (2H, d, J ) 8.7
Hz, H-2′, H-6′), 7.32-7.46 (5H, m, H-OBz), 7.06 (2H, d, J ) 8.7
5-Hydroxy-2-(4-hydroxyphenyl)-7-(methoxymethoxy)chromen-
4-one (12). 86% yield; 1H NMR (pyridine-d5) 7.93 (2H, d, J ) 8.7
Hz, H-2′, H-6′), 7.27 (2H, d, J ) 8.7 Hz, H-3′, H-5′), 6.96 (1H, s,
H-3), 6.91 (1H, d, J ) 2.1 Hz, H-8), 6.78 (1H, d, J ) 2.1 Hz,
H-6), 5.35 (2H, s, CH2-OMOM), 3.45 (3H, s, OMe); 13C NMR
(CDCl3) δ 182.4 (C-4), 164.4 (C-7), 162.8 (C-2), 163.4 (C-5), 162.1
(C-9), 157.4 (C-4′), 128.5 (C-2, C-6), 121.5 (C-1′), 116.4 (C-3,
C-5), 106.0 (C-10), 103.6 (C-3), 99.7 (C-6), 94.3 (C-8), 94.2 (CH2-
MOM), 55.8 (OMe).
2-(4-Hydroxyphenyl)-5,7-dimethoxychromen-4-one (13). 83%
yield; 1H NMR (pyridine-d5) δ 7.93 (2H, d, J ) 9.0 Hz, H-2′, H-6′),
7.27 (2H, d, J ) 9.0 Hz, H-3′, H-5′), 6.95 (1H, s, H-3), 6.82 (1H,
d, J ) 2.4 Hz, H-8), 6.56 (1H, d, J ) 2.4 Hz, H-6), 3.84 (6H, s,
OMe); 13C NMR (pyridine-d5) δ 176.7 (C-4), 164.2 (C-7), 162.1
(C-2), 161.3 (C-5), 161.1 (C-4′), 160.2 (C-9), 128.4 (C-2′, C-6′),
122.6 (C-1′), 116.8 (C-3′, C-5′), 109.7 (C-10), 107.5 (C-3), 96.7
(C-6), 93.7 (C-8), 56.2, 55.9 (OMe).
General Procedure for Oxidation Reaction with TAIB.
Synthesis of Compounds 14-16, 20-24, 26, 27, and 29. The
precusor flavonoid [for example, 12 (90 mg)] was dissolved in
acetonitrile/H2O (9:1) or MeOH (5 mL) individually, and a catalytic
amount of TEMPO (0.2 eq) was added to the flask, followed by 2
equiv of TAIB. The reaction mixture was stirred vigorously at
25 °C for 90 min. The reaction mixture was then evaporated to
dryness under reduced pressure and the residue purified on a silica
gel column (isocratic elution, 2.5% MeOH/97.5% CH2Cl2) to give
14 in 22% yield. Corresponding oxidations of the remaining
flavonoids (13, 17-19, 25, and 28) gave 15/16, 20/21, 22/23, 24,
26/27, and 29, respectively, with different solvents.
7-Methoxymethoxyprotoapigenone (14). 22% yield from 12;
1H NMR (CDCl3) δ 12.34 (1H, s, OH-5), 6.87 (2H, d, J ) 9.9 Hz,
H-2′, H-6′), 6.66 (1H, s, H-3), 6.46 (2H, s, H-6, H-8), 6.41 (2H, d,
J ) 9.9 Hz, H-3′, H-5′), 5.20 (2H, s, CH2-OMOM), 3.46 (6H, s,
OMe); 13C NMR (CDCl3) δ 184.5 (C-4′), 182.4 (C-4), 165.6 (C-
7), 163.4 (C-2), 162.0 (C-5), 157.6 (C-9), 145.5 (C-2′, C-6′), 130.2
(C-3′, C-5′), 107.5 (C-3, C-10), 100.6 (C-6), 94.4 (C-8), 94.2 (CH2-
MOM), 69.5 (C-1′), 56.5 (OMe).
5,7-Dimethoxyprotoapigenone (15). 33% yield from 13; ESI-
1
MS (m/z, %): 313 (M- - H, 100); H NMR (pyridine-d5) δ 7.26
(2H, d, J ) 10.2 Hz, H-2′, H-6′), 7.10 (1H, s, H-3), 6.56 (2H, d,
J ) 10.2 Hz, H-3′, H-5′), 6.55 (2H, s, H-6, H-8), 3.82, 3.68 (3H
each, s, OMe); 13C NMR (pyridine-d5) δ 185.3 (C-4′), 176.4 (C-
4), 164.5 (C-7), 164.3 (C-2), 161.3 (C-5), 160.3 (C-9), 148.8 (C-

3926 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 16
Lin et al.
2′, C-6′), 129.4 (C-3′, C-5′), 110.9 (C-3), 109.8 (C-10), 96.9 (C-
6), 93.4 (C-8), 69.4 (C-1′), 56.2, 55.8 (OMe).
3-(1-Methoxy-4-oxocyclohexa-2,5-dienyl)-1H-benzo[f]chromen-
1-one (27). 15% yield from 25; ESI-MS (m/z, %): 288 (M+ + H
- OCH3, 100), 319 (M+ + H, 95); 1H NMR (CDCl3) δ 9.98 (1H,
dd, J ) 9.0, 1.2 Hz, H-5), 8.07 (1H, d, J ) 9.0 Hz, H-9), 7.94 (1H,
dd, J ) 8.4, 1.5 Hz, H-8), 7.76 (1H, ddd, J ) 8.4, 8.4, 1.2 Hz,
H-7), 7.62 (1H, ddd, J ) 9.0, 8.4, 1.2 Hz, H-6), 7.40 (1H, d, J )
9.0 Hz, H-10), 6.88 (1H, s, H-3), 6.88 (2H, d, J ) 9.9 Hz, H-2′,
H-6′), 6.60 (2H, d, J ) 9.9 Hz, H-3′, H-5′), 3.45 (3H, s, OMe);
13C NMR (CDCl3) δ 184.5 (C-4′), 179.8 (C-4), 161.1 (C-2), 157.4
(C-10a), 145.9 (C-2′, C-6′), 133.1 (C-3′, C-5′), 135.8, 130.6, 130.3,
129.4, 128.2, 127.0, 126.8 (C-4b, C-5, C-6, C-7, C-8, C-8a, C-9),
117.3 (C-10), 117.3 (C-4a), 112.6 (C-3), 74.7 (C-1′), 52.8 (OMe).
5,7,1′-Trimethoxyprotoapigenone (16). 33% yield from 13;
ESI+MS (m/z, %): 328 (M+ + H, 100); 1H NMR (pyridine-d5) δ
6.95 (2H, d, J ) 10.2 Hz, H-2′, H-6′), 6.76 (1H, s, H-3), 6.67 (2H,
d, J ) 10.2 Hz, H-3′, H-5′), 6.55 (1H, s, H-8), 6.52 (1H, s, H-6),
3.81, 3.68, 3.25 (3H each, s, OMe); 13C NMR (pyridine-d5) δ 184.6
(C-4′), 176.0 (C-4), 164.6 (C-7), 161.4 (C-2), 161.3 (C-5), 160.1
(C-9), 146.0 (C-2′, C-6′), 133.2 (C-3′, C-5′), 111.5 (C-3, C-10),
97.0 (C-6), 93.4 (C-8), 74.7 (C-1′), 56.2, 55.8, 52.4 (OMe).
2-(1-Hydroxy-4-oxocyclohexa-2,5-dienyl)-4H-chromen-4-
-
one (20). 36% yield from 17; ESI-MS (m/z, %): 254 (M- H,
1
100); H NMR (CDCl3) δ 8.14 (1H, dd, J ) 7.8, 1.5 Hz, H-5),
2-(1-Methoxy-4-oxocyclohexa-2,5-dienyl)-4H-benzo[h]chromen-
4-one (29). 23% yield from 28; ESI-MS (m/z, %): 319 (M+ + H,
7.66 (1H, ddd, J ) 8.4, 7.2, 1.5 Hz, H-7), 7.40 (1H, ddd, J ) 7.8,
7.2, 0.9 Hz, H-6), 7.36 (1H, dd, J ) 8.4, 0.9 Hz, H-8), 6.95 (2H,
d, J ) 10.2 Hz, H-2′, H-6′), 6.88 (1H, s, H-3), 6.41 (2H, d, J )
10.2 Hz, H-3′, H-5′); 13C NMR (CDCl3) δ 185.0 (C-4′), 178.9 (C-
4), 166.3 (C-2), 156.2 (C-9), 146.2 (C-2′, C-6′), 134.3 (C-7), 129.9
(C-3′, C-5′), 125.7 (C-5, C-6), 123.5 (C-7), 118.1 (C-8), 108.8 (C-
3), 69.8 (C-1′).
1
100), 288 (M+ + H - OCH3, 17); H NMR (CDCl3) δ 8.12 (1H,
dd, J ) 7.8, 1.2 Hz, H-10), 8.10 (1H, d, J ) 8.7 Hz, H-5), 7.91
(1H, dd, J ) 7.2, 1.5 Hz, H-7), 7.76 (1H, d J ) 8.7 Hz, H-6), 7.69
(1H, ddd, J ) 8.4, 7.8, 1.5 Hz, H-9), 7.62 (1H, ddd, J ) 8.4, 7.2,
1.2 Hz, H-8), 6.94 (1H, s, H-3), 6.93 (2H, d, J ) 10.2 Hz, H-2′,
H-6′), 6.67 (2H, d, J ) 10.2 Hz, H-3′, H-5′), 3.48 (3H, s, OMe);
13C NMR (CDCl3) δ 184.6 (C-4′), 177.9 (C-4), 162.9 (C-2), 160.6
(C-10b), 146.2 (C-2′, C-6′), 133.1 (C-3′, C-5′), 136.0, 129.5, 128.1,
127.5, 125.7, 123.7, 122.0, 120.5, 120.3 (C-4a, C-5, C-6, C-6a,
C-7, C-8, C-9, C-10, C-10a), 110.8 (C-3), 75.1 (C-1′), 52.9 (OMe).
2-(1-Methoxy-4-oxocyclohexa-2,5-dienyl)-4H-chromen-4-
one (21). 28% yield from 17; ESI+MS (m/z, %): 268 (M+, 100);
1H NMR (CDCl3) δ 8.17 (1H, dd, J ) 7.8, 1.5 Hz, H-5), 7.65 (1H,
ddd, J ) 8.4, 7.2, 1.5 Hz, H-7), 7.40 (1H, ddd, J ) 7.8, 7.2, 0.9
Hz, H-6), 7.35 (1H, dd, J ) 8.4, 0.9 Hz, H-8), 6.82 (2H, d, J )
10.2 Hz, H-2′, H-6′), 6.76 (1H, s, H-3), 6.58 (2H, d, J ) 10.2 Hz,
H-3′, H-5′), 3.42 (3H, s, OMe); 13C NMR (CDCl3) δ 185.5 (C-4′),
178.0 (C-4), 163.8 (C-2), 156.0 (C-9), 145.5 (C-2′, C-6′), 134.0
(C-7), 133.1 (C-3′, C-5′), 125.7, 125.5 (C-5, C-6), 123.9 (C-7), 118.0
(C-8), 109.5 (C-8), 74.8 (C-1′), 52.7 (OMe).
Acknowledgment. This investigation was supported by
Grants CA 17625 from National Cancer Institute (K. H. Lee),
National Science Council, Taiwan, and National SunYat-Sen
University-Kaohsiung Medical University Joint Research Center
(Y.-C. Wu).
5-Hydroxy-2-(1-hydroxy-4-oxocyclohexa-2,5-dienyl)-4H-
chromen-4-one (22). 16% yield from 18; ESI-MS (m/z, %): 270
1
(M- - H, 100); H NMR (CDCl3) δ 12.24 (1H, s, OH-5), 7.52
Supporting Information Available: Additional information on
compound purity, high-resolution mass spectral data, and HPLC
analysis results of the target compounds. This material is available
free of charge via the Internet at http://pubs.acs.org.
(1H, dd, J ) 8.4, 8.4 Hz, H-7), 6.99 (2H, d, J ) 9.9 Hz, H-2′,
H-6′), 6.81 (1H, d, J ) 8.4 Hz, H-8), 6.80 (1H, d, J ) 8.4 Hz,
H-6), 6.73 (1H, s, H-3), 6.42 (2H, d, J ) 9.9 Hz, H-3′, H-5′); 13C
NMR (CDCl3) δ 184.4 (C-4′), 183.5 (C-4), 166.4 (C-2), 160.8 (C-
5), 156.3 (C-9), 145.3 (C-2′, C-6′), 135.9 (C-7), 130.4 (C-3′, C-5′),
112.0 (C-6), 110.7 (C-10), 107.7 (C-3), 107.0 (C-8), 69.6 (C-1′).
5-Hydroxy-2-(1-methoxy-4-oxocyclohexa-2,5-dienyl)-4H-
chromen-4-one (23). 15% yield from 18; ESI-MS (m/z, %): 283
(M- - H, 25), 268 (M- - CH3, 8), 253 (M- - CH3 - OH, 100);
1H NMR (CDCl3) δ 12.29 (1H, s, OH-5), 7.50 (1H, dd, J ) 8.4,
8.4 Hz, H-7), 6.79 (2H, m, H-6, H-8), 6.78 (2H, d, J ) 9.9 Hz,
H-2′, H-6′), 6.70 (1H, s, H-3), 6.58 (2H, d, J ) 9.9 Hz, H-3′, H-5′),
3.41 (3H, s, OMe); 13C NMR (CDCl3) δ 184.3 (C-4′), 183.5 (C-
4), 165.1 (C-2), 160.7 (C-5), 156.2 (C-9), 145.1 (C-2′, C-6′), 135.7
(C-7), 133.4 (C-3′, C-5′), 111.8 (C-6), 110.8 (C-10), 108.2 (C-3),
107.0 (C-8), 74.8 (C-1′), 52.8 (OMe).
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6.61 (1H, d, J ) 2.7 Hz, H-8), 6.59 (2H, d, J ) 10.2 Hz, H-3′,
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1
100); H NMR (CDCl3) δ 9.92 (1H, dd, J ) 9.0, 1.0 Hz, H-5),
8.04 (1H, d, J ) 9.0 Hz, H-9), 7.88 (1H, dd, J ) 8.4, 1.2 Hz, H-8),
7.76 (1H, ddd, J ) 8.4, 8.4, 1.0 Hz, H-7), 7.63 (1H, ddd, J ) 9.0,
8.4, 1.2 Hz, H-6), 7.36 (1H, d, J ) 9.0 Hz, H-10), 7.02 (1H, s,
H-3), 7.00 (2H, d, J ) 10.2 Hz, H-2′, H-6′), 6.43 (2H, d, J ) 10.2
Hz, H-3′, H-5′); 13C NMR (CDCl3) δ 184.9 (C-4′), 180.4 (C-4),
163.3 (C-2), 157.6 (C-10a), 146.1 (C-2′, C-6′), 130.0 (C-3′, C-5′),
136.1, 130.7, 130.1, 129.6, 128.3, 127.0, 126.9 (C-4b, C-5, C-6,
C-7, C-8, C-8a, C-9), 117.2 (C-10), 117.1 (C-4a), 111.8 (C-3), 69.6
(C-1′).

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