Synthesis and anti-oxidant activity evaluation of (±)-Anastatins A, B and their analogs

Article abstract of DOI:10.1016/j.ejmech.2017.06.054

Two novel flavonoids (±)-Anastatins A and B as well as 14 analogs, which containing a benzofuran moiety, were synthesized by using halogenation, Suzuki coupling reaction and an oxidation/Oxa-Michael reaction cascade as the key steps. The structures of the new flavonoids were confirmed by ¹H NMR, ¹³C NMR and HRMS. The antioxidant activities of them as well as the key intermediates were evaluated by ferric reducing antioxidant power (FRAP) assay and the active compounds were evaluated in the PC12 cell model of hydrogen peroxide (H₂O₂)-induced oxidative damage. SAR studies suggested that, for in vitro antioxidant activity, aurone derivatives showed better bioactivity than flavone counterparts. However, cyclization to benzofuran and connecting the two conjugated parts as a whole conjugated system by a double bond diminished the in vitro antioxidant activity. Among them, the most potent compound 24c was significantly decreased H₂O₂-caused cell injury. The apoptotic rate (Annexin V⁺) of H₂O₂-damaged PC12 cells was 60.7% while that of the compound 24c-treated cells decreased to 5.9% and 4.1% at 10 μM and 100 μM respectively.

Full text of DOI:10.1016/j.ejmech.2017.06.054

Accepted Manuscript
Synthesis and anti-oxidant activity evaluation of (±)-Anastatins A, B and their analogs
Guojun Pan, Xuehui Li, Long Zhao, Meng Wu, Chao Su, Xuzhe Li, Yongmin Zhang,
Peng Yu, Yuou Teng, Kui Lu
PII:
S0223-5234(17)30508-1
10.1016/j.ejmech.2017.06.054
EJMECH 9549
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 19 April 2017
Revised Date: 23 June 2017
Accepted Date: 25 June 2017
Please cite this article as: G. Pan, X. Li, L. Zhao, M. Wu, C. Su, X. Li, Y. Zhang, P. Yu, Y. Teng, K. Lu,
Synthesis and anti-oxidant activity evaluation of (±)-Anastatins A, B and their analogs, European Journal
of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.06.054.
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ACCEPTED MANUSCRIPT
Graphical Abstract
Two novel flavonoids Anastatins A and B as well as their analogs were synthesized. The
antioxidant activities of these flavonoids and the key intermediates were evaluated by ferric
reducing antioxidant power (FRAP) assay and PC12 cell-based antioxidant assay.

ACCEPTED MANUSCRIPT
Synthesis and anti-oxidant activity evaluation of
(
±)-Anastatins A, B and their analogs
a
a
a
a
a
a
Guojun Pan , Xuehui Li , Long Zhao , Meng Wu , Chao Su , Xuzhe Li , Yongmin
Zhang , Peng Yu , Yuou Teng *, Kui Lu *
a,b
a
a
a
a
China International Science and Technology Cooperation Base of Food
Nutrition/Safety and Medicinal Chemistry, Sino-French Joint Lab of Food
Nutrition/Safety and Medicinal Chemistry, Key Laboratory of Industrial
Fermentation Microbiology of Ministry of Education, Tianjin Key Laboratory of
Industry Microbiology, Tianjin University of Science and Technology, Tianjin
3
b
00457, China
Universite Pierre et Marie Curie-Paris 6, Institut Parisien de Chimie Moleculaire
UMR CNRS 8232, 4 place Jussieu, 75005, Paris, France
*
Corresponding author.
Tel.: +86 22 60912562, Fax:+86 22 60602298
E-mail: tyo201485@tust.edu.cn (Yuou Teng), lukui@tust.edu.cn (Kui Lu)
1

ACCEPTED MANUSCRIPT
Abstract:
Two novel flavonoids (±)-Anastatins A and B as well as 14 analogs,
which containing a benzofuran moiety, were synthesized by using
halogenation, Suzuki coupling reaction and an oxidation/Oxa-Michael
reaction cascade as the key steps. The structures of the new flavonoids
1
13
were confirmed by H NMR, C NMR and HRMS. The antioxidant
activities of them as well as the key intermediates were evaluated by ferric
reducing antioxidant power (FRAP) assay and the active compounds were
evaluated in the PC12 cell model of hydrogen peroxide (H O )-induced
2
2
oxidative damage. SAR studies suggested that, for in vitro antioxidant
activity, aurone derivatives showed better bioactivity than flavone
counterparts. However, cyclization to benzofuran and connecting the two
conjugated parts as a whole conjugated system by a double bond
diminished the in vitro antioxidant activity. Among them, the most potent
compound 24c was significantly decreased H O -caused cell injury. The
2
2
+
apoptotic rate (Annexin V ) of H O -damaged PC12 cells was 60.7%
2
2
while that of the compound 24c-treated cells decreased to 5.9% and 4.1%
at 10 µM and 100 µΜ respectively.
Keywords: Anastatins A, B; Flavone; Aurone; Antioxidant
2

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1
. Introduction
Flavonoids are naturally occurring polyphenolic compounds exists in
plants such as parsley, onion, orange, chamomile and tea.[1] Many of
them are reported to have a variety of pharmacological activities
including antioxidant, anti-cancer, anti-viral and anti-inflammatory
properties.[2-9] Two novel flavonoids Anastatins A (1) and Anastatins B
(
2), which containing a benzofuran moiety and an S-configuration, were
isolated from Anastatica hierochuntica in Egypt by Yoshikawa et al. in
003 (Scheme 1). Preliminary biological evaluation revealed that these
2
two compounds showed hepatoprotective effects on D-galactosamine
induced cytotoxicity in primary cultured mouse hepatocytes. Moreover,
their hepatoprotective activities were stronger than the well-known and
commercial hepatoprotective drug silybin.[10]
Liver injury can cause by many factors such as oxidative stress,
inflammatory, drugs, excessive intake of ethanol, infection of
virus.[11-13] The accumulation of free radicals is a major pathogenetic
event occuring in the pathophysiology of various liver injuries. The main
sources of the reactive oxygen species (ROS) are represented by the
mitochondria and cytochrome P450 enzymes in the hepatocyte, Kupffer
cells and neutrophils.[14-16] Overproduction of ROS results in oxidative
stress, a deleterious process that can cause membrane lipid peroxidation
3

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(a hallmark of hepatotoxicity), decrease membrane fluidity, suppress
enzyme and receptor activity, and damage membrane proteins, which
finally triggers cell inactivation and death.[17-19] It is important to
develop new antioxidant drugs to prevent or impair the accumulation of
free radicals for hepatoprotection.
As mentioned above, Anastatins A and B showed hepatoprotective
activities which may be caused by their antioxidant activity. Some
aurones, also belong to flavonoids, have been reported to exhibit better
antioxidant activity than their flavone counterparts do.[20] Therefore,
replace the flavone with aurone might increase their antioxidant activities.
In this study, we synthesized Anastatins A and B as well as their
analogs by using halogenation, Suzuki coupling reaction and an
oxidation/Oxa-Michael reaction cascade as the key steps and subsequently
evaluated the antioxidant activity by ferric reducing antioxidant power
(
FRAP) assays and H O -induced PC12 cell oxidative damage model.
2
2
Based on this data, preliminary SAR was analyzed.
2
. Chemistry
Anastatins A and Anastatins B were synthesized according to our
previous strategy (Scheme 2 and 3).[21] The 8-iodo substituted apigenin
derivative (5) and 6-iodo apigenin derivative (7) were readily
4

ACCEPTED MANUSCRIPT
synthesized from commercially available apigenin (3) by protection and
iodination sequence according to our previous procedures.[22] With the
iodo compounds 5 and 7 in hand, benzo[d][1,3] dioxol-5-ylboronic acid
(
8) was coupled with 5 and 7 by using Pd(PPh ) as the catalyst and
3
4
Cs CO as the base in DMF at 100 °C to give compounds 12 and 9
2
3
respectively. After dealkyation, oxidation/Oxa-Michael reaction cascade
and hydrogenation, (±)-Anastatins A and B were obtained in 54% and
6
6% overall yield from 12 and 9 respectively (Scheme 3). Then we put
our efforts to synthesize the analogs of (±)-Anastatins A and B by
replacing a flavone core with aurone. 5-bromo aurone derivatives
(
17a-17c) and 7-bromo aurone derivatives (18a-18c) were prepared from
the commercial available aurones (15a-15c) by isopropyl protection and
bromination (Scheme 4). With the bromo compounds (17a-18c) in hand,
the synthesis of the analogs of (±)-Anastatins A and B (22a-22c and
2
6a-26c) was carried out by similar approach (Scheme 5 and 6). Notably,
in order to investigate the influence of cyclization (20c' versus 22c and
2
2
4c' versus 26c), compounds 20c and 24c were reduced to compounds
0c' and 24c' by hydrogenation.
Results and discussion
The antioxidant activity of our newly synthesized analogs of Anastatins
A and B was evaluated using the ferric reducing antioxidant power (FRAP)
5

ACCEPTED MANUSCRIPT
assays using Gallic acid as positive control and the results are shown in
Table 1. Compound 24c showed similar activity in the FRAP assay as
positive control did. Moreover, compounds 20c, 20c', 22c, 24c' and 26c
exhibited better activity in the FRAP assay than positive control did.
Before the cell-based antioxidant test of these six compounds, the toxicity
of them were evaluated using rat pheochromocytoma cells (PC12). The
result revealed that compound 22c showed cytotoxicity and 26c could
promote cell growth (Figure 1 A).
Based on this information, antioxidant activity of other four compounds
(
(
20c, 20c', 24c and 24c') was evaluated in H O -damaged PC12 cells
2
2
Figure 1B) at a concentration of 100 µΜ and 10 µΜ. Gallic acid was used
as a positive control, and the results are illustrated in Figure 1C and 1D. To
our delight, 20c, 20c', 24c and 24c' showed better antioxidant activity than
Gallic acid. The viability of PC12 cells was nearly 100% after a 2 h
exposure to 100 µM H O when treated with these four compounds at 100
2
2
µM level. Moreover, compounds 20c' and 24c showed better antioxidant
activity than Gallic acid at 10 µM level. The viability of the
2
0c'-pretreated and 24c-pretreated PC12 cells was 61.46% and 85.94%
respectively.
Oxidative stress induces apoptosis rather than necrosis in PC12 cells.
The effect of compound 24c on the morphological alterations was
6

ACCEPTED MANUSCRIPT
observed in H O -injured PC12 cells. As shown in Figure 2A, the typical
2
2
apoptotic cells such as cell shrinkage, blebbing were clearly observed in
PC12 cells after treatment with H O (100 µM) for 2 h. Moreover,
2
2
morphological changes in the gallic acid and compound 24c-pretreated
PC12 cells suggested that both gallic acid and compound 24c significantly
protected them against H O -induced cell injury. These results indicate
2
2
that compound 24c might protect PC12 cells against oxidative stress injury
caused by H O .
2
2
To further confirm the effects of compound 24c on apoptosis of
pretreated H O -damaged PC12 cells, they were double-labeled with
2
2
Annexin V-FITC and propidium iodide (PI) and then analyzed using flow
+
cytometry (Figure 2B). The apoptotic rate (Annexin V ) of the compound
-
untreated H O -damaged PC12 cells was 60.7% while that of the
2
2
compound 24c -treated cells decreased to 5.9% of the total cells compared
to 12.5% observed in the gallic acid-treated control cells (10 µM).
Pretreated with compound 24c protected the PC12 cell against H O2
2
oxidation and the apoptotic rate decreased to 4.1% at 100 µΜ, similar to
that of gallic acid (2.8%). These results suggest that compound 24c
protected the H O -injured PC12 cells against apoptosis.
2
2
Based on these data, the preliminary structure-activity relationships
SAR) was analyzed. The results are illustrated as following: 1) As we
(
7

ACCEPTED MANUSCRIPT
expected, compound bearing an aurone core showed better in vitro
antioxidant activity than compound bearing a flavone core (21b versus 11
and 25b versus 14). 2) In most of the case, the 7-hydroxy group for flavone
derivatives and 6-hydroxy group for aurone derivatives could improve the
in vitro antioxidant activity (10 versus 11, 13 versus 14, 20a versus 21a,
2
0c versus 21c, 24a versus 25a and 24c versus 25c). 3) In most of the case,
the α, β-unsaturated double bond, which connected the two conjugated part
as a whole conjugated system, could diminish the in vitro antioxidant
activity (22a versus 21a, 20c' versus 20c, 22c versus 21c, Anastatins A
versus 11, 26a versus 25a, 24c' versus 24c, 26c versus 25c and
Anastatins B versus 14).
4
. Conclusions
In summary, Anastatins A and B as well as their analogs (22a-c and
6a-c) were synthesized from the commercially available apigenin and
2
aurone derivatives respectively. The in vitro antioxidant activity as well
as PC12 cell-based antioxidant activity evaluation of these compounds
and the key intermediates illustrated the following. For in vitro
antioxidant activity, aurone derivatives showed better bioactivity than
flavone counterparts. However, cyclization to benzofuran and connecting
the two conjugated part as a whole conjugated system by a double bond
diminished the bioactivity. For PC12 cell-based antioxidant activity,
8

ACCEPTED MANUSCRIPT
compound 20c, 20c', 24c and 24c' showed better activity than Gallic acid
at 100 µM level. Moreover, the most potent compound, 24c protected the
PC12 cell against H O oxidation by 85.94% at 10 µΜ. Clarification the
2
2
mechanism of action of compound 24c and identify its target molecule is
underway in our lab.
5
5
. Experimental section
.1. Chemistry
All commercial materials and reagents were used without further
purification, unless otherwise stated. All solvents were distilled prior to use.
The solvents for reaction were distilled to remove water over Na or CaH2.
All reactions were carried out in oven-dried glassware under an inert
atmosphere (nitrogen or argon). For chromatography, 200-300 mesh silica
1
13
gel (Qingdao, China) was employed. H and C NMR spectra were
recorded at 400 MHz and 100 MHz with a Brucker ARX 400 spectrometer.
1
The chemical shifts (δ) for H NMR spectra were given in parts per million
(ppm) referenced to the residual proton signal of the duterated solvent
(
CDCl at δ = 7.26 ppm, DMSO-d at δ = 2.50 ppm and Acetone-d at δ =
3
6
6
2
.05 ppm); coupling constants were expressed in hertz l= 77.0 ppm),
DMSO-d (δ = 40.0 ppm) and Acetone-d (δ = 30.0 ppm and 206.0 ppm).
6
6
The following abbreviations are used to describe NMR signals: s = singlet,
d = doublet, t = triplet, m = multiple, and dd = doublet of doublets. HRMS
9

ACCEPTED MANUSCRIPT
were recorded on Bruker Daltonics, Inc. APEXIII 7.0 TESLA FTMS (ESI).
Known products were characterized by comparing to their corresponding
1
H-NMR reported in the literatures.
5
.1.1 (±)-Anastatins B
Pd/C (10%) 300 mg was added to a solution of 14 (376 mg, 1.0
mmol) in Tetrahydrofuran (THF) (10 mL) and the mixture was stirred at
0℃ under hydrogen at balloon pressure for 12 h. Then the suspension
3
was filtrated by Celite and concentrated under reduced pressure to afford
1
(
±)-Anastatins B (351 mg, 93%) as a yellow solid. H NMR (400 MHz,
Acetone-d ) δ 12.18 (s, 1H), 8.64 (s, 1H), 8.20 (s, 2H), 7.54 (d, J = 8.0 Hz,
6
2
5
H), 7.27 (s, 1H), 7.05 (s, 1H), 6.98 (d, J = 8.0 Hz, 2H), 6.58 (s, 1H),
.77 (dd, J = 13.2, 2.8 Hz, 1H), 3.41 (dd, J = 17.2, 13.2 Hz, 1H), 2.92 (dd,
13
J = 17.2, 2.8 Hz, 1H); C NMR (100 MHz, Acetone-d ) δ 198.6, 163.9,
6
1
1
62.6, 159.0, 157.4, 151.0, 146.0, 143.4, 130.7, 129.2, 116.5, 114.9,
07.6, 106.9, 105.0, 99.3, 93.0, 80.9, 43.9; HRMS (ESI) m/z: Calcd for
-
C H O [M-H] 377.0667; found, 377.0688.
2
1
13
7
5
.1.2 (±)-Anastatins A
The procedure is same as the preparation of (±)-Anastatins B by
using 11 (100 mg) as substrate to give compound (±)-Anastatins A (91
1
mg, 91%) as a yellow solid. H NMR (400 MHz, Acetone-d ) δ 12.93 (s,
6
1
H), 8.59 (s, 1H), 8.32 (s, 1H), 8.18 (s, 1H), 7.46(s, 1H), 7.44 (d, J = 8.4
1
0

ACCEPTED MANUSCRIPT
Hz, 2H), 7.07 (s, 1H), 6.92 (d, J = 8.4 Hz, 2H), 6.64 (s, 1H), 5.57 (dd, J =
1
3.2, 2.8 Hz, 1H), 3.34 (dd, J = 17.2, 13.2 Hz, 1H), 2.87 (dd, J = 17.2, 2.8
13
Hz, 1H); C NMR (100 MHz, Acetone-d ) δ 199.6, 163.6, 161.6, 158.8,
6
1
1
58.4, 151.0, 145.9, 143.5, 130.7, 129.1, 116.2, 115.0, 108.0, 107.8,
05.0, 99.3, 92.4, 80.4, 43.9; HRMS (ESI) m/z: Calcd for C H O
2
1
13
7
-
[
M-H] 377.0667; found, 377.0676.
5
.1.3 5,7-diisopropoxy-2-(4-isopropoxyphenyl)-4H-chromen-4-one (4)
K CO (5.39 g, 39 mmol) and (CH ) CHI (6.63 g, 39 mmol) were
2
3
3 2
added to a stirred solution of Apigenin 3 (2.70 g, 10 mmol) in dry N,
N-Dimethylformamide (DMF) (10 mL). After the addition, the mixture
was heated to 45 °C and stirred for 24 h. The reaction was cooled to room
temperature, filtered and diluted with ethyl acetate (100 mL) and the
resulting solution was poured into aqueous HCl (1 M, 100 mL). The
organic layer was separated, washed with saturated NaCl (3×100 mL) and
dried over anhydrous Na SO . After concentrated to dryness under
2
4
reduced pressure, the crude product was purified by column
chromatography on silica gel (petroleum ether/ethyl acetate 40:1) to
1
afford the known compound 4 (3.25 g, 82%) as a yellow solid. H NMR
(
400 MHz, CDCl ) δ 7.79 (d, J = 8.8 Hz, 2H), 6.96 (d, J = 8.8 Hz, 2H), 6.52
3
(d, J = 2.4 Hz, 1H), 6.50 (s, 1H), 6.35 (d, J = 2.4 Hz, 1H), 4.69 ~ 4.55 (m,
3
H), 1.45 (d, J = 6.0 Hz, 6H), 1.40 (d, J = 6.0 Hz, 6H), 1.38 (d, J = 6.0 Hz,
1
1

ACCEPTED MANUSCRIPT
1
3
6
1
2
H); C NMR (100 MHz, CDCl ) δ 177.4, 162.0, 160.5, 160.4, 159.9,
3
59.4, 127.6, 123.7, 115.8, 110.1, 107.6, 100.8, 94.5, 72.5, 70.5, 70.1,
+
2.0, 21.9; LRMS (ESI) m/z 397 [M+H] .
5
.1.4
8
-iodo-5,7-diisopropoxy-2-(4-isopropoxyphenyl)-4H-chromen-4-one(5)
N-Iodosuccinimide (NIS) (2.47 g, 10.99 mmol) was added to a
solution of 4 (3.97 g, 10 mmol) in dry DMF (25 mL) and the resulting
solution was stirred at 70℃ for 10h. The reaction mixture was then
poured into water (200 mL) at 0 °C and extracted with Dichloromethane
(
DCM) (3×50 mL), the combined organic layers were washed with brine
3×100 mL), dried over Na SO and concentrated under reduced pressure.
(
2
4
The crude products was purified by column chromatography on silica
(petroleum ether/ethyl acetate 30:1) to afford compound 5 (4.49 g, 86%)
1
as yellow solid. H NMR (400 MHz, CDCl ) δ 7.93 (d, J = 8.8 Hz, 2H),
3
6
1
.99 (d, J = 8.8 Hz, 2H), 6.56 (s, 1H), 6.49 (s, 1H), 4.74 ~ 4.53 (m, 3H),
1
3
.46 (d, J = 6.0Hz, 12H), 1.38 (d, J = 6.0 Hz, 6H); C NMR (100 MHz,
CDCl ) δ 177.1, 160.8, 160.7, 158.6, 158.4, 155.6, 128.0, 123.2, 115.9,
3
1
11.5, 106.8, 100.1, 93.5, 73.7, 72.5, 70.1, 22.04, 21.95, 21.90; HRMS
+
(
ESI) m/z: Calcd for C H O INa [M+Na] 545.0795; found, 545.0820.
2
4
27
5
5
.1.5
-hydroxy-7-isopropoxy-2-(4-isopropoxyphenyl)-4H-chromen-4-one(6)
5
1
2

ACCEPTED MANUSCRIPT
K CO (3.31 g, 24 mmol) and (CH ) CHI (4.08 g, 24 mmol) were
2
3
3 2
added to a stirred solution of Apigenin 3 (2.70 g, 10 mmol) in dry DMF
10 mL). After the addition, the mixture was stirred at room temperature
(
for 30 h. The reaction was filtered and diluted with ethyl acetate (100
mL) and the resulting solution was poured into aqueous HCl (1 M, 100
mL). The organic layer was separated, washed with saturated NaCl
(
3×100 mL) and dried over anhydrous Na SO . After concentrated to
2
4
dryness under reduced pressure, the crude product was purified by
column chromatography on silica gel (petroleum ether/ethyl acetate 10:1)
to afford the known compound 6 (3.36 g, 95%) as a yellow solid.
5
.1.6
6
-iodo-5,7-diisopropoxy-2-(4-isopropoxyphenyl)-4H-chromen-4-one(7)
NIS (2.47 g, 10.99 mmol) was added to a solution of 6 (3.54 g, 10
mmol) in dry DMF (25 mL) and the resulting solution was stirred at
ambient temperature for 10h. The reaction mixture was then poured into
water (200 mL) and extracted with DCM (3×50 mL), the combined
organic layers were washed with brine (3×100 mL), dried over Na SO and
2
4
concentrated under reduced pressure. The crude products was used in the
next step without further purification. K CO (1.04 g, 7.5 mmol) and
2
3
(
CH ) CHI (1.27 g, 7.5 mmol) were added to a stirred solution of rude
3
2
product (2.40 g, 5 mmol) in dry DMF (10 mL). After the addition, the
1
3

ACCEPTED MANUSCRIPT
mixture was heated to 45 °C and stirred for 24 h. The reaction was cooled
to room temperature, filtered and diluted with ethyl acetate (100 mL) and
the resulting solution was poured into aqueous HCl (1 M, 100 mL). The
organic layer was separated, washed with saturated NaCl (3×100 mL) and
dried over anhydrous Na SO . After concentrated to dryness under
2
4
reduced pressure, the crude product was purified by column
chromatography on silica gel (petroleum ether/ethyl acetate 80:1) to
1
afford the compound 7 (1.12 g, 43%) as a white solid. H NMR (400
MHz, CDCl ) δ 7.80 (d, J = 9.2 Hz, 2H), 6.98 (d, J = 9.2 Hz, 2H), 6.71 (s,
3
1
6
1
8
H), 6.55 (s, 1H), 4.78 ~ 4.62 (m, 3H), 1.49 (d, J = 6.0 Hz, 6H), 1.43 (d, J =
1
3
.0 Hz, 6H), 1.38 (d, J = 6.0Hz, 6H); C NMR (100 MHz, CDCl ) δ 176.1,
3
61.0, 160.7, 160.5, 159.9, 158.4, 127.7, 123.2, 115.9, 112.7, 107.1, 96.8,
6.6, 79.4, 72.6, 70.2, 22.5, 22.0, 21.9; HRMS (ESI) m/z: Calcd for
+
C H O INa [M+Na] 545.0795; found, 545.0820.
2
4
27
5
5
.1.7
-(benzo[d][1,3]dioxol-5-yl)-5,7-diisopropoxy-2-(4-isopropoxyphenyl)-4
6
H-chromen-4-one (9)
A mixture of compounds 7 (5.22 g, 10 mmol), 8 (2.16 g, 13 mmol),
Pd(PPh ) (0.346 g, 0.3 mmol) and Cs CO (6.53 g, 20 mmol) in DMF
3
4
2
3
(20 mL) was heated to 100 °C and stirred for 12 h under argon. After
cooled to room temperature, the reaction mixture was poured into
1
4

ACCEPTED MANUSCRIPT
ice-water (200 mL) and extracted with DCM (3×50 mL). The combined
organic layers were washed with brine (100 mL), dried over Na SO and
2
4
concentrated under reduced pressure. The crude product was purified by
column chromatography on silica gel (petroleum ether/ethyl acetate 30:1)
1
to afford compound 9 (3.57 g, 69%) as a yellow solid. H NMR (400
MHz, CDCl ) δ 7.82 (d, J = 8.8 Hz, 2H), 6.98 (d, J = 8.8 Hz, 2H), 6.91 ~
3
6
4
1
1
1
2
5
.89 (m, 3H), 6.78 (s, 1H), 6.56 (s, 1H), 6.00 (s, 2H), 4.68 ~ 4.57 (m, 2H),
.17 ~ 4.11 (m, 1H), 1.38 (d, J = 6.0 Hz, 6H), 1.33 (d, J = 6.0 Hz, 6H),
1
3
.01 (d, J = 6.0 Hz, 6H); C NMR (100 MHz, CDCl ) δ 177.3, 160.7,
3
60.5, 159.6, 158.3, 155.8, 146.6, 146.2, 127.7, 127.3, 125.4, 124.3,
23.6, 115.9, 112.9, 112.2, 107.4, 107.4, 100.8, 97.1, 76.7, 71.2, 70.2,
+
2.0, 22.0, 21.8; HRMS (ESI) m/z: Calcd for C H O Na [M+Na]
31
32
7
39.2040; found, 539.2063.
5
.1.8
6
-(3,4-dihydroxyphenyl)-5,7-dihydroxy-2-(4-hydroxyphenyl)-4H-chromen
-4-one (10)
Boron tri-chloride (70 mL, 70 mmol, 1M solution in hexane) was
added to a stirred solution of 9 (5.17 g, 10 mmol) in anhydrous DCM (20
mL) under 0 °C for 30 min. After addition, the reaction mixture was
heated to reflux for 4h. Then the reaction was cooled to room temperature,
excess ice-water was added and stirred for 10 min. Then the suspension
1
5

ACCEPTED MANUSCRIPT
was filtrated and the residue was dried to afford compound 10 (3.62 g,
1
9
1
2
6
1
1
6%) as a yellow solid. H NMR (400 MHz, DMSO-d ) δ 13.30 (s, 1H),
6
0.50 (s, 2H), 8.82 (s, 2H), 7.94 (d, J = 8.0 Hz, 2H), 6.93 (d, J = 8.0 Hz,
H), 6.79 (s, 1H), 6.75 (s, 1H), 6.74 (d, J = 5.2 Hz, 1H), 6.61 (s, 1H),
1
3
.60 (d, J = 5.2 Hz, 1H); C NMR (100 MHz, DMSO-d ) δ 182.0, 163.6,
6
61.7, 161.2, 158.5, 155.6, 144.4, 144.2, 128.5, 123.1, 122.0, 121.3,
18.5, 116.0, 115.0, 112.8, 103.7, 102.9, 93.6; HRMS (ESI) m/z: Calcd
-
for C H O [M-H] 377.0667; found, 377.0673.
21
13
7
5
.1.9
,7,8-trihydroxy-2-(4-hydroxyphenyl)-4H-benzofuro[3,2-g]chromen-4-on
5
e(11)
Ag O (463 mg, 2.0 mmol) was added to a solution of 10 (378 mg,
2
1
5
.0 mmol) in DMF (10 mL) and the resulting mixture was heated to
0 °C and stirred for 6 h. After cooled to room temperature, EA (50 mL)
was added to the reaction mixture and stirred for 30 min. The reaction
suspension was filtered through celite and washed with EtOAc, the
organic phase was poured into water (100 mL) and extracted with EtOAc
(5×100 mL). The combined organic layer was washed with brine (5×50
mL), dried over Na SO and concentrated under reduced pressure to
2
4
1
afford compound 11 (232 mg, 62%) as a black green solid. H NMR (400
MHz, DMSO-d ) δ 13.88 (s, 1H), 10.41 (s, 1H), 9.31 (s, 2H), 8.02 (d, J =
6
1
6

ACCEPTED MANUSCRIPT
8
2
1
1
.4 Hz, 2H), 7.46 (s, 1H), 7.41 (s, 1H), 7.09 (s, 1H), 6.96 (d, J = 7.2 Hz,
1
3
H), 6.95 (s, 1H); C NMR (100 MHz, DMSO-d ) δ 183.8, 165.0, 161.9,
6
60.1, 154.7, 154.6, 150.1, 146.6, 143.5, 129.2, 121.6, 116.5, 112.9,
09.5, 107.5, 106.0, 102.9, 99.2, 91.7; HRMS (ESI) m/z: Calcd for
-
C H O [M-H] 375.0510; found, 375.0526.
2
1
11
7
5
.1.10
-(benzo[d][1,3]dioxol-5-yl)-5,7-diisopropoxy-2-(4-isopropoxyphenyl)-4
8
H-chromen-4-one (12)
The procedure is same as the preparation of 9 by using 5 (5.22 g, 10
mmol) as substrate to give compound 12 (3.82 g, 74%) as a yellow solid.
1
H NMR (400 MHz, CDCl ) δ 7.50 (d, J = 8.8 Hz, 2H), 6.95 ~ 6.89 (m, 3H),
3
6
.84 (d, J = 8.8 Hz, 2H), 6.52 (s, 1H), 6.51 (s, 1H), 6.05 (s, 2H), 4.65 ~ 4.53
(
m, 3H), 1.49 (d, J = 6.0 Hz, 6H), 1.34 (d, J = 6.0 Hz, 6H), 1.29 (d, J = 6.0
1
3
Hz, 6H); C NMR (100 MHz, CDCl ) δ 177.8, 160.6, 160.4, 159.0, 158.2,
3
1
1
56.3, 147.1, 146.5, 127.6, 126.2, 124.7, 123.5, 115.8, 113.3, 111.8, 110.6,
07.8, 106.5, 101.0, 100.1, 73.5, 71.4, 70.0, 22.02, 22.01, 21.95; HRMS
+
(
ESI) m/z: Calcd for C H O Na [M+Na] 539.2040; found, 539.2065.
3
1
32
7
5
8
.1.11
-(3,4-dihydroxyphenyl)-5,7-dihydroxy-2-(4-hydroxyphenyl)-4H-chromen
-4-one (13)
1
7

ACCEPTED MANUSCRIPT
The procedure is same as the preparation of 10 by using 12 (5.17 g,
1
0 mmol) as substrate to give compound 13 (3.66 g, 97%) as a black-red
1
solid. H NMR (400 MHz, DMSO-d ) δ 13.12 (s, 1H), 10.65 (s, 1H),
6
1
6
0.37 (s, 1H), 8.98 (s, 2H), 7.67 (d, J = 8.8 Hz, 2H), 6.85 ~ 6.80 (m, 5H),
13
.72 (dd, J = 8.4, 2.0 Hz, 1H), 6.38 (s, 1H); C NMR (100 MHz,
DMSO-d ) δ 182.3, 163.7, 161.3, 161.1, 159.7, 154.0, 144.7, 144.4, 128.3,
6
1
22.5, 122.1, 121.3, 118.5, 115.8, 115.1, 108.3, 103.7, 102.4, 98.7;
-
HRMS (ESI) m/z: Calcd for C H O [M-H] 377.0667; found, 377.0688.
2
1
13
7
5
.1.12
,9,10-trihydroxy-2-(4-hydroxyphenyl)-4H-benzofuro[2,3-h]chromen-4-o
5
ne (14)
The procedure is same as the preparation of 11 by using 13 (378 mg)
1
as substrate to give compound 14 (274 mg, 73%) as a red solid. H NMR
(
400 MHz, DMSO-d ) δ 13.20 (s, 1H), 10.56 (s, 1H), 9.52 (s, 2H), 8.10 (d,
6
J = 8.8 Hz, 2H), 7.56 (s, 1H), 7.12 (s, 1H), 7.06 (d, J = 8.8 Hz, 2H), 7.05
13
(
s, 1H); C NMR (100 MHz, DMSO-d ) δ 182.4, 163.9, 161.4, 159.7,
6
1
1
58.5, 149.5, 149.1, 145.6, 143.2, 128.4, 120.9, 116.1, 111.8, 106.1,
05.9, 104.8, 103.8, 98.8, 95.0; HRMS (ESI) m/z: Calcd for C H O
2
1
11
7
-
[
M-H] 375.0510; found, 375.0525.
5
.1.13 (Z)-2-benzylidene-4,6-diisopropoxybenzofuran-3(2H)-one(16a)
1
8

ACCEPTED MANUSCRIPT
K CO (3.59 g, 26 mmol) and (CH ) CHI (4.42 g, 26 mmol) were
2
3
3 2
added to a stirred solution of 15a (2.54 g, 10 mmol) in dry DMF (10 mL).
After the addition, the mixture was heated to 45 °C and stirred for 24 h.
The reaction was cooled to room temperature, filtered and diluted with
ethyl acetate (100 mL) and the resulting solution was poured into aqueous
HCl (1 M, 100 mL). The organic layer was separated, washed with
saturated NaCl (3×100 mL) and dried over anhydrous Na SO . After
2
4
concentrated to dryness under reduced pressure, the crude product was
purified by column chromatography on silica gel (petroleum ether/ethyl
acetate 40:1) to afford the known compound 16a (2.98 g, 88%) as a
1
yellow solid. H NMR (400 MHz, CDCl ) δ 7.88 ~ 7.85 (m, 2H), 7.50 ~
3
7
1
6
.48 (m, 3H), 6.61 (s, 1H), 6.54 (d, J = 2.0 Hz, 1H), 6.36 (d, J = 2.0 Hz,
H), 4.69 ~ 4.55 (m, 2H), 1.46 (d, J = 6.0 Hz, 6H), 1.41 (d, J = 6.0 Hz,
H).
5
.1.14
(Z)-4,6-diisopropoxy-2-(4-isopropoxybenzylidene)benzofuran-3(2H)-one
(
16b)
The procedure is same as the preparation of 16a by using 15b (2.70 g,
1
0 mmol) as substrate to give known compound 16b (3.09 g, 78%) as a
1
yellow solid. H NMR (400 MHz, CDCl ) δ 7.81 (d, J = 8.8 Hz, 2H), 6.93
3
(d, J = 8.8 Hz, 2H), 6.68 (s, 1H), 6.32 (d, J = 1.6 Hz, 1H), 6.09 (d, J = 1.6
1
9

ACCEPTED MANUSCRIPT
Hz, 1H), 4.75 ~ 4.59 (m, 3H), 1.43 (d, J = 6.0 Hz, 6H), 1.40 (d, J = 6.0 Hz,
6
H), 1.36 (d, J = 6.0 Hz, 6H).
5
.1.15
(Z)-2-(3,4-diisopropoxybenzylidene)-4,6-diisopropoxybenzofuran-3(2H)-
one(16c)
The procedure is same as the preparation of 16a by using 15c (2.86 g, 10
mmol) as substrate to give known compound 16c (3.77 g, 83%) as a
1
yellow solid. H NMR (400 MHz, CDCl ) δ 7.41 ~ 7.36 (m, 2H), 7.46 ~
3
7
1
6
.38 (m, 3H), 6.87 (d, J = 8.4 Hz, 1H), 6.57 (s, 1H), 6.23 (d, J = 1.6 Hz,
H), 6.02 (d, J = 1.6 Hz, 1H), 4.69 ~ 4.39 (m, 4H), 1.36 (d, J = 6.0 Hz,
H), 1.33 (d, J = 6.0 Hz, 6H), 1.31 (d, J = 6.0 Hz, 6H), 1.30 (d, J = 6.4
1
3
Hz, 6H); C NMR (101 MHz, CDCl ) δ 179.3, 167.9, 165.9, 157.2,
3
1
7
49.6, 147.7, 146.1, 125.1, 124.8, 120.1, 115.8, 109.4, 104.8, 96.4, 89.3,
1.7, 71.1, 70.8, 70.0, 21.2, 21.1, 20.9, 20.9.
5
.1.16
(
Z)-5-bromo-2-benzylidene-4,6-diisopropoxy-benzofuran-3(2H)-one (17a)
and (Z)-7-bromo-2-benzylidene-4,6-diisopropoxy-benzofuran-3(2H)-one
18a)
N-Bromosuccinimide (NBS) (1.1 mmol) was added to a solution of
6a (3.38 g, 10 mmol) in dry DCM (25 mL) and the resulting solution
was stirred at room temperature for 30 min. The reaction mixture was
(
1
2
0

ACCEPTED MANUSCRIPT
then poured into water (200 mL) at 0 °C and extracted with DCM (3×50
mL), the combined organic layers were washed with brine (100 mL),
dried over Na SO and concentrated under reduced pressure. The crude
2
4
products was purified by column chromatography on silica gel
petroleum ether/ethyl acetate 40:1) to afford compound 18a (2.13 g,
(
5
1%) and compound 17a (1.83 g, 44%) as yellow solids respectively.
1
Data for 17a: H NMR (400 MHz, CDCl ) δ 7.87 (d, J = 8.4 Hz, 2H),
3
7
.46 ~ 7.36 (m, 3H), 6.77 (s, 1H), 6.55 (s, 1H), 5.25 ~ 5.14 (m, 1H), 4.76
~4.66 (m, 1H), 1.48 (d, J = 6.0 Hz, 6H), 1.42 (d, J = 6.0 Hz, 6H). Data for
1
1
8a: H NMR (400 MHz, CDCl ) δ 7.93 (d, J = 8.4 Hz, 2H), 7.48 ~ 7.36
3
(
m, 3H), 6.77 (s, 1H), 6.21 (s, 1H), 4.89 ~ 4.81 (m, 1H), 4.76 ~ 4.64 (m,
1
H), 1.46 (d, J = 6.0 Hz, 6H), 1.44 (d, J = 6.0 Hz, 6H).
5
.1.17
(
Z)-5-bromo-4,6-diisopropoxy-2-(4-isopropoxybenzylidene)benzofuran-3(
H)-one (17b)
Z)-7-bromo-4,6-diisopropoxy-2-(4-isopropoxybenzylidene)benzofuran-3(
H)-one (18b)
The procedure is same as the preparation of 18a by using 16b (3.97
2
(
2
g, 10 mmol) as substrate to give known compound 18b (2.52 g, 53%) and
compound 17b (2.14 g, 45%) as yellow solids respectively. Data for 17b:
1
H NMR (400 MHz, CDCl ) δ 7.81 (d, J = 8.8 Hz, 2H), 6.94 (d, J = 8.8
3
2
1

ACCEPTED MANUSCRIPT
Hz, 2H), 6.74 (s, 1H), 6.54 (s, 1H), 5.30 - 5.12 (m, 1H), 4.77 - 4.55 (m,
2
H), 1.48 (d, J = 6.1 Hz, 6H), 1.41 (d, J = 6.1 Hz, 6H), 1.37 (d, J = 6.0
1
3
Hz, 6H); C NMR (101 MHz, CDCl ) δ 179.7, 167.3, 162.4, 159.3,
3
1
7
54.9, 146.3, 133.1, 124.7, 116.0, 111.9, 108.2, 102.7, 92.2, 79.1, 72.9,
+
0.0, 22.7, 22.0, 21.9. LRMS (ESI) m/z 475 [M+H] ; HRMS (ESI) m/z:
+
Calcd for C H O Br [M+H] 475.1115; found, 475.1119. Data for 18b:
2
4
28
5
1
H NMR (400 MHz, CDCl ) δ 7.87 (d, J = 8.8 Hz, 2H), 6.96 (d, J = 8.8
3
Hz, 2H), 6.75 (s, 1H), 6.20 (s, 1H), 4.91 – 4.79 (m, 1H), 4.72 – 4.58 (m,
H), 1.45 (d, J = 6.1 Hz, 6H), 1.43 (d, J = 6.1 Hz, 6H), 1.37 (d, J = 6.0
2
1
3
Hz, 6H); C NMR (101 MHz, CDCl ) δ 180.2, 164.7, 162.5, 159.3,
3
1
7
57.3, 146.4, 133.3, 124.8, 116.1, 111.9, 107.5, 96.5, 86.9, 73.4, 72.9,
+
0.0, 22.0. LRMS (ESI) m/z 475 [M+H] ; HRMS (ESI) m/z: Calcd for
+
C H O Br [M+H] 475.1115; found, 475.1111.
2
4
28
5
5
.1.18
Z)-5-bromo-4,6-diisopropoxy-2-(3,4-diisopropoxybenzylidene)benzofura
n-3(2H)-one (17c)
Z)-7-bromo-4,6-diisopropoxy-2-(3,4-diisopropoxybenzylidene)benzofura
n-3(2H)-one (18c)
The procedure is same as the preparation of 18a by using 16c (4.54 g,
0 mmol) as substrate to give known compound 18c (2.61 g, 49%) and
compound 17c (2.13 g, 40%) as yellow solids respectively. Data for 17c:
(
(
1
2
2

ACCEPTED MANUSCRIPT
1
H NMR (400 MHz, CDCl ) δ 7.46 (d, J = 2.0 Hz, 1H), 7.45 (d, J = 2.0 Hz,
3
1
4
H), 6.95 (d, J = 9.0 Hz, 1H), 6.70 (s, 1H), 6.52 (s, 1H), 5.25-5.18 (m, 1H),
.74-4.67 (m, 1H), 4.62-4.56 (m, 1H), 4.54-4.48 (m, 1H), 1.48 (d, J = 6.0
Hz, 6H), 1.41 (d, J = 6.0 Hz, 6H), 1.38 (d, J = 6.0 Hz, 6H), 1.37 (d, J = 6.0
13
Hz, 6H). C NMR (101 MHz, CDCl ) δ 179.65, 167.35, 162.45, 154.89,
3
1
1
51.24, 148.63, 146.46, 126.33, 125.58, 121.71, 116.44, 112.04, 108.16,
02.78, 92.25, 79.07, 72.96, 72.88, 71.72, 22.63, 22.27, 22.15, 21.90;
+
LRMS m/z Calcd for C H BrO [M+H] 533.1; found:533.1. Data for
2
7
33
6
1
1
8
4
6
1
8c: H NMR (400 MHz, CDCl ) δ 7.73 (d, J = 2.0 Hz, 1H), 7.37 (dd, J =
3
.4, 2.0 Hz, 1H), 6.95 (d, J = 8.4 Hz, 1H), 6.72 (s, 1H), 6.20 (s, 1H),
.88-4.82 (m, 1H), 4.69-4.54 (m, 3H), 1.44 (t, J = 6.0 Hz, 12H), 1.41 (d, J =
1
3
.0 Hz, 6H), 1.37 (d, J = 6.0 Hz, 6H). C NMR (101 MHz, CDCl ) δ
3
80.21, 164.67, 162.48, 157.32, 150.71, 149.07, 146.54, 126.09, 125.79,
119.47, 116.93, 112.12, 107.44, 96.50, 86.82, 73.43, 72.90, 72.11, 72.06,
+
2
2.37, 22.20, 22.00; LRMS m/z Calcd for C H BrO [M+H] 533.1;
27
33
6
found:533.1.
5
.1.19
Z)-5-(benzo[d][1,3]dioxol-5-yl)-2-benzylidene-4,6-diisopropoxybenzofur
an-3(2H)-one (19a)
The procedure is same as the preparation of 9 by using 17a (4.17 g,
0 mmol) as substrate to give compound 19a (3.16 g, 69%) as a yellow
(
1
2
3

ACCEPTED MANUSCRIPT
solid. H NMR (400 MHz, CDCl ) δ 7.88 (d, J = 8.4 Hz, 2H), 7.46 ~ 7.35
1
3
(
m, 3H), 6.84 ~ 6.74 (m, 4H), 6.57 (s, 1H), 5.99 (s, 2H), 4.77 (hept, J =
.0 Hz, 1H), 4.62 (hept, J = 6.0 Hz, 1H), 1.32 (d, J = 6.0 Hz, 6H), 1.11 (d,
6
13
J = 6.0 Hz, 6H); C NMR (100 MHz, CDCl ) δ 180.7, 168.0, 164.5,
3
1
1
55.5, 147.9, 146.7, 146.1, 132.7, 131.1, 129.3, 128.8, 127.1, 124.8,
20.7, 111.8, 110.7, 107.8, 107.5, 100.8, 91.8, 77.6, 71.7, 22.4, 21.8.
+
HRMS (ESI) m/z: Calcd for C H NaO [M+Na] 481.1622; found,
28
26
6
4
81.1618.
5
.1.20
(Z)-5-(benzo[d][1,3]dioxol-5-yl)-4,6-diisopropoxy-2-(4-isopropoxybenzyl
idene)benzofuran-3(2H)-one (19b)
The procedure is same as the preparation of 9 by using 17b (4.76 g,
1
0 mmol) as substrate to give compound 19b (3.26 g, 63%) as a yellow
1
solid. H NMR (400 MHz, CDCl ) δ 7.83 (d, J = 8.4 Hz, 2H), 6.94 (d, J =
3
8
.4 Hz, 2H), 6.84 ~ 6.74 (m, 4H), 6.56 (s, 1H), 5.98 (s, 2H), 4.80 ~ 4.73
(
m, 1H), 4.65 ~ 4.58 (m, 2H), 1.37 (d, J = 6.0 Hz, 6H), 1.31 (d, J = 6.0
13
Hz, 6H), 1.10 (d, J = 6.0 Hz, 6H); C NMR (100 MHz, CDCl ) δ 180.6,
3
1
1
2
5
67.7, 164.2, 159.1, 155.3, 146.7, 146.1, 132.9, 127.3, 125.0, 124.8,
20.5, 116.0, 111.8, 111.1, 108.1, 107.4, 100.8, 91.8, 77.5, 71.7, 70.0,
+
2.4, 22.0, 21.8; HRMS (ESI) m/z: Calcd for C H NaO [M+Na]
31
32
7
39.2040; found, 539.2065.
2
4

ACCEPTED MANUSCRIPT
5
.1.21
(Z)-5-(benzo[d][1,3]dioxol-5-yl)-2-(3,4-diisopropoxybenzylidene)-4,6-dii
sopropoxybenzofuran-3(2H)-one (19c)
The procedure is same as the preparation of 9 by using 17c (5.33 g,
1
0 mmol) as substrate to give compound 19c (3.9 g, 68%) as a yellow
1
solid. H NMR (400 MHz, CDCl ) δ 7.49 ~ 7.47 (m, 2H), 6.96 (d, J = 8.8
3
Hz, 1H), 6.84 ~ 6.74 (m, 3H), 6.70 (s, 1H), 6.54 (s, 1H), 5.99 (s, 2H),
4
1
.76 (hept, J = 6.0 Hz, 1H), 4.66 ~ 4.48 (m, 3H), 1.38 (d, J = 6.0 Hz,
13
2H), 1.32 (d, J = 6.0 Hz, 6H), 1.10 (d, J = 6.0 Hz, 6H); C NMR (100
MHz, CDCl ) δ 180.5, 167.7, 164.2, 155.3, 151.0, 148.7, 146.8, 146.7,
3
1
1
46.1, 127.2, 126.1, 125.9, 124.8, 121.6, 120.6, 116.6, 111.8, 111.2,
08.0, 107.4, 100.8, 91.9, 77.5, 72.9, 71.8, 71.7, 22.4, 22.3, 22.2, 21.7;
+
HRMS (ESI) m/z: Calcd for C H NaO [M+Na] 597.2459; found,
34
38
8
5
97.2449.
5
.1.22
(
Z)-2-benzylidene-5-(3,4-dihydroxyphenyl)-4,6-dihydroxybenzofuran-3(2
H)-one (20a)
The procedure is same as the preparation of 10 by using 19a (4.58 g,
0 mmol) as substrate to give compound 20a (3.51 g, 97%) as a yellow
1
1
solid. H NMR (400 MHz, DMSO-d ) δ 10.87 (s, 1H), 9.51 (s, 1H), 8.81
6
(s, 1H), 7.93 (d, J = 8.0 Hz, 2H), 7.52 ~ 7.40 (m, 3H), 6.75 (d, J = 8.0 Hz,
2
5

ACCEPTED MANUSCRIPT
1
6
1
1
H), 6.68 (s, 1H), 6.64 (d, J = 2.0 Hz, 1H), 6.50 (dd, J = 8.0, 2.0 Hz, 1H),
1
3
.44 (s, 1H); C NMR (100 MHz, DMSO-d ) δ 180.7, 165.8, 155.1,
6
48.1, 145.1, 144.8, 132.8, 131.3, 129.8, 129.4, 123.5, 122.6, 119.1,
15.7, 113.3, 109.1, 103.1, 91.4; HRMS (ESI) m/z: Calcd for C21H13O6
-
[
M-H] 361.0718; found, 361.0705.
5
.1.23
(Z)-5-(3,4-dihydroxyphenyl)-4,6-dihydroxy-2-(4-hydroxybenzylidene)benz
ofuran-3(2H)-one (20b)
The procedure is same as the preparation of 10 by using 19b (5.17 g,
1
0 mmol) as substrate to give compound 20b (3.74 g, 99%) as a yellow
1
solid. H NMR (400 MHz, DMSO-d ) δ 10.74 (s, 1H), 10.07 (s, 1H), 9.36
6
(s, 1H), 8.79 (s, 2H), 7.80 (d, J = 8.4 Hz, 2H), 6.89 (d, J = 8.4 Hz, 2H),
6
1
1
1
.73 (d, J = 8.0 Hz, 1H), 6.64 (s, 1H), 6.62 (s, 1H), 6.50 (d, J = 8.0 Hz,
1
3
H), 6.41 (s, 1H); C NMR (100 MHz, DMSO-d ) δ 180.8, 165.4, 165.3,
6
59.4, 154.8, 146.3, 145.0, 144.7, 133.4, 123.7, 123.6, 122.6, 119.1,
16.5, 115.6, 113.0, 110.2, 103.4, 91.2; HRMS (ESI) m/z: Calcd for
+
C H NaO [M+Na] 401.0632; found, 401.0627.
2
1
14
7
5
.1.24
Z)-2-(3,4-dihydroxybenzylidene)-5-(3,4-dihydroxyphenyl)-4,6-dihydroxy
benzofuran-3(2H)-one (20c)
(
2
6

ACCEPTED MANUSCRIPT
The procedure is same as the preparation of 10 by using 19c (5.74 g,
1
0 mmol) as substrate to give compound 20c (3.82 g, 97%) as a yellow
1
solid. H NMR (400 MHz, DMSO-d ) δ 10.80 (s, 1H), 9.29 (bs, 3H), 7.46
6
(d, J = 2.0 Hz, 1H), 7.21 (dd, J = 8.0, 2.0 Hz, 1H), 6.85 (d, J = 8.0 Hz,
1
H), 6.75 (d, J = 8.0 Hz, 1H), 6.65 (d, J = 2.0 Hz, 1H), 6.53 (s, 1H), 6.51
13
(
dd, J = 8.0, 2.0 Hz, 1H), 6.42 (s, 1H); C NMR (100 MHz, DMSO-d )
6
δ180.8, 165.3, 154.8, 148.1, 146.2, 145.9, 145.0, 144.7, 124.7, 124.0,
1
23.6, 122.6, 119.1, 118.0, 116.5, 115.6, 112.9, 110.7, 103.4, 91.1;
-
HRMS (ESI) m/z: Calcd for C H O [M-H] 393.0616; found, 393.0638.
2
1
13
8
5
.1.25
-(3,4-dihydroxybenzyl)-5-(3,4-dihydroxyphenyl)-4,6-dihydroxybenzofura
2
n-3(2H)-one(20c')
The procedure is same as the preparation of (±)-Anastatins A by
using 20c (100 mg) as substrate to give compound 20c' (95 mg, 95%) as
1
a pale yellow solid. H NMR (400 MHz, DMSO-d ) δ 8.86 (m, 6H), 6.72
6
(d, J = 8.4 Hz, 1H), 6.67 (d, J = 2.4 Hz, 1H), 6.64 (d, J = 8.0 Hz, 1H),
6
2
1
1
.59 (d, J = 2.0 Hz, 1H), 6.53 (dd, J = 8.0, 2.0 Hz, 1H), 6.46 (dd, J = 8.0,
.0 Hz, 1H), 4.81 (dd, J = 8.4, 4.0 Hz, 1H), 3.04 (dd, J = 14.8, 4.0 Hz,
1
3
H), 2.72 (q, J = 14.8, 8.4 Hz, 1H). C NMR (101 MHz, DMSO-d ) δ
6
97.0, 172.0, 166.3, 154.2, 145.3, 144.9, 144.5, 144.3, 127.7, 123.7,
2
7

ACCEPTED MANUSCRIPT
1
22.6, 120.6, 119.0, 117.2, 115.8, 115.6, 111.3, 102.8, 90.7, 86.3, 36.6;
-
HRMS (ESI) m/z: Calcd for C H O [M-H] 395.0772; found, 395.0770.
2
1
15
8
5
.1.26
(Z)-4,6,7-trihydroxy-2-benzylidene-2H-benzofuro
[3,2-f]
benzofuro-3-one(21a)
The procedure is same as the preparation of 11 by using 20a (100
1
mg) as substrate to give compound 21a (61 mg, 61%) as a black solid. H
NMR (400 MHz, DMSO) δ 9.42 (s, 1H), 9.18 (s, 1H), 7.97 (d, J = 7.6 Hz,
2
1
1
1
3
H), 7.55 ~ 7.42 (m, 3H), 7.40 (s, 1H), 7.16 (s, 1H), 6.82 (s, 1H), 6.75 (s,
13
H); C NMR (100 MHz, DMSO-d ) δ 178.6, 166.9, 162.3, 151.8, 149.8,
6
48.0, 145.7, 143.7, 132.6, 131.5, 130.1, 129.5, 113.4, 110.5, 110.2,
-
07.0, 100.6, 99.2, 93.6; HRMS (ESI) m/z: Calcd for C H O [M-H]
2
1
11
6
59.0561; found, 359.0550.
.1.27 (Z)-4,6,7-trihydroxy-2-(4-hydroxybenzylidene)-2H-benzofuro [3,2-f]
5
benzofuro-3-one (21b)
The procedure is same as the preparation of 11 by using 20b (100
1
mg) as substrate to give compound 21b (63 mg, 63%) as a red solid. H
NMR (400 MHz, DMSO-d ) δ 12.24 (s, 1H), 10.26 (s, 1H), 9.44 (s, 1H),
6
9
.23 (s, 1H), 7.84 (d, J = 8.8 Hz, 2H), 7.40 (s, 1H), 7.16 (s, 1H), 6.93 (d,
1
3
J = 8.8 Hz, 2H), 6.86 (s, 1H), 6.76 (s, 1H). C NMR (100 MHz,
DMSO-d ) δ 178.5, 166.5, 162.0, 159.8, 151.7, 149.7, 146.2, 145.6, 143.6,
6
2
8