Syntheses of prodelphinidin B3 and C2, and their antitumor activities through cell cycle arrest and caspase-3 activation

Article abstract of DOI:10.1016/j.tet.2013.02.087

Total synthesis of prodelphinidin B3 and C2 have been accomplished. The key step is Lewis acid-mediated equimolar condensations between a catechin and/or gallocatechin nucleophile and a gallocatechin electrophile. The antitumor effects of synthetic prodel

Full text of DOI:10.1016/j.tet.2013.02.087

Tetrahedron 69 (2013) 3543e3550
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Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Syntheses of prodelphinidin B3 and C2, and their antitumor
activities through cell cycle arrest and caspase-3 activation
Wataru Fujii a, Kazuya Toda b, Koichiro Kawaguchi b, Sei-ichi Kawahara c, Miyuki Katoh a,
,
a,
*
Yasunao Hattori d, Hiroshi Fujii b , Hidefumi Makabe
*
a Graduate School of Agriculture, Sciences of Functional Foods, Shinshu University, 8304 Minami-minowa Kami-ina, Nagano 399-4598, Japan
b Department of Bioscience and Biotechnology, Faculty of Agriculture, Shinshu University, 8304 Minami-minowa Kami-ina, Nagano 399-4598, Japan
c St. Cousair Co., Ltd, 1260 Imogawa, Kamiminochi, Nagano 389-1201, Japan
d Department of Medicinal Chemistry, Kyoto Pharmaceutical University, Yamashina-ku, Kyoto 607-8412, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 25 January 2013
Received in revised form 19 February 2013
Accepted 23 February 2013
Available online 28 February 2013
Total synthesis of prodelphinidin B3 and C2 have been accomplished. The key step is Lewis acid-
mediated equimolar condensations between a catechin and/or gallocatechin nucleophile and a galloca-
techin electrophile. The antitumor effects of synthetic prodelphidin B3 and C2 against PC-3 prostate
cancer cell lines have been investigated. Both compounds showed significant antitumor effects. Their
activity was almost the same as that of EGCG, a known antitumor agent.
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
Polyphenols
Synthesis
Natural product
Anticancer agents
1. Introduction
we accomplished equimolar coupling between nucleophilic and
electrophilic partners for the first time using Yb(OTf)3 as a Lewis
Recently proanthocyanidins have been receiving much attention
due to their significant bioactivities.1,2 However, because identifi-
cation and purification from nature are extremely difficult, the
mechanism for their biological activities remains unsolved. Thus,
researchers have sought to synthesize proanthocyanidins.3e5 Syn-
theses of procyanidins (oligomeric catechin and/or epicatechin)
have been reported in the past decade for the end goal of obtaining
proanthocyanidins in a pure state.6e28 However synthetic studies
on prodelphinidins, which contains (ꢀ)-gallocatechin or (þ)-epi-
gallocatechin are quite limited.29 Until now no total synthesis of
prodelphinidins has been reported. The typical synthesis of catechin
derived oligomers can be accomplished using Lewis acid-mediated
condensation of catechin nucleophiles and electrophiles. The dis-
advantage of this reaction is that using an excess amount of nu-
cleophile is necessary to avoid polymerization. Recently, we have
developed an efficient synthesis of procyanidins through equimolar
condensation of catechin nucleophiles with electrophiles. The typ-
ical examples are as follows: (1) For synthesis of a catechin dimer,
acid.7,9,10,13 (2) For synthesis of a catechin trimer, equimolar coupling
between a nucleophilic catechin dimer and monomeric electro-
philic partner (2þ1 coupling) was accomplished using silver Lewis
acids, such as AgBF4 or AgOTf.22,30 Herein we demonstrate equi-
molar condensation of a catechin and/or a gallocatechin nucleophile
with a gallocatechin-derived electrophile and the first total syn-
thesis of prodelphinidin B3 (PDB3, 1) and C2 (PDC2, 2) (Fig. 1).
* Corresponding authors. Tel.: þ81 265 77 1630; fax: þ81 265 77 1700 (H.M.);
tel./fax: þ81 265 77 1629 (H.F.); e-mail addresses: hfujii@shinshu-u.ac.jp (H. Fujii),
makabeh@shinshu-u.ac.jp (H. Makabe).
Fig. 1. The structures of prodelphinidin B3 (1) and C2 (2).
0040-4020/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tet.2013.02.087

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W. Fujii et al. / Tetrahedron 69 (2013) 3543e3550
2. Results and discussion
although electrophile 6 with a related nucleophile, the yield was
85% as we have reported earlier.30 We found the combination of
the C4 leaving group and silver Lewis acid was very important
(Table 2).
2.1. Synthesis
The gallocatechin-derived building block 5 was constructed as
Chan and co-workers reported with slight modification.31 DDQ
oxidation in the presence of methanol or ethoxyethanol gave
electrophiles 6 and 7, respectively (Scheme 1).
The condensed product 12 was transformed into triol 13 using
n-Bu4NOH.28 Finally deprotection of the benzyl ethers of 13 and
subsequent lyophilization afforded prodelphinidin C2 (2) in good
yield. We confirmed that lyophilized prodelphinidin C2 (2) was
Scheme 1. Synthesis of gallocatechin electrophiles 6 and 7. Reagents and conditions: (a) (i) DDQ, ROH; (ii) Ac2O, pyridine, DMAP, 37% for 6, 51% for 7.
Since the gallocatechin electrophile 6 and 7 were in hand, we
examined the condition of equimolar condensation of catechin
nucleophile 8 with gallocatechin electrophile 6 or 7. As shown in
Table 1, 4-(200-ethoxyethoxy) derivative 7 afforded condensed
product in high yield when Yb(OTf)3 was used as Lewis acid. To our
surprise, the reaction using methoxy derivative 6 gave 9 in very
poor yield although nucleophile 8 with a related electrophile, the
yield was 64% as we have reported earlier.13 We confirmed that
leaving the group at the C4 position was crucial in the Yb(OTf)3
mediated condensation (Table 1).9
pure (>95%) by HPLC analysis.34 The 1H spectral data of per-
acetate 14 were in good agreement with the reported values
(Scheme 3).32,35
2.2. Cytotoxic effects on PC-3 prostate cancer cell
Our interest was focused on examining the antitumor activities
of the newly synthesized prodelphinidins. The synthesis of pro-
delphinidin B3 (1) and C2 (2) allowed us to obtain sufficient
quantities of purified compounds to screen against PC-3 prostate
Table 1
Equimolar condensation of gallocatechin electrophile 6 or 7 with catechin nucleophile 8a
Electrophile
Lewis acid
Time (h)
Yield (%)
6
6
7
7
AgBF4
Yb(OTf)3
AgBF4
7
7
7
3
48
4
45
86
Yb(OTf)3
a
The reaction was carried out in CH2Cl2 at room temperature.
The condensed product 9 was transformed into diol 10 using n-
Bu4NOH.28 The 1H and 13C NMR spectral data of 10 were in good
agreement with the reported values.29 The 3,4-cis diastereomer
was not detected. Finally deprotection of the benzyl ethers of 10
and subsequent lyophilization afforded prodelphinidin B3 (1) in
good yield. The 1H spectral data of peracetate of 1 (11) were in good
agreement with the reported values (Scheme 2).32,33
cancer cell lines together with procyanidin B3, C1 and C2, which
were prepared by us previously (Fig. 2).9,13,22
Results were obtained by two independent methods: cell count
measurement and MTT assay. Epigallocatechin gallate (EGCG) was
used as a positive control. As shown in Fig. 3, EGCG, prodelphi-
nidin B3 (1) and C2 (2) exhibited significant cytotoxic activity with
IC50 values below 50 mM. A comparison of the potencies of 1 and
Because diol 10 could be used as a nucleophile for the synthesis
of prodelphinidin C2 (2), we focused on the equimolar conden-
sation of 10 with electrophile 6 or 7. In the previous report, we
found that silver Lewis acids were effective for the construction of
catechin trimer derivatives.22,30 Thus we used AgBF4 and AgOTf as
Lewis acid. As shown in Table 2, the 4-methoxy derivative 6
afforded condensed product 12 in good yield when AgOTf was
used as a Lewis acid. The reaction using AgBF4 as Lewis acid and 4-
methoxy derivative 6 as an electrophile afforded 12 in poor yield
procyanidin B3, suggested that the cytotoxic effects were clearly
associated with the presence of the pyrogallol moiety of the B ring.
PDB3 (1) and PCB3 have the same carbon skeleton. The only dif-
ference is that PDB3 has an additional hydroxy group at the B ring.
We found that this hydroxy group greatly affected the level of the
cytotoxic effect. As for 2 and procyanidin C1 or C2, the pyrogallol
moiety was essential for their activity.36 Comparisons between 1
and either 2 or EGCG suggested that the number of pyrogallol
moieties did not seem to affect the activity. This tendency was also

W. Fujii et al. / Tetrahedron 69 (2013) 3543e3550
3545
Scheme 2. Synthesis of prodelphinidin B3 (1) and its peracetate 11. Reagents and conditions: (a) n-Bu4NOH, 82%; (b) H2, Pd(OH)2/C, 96%; (c) Ac2O, pyridine, DMAP, 28%.
Table 2
Condensation of gallocatechin electrophile 6 or 7 with nucleophile 10a
OBn
OBn
BnO
O
OBn
OBn
OBn
OBn
OBn
OBn
OBn
OAc
OBn
OBn
OBn
BnO
BnO
O
BnO
O
OBn
Lewis acid
O
+
OH
O
OAc
OBn
OH
OBn
OBn
OBn
BnO
OBn OR
OBn
BnO
OBn
O
R = Me: 6
EE:
7
OH
OH
OBn
OBn
10
12
Electrophile
Lewis acid
Time (h)
Yield (%)
6
6
7
7
AgBF4
AgOTf
AgBF4
AgOTf
3
5
1
1
14
73
63
11
a
The reaction was carried out in CH2Cl2 at room temperature.
OH
OBn
OBn
OBn
OAc
OH
OBn
OAc
HO
O
BnO
O
BnO
O
AcO
O
OH
OH
OBn
OBn
OBn
OBn
OAc
OAc
OH
O
OAc
O
OH
OAc
OH
OH
OBn
OBn
OBn
OBn
OAc
OAc
a
OH
HO
OBn
BnO
OBn
BnO
OAc
AcO
c
b
O
O
OH
OH
OH
OAc
OH
OH
OBn
OBn
OBn
OBn
OAc
OAc
OH
HO
OBn
BnO
OBn
BnO
OAc
AcO
O
O
O
O
OH
OH
OH
OAc
OH
OBn
OBn
OAc
12
13
prodelphinidin C2 (2)
14
Scheme 3. Synthesis of prodelphinidin C2 (2) and its peracetate 14. Reagents and conditions: (a) n-Bu4NOH, 77%; (b) H2, Pd(OH)2/C, 96%; (c) Ac2O, pyridine, DMAP, 27%.
observed in the MTT assay. This finding might be useful in
searching for antitumor molecules among the proanthocyanidins
(Fig. 3).
2.3. Effects on cell cycle distribution
Cell growth and proliferation are mediated via cell cycle pro-
gression. Loss of cell cycle control can initiate the apoptotic pro-
gram.37 In the present studies, treatment of PC-3 prostate cancer
After treatment of cells with EGCG, PCB3, PCC1, PCC2, PDB3,
PDC2, or CPT for 48 h, the cell proliferation was determined by cell
count (A) and MTT assay (B) as shown in Supplementary data. The
values were represented as the rate of inhibition of cell pro-
liferation by the treated sample compared to the untreated control
(vehicle). Values are meansꢁS.Ds. for three independent experi-
ments. Asterisks indicated a significant difference between the
control- and test-compound-treated cells, as analyzed by Student’s
test (p<0.001).
cells with 50 mM of prodelphinidin B3 (1) for 48 h induced a G1/G0
phase population increase from 62.88% to 74.50% and an S phase
fraction decrease from 16.06% to 8.67%. Obviously, prodelphinidin
B3 (1) blocked the PC-3 prostate cancer cell cycle partly at the G1/
G0 phase within 48 h. EGCG and prodelphinidin C2 (2) showed
a similar effect. On the other hand, no effect from procyanidin B3,
C1 or C2 was observed. A lower S phase population indicated

3546
W. Fujii et al. / Tetrahedron 69 (2013) 3543e3550
Fig. 2. The structures of test compounds for PC3 prostate anticancer activity.
Fig. 3. Effects of various concentrations of test compounds on cell proliferation.
a slower cell division and growing tumor. Compounds, which
promote cell apoptosis and inhibit proliferation of cancer cells are
likely to be good candidates as antitumor agents.38 Our findings
suggest that prodelphinidins might be promising chemopreventive
agents against prostate cancer (Fig. 4).
The cells treated with test compounds (EGCG, PCB3, PDB3, PCC1,
PCC2, or PDC2) for 48 h were collected and stained with propidium
iodide using a BD CycletestÔ Plus DNA Reagent Kit (Becton Dick-
inson and Company BD Biosciences) obtained from Phoenix Flow
Systems. Following FACS analysis, cell cycle distributions were
further analyzed by Cell Quest software. The phase fraction (%) is
shown in the graph. The experimental data are shown in
Supplementary data.
2.4. Effects on caspase-3 activity
It has been well known that the caspases initiate cell apoptosis
caused by some stimuli.39 Caspases are synthesized as inactive
proenzymes, which need proteolytic cleavage for activation.
Caspase-3 is related to the enforcement phase of apoptosis, where
Fig. 4. Effects of test compounds on cell cycle distribution.

W. Fujii et al. / Tetrahedron 69 (2013) 3543e3550
3547
cells undergo morphological changes, such as chromatin conden-
sation, apoptotic body formation, and DNA fragmentation.40 As
caspase-3 is the most important enzyme involved in execution of
apoptosis, its activation, and formation of cleaved caspase-3, is
a biochemical marker of early apoptosis. Therefore, the detection of
active caspase-3 in cells is a reliable method of measuring apo-
ptosis. Thus, we next examined the caspase-3 activity to confirm
that the cells exposed to prodelphinidin were actually undergoing
apoptosis, and to determine whether caspase-3 was involved in the
cell death pathway. We found that EGCG, prodelphinidin B3 (1),
and prodelphinidin C2 (2) activated caspase-3 up to 1.5e1.8 times
compared to the control. Thus, we concluded that the cell death
caused by prodelphinidins was attributable to a kind of apoptosis
(Fig. 5).
spectrometer in CDCl3 or CD3OD with residual tetramethylsilane as
the standard. The coupling constants were given in Hertz. Mass
spectra were obtained on JEOL JMS-SX102A and Waters Xevo QTOF
mass spectrometers. IR spectra were recorded with JASCO FT-IR 480
Plus infrared spectrometer. Optical rotations were determined with
a JASCO DIP-1000 polarimeter.
4.1.1. (2R,3S,4S)-3-Acetoxy-4-methoxy-5,7-30,40,50-pentabenzyloxy-
flavan (6). To a solution of 5 (208 mg, 0.280 mmol) in MeOH/CH2Cl2
(1/8, 2.0 mL) was added DDQ (250 mg, 1.10 mmol) at room tem-
perature. After the resulting mixture had been stirred for 1 h at
room temperature, the mixture was cooled to 0 ꢂC and the reaction
was quenched with water. The mixture was extracted with Et2O
(3ꢃ20 mL) and the combined organic layer was washed with brine,
dried over MgSO4, filtered, and concentrated. The crude product
was purified with silica gel column chromatography (hexane/
AcOEt/CH2Cl2¼6:1:3) to afford colorless solid. Acetylation of this
compound using general procedure gave 6 (84 mg, 37%, two steps)
23
as a colorless oil. [
a]
þ32.4 (c 1.15, CHCl3); IR (film) nmax
D
cmꢀ1:3088, 3063, 3032, 2931, 2829, 1742, 1616, 1593, 1230, 1151,
1108, 736, 696; 1H NMR (CDCl3)
d: 7.43e7.21 (25H, m), 6.81 (2H, s),
6.28 (1H, d, J¼2.5 Hz), 6.18 (1H, d, J¼2.0 Hz), 5.28e5.22 (2H, m),
5.15e4.97 (10H, m), 4.77 (1H, d, J¼2.5 Hz), 3.45 (3H, s), 1.77 (3H, s);
13C NMR (CDCl3)
d:169.4, 160.9, 158.4, 155.5, 152.5, 138.4, 137.7,
137.0,136.9,136.4,136.3,132.7,128.5,128.4,128.1,128.0,127.8,127.7,
127.6, 127.4, 107.3, 103.1, 94.2, 93.7, 75.0, 74.5, 72.5, 71.3, 71.1, 70.4,
70.0, 69.4, 69.1, 59.0, 20.7; HRFABMS (MþNa)þ: calcd for
C53H48NaO9, 851.3196, found; 851.3201.
4.1.2. (2R,3S,4S)-3-Acetoxy-4-ethoxyethoxy-5,7,30,40,50-pentabenzy-
loxyflavan (7). To a solution of 5 (118 mg, 0.160 mmol) in 2-
ethoxyethanol/CH2Cl2 (1/8, 2.7 mL) was added DDQ (106 mg,
0.470 mmol) at room temperature. After the resulting mixture had
been stirred for 1 h at room temperature, the mixture was cooled to
ꢂC and the reaction was quenched with water. The mixture was
Fig. 5. Effects of test compounds on caspase-3 activities assessed by FACS.
0
extracted with Et2O (3ꢃ10 mL) and the combined organic layer was
washed with brine, dried over MgSO4, filtered, and concentrated.
The crude product was purified with silica gel column chroma-
tography (hexane/AcOEt/CH2Cl2¼6:1:3) to afford colorless solid.
Acetylation of this compound using general procedure gave 7
Assay for caspase-3 activities after treatment with test com-
pounds (5 mmol/L of CPT and 50 mmol/L of EGCG, PCB3, PDB3, PCC1,
PCC2, or PDC2) were performed. The values represented are the
rate of induction of apoptosis compared to the untreated control
(vehicle). Experimental data are shown in the Supplementary data.
(70 mg, 51%, two steps) as a colorless oil. [
a
]
23 þ31 (c 1.9, CHCl3); IR
D
(film) nmax cmꢀ1: 3032, 2925, 1741, 1616, 1592, 1230, 1151, 1110, 736,
697; 1H NMR (CDCl3)
d
: 7.48e7.20 (25H, m), 6.77 (2H, d, J¼1.5 Hz),
3. Conclusion
6.26 (1H, d, J¼2.0 Hz), 6.16 (1H, d, J¼1.5 Hz), 5.28e4.95 (12H, m),
4.88 (1H, d, J¼3.0 Hz), 3.82e3.70 (2H, m), 3.50e3.40 (4H, m), 1.79
The first total synthesis of prodelphinidin B3 (1) and C2 (2) has
been achieved via Lewis acid-mediated equimolar condensation of
a catechin and/or catechin-gallocatechin nucleophile with gallo-
catechin electrophiles. In addition to demonstrating the total syn-
thesis, we examined their antitumor activities against PC-3
prostate cancer cells. We found that cytotoxic effects are clearly
associated with the presence of the pyrogallol moiety of the B ring;
procyanidins, which lacked the pyrogallol moiety of the B ring did
not show any potency. This activity of prodelphinidins might be
ascribed to their blocking cell cycle partly at the G1/G0 phase and
activating caspase-3. Prodelphinidins might be potential chemo-
preventing agents for prostate cancer cells. Based on this study’s
promising results, we are currently synthesizing various proan-
thocyanidins, which contain pyrogallol moieties to clarify the
mechanism of antitumor activity.
(3H, s), 1.15 (3H, t, J¼7.0 Hz); 13C NMR (CDCl3)
d: 169.6, 160.9, 158.5,
155.6, 152.7, 138.6, 137.8, 137.0, 136.6, 136.5, 132.9, 128.6, 128.5,
128.4, 128.1, 128.0, 127.8, 127.7, 127.6, 127.5, 107.6, 103.6, 94.3, 93.8,
75.1, 74.5, 72.6, 71.2, 70.7, 70.5, 70.3, 70.1, 68.2, 66.4, 20.7, 15.2;
HRFABMS (MþNa)þ: calcd for C56H54NaO10, 909.3615, found;
909.3610.
4.1.3. [4,80]-2,3-trans-3,4-trans:20,30-trans-3-Acetoxy-nonabenzyloxy-
(þ)-gallocatechin-(þ)-catechin (9). To a solution of nucleophile 8
(28 mg, 43 mmol) and electrophile 7 (38 mg, 43 mmol) in CH2Cl2
(4.0 mL) was added Yb(OTf)3 (27 mg, 43 mmol). After the resulting
mixture had been stirred for 3 h at room temperature, the reaction
was quenched with water. The mixture was extracted with EtOAc
(2ꢃ10 mL) and the combined organic layer was washed with brine,
dried over MgSO4, filtered, and concentrated. The crude product was
purified with preparative TLC (hexane/AcOEt/CH2Cl2¼6:1:3) to af-
4. Experimental
4.1. General
ford 9 (53 mg, 86%) as pale yellow oil. [
a
]
20 ꢀ114 (c 1.15, CHCl3); IR
D
(film) nmax cmꢀ1:3524, 3062, 3031, 2928, 2870,1741, 1593,1231, 1114,
735, 696; 1H NMR (CDCl3, 1:1 rotational isomer, major isomer)
d:
All melting points were uncorrected. 1H (500 MHz) and 13C NMR
(125 MHz) spectra were measured with a Bruker DRX 500 FT-NMR
7.48e7.10 (43.5H, m), 6.94e6.60 (7H, m), 6.43 (0.5H, dd, J¼8.5,
1.5 Hz), 6.24 (1H, s), 6.22 (0.5H, d, J¼2.0 Hz), 6.19 (0.5H, d, J¼2.0 Hz),

3548
W. Fujii et al. / Tetrahedron 69 (2013) 3543e3550
6.15 (0.5H, d, J¼2.0 Hz), 6.13 (0.5H, d, J¼2.0 Hz), 5.98 (0.5H, t,
J¼9.5 Hz), 5.96 (0.5H, s), 5.85 (0.5H, t, J¼9.5 Hz), 5.22e4.50 (18.5H,
m), 3.84 (0.5H, m), 3.56 (0.5H, m), 3.27 (0.5H, d, J¼9.0 Hz), 3.01 (0.5H,
dd, J¼16.5, 6.0 Hz), 2.86 (1H, dd, J¼16.5, 5.5 Hz), 2.72 (0.5H, dd,
J¼16.5, 7.5 Hz), 2.35 (0.5H, dd, J¼16.5, 9.8 Hz), 2.05 (0.5H, m), 1.58
(0.67H, dd, J¼16.5, 8.0 Hz); 13C NMR (CD3OD)
d: 158.7, 157.2, 155.9,
155.8, 146.7, 146.6, 146.2, 145.6, 131.9, 131.7, 119.5, 116.3, 116.1, 115.1,
108.3, 107.9, 107.1, 101.7, 97.4, 96.9, 95.9, 84.3, 82.9, 82.0, 73.5, 68.6,
38.7, 38.5, 27.9; HRFABMS (MþNa)þ: calcd for C30H26NaO13
,
617.1271, found; 617.1276.
(1.5H, s), 1.49 (1.5H, s), 1.27 (0.5H, d, J¼1.5 Hz); 13C NMR (CDCl3)
d:
169.3, 168.8, 158.2, 157.8, 156.8, 156.5, 155.8, 155.6, 154.9, 153.5, 152.6,
149.2, 148.9, 148.7, 148.6, 138.4, 138.2, 137.9, 137.8, 137.4, 137.3, 137.2,
137.1, 137.0, 136.9, 136.8, 136.5, 133.0, 132.7, 132.6, 132.3, 128.3, 128.1,
128.0, 127.9, 127.8, 127.7, 127.6, 127.5, 127.4, 127.3, 127.2, 127.1, 126.9,
126.8, 120.3, 120.0, 114.8, 114.6, 114.2, 113.3, 110.6, 110.5, 110.0, 108.5,
108.2, 107.9, 107.7, 107.2, 107.0, 102.4, 102.3, 94.8, 94.7, 94.5, 94.4, 91.1,
81.3, 80.7, 80.2, 76.7, 72.8, 72.1, 71.5, 71.3, 71.2, 71.1, 71.0, 70.9, 70.5,
70.4, 70.1, 70.0, 69.9, 69.8, 68.3, 67.9, 35.3, 35.1, 28.6, 26.7, 20.6, 20.4;
HRFABMS (MþNa)þ: calcd for C95H82NaO14, 1469.5602, found;
1469.5597.
4.1.6. Peracetate of 1 (11). A mixture of pyridine (50
anhydride (50 L), and DMAP (1.0 mg) was added to 1 (4.0 mg,
6.7 mol). After the reaction mixture had been stirred for 12 h,
mL), acetic
m
m
saturated aqueous NaHCO3 (5 mL) was added, and the product
was extracted with EtOAc (2ꢃ10 mL). The organic layers were
washed with H2O (5.0 mL) and brine, and dried over MgSO4, fil-
tered, and concentrated. The crude product was purified with
preparative TLC (hexane/AcOEt/CH2Cl2¼6:1:3) to afford per-
acetate 11 (2.0 mg, 28%) as colorless glass. IR (film) nmax cmꢀ1
:
1924, 2851, 1772, 1617, 1505, 1431, 1371, 1206, 1127, 1046, 896, 736;
1H NMR (CDCl3)
d
: 7.12 (1H, d, J¼8.5 Hz), 6.96 (2H, s), 6.90 (1H, d,
4.1.4. [4,80]-2,3-trans-3,4-trans:20,30-trans-Nonabenzyloxy-(þ)-gal-
J¼2.0 Hz), 6.70 (1H, dd, J¼8.5, 2.0 Hz), 6.65 (1H, s), 6.49 (2H, s),
5.61 (1H, dd, J¼10.0, 8.0 Hz), 5.08e4.99 (2H, m), 4.71 (1H, d,
J¼10.0 Hz), 4.50 (1H, d, J¼9.5 Hz), 2.86 (1H, m), 2.66 (1H, m), 2.35
(3H, s), 2.29e2.24 (21H, m), 1.98 (3H, s), 1.95 (3H, s), 1.70 (3H, s);
locatechin-(þ)-catechin (10). To a solution of 9 (44 mg, 30
mmol) in
THF (3.0 mL) was added 40% aqueous n-Bu4NOH (0.59 mL,
0.91 mmol). The reaction mixture was allowed to be stirred for 72 h
at room temperature, then partially evaporated to remove THF. The
residue was diluted with H2O (5.0 mL), and the product was
extracted with EtOAc (2ꢃ5.0 mL). The combined organic layers
were washed with brine and concentrated. The residue was puri-
13C NMR (CDCl3)
d: 170.1, 169.1, 168.6, 168.4, 168.1, 167.7, 167.5,
166.6, 155.8, 152.6, 149.6, 149.2, 149.0, 147.9, 147.6, 143.3, 143.2,
142.0, 141.6, 135.2, 135.0, 134.7, 134.6, 124.5, 123.4, 122.2, 121.7,
120.0, 119.6, 116.7, 115.2, 111.3, 110.2, 110.0, 109.6, 109.4, 108.2,
107.9, 78.8, 77.9, 77.7, 70.4, 68.3, 68.1, 36.7, 29.7, 25.2, 21.1, 20.9,
20.7, 20.6, 20.4, 20.3, 20.1.
fied with preparative TLC (hexane/AcOEt/CH2Cl2¼4:1:2) to afford
19
10 (35 mg, 82%) as pale yellow oil. [
a
]
ꢀ106 (c 1.60, CHCl3); IR
D
(film) nmax cmꢀ1:3566, 3062, 3031, 2926, 1592, 1112, 736, 696; 1H
NMR (CDCl3, 2:1 rotational isomer, major isomer) : 7.47e7.20
d
4.1.7. [4,8:400,800]-2,3-trans-3,4-trans: 200,300-trans-300,400-trans:2000,3000-
trans-Acetoxytetradecabenzyloxy-(þ)-gallocatechin-(þ)-galloca-
techin-(þ)-catechin (12). To a solution of nucleophile 10 (43 mg,
(40H, m), 6.94e6.84 (7H, m), 6.94 (1H, d, J¼2.0 Hz), 6.80 (1H, dd,
J¼8.5, 2.0 Hz), 6.26 (1H, s), 6.18 (1H, d, J¼2.0 Hz), 6.13 (1H, d,
J¼2.0 Hz), 5.20e4.60 (18H, m), 4.57 (2H, d, J¼11.0 Hz), 4.48 (1H, d,
J¼9.5 Hz), 4.21 (1H, dd, J¼9.5, 8.5 Hz), 3.75e3.60 (1H, m), 3.59 (1H,
d, J¼8.0 Hz), 3.02 (1H, dd, J¼16.5, 5.8 Hz), 2.39 (1H, dd, J¼16.0,
31
mmol) and electrophile 6 (26 mg, 31
mmol) in CH2Cl2 (4.0 mL)
under an argon atmosphere was added AgOTf (7.8 mg, 31
mmol).
After the resulting mixture had been stirred for 5 h at room tem-
perature, the reaction was quenched with water. The mixture was
extracted with AcOEt, and the combined organic layer was washed
with brine, dried over MgSO4, filtered, and concentrated. The crude
product was purified with silica gel column chromatography
(hexane/AcOEt/CH2Cl2¼6:1:3) to give 12 (49 mg, 73%) as a pale
9.0 Hz), 1.60e1.28 (2H, m); minor isomer: d: 7.47e7.27 (20H, m),
7.16e7.12 (3.5H, m), 6.56 (0.5H, d, J¼2.0 Hz), 6.48 (0.5H, dd, J¼8.5,
2.0 Hz), 6.35 (0.5H, s), 6.09 (0.5H, d, J¼2.0 Hz), 6.05 (0.5H, d,
J¼2.0 Hz), 5.16e4.65 (10H, m), 4.50 (0.5H, d, J¼9.5 Hz), 4.45 (0.5H,
dd, J¼9.5, 8.5 Hz), 4.18 (0.5H, d, J¼8.5 Hz), 3.75e3.60 (0.5H, m), 3.16
(0.5H, dd, J¼16.5, 6.0 Hz), 2.64 (0.5H, dd, J¼16.5, 9.0 Hz), 1.70e1.35
yellow oil. [
a
]
19 ꢀ80 (c 0.23, CHCl3); IR (film) nmax cmꢀ1:3566, 3062,
D
(1H, m); 13C NMR (CDCl3)
d
: 158.0, 157.8, 157.6, 156.7, 155.6, 155.5,
3031, 2927, 2829, 1741, 1593, 1113, 735, 696; HRFABMS (MþNa)þ:
154.0, 152.9, 152.8, 149.1, 149.0, 138.7, 137.9, 137.3, 137.2, 137.1, 137.0,
136.7, 134.2, 131.6, 130.6, 128.6, 128.5, 128.4, 128.3, 128.2, 128.1,
127.9, 127.8, 127.7, 127.6, 127.5, 127.4, 127.2, 127.1, 120.7, 120.2, 114.9,
114.3, 113.9, 112.0, 107.2, 106.9, 103.3, 102.6, 94.9, 91.9, 91.5, 82.3,
82.1, 81.3, 80.7, 77.6, 75.2, 73.4, 73.2, 71.3, 71.0, 70.3, 70.1, 70.0, 69.9,
68.6, 68.4, 37.2, 33.7, 31.9; HRFABMS (MþNa)þ: calcd for
C93H80NaO13, 1427.5497, found; 1427.5493.
calcd for C145H124NaO21, 2223.8533, found; 2223.8521.
4.1.8. [4,8:400,800]-2,3-trans-3,4-trans: 200,300-trans-300,400-trans:2000,3000-
trans-Tetradecabenzyloxy-(þ)-gallocatechin-(þ)-gallocatechin-
(þ)-catechin (13). To a solution of 12 (60 mg, 0.025 mmol) in THF
(3.0 mL) was added n-Bu4NOH (0.46 mL, 0.76 mmol). The reaction
mixture was allowed to be stirred for 72 h at room temperature,
then partially evaporated to remove THF. The residue was diluted
with H2O (5.0 mL), and the product was extracted with EtOAc
(2ꢃ5.0 mL). The combined organic layers were washed with brine
and concentrated. The residue was purified with preparative TLC
(hexane/AcOEt/CH2Cl2¼6:1:3) to afford 13 (44 mg, 77%) as a pale
4.1.5. Prodelphinidin B3 (1). A solution of 10 (32 mg, 23 mmol) in
THF/MeOH/H2O (20/20/1) (4.0 mL) was hydrogenated over 20%
Pd(OH)2/C (28 mg) for 3 h at room temperature. The mixture was
filtered and the filtration residue was washed with MeOH (10 mL).
The combined filtrates were evaporated, and the residue was taken
up in distilled water (5.0 mL). The solution was filtered and ly-
yellow oil. [
a
]
D
22 ꢀ91 (c 0.14, CHCl3); IR (film) nmax cmꢀ1:3573, 3062,
3031, 2869, 1742, 1593, 1113, 735, 696; 1H NMR (CDCl3, 0.82:0.18
ophilized to give 1 (13 mg, 96%) as a fluffy amorphous solid. Mp
mixture of rotational isomer, major isomer)
d
¼7.44e5.90 (81H, m),
19
212e213 ꢂC (decomp.); [
a
]
ꢀ207 (c 0.350, MeOH); IR (KBr) nmax
5.57e4.40 (30H, m), 4.25 (1H, d, J¼8.8 Hz), 4.05e3.95 (2H, m),
3.81e3.75 (1H, m), 3.57 (1H, d, J¼9.0 Hz), 3.10 (1H, dd, J¼16.0,
6.0 Hz), 2.92 (1H, d, J¼9.5 Hz), 2.37 (1H, dd, J¼16.5, 9.8 Hz),1.52 (2H,
D
cmꢀ1: 3350, 2924, 1614, 1520, 1452, 1365, 1283, 1233, 1144, 1032,
821, 628; 1H NMR (CD3OD, 2:1 rotational isomer)
d
: 6.94e6.05 (5H,
m), 5.88 (0.67H, d, J¼2.0 Hz), 5.82 (0.33H, d, J¼2.0 Hz), 5.82 (0.33H,
d, J¼2.5 Hz), 5.74 (0.67H, d, J¼2.5 Hz), 4.73 (0.33H, d, J¼7.0 Hz), 4.65
(0.67H, d, J¼7.0 Hz), 4.48 (1H, dd, J¼9.5, 8.5 Hz), 4.42 (1H, d,
J¼8.0 Hz), 4.34 (0.67H, dd, J¼10.0, 8.0 Hz), 4.28 (0.33H, dd, J¼10.0,
8.0 Hz), 4.23 (0.67H, d, J¼9.5 Hz), 4.17 (0.33H, d, J¼9.0 Hz), 4.07
(0.33H, m), 3.78 (0.67H, m), 2.68 (0.67H, dd, J¼16.0, 5.0 Hz), 2.80
(0.33H, dd, J¼16.0, 5.0 Hz), 2.58 (0.33H, dd, J¼16.0, 7.8 Hz), 2.48
br s, eOH), 1.12 (1H, br s, eOH); 13C NMR (CDCl3)
d: 158.0, 157.9,
156.6, 156.5, 156.0, 155.6, 155.5, 155.3, 155.2, 155.0, 153.9, 152.9,
152.7, 152.5, 149.4, 149.1, 138.4, 138.3, 138.1, 138.0, 137.8, 137.4, 137.2,
137.1, 136.0, 134.7, 134.6, 128.9, 128.5, 128.4, 128.3, 128.2, 128.1,
127.9, 127.8, 127.7, 127.6, 127.5, 127.4, 127.3, 127.2, 127.1, 127.0, 120.7,
115.2, 114.6, 112.5, 109.5, 108.9, 108.0, 107.5, 107.1, 107.0, 102.6, 94.9,
94.1, 92.2, 91.9, 82.2, 81.1, 75.2, 75.0, 73.0, 72.9, 71.4, 71.3, 71.1, 70.6,

W. Fujii et al. / Tetrahedron 69 (2013) 3543e3550
3549
70.4, 70.3, 70.0, 69.9, 68.4, 37.7, 37.4, 29.7; HRFABMS (MþNa)þ:
EGCG, PCB3, PCC1, PCC2, PDB3, PDC2, or CPT for 48 h. Absorbance at
595 nm was measured using the microplate reader after the addi-
tion of the MTT solvent.
calcd for C143H122NaO20, 2181.8427, found; 2181.8440.
4.1.9. Prodelphinidin C2 (2). A solution of 13 (32 mg, 15 mmol) in
THF/MeOH/H2O (20/20/1) (4.0 mL) was hydrogenated over 20%
Pd(OH)2/C (28 mg) for 3 h at room temperature. The mixture was
filtered and the filtration residue was washed with MeOH (10 mL).
The combined filtrates were evaporated, and the residue was taken
up in distilled water (5.0 mL). The solution was filtered and ly-
4.2.4. Measurement of apoptosis by assay for caspase-3 activity
(Fig. 5). Assay for caspase-3 activities were carried out using BD
Cytofix/CytopermÔ Kit (BD Biosciences, No. 554714), according to
the manufacturer’s protocol. Purified rabbit anti-active caspase-3
(BD PharmingenÔ, No. 559565) was used for the first antibody (1st
Ab) and FITC-conjugate anti-rabbit Ig G (Jackson ImmunoResearch,
No. 711-096-152) was used for the second antibody (2nd Ab).
ophilized to give 2 (13 mg, 96%) as a fluffy amorphous solid. Mp
21
218e220 ꢂC (decomp.); [
a
]
D
ꢀ283 (c 0.350, MeOH); IR (KBr) nmax
cmꢀ1: 3350, 2925, 1616, 1523, 1453, 1367, 1281, 1238, 1140, 1035,
821, 629; 1H NMR (CD3OD)
¼7.05e5.75 (11H, m), 4.80e4.05 (6H,
m), 2.90e2.40 (4H, m); 13C NMR (CD3OD)
¼158.6, 157.7, 157.2,
Briefly, after treatment of cells with 50 mM of EGCG, PCB3, PCC1,
d
PCC2, PDB3, PDC2, or 500 nM of CPT for 48 h, the cells were col-
lected and prepared by the same method as cell cycle analysis
described in Materials and methods. The cells were diluted in PBS
and fixed with BD Cytofix/CytopermÔ Fixation and Permeabiliza-
tion Solution for 20 min on ice in the dark. The cells were washed
with the washing buffer and then reacted with 1st Ab at room
temperature. Next, the cells were washed with the washing buffer
and then reacted with 2nd Ab at room temperature. After the re-
action, the cells were diluted in PBS, and flow cytometry was per-
formed with a FACScan (Becton Dickinson, Japan), and the data
obtained were analyzed utilizing Cell Quest software. For each
sample, 1ꢃ104 cells were recorded.
d
155.9, 155.8, 155.6, 155.5, 155.4, 155.3, 155.2, 155.1, 155.0, 146.7,
146.2, 146.0, 145.9, 134.2, 130.8, 124.5, 120.2, 117.6, 116.2, 115.3,
108.7, 108.5, 108.3, 97.4, 96.1, 95.7, 84.0, 83.2, 81.9, 73.0, 69.8, 68.6,
68.5, 68.3, 38.9, 38.8, 30.8, 21.3; ESI-TOFMS calcd for C45H38O20Na
(MþNa)þ; 921.1854; found 921.1941.
4.1.10. Peracetate of 2 (14). A mixture of pyridine (50
anhydride (50 L), and DMAP (1.0 mg) was added to 2 (4.0 mg,
4.5 mol). After the reaction mixture had been stirred for 12 h,
mL), acetic
m
m
saturated aqueous NaHCO3 (5.0 mL) was added, and the product
was extracted with EtOAc (2ꢃ10 mL). The organic layers were
washed with H2O (5.0 mL) and brine, and dried over MgSO4, fil-
tered, and concentrated. The crude product was purified with
preparative TLC (hexane/AcOEt/CH2Cl2¼6:1:3) to afford peracetate
14 (2.0 mg, 27%) as colorless glass. IR (film) nmax cmꢀ1: 2925, 2850,
1773, 1619, 1502, 1433, 1370, 1207, 1128, 1042, 896, 736; 1H NMR
4.2.5. Statistical analysis. Each experiment was performed at least
three times. Data were expressed as the meansꢁstandard deviation
(S.D.). Statistical analysis was performed using Student t-test.
P<0.05 or P<0.01 was considered to be significant.
(CDCl3)
d
: 7.05 (1H, d, J¼8.5 Hz), 6.96 (2H, s), 6.86 (1H, d, J¼2.0 Hz),
Acknowledgements
6.67 (2H, s), 6.64 (1H, s), 6.63 (1H, d, J¼6.5 Hz), 6.54 (1H, d,
J¼2.5 Hz), 6.25 (1H, d, J¼2.5 Hz), 5.57 (1H, dd, J¼19.0, 9.5 Hz), 5.47
(1H, dd, J¼19.0, 9.5 Hz), 5.30e5.15 (2H, m), 4.78 (1H, d, J¼10.0 Hz),
4.66 (1H, d, J¼10.0 Hz), 4.58 (1H, d, J¼8.0 Hz), 4.18 (1H, d, J¼9.0 Hz),
2.56 (2H, s), 2.38e1.61 (OAc-groups).
This work was supported in part by Grant-in-Aid for Scientific
Research from the Ministry of Education, Science, Culture, Sports,
and Technology of Japan (22570112 to H.F.) and by Grant
from Uehara Memorial Foundation (to H.F.). We also thank
Prof. Dr. Toshiyuki Kan for providing EGCG and Mr. Hideyuki Kar-
asawa at Nagano Prefecture General Industrial Technology Center
for obtaining the ESI-TOF mass spectrum of prodelphinidin C2 (2).
4.2. Biochemical methods
4.2.1. Cell lines, cell culture and reagents. THuman prostate cancer
cell, PC-3, was purchased from the Health Science Research Re-
sources Bank. The cells were maintained in monolayer culture at
37 ꢂC and 5% CO2 in RPMI-1640 (SIGMA, R8755) supplemented
with 10% charcoal-stripped fetal bovine serum (Biological In-
dustries, No. 04-201-1), 1% antibiotic-antimycotic mixed stock so-
lution (Nacalai Tesque, No. 09366-44). The cells were treated with
various concentrations of epigallocatechine-3-gallate (EGCG), pro-
cyanidin B3 (PCB3), procyanidin C1 (PCC1), procyanidin C2 (PCC2),
prodelphinidin B3 (PDB3), prodelphinidin C2 (PDC2) or campto-
thecin (CPT) for 48 h. CPT (WAKO, No. 038-18191) was used for the
index of apoptosis. EGCG was generously gifted from Prof. Dr.
Toshiyuki Kan in University of Shizuoka.
Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.tet.2013.02.087.
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