Study of the green tea polyphenols catechin-3-gallate (CG) and epicatechin-3-gallate (ECG) as proteasome inhibitors

Article abstract of DOI:10.1016/j.bmc.2004.04.033

The green tea polyphenol catechin-3-gallate (CG) and epicatechin-3-gallate (ECG) were synthesized enantioselectively via a Sharpless hydroxylation reaction followed by a diastereoselective cyclization. Their potencies to inhibit the proteasome activity were measured. The unnatural enantiomers were found to be equally potent to the natural compounds.

Full text of DOI:10.1016/j.bmc.2004.04.033

Bioorganic & Medicinal Chemistry 12 (2004) 3521–3527
Study of the green tea polyphenols catechin-3-gallate (CG) and
epicatechin-3-gallate (ECG) as proteasome inhibitors
Sheng Biao Wan,a Di Chen,b, Q. Ping Doub, and Tak Hang Chana,*
aDepartment of Applied Biology and Chemical Technology and the Open Laboratory of Chirotechnology,
Institute of Molecular Technology for Drug Discovery and Synthesis,
The Hong Kong Polytechnic University, Hung Hom, Hong Kong, SAR, China
bThe Prevention Program, Barbara Ann Karmanos Cancer Institute, and Department of Pathology,
School of Medicine, Wayne State University, Detroit, MI, USA
Received 2 April 2004; revised 26 April 2004; accepted 26 April 2004
Available online 25 May 2004
Abstract—The green tea polyphenol catechin-3-gallate (CG) and epicatechin-3-gallate (ECG) were synthesized enantioselectively via
a Sharpless hydroxylation reaction followed by a diastereoselective cyclization. Their potencies to inhibit the proteasome activity
were measured. The unnatural enantiomers were found to be equally potent to the natural compounds.
Ó 2004 Elsevier Ltd. All rights reserved.
1. Introduction
containing green tea polyphenols, such as EGCG, GCG,
and ECG, possess the ability to inhibit proteasome
activity in vitro and in vivo.6 In contrast, the polyphe-
nols without the gallate ester function, such as EGC,
GC, and EC, are not inhibitors of proteasome.
According to a number of epidemiological studies, reg-
ular drinking of green tea has been associated with re-
duced risk of several forms of cancer.1 In 1996, a
development plan with respect to tea as a possible agent
for cancer prevention has been formulated by the Na-
tional Cancer Institute of the United States.2 The bio-
logical effects of green tea are often attributed to the
polyphenols, in particular, the catechins.3 Using liquid
chromatography combined with atmospheric pressure
chemical ionization-mass spectrometry, at least 12
catechins have been identified in green tea infusions.4
They are ())-epiafzelechin (EZ, 1), (+)-catechin (C, 2),
())-epicatechin (EC, 3), ())-gallocatechin (GC, 4), ())-
epigallocatechin (EGC, 5), their respective 3-gallate es-
ters ())-EZG (6), (+)-CG (7), ())-ECG (8), ())-GCG
(9), and ())-EGCG (10), as well as two 3-(30-
methyl)gallate esters ())-ECMG (11) and ())-EGCMG
(12). Although a number of cancer-related proteins are
affected by tea polyphenols, the exact mechanism of tea-
mediated cancer prevention is not known.5 Recently, it
was reported that the naturally occurring ester bond-
The proteasome is a multicatalytic protease responsible
for the degradation of most cellular proteins. The
eukaryotic proteasome contains three known activities:
chymotrypsin-like mediated by the b5-subunit, trypsin-
like by the b2-subunit and the caspase-like by the b1-
subunit.7 Since the ubiquitin/proteasome-dependent
degradation pathway plays an important role in the up-
regulation of cell proliferation and down-regulation of
cell death, proteasome inhibitors have been considered
as potential anticancer drugs.8 Indeed, MLN-341 (for-
merly PS-341), a dipeptidyl boronic compound and a
potent and selective inhibitor of the chymotrypsin-like
activity of the 20S proteasome, has recently been ap-
proved for the treatment of hematological malignant
neoplasms.9 In the case of natural green tea polyphe-
nols, the selective inhibition of the chymotrypsin-like
activity of proteasome by EGCG and other gallate es-
ters of catechins may offer an approach to develop drug
candidates for cancer prevention or therapy.6 Through
our synthetic efforts, we had recently prepared the nat-
ural ())-EGCG and ())-GCG as well as the enantiomers
(+)-EGCG and (+)-GCG.10 Surprisingly, it was found
that the unnatural enantiomers were equally or more
* Corresponding author. Tel.: +1-852-2766-5605; fax: +1-852-2364-
9932; e-mail address: bcchanth@polyu.edu.hk
Tel.: +1-313-966-0641; fax: +1-313-966-7368; e-mail address: doup@
karmanos.org
0968-0896/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.04.033

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S. B. Wan et al. / Bioorg. Med. Chem. 12 (2004) 3521–3527
effective as the natural compounds in inhibiting the
chymotrypsin-like activity of proteasome.11 Based on
these and similar in vitro results, a mechanistic model to
account for their inhibition of proteasome has been
proposed.12 In this model, the green tea polyphenol
gallate ester, for example, ())-EGCG, binds to the active
site of the b5-subunit of 20S proteasome. The A ring of
())-EGCG acts as the phenyl ring of a phenylalanine
mimic, binding to the hydrophobic S1 pocket of the b5-
subunit. The ester bond of ())-EGCG is then in rea-
sonable proximity (about 3.18) from the N-terminal Thr
(Thr 1) OH function, which is responsible for the pro-
tease catalytic activity. Inhibition is then due to the
irreversible transfer of the gallate moiety from ())-
EGCG to the hydroxy oxygen of Thr 1. This model was
evaluated using in silico docking calculations in con-
junction with biological activities of a number of natural
occurring or synthetic analogues. It was found that the
calculated binding free energies between the enzyme and
the inhibitors correlated quite well with the measured in
vitro IC50 values (Fig. 1).12
their inhibition of proteasome. As far as we are aware,
there has been no literature report on the enantioselec-
tive synthesis of these compounds.13
2. Results and discussion
2.1. Enantioselective synthesis of CG (7) and ECG (8)
The syntheses and 7 and 8 followed the sequence of
reactions outlined in Scheme 1. Friedel–Craft alkylation
of 3,5-bis(benzyloxy)phenol (13) with 30,40-bis(benzyl-
oxy)cinnamyl alcohol (14) under acidic conditions gave
compound 15. The double bond retained the trans ste-
reochemistry as evident from the coupling constant of
the vinyl hydrogens. Protection of the phenolic hydroxy
group as the t-butyldimethylsilyl (TBS) ether was fol-
lowed by Sharpless asymmetric dihydroxylation of 15
with AD-mix-a. Subsequent deprotection of the TBS
group gave the optically active compound (+)-16 in 48%
yield. The configurations of the two newly created chiral
centres were assigned to have the 1S, 2S stereochemistry
on the basis of the model normally used for Sharpless
asymmetric hydroxylation, and was later confirmed by
identity of the final product CG (7). Cyclization of (+)-
16 with ethyl orthoformate under catalytic pyridinium
p-toluenesulfonate conditions, followed by methanolysis
of the intermediate formate ester, gave the cyclized
compound (+)-17 in 73% yield. Compound 17 had the
trans stereochemistry as evident from its 1H NMR
spectrum. Furthermore, since there was an inversion at
the benzylic position, the stereochemistry of (+)-17 must
be 2R, 3S (chromane numbering). Acylation of (+)-17
with 3,4,5-tris(benzyloxy)galloyl chloride (18) gave the
protected gallate ester (+)-19. Hydrogenolysis of (+)-19
with Pd(OH)2/C gave (+)-CG (7) with 2R, 3S configu-
ration whose optical activity and NMR spectra were in
agreement with values reported for the natural com-
pound.14 On the other hand, when compound 15 was
converted to the dihydroxy compound ())-16 with AD-
In order to further validate this model, we have em-
barked on the synthesis of the natural (+)-CG (7) and
())-ECG (8) as well as their enantiomers and examined
R1
R1
R2
R3
R2
R3
HO
O
HO
O
OR
OR
OH
OH
1: R=R1=R3=H, R2=OH
2: R=R1=H, R2=R3=OH
3: R=R1=H, R2=R3=OH
4: R=H, R1=R2=R3=OH
5: R=H, R1=R2=R3=OH
7: R=gallate, R1=H, R2=R3=OH
9: R=gallate, R1=R2=R3=OH
6: R=gallate, R1=R3=H, R2=OH
8: R=gallate, R1=H, R2=R3=OH
10:R=gallate, R1=R2=R3=OH
11:R=3'-methylgallate, R1=H, R2=R3=OH
12:R=3'-methylgallate, R1=R2=R3=OH
Figure 1. Structure of catechins and analogs.
OBn
OBn
OBn
OBn
OBn
OBn
BnO
OH
BnO
OH
OBn
OH
BnO
OH
b,c,d
a
+
OH
OH
OBn
OBn
OH
16
15
14
13
OBn
OBn
OBn
OH
OBn
HO
O
BnO
O
BnO
O
OH
OH
OH
OBn
OBn
OBn
g
h
e, f
O
O
O
O
OH
OH
OBn
OBn
7
19
17
i
OH
OBn
OBn
OBn
OBn
OH
HO
g,h
O
BnO
O
BnO
O
OH
OH
OH
j
O
OH
21
O
OH
OBn
O
OBn
20
8
Scheme 1. Synthesis of CG and ECG. (a): H2SO4(SiO2)/CH2Cl2, rt; (b): TBSCl/imidazole/DMF, rt; (c): AD-mix/CH3SO2NH2/H2O/t-BuOH/
CH2Cl2, 0 °C; (d): TBAF/THF, rt; (e): CH(OEt)3/PPTS/(CH2Cl)2, rt; (f): K2CO3/MeOH/DME, rt; (g): 3,4,5-tris(benzyloxy)benzoyl chloride/DMAP/
CH2Cl2, rt; (h): H2/Pd(OH)2 on charcoal/MeOH/THF, rt; (i): Dess–Martin periodinane/CH2Cl2, rt; (j): L-Selectride/THF, )78 °C–rt.

S. B. Wan et al. / Bioorg. Med. Chem. 12 (2004) 3521–3527
3523
mix-b, and the same reaction sequence was followed,
())-CG (7) was obtained. It had the same NMR spectra
as the (+)-enantiomer but with opposite optical rota-
tion.
ranging from 3.7 to 5.3 lM. However, these four com-
pounds had much less inhibitory activities to the tryp-
sin-like activity: only 3–10% inhibition at 10 lM (Table
1). Therefore, these new synthetic polyphenol com-
pounds are potent inhibitors of the proteasomal chy-
motrypsin-like (and caspase-like) activities.
Dess–Martin oxidation of compound (+)-17 with the
2R, 3S configuration gave the ketone compound (+)-20
with 2R stereochemistry. Selective hydride reduction of
(+)-20 gave the cis epimer ())-21, now with the 2R, 3R
stereochemistry. Acylation of ())-21 with 18 gave 22,
which was followed by hydrogenolysis to give ())-ECG
with the same optical rotation as the natural ())-ECG
(8), with 2R, 3R configuration. The ent-(+)-ECG was
obtained by the same reaction sequence starting from
())-17.
The nearly equal potencies of the synthetic enantiomeric
())-CG and (+)-ECG as their natural compounds in
proteasome inhibition corroborate our previous obser-
vations on GCG and EGCG where similar potencies
were observed for both the natural and the synthetic
enantiomers.11;12 Any mechanistic model to account for
the proteasome inhibition activities of the green tea
catechin gallate esters will have to take these observa-
tions into consideration. Our previous in silico docking
calculation suggested that the natural ())-EGCG binds
to the b5-chymotrypsin active site of proteasome. In this
model, the fairly hydrophobic A–C rings of ())-EGCG
are oriented in the S1 pocket of the b5-subunit, the B
ring projected up into solvent, and the gallate (G) ring
2.2. Inhibition of proteasome
We then tested whether these synthetic compounds,
())-ECG, (+)-ECG, ())-CG and (+)-CG, can inhibit
the proteasomal chymotrypsin-like activity, using
())-EGCG as a positive control.6;11;12 We first used a
purified 20S prokaryotic proteasome. The chymotryp-
sin-like activity of purified 20S prokaryotic proteasome
was significantly inhibited by the synthetic ())-ECG and
(+)-CG with IC50 values of 0.58 and 0.73 lM, respec-
tively (Table 1). Furthermore, the synthetic ent-(+)-
ECG and ent-())-CG were also equally potent to inhibit
the chymotrypsin-like activity of purified proteasome
(Table 1). As expected, ())-EGCG acts as a potent
inhibitor of the proteasomal chymotrypsin activity
(IC50 ¼ 0.30 lM in Table 1), similar to what we reported
previously.6;12 In our previous study,12 we have shown
that ())-ECG and ())-CG were able to inhibit the chy-
motrypsin-like activity of a purified rabbit 20S protea-
some with similar potencies (IC50 values of 0.71 and
0.51 lM, respectively). When both (+)-ECG and (+)-CG
were tested using the purified rabbit 20S proteasome, we
observed similar potencies to those reported in Table 1.
ꢀ
ester bond-carbon located 3.18 A away from the hy-
droxyl of Thr 1.12 Experimentally, the synthetic ent-(+)-
EGCG was found to be slightly more potent than the
natural ())-EGCG with regard to purified 20S protea-
some.11 The docking studies revealed that (+)-EGCG
was also oriented in the proteasome b5-subunit, with the
A–C rings in the S1 pocket and the B ring in solvent.
However, in order that the gallate (G) group of (+)-
EGCG was to bind in the same relative position to the
hydroxyl of Thr 1 as ())-EGCG, the A–C rings of (+)-
EGCG had to flip 180° (in relation to the A–C rings) to
attain similar orientation/conformation. Due to the
partial symmetry of the A–C rings, with O1 being iso-
stere of C4 (in C ring) and B ring similar to G ring, the
model successfully accounted for the similar activities of
(+)-EGCG and the natural ())-EGCG.12 This mecha-
nistic and binding model could also be used to interpret
the proteasome inhibition properties of the natural
occurring GCG, ECG, and CG, which are less potent
than EGCG in proteasome inhibition.12 Their binding
energies to the proteasome’s active site have all in-
creased relative to that of EGCG due to either the
change of relative stereochemistry of the B and G rings,
or the absence of one hydroxyl group in the B ring. The
calculated binding free energies between the enzyme and
these compounds correlated reasonably well with the
measured in vitro IC50 values.12 Furthermore, since the
partial symmetry of the A–C rings is preserved between
the natural compounds and the synthetic enantiomers,
the model can also be used to explain the nearly equal
potencies of the unnatural enantiomers.
We next tested the potencies of these compounds in
inhibiting the 26S proteasome activities in a leukemia
Jurkat T cell extract. All the four compounds, ())-ECG,
(+)-ECG, ())-CG, and (+)-CG, greatly inhibited the
chymotrypsin-like activity of 26S proteasome, with IC50
values of 3.5, 3.0, 4.5, and 3.5 lM, respectively (Table 1).
As a positive control, ())-EGCG at 2.0 lM inhibited
50% of the 26S proteasomal chymotrypsin-like activity
(Table 1). Furthermore, ())-ECG, (+)-ECG, ())-CG
and (+)-CG also inhibited the caspase-like activity of the
26S proteasome in the cell extract, with IC50 values
Table 1. Effects of tea polyphenols on various proteasome activities
Compound
IC50 for chymotrypsin-like
activity of the purified 20S
prokaryotic proteasome (lM)
IC50 for chymotrypsin-like
activity of Jurkat T cell
extract (lM)
IC50 for caspase-like activity
of Jurkat T cell extract (lM)
Inhibition of trypsin-like
activity of Jurkat T cell
extract at 10 lM (%)
())-ECG
(+)-ECG
())-CG
(+)-CG
())-EGCG
0.58
0.73
0.75
0.73
0.30
3.5
3.0
4.5
3.5
2.0
5.3
4.2
3.9
3.7
3.1
3
3
10
10
2

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S. B. Wan et al. / Bioorg. Med. Chem. 12 (2004) 3521–3527
3. Experimental
148.8, 148.6, 137.0, 130.0, 128.4, 127.7, 127.2, 127.1,
126.7, 120.1, 114.7, 112.7, 71.2, 71.1, 63.5.
3.1. General
The starting materials and reagents, purchased from
commercial suppliers, were used without further purifi-
cation. Literature procedures were used for preparation
of 3,5-bis(benzyloxy)phenol (13),15 3,4,5-tris(benzyloxy)-
benzoic acid,16 silica gel supported H2SO4,10 and (E)-
3,4-bis(benzyloxy)cinnamyl alcohol (14).17 Anhydrous
THF was distilled under nitrogen from sodium benzo-
phenone ketyl. Anhydrous methylene chloride was dis-
tilled under nitrogen from CaH2. Anhydrous DMF was
distilled under vacuum from CaH2. Reaction flasks were
flame-dried under a stream of N2. All moisture-sensitive
reactions were conducted under a nitrogen atmosphere.
Flash chromatography was carried out using silica-gel
60 (70-230 mesh). The melting points were uncorrected.
1H NMR and 13C NMR (400 MHz) spectra were mea-
sured with TMS as an internal standard when CDCl3
and acetone-d6 were used as a solvent. High-resolu-
tion (ESI) MS spectra were recorded using a QTOF-2
Micromass spectrometer.
3.3. (E)-3-[2,4-Bis(benzyloxy)-6-hydroxyphenyl]-1-[3,4-
bis(benzyloxy)phenyl]propene (15)
At rt under an N2 atmosphere, 25% H2SO4/SiO2 (1.6 g,
4 mmol) was added in one batch to the stirred mixture of
3,5-bis(benzyloxy)phenol (3.06 g, 10 mmol) and (E)-3,4-
bis (benzyloxy)cinnamyl alcohol (3.46 g, 10 mmol) in dry
CH2Cl2 (50 mL). The resulting mixture was stirred at rt
overnight. After filtration and evaporation, the residue
was purified by column chromatography on silica gel
(benzene) to afford the desired compound as white
amorphous mass (3.0 g, 47.3% yield): 1H NMR (CDCl3,
400 MHz): d 7.43–7.32 (m, 20H), 6.95 (d, J ¼ 1:2 Hz,
1H), 6.84–6.81 (m, 2H), 6.37 (A of AB, J ¼ 15:5 Hz,
1H), 6.27 (d, J ¼ 1:7 Hz, 1H), 6.16 (d, J ¼ 1:7 Hz, 1H),
6.14–6.10 (B of ABt, J ¼ 15:5, 5.8 Hz, 1H), 5.12 (s, 2H),
5.11 (s, 2H), 5.01 (s, 2H), 4.98 (s, 2H), 3.55 (d,
J ¼ 5:8 Hz, 2H); 13C NMR (CDCl3, 400 MHz): d 158.5,
157.6, 155.5, 148.7, 148.1, 137.0, 136.8, 136.6, 130.9,
129.8, 128.3, 128.2, 128.1, 127.7, 127.5, 127.4, 127.2,
127.1, 127.0, 126.4, 119.6, 114.8, 112.3, 106.7, 94.8, 93.4,
76.9, 71.1, 70.1, 69.8, 26.1; HRMS (ESI) calcd for
C43H38O5 (M+Na) 657.2617, found 657.2631.
3.2. (E)-3,4-Bis(benzyloxy)cinnamyl alcohol (14)
3,4-Bis(benzyloxy)benzaldehyde (9.0 g, 42.4 mmol) was
dissolved in dry THF (150 mL) under a nitrogen atmo-
sphere and cooled in ice bath. To this solution triethyl
phosphonoacetate (9.5 g, 42.4 mmol) was added. So-
dium hydride (1.7 g, 60% dispersion in mineral oil,
42.4 mmol) was then added in 10 batches. The mixture
was allowed to be stirred at rt for 2 h. Saturated aqueous
NaHCO3 solution was added. The organic phase was
separated, and the aqueous layer was extracted with
EtOAc. The organic phases were combined, dried
(MgSO4) and evaporated to afford a solid. The solid was
washed with hexane to remove the mineral oil to yield
ethyl (E)-3,4-bis(benzyloxy)cinnamate (10.1 g, 92.0%
yield): mp 80–81 °C.
3.4. ())-(1R,2R)-3-[2,4-Bis(benzyloxy)-6-hydroxyphenyl]-
1-[3,4-bis(benzyloxy)phenyl]propane-1,2-diol (())-16)
The propene 15 (3.0 g, 4.7 mmol) was dissolved in dry
DMF (50 mL), and to this solution imidazole (1.4 g) and
TBSCl (1.7 g) were added successively. The resulting
mixture was stirred at rt overnight, and then saturated
Na2CO3 solution was added to quench the reaction. The
mixture was extracted with EtOAc. The organic layers
were combined, dried (MgSO4), and evaporated. The
residue was purified by flash chromatograph on silica gel
(n-hexane and EtOAc 6/1 v/v) to afford [3,5-bis(benzyl-
oxy)-2-[3-[3,4-bis(benzyloxy)phenyl]allyl]-phenoxy]-tert-
butyldimethyl-silane (3.1 g). This material was used in
the next step without further purification.
To a solution of ethyl (E)-3,4-bis(benzyloxy)cinnamate
(9.5 g, 24.4 mmol) in dry THF (120 mL) at )78 °C under
a nitrogen atmosphere, DIBAL (36 mL, 1 M solution in
hexane, 54 mmol) was added dropwise. The mixture was
stirred at )78 °C for 1 h and then at rt for another 1 h.
The mixture was cooled to 0 °C and poured into a stirred
mixture of hexane (250 mL) and saturated aqueous
Na2SO4 solution (5 mL). The resulting mixture was
stirred until a large quantity of solid was formed. The
mixture was filtered, and the solid was thoroughly
washed with EtOAc. The organic solutions were com-
bined and dried (MgSO4). The residue after evaporation
of the solvent was washed again with hexane, and the
solid was collected and recrystallized in EtOAc and
hexane to afford (E)-3, 4-bis(benzyloxy)cinnamyl alco-
hol (7.9 g, 71% overall yield based on 3,4-dihydroxy-
bezaldehyde): mp 75–76 °C; 1H NMR (CDCl3,
400 MHz): d 7.44–7.29 (m, 10H), 6.98 (s, 1H), 6.84 (bs,
2H), 6.43 (d, J ¼ 15 Hz, 1H), 6.12 (dt, J ¼ 15, 5.8 Hz,
1H), 5.12 (s, 2H), 5.11 (s, 2H), 4.20 (dd, J ¼ 5:8, 1.3 Hz,
2H), 1.90 (br s, 1H); 13C NMR (CDCl3, 400 MHz): d
AD-mixb (12.8 g) and methanesulfonamide (0.85 g) were
dissolved in a solvent mixture of t-BuOH (60 mL) and
H2O (60 mL). The resulting mixture was stirred at rt for
5 min, then the mixture was cooled to 0 °C and a solu-
tion of [3,5-bis(benzyloxy)-2-[3-[3,4-bis(benzyloxy)-
phenyl]-allyl]phenoxy]-tert-butyldimethylsilane (3.1 g) in
dichloromethane (60 mL) was added. After the mixture
had been stirred overnight, a total of four batches of
AD-mixb (6.4 g each) and methanesulfonamide (0.43 g
each) were added in 24 h intervals. After another 24 h of
stirring at 0 °C, TLC showed that the reaction was
completed. Then a 10% Na2S2O3 aqueous solution
was added to quench the reaction. The mixture was
extracted with EtOAc. The organic phases were com-
bined, dried (MgSO4) and evaporated. The residue was
redissolved in THF (30 mL), and TBAF (10 mL, 1 M in
THF) was added. The resulting mixture was stirred at rt
for 4 h, and saturated NaHCO3 solution was added. The

S. B. Wan et al. / Bioorg. Med. Chem. 12 (2004) 3521–3527
3525
mixture was extracted with EtOAc and the organic
3.6. ())-(2S,3R)-5,7-Bis(benzyloxy)-2-[3,4-bis(benzyl-
oxy)-phenyl]chroman-3-yl 3,4,5-tris(benzyloxy)benzoate
(())-19)
layers were combined, dried (MgSO4), and evaporated.
The residue was purified by flash chromatography on
silica gel (5% EtOAc/CH3Cl) and then recrystallized in
EtOAc to give a white solid (2.1 g, 32% yield based on
Under an N2 atmosphere, a solution of 3,4,5-tris(benzyl-
oxy)benzoic acid (270 mg, 0.61 mmol) was refluxed with
oxally chloride (1 mL) in dry CH2Cl2 (10 mL) and one
drop of DMF for 3 h. The excess oxally chloride and
solvent were removed by distillation and the residue was
dried under vacuum for 3 h and dissolved in CH2Cl2
(2 mL). This solution was added dropwise to a solution
of ())-17 (200 mg, 0.3 mmol) and DMAP (75 mg,
0.62 mmol) in CH2Cl2 (15 mL) at 0 °C. The mixture was
stirred at rt overnight, then saturated NaHCO3 aqueous
solution was added. The organic layer was separated,
and the aqueous layer was extracted with CH2Cl2. The
organic phases were combined, dried (MgSO4) and
evaporated. The residue was purified by flash chroma-
tography on silica gel (5% EtOAc/benzene) to afford the
desired compound (260 mg, 81.0%). Recrystallization in
CHCl3 and ether gave a white powder: mp 149–151 °C;
compound 13): mp 160–162 °C; ½aꢀ )12.8 (c 0.5,
D
1
CH3Cl/MeOH 3/1 v/v); H NMR (CDCl3, 400 MHz): d
7.41–7.25 (m, 18H), 7.10 (d, J ¼ 6:9 Hz, 2H), 6.92 (d,
J ¼ 1:5 Hz, 1H), 6.80 (AB, J ¼ 8 Hz, 2H), 6.27 (d,
J ¼ 2:2 Hz, 1H), 6.19 (d, J ¼ 2:2 Hz, 1H), 5.10 (AB,
J ¼ 12:2 Hz, 2H), 5.05 (s, 2H), 5.02 (s, 2H), 4.88 (AB,
J ¼ 11:8 Hz, 2H), 4.44 (d, J ¼ 6:8 Hz, 1H), 3.97–3.93
(m, 1H), 2.92 (A of AB, J ¼ 14:5, 3.6 Hz, 1H), 2.74 (B of
AB, J ¼ 14:5, 8.2 Hz, 1H); 13C NMR (CDCl3,
400 MHz); d 158.8, 157.5, 157.2, 148.6, 148.5, 136.8,
136.7, 136.6, 136.5, 133.1, 128.3, 128.2, 128.1, 127.7,
127.5, 127.4, 127.3, 127.1, 126.9, 126.4, 119.7, 114.2,
113.3, 105.8, 95.6, 93.1, 76.9, 70.9, 70.7, 69.8, 69.7, 26.1;
HRMS (ESI) calcd for C43H40O7 (M+Na) 691.2672,
found 691.2677.
Using the same procedure with AD-mixa instead of
AD-mixb, (+)-(1S,2S)-3-[2,4-bis(benzyloxy)-6-hydroxy-
phenyl]-1-[3,4-bis(benzyloxy)phenyl]propane-1,2-diol
½aꢀ )36.8 (c 2.0, CHCl3); 1H NMR (CDCl3, 400 MHz):
D
d 7.38–7.30 (m, 35H), 7.19 (s, 2H), 6.99 (d, J ¼ 1:8 Hz,
1 H), 6.90 (Ab, J ¼ 8:3, 1.8 Hz, 2H), 6.29 (AB,
J ¼ 2:1 Hz, 2H), 5.47 (q, J ¼ 6:6 Hz, 1H), 5.10 (s, 1H),
5.09 (s, 2H), 5.06 (s, 2H), 5.03–5.02 (m, 4H), 5.00 (d,
J ¼ 2:7 Hz, 2H), 3.08 (AB, J ¼ 16:5, 5.4 Hz, 1H), 2.86
(AB, J ¼ 16:5, 6.7 Hz, 1H); 13C NMR (CDCl3,
400 MHz): d 158.7, 158.2, 155.2, 148.9, 148.8, 137.2,
137.1, 136.9, 136.8, 131.4, 128.5, 128.4, 128.3, 128.2,
128.1, 127.9, 127.8, 127.7, 127.5, 127.4, 127.2, 127.1,
119.4, 115.0, 113.4, 100.9, 94.6, 78.3, 71.3, 70.0, 69.8,
66.2, 28.1; HRMS (ESI) calcd for C71H61O10 (M+Na)
1095.4084, found 1095.4080.
((+)-16) (½aꢀ +12.8 (c 0.5, CH3Cl/MeOH 3/1 v/v)) was
D
prepared and it had identical NMR spectra as the
())-isomer.
3.5. ())-(2S,3R)-5,7-Bis(benzyloxy)-2-[3,4-bis(benzyloxy)-
phenyl]chroman-3-ol (())-17)
To a suspension of (1R,2R)-3-[2,4-bis(benzyloxy)-6-hy-
droxyphenyl]-1-[3,4-bis(benzyloxy)phenyl]propane-1,2-
diol (2.1 g, 3.1 mmol) in 1,2-dichloroethane (50 mL) was
added triethyl orthoformate (2 mL), followed by PPTS
(500 mg, 2.0 mmol). The mixture was stirred at rt for
20 min and the solid dissolved. The mixture was heated
to 60 °C for 5 h until TLC showed the reaction had been
completed. After evaporation of the solvent, the residue
was redissolved in DME (30 mL) and MeOH (30 mL),
K2CO3 (500 mg) was added, and the mixture was stirred
at rt overnight. The solvent was evaporated, and the
residue was purified by flash chromatography on silica
gel (EtOAc/hexane, 1/3 v/v) to afford the desired product
Using the same procedure with (+)-17 as the starting mate-
rial, (+)-(2R,3S)-5,7-bis(benzyloxy)-2-[3,4-bis(benzyl-
oxy)phenyl]chroman-3-yl 3,4,5-tris(benzyloxy)-benzoate
((+)-19), ½aꢀ +36.8 (c 2.0, CHCl3) was prepared with
D
identical NMR spectra as the ())-isomer.
3.7. ())-3-O-Galloylcatechin (())-7)
Under an H2 atmosphere, Pd(OH)2/C (20%, 400 mg)
was added to a solution of ())-19 (260 mg, 0.24 mmol) in
a solvent mixture of THF/MeOH (1:1 v/v, 50 mL). The
resulting reaction mixture was stirred at rt under H2 for
6 h, TLC showed that the reaction was completed. The
reaction mixture was filtered to remove the catalyst. The
filtrate was evaporated, and the residue was rapidly
purified by flash chromatograph on silica gel (2%AcOH/
10% MeOH/CH2Cl2, then 2%AcOH/20% MeOH/
CH2Cl2) to afford ())-CG (80 mg, 74% yield): mp 248–
251 °C (decompose); ½aꢀ )54 (c 0.9, EtOH), lit. ½aꢀ
as white solid (1.5 g, 73% yield): mp 136–138 °C; ½aꢀ )3
D
1
(c 1.0, CH2Cl2); H NMR (CDCl3, 400 MHz): d 7.43–
7.31 (m, 20H), 7.02 (s, 1H), 6.94 (d, J ¼ 0:9 Hz, 2H),
6.27 (d, J ¼ 2:2 Hz, 1H), 6.20 (d, J ¼ 2:2 Hz, 1H), 5.19
(m, 4H), 5.02 (s, 2H), 4.98 (s, 2H), 4.62 (d, J ¼ 8:2 Hz,
1H), 3.99–3.97 (m, 1H), 3.13 (A of AB, J ¼ 16:4, 5.6 Hz,
1H), 2.67 (B of AB, J ¼ 16:4, 8.8 Hz, 1H); 13C NMR
(CDCl3, 400 MHz): d 158.5, 157.5, 155.0, 149.0, 136.8,
136.7, 136.6, 128.3, 128.2, 127.7, 127.6, 127.5, 127.3,
127.2, 126.9, 126.8, 120.3, 114.7, 113.6, 102.0, 94.1, 93.5,
81.3, 71.0, 70.9, 69.8, 69.6, 67.9, 27.4; HRMS (ESI)
calcd for C43H39O6 (M+H) 651.2747, found 651.2712.
D
D
)51.5 (c 0.2, EtOH);18 1H NMR (acetone-d6/D2O, 3/1,
v/v, 400 MHz): d 7.11 (s, 2H), 7.03 (s, 1H), 6.87 (AB,
J ¼ 8:1 Hz, 2H), 6.16 (d, J ¼ 2 Hz, 1H), 6.07 (d,
J ¼ 2 Hz, 1H), 5.44 (q, J ¼ 6:6 Hz, 1H), 5.15 (d,
J ¼ 6:6 Hz, 1H), 3.03 (A of AB, J ¼ 16, 5.3 Hz, 1H),
2.82 (B of AB, J ¼ 16, 6.6 Hz, 1H); 13C NMR (acetone-
d6/D2O, 3/1, v/v, 400 MHz): d 165.7, 156.3, 155.9, 154.8,
144.7, 144.4, 138.1, 129.6, 119.8, 118.1, 115.0, 113.5,
Using the same procedure with (+)-16 as the starting
material, (+)-(2R,3S)-5,7-bis(benzyloxy)-2-[3,4-bis(ben-
zyloxy)phenyl]chroman-3-ol ((+)-17), ½aꢀ +3 (c 1.0,
D
CH2Cl2), was prepared with identical NMR spectra as
the ())-isomer.

3526
S. B. Wan et al. / Bioorg. Med. Chem. 12 (2004) 3521–3527
108.9, 98.2, 95.3, 94.3, 77.7, 69.6, 23.7; HRMS (ESI)
calcd for C22H19O10 (M+H) 443.0978, found 443.1016.
afford the desired product (450 mg, 90%) as a white
solid: mp 129–130 °C, lit 129.5–130;18 ½aꢀ )27 (c 2.2,
D
EtOAc), lit. ½aꢀ )27.7 (c 2.16, EtOAc);18 1H NMR
D
Using the same procedure with (+)-19 as the starting
material. the naturally occurring compound (+)-3-O-
(CDCl3, 400 MHz): d 7.43–7.30 (m, 20H), 7.13 (d,
J ¼ 1:6 Hz, 1H), 6.95 (AB, J ¼ 8, 1.6 Hz, 2H), 6.26 (s,
2H), 5.17 (s, 2H), 5.15 (s, 2H), 5.02 (s, 4H), 4.88 (s, 1H),
4.17 (b, 1H), 3.00 (A of AB, J ¼ 17:2, 1.3 Hz, 1H), 2.92
(B of AB, J ¼ 17:2, 4.3 Hz, 1H); 13C NMR (CDCl3,
400 MHz): d 165.0, 158.8, 157.6, 154.9, 152.2, 148.9,
148.8, 142.3, 137.3, 136.9, 136.8, 136.6, 136.4, 130.9,
128.5, 128.4, 128.3, 128.1, 127.9, 127.8, 127.7, 127.5,
127.4, 127.3, 127.1, 124.8, 119.9, 114.7, 113.2, 108.9,
101.3, 94.2, 93.6, 78.2, 75.0, 71.2, 71.0, 70.0, 69.8, 24.2;
HRMS (ESI) calcd for C43H39O6 (M+H) 651.2747,
found 651.2730.
galloylcatechin (½aꢀ +54 (c 0.9, EtOH); Lit +56 (c 0.9,
D
EtOH))18 was prepared with identical NMR spectra as
the ())-isomer.
3.8. (+)-(2R)-5,7-Bis(benzyloxy)-2-[3,4-bis(benzyloxy)-
phenyl]chroman-3-one ((+)-20))
Dess–Martin periodinane (6.3 mL, 15% g/mL in
CH2Cl2, 2.2 mmol) was added in one batch to a stirred
solution of (+)-17 (650 mg, 1.0 mmol) in CH2Cl2
(30 mL) under an N2 atmosphere. The mixture was
stirred at rt for about 2 h till TLC showed the absence of
starting material. Subsequently, saturated NaHCO3
solution (15 mL) and 10% Na2S2O3 aqueous solution
(15 mL) were added to quench the reaction. The organic
layer was separated, and the aqueous layer was ex-
tracted with CH2Cl2. The combined organic phases were
dried (MgSO4) and evaporated. The residue was purified
by flash chromatography on silica gel (benzene) and
then recrystallized in CHCl3 and ether to afford the
desired compound (500 mg, 76.0%): mp 141–143 °C, lit
142–143.5 °C;18 ½aꢀ +38 (c 2.1, CHCl3); lit. ½aꢀ +38.5 (c
Using the same procedure with ())-20 as the starting
material, (+)-(2S,3S)-5,7-bis(benzyloxy)-2-[3,4-bis(benzyl-
oxy)phenyl]chroman-3-ol ((+)-21), ½aꢀ +27 (c 2.2,
D
EtOAc), was prepared with identical NMR spectra as
the ())-isomer.
3.10. ())-(2R,3R)-5,7-Bis(benzyloxy)-2-[3,4-bis(benzyl-
oxy)-phenyl]chroman-3-yl 3,4,5-tris(benzyloxy)benzoate
(())-22)
Following the procedure used for the preparation of ())-
19 but with ())-21 as starting material, ())-(2R,3R)-5,7-
bis(benzyloxy)-2-[3,4-bis(benzyloxy)phenyl]-chroman-3-
yl 3,4,5-tris(benzyloxy)benzoate was obtained (80%
yield) as a white solid: mp 122–124 °C, lit 122–
123.5 °C;18 ½aꢀ )71 (c 2.2, CH2Cl2), lit. ½aꢀ )70.7 (c
D
D
2.08, CHCl3);18 1H NMR (CDCl3, 400 MHz): d 7.42–
7.25 (m, 20H), 6.95 (d, J ¼ 1:4 Hz, 1H), 6.89 (AB,
J ¼ 8:2 Hz, 2H), 6.35 (AB, J ¼ 1:9 Hz, 2H), 5.23 (s, 1H),
5.13 (s, 2H), 5.10 (d, J ¼ 2:9 Hz, 2H), 5.01 (s, 2H), 5.00
(s, 2H), 3.64 (AB, J ¼ 21:5 Hz, 2H); 13C NMR (CDCl3,
400 MHz) d 205.0, 159.4, 157.0, 154.4, 149.1, 148.9,
137.0, 136.9, 136.4, 128.6, 128.5, 128.4, 128.1, 128.0,
127.8, 127.7, 127.5, 127.3, 127.2, 127.1, 119.9, 114.6,
113.2, 101.9, 95.7, 95.0, 83.0, 71.1, 71.0, 70.2, 70.0, 33.6;
HRMS (ESI) calcd for C43H37O6 (M+H) 649.2590,
found 649.2580.
D
D
1.94, CH2Cl2);2 1H NMR (CDCl3, 400 MHz): d 7.37–
7.15 (m, 37H), 7.03 (s, 1H), 6.89 (AB, J ¼ 8 Hz, 2H),
6.37 (s, 1H), 6.31 (s, 1H), 5.60 (bs, 1H), 5.04 (bs, 1H),
5.00 (m, 12H), 4.74 (AB, J ¼ 11 Hz, 2H), 3.07 (bs, 2H);
13C NMR (CDCl3, 400 MHz): d 164.8, 158.7, 157.9,
155.6, 152.2, 148.8, 148.7, 142.3, 137.3, 137.0, 136.9,
136.7, 136.3, 130.9, 128.5, 128.4, 128.3, 128.2, 128.0,
127.9, 127.8, 127.6, 127.3, 127.2, 127.1, 124.8, 119.9,
114.5, 113.4, 108.9, 100.8, 94.5, 93.8, 77.5, 74.9, 71.0,
70.8, 70.0, 69.8, 68.4, 26.0; HRMS (ESI) calcd for
C71H61O10 (M+H) 1073.4265, found 1073.4230.
Using the same procedure with ())-17 as the starting
material, ())-(2S)-5,7-bis(benzyloxy)-2-[3,4-bis(benzyl-
oxy)phenyl]chroman-3-one (())-20), ½aꢀ )38 (c 2.1,
D
CHCl3), was prepared with identical NMR spectra as
the (+)-isomer.
Using the same procedure with (+)-21 as starting
material, (+)-(2S,3S)-5,7-bis(benzyloxy)-2-[3,4-bis(benzyl-
oxy)-phenyl]-chroman-3-yl 3,4,5-tris(benzyloxy)benzo-
3.9. ())-(2R,3R)-5,7-Bis(benzyloxy)-2-[3,4-bis(benzyloxy)-
phenyl]chroman-3-ol (())-21)
ate ((+)-22), ½aꢀ +71 (c 2.2, CH2Cl2), was prepared with
D
identical NMR spectra as the ())-isomer.
Under an N2 atmosphere, the ketone (+)-20 (500 mg,
0.77 mmol) was dissolved in dry THF (10 mL), and the
solution was cooled to )78 °C. Then L-Selectride
(1.5 mL, 1 M solution in THF, 1.5 mmol) was added
dropwise. The resulting solution was stirred at )78 °C
overnight. When TLC showed the reaction was com-
pleted, saturated NaHCO3 aqueous solution (10 mL)
was added to quench the reaction. The organic layer was
separated and the aqueous layer was extracted with
EtOAc. The combined organic phases were dried
(MgSO4) and evaporated. The residue was purified by
flash chromatograph on silica gel (5% EtOAC/benzene)
and then recrystallized with ethanol and EtOAC to
3.11. ())-3-O-Galloylepicatechin (())-8)
Following the procedure for the preparation ())-7, but
with ())-21 as starting material, ())-3-O-galloylepi cat-
echin was obtained (80% yield): mp 253–255°C
(decompose), lit 257–259 °C;18 ½aꢀ )157 (c 0.3, acetone),
D
lit. 160.6 (c 0.2, acetone);18 1H NMR (acetone-d6/D2O,
3/1, v/v, 400 MHz): d 7.12 (d, J ¼ 1:9 Hz, 1H), 7.06 (s,
2H), 6.94 (A of AB, J ¼ 8:2, 1.9 Hz, 1 H), 6.83 (B of AB,
J ¼ 8:2 Hz, 1H), 6.09 (AB, J ¼ 2:2 Hz, 2H), 5.49 (bs, 1
H), 5.13 (s, 1H), 3.09 (A of AB, J ¼ 17:3, 4.5 Hz, 1H),

S. B. Wan et al. / Bioorg. Med. Chem. 12 (2004) 3521–3527
3527
2.97 (B of AB, J ¼ 17:3 Hz, 1H); 13C NMR (acetone-d6/
D2O, 3/1, v/v, 400 MHz): d 166.9, 157.1, 157.0, 156.6,
145.7, 145.1, 139.1, 130.8, 120.9, 118.9, 115.8, 114.8,
109.9, 98.7, 96.3, 95.5, 77.8, 69.9, 26.3; HRMS (ESI)
calcd for C22H19O10 (M+H) 443.0978, found 443.1016.
T.H.C. acknowledges the support of McGill University
for the granting of a leave.
References and notes
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Using the same procedure with (+)-21 as starting
material, (+)-3-O-galloylepi catechin, ½aꢀ +155 (c 0.3,
D
acetone), was prepared with identical NMR spectra as
the ())-isomer.
Inhibition of purified 20S proteasome activity––The chy-
motrypsin-like activity of a purified 20S prokaryotic
proteasome (Methanosarcina thermophile, recombinant,
E. coli; from Calbiochem) or a purified rabbit 20S pro-
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quantity of 0.5 g of purified 20S proteasome was pre-
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96-well plate. This was followed by an additional
incubation with 20 lM fluorogenic peptide substrate,
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motrypsin-like activity), for 1 h at 37 °C in 100 lL of
assay buffer (20 mM Tris–HCl, pH 8.0). After that, the
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This work was supported by the Areas of Excellence
Scheme established under the University Grants com-
mittee of the Hong Kong Special Administrative Re-
gion, China (to T.H.C., Project No. AoE/P-10/01), and
research grants from the National Cancer Institute-
National Institutes of Health and Barbara Ann Karm-
anos Cancer Institute Prevention Program (to Q.P.D.).