Semi-synthesis and proteasome inhibition of D-ring deoxy analogs of (-)-epigallocatechin gallate (EGCG), the active ingredient of green tea extract

Article abstract of DOI:10.1139/V07-141

A semi-synthetic route to the D-ring analogs of (-)-epigallocatechin gallate (EGCG) from the relatively abundant (-)-epigallocatechin (EGC), isolated from, green tea leaves, is described. A natural product (13), found in Cistus salvifolius, its acetate (14) and analog (17) were synthesized by this method. Their inhibitory activities against proteasomes were investigated.

Full text of DOI:10.1139/V07-141

495
Semi-synthesis and proteasome inhibition of
D-ring deoxy analogs of (–)-epigallocatechin gallate
(EGCG), the active ingredient of green tea extract1
Congde Huo, Guoqing Shi, Wai Har Lam, Di Chen, Quizhi Cindy Cui, Q. Ping Dou,
and Tak Hang Chan
Abstract: A semi-synthetic route to the D-ring analogs of (–)-epigallocatechin gallate (EGCG) from the relatively abun-
dant (–)-epigallocatechin (EGC), isolated from green tea leaves, is described. A natural product (13), found in Cistus
salvifolius, its acetate (14) and analog (17) were synthesized by this method. Their inhibitory activities against
proteasomes were investigated.
Key words: green tea, (–)-epigallocatechin gallate (EGCG), (–)-epigallocatechin (EGC), proteasome inhibition.
Résumé : On a développé une voie semisynthétique vers des analogues du cycle D du gallate de (–)-épigallocatéchine
(GEGC) à partir de la (–)-épigallocatéchine (EGC) qui est relativement abondante et qu’on a isolé des feuilles de thé
vert. Faisant appel à cette méthode, on a réalisé la synthèse du produit naturel 13 qu’on retrouve dans Cistus Salvifo-
lius, son acétate 14 et son analogue 17. On a évalué leurs activités inhibitrices vis-a-vis des protéasomes.
Mots-clés : thé vert, gallate de (–)-épigallocatéchine (GEGC), (–)-épigallocatéchine (EGC), inhibition des protéasomes.
[Traduit par la Rédaction] Huo et al. 502
Introduction
availability (6). The low bioavailability was partly due to
poor cellular uptake as well as the instability of EGCG in
alkaline or neutral solutions (7, 8). To improve the bio-
availability of EGCG, we recently proposed the use of
EGCG octaacetate (pro-EGCG, 5) as a prodrug (9). Com-
pound 5 was found to be more stable to alkaline than EGCG
(9) but inactive in vitro as an inhibitor of proteasomes, a
presumed key mechanism for the cancer cytotoxicity of
Tea, produced from the plant Camellia sinensis, has been
consumed by humans for thousands of years. Green tea
contains many polyphenols, commonly known as catechins,
which constitute about 30%–40% of the water-soluble compounds
in brewed green tea (1). The major catechins are: (–)-
epigallocatechin-3-gallate (EGCG, 1), (–)-epigallocatechin
(EGC, 2), (–)-epicatechin-3-gallate (ECG, 3), and (–)-
epicatechin (EC, 4) (Fig. 1). Of these, EGCG is by far the
most abundant and has been reported to have various biolog-
ical activities that may account for the beneficial effects at-
tributed to green tea (2, 3). Regular drinking of green tea has
been associated with reduced risk of several forms of cancer
(4), and the effect is attributed mainly to tea polyphenols, in
particular EGCG (5).
EGCG (9). Compound
5
was, however, converted
intracellularly into EGCG and active in proteasome inhibi-
tion and cancer cell apoptosis (10, 11). Furthermore,
intragastric administration of 5 to CF-1 mice resulted in
higher bioavailability compared to the administration of
equimolar doses of EGCG (10). More importantly, we
showed that in vivo for either breast cancer animal model
(11) or the androgen-independent prostate cancer animal
model (12), 5 showed a significant inhibition of cancer
growth compared to EGCG itself (11, 12).
A critical issue concerning the potential application of
EGCG as an anti-cancer agent is its known poor bio-
Received 10 October 2007. Accepted 30 November 2007. Published on the NRC Research Press Web site at canjchem.nrc.ca on
22 March 2008.
C. Huo2 and T.H. Chan.2,3 Department of Chemistry, McGill University, Montreal, QC H3A 2K6, Canada.
W.H. Lam. Department of Applied Biology and Chemical Technology and the Open Laboratory for Chiral Technology, the
Institute of Molecular Technology for Drug Discovery and Synthesis, The Hong Kong Polytechnic University, Hung Hom,
Kowloon, Hong Kong SAR, China.
G. Shi,4 D. Chen, Q.C. Cui, and Q.P. Dou. The Prevention Program, Barbara Ann Karmanos Cancer Institute, and Department of
Pathology, School of Medicine, Wayne State University, Detroit, MI 48201, USA.
1This article is part of a Special Issue dedicated to Professor R. Marchessault.
2Alternative address: Department of Applied Biology and Chemical Technology and the Open Laboratory for Chiral Technology, the
Institute of Molecular Technology for Drug Discovery and Synthesis, The Hong Kong Polytechnic University, Hung Hom,
Kowloon, Hong Kong SAR, China.
3Corresponding author (e-mail: tak-hang.chan@mcgill.ca).
4Present address: School of Applied Science, University of Science and Technology Beijing, Beijing, China.
Can. J. Chem. 86: 495–502 (2008)
doi:10.1139/V07-141
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Can. J. Chem. Vol. 86, 2008
Fig. 1. Four major green tea catechins, 1, 2, 3, and 4 and their
peracetates, 5, 6, 7, and 8.
Scheme 1.
compound 9 is simply the benzylated derivative of EGC, it
seems reasonable that EGC can serve as the starting material
for the preparation of 9 in a semi-synthesis approach.
Results and discussion
Even though the amounts of catechins, especially that of
EGCG and EGC, in green tea leaves are quite high, the puri-
fication of individual catechins to high purity (>98%) has
not been easy due to the presence of many components with
similar water solubility and the structural similarities of the
catechins. A US patent described a four-step process involv-
ing three column chromatographic separations to isolate
catechins from green tea, using expensive reverse phase col-
umn fillings (22). A more recent patent application de-
scribed a process of separating catechins by subjecting a
green tea extract to chromatography on a macroporous polar
resin with a polar elution solvent under pressure (23). We
have devised an alternative method of purifying catechins to
high purity by treating comminuted green tea leaves directly
or green tea extracts with acetic anhydride in pyridine. This
acetylation reaction converted the mixture of catechins into
fully acetylated catechins (Fig. 1) and rendered them less
hydrophilic and separable by simple column chromatogra-
phy over silica gel with ethyl acetate/hexane as eluent (see
experimental). In this way, EGCG octaacetate (5), EGC
hexaacetate (6), ECG heptaacetate (7), and EC pentaacetate
(8) were obtained as solids with good analytical data. HPLC
analysis showed that they can be obtained in >98% purity.
The amounts of the four acetates depended on the source of
green tea. Using Japanese green tea bags with OSK trade-
mark, EGCG octaacetate (5) and EGC hexaacetate (6) were
obtained respectively in 4.3% and 1.5% of the dry weight of
the green tea leaves used. The yields of compounds 7 and 8
were generally less than 1% of dry weight.
With EGC hexaacetate (6) readily at hand, we examined
the selective hydrolysis of the acetate moieties. We found
that ammonium acetate in aqueous methanol (24) was able
to selectively hydrolyze the aromatic acetate esters of EGC
hexaacetate (6) to give compound 10. Benzylation of 10
gave the protected benzyl ether 11. Base hydrolysis of 11 re-
moved the aliphatic acetate group to give the key intermedi-
ate 9 (Scheme 2).
After benzylated EGC (9) was obtained by the above
method, esterification of 9 with 4-benzyloxybenzoic acid
gave the ester 12. Catalytic hydrogenolysis of 12 gave com-
pound 13. Compound 13 is a natural product, which has
been identified and isolated in small quantities from C.
salvifolius, a plant indigenous in the Mediterranean area
(25). In Turkey, tea prepared from the leaves of C.
salvifolius has been used for the treatment of cancer (26).
Using this semi-synthetic method, we can obtain 13 from
Another factor that affects the bioavailability of EGCG is
due to metabolic biotransformation reactions, including
methylation, glucuronidation, and sulfonation, resulting in
reduced biological activities of EGCG in vivo (13–15). Re-
cently, an epidemiological study of women found that risk of
breast cancer did not differ between tea drinkers and non-tea
drinkers among those who were homozygous for the high
activity catechol-O-methyltransferase (COMT) (16). Only
women who carried at least one low activity COMT allele
and were tea drinkers had a significantly reduced risk of
breast cancer compared to non-tea drinkers (16). The
hypothesis to explain these results is that tea polyphenols are
methylated by COMT and that the methylated tea
polyphenols are less cancer-protective. Indeed, EGCG is a
substrate of COMT and is converted to methylated EGCGs
(17). In support of this hypothesis, we found that methylated
EGCGs are much weaker inhibitors of proteasomes (18). A
possible approach to circumvent the effect of metabolic
methylation is to use analogs of EGCG, which may be
poorer substrates of COMT. In this connection, we have re-
cently synthesized compound 13, a natural compound found
in Cistus salvifolius and a D-ring deoxy analog of EGCG
(19). Interestingly, even though 13 is a poorer inhibitor of
proteasome than EGCG itself, the prodrug of 13 (acetate 14)
was found to be a more potent cancer cytotoxic agent than
EGCG itself and comparable to 5 (19). A possible explana-
tion of the enhanced activity of 13 (via its prodrug 14) is
that 13 is probably a poorer substrate of COMT than EGCG.
To validate this explanation and to perform animal studies,
we need substantial quantities of 13 and 14, which cannot be
easily fulfilled with total synthesis (19, 20). We therefore
sought a semi-synthetic approach to prepare 13 and 14 as
well as the isomeric acetate (17).
In 2001, we reported the first total enantioselective syn-
thesis of EGCG (20). Subsequently, many analogs of EGCG
have been prepared utilizing the established total synthetic
routes (21), including compound 13, which was prepared via
the key intermediate 9 in 12 steps (Scheme 1) (19). Since
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Huo et al.
497
Scheme 2.
Scheme 4.
Fig. 2. The inhibition potency of 14 and 17 to the proteasomal
chymotrypsin-like activity in Jurkat T cells. Jurkat T cells were
treated with each of the indicated compounds at 50 µmol/L for
48 h, and then the proteasomal chymotrypsin-like activity of cell
lysate was determined.
Scheme 3.
Table 1. Inhibition of the CT-like activity of
purified 20S proteasome by D-ring analogs of
EGCG.
Compound
IC50 (µmol/L)
13
14
17
6.61 0.76a
n.ab
n.a.b
Note: Compounds at 2, 5, 10, 20, and 50 µmol/L
were incubated with a purified 20S proteasome
(0.1 µg) and the substrate Suc-LLVY-AMC
(40 µmol/L) for 2 h, followed by the measurement
of the proteasomal CT-like activity.
human leukemia Jurkat T cells were treated with each of
these compounds at 50 µmol/L for 48 h, using pro-EGCG 5
as a control. As shown in Fig. 2, 14 and 17 exhibit 43% and
37% of proteasome inhibition, respectively, while pro-
EGCG 5 inhibited ~50% of the proteasome activity (Fig. 2).
Because the per-acetylated EGCG was easier to get into
cells than EGCG and the per-acetylated EGCG can be con-
verted to EGCG by the cellular esterase enzymes (9, 11, 27),
we believe that 14 and 17 behave as prodrugs in a manner
similar to pro-EGCG.
aThe data are representatives of independent tripli-
cate experiments.
bn.a. represents the inhibition of the CT-like activ-
ity of purified 20S proteasome by the indicated com-
pounds at 50 µmol/L to be lower than 50%. Similar
results were obtained from three independent
experiments.
Consistent with the inhibition of proteasomal
chymotrypsin-like activity, the accumulation of the
proteasome target protein Bax (p21) and ubiquitinated Bax
[Bax/(Ub)n, mainly by 5] by 14, 17 and 5 were also ob-
served, while expression of Bax (p36), a calpain cleavage
product homodimer, was not increased (Fig. 3), indicating
that proteasome inhibition by these EGCG analog prodrugs
activates the caspase, but not calpain enzyme (see the fol-
lowing paragraph).
In various cancer cells, induction of apoptotic cell death is
the consequence of proteasomal CT-like activity inhibition.
We thus investigated apoptosis by measuring caspase-3 ac-
tivity and PARP cleavage. Figure 4 shows that 14 and 17 in-
duced a 13.5- and 15-fold increase of caspase-3 activity,
respectively, while pro-EGCG 5 also induced a ~15-fold in-
crease of caspase activity (Fig. 4). Trypan blue assay shows
that 14 and 17 induced 36% and 34% of cell death after
treatment for 24 h, while pro-EGCG induced 40% of cell
death (Fig. 5). Consistent with apoptotic cell death induc-
tion, the PARP cleavage fragment p85 was produced by 14,
17, and pro-EGCG 5 (Fig. 3). It has been shown that
caspase-3/-7 cleaves PARP, generating the p85 PARP frag-
EGC hexaacetate (6) isolated from green tea leaves in 5
steps with an overall yield of 38%. Acetylation of compound
13 afforded the fully acetylated derivative 14 (Scheme 3).
Esterification of 9 with 3-acetoxybenzoyl chloride gave
the ester 15. Catalytic hydrogenolysis followed by
acetylation gave compound 17, the positional isomer of 14
(Scheme 4).
With the synthesis accomplished, the inhibition potency of
compound 13, compound 14, and compound 17 against the
chymotrypsin (CT)-like activity of purified 20S proteasome
were investigated. As shown in Table 1, compound 13 was
an inhibitor to the CT-like activity of purified 20S
proteasome with IC50 of 6.61 µmol/L, while 14 and 17 had
much less inhibitory effect and could not inhibit 50% of the
CT-like activity of purified 20S proteasome at 50 µmol/L.
Those results were in accordance with our previous observa-
tions (19, 27).
We have previously reported that compound 13 has no ob-
vious inhibition to proteasomal CT-like activity in intact tu-
mor cells (19). To investigate if the acetylated derivative 14
and 17 could exhibit proteasomal inhibition in whole cells,
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Can. J. Chem. Vol. 86, 2008
Fig. 3. Compounds 14 and 17 inhibit the proteasome and acti-
vate the caspase pathway. Jurkat T cells were treated with
50 µmol/L of each indicated compound or solvent DMSO for
48 h, followed by Western blot analysis of the Bax and PARP.
DMSO- and pro-EGCG-treated cells were used as solvent control
and positive control, respectively, and Actin was used as a load-
ing control.
Fig. 5. Induction of cell death. Jurkat T cells were treated with
each of the compounds at 50 µmol/L for 24 h, and then the per-
centage of death cells was determined.
market in Hong Kong. Alternately, Shou Mee green tea
leaves were purchased from Ying Kee Tea House, Hong
Kong. Anhydrous DMF was distilled under vacuum from
CaH2. Anhydrous DCM was distilled under nitrogen from
CaH2. Anhydrous THF was distilled under nitrogen from so-
dium benzophenone ketyl. Flash chromatography was car-
ried out using silica gel 60 (70–230 mesh). The melting
points were uncorrected. The spectra were run on the fol-
1
lowing instruments: Varian AS500 spectrometer for H and
13C NMR and VG Micromass 7070F mass spectrometer for
MS and HR-MS.
To investigate their proteasome inhibition, compounds 13,
14, and 17 were diluted in DMSO to give stock solution of
50 mmol/L and stored in aliquots at –20 °C. Purified rabbit
20S proteasome and fluorogenic peptide substrates, Suc-
LLVY-AMC for the proteasomal chymotrypsin-like activity
and Ac-DEVD-AMC for caspase-3/-7 activity, were from
Calbiochem Inc. (San Diego, Calif.). RPMI 1640, penicillin,
and streptomycin were purchased from Invitrogen (Carlsbad,
Calif.). Mouse monoclonal antibody against human poly
(ADP-ribose) polymerase (PARP) was purchased from
BIOMOL International LP (Plymouth Meeting, Penn.).
Mouse monoclonal antibodies against Bax (B-9), goat
polyclonal antibody against actin (C-11), and HRP labeled
secondary antibodies were from Santa Cruz Biotechnology,
Inc. (Santa Cruz, Calif.).
Fig. 4. Induction of caspase-3 activity of Jurkat T cells. These
cells were treated with compounds at 50 µmol/L for 48 h, and
then the caspase-3 activity of cell lysate was determined.
Extraction and isolation of the four major green tea
catechins peracetates (5, 6, 7, and 8).
ment (28). The PARP cleavage is consistent with caspase-3
activation.
Method 1
Comminuted green tea leaves (100 g) were added to a
mixture of pyridine (200 mL) and acetic anhydride
(100 mL). The resulting mixture was stirred at room temper-
ature for 20 h. The mixture was filtered through a Buchner
funnel and washed with ethyl acetate. The filtrate was
washed with 0.1 mol/L HCl, saturated NaHCO3 solution,
water, and brine. The organic solution was dried over anhy-
drous magnesium sulfate and evaporated by rotary evapora-
tor. The residue was subjected to chromatography as
described later.
Conclusion
We have developed an efficient method for the isolation
and purification of various catechin acetates from green tea
leaves. Using EGC hexaacetate (6) as starting material,
semi-synthesis of D-ring deoxy analogs of EGCG can be ob-
tained in gram quantities. The acetates of these analogs
show cytotoxic activities and proteasomal inhibition after
cellular intake.
Method 2
Experimental
Green Tea leaves (100 g) were extracted with water (1 L)
at 70 °C for 30 min four times. The combined water solution
was washed with chloroform three times to remove the low
polarity components of green tea. Then the water phase was
extracted with ethyl acetate five times. The ethyl acetate so-
General
The starting materials and reagents, purchased commer-
cially, were used without further purification. Green tea bags
with OSK trademark were purchased from SEIYU super-
© 2008 NRC Canada

Huo et al.
499
lution was dried over anhydrous magnesium sulfate and
evaporated by rotary evaporator to give green tea extract as
a pale yellow powder after vacuum (10 g, 10%). The powder
(10 g) was added to a mixture of pyridine (70 mL) and ace-
tic anhydride (80 mL) with stirring. And the mixture was
kept for 20 h at 45 °C in an oil bath. The reaction mixture
was then poured into ice-cold water and extracted with ethyl
acetate three times. The combined organic layer was washed
with 0.1 mol/L HCl, saturated NaHCO3 solution, water, and
brine. The organic solution was dried over anhydrous mag-
nesium sulfate and evaporated by rotary evaporator.
The concentrate from method 1 or method 2 was purified
by column chromatography over silica gel (EA/n-Hex from
2:3 to 3:2) to afford the EC pentaacetate (8), EGC hexa-
acetate (6), ECG heptaacetate (7), and EGCG octaacetate
(5), sequentially. TLC performed with Merck pre-coated sil-
ica gel 60 F254 plates and with EA/n-Hex (v/v = 2:1) as
eluent gave the following Rf values: 8 0.68, 6 0.58, 7 0.46,
and 5 0.32.
EC pentaacetate (8)
1
Yield: 0.1%–1.2%. [α]2D0 –12.6 (c 2.0, CHCl3). H NMR
(CDCl3, 500 MHz) δ: 7.35 (s, 1H), 7.27 (d, J = 8.5 Hz, 1H),
7.20 (d, J = 8.5 Hz, 1H), 6.67 (d, J = 1.0 Hz, 1H), 6.57 (d,
J = 1.0 Hz, 1H), 5.39 (s, 1H), 5.11 (s, 1H), 2.98 (dd, J =
17.5 Hz, J = 4.0 Hz, 1H), 2.88 (d, J = 17.5 Hz, 1H), 2.29
(m, 12H), 1.92 (s, 3H). 13C NMR (CDCl3, 125 MHz) δ:
170.7, 169.3, 168.7, 168.4, 168.3, 155.2, 150.0, 149.9,
142.3, 142.1, 136.1, 124.6, 123.4, 122.3, 109.8, 109.0,
108.3, 76.9, 66.9, 26.3, 21.3, 21.0, 21.0, 20.9. LR-MS (ESI)
m/z: 523.11 (M+ + Na, 100). HR-MS m/z calcd. for
C25H24O11Na: 523.1216; found: 523.1206.
(–)-(2R,3R)-5,7-Dihydroxy-2-(3,4,5-
trihydroxyphenyl)chroman-3-yl acetate (10)
To a solution of 6 (558 mg, 1 mmol) in water/methanol
(1:4 v/v, 50 mL), ammonium acetate (1.54 g, 20 mmol) was
added. The resulting mixture was stirred at room tempera-
ture and the progress of the reaction was monitored by TLC.
After 24 h the mixture was concentrated and water was
added, then the mixture was extracted with ethyl acetate.
The organic extract was then dried over sodium sulfate and
evaporated. The residue was purified by column chromatog-
raphy over silica gel (EA/n-Hex = 3:1) to afford compound
EGCG octaacetate (5)
Yield: 4.2%–4.9%. Mp 114.3–115.1 °C. [α]2D0 –60.8 (c
1
2.0, CHCl3). H NMR (CDCl3, 500 MHz) δ: 7.63 (s, 2H),
7.24 (s, 2H), 6.73 (d, J = 2.0 Hz, 1H), 6.61 (d, J = 2.0 Hz,
1H), 5.64 (s, 1H), 5.18 (s, 1H), 3.06 (dd, J = 18.0 Hz, J =
4.5 Hz, 1H), 2.99 (dd, J = 18.0 Hz, J = 2.5 Hz, 1H), 2.31–
2.23 (m, 24H). 13C NMR (CDCl3, 125 MHz) δ: 169.1,
168.7, 167.9, 167.7, 167.0, 166.5, 163.8, 155.0, 150.0,
149.9, 143.7, 143.6, 139.2, 135.3, 134.6, 127.7, 122.6,
119.1, 109.7, 109.3, 108.3, 76.8, 68.3, 26.1, 21.3, 21.0, 20.8,
20.8, 20.4, 20.4. LR-MS (ESI) m/z: 817.15 (M+ + Na, 100).
HR-MS m/z calcd. for C38H34O19Na: 817.1592; found:
817.1605.
1
10 (296 mg, 85% yield) as a white solid. H NMR (acetone-
d6, 500 MHz) δ: 6.56 (s, 1H), 6.06 (d, J = 2.0 Hz, 1H), 5.96
(d, J = 2.0 Hz, 1H), 5.37 (s, 1H), 4.96 (s, 1H), 2.96 (dd, J =
17.5 Hz, J = 4.5 Hz, 1H), 2.78 (dd, J = 17.5 Hz, J = 2.0 Hz,
1H), 1.88 (s, 3H). 13C NMR (acetone-d6, 125 MHz) δ:
169.7, 157.1, 156.8, 156.3, 145.6, 132.6, 129.9, 106.1, 98.2,
95.7, 95.1, 77.1, 68.3, 30.0, 25.7, 20.3.
(–)-(2R,3R)-5,7-Bis(benzyloxy)-2-(3,4,5-
tris(benzyloxy)phenyl)chroman-3-yl acetate (11)
A solution of 10 (256 mg, 0.73 mmol) and benzyl bro-
mide (0.48 mL, 4 mmol) in acetone (20 mL) was treated
with potassium carbonate (554 mg, 4 mmol), and the result-
ing mixture was heated under reflux for 6 h. After cooling,
the mixture was concentrated and water was added. The
mixture was extracted with ethyl acetate. The organic phase
was then dried over sodium sulfate and evaporated. The resi-
due was purified by column chromatography over silica gel
(EA/n-Hex = 1:6) to afford the benzyl protected product 11
EGC hexaacetate (6)
Yield of dry weight of green tea leaves: 1.5%–2.1%. Mp
1
192–193 °C. [α]2D0 –15.6 (c 2.0, CHCl3). H NMR (CDCl3,
500 MHz) δ: 7.22 (s, 2H), 6.67 (d, J = 2.0 Hz, 1H), 6.57 (d,
J = 2.0 Hz, 1H), 5.38 (s, 1H), 5.09 (s, 1H), 2.95 (dd, J =
18.0 Hz, J = 4.5 Hz, 1H), 2.88 (dd, J = 18.0 Hz, J = 1.5 Hz,
1H), 2.28–2.27.94 (m, 15H), 1.94 (s, 3H). 13C NMR (CDCl3,
125 MHz) δ: 170.8, 169.3, 168.7, 168.0, 167.1, 155.0, 150.0,
149.9, 143.5, 135.7, 134.5, 119.2, 109.8, 109.1, 108.3, 76.7,
66.7, 26.1, 21.3, 21.0, 21.0, 20.9, 20.4. LR-MS (EI) m/z:
558.14 (M+, 15). HR-MS m/z calcd. for C27H26O13Na:
581.1271; found: 581.1252.
1
(382 mg, 65% yield) as a white solid. Mp 116–118 °C. H
NMR (CDCl3, 500 MHz) δ: 7.48–7.30 (m, 25H), 6. 82 (s,
2H), 6.35 (s, 1H), 6.34 (s, 1H), 5.47 (s, 1H), 5.19–5.06 (m,
10H), 4.99 (s, 1H), 3.06 (dd, J = 17.5 Hz, J = 4.5 Hz, 1H),
3.00 (d, J = 17.5 Hz, 1H), 1.84 (s, 3H). 13C NMR (CDCl3,
125 MHz) δ: 170.1, 158.7, 157.9, 155.3, 152.7, 138.1, 137.8,
137.0, 136.8, 136.8, 133.3, 128.6, 128.6, 128.5, 128.5,
128.4, 128.1, 128.0, 127.9, 127.9, 127.8, 127.5, 127.4,
127.1, 106.3, 100.8, 94.7, 93.9, 77.3, 75.1, 71.3, 70.1, 69.9,
67.7, 26.0, 21.0.
ECG heptaacetate (7)
1
Yield: 0.1%–0.8%. [α]2D0 –58.8 (c 2.0, CHCl3). H NMR
(CDCl3, 500 MHz) δ: 7.63 (s, 2H), 7.34 (s, 1H), 7.30 (d, J =
8.5 Hz, 1H), 7.20 (d, J = 8.5 Hz, 1H), 6.73 (d, J = 2.0 Hz,
1H), 6.61 (d, J = 2.0 Hz, 1H), 5.64 (s, 1H), 5.21 (s, 1H),
3.08 (dd, J = 18.0 Hz, J = 4.5 Hz, 1H), 2.99 (d, J = 18.9 Hz,
1H), 2.29–2.24 (m, 21H). 13C NMR (CDCl3, 125 MHz) δ:
168.9, 168.4, 168.0, 167.9, 167.4, 166.2, 163.5, 154.9,
149.7, 149.6, 143.3, 142.1, 142.0, 138.9, 135.4, 127.4,
124.4, 123.5, 122.3, 121.8, 109.5, 108.9, 108.1, 68.1, 26.0,
21.1, 20.8, 20.6, 20.6, 20.5, 20.1. LR-MS (ESI) m/z: 759.14
(M+ + Na, 100). HR-MS m/z calcd. for C36H32O17Na:
759.1537; found: 759.1539.
(–)-(2R,3R)-5,7-Bis(benzyloxy)-2-(3,4,5-tris(benzyl-
oxy)phenyl)chroman-3-ol (9)
To a solution of 11 (342 mg, 0.43 mmol) in methanol
(50 mL), potassium carbonate (119 mg, 0.86 mmol) was
added. The resulting mixture was stirred at room tempera-
ture and the progress of the reaction was monitored by TLC.
After 2 h the mixture was concentrated and water was
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Can. J. Chem. Vol. 86, 2008
added. The mixture was extracted with ethyl acetate. Evapo-
ration of the organic solvent afforded compound 9 (308 mg,
95% yield) as a white solid. Mp 135–137 °C. 1H NMR
(CDCl3, 500 MHz) δ: 7.45–7.27 (m, 25H), 6.83 (s, 2H), 6.30
(s, 2H), 5.15 (s, 4H), 5.08 (s, 2H), 5.04 (s, 4H), 4.91 (s, 1H),
4.23 (s, 1H), 3.03 (d, J = 17.0 Hz, 1H), 2.93 (dd, J =
17.0 Hz, J = 4.5 Hz, 1H). 13C NMR (CDCl3, 125 MHz) δ:
159.0, 158.6, 155.3, 153.3, 138.6, 138.1, 137.2, 137.2,
134.0, 128.9, 128.8, 128.8, 128.7, 128.7, 128.7, 128.4,
128.3, 128.2, 128.2, 128.1, 128.1, 127.8, 127.8, 127.5,
106.4, 101.2, 95.0, 94.4, 78.8, 75.5, 71.6, 70.4, 70.2, 66.7,
28.4. LR-MS (ESI) m/z: 779.28 (M+ + Na, 100). HR-MS m/z
calcd. for C50H44O7H: 757.3165; found: 757.3177.
MS (ESI) m/z: 989.37 (M+ + Na, 69). HR-MS m/z calcd. for
C64H54O9H: 967.3846; found: 967.3874.
(2R,3R)-5,7-Dihydroxy-2-(3,4,5-trihydroxy-
phenyl)chroman-3-yl 4-hydroxy-benzoate (13)
Under a hydrogen atmosphere, palladium hydroxide
(5 mg, 20% on carbon) was added to a solution of 12
(48.4 mg, 0.05 mmol) in ethyl acetate (5 mL). The resulting
mixture was stirred at room temperature until TLC showed
that the reaction was completed in about 2 h. The reaction
mixture was filtered to remove the catalyst. The filtrate was
evaporated to afford compound 13 (20.4 mg, 96% yield) as
1
white foam. H NMR (CDCl3, 500 MHz) δ: 7.78 (d, J =
8.0 Hz, 2H), 6.83 (d, J = 8.0 Hz, 2H), 6.63 (s, 2H), 6.05 (d,
J = 2.0 Hz, 1H), 6.03 (d, J = 1.5 Hz, 1H), 5.54 (m, 1H), 5.09
(s, 1H), 3.05 (dd, J = 17.0 Hz, J = 4.5 Hz, 1H), 2.95 (dd, J =
17.0 Hz, J = 2.5 Hz, 1H). 13C NMR (CDCl3, 125 MHz) δ:
165.3, 161.9, 157.1, 156.8, 156.4, 145.6, 131.9, 130.1,
121.9, 115.3, 105.9, 98.2, 95.7, 95.0, 77.4, 68.9, 25.8. LR-
MS (ESI) m/z: 449.08 (M+ + Na, 74). HR-MS m/z calcd. for
C22H18O9H: 427.1029; found: 427.1028.
4-Benzyloxybenzoic acid
To a suspension of 4-hydroxybenzoic acid (2.0 g,
15.2 mmol) and potassium carbonate (4 g) in dry DMF
(40 mL), benzyl bromide (3.6 mL, 30.5 mmol) was added
dropwise under a nitrogen atmosphere. The reaction mixture
was heated at 80 °C for 6 h. The mixture was evaporated,
added to ice water (300 mL) and acidified with 1:1 HCl to a
pH of 1. The suspension was filtered, washed three times
with water, then dried to give benzyl 4-(benzyloxy)benzoate
(3.5 g, 76%) as an off-white solid. To the suspension of the
dibenzylated product (2 g, 6.3 mmol) in methanol (20 mL)
was added 30% aqueous solution of potassium hydroxide
(4 mL) and heated at reflux for 6 h. The solvent was evapo-
rated, added to ice water (100 mL) and acidified to a pH of
1. The white precipitate was filtered, washed thoroughly
with water, and dried to give the acid (1.3 g, 91%) as white
(2R,3R)-5,7-Bis(acetoxy)-2-(3,4,5-tris(acetoxy)-
phenyl)chroman-3-yl 4-acetoxy-benzoate (14)
To a solution of 13 (17.1 mg, 0.04 mmol) and acetic anhy-
dride (28.4 µL, 0.30 mmol) in dry MeCN (5 mL) at –40 °C,
DMAP (5 mg, 0.04 mmol) was added. The resulting mixture
was stirred for 1 h. Saturated NaHCO3 solution was added
and the mixture was allowed to warm to room temperature.
The mixture was extracted three times with ethyl acetate.
The combined organic phase was washed with water and
brine, dried over sodium sulfate, and evaporated. The resi-
due was purified by column chromatography over silica gel
(EA/n-Hex 1:1) to afford the title product 14 (24.4 mg, 90%
yield) as a white solid. Mp 109–111 °C. [α]2D0 = –60.0 (c 0.2,
acetone). 1H NMR (CDCl3, 500 MHz) δ: 7.87 (d, J =
8.0 Hz, 2H), 7.27 (s, 2H), 7.09 (d, J = 8.0 Hz, 2H), 6.74 (d,
J = 2.0 Hz, 1H), 6.59 (d, J = 2.0 Hz, 1H), 5.62 (s, 1H), 5.20
(s, 1H), 3.06 (m, 2H), 2.28 (s, 6H), 2.27 (s, 3H), 2.24 (s,
3H), 2.23 (s, 6H). 13C NMR (CDCl3, 125 MHz) δ: 168.9,
168.6, 168.4, 167.5, 166.7, 165.0, 154.7, 154.4, 149.7,
149.7, 143.4, 135.4, 134.2, 131.4, 126.9, 121.5, 118.6,
109.5, 108.9, 108.0, 76.4, 67.5, 25.9, 21.1, 20.7, 20.6, 20.1.
LR-MS (ESI) m/z: 701.12 (M+ + Na, 67). HR-MS m/z calcd.
for C34H30O15Na: 701.1482; found: 701.1472.
1
solid. H NMR (CDCl3, 500 MHz) δ: 8.00 (d, J = 8.5 Hz,
2H), 7.51 (d, J = 8.0 Hz, 2H), 7.42 (t, J = 8.0 Hz, 2H), 7.35
(t, J = 8.0 Hz, 1H), 7.12 (d, J = 8.0 Hz, 2H), 5.23 (s, 2H).
(2R,3R)-5,7-Bis(benzyloxy)-2-(3,4,5-tris(benzyl-
oxy)phenyl)chroman-3-yl 4-(benzyl-oxy)-benzoate (12)
To a solution of 4-benzyloxylbenzoic acid (34.2 mg,
0.15 mmol) in dry CH2Cl2 (10 mL), dicyclohexyl-
carbodiimide (DCC, 31 mg, 0.15 mmol) was added. The re-
sulting mixture was stirred at room temperature for 4 h and
cooled down to 0 °C. 4-Dimethylaminopyridine (DMAP,
1.2 mg, 0.01 mmol.) was added, then a solution of 9
(75.7 mg, 0.1 mol) in dry CH2Cl2 (2 mL) was added
dropwise. The mixture was refluxed and the progress of the
reaction was monitored by TLC. After 24 h the mixture was
concentrated and ethyl acetate was added. The resulting
mixture was cooled in a refrigerator and the urea byproduct
was filtered. The filtrate was evaporated. The resulting resi-
due was purified by column chromatography (EA/n-Hex 1:6)
to afford compound 12 as a pale yellow amorphous solid
3-Acetoxybenzoyl chloride
A suspension of 3-hydroxybenzoic acid (3.5 g, 25 mmol)
was heated in acetic anhydride (5 mL) under reflux for 6 h.
The acetic anhydride was evaporated and the resulting resi-
due was triturated in ethyl ether, filtered, and washed with
ethyl ether to give 3-acetoxybenzoic acid as a white solid
(3.4 g, 74% yield). 1H NMR (CDCl3, 500 MHz) δ: 9.50 (brs,
1H), 8.00 (d, J = 7.5 Hz, 1H), 7.84 (s, 1H), 7.50 (t, J =
7.5 Hz, 1H), 7.36 (d, J = 8.5 Hz, 1H), 2.34 (s, 3H).
To a solution of 3-acetoxybenzoic acid (1 g, 5.5 mmol) in
methylene chloride (20 mL), thionyl chloride (10 mL) was
added. After the solution was stirred overnight, the solvents
were removed under vacuum to give 3-acetoxybenzoyl chlo-
1
(72.5 mg, 75% yield). [α]2D0 –42.1 (c 0.2, CHCl3). H NMR
(CDCl3, 500 MHz) δ: 7.96 (d, J = 8.0 Hz, 2H), 7.47–7.22
(m, 30H), 6.93 (d, J = 8.0 Hz, 2H), 6.81 (s, 2H), 6.36 (d, J =
1.5 Hz, 1H), 6.32 (d, J = 1.5 Hz, 1H), 5.68 (m, 1H), 5.09–
4.96 (m, 11H), 4.79 (d, J = 11.5 Hz, 2H), 3.11 (m, 2H). 13C
NMR (CDCl3, 125 MHz) δ: 165.3, 162.9, 159.0, 158.2,
155.8, 153.1, 138.5, 138.1, 137.3, 137.2, 137.1, 136.3,
133.6, 132.1, 128.9–122.9 (m, Bn), 114,7, 106.9, 101.2,
95.0, 94.2, 78.1, 75.4, 71.4, 70.4, 70.3, 70.2, 68.2, 26.4. LR-
© 2008 NRC Canada

Huo et al.
501
ride as a colorless oil. This was used for the next step with-
out further purification.
NMR (CDCl3, 125 MHz) δ: 169.3, 169.2, 168.7, 167.8,
167.0, 165.1, 155.0, 150.7, 150.0, 149.9, 143.7, 135.6,
134.5, 131.1, 129.6, 127.6, 126.9, 123.1, 119.0, 109.8,
109.2, 108.3, 76.8, 67.9, 26.2, 21.4, 21.3, 21.0, 20.8, 20.4.
LR-MS (ESI) m/z: 701.03 (M+ + Na, 100). HR-MS m/z
calcd. for C34H30O15Na: 701.1482; found: 701.1467.
(–)-(2R,3R)-5,7-Bis(benzyloxy)-2-(3,4,5-tris(benzyl-
oxy)phenyl)chroman-3-yl 3-acetoxybenzoate (15)
A solution of 3-acetoxybenzoyl chloride (164 mg,
0.8 mmol) in methylene chloride (2 mL) was added
dropwise to a solution of 9 (308 mg, 0.407 mmol) and
DMAP (122 mg, 1 mmol) in methylene chloride (15 mL) at
0 °C. The mixture was stirred at room temperature overnight
and then saturated NaHCO3 solution was added. The organic
layer was separated and the aqueous layer was extracted
with methylene chloride. The organic layers were combined,
dried over sodium sulfate, and evaporated. The residue
was purified by column chromatography over silica gel
(EA/n-Hex 1:5) to afford the desired compound, 15
Cell culture and cell extract preparation
Jurkat T cells were from American Type Culture Collec-
tion (Manassas, Va.). Cells were grown in RPMI 1640 sup-
plemented with 10% fetal bovine serum, 100 units/mL of
penicillin, and 100 µg/mL of streptomycin and were incu-
bated at 37<C in a humidified incubator with an atmosphere
of 5% CO2. A whole extract was prepared as previously de-
scribed (29).
1
Inhibition of chymotrypsin-like activity of purified 20S
proteasome
(114 mg, 78% yield), as a colorless oil. H NMR (CDCl3,
500 MHz) δ: 7.84 (d, J = 7.5 Hz, 1H), 7.68 (s, 1H), 7.45–
7.19 (m, 27H), 6. 78 (s, 2H), 6.34 (d, J = 2.0 Hz, 1H), 6.30
(d, J = 2.0 Hz, 1H), 5.66 (s, 1H), 5.13 (s, 1H), 5.05–4.94 (m,
9H), 4.85 (d, J = 11.5 Hz, 2H), 3.12–3.10 (m, 2H), 2.21 (s,
3H). 13C NMR (CDCl3, 125 MHz) δ: 169.0, 164.5, 158.8,
157.9, 155.4, 152.8, 150.6, 138.2, 137.7, 136.9, 136.8,
136.7, 133.2, 131.5, 129.3, 128.7, 128.6, 128.5, 128.5,
128.4, 128.0, 128.0, 127.9, 127.8, 127.7, 127.5, 127.5,
127.5, 127.5, 127.2, 126.6, 122.8, 106.4, 100.8, 94.8, 93.9,
77.6, 75.1, 71.2, 70.1, 70.0, 68.8, 26.0, 20.9. LR-MS (ESI)
m/z: 941.34 (M+ + Na, 17). HR-MS m/z calcd. for
C59H51O10: 919.3482; found: 919.3480.
Two µL of each compound’s stock DMSO solution was
added to 100 µL of 20S proteasome solution (25 mmol/L
Tris-HCl buffer, pH 7.5) on the 96-well plate, in the pres-
ence of Suc-Leu-Leu-Val-Try-7-amido-4-methyl-coumarin
(AMC) (40 µmol/L) fluogenic substrate for 2 h at 37 °C, fol-
lowed by measurement of hydrolyzed AMC groups using a
Victor 3 Multilabel counter with an excitation filter of
380 nm and an emission filter of 460 nm (PerkinElmer).
Proteasome CT-like activity and caspase-3 activity assay
in whole cell extracts
The Jurkat T cells were treated with 50 µmol/L of com-
pounds or solvent DMSO for 48 h, harvested, and lysed.
Whole cell extracts (10 µg) were incubated with 40 µmol/L
of Suc-Leu-Leu-Val-Try-AMC (for the proteasomal CT-like
activity) for 2 h or Ac-DEVD-AMC (for caspase-3 activity)
for 3 h at 37 °C. The release of the AMC groups was mea-
sured as described above.
(–)-(2R,3R)-5,7-Dihydroxy-2-(3,4,5-trihydroxy-
phenyl)chroman-3-yl 3-acetoxy-benzoate (16)
Under a hydrogen atmosphere, palladium hydroxide
(52 mg, 20% on carbon) was added to a solution of 15
(48 mg, 0.052 mmol) in a solvent mixture of THF/MeOH
(1:1 v/v, 8 mL). The resulting mixture was stirred at room
temperature until TLC showed that the reaction was com-
pleted. The reaction mixture was filtered to remove the cata-
lyst. The filtrate was evaporated to afford compound 16
Trypan blue assay
The trypan blue dye exclusion assay was performed as de-
scribed previously (30).
1
(22.5 mg, 91% yield) as a white solid. H NMR (CDCl3,
500 MHz) δ: 7.77 (d, J = 7.5 Hz, 1H), 7.61 (s, 1H), 7. 48 (t,
J = 7.5 Hz, 1H), 7.34 (d, J = 7.5 Hz, 1H), 6.63 (s, 2H), 6.05
(s, 1H), 6.03 (s, 1H), 5.60 (s, 1H), 5.12 (s, 1H), 3.09 (dd, J =
18.0 Hz, J = 4.5 Hz, 1H), 2.78 (d, J = 18.0 Hz, 1H), 2.27 (s,
3H).
Western blot analysis
Western blot analysis using the enhanced chemilumine-
scence reagent was done as previously described (11).
Acknowledgements
(–)-(2R,3R)-5,7-Bis(acetoxy)-2-(3,4,5-
tris(acetoxy)phenyl)chroman-3-yl 3-acetoxy-benzoate (17)
Compound 16 (20 mg, 0.043 mmol) was added to a mix-
ture of pyridine (0.7 mL) and acetic anhydride (0.8 mL) with
stirring, and the mixture was kept for 6 h at 45 °C in an oil
bath. The solvent was removed under vacuum. The crude
product was purified by column chromatography on silica
gel with EA/n-Hex (1:1) to afford the desired compound, 17
We thank the Areas of Excellence Scheme established un-
der the University Grants Committee of the Hong Kong Ad-
ministrative Region, China (Project No. AoE/P-10/01) for
financial support. Funding support from the Natural Sci-
ences and Engineering Research Council of Canada
(NSERC) and the National Cancer Institute (Grant Number:
1R01CA120009 and 5R03CA112625) is gratefully acknowl-
edged.
1
(22 mg, 75% yield), as a white solid. Mp 72–73 °C. H
NMR (CDCl3, 500 MHz) δ: 7.73 (d, J = 7.5Hz, 1H), 7.58 (s,
1H), 7.38 (t, J = 7.5 Hz, 1H), 7.27 (s, 2H), 7.25 (d, J =
7.5 Hz,1H), 6.75 (d, J = 2.5 Hz, 1H), 6.60 (d, J = 2.5 Hz,
1H), 5.64 (s, 1H), 5.21 (s, 1H), 3.06 (m, 1H), 2.30 (s, 3H),
2.29 (s, 3H), 2.28 (s, 3H), 2.25 (s, 3H), 2.24 (s, 6H). 13C
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