Proteasome inhibition in human breast cancer cells with high catechol-O-methyltransferase activity by green tea polyphenol EGCG analogs

Article abstract of DOI:10.1016/j.bmc.2009.12.034

A pro-drug 8 of a synthetic analog 7 is more active in its antiproliferative activity against human breast cancer MDA-MB-231 cells possessing high catechol-O-methyltransferase (COMT) activity than the pro-drugs of EGCG and the analog 5. The higher activity of 8 is attributed to it not being a substrate of COMT.

Full text of DOI:10.1016/j.bmc.2009.12.034

Bioorganic & Medicinal Chemistry 18 (2010) 1252–1258
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Proteasome inhibition in human breast cancer cells with high
catechol-O-methyltransferase activity by green tea polyphenol EGCG analogs
a,c,
Congde Huo a, , Huanjie Yang b, , Qiuzhi Cindy Cui b, Q. Ping Dou b, , Tak Hang Chan
*
*
a Department of Chemistry, McGill University, Montreal, Quebec, Canada
b The Prevention Program, Barbara Ann Karmanos Cancer Institute, and Department of Pathology, School of Medicine, Wayne State University, Detroit, MI, USA
c Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Hong Kong, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A pro-drug 8 of a synthetic analog 7 is more active in its antiproliferative activity against human breast
cancer MDA-MB-231 cells possessing high catechol-O-methyltransferase (COMT) activity than the pro-
drugs of EGCG and the analog 5. The higher activity of 8 is attributed to it not being a substrate of COMT.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 22 October 2009
Revised 8 December 2009
Accepted 10 December 2009
Available online 16 December 2009
1. Introduction
to the same active site in proteasome suggested that the active site
may be nearly symmetrical and can accommodate both enantio-
Regular drinking of green tea has been associated with reduced
incidence of a variety of cancers. (À)-Epigallocatechin-3-gallate [1,
(À)-EGCG] is the most abundant constituent in green tea extract
and the most biologically active among the green tea polyphenols
(GTPs). A number of epidemiological and biological studies have
shown that (À)-EGCG can reduce or inhibit tumor growth in
breast,1–3 lung,4 GI5 and urinary6,7 tracts.
We previously reported that the tumor cellular proteasome is
an important target of (À)-EGCG.8 The eukaryotic proteasome is
a large multi-catalytic, multi-subunit protease complex. Inhibition
of the chymotrypsin-like activity of the proteasome has been asso-
ciated with induction of tumor cell apoptosis.9–11 We had previ-
ously found that (À)-EGCG inhibits the chymotrypsin-like
activity of the proteasome in vitro (IC50 = 86–194 nM) and in intact
mers equally well.12 On that basis, we designed a simple synthetic
analog 5 which is symmetrical and contains the gallate ester func-
tion. Interestingly, compound 5 was found to be nearly as potent a
proteasome inhibitor as either 1 or 3, with an IC50 = 340 nM.14
The challenge in developing (À)-EGCG for cancer prevention
and therapy is its low bioavailability, partly due to its poor absorp-
tion and its instability under neutral or alkaline conditions (i.e.,
physiologic pH) and partly due to biologically inactivating pro-
cesses such as methylation.16,17 We have previously tried to im-
prove the bioavailability of (À)-EGCG through
a pro-drug
approach.18 Acetylation of (À)-EGCG gave the peracetate of (À)-
EGCG (4, pro-EGCG). Even though 4 itself is not active as a protea-
some inhibitor, it exhibited enhanced growth inhibitory activity
relative to EGCG in a number of cancer cell lines.19 Improved bio-
availability was observed in vivo: intragastric administration of 4
to CF-1 mice led to higher concentration of EGCG in plasma, small
intestinal and colonic tissues compared with administration of
equimolar doses of EGCG.19 More importantly, the enhanced bio-
availability also manifested in enhanced bioactivity in vivo. In ani-
mal xenograft models, Pro-EGCG (4) was found to be more effective
than EGCG (1) at equivalent dosages in inhibiting tumor growth for
breast tumors20 and for androgen-independent prostate cancer.21
Methylation of (À)-EGCG occurs by catechol-O-methyltransfer-
ase (COMT), an enzyme widely distributed throughout the body.22
In humans, a single gene for COMT encodes both a soluble COMT
(S-COMT) and a membrane-bound COMT (MB-COMT). A single
nucleotide polymorphism (G to A) in codon 108 (S-COMT) or 158
(MB-COMT) results in a valine to methionine (Val to Met) substitu-
tion, leading to a high- (Val/Val [H/H]), intermediate- (Val/Met [H/
L]), or low-activity (Met/Met [L/L]) form of COMT.23 There is a
three-to-four-fold difference in enzyme activity between the high-
and low-activity expressed genes.24 A recent case-control study of
tumor cells (1–10 lM) (8). In silico docking studies have indicated
that (À)-EGCG binds to the N-terminal threonine (Thr1) of the pro-
teasomal chymotrypsin active site thus inhibiting the proteasomal
chymotrypsin-like activity.12 Acylation of the threonine hydroxy
group by the gallate ester function in EGCG accounts for the inhi-
bition. This explains why green tea polyphenols without the ester
function, such as epigallocatechin (EGC, 2), is essentially inactive
(IC50 = 12 mM).8 Synthetic (+)-EGCG (3)13, the enantiomer of the
natural (À)-EGCG, and other synthetic analogs of green tea cate-
chins with an ester bond have also been found to inhibit the prote-
asomal chymotrypsin-like activity.14,15 The fact that the synthetic
(+)-EGCG (3) is equally potent
a
proteasome inhibitor
(IC50 = 170 nM) as the natural (À)-EGCG and that they both bind
* Corresponding author. Tel.: +313 576 8301 (Q.P.D.); +852 3400 8670 (T.H.C.).
E-mail addresses: doup@karmanos.org (Q.P. Dou), bcchanth@polyu.edu.hk (T.H.
Chan).
These two authors made equal contributions to this work.
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.12.034

C. Huo et al. / Bioorg. Med. Chem. 18 (2010) 1252–1258
1253
breast cancer in Asian–American women revealed that women
who consumed green tea and who also carried the low activity
COMT polymorphism had a reduced risk of breast cancer.25 In con-
trast, among those who were homozygous for the high activity
COMT allele, breast cancer risk did not differ between tea drinkers
and non-tea drinkers. These data suggest that EGCG and other tea
polyphenols may be less cancer-protective upon methylation. In
support of this possibility, catechins were known to be substrates
of human COMT.26,27 Recently, we found that, using synthetic
methylated catechins which are metabolites or potential metabo-
lites of tea EGCG in biomethylation,28 the proteasome inhibition
potency decreased as the number of methyl groups in methylated
EGCG increased.29 Metabolic O-methylation of EGCG may therefore
reduce the effectiveness of EGCG in its anti-cancer activity29 in
support of the human study.25
In this study, we wish to build on the observation that the sim-
ple analog 5 is almost as effective a proteasome inhibitor as the
natural EGCG. We want to know if acetylation of 5 would enhance
the cytotoxic activity of 5 just as pro-EGCG exhibited enhanced
growth inhibitory activity relative to EGCG. We also want to see
if the problem of metabolic O-methylation can be circumvented
by using analogs of EGCG. We hypothesize that an analog such as
7, being devoid of the o-catechol structure, should not be a sub-
strate of COMT. We report here the proteasome inhibition and
cytotoxic activity of 7 as well as its peracetate 8 against breast can-
cer MDA-MB-231 cells possessing high COMT activity. Their activ-
ities are compared with the natural EGCG (1) and the analogs 5 as
well as their peracetates 4 and 6, respectively.
obtained. Removal of the benzyl protection of 14 and 15 by palla-
dium catalyzed hydrogenolysis gave compounds 16 and 5, respec-
tively. Acetylation of 16 and 5 gave the corresponding acetates 17
and 6. Similar sequence of reactions of 12 with 3,5-dibenzyloxy-
benzoic acid (18) gave the series 7 and 21 as well as their acetates
8 and 22.
2.2. Bioassays
We first examined the ability of the analogs to inhibit the chy-
motrypsin-like activity of purified 20S proteasome (Fig. 2). EGCG
potently inhibited the proteasomal chymotryptic activity consis-
tent with our previous observation.8 As shown in Fig. 2, compound
5 which can be considered as an analog of EGCG, inhibited the pro-
teasomal chymotrypsin-like activity with an IC50 value of 19
(Fig. 2A). Compound 16, which can be considered as the analog
of (À)-EGC, is not active even at a concentration of 50 M. On
the other hand, compound 7, with an IC50 of 29 M, is less active
lM
l
l
than EGCG or 5 (Fig. 2A). This is consistent with our previous
observation that for EGCG, when the number of hydroxyl groups
in the B ring or D ring is reduced, their proteasomal inhibition
activity is also reduced.15,30 Not surprisingly, compound 21 is not
active in proteasome inhibition even at 50 lM. As expected, the
peracetates 4, 6, 8, 17 and 22 are all not active in proteasome inhi-
bition under in vitro conditions (Fig. 2B).
To test whether COMT could affect the proteasome inhibitory
effects of the two EGCG analogs 5 and 7, we tested proteasome
inhibition by compounds 5 and 7 in cells containing COMT. Human
breast cancer MDA-MB-231 cell lysates that contains high COMT
activity were treated with different concentrations of compound
5 or 7. Compound 7 inhibited 18–51% proteasomal activity at 1–
2. Results
10 lM while compound 5 only inhibited 10–16% proteasomal
2.1. Chemical synthesis
activity at the same conditions (Fig. 3A). Since compound 5 was
more potent in inhibiting purified 20S proteasome than compound
7 (Fig. 2) and compound 5 but not 7 contains COMT-recognizable
structure (Fig. 1), these results suggested that compound 5 may
be more susceptible to COMT modification than compound 7. As
The synthesis of compounds 5 and 7 as well as their peracetates
6 and 8 is outlined in Scheme 1. 1,4-Dihydronaphthalene (11) was
dihydroxylated with osmium tetraoxide to give the cis-diol 12.
Acylation of 12 with DCC/DMAP and an equivalent of benzyl-pro-
tected gallic acid 13 gave the corresponding monobenzoate 14.
When more than 2 equiv of 13 were used, the dibenzoate 15 was
a control, EGCG at 10
lM only inhibited ꢀ22% of the chymotryp-
sin-like activity in these cells, also not as active as compound 7
OR
O
O
OR
19:
R'=H, R=Bn
f
21
: R'=R=H
g
22
: R'=R=Ac
OR'
OR
OR
OR
OR
OR
OR
d
O
O
O
OH
OH
O
O
O
b
c
OR'
OR
12
15:
R=Bn
R=H
R=Ac
14:
R'=H, R=Bn
f
f
5
:
:
16
17
: R'=R=H
: R'=R=Ac
OR
g
g
6
OR
OR
a
e
O
OR
O
O
20:
R=Bn
R=H
R=Ac
f
OR
O
7
8
:
g
11
:
OR
Scheme 1. Reagents and conditions: (a) OsO4, NMO, acetone/H2O; (b) 3,4,5-tris(benzyloxy)benzoic acid (1.1 equiv), DCC, DMAP, CH2Cl2; (c) 3,4,5-tris(benzyloxy)benzoic acid
(2.1 equiv), DCC, DMAP, CH2Cl2; (d) 3,5-bis(benzyloxy)benzoic acid (1.1 equiv), DCC, DMAP, CH2Cl2; (e) 3,5-bis(benzyloxy)benzoic acid (2.1 equiv), DCC, DMAP, CH2Cl2; (f) Pd/
C, H2, THF/MeOH; (g) Ac2O, Py.

1254
C. Huo et al. / Bioorg. Med. Chem. 18 (2010) 1252–1258
OH
OH
OH
OH
OH
OH
OH
OH
HO
O
HO
O
HO
O
OH
O
O
OH
OH
OH
OH
OH
OH
OH
O
O
OH
OH
OH
3, (+)-EGCG
1, (-)-EGCG
2, (-)-EGC
OH
OH
OH
OH
OAc
O
O
O
O
OH
OAc
OAc
AcO
O
O
O
O
O
O
OH
OH
OH
OAc
OAc
OAc
O
OH
OH
OAc
4, pro-EGCG
7
5
Figure 1. Structures of EGCG and analogs.
0.1 μΜ
160
1 μΜ
10 μΜ
25 μΜ
50 μΜ
0.1 μΜ
1 μΜ
10 μΜ
140
140
120
100
80
60
40
120
100
80
60
40
20
0
20
0
DMSO
5
16
7
21
EGCG
0.1 μΜ
DMSO
5
7
EGCG
1 μΜ
160
140
120
100
80
10 μΜ
25 μΜ
50 μΜ
140
120
100
80
60
60
40
40
20
20
0
0
DMSO
6
17
8
22
4
EGCG
DMSO DNC
5
5+
DNC
7
7 + EGCG EGCG
DNC + DNC
Figure 2. Proteasome inhibition by EGCG analogs. Purified 20S proteasome (35 ng)
was incubated with compound 5, 7, 16, or 21 (A) or MDA-MB-231 cell extract
(5.7 lg) was incubated with compound 6, 8, 17, or 22 (B) at indicated concentra-
tions for 2 h, followed by measuring the proteasomal chymotrypsin-like activity.
EGCG was used as a comparison.
Figure 3. Inhibition on cellular proteasome by EGCG and EGCG analogs. (A) MDA-
MB-231 cell extracts (5.7 g) were incubated with different concentrations of
compound or or EGCG for 2 h, followed by performance of proteasomal
chymotrypsin-like activity assay. EGCG was used as a control. (B) MDA-MB-231 cell
extracts (5.7 g) were pre-treated with 10 M DNC for 20 min, followed by co-
l
5
7
l
l
incubation with 5, or 7 or EGCG for 2 h. The proteasomal chymotrypsin-like activity
was measured.
(Fig. 3A). Presumably, EGCG is also susceptible to COMT from the
lysate. Indeed, compound 5 or EGCG activity was improved when
the lysate was pre-treated with a COMT inhibitor, 3,5-dinitrocate-
chol (DNC). In contrast, compound 7’s activity was only slightly in-
creased at the same condition (Fig. 3B), further confirming that
compound 7 is resistant to COMT modification.
It was previously reported that pro-EGCG (4) exhibited en-
hanced growth inhibitory activity relative to EGCG (1) in a number
of cancer cell lines.18,20,21,30 We now found that the peracetates 6

C. Huo et al. / Bioorg. Med. Chem. 18 (2010) 1252–1258
1255
and 8 were also more potent in inhibiting cell growth compared to
120
100
80
60
40
20
0
their non-acetylated precursors 5 and 7 (Fig. 4). It was reasonable
to assume that the peracetates 6 and 8 function similarly as pro-
drugs to release 5 and 7 intracellularly after absorption. We next
compared the growth inhibitory activity of 4 with the peracetates
6 and 8 on human breast cancer MDA-MB-231 cells. The results are
summarized in Fig. 4. Among these pro-drugs, compound 8, the
pro-drug of compound 7, was the most potent analog, showing
70–79% inhibition in MDA-MB-231 cells (Fig. 4) at 25–50 lM. Next
to compound 8, compound 6, the pro-drug of compound 5 induced
about 50% inhibition in MDA-MB-231 cells (Fig. 4). Both are more
potent than pro-EGCG (4) itself which showed 0–32% inhibition at
these concentrations. It is tempting therefore to suggest that the
higher potency of compound 8 in inhibition of MDA-MB-231 pro-
liferation is due to the resistance of compound 7 to COMT methyl-
ation. By contrast, EGCG, though more potent an inhibitor for
purified 20S proteasome, is less potent in the inhibition of MDA-
MB-231 proliferation because it is readily subject to biomethyla-
tion by the COMT in MDA-MB-231 cells.29
To test the validity of the above argument, we examined
whether the inhibitory activity of compounds 8 (as pro-drug of
7) or 6 (as pro-drug of 5) as well as that of pro-EGCG (4) is affected
by the addition of 3,5-dinitrocatechol (DNC), a tight-binding inhib-
itor of COMT.37 We hypothesize that if DNC inhibits COMT-medi-
ated methylation of compound 5 or EGCG, we would observe
increased growth inhibitory activity of compound 6 or pro-EGCG
(4) on the addition of DNC. On the other hand, the growth inhibi-
tory activity of compound 8 would not be significantly affected
by the addition of DNC as compound 7 presumably should not be
a substrate of COMT. To test this idea, MDA-MB-231 cells were
treated with compounds 8 or 6, the pro-drugs of compounds 7 or
5, in the presence or absence of DNC. Compound 6 alone at
Figure 5. Effects of DNC on EGCG analogs efficacy against cell proliferation. Human
breast cancer MDA-MB-231 cells with high COMT activity were treated with 50
lM
EGCG analogs for 24 h in the absence or presence of 10
assay. Pro-EGCG was used as a comparison.
lM DNC, followed by MTT
6
8
Pro-E (4)
D 10 25 50 10 25 50 10 25 50
Ub-prs
actin
Figure 6. Accumulation of proteasome substrates. MDA-MB-231 cells were treated
with 50 M EGCG analogs for 22 h. Extracted proteins were subject to Western
blotting analysis using antibodies against ubiquitinated proteins and actin.
50
10
l
M inhibited 48% cell proliferation. With the co-addition of
l
lM DNC, compound 6 efficacy was increased, showing 88% inhi-
bition on cell proliferation (Fig. 5). Similarly, Pro-EGCG (4) efficacy
was greatly improved from 42% inhibition to 89% inhibition when
combined with DNC. However, compound 8 efficacy was not
greatly improved by co-treatment with DNC, showing 69% and
84% inhibition on cell proliferation without or with the presence
of DNC. These results are consistent with the hypothesis and sug-
gest that for the compounds which could be methylated by COMT
in cells, inhibition of COMT could increase their activity by reduc-
ing biomethylation. Compared with compound 7, compound 5 and
Pro-E are more susceptible towards COMT modification.
Next, we would like to see if these pro-drugs indeed function by
proteasome inhibition and if compound 8 is more potent to hit the
target, the proteasome. Indeed, higher levels of ubiquitinated pro-
teins were accumulated by compound 8 compared with compound
6 and Pro-EGCG (4) in MDA-MB-231 cells (Fig. 6), indicating that
more proteasome activity was inhibited by compound 8.
3. Discussion
The health benefits of green tea and its main constituent (À)-
EGCG have been widely supported by results from epidemiological,
cell culture, and animal studies.31 Since there are no toxic effects
associated with tea drinking, the attraction of using green tea ex-
tract or EGCG as therapeutic agents is considerable. Yet, the U. S.
Food and Drug Administration (FDA), after reviewing the human
data, concluded in 2005 that ‘it is highly unlikely that green tea re-
duces the risk of breast cancer or prostate cancer’.32 A major chal-
lenge in extrapolating the biological activities of green tea
polyphenols in vitro to possible benefits in vivo is its bioavailabil-
ity,33,35,36 The poor bioavailability of EGCG can be attributed to the
following factors: (a) the ease of degradation of EGCG in alkaline or
neutral conditions,34 (b) poor cellular uptake because of the high
aqueous solubility of EGCG and poor hydrophobicity to be ab-
sorbed by cells and (c) metabolic transformations such as methyl-
ation, glucuronidation and sulfation after absorption.16
25 μΜ
50 μΜ
140
120
100
80
60
40
20
0
In the present study, we demonstrated that a pro-drug of a
simple synthetic analog 5, the acetate 6, enhanced the cytotoxic
activity of 5 just as pro-EGCG (4) enhanced the growth inhibitory
activity relative to EGCG (Fig. 4). An even simpler analog 7,
though not as potent an inhibitor of purified proteasome as EGCG
or 5 (Fig. 2), was found to be a better proteasome inhibitor when
incubated with MDA-MB-231 cell extracts (Fig. 3). Furthermore,
the pro-drug of 7, the acetate 8, was found to be more potent
in growth inhibition than either 6 or pro-EGCG (Fig. 4). In order
to explain the enhanced cytotoxicity of 7 (from 8) towards
MDA-MB-231cells, we postulated that this is due to the biome-
thylation of 5 or EGCG by COMT in MDA-MB-231cells. We have
DMSO
6
5
8
7
Pro-E (4)
Figure 4. Inhibition on cell proliferation by EGCG analogs. Human breast cancer
MDA-MB-231 cells were treated with 25 or 50 M compound 5 or its pro-drug 6, or
compound 7 or its pro-drug 8 for 24 h, followed by MTT assay. Pro-EGCG (4) was
l
used as a comparison.

1256
C. Huo et al. / Bioorg. Med. Chem. 18 (2010) 1252–1258
recently assayed the COMT genotype of various breast cancer cell
lines via DNA sequencing and MDA-MB-231 was found to express
the COMT-H allele.37 Biomethylation of 5 or EGCG by COMT
would lead to methylated metabolites. We have previously
showed that the proteasome inhibition potency decreased as
the number of methyl groups in methylated EGCG increased.30
Compound 7, in contrast, does not have the o-catechol structure
and should not be a substrate of COMT and biomethylated. Sup-
porting evidence for the role of COMT in reducing the proteasome
inhibition activity of EGCG and 5 is provided by the addition of
DNC, a known COMT inhibitor37 (Fig. 3B). The activity of either
EGCG or 5 was enhanced in the presence of DNC. By contrast,
the proteasome inhibition activity of 7 was not affected by the
addition of DNC. Similarly, the antiproliferative activities of pro-
EGCG and 6 but not 8 are affected by co-treatment with DNC. Fig-
ure 5 showed that addition of DNC potently enhanced the anti-
4.1.2. Preparation of monobenzoates 14 and 19
To a solution of the corresponding benzoic acid (0.22 mmol) in
dry CH2Cl2 (20 mL), dicyclohexylcarbodiimide (DCC, 45 mg,
0.22 mmol) was added. The resulting mixture was stirred at room
temperature for 4 h, 4-dimethylaminopyridine (DMAP, 3 mg,
0.025 mmol.) was added, then a solution of diol 12 (33 mg,
0.2 mol) in CH2Cl2 (5 mL) was added dropwise. The mixture was
stirred at room temperature overnight. Then the mixture was con-
centrated, EA (1 mL) was added and cooled in fridge, the urea
byproduct was filtered and the filtrate was evaporated. The result-
ing residue was purified by column chromatography (EA/n-
hex = 1:3) to afford the desired compound as a pale yellow amor-
phous solid.
4.1.2.1. Compound 14. White solid (61% yield). 1H NMR (CDCl3,
300 MHz) d 7.43–7.13 (m, 22H), 5.48 (br s, 1H), 5.15 (s, 2H), 5.11
(s, 4H), 4.33 (s, 1H), 3.28–3.03 (m, 4H); 13C NMR (CDCl3, 75 MHz)
d 166.4, 152.7, 142.7, 137.6, 136.9, 133.3, 132.7, 129.5, 129.3,
128.9, 128.8, 128.5, 128.3, 127.8, 126.7, 126.6, 125.3, 109.4, 75.4,
73.4, 71.4, 69.8, 68.1, 35.0, 32.2.
proliferative activities of both pro-EGCG and
6 but not 8.
Inhibition of COMT by DNC restored the potencies of EGCG and
5 because they are better proteasome inhibitors than 7.
These results suggest that COMT does play an important role in
affecting the antiproliferative activities of EGCG and its analogs.
This is especially critical in cells having high COMT activities, such
as the MDA-MB-231 breast cancer cells. The results are also in
agreement with the epidemiological study which showed that for
Asian–American women who were homozygous for the high activ-
ity COMT allele, breast cancer risk did not differ between tea drink-
ers and non-tea drinkers.25 Such information will have to be taken
into consideration for any effort to develop green tea polyphenols
or analogs into therapeutics for cancer treatment.
4.1.2.2. Compound 19. White solid (65% yield). 1H NMR (CDCl3,
300 MHz) d 7.44–7.13 (m, 16H), 6.82 (s, 1H), 5.50 (br s, 1H), 5.14
(s, 1H), 5.05 (s, 4H), 4.35 (s, 1H), 3.31–3.08 (m, 4H); 13C NMR
(CDCl3, 75 MHz) d 166.6, 160.0, 136.8, 133.4, 132.8, 132.3, 129.6,
129.3, 129.0, 128.5, 128.0, 126.7, 126.7, 108.9, 107.4, 73.7, 70.6,
68.0, 35.0, 32.1.
4.1.3. Preparation of dibenzoates 15 and 20
To a solution of 12 (33 mg, 0.2 mmol) in CH2Cl2 (3.0 mL) were
added the corresponding benzoic acid (0.42 mmol), 4-dimethyl-
aminopyridine (DMAP, 6 mg, 0.05 mmol) and dicyclohexylcarbodi-
imide (DCC, 87 mg, 0.42 mmol). The mixture was stirred at room
temperature overnight. Then the mixture was concentrated, EA
(1 mL) was added and cooled in fridge, the urea byproduct was fil-
tered and the filtrate was evaporated. The resulting residue was
purified by column chromatography (EA/n-hex = 1:6) to afford
the desired compound as white amorphous solid.
4. Experimental
4.1. General experimentals for chemical synthesis
The starting materials and reagents, purchased from commer-
cial suppliers, were used without further purification. Anhydrous
methylene chloride was distilled under nitrogen from CaH2. Anhy-
drous DMF was distilled under vacuum from CaH2. Reaction flasks
were flame-dried under a stream of N2. All moisture-sensitive reac-
tions were conducted under a nitrogen atmosphere. Flash chroma-
tography was carried out using Silica Gel 60 (70–230 mesh). The
melting points were uncorrected. 1H NMR and 13C NMR
(300 MHz) spectra were measured with TMS as an internal stan-
dard when CDCl3, CD3OD and acetone-d6 were used as solvent.
High-resolution (ESI) MS spectra were recorded using a QTOF-2
Micromass spectrometer.
4.1.3.1. Compound 15. White solid (59% yield). 1H NMR (CDCl3,
300 MHz) d 7.43–7.22 (m, 38H), 5.72 (br s, 1H), 5.01–4.96 (m,
12H), 3.38 (dd, J = 17.4 Hz, J = 4.8 Hz, 1H), 3.25 (dd, J = 17.4 Hz,
J = 6.9 Hz, 1H); 13C NMR (CDCl3, 75 MHz) d 165.7, 152.7, 142.7,
137.7, 136.7, 132.6, 129.4, 128.8, 128.7, 128.5, 128.3, 128.2,
127.8, 126.9, 125.2, 109.2, 75.3, 71.2, 70.5, 32.5.
4.1.3.2. Compound 20. White solid (71% yield). 1H NMR (CDCl3,
300 MHz) d 7.42–7.18 (m, 28H), 6.74 (s, 1H), 5.72 (br s, 1H), 4.91
(m, 9H), 3.34 (m, 4H); 13C NMR (CDCl3, 75 MHz) d 165.9, 160.0,
136.6, 132.5, 132.2, 129.4, 128.8, 128.4, 127.9, 126.8, 108.6,
107.8, 70.6, 70.4, 32.3.
4.1.1. Preparation of cis-1,2,3,4-tetrahydro-naphthalene-2,3-
diol (12)
To a solution of 1,4-dihydronaphthalene (500 mg, 3.84 mmol)
in acetone/H2O (3.0/1.0 mL) was added a solution of NMO in H2O
(1.43 mL, 50 wt %, 6.90 mmol) and a solution of OsO4 in 2-
4.1.4. General procedures for palladium catalyzed
methyl-2-propanol (313 lL, 2.5 wt %, 25 lmol). The mixture was
hydrogenolysis: preparation of 5, 7, 16 and 21
stirred at room temperature for 16 h. Saturated Na2SO3 aqueous
solution (10 mL) was added and stirred for an additional 15 min.
H2O (10 mL) and EtOAc (30 mL) was added and stirred for 5 min.
The aqueous phase was extracted with EtOAc (4 Â 30 mL). The
combined organic phase was washed with brine (20 mL) and dried
over anhydrous Na2SO4. The solution was concentrated by rotary
evaporator and vacuum drying to give the crude product which
was purified by silica gel chromatography (hexane/EtOAc/
CH2Cl2 = 5/1/1) to afford 521.7 mg (83%) of the title compound as
a white solid. 1H NMR (acetone-d6, 300 MHz) d 7.13 (m, 2H), 7.09
(m, 2H), 4.11 (t, J = 5.4, 2H), 3.01 (m, 4H), 2.36 (s, 2H); 13C NMR
(acetone-d6, 75 MHz) d 132.87, 129.11, 126.25, 69.24, 34.32.
To a solution of benzylated substrate (0.1 mmol) in THF/MeOH
(3 mL, 1:2) was added palladium hydroxide (20 mg, 20% on car-
bon). The resulting mixture was stirred at room temperature until
TLC showed that the reaction was completed (within 6 h). Then the
reaction mixture was filtered to remove the catalyst. The filtrate
was evaporated to afford product as a white solid.
4.1.4.1. Compound 16. White solid (95% yield). 1H NMR (CD3OD,
300 MHz) d 7.13–7.01 (m, 6H), 5.39 (m, 1H), 4.25 (m, 1H), 3.14–
3.06 (m, 4H); 13C NMR (CD3OD, 75 MHz) d 167.1, 145.2, 138.6,
133.5, 132.8, 129.0, 128.8, 126.1, 126.0, 120.6, 109.0, 72.5, 67.4,

C. Huo et al. / Bioorg. Med. Chem. 18 (2010) 1252–1258
1257
34.4, 32.1; HRMS m/z calcd for C17H16O6Na 339.0840, found
339.0839.
chased from Invitrogen Co. (Carlsbad, CA). RPMI 1640 medium
was purchased from Invitrogen Co. (Carlsbad, CA). MTT (3-4, 5-
dimethyltiazol-2-yl-2.5-diphenyl-tetrazolium bromide) was pur-
chased from Sigma-Aldrich.
4.1.4.2. Compound 5. White solid (95% yield). 1H NMR (CD3OD,
300 MHz) d 7.21–7.16 (m, 4H), 7.09 (s, 4H), 5.61 (m, 2H), 3.37–
3.25 (m, 4H); 13C NMR (CD3OD, 75 MHz) d 165.3, 145.1, 138.0,
132.8, 129.0, 126.3, 120.9, 109.0, 69.7, 31.9; HRMS m/z calcd for
C24H20O10Na 491.0947, found 491.0949.
4.1.6.2. Cell culture. Human breast cancer MDA-MB-231 cells
were purchased from American Type Culture Collection (Manassas,
VA) and grown in RPMI 1640 medium supplemented with 10% FBS,
100 units/mL of penicillin, and 100
were maintained at 37 °C and 5% CO2.
lg/mL of streptomycin. Cells
4.1.4.3. Compound 21. White solid (95% yield). 1H NMR (CD3OD,
300 MHz) d 7.14–7.07 (m, 4H), 6. 92 (s, 2H), 6.44 (s, 1H), 5.43 (m,
1H), 4.27 (m, 1H), 3.17–3.10 (m, 4H); 13C NMR (CD3OD, 75 MHz)
d 170.6, 166.0, 134.2, 133.2, 132.9, 129.3, 129.1, 126.3, 126.2,
108.2, 107.4, 73.2, 67.2, 35.0, 32.1; HRMS m/z calcd for C17H16O5Na
323.0891, found 323.0890.
4.1.6.3. Inhibition of purified 20S proteasome activity by EGCG
and its analogs. A purified rabbit 20S proteasome (35 ng) was
incubated with 20 lM of substrate Suc-LLVY-AMC in 100 lL assay
buffer (20 mM Tris–HCl, pH 7.5), in the presence of EGCG or EGCG
analogs at different concentrations or the solvent for 2 h at 37 °C,
followed by measurement of hydrolysis of the fluorogenic sub-
strates using a Wallac Victor3™ multi-label counter with 355-nm
excitation and 460-nm emission wavelengths.
4.1.4.4. Compound 7. White solid (95% yield). 1H NMR (CD3OD,
300 MHz) d 7.16 (s, 4H), 6.88 (s, 4H), 6.46 (s, 2H), 5.66 (m, 2H),
3.31 (m, 4H); 13C NMR (CD3OD, 75 MHz) d 166.3, 158.6, 132.5,
131.9, 128.9, 126.4, 107.7, 107.3, 70.4, 31.8; HRMS m/z calcd for
C24H20O8 Na 459.1047, found 459.1050.
4.1.6.4. Inhibition of 26S proteasome activity by EGCG and its
analogs in vitro. Human breast cancer MDA-MB-231 cell extract
4.1.5. Preparation of the acetates 6, 8, 17 and 22
(5.7
100
l
g) was incubated in 20
lM of substrate Suc-LLVY-AMC in
To a solution of the corresponding substrate (0.1 mmol) and
Ac2O (0.5 mL) in pyridine (0.5 mL) at room temperature. The
resulting mixture was stirred overnight. Then EA (50 mL) was
added and 1 N HCl (1 mL) and washed with CuSO4 solution
(3 Â 10 mL), water (2 Â 10 mL) and brine (10 mL), dried over so-
dium sulfate and evaporated. The residue was purified by column
chromatography over silica gel (EA/n-hex = 3:2) to afford the title
product as white solid.
lL assay buffer (20 mM Tris–HCl, pH 7.5) in the presence of
EGCG or EGCG analogs at different concentrations or the solvent
for 2 h at 37 °C, followed by measurement of hydrolysis of the fluo-
rogenic substrates as described above.
4.1.6.5. Inhibition of cellular 26S proteasome by EGCG and its
analogs. Human breast cancer MDA-MB-231 cells were treated
with compound 5 or 7 for 24 h. Cell lysates were subjected to chy-
motrypsin activity assay and Western blotting analysis as de-
scribed before.20
4.1.5.1. Compound 17. White solid (92% yield). 1H NMR (CDCl3,
300 MHz) d 7.74 (s, 2H), 7.21–7.11 (m, 4H), 5.64 (m, 1H), 5.42
(m, 1H), 3.26–3.16 (m, 4H), 2.30 (s, 9H), 2.07 (s, 3H); 13C NMR
(CDCl3, 75 MHz) d 170.9, 167.8, 166.6, 164.1, 143.7, 139.0, 132.6,
132.3, 129.3, 128.6, 126.8, 126.7, 122.5, 70.9, 69.7, 32.5, 31.7,
21.4, 20.8, 20.4; HRMS m/z calcd for C25H24O10Na 507.1266, found
507.1262.
4.1.6.6. MTT assay. Cells were grown in a 96-well plate. Triplicate
wells of cells were treated with indicated concentrations of EGCG
or EGCG analogs for 24 h. After aspiration of medium, MTT
(1 mg/mL) was then added to the cell cultures, followed by incuba-
tion for 3 h at 37 °C. After cells were crystallized, MTT was re-
moved and DMSO was added to dissolve the metabolized MTT
product. The absorbance was then measured on a Wallac Victor3
1420 Multi-label counter at 540 nm.
4.1.5.2. Compound 6. White solid (94% yield). 1H NMR (CDCl3,
300 MHz) d 7.73–7.70 (m, 4H), 7.22–7.14 (m, 4H), 5.68 (m, 2H),
3.31 (m, 4H), 2.28 (s, 18H); 13C NMR (CDCl3, 75 MHz) d 167.9,
166.6, 164.1, 143.7, 139.1, 132.2, 129.4, 128.4, 126.9, 122.6, 71.1,
32.1, 20.8, 20.4; HRMS m/z calcd for C36H32O16Na 743.1576, found
743.1583.
Acknowledgements
Funding support from the Natural Sciences and Engineering Re-
search Council of Canada (NSERC) and the National Cancer Institute
(Grant Nos. 1R01CA120009 and 3R01CA120009-04S1) is gratefully
acknowledged.
4.1.5.3. Compound 22. White solid (90% yield). 1H NMR (CDCl3,
300 MHz) d 7.60 (s, 2H), 7.21–7.11 (m, 5H), 5.64 (m, 1H), 5.44
(m, 1H), 3.26–3.17 (m, 4H), 2.31 (s, 6H), 2.08 (s, 3H),; 13C NMR
(CDCl3, 75 MHz) d 170.9, 169.0, 164.6, 151.1, 132.6, 132.5, 132.3,
129.3, 126.8, 126.7, 120.7, 120.6, 70.8, 69.7, 32.4, 31.8, 21.4, 21.3;
HRMS m/z calcd for C23H22O8Na 449.1201, found 449.1207.
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