Synthesis of phosphates and phosphates-acetates hybrids of green tea polyphenol (-)-epigallocatechine-3-gallate (EGCG) and its G ring deoxy analogs as potential anticancer prodrugs

Article abstract of DOI:10.1016/j.tetlet.2011.08.061

A series of phosphate or phosphate-acetate hybrid modified EGCG or EGCG G ring deoxy analogs were synthesized by a convenient semi-synthesis strategy from the abundant natural compound EGCG.

Full text of DOI:10.1016/j.tetlet.2011.08.061

Tetrahedron Letters 52 (2011) 5478–5483
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Tetrahedron Letters
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Synthesis of phosphates and phosphates–acetates hybrids of green tea
polyphenol (ꢀ)-epigallocatechine-3-gallate (EGCG) and its G ring deoxy
analogs as potential anticancer prodrugs
Congde Huo a,b,e, , Q. Ping Dou c,d, Tak Hang Chan e
⇑
a Key Laboratory of Eco-Environment-Related Polymer Materials, Ministry of Education, Northwest Normal University, Lanzhou, Gansu 730070, China
b Gansu Key Laboratory of Polymer Materials, College of Chemistry and Chemical Engineering, Northwest Normal University, Lanzhou, Gansu 730070, China
c The Developmental Therapeutics Program, Barbara Ann Karmanos Cancer Institute, Detroit, MI 48201, USA
d Departments of Oncology, Pharmacology and Pathology, School of Medicine, Wayne State University, Detroit, MI 48201, USA
e Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Hung Hum, 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 series of phosphate or phosphate–acetate hybrid modified EGCG or EGCG G ring deoxy analogs were
synthesized by a convenient semi-synthesis strategy from the abundant natural compound EGCG.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 20 June 2011
Revised 23 July 2011
Accepted 9 August 2011
Available online 18 August 2011
Keywords:
Green tea polyphenols
Prodrugs
Anticancer
Phosphates
Acetates
Green tea, produced from the unfermented dried leaves of the
plant Camellia sinensis, originated in China and has been consumed
by humans for thousands of years. Regular drinking of green tea
has been associated with many health benefits.1,2 Since tea
consumption is generally not associated with any toxic effect, the
attraction of using green tea extract as potential therapeutic agents
is considerable.3 Polyphenolic catechins constituents are thought
to contribute to the biological effects of green tea. A number of cat-
echins have been identified and the major ones are (ꢀ)-epicatechin
(EC), (ꢀ)-epicatechin gallate (ECG), (ꢀ)-epigallocatechin (EGC) and
(ꢀ)-epigallocatechin gallate (EGCG). EGCG is found to be the most
abundant and significant active compound among them.4,5 It is
known that a number of cancer-related proteins are affected by
tea polyphenols, in particular by EGCG. However, the exact mech-
anism of tea-mediated cancer prevention is under active investiga-
tions.6 Several years ago, we proposed that inhibition of
proteasome may be a key mechanism in the cancer prevention
activity of green tea.7 Furthermore, it is the gallate ester bond-con-
taining tea polyphenols (e.g., ECG, EGCG) but not the flavan-3-ols
(e.g., EC, EGC), which inhibit the proteasomal chymotrypsin-like
(b5) activities of the proteasome8 and are responsible for the can-
cer prevention activity.
A major challenge in extrapolating the biological activities of
green tea polyphenols in vitro to possible effects in vivo is bioavail-
ability. In this respect, it is known that EGCG itself has poor
bioavailability.9 EGCG (Fig. 1) is relatively unstable under neutral
or alkaline conditions and could be rapidly degraded, involving
deprotonation of hydroxyl groups on the phenol rings. Moreover,
the hydroxyl groups of EGCG could be modified through biotrans-
formation reactions, such as methylation, glucuronidation, and
sulfate formation, resulting in reduced biological activities
in vivo. We have suggested that EGCG peracetate (1, Fig. 1) which
can be converted to EGCG under cellular conditions by esterases
with enhanced bioavailability in vivo can act as a prodrug.10 Even
though it is not an inhibitor of proteasome in cell-free system, 1 is
more potent than EGCG at inhibiting the proteasomal chymotryp-
sin-like activity in human breast cancer MDA-MB-231 cells.11 More
importantly, the enhanced bioactivity also manifested in animal
xenograft models.11,12 This progress has encouraged us to screen
other potential EGCG prodrugs in order to enhance and promote
more desirable qualities, such as chemical stability, bioavailability
and site selectivity.
Phosphate esters of alcohol or phenol functionalities have been
used as a prodrug approach. For example, a combretastatin A-4
⇑
Corresponding author. Tel.: +86 0931 7971533; fax: +86 0931 7971989.
E-mail address: huocongde1978@hotmail.com (C. Huo).
0040-4039/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2011.08.061

C. Huo et al. / Tetrahedron Letters 52 (2011) 5478–5483
5479
An intriguing possibility is to use the cyclic phosphate prodrug ap-
proach to direct the parent drug selectively to the liver by taking
advantage of the cytochrome P-450 catalyzed oxidation predomi-
nantly in hepatocytes.17–19 Such liver-targeted drug delivery
approach has been applied to a collection of nucleosides.19
These considerations have prompted us to examine the poten-
tial of phosphates or phosphate–acetate hybrids of EGCG and
analogs as prodrugs.
A challenge in EGCG chemistry is to differentiate the reactivity
of the eight phenolic OH groups. When EGCG was treated with ex-
cess dibenzyl phosphite with carbon tetrachloride (CCl4) together
with DMAP/DIPEA in CH3CN at ꢀ10 °C, EGCG was converted to
the corresponding dibenzyl phosphate ester 11 (38% yield) with
all eight OH phosphorylated (Scheme 1). The phosphorylation
was believed to occur through the intermediate dibenzyl chloro-
phosphate which was generated in situ from the reaction of diben-
zyl phosphite with CCl4.20 The 31P NMR of 11 showed the expected
six signals in the relative intensity ratios of 1:1:1:2:1:2 for the
phosphate groups in A, B and G rings. The 1H NMR of 11 also
showed the aromatic protons to be well separated and easily
assigned to those in A (d 6.94 and 6.61), B (7.79) and G (7.52) rings.
Their chemical shifts are all shifted downfield about 0.8 ppm
compared with the same protons in EGCG due to the electron
OAc
OH
OAc
OAc
OH
OH
B
AcO
O
HO
O
C
2
A
3
O
O
4
OAc
OAc
OAc
OH
OH
OH
O
O
G
OAc
OH
(-)-epigallocatechin gallate
(EGCG)
EGCG peracetate (1)
Figure 1. EGCG and EGCG peracetate (1).
phosphate prodrug is now undergoing phase 1 clinical trial with
the potential for combination with other conventional antitumour
drugs and radiotherapy.13–15 The phosphate salt itself is inactive
but there is rapid phosphate hydrolysis in vivo to produce combre-
tastatin A-4.15 Another example is the successful development of
Amifostine as the first broad-spectrum cytoprotective agent, on
the basis of higher concentration and activity of alkaline phospha-
tase (which dephosphorylates phosphate esters) in normal cell.16
ODBP
ODBP
OPO3H2
OPO3H2
DBPO
a
O
H2O3PO
b
O
ODBP
OPO3H2
EGCG
O
O
ODBP
11
ODBP
OPO3H
OPO3H2
O
2O
2
ODBP
ODBP
OPO3H2
OPO3H2
DBP=dibenzyl phosphate
Scheme 1. Reagents and conditions: (a) Dibenzyl phosphite (8.5 equiv), CCl4 (40 equiv), DMAP (0.8 equiv), DIPEA (16 equiv), MeCN, 0 °C, 2 h; (b) Pd/C, H2, THF/MeOH, 4 h.
OH
ODBP
OH
OH
ODBP
HO
O
HO
O
ODBP
O
a
O
OH
OH
OH
O
OH
12
ODBP
O
EGCG
ODBP
ODBP
OH
OBn
DBP=
P
OBn
O
ODBP
OPO H
3
2
ODBP
OPO H
3
2
2
AcO
O
AcO
O
ODBP
OPO H
3
c
b
O
O
OAc
13
ODBP
OAc
OPO H
3
2
2
O
O
ODBP
ODBP
3
OPO H
3
OPO H
3
2
Scheme 2. Reagents and conditions: (a) Dibenzyl phosphate (6 equiv), CCl4 (30 equiv), DMAP (0.6 equiv), DIPEA (12 equiv), MeCN, 0 °C, 1 h; (b) DMAP (2.1 equiv), Ac2O
(2.1 equiv), MeCN, 5 min; (c) Pd/C, H2, THF/MeOH, 4 h.

5480
C. Huo et al. / Tetrahedron Letters 52 (2011) 5478–5483
OAc
OAc
OAc
OAc
OAc
AcO
O
AcO
O
OAc
a
b
O
O
OAc
15
OAc
ODBP
OAc
14
OAc
OH
O
O
OAc
OAc
OAc
c
OAc
OAc
OAc
OAc
AcO
O
AcO
O
OAc
a
1
O
O
OAc
16
OAc
OH
OAc
OAc
OPO3H2
O
O
4
OH
OAc
OAc
b
OAc
OAc
OAc
OAc
AcO
O
AcO
O
OAc
c
O
O
OAc
OAc
OAc
17
OAc
O
O
5
OPO3H2
OPO3H2
ODBP
ODBP
OAc
OAc
OAc
OAc
OAc
OAc
AcO
O
AcO
O
a
b
O
O
OH
OH
ODBP
OAc
18
OAc
O
O
19
ODBP
ODBP
OH
c
OAc
OAc
OAc
AcO
O
O
OPO3H2
OAc
O
6
OPO3H2
OPO3H2
Scheme 3. Reagents and conditions: (a) NaHCO3, MeCN, reflux, 2.5 h or Li2CO3, MeCN, reflux, 4 h or Lipase, n-butanol, THF, 24 h; (b) dibenzyl phosphite, CCl4, DMAP, DIPEA,
MeCN, 0 °C, 1 h; (c) Pd/C, H2, THF/MeOH, 4 h.
withdrawing effect of the phosphate groups. These assignments
are useful in deducing the structures of subsequent compounds.
Removal of the benzyl groups by catalytic hydrogenation over
Pd/C afforded EGCG octaphosphate 2 (75% yield).
When the same reaction of EGCG was carried out with only
6 equiv of dibenzyl phosphite, EGCG hexa-dibenzyl phosphate 12
was obtained as the major product in 45% yield. The assignment
of structure 12 was based on the following considerations. In the
31P NMR of 12, there were only four signals in the ratio of
1:2:1:2 consistent with the structure. Equally instructive is the
1H NMR: the A ring protons at 5.98 and 5.97 ppm are nearly the
same chemical shifts of the A ring protons of EGCG at 6.08 and
6.05 ppm; whereas the B ring protons at 7.71 ppm and G ring
protons at 7.50 ppm are similar to those of compound 11 at 7.79

C. Huo et al. / Tetrahedron Letters 52 (2011) 5478–5483
5481
OAc
OAc
OAc
OMe
AcO
O
AcO
O
OAc
OAc
O
O
OAc
20
OAc
OAc
21
OAc
O
O
O
O
OMe
OAc
OAc
OAc
OAc
OAc
OAc
OAc
OAc
OAc
AcO
AcO
a
1
b
O
O
OAc
14
OAc
OH
OAc
20
OAc
OMe
O
O
OAc
OAc
Scheme 4. Reagents and conditions: (a) NaHCO3, MeCN, reflux, 2.5 h or Li2CO3, MeCN, reflux, 4 h or Lipase, n-butanol, THF, 24 h; (b) (i)TBDMSCl, Im, DMF; (ii) MeI, KF, DMF,
rt.
Figure 2. HNMR spectra of compounds 19, 15, 17.
and 7.52, respectively. Acetylation of 12 gave the diacetate 13 (92%
yield) which on hydrogenation gave the phosphate–acetate hybrid
3 (85% yield) (Scheme 2).
These results suggest that of the eight OH groups in EGCG, the
two OH groups in the A-ring are less reactive toward phosphoryla-
tion under the reaction conditions. When less than 6 equiv of
dibenzyl phosphite was used to see if selectivity among the
remaining six OH groups could be achieved, a complex mixture
of products was found.
time and phosphorylate the intermediates, it was possible to
obtain EGCG heptaacetate monophosphate 4 (23% yield) and hexa-
acetate di-phosphate 5 (18% yield) after phosphorylation and
hydrogenolysis. If we prolonged the hydrolysis time properly,
EGCG pentaacetate triphosphate 6 (11% yield) was obtained as well
(Scheme 3).
The assignment of the structures of 4, 5, 6, is based on the fol-
lowing argument. Firstly, our group had previously synthesized
both 400-O-methyl-EGCG heptaacetate 20 and 40-O-methyl-EGCG
heptaacetate 21.21 In the sequential hydrolysis of 1, it was possible
to methylate the first intermediate to give a EGCG heptaacetate
monomethyl ether, identical to 400-O-methyl-EGCG heptaacetate
In order to see if phosphorylation can be selectively introduced
into the B or G ring, we examined the controlled hydrolysis of
EGCG peracetate 1. By stopping the reaction at the appropriate

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C. Huo et al. / Tetrahedron Letters 52 (2011) 5478–5483
OBn
OBn
OBn
OBn
OBn
BnO
O
BnO
O
OBn
a
OH
O
OBn
OBn
O
24
OMOM
25, 26
OH
OAc
OH
OH
OAc
OAc
HO
O
AcO
O
b
c
O
O
OH
OAc
O
O
OMOM
OMOM
27, 28
29, 30
OAc
OAc
OAc
OAc
OAc
OAc
AcO
d
O
AcO
O
e
O
O
OAc
OAc
O
O
OH
ODBP
33, 34
31, 32
OAc
OAc
OAc
AcO
O
25,27,29,31,33,7
26,28,30,32,34,8
: p-OR
: m-OR
O
f
OAc
O
OPO3H2
7, 8
Scheme 5. Reagents and conditions: (a) 3-(Methoxymethoxy)benzoic acid or 4-(methoxymethoxy)benzoic acid, DCC, DMAP, CH2Cl2, 0 °C to rt, 48 h; (b) Pd/C, H2, EtOAc, 4 h;
(c) Ac2O, DMAP, MeCN, 1 h; (d) TMSBr, CH2Cl2, rt 4 h; (e) dibenzylphosphite, CCl4, DMAP, DIPEA, MeCN, 2 h; (f) Pd/C, H2, EtOAc, 4 h.
20 but distinctly different from 21 according to their NMR spectra.
It follows therefore that the first EGCG heptaacetate monophos-
phate must have structure 4 (Scheme 4).
is an electron-withdrawing carbonyl group in the p-position. Once
this acetate is hydrolyzed, the generated phenoxide anion would
have a neighboring group effect in facilitating the hydrolysis of
its adjacent acetates, leading to the hydrolysis of 300- and 500-
acetates.
Previously, we had synthesized (2R,3R)-epigallocatechin-3-O-
(4-hydroxybenzoate), compound 22, which occurs naturally in
the plant Cistus salvifolius.22 Compound 22, together with its regio-
isomer (2R,3R)-epigallocatechin-3-O-(3-hydroxybenzoate) (23)
was found to be an inhibitor of proteasome and has the cytotoxic-
ity comparable to EGCG. We thus want to prepare their phosphate
derivatives as well.
Secondly, as shown in Figure 2, in the spectrum of 15, we can
see that there are five singlets at about 2.3 ppm, which are attrib-
uted to five different acetate methyl hydrogens. Two of these five
singlets are about twice as high as the others, and should be the
two B ring meta acetates and the two G ring meta acetates. In the
spectrum of 17, there are five singlets at similar positions, but only
one singlet has twice the intensity of the others, so we can
conclude that the second hydrolyzed acetate should be on either
the B ring or the G ring meta position. Thirdly, the two aromatic
hydrogens of G ring of 15 are equivalent and they show up as a sin-
glet at 7.56 ppm. In 17, the two aromatic protons of G ring show as
two distinct singlets at 7.82 ppm and at 7.46 ppm suggesting that
they are no longer equivalent. On the other hand, the two aromatic
protons of B ring remain as a singlet. We conclude therefore that
the second hydrolyzed acetate occurred on G ring. In the spectrum
of 19, only four acetate singlets remain at around 2.3 ppm and the
G ring aromatic hydrogens become equivalent again as a singlet so
the third hydrolyzed acetate also occurred on G ring. Finally, the
31P NMR of 15 showed only one signal, whereas that of 17 and
19 showed the expected two signals with relative intensity ratios
of 1:1 and 2:1, respectively. These results are consistent with the
above structural assignment.
OH
OH
OH
OH
OH
OH
HO
O
HO
O
O
O
OH
OH
O
O
OH
23
22
OH
We started with the benzylated EGC compound 24 which was
readily prepared semi-synthetically from EGCG.23 Compound 24
was benzoated with MOM protected 3- or 4-hydroxylbenzoic acid
to give 25 (71%) or 26 (69%), respectively. The benzyl groups of 25/
It is reasonable to expect the 400-acetate to be hydrolyzed first
among all the acetates in 1 because this is the only one where there

C. Huo et al. / Tetrahedron Letters 52 (2011) 5478–5483
5483
OBn
OBn
OBn
OBn
OBn
BnO
O
BnO
O
OBn
a
O
O
OBn
OBn
O
O
O
OMOM
OH
35, 36
25, 26
OBn
OH
OBn
OBn
OH
OH
BnO
HO
O
b
c
O
O
OBn
OH
9, 10
36,38,10:
O
O
OPO3H2
ODBP
37, 38
35,37,9:
p-OR
m-OR
Scheme 6. Reagents and conditions: (a) TMSBr, CH2Cl2, rt 4 h; (b) dibenzylphosphite, CCl4, DMAP, DIPEA, MeCN, 2 h; (c) Pd/C, H2, EtOAc, 4 h.
3. Huo, C.; Wan, S. B.; Lam, W. H.; Li, L.; Wang, Z.; Landis-Piwowar, K. R.; Chen, D.;
Dou, Q. P.; Chan, T. H. Inflammopharmacology 2008, 16, 248.
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7. Landis-Piwowar, K. R.; Milacic, V.; Chen, D.; Yang, H.; Zhao, Y.; Chan, T. H.; Yan,
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26 were removed by catalytic hydrogenolysis and followed by
acetylation to give 29/30 (91%/90%). The MOM group of 29/30
was then removed under acidic condition to give compound 31
(93%) or 32 (93%), after phosphorylation followed by catalytic
hydrogenolysis, compounds 7 (92%) and 8 (90%) were obtained
(Scheme 5).
As shown in Scheme 6, when the MOM group was removed
from 25/26, 35 (87%) and 36 (88%) were obtained. Also, the follow-
ing phosphorylation and catalytic hydrogenolysis gave the corre-
sponding phosphate 9 (80%) and 10 (82%), respectively.
In conclusion, we have therefore prepared regioselectively
several phosphate and phosphate–acetate hybrid derivatives of
EGCG and analogs. The bioactivity studies on these compounds
are being evaluated and will be reported elsewhere.
12. Lee, S.-C.; Chan, W.-K.; Lee, T.-W.; Lam, W.-H.; Wang, X.; Chan, T.-H.; Wong, Y.-
C. Nutr. Cancer 2008, 60, 483.
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Rafie, N.; Overmoyer, B.; Makkar, V.; Stambler, B.; Taylor, A.; Waas, J.; Lewin, J.
S.; McCrae, K. R.; Remick, S. C. Cancer Res. 2002, 62, 3408.
14. Cirla, A.; Mann, J. Nat. Prod. Rep. 2003, 20, 558–564.
15. Chaplin, D. J.; Pettit, G. R.; Parkins, C. S.; Hill, S. A. Br. J. Cancer Suppl. 1996, 27,
S86.
16. Hensley, M. L.; Schuchter, L. M.; Lindley, C.; Meropol, N. J.; Cohen, G. I.; Broder,
G.; Gradishar, W. J.; Green, D. M.; Langdon, R. J., Jr.; Mitchell, R. B.; Negrin, R.;
Szatrowski, T. P.; Thigpen, J. T.; Von Hoff, D.; Wasserman, T. H.; Winer, E. P.;
Pfister, D. G. J. Clin Oncol. 1999, 17, 3333.
Acknowledgments
We thank the Key project (210232) of the Education Ministry of
China and HKRGC (Project No. 5003/05P), the Areas of Excellence
Scheme established under the University Grants Committee of
the Hong Kong Special Administrative Region, China (Project No.
AoE/P-10/01) for financial support.
17. Erion, M. D.; Van Poelje, P. D.; MacKenna, D. A.; Colby, T. J.; Montag, A. C.;
Fujitaki, J. M.; Linemeyer, D. L.; Bullough, D. A. J. Pharmacol. Exp. Ther. 2005, 312,
554–560.
18. Huttunen, K. M.; Maehoenen, N.; Leppaenen, J.; Vepsaelaeinen, J.; Juvonen, R.
O.; Raunio, H.; Kumpulainen, H.; Jaervinen, T.; Rautio, J. Pharm. Res. 2007, 24,
679.
Supplementary data
19. Bookser, B. C.; Raffaele, N. B. J. Comb. Chem. 2008, 10, 567–572.
20. Silverberg, L. J.; Dillon, J. L.; Vemishetti, P. Tetrahedron Lett. 1996, 37,
771–774.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.tetlet.2011.08.061.
21. Wan, S. B.; Dou, Q. P.; Chan, T. H. Tetrahedron 2006, 62, 5897.
22. Osanai, K.; Huo, C.; Landis-Piwowar, K. R.; Dou, Q. P.; Chan, T. H. Tetrahedron
2007, 63, 7565.
References and notes
23. Huo, C.; Shi, G.; Lam, W. H.; Chen, D.; Cui, Q. C.; Dou, Q. P.; Chan, T. H. Can. J.
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