Synthesis and biological activity of the tea catechin metabolites, M4 and M6 and their methoxy-derivatives

Article abstract of DOI:10.1016/j.bmcl.2004.12.070

Syntheses are reported for metabolites M4 (1) and M6 (2) of the green tea polyphenols epicatechin (EC) and epigallocatechin (EGC) and their gallate derivatives. Several methoxy-derivatives of 1 and 2 were also prepared. Compounds 1 and 2 were evaluated for growth inhibitory activity against a panel of immortalized and malignant human cell lines with 1 being the more active compound. The possible antiinflammatory activity of 1 and its trimethoxy derivative was also evaluated. Neither compound inhibited the release of arachidonic acid, although 1 inhibited NO production by 50% at 20 μM.

Full text of DOI:10.1016/j.bmcl.2004.12.070

Bioorganic & Medicinal Chemistry Letters 15 (2005) 873–876
Synthesis and biological activity of the tea catechin metabolites,
M4 and M6 and their methoxy-derivatives
Joshua D. Lambert,a Joseph E. Rice,b,* Jungil Hong,a Zhe Houa and Chung S. Yanga
aDepartment of Chemical Biology, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey,
Piscataway, NJ08854-8020, USA
bDepartment of Pharmaceutical Chemistry, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey,
Piscataway, NJ08854-8020, USA
Received 29 November 2004; revised 21 December 2004; accepted 22 December 2004
Available online 18 January 2005
Abstract—Syntheses are reported for metabolites M4 (1) and M6 (2) of the green tea polyphenols epicatechin (EC) and epigalloca-
techin (EGC) and their gallate derivatives. Several methoxy-derivatives of 1 and 2 were also prepared. Compounds 1 and 2 were
evaluated for growth inhibitory activity against a panel of immortalized and malignant human cell lines with 1 being the more active
compound. The possible antiinflammatory activity of 1 and its trimethoxy derivative was also evaluated. Neither compound inhib-
ited the release of arachidonic acid, although 1 inhibited NO production by 50% at 20 lM.
ꢀ 2005 Elsevier Ltd. All rights reserved.
The green tea (Camellia sinenesis Theaceae) catechins
have shown growth inhibitory activity against a number
of human tumor cell lines (Fig. 1).1 Epigallocatechin-3-
gallate (EGCG) and green tea have shown cancer pre-
ventive activity in a number of animal models of carci-
nogenesis including: lung, liver, small intestine, colon,
skin, and prostate.1,2 We and others have previously
shown that the catechins undergo extensive metabolism
including methylation, glucuronidation, and sulfation.3–5
These compounds also undergo microbial degradation
in the colon resulting in the formation of, among other
compounds, (ꢀ)-5-(30,40,50-trihydroxyphenyl)-c-valero-
lactone (M4, 1) and (ꢀ)-5-(30,40-dihydroxyphenyl)-c-
valerolactone (M6, 2), which are detectable in the plas-
ma and urine following consumption of green tea.6–8
Given that 1 and 2 retain the polyphenolic character of
the catechins, it is possible that they also retain some of
the biological activities of the parent compounds. In
order to characterize the potential biological activities
of 1 and 2 we have developed methods for their synthe-
sis. Herein we report these synthetic methods and data
from preliminary studies of the antiinflammatory and
growth inhibitory activities of 1 and 2 and several of
their methoxylated derivatives.
OH
O
OH
HO
HO
OH
R1
O
HO
O
1
OR2
OH
O
OH
HO
O
EC
ECG
R1, R2 = H
1 = H, R2 =gallate
EGC R1 = OH, R2 = H
EGCG R1 = OH, R2 = gallate
R
Compound 2 was synthesized as a racemic mixture
(Scheme 1) starting from guaiacol 3 by acetylation of
the free hydroxy group and iodination para to the meth-
oxy group with iodine monochloride.9 The pure product
5 was obtained in 46% yield after recrystallization from
ethanol. Heck reaction of 5 with unsaturated ester 6
(legend to Scheme 1, prepared in 83% yield from 4-pent-
enoic acid by conversion to the acid chloride with oxalyl
chloride followed by reaction with methanol) was medi-
ated by palladium(II) acetate in the presence of silver
2
Figure 1. Structures of the major tea catechins and metabolites M4 (1)
and M6 (2).
Keywords: Synthesis; Growth inhibition; Antiinflammatory; Lactones;
Green tea.
*
Corresponding author. Tel.: +1 732445 538;2 fax: +1 732445
6312; e-mail: jrice@rci.rutgers.edu
0960-894X/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.12.070

874
J. D. Lambert et al. / Bioorg. Med. Chem. Lett. 15 (2005) 873–876
OH
OAc
OAc
OMe
OMe
MeO
MeO
MeO
OAc
MeO
MeO
MeO
MeO
a
b
a
b
c
97 %
46 %
73 %
(as a mixture
of isomers)
88 %
58 %
I
CHO
3
4
5
12
OH
OAc
OMe
OMe
MeO
MeO
d
MeO
MeO
MeO
MeO
O
c
O
CO2Me
CO2Me
CO2Et
CO2Et
7
8
14
13
OAc
OH
O
O
OMe
OMe
MeO
MeO
f,g
e
OH
O
MeO
MeO
MeO
MeO
98 %
(2 steps)
OH
OH
e
d
45 %
(2 steps)
CO2Me
10
9
CO2H
CO2Et
O
49%
(2 steps)
16
h
i
15
34%
O
20%
HO
OH
OMe
O
OMe
OH
MeO
O
O
MeO
MeO
HO
HO
g
f
O
O
87%
(2 steps)
47%
2
11
17
1
Scheme 1. Reagents and conditions: (a) AcCl, pyr; (b) ICl, CH2Cl2,
0 ꢁC; (c) H2C@CHCH2CH2CO2Me (6), Pd(OAc)2, PPh3, Ag2CO3,
CH3CN, D; (d) mCPBA, NaHCO3 (aq), CH2Cl2; (e) H2, 10% Pd–C; (f)
KOH, MeOH; (g) PhH, CuSO4, D; (h) BBr3, CH2Cl2, ꢀ78 ! 0 ꢁC; (i)
MeI, K2CO3, acetone, D.
Scheme 2. Reagents and conditions: (a) H2C@CHMgBr, THF; (b)
CH3C(OEt)3, CH3CH2CO2H, 140 ꢁC; (c) mCPBA, NaHCO3 (aq),
CH2Cl2; (d) H2, 10% Pd–C; (e) KOH, MeOH; (f) PhH, CuSO4, D; (g)
BBr3, CH2Cl2, ꢀ78 ! 0 ꢁC.
carbonate and triphenylphosphine in refluxing acetoni-
trile.10 Product 7 was obtained in 73% yield as an insep-
arable mixture of the E-, Z-, and several other isomers.
This mixture was epoxidized with m-chloroperoxyben-
zoic acid (mCPBA) buffered with sodium bicarbonate
and the epoxide mixture 8 was then directly subjected
to hydrogenolysis over 10% Pd–C to cleave the benzylic
C–O bond. The resulting alcohol mixture was purified
by flash chromatography to give the desired secondary
alcohol 9 in 45% yield for the two-step sequence. Simul-
taneous hydrolysis of the acetate and methyl ester with
KOH in methanol gave a diol acid in 98% yield that
was converted to 4-methoxy M6 10 quantitatively by
refluxing in benzene over anhydrous CuSO4 to sequester
evolved water. A portion of 10 was converted into 2 in
34% yield by demethylation using boron tribromide.
Another portion of 10 was methylated using methyl io-
dide and potassium carbonate in refluxing acetone to
give 3,4-dimethoxy M6 11 in 20% yield after purification
by preparative TLC.
87% yield. The removal of all three methoxy groups
was achieved upon treatment with BBr3 in methylene
chloride to give racemic 1 in 47% yield.12
Compounds 1 and 2 and their methoxy-derivatives, were
first assessed for their ability to inhibit the growth of a
panel of immortalized and malignant human cell lines
using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltet-
razolium bromide (MTT) assay (Fig. 2).13 Compound
1 was the most active metabolite tested with IC50 values
of 15–73 lM for human esophageal squamous cell carci-
noma cells (KYSE150), human colon adenocarcinoma
cells (HT-29 and HCT-116), immortalized human intes-
tinal epithelial cells (INT-407), and an immortalized rat
intestinal epithelial cell line (IEC-6) (Fig. 2A). Com-
pound 17 inhibited cell growth less than 20% at concen-
trations up to 50 lM (Fig. 2B). Compounds 2, 10, and 11
were significantly less potent than compound 1 and
inhibited the growth of KYSE150 cells by 20–40% at
50 lM, but had no effect on HT-29 cells (Fig. 2C and D).
A modification of the synthesis outlined above was em-
ployed for the preparation of 1 so as to avoid the mix-
ture of isomers obtained from the Heck reaction
(Scheme 2). Reaction of 3,4,5-trimethoxybenzaldehyde
with vinylmagnesium bromide gave allylic alcohol 12
in 88% yield. Upon heating 12 with triethyl ortho ace-
tate in the presence of propanoic acid an ortho ester Cla-
isen rearrangement was initiated, furnishing unsaturated
ester 13 in 58% yield as the E-isomer.11 Epoxidation
with mCPBA in the presence of sodium bicarbonate
gave epoxide 14, which was used without further purifi-
cation. Hydrogenolysis over 10% Pd–C gave hydroxy es-
ter 15 in 49% yield for the two-steps. The ester was
hydrolyzed with KOH in methanol (quantitative yield)
and hydroxy acid 16 was cyclodehydrated in refluxing
benzene over CuSO4 to give 3,4,5-trimethoxy M4 17 in
To assess the possible antiinflammatory activity of 1 and
17, the ability of the compounds to inhibit the release of
arachidonic acid and the production of nitric oxide
(NO) by lipopolysaccharide (LPS)-stimulated murine
macrophages (RAW264.7) was evaluated. The assay
was performed using previously described methods.14,15
Neither test compound inhibited the release of arachi-
donic acid from stimulated cells, however 1 inhibited
NO production by 50% at 20 lM (Fig. 3). Compound
17 had no effect on NO production.
The growth inhibitory activity of 1 against immortalized
and cancer cell lines suggests that this metabolite may
contribute to the cancer preventive effect of green tea
in vivo. The greater sensitivity of INT-407 immortalized
intestinal cells relative to the other cell lines may indicate

J. D. Lambert et al. / Bioorg. Med. Chem. Lett. 15 (2005) 873–876
875
Figure 2. Effects of compounds 1 (A) and 17 (B) on the proliferation of normal immortalized intestinal cell (INT-407 and IEC-6), human colon
cancer cell (HT-29 and HCT-116), and human esophageal squamous cell carcinoma (KYSE150). Effects of compounds 2, 10, and 11 on the
proliferation of HT-29 cells (C) and KYSE150 cells (D). n = 5–12, error bars represent SEM.
Figure 3. Effects of 20 lM M4 (1) or trimethoxy-M4 (M3M4, 17) on arachidonic acid release and NO formation by LPS-stimulated RAW264.7
murine macrophages. N = 4, error bars represent the SD. * = p < 0.05.
that this compound has some selectivity for pre-malig-
nant cells, but this remains to be more thoroughly inves-
tigated. Likewise, the ability of 1 to inhibit the
production of LPS-stimulated macrophages may indi-
cate that this metabolite can contribute to the antiinflam-
matory and cancer preventive activities of green tea. The
assays described above were performed using racemic
compounds. It remains for further evaluation whether
the individual enantiomers might display enhanced
growth inhibitory and/or antiinflammatory activity.
Acknowledgements
This study was supported by NIH Grant CA88961
(CSY).
References and notes
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In summary, we describe methods for the synthesis of two
previously-reported metabolites of the green tea catechins
M4 (1) and M6 (2) and several of their methoxylated
derivatives. Furthermore, we have determined the growth
inhibitory and antiinflammatory activity of compounds 1
and 2 and their methoxy-derivatives. The results suggest
that 1 may contribute slightly to the cancer preventive
and antiinflammatory activity of green tea.
5. Lambert, J. D.; Lee, M. J.; Lu, H.; Meng, X.; Ju, J.; Hong,
J.; Seril, D. N.; Sturgill, M. G.; Yang, C. S. J. Nutr. 2003,
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J. D. Lambert et al. / Bioorg. Med. Chem. Lett. 15 (2005) 873–876
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plates (2–10 · 103 cells per well) and allowed to attach
overnight. The medium was replaced with serum-free
medium containing the test compounds (0–50 lM). Cells
were incubated for 24 h at 37 ꢁC. The compound-contain-
ing medium was removed, fresh serum-complete medium
was added, and the cells were incubated for an additional
24 h. Growth inhibition was determined using 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide
9. Feldman, K. S. J. Org. Chem. 1997, 62, 4983.
10. Sonesson, C.; Larhed, M.; Nyqvist, C.; Hallberg, A. J.
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(MTT). Cell growth is correlated with mitochondria-
mediated reduction of MTT to a purple formazan
precipitate. Growth of treated cells was compared to the
growth of the untreated controls to determine percent
growth inhibition.
11. Fox, D. N. A.; Lathbury, D.; Mahon, M. F.; Molloy, K.
C.; Gallagher, T. J. Am. Chem. Soc. 1991, 113, 2652.
12. The structures of all compounds were elucidated using 1H
(200 MHz) and 13C NMR (50 MHz) in CDCl3 unless
otherwise noted. Results are reported as ppm downfield
14. Sang, S.; Lambert, J. D.; Tian, S.; Hong, J.; Hou, Z.; Ryu,
J. H.; Stark, R. E.; Rosen, R. T.; Huang, M. T.; Yang, C.
S.; Ho, C. T. Bioorg. Med. Chem. 2004, 12, 459. RAW
264.7 murine macrophages were plated in 24-well plates
(3 · 105 cells per well) and allowed to attach overnight.
The medium was replaced with fresh, serum-free DMEM
medium containing 0.1 lCi/mL [5,6,8,9,11,12,14,15-3H](N)
arachidonic acid (NEN Life Science, Boston, MA, USA).
The cells were incubated overnight to allow absorption of
the labeled arachidonic acid, washed twice with PBS
containing 0.1% BSA, and stimulated for 1 h with 2 lg/mL
lipopolysaccharide (LPS). The medium was replaced with
new, serum-free medium containing test compounds
(20 lM) and the cells were incubated for an additional
18 h at 37 ꢁC. After incubation, the medium was collected,
centrifuged at 12,000 rpm for 10 min and radioactivity in
the extracellular fluid was measured with a scintillation
counter.
15. Ryu, J. H.; Ahn, H.; Jin Lee, H. Fitoterapia 2003, 74, 350,
RAW 264.7 cells were determined by stimulating cells
(plated in 24-well plates at 3 · 105 cells per well) for 1 h
with 2 lg/mL LPS. The medium was then replaced with
serum-free medium containing the test compounds
(20 lM) and cells were cultured for 18 h. NO production
was determined spectrophotometrically using previously-
reported methods.
1
from internal TMS: (1) H NMR (CD3CN) d 6.53 (br s,
3H), 6.33 (s, 2H), 4.68 (m, 1H), 2.84 (dd, 1H, J = 14, 6),
2.74 (dd, 1H, J = 14, 6), 2.45 (m, 2H), 2.26 (m, 1H), 1.93
(m, 1H); 13C NMR d 176.7, 144.6, 130.2, 128.0, 107.9,
80.4, 39.6, 27.6, 26.1. (2) 1H NMR (CD3CN) d 6.80 (d, 1H,
J = 8), 6.77 (d, 1H, J = 2), 6.65 (dd, 1H, J = 8, 2), 4.69 (m,
1H), 2.91 (dd, 1H, J = 14, 6), 2.81 (dd, 1H, J = 14, 6),
2.58–2.17 (m, 3H), 2.03–1.84 (m, 1H); 13C NMR (CD3CN)
d 176.6, 143.7, 142.6, 128.4, 120.5, 115.7, 114.6, 80.5, 39.5,
27.6, 26.1. (10) 1H NMR d 6.82(d, 1H, J = 8), 6.81 (d, 1H,
J = 1.8), 6.72(dd, 1H, J = 8, 1.8), 4.71 (m, 1H), 3.89 (s,
3H), 3.01 (dd, 1H, J = 14, 6), 2.83 (dd, 1H, J = 14, 6),
2.60–2.18 (m, 3H), 2.05–1.86 (m, 1H); 13C NMR d 177.1,
145.8, 145.7, 129.1, 121.1, 115.6, 110.9, 80.9, 56.1, 40.8,
28.7, 27.1. (11) 1H NMR d 6.90–6.75 (m, 3H), 4.75 (m,
1H), 3.89 (s, 3H), 3.88 (s, 3H), 3.01 (dd, 1H, J = 14, 6),
2.91 (dd, 1H, J = 14, 6), 2.60–2.20 (m, 3H), 2.01–1.86 (m,
1
1H). (17) H NMR d 6.46 (s, 2H), 4.76 (m, 1H), 3.87 (s,
6H), 3.85 (s, 3H), 3.00 (dd, 1H, J = 14, 6), 2.91 (dd, 1H,
J = 14, 6), 2.60–2.21 (m, 3H), 1.97 (m, 1H); 13C NMR d
177.0, 153.4, 131.7, 106.8, 80.7, 60.9, 56.3, 41.7, 28.7, 27.2.
13. Bold, R. J.; Virudachalam, S.; McConkey, D. J. J. Surg.
Res. 2001, 100, 11. Cells were plated in 96-well microtiter