Tumor chemopreventive activity of 3-O-acylated (-)-epigallocatechins

Article abstract of DOI:10.1016/j.bmc.2003.08.016

In order to seek promising cancer chemopreventive agents, we assessed the antitumor promoting activities of 3-O-octanoyl- or 3-O-(2-methyloctanoyl)-(-)- epigallocatechins, inhibiting markedly the activation of Epstein-Barr virus early antigen, in a two-stage mouse skin carcinogenesis assay. As a result, these derivatives inhibited a papilloma formation 1.3-1.6-fold more strongly than (-)-epigallocatechin gallate well established as anti-tumor promoter.

Full text of DOI:10.1016/j.bmc.2003.08.016

Bioorganic & Medicinal Chemistry 11 (2003) 5143–5148
Tumor Chemopreventive Activity of 3-O-Acylated
(ꢀ)-epigallocatechins
Ayako Kumagai,a Yasuo Nagaoka,a,b Tomoko Obayashi,a Yasuhiro Terashima,a
Harukuni Tokuda,c Yukihiko Hara,d Teruo Mukainaka,c Hoyoku Nishino,c
Hiroshi Kuwajimae and Shinichi Uesatoa,b,
*
aDepartment of Biotechnology, Faculty of Engineering, Kansai University, Suita, Osaka 564-8680, Japan
bOrganization for Research and Development of Innovative Science and Technology, Kansai University,
Suita, Osaka 564-8680, Japan
cDepartment of Biochemistry and Molecular Biology, Kyoto Prefectural University of Medicine,
Kawaramachi-Hirokoji, Kamigyo-ku, Kyoto 602-0841, Japan
dCentral Research Laboratories, Tokyo Food techno Co., LTD., Miyabara, Fujieda, Shizuoka 426-0133, Japan
eFaculty of Pharmaceutical Sciences, Kinki University, 3-4-1 Kowakae, Higashiosaka, Osaka 577-8502, Japan
Received 17 July 2003; accepted 15 August 2003
Abstract—In order to seek promising cancer chemopreventive agents, we assessed the antitumor promoting activities of 3-O-octa-
noyl- or 3-O-(2-methyloctanoyl)-(ꢀ)-epigallocatechins, inhibiting markedly the activation of Epstein–Barr virus early antigen, in a
two-stage mouse skin carcinogenesis assay. As a result, these derivatives inhibited a papilloma formation 1.3–1.6-fold more strongly
than (ꢀ)-epigallocatechin gallate well established as anti-tumor promoter.
# 2003 Elsevier Ltd. All rights reserved.
Introduction
chain acyl bearing derivatives: 3-O-[(RS)-2-methylocta-
noyl]-(4) and 3-O-[(RS)-2-methyldecanoyl]-(ꢀ)-EGC (5)
were prepared by the same method in expectation that
the 2-methyl group could shield the ester bond at the C-
3 acyloxy moiety from esterase-mediated cleavage in
body, leading to maintenance of a high tissue concen-
tration. These synthetic 3-O-acyl-(ꢀ)-EGCs were asses-
sed for the inhibitory effects on the Epstein–Barr virus
early antigen (EBV-EA) activation in Raji cells induced
by 12-O-tetradecanoylphorbol-13-acetate (TPA) in
order to seek promising antitumor promoting candi-
dates for cancer chemoprevention. As a result, the (ꢀ)-
EGCs possessing an acyl group of carbon number (C8–
C11) showed the strong inhibitions.14 In the present
work, we therefore tried to examine the antitumor pro-
moting activities of the promising compounds in a two-
stage mouse skin carcinogenesis test (Fig. 1).
Green tea catechins have preventive activities against
cancer,1,2 atherosclerosis,3,4 diabetes,5 cold syndrome,6
and so on. Recently, it was proposed that the catechins
contribute to cancer prevention by multiple pathways
involving antioxidative7 and antiangiogenic8 actions as
well as ulokinase-9 and telomerase-inhibiting activ-
ities.10 In order to produce the satisfactory effects for
human diseases, however, the structures of catechins
need to be modified because of their low plasma and
tissue concentrations in body.11 We therefore tried to
synthesize 3-O-acylated (ꢀ)-epigallocatechins (EGCs) in
order to improve their pharmacokinetic profile such as
cell membrane- and tissue-permeability. Straight-chain
acids of C4–C18 were introduced after acid chloride for-
mation at the C-3 hydroxy group of (ꢀ)-EGC (1), being
less responsible for radical scavenging action12,13 than
the phenolic hydroxyl groups, yielding 3-O-acyl-(ꢀ)-
EGCs (3a–3h),14 respectively. Furthermore, branched-
Results and Discussion
The EBV-EA activation assay of the synthetic 3-O-acyl-
(ꢀ)-EGCs indicated that the following derivatives with a
fatty acid (C8–C11) have ca. 1.7–3-fold higher inhibitory
*Corresponding author. Tel.: +81-6-6368-0834; fax: +81-6-6388-
8609; e-mail: uesato@ipcku.kansai-u.ac.jp
0968-0896/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2003.08.016

5144
A. Kumagai et al. / Bioorg. Med. Chem. 11 (2003) 5143–5148
Figure 1. Structures of catechins and 3-O-acyl-(ꢀ)-epigallocatechins.
effects than (ꢀ)-EGCG (2) at 1ꢁ103 mol ratios/TPA: 3-
O-octanoyl-(ꢀ)-EGC (3c), 3-O-decanoyl-(ꢀ)-EGC (3d),
3-O-[(RS)-2-methyloctanoyl]-(ꢀ)-EGC (4) and 3-O-
[(RS)-2-methyldecanoyl]-(ꢀ)-EGC (5).14 Since the 3-O-
octanoyl- and 3-O-decanoyl-derivatives showed almost
the same efficacies, we focused on the octanoyl-series
such as 3c and 4 to compare with (ꢀ)-EGC (1) and (ꢀ)-
EGCG (2) for the antitumor promoting activities in a
two-stage mouse skin carcinogenesis test. Furthermore,
since 4 was found to consist of (S)-2-methyl- and (R)-2-
methyl-diastereomers in a 1:1 ratio in its 1H NMR
spectrum (see Experimental), 3-O-[(S)-2-methylocta-
noyl]-(ꢀ)-EGC (6) and 3-O-[(R)-2-methyloctanoyl]-(ꢀ)-
EGC (7) were separately prepared and subjected to this
test for the purpose of examining whether or not there
exists discrepancy between the diastereomers in the
inhibitory activity. Thus, (S)-(12) and (R)-2-methyl-
octanoic acid (13) were synthesized using chiral aux-
iliary 4-benzyl-2-oxazolidinones15,16 through sequential
diastereoselective a-methylation of (4S)- (8) and (4R)-3-
(octanoyl)-4-benzyl-2-oxazolidinone (9) to [3(20S),4S]-
(10) and [3(20R),4R]-3-(2-methyloctanoyl)-isomer (11),
chromatographic purification and LiOOH-catalyzed
hydrolysis, respectively. Acids 12 and 13 indicated the
specific optical rotations of mutually opposite figures
([a]D +16.2ꢂ; ꢀ15.3ꢂ),17 respectively. They were in turn
converted, after acid chloride formation, to 3-O-[(S)-2-
methyloctanoyl]-(ꢀ)-EGC (6) and 3-O-[(R)-2-methyl-
octanoyl]-(ꢀ)-EGC (7), respectively, in the conventional
way (see Experimental). In the 400 MHz 1H NMR
spectra, neither of 6 and 7 showed any signals due to the
respective 2-methyl-isomers 7 and 6 (Fig. 2).
The two-stage mouse skin carcinogenesis test was per-
formed with 7,12-dimethylbenz[a]anthracene (DMBA)
as an initiator and TPA as a tumor promoter according
to the reported procedure.18
Figures 3(a) and 4(a) showed the time-course percentage
of mice bearing papillomas which were treated with an
acetone solution of the 3-O-acylated (ꢀ)-EGC, (ꢀ)-
EGC (1), (ꢀ)-EGCG (2) or acetone only (control).
Papillomas were observed on 100% mice of the control
group in 9 weeks after TPA treatment. On the other
hand, papillomas were detected on 20, 10 and 30% mice
treated with (ꢀ)-EGC (1), (ꢀ)-EGCG (2) and 3-O-
octanoyl-(ꢀ)-EGC (3c), respectively, whereas they were
not observed on the mice operated with 3-O-[(RS)-2-
methyloctanoyl]-(ꢀ)-EGC (4) in 9 weeks. As shown in
Figure 3(b), (ꢀ)-EGC (1) and (ꢀ)-EGCG (2) achieved
Figure 2. Synthesis of (S)-2- (12) and (R)-2-methyloctanoic acid (13) using chiral auxililary.

A. Kumagai et al. / Bioorg. Med. Chem. 11 (2003) 5143–5148
5145
Figure 3. Antitumor promoting activities of 1, 2, 3c and 4 in a two-
stage mouse skin carcinogenesis test: (a) percentage of mice bearing
papillomas (b) average number of papillomas per mouse [^, positive
control, TPA alone; &, TPA + 1 (p<0.05); *, TPA + 2 (p<0.05);
&, TPA + 3c (p<0.05); ~, TPA + 4 (p<0.005)].
Figure 4. Antitumor promoting activities of 4, 6 and 7 in a two-stage
mouse skin carcinogenesis test: (a) percentage of mice bearing papil-
lomas (b) average number of papillomas per mouse [^, positive con-
trol, TPA alone; ~, TPA + 4 (p<0.005); ^, TPA + 6 (p<0.005); ꢃ,
TPA + 7 (p<0.05)].
almost the same efficacies. Moreover, the average
papilloma number per mouse treated with 1, 2 and 4,
were, 3.8, 4.0 and 2.3, respectively, to 7.2 of control
mouse in 20 weeks, indicating that they inhibited the
papilloma formation by 47, 44 and 68%, respectively.
Thus, 4 was ca. 1.5-fold more effective than 1 and 2. 3-
O-octanoyl-(ꢀ)-EGC (3c) showing 53% inhibition
seems a little more active than 1 and 2 though it was
inferior to 4. It is therefore most likely that the inhibi-
tory effect of papilloma fomation is enhanced by intro-
ducing an acyl group to (ꢀ)-EGC (1). Interestingly,
there existed a difference in the inhibition rate of papil-
loma formation between the two diastereomers 6 and 7
in 20 weeks, that is the inhibition rate (72%) of 3-O-
[(S)-2-methyloctanoyl]-(ꢀ)-EGC (6) was higher than the
rate (50%) of the (R)-isomer 7 [Fig. 4(b)]. The reason
for such a difference in the inhibition remains elusive.
The most stable conformations of 6 and 7 were searched
using the molecular mechanics method with MMFF94
force field19 (Fig. 5).
Figure 5. The global minimum-energy conformations of 3-O-[(S)-2-
methylocatanoyl]-(ꢀ)-EGC (6) and 3-O-[(R)-2-methyloctanoyl]-(ꢀ)-
EGC (7). They were generated using the molecular mechanics method
with MMFF94 force field by repeating rotations (360ꢂ at 60ꢂ intervals)
of rotatable ester C–C and C–O bonds in the side chain followed by
optimization.
It was indicated that the (2S)-methyl group is displaced
on the O–CO–C plane of the C-3 acyloxy group in 6,
whereas the (2R)-methyl group is oriented perpendicu-
larly to the O–CO–C plane in 7. We speculated that the
differential spatial proximity of the 2-methyl group and
the ester bond moiety between 6 and 7 might be asso-
ciated with their resistivity toward catalytic cleavage by
an esterase in the dermal tissue of mice.
Conclusion
The introduction of the octanoyl group or 2-methyl-
octanoyl group at C-3 of (ꢀ)-EGC (1) enhanced the
antitumor promoting activity of 1 on DMBA-TPA two-
stage mouse skin carcinogenesis test. As exemplified,

5146
A. Kumagai et al. / Bioorg. Med. Chem. 11 (2003) 5143–5148
(2S)-isomer 6 was 1.6-fold more active than (ꢀ)-EGCG
(2) well established as an antitumor promoter. Thus, 3-
O-acyl-(ꢀ)-EGCs such as 3c, 4 and 6 could be good
candidates for cancer chemoprevention.
concentrated in vacuo to give a residue, which was
purified by preparative TLC with CHCl3/MeOH (13:1)
two developments) as an eluent, followed by freeze-
drying to afford a white powder.
3-O-Butyryl-(ꢀ)-EGC (3a).20 46.4% yield. [a]D19 ꢀ97.1ꢂ
(EtOH, c 0.58); IR nmax (KBr) 3401, 2964, 1718, 1701,
1637, 1625, 1541, 1523, 1509, 1460, 1340, 1259, 1186,
Experimental
1
1148, 1096, 1017, 825, 737 cmꢀ1; H NMR(300 MHz,
Melting points were determined on a Yanagimoto MP-
32 micromelting point apparatus and are uncorrected.
IRspectra were recorded on Shimadzu FTIR-8400
infrared spectrophotometer. Low-resolution (LR)-
FABMS spectra measured on a JEOL JMS-HX 100
instrument, whereas high resolution (HR)- and LR-
electron impact (EI) MS spectra, on JEOL The Tandem
CD3OD) d: 0.76 (3H, t, J=7.3 Hz, –COCH2CH2CH3)),
1.47 (2H, st, J=7.4 Hz, –COCH2CH2CH3), 2.18 (2H, t,
J=7.3 Hz, –COCH2CH2CH3), 2.76 (1H, AB of ABX,
J=2.3 and 17.5 Hz, H-4), 2.91 (1H, AB of ABX, J=4.5
and 17.5 Hz, H-4), 4.82 (1H, s, H-2), 5.35 (1H, m, H-3),
5.91 (1H, d, J=2.3 Hz, H-6 or H-8), 5.94 (1H, d, J=2.3
Hz, H-8 or H-6), 6.47 (2H, s, H-20 and H-60); FABMS:
m/z 377.13 [M+H]+; FABHRMS m/z: 377.1261
([M+H]+, calcd for C19H21O8: 377.1236).
1
MStation JMS-700. H NMRspectra were recorded on
JEOL EX-270 (270 MHz), Bruker AX-300 (300 MHz),
JEOL EX-400 (400 MHz) and JEOL JNM-GX 500
(500 MHz) instruments using tetramethylsilane (TMS)
as an internal standard. Multiplicities were abbreviated
as s=singlet, d=doublet, t=triplet, q=quartet,
st=sextet, m=multiplet, dd=doublet of doublet,
brs=broad singlet, brd=broad doublet. Analytical
TLC and preparative TLC were performed using Silica
gel 60 F254 (Merck, 0.25 mm) and Silica gel 60 F254
(Merck, 1 mm) glass plates, respectively. Preparative
HPLC was performed with LC-908 (Japan Analytical
Industry, Co. Ltd.) using a GS-320 column (21.5 mm
IDꢁ500 mm) and MeOH as an eluent. All extracted
solvents were dried over Na2SO4, followed by evapora-
tion in vacuo. Specific-pathogen-free (SPF) 6-week-old
female ICRmice were purchased from Japan SLC Inc.
(Shizuoka, Japan).
3-O-Hexanoyl-(ꢀ)-EGC (3b). 37.9% yield. [a]2D0 ꢀ47.3ꢂ
(EtOH, c 1.27); IR nmax (KBr) 3398, 2932, 1706, 1636,
1466, 1340, 1252, 1182, 1146, 1098, 1016, 824, 735 cmꢀ1
;
1H NMR(300 MHz, CD 3OD) d: 0.82 (3H, t, J=7.1
Hz, –COCH2CH2CH2CH2CH3), 1.11 (2H, m, –COCH2
CH2CH2CH2CH3), 1.19 (2H, m, –COCH2CH2CH2
CH2CH3), 1.43 (2H, m, –COCH2CH2CH2CH2CH3),
2.19 (2H, t, J=7.1 Hz, –COCH2CH2CH2CH2CH3),
2.77 (1H, AB of ABX, J=2.2 and 17.2 Hz, H-4), 2.90
(1H, AB of ABX, J=4.3 and 17.2 Hz, H-4), 5.33 (1H,
m, H-3), 5.90 (1H, d, J=2.3 Hz, H-6 or H-8), 5.94 (1H,
d, J=2.3 Hz, H-8 or H-6), 6.46 (2H, s, H-20 and H-60);
EIMS: m/z 404.2 [M]+; EIHRMS m/z: 404.1493 ([M]+,
calcd for C21H24O8 : 404.1471).
3-O-Octanoyl-(ꢀ)-EGC (3c). 43.7% yield. [a]1D9 ꢀ96.6ꢂ
(EtOH, c 0.45); IR nmax (KBr) 3399, 2928, 1718, 1702,
1629, 1618, 1541, 1522, 1458, 1378, 1344, 1256, 1187,
Two-stage carcinogenesis test on mouse skin
1
The ICRmice were housed in a SPF mouse room at five
per polycarbonate cage and were given food and water
at all the times throughout the experiment. Animals
were divided into seven experimental groups 10 mice
each. The back of each mouse was shaved with surgical
clippers one day before starting the test, and the mice
were topically treated with 100 mg of DMBA (0.1 mL of
a 390 nmol solution in acetone) as an initiating treat-
ment. One week after the initiation, papilloma forma-
tion was promoted by application twice a week of 1 mg
of TPA (0.1 mL of a 1.7 nmol solution in acetone) to
the skin. One h before each TPA treatment, the mice
were treated with 0.1 mL of the test compounds (85
nmol in acetone). The incidence and numbers of papil-
lomas were observed weekly over the course of 20
weeks.
1145, 1099, 1016, 828, 733 cmꢀ1; H NMR(270 MHz,
CD3OD) d: 0.66 (3H, t, J=7.3 Hz, –COCH2CH2
(CH2)4CH3), ca. 0.98 (8H, m, –COCH2CH2(CH2)4CH3),
1.24 (2H, st, J=7.3 Hz, –COCH2CH2(CH2)4CH3), 2.02
(2H, t, J=7.3 Hz,–COCH2CH2(CH2)4CH3), 2.57 (1H,
AB of ABX, J=2.2 and 17.3 Hz, H-4), 2.70 (1H, AB of
ABX, J=4.6 and 17.3 Hz, H-4), 4.67 (1H, s, H-2), 5.13
(1H, m, H-3), 5.70 (1H, d, J=2.4 Hz, H-6 or H-8), 5.74
(1H, d, J=2.4 Hz, H-8 or H-6), 6.26 (2H, s, H-20 and H-
60); EIMS: m/z 432.2 [M]+; EIHRMS m/z: 432.1776
([M]+, calcd for C23H28O8 : 432.1784).
3-O-Decanoyl-(ꢀ)-EGC (3d). 31.0% yield. [a]2D0 ꢀ53.6ꢂ
(EtOH, c 0.63); IR nmax (KBr) 3418, 2929, 2860, 1714,
1633, 1516, 1476, 1324, 1260, 1191, 1144, 1103, 1010,
1
824, 719 cmꢀ1; H NMR(270 MHz, CD OD) d: 0.69
3
(3H, t, J=6.6 Hz, –COCH2CH2(CH2)6CH3), 1.05 (12H,
m, –COCH2CH2(CH2)6CH3), 1.24 (2H, m, –COCH2
CH2(CH2)6CH3), 1.94 (2H, t, J=7.6 Hz, –COCH2CH2–
(CH2)6CH3), 2.58 (1H, AB of ABX, J=2.1 and 17.6 Hz,
H-4), 2.71 (1H, AB of ABX, J=4.6 and 17.6 Hz, H-4),
4.68 (1H, s, H-2), 5.14 (1H, m, H-3), 5.75 (1H, d, J=1.9
Hz, H-6 or H-8), 5.75 (1H, d, J=1.9 Hz, H-8 or H-6),
6.27 (2H, s, H-20 and H-60); FABMS: m/z 461.22
[M+H]+; FABHRMS m/z: 461.2178 ([M+H]+, calcd
for C25H33O8: 461.2175).
General procedure for the synthesis of (ꢀ)-EGCs posses-
sing a straight-chain acyl group
(ꢀ)-EGC (3.27 mmol), acid chloride (3.60 mmol) and
trifluoroacetic acid (6.54 mmol) were dissolved in tetra-
hydrofuran (10 mL), and the solution was stirred for 24
h under an Ar gas. After concentration of the reaction
mixture, the residue was extracted with AcOEt, which
was washed with satd NaCl. The organic layer was

A. Kumagai et al. / Bioorg. Med. Chem. 11 (2003) 5143–5148
5147
3-O-Dodecanoyl-(ꢀ)-EGC (3e). 35.6% yield. [a]D20
ꢀ40.8ꢂ (EtOH, c 0.98); IR nmax (KBr) 3422, 2926, 2857,
1702, 1634, 1515, 1462, 1307, 1184, 1143, 1013, 825, 724
;
cmꢀ1 1H NMR(300 MHz, CD 3OD) d: 0.89 (3H, t,
61% yield), respectively, through diastereoselective a-
methylation with MeI and sodium bis(trimethylsilyl)amide
followed by chromatographic purification according to
the reported procedure.14,15
J=6.8 Hz, –COCH2CH2(CH2)8CH3), 1.20–1.27 (16H,
m, –COCH2CH2(CH2)8CH3), 1.43 (2H, m, –COCH2
CH2(CH2)8CH3), 2.77 (1H, AB of ABX, J=2.2 and
17.4 Hz, H-4), 2.90 (1H, AB of ABX, J=4.5 and 17.4
Hz, H-4), 5.33 (1H, m, H-3), 5.90 (1H, d, J=2.3 Hz, H-
6 or H-8), 5.93 (1H, d, J=2.3 Hz, H-8 or H-6), 6.46
(2H, s, H-20 and H-60); FABMS: m/z 489.3 [M+H]+;
FABHRMS m/z: 489.2465 ([M+H]+, calcd for
C27H37O8 : 489.2488).
[3(20S),4S]-isomer (10). [a]2D0 +68.2ꢂ (MeOH, c 0.80); IR
nmax (neat) 2930, 1778, 1693, 1454, 1385, 1348, 1196,
1099, 1016, 972, 702 cmꢀ1; 1H NMR(270 MHz, CDCl 3)
d: 0.88 (3H, t, J=7.0 Hz), 1.22 (3H, d, J=6.8 Hz), 1.27
(8H, m), 1.71 (1H, m), 2.77 (1H, dd, J=9.5 and 13.2
Hz), 3.27 (1H, dd, J=3.2 and 13.2 Hz), 3.70 (1H, m),
4.07–4.23 (2H, m), 4.62–4.72 (1H, m), 7.19–7.35 (5H,
m); EIMS: m/z 317 [M]+; EIHRMS m/z: 317.1988
([M]+, calcd for C19H27NO3: 317.1991).
3-O-Myristoyl-(ꢀ)-EGC (3f). 44.7% yield. [a]1D9 ꢀ39.4ꢂ
(EtOH, c 1.1); IR nmax (KBr) 3433, 2925, 2857, 1718,
1630, 1515, 1467, 1317, 1184, 1144, 1098, 1018, 821, 724
;
cmꢀ1 1H NMR(300 MHz, CD 3OD) d: 0.89 (3H, t,
J=6.7 Hz, –COCH2CH2(CH2)10CH3), 1.20–1.27 (20H,
m, –COCH2CH2–(CH2)10CH3), 1.43 (2H, m, –COCH2
CH2(CH2)10CH3), 2.19 (2H, t, J=7.3 Hz, –COCH2–
(CH2)10CH3), 2.77 (1H, AB of ABX, J=2.2 and 17.5
Hz, H-4), 2.90 (1H, AB of ABX, J=2.2 and 17.5 Hz, H-
4), 5.33 (1H, m, H-3), 5.90 (1H, d, J=2.3 Hz, H-6 or H-
8), 5.94 (01H, d, J=2.3, H-8 or H-6), 6.46 (2H, s, H-20
and H-6 ); EIMS: m/z 516.3 [M]+; EIHRMS m/z:
516.2692 ([M]+, calcd for C29H40O8: 516.2723).
[3(20R),4R]-isomer (11). [a]2D0 ꢀ63.2ꢂ (MeOH, c 1.1); IR
nmax (neat) 2930, 1780, 1697, 1454, 1385, 1348, 1195,
1099, 1016, 972, 702 cmꢀ1; 1H NMR(270 MHz, CDCl 3)
d: 0.87 (3H, t, J=7.0 Hz), 1.22 (3H, d, J=7.0 Hz), 1.27
(8H, m), 1.69 (1H, m), 2.76 (1H, dd, J=9.5 and 13.2
Hz), 3.26 (1H, dd, J=3.2 and 13.2 Hz), 3.70 (1H, m),
4.07–4.23 (2H, m), 4.62–4.71 (1H, m), 7.19–7.35 (5H,
m); EIMS: m/z 317.0 [M]+; EIHRMS m/z: 317.1981
([M]+, calcd for C19H27NO3: 317.1991).
(S)-2-(12) and (R)-2-methyloctanoic acid (13). Com-
pound 10 or 11 (each 1.2 mmol) was dissolved in a
mixture of tetrahydrofuran (18 mL) and dist. Water (6
3-O-Palmitoyl-(ꢀ)-EGC (3g). 44.9% yield. [a]2D2 ꢀ32.5ꢂ
(EtOH, c 2.51); IR nmax (KBr) 3423, 2924, 2852, 1718,
1627, 1522, 1466, 1344, 1253, 1184, 1144, 1093, 1016,
mL) containing LiOH H2O (2.0 mmol) and 30% H2O2
.
(9.9 mmol). After stirring at rt for 4 h, the reaction was
quenched with 1 M Na2S2O3. The water layer was
adjusted to pH 1 with 10% HCl and extracted with
Et2O. The Et2O layer was washed with satd NaCl, dried
and concentrated in vacuo to give a brownish oily acid.
It was purified by SiO2 column chromatography with
AcOEt/n-hexane (1:4) followed by vacuum distillation
at 120–123 ꢂC.
1
825, 719 cmꢀ1; H NMR(270 MHz, CD OD) d: 0.91
3
(3H, t J=6.9 Hz, –COCH2CH2(CH2)12CH3), 1.01–1.37
(24H, m, –COCH2CH2(CH2)12CH3), 1.44 (2H, m, –CO
CH2CH2(CH2)12CH3), 2.19 (2H, t, J=6.8 Hz, –CO
CH2CH2(CH2)12CH3), 2.76 (1H, brd, J=17.8 Hz, H-4),
2.89 (1H, brd, J=17.8 Hz, H-4), 4.97 (1H, s, H-2), 5.31
(1H, m, H-3), 5.90 (1H, brs, H-6 or H-8), 5.92 (1H, brs,
H-8 or H-6), 6.45 (2H, s); EIMS: m/z 544.3 [M]+;
EIHRMS m/z: 544.2979 ([M]+, calcd for C31H44O8:
544.3036).
(S)-2-Methyloctanoic acid (12). 69.0% yield. [a]D20
+16.2ꢂ (MeOH, c 1.1); IR nmax (neat) 3100, 2930, 1713,
1467, 1418, 1238, 941 cmꢀ1 1H NMR(270 MHz,
;
CDCl3) d: 0.88 (3H, t, J=6.8 Hz), 1.15 (3H, d, J=1.68
Hz), 1.21–1.39 (10H, m), 2.45 (1H, m); EIMS: m/z:
158.1 [M]+; EIHRMS m/z: 158.1309 ([M]+, calcd for
C9H18NO2: 158.1307).
3-O-Stearoyl-(ꢀ)-EGC (3h). 41.7% yield. [a]2D1 ꢀ25.8ꢂ
(EtOH, c 0.91); IR nmax (KBr) 3420, 2925, 2851, 1718,
1618, 1541, 1458, 1312, 1180, 1141, 1085, 1016, 822, 711
cmꢀ1; 1H NMR (300 MHz, CD3OD) d: 0.94 (3H, t, J=6.9
Hz, –COCH2CH2(CH2)14CH3), 1.33 (28H, m, –CO
CH2CH2(CH2)14CH3), 1.48 (2H, m, –COCH2CH2
(CH2)14CH3), 2.24 (2H, t, J=7.1 Hz, –COCH2
CH2(CH2)14CH3), 2.82 (1H, AB of ABX, J=2.3 and
17.4 Hz, H-4), 2.95 (1H, AB of ABX, J=4.7 and 17.4
Hz, H-4), 4.93 (1H, s, H-2), 5.38 (1H, m, H-3), 5.96 (1H,
d, J=2.3 Hz, H-6 or H-8), 5.99 (1H, d, J=2.3 Hz, H-8
or H-6), 6.52 (2H, s, H-20 and H-60); FABMS: m/z 573.3
[M+H]+. FABHRMS m/z: 573.3464 ([M+H]+, calcd
for C33H49O8: 573.3427).
(R)-2-methyloctanoic acid (13). 53.5% [a]2D0 ꢀ15.3ꢂ
(MeOH, c 1.6); IR nmax (neat) 3100, 2930, 1710, 1465,
1417, 1236, 941 cmꢀ1; 1H NMR(270MHz, CDCl 3) d: 0.85
(3H, t, J=6.8 Hz), 1.17 (3H, d, J=7.0 Hz), 1.21–1.39
(10H, m), 2.45 (1H, m); EIMS m/z 158.1 [M]+; EIHR MS
m/z: 158.1303 ([M]+, calcd for C9H18NO2: 158.1307).
General procedure for the synthesis of (ꢀ)-EGCs posses-
sing a 2-methyl-substituted acyl group
(RS)-2-Methyloctanoic acid,21 (RS)-2-methyldecanoic
acid,21 12 or 13 (0.360 mmol) was converted to the cor-
responding acid chloride with SOCl2. It was then dis-
solved in a tetrahydrofuran solution (1 mL) containing
(ꢀ)-EGC (0.327 mmol) and trifluoroacetic acid (0.65
mmol), and the solution was stirred for 24 h under an
Ar gas. After concentration of the reaction mixture, the
[3(20S),4S]-3-(2-Methyloctanoyl)-4-benzyl-2-oxazolidi-
none (10) and [3(20R),4R]-3-(2-methyloctanoyl)-4-benzyl-
2-oxazolidinone (11). (4S)-3-(8) (3.5 g) and (4R)-3-
(octanoyl)-4-benzyl-2-oxazolidinone (9) (2.5 g) were
converted to [3(20S),4S]-3- (10) (2.9 g, 85% yield) and
[3(20R),4R]-3-(2-methyloctanoyl)-isomer (11) (1.6 g,

5148
A. Kumagai et al. / Bioorg. Med. Chem. 11 (2003) 5143–5148
residue was extracted with AcOEt, which was washed
with satd. NaCl. The organic layer was dried and con-
centrated in vacuo to give a residue, which was purified
by preparative HPLC, followed by freeze-drying, yield-
ing a white powder.
Conformational analysis
The conformations of 11 and 16 were analyzed by the
software MacSPARTAN Pro (version 2.0, Wavefunc-
tion, Inc.) using the molecular mechanics method with
MMFF94 force field.19 The global minimum-energy
conformation of each compound was generated by
repeating rotations (360ꢂ at 60ꢂ intervals) of rotatable
ester C–C and C–O bonds in the side chain followed by
optimization.
3-O-[(RS)-2-Methyloctanoyl]-(ꢀ)-EGC (4). 22.0% yield.
IR nmax (KBr) 3436, 2931, 2861, 1718, 1637, 1508, 1467,
1
1343, 1190, 1143, 1102, 1008, 879, 826, 726 cmꢀ1. H
NMR(270 Hz, CD 3OD) d: 0.81 (3Hꢁ0.5, t, J=6.9 Hz, –
COCH(CH3)CH2(CH2)4CH3), 0.82 (3Hꢁ0.5, t, J=6.9
Hz, –COCH(CH3)CH2(CH2)4CH3), 0.88 (3Hꢁ0.5, d,
J=6.8 Hz, –COCH(CH3)CH2(CH2)4CH3), 0.92
(3Hꢁ0.5, d, J=7.3 Hz, –COCH(CH3)CH2(CH2)4CH3),
0.9–1.35 (10H, m, –COCH(CH3)CH2(CH2)4CH3), 2.25
(1H, m, –COCH(CH3)CH2(CH2)4CH3 ), 2.75 (1H, brd,
J=17.3 Hz, H-4), 2.85 (1H, AB of ABX, J=4.3 and
17.3 Hz, H-4), 5.25 (1H, m, H-3), 5.88 (2H, m, H-6 and
H-8), 6.40 (2H, s, H-20 and H-60); FABMS: m/z 447.20
[M+H]+; FABHRMS m/z: 447.2036 ([M+H]+, calcd
for C24H31O8: 447.2019).
Acknowledgements
We are grateful to Dr. Masanori Morita (Research
Institute for Technology and Science, Kinki University)
for measurement of MS spectra. Part of this research was
financially supported by the Kansai University Special
Aid for Promotion of Research and Education, 2002.
References and Notes
3-O-[(RS)-2-Methyldecanoyl]-(ꢀ)-EGC (5). 16.7% yield.
IR nmax (KBr) 3436, 2931, 2861, 1700, 1616,
1522, 1459, 1342, 1265, 1195, 1144, 1098, 1015,
1. Xu, Y.; Ho, C.-T.; Amin, S. G.; Han, C.; Chung, F.-L.
Cancer Res. 1992, 52, 3875.
2. Matsumoto, N.; Kohri, T.; Okushio, K.; Hara, Y. Jpn. J.
Cancer Res. 1996, 56, 1034.
824, 723 cmꢀ1
.
1H NMR(CD 3OD) d: 0.81–0.98
(6H, m, –COCH(CH3)CH2(CH2)6CH3), 1.01–1.41
(14H, m, –COCH(CH3)CH2(CH2)6CH3), 2.22–2.23
(1H, m –COCH(CH3)CH2(CH2)6CH3), 2.79 (1H,AB
of ABX, J=5.4 and 13.5 Hz, H-4), 2.88 (1H, AB and
ABX J=4.3 and 17.6 Hz, H-4), 4.87 (1H, s, H-2), 5.27
(1H, m, H-3), 5.90 (1H, d, J=1.90 Hz, H-6 and H-8),
5.92 (1H, d, J=2.4 Hz, H-8 and H-6), 6.46 (2H, s, H-20
and H-60); FABMS: m/z: 475.24 [M+H]+; FABHRMS
m/z: 475.2355 ([M+H]+, calcd for C26H35O8: 475.2332).
3. Muramatsu, K.; Fukuyo, M.; Hara, Y. J. Nutr. Sci. Vita-
minol. 1986, 32, 613.
4. Chisaka, T.; Matsuda, H.; Kubomura, Y.; Mochizuki, M.;
Yamahara, J.; Fujimura, H. Chem. Pharam. Bull. 1988, 36,
227.
5. Matsumoto, N.; Ishigaki, F.; Ishigaki, A.; Iwashina, H.;
Hara, Y. Biosci. Biotechnol. Biochem. 1993, 57, 525.
6. Nakayama, M.; Suzuki, K.; Toda, M.; Okubo, S.; Hara,
Y.; Shimamura, T. Antiviral Res. 1993, 21, 289.
7. Nanjo, F.; Honda, M.; Okushio, K.; Matsumoto, N.; Ishi-
gaki, F.; Ishigami, T.; Hara, Y. Biol. Pharm. Bull. 1993, 16,
1156.
8. Cao, Y.; Cao, R. Nature 1999, 398, 381.
9. Jankun, J.; Selman, S. H.; Swiercz, R.; Skrzypczak-Jankun,
E. Nature 1997, 387, 561.
10. Naasani, I.; Seimiya, H.; Tsuruom, T. Biochem. Biophys.
Res. Commun. 1998, 249, 391.
11. Kohri, T.; Matsumoto, N.; Yamakawa, M.; Suzuki, M.;
Nanjo, F.; Hara, Y.; Oku, N. J. Agr. Food Chem. 2001, 49,
4102.
12. Nanjo, F.; Goto, K.; Seto, R.; Suzuki, M.; Sakai, M.;
Hara, Y. Free Radic. Biol. Med. 1996, 21, 895.
13. Nanjo, F.; Mori, M.; Goto, K.; Hara, Y. Biosci. Bio-
technol. Biochem. 1999, 63, 1621.
3-O-[(S)-2-Methyloctanoyl]-(ꢀ)-EGC (6). 13.4% yield.
[a]2D7 ꢀ23.2ꢂ (EtOH, c 0.11); IR nmax (KBr) 1706, 1608,
1
1458, 1299, 1145, 826; H NMR(500 MHz, CD OD) d:
3
0.84 (3H, t, J=6.9 Hz, –COCH(CH3)CH2(CH2)4CH3),
0.91 (3H, d, J=7.3 Hz, –COCH(CH3)CH2(CH2)4CH3),
0.9–1.35 (10H, m, –COCH(CH3)CH2(CH2)4CH3), 2.34
(1H, m, –COCH(CH3)CH2(CH2)4CH3), 2.75 (1H, brd,
J=17.2 Hz, H-4), 2.85 (1H, AB of ABX, J=3.7 and
17.2 Hz, H-4), 5.29 (1H, m, H-3), 5.94 (2H, m, H-6 and
H-8), 6.57 (2H, s, H-20 and H-60); FABMS: m/z 447.21
[M+H]+; FABHRMS m/z: 447.2036 ([M+H]+, calcd
for C24H31O8: 447.2019).
14. Uesato, S.; Kitagawa, Y.; Hara, Y.; Tokuda, H.; Okuda,
M.; Mou, X. Y.; Mukainaka, T.; Nishino, H. Bioorg. Med.
Chem. Lett. 2000, 10, 1673.
15. Wipf, P.; Kim, Y.; Fritch, P. C. J. Org. Chem. 1993, 58,
7195.
16. Mero, C. L.; Poter, N. A. J. Am. Chem. Soc. 1999, 121,
5155.
17. Sonnet, P. E.; Gazzillo, J. Org. Prep. Proc. Int. 1990, 22,
203.
18. Ito, H.; Kobayashi, E.; Li, S.-H.; Hatano, T.; Sugita, D.;
Kubo, N.; Shimura, S.; Itoh, Y.; Tokuda, H.; Nishino, H.;
Yoshida, T. J. Agr. Food Chem. 2002, 50, 2400.
19. Halgren, T. A. J. Computational Chem. 1996, 17, 490.
20. Sakai, M.; Suzuki, M.; Nanjo, F.; Hara, Y. JP 93-91934.
21. Meng, Y.; Chen, L.-G.; Gu, X.-F.; Song, Y. Huaxue
Gongye Yu Gongcheng 2001, 18, 303.
3-O-[(R)-2-Methyloctanoyl]-(ꢀ)-EGC (7). 8.6% yield.
[a]2D8 ꢀ21.7ꢂ (EtOH, c 0.03); IR nmax (KBr) 1706, 1608,
1
1458, 1299, 1145, 826; H NMR(400 MHz, CD OD) d:
3
0.77 (3H, t, J=7.0 Hz, –COCH(CH3)CH2(CH2)4CH3),
0.87 (3H, d, J=7.2 Hz, –COCH(CH3)CH2(CH2)4CH3),
0.9–1.35 (10H, m, –COCH(CH3)CH2(CH2)4CH3), 2.17
(1H, q, J=7.2 Hz, –COCH(CH3)CH2(CH2)4CH3), 2.71
(1H, AB of ABX, J=2.0 and 17.6 Hz, H-4), 2.80 (1H,
AB of ABX, J=4.4 and 17.6 Hz, H-4), 4.81 (1H, s, H-
2), 5.19 (1H, brs, H-3), 5.82 (1H, d, J=2.2 Hz, H-6 or
H-8), 5.84 (1H, d, J=2.2 Hz, H-8 or H-6), 6.40 (2H, s,
H-20 and H-60); FABMS: m/z 445.19 [MꢀH]ꢀ,
FABHRMS m/z: 445.1859 ([MꢀH]ꢀ, calcd for
C24H29O8: 445.1863).