Structure-activity relationship of bis-galloyl derivatives related to (-)-epigallocatechin gallate

Article abstract of DOI:10.1248/cpb.57.190

Green tea and (-)-epigallocatechin gallate (EGCG: one of main components of green tea) are well known to have preventive activities against human cancers. Previously, using a galloyl group as a core structure derived from EGCG, we developed alkyl gallate and gallamide derivatives, which showed strong antiproliferative activity towards human leukemia HL-60 cells by inducing apoptosis. Here, as a further structural development study, we planned to introduce an additional galloyl group into alkyl gallates and gallamides. According to this strategy, various bisgallate and bisgallamide derivatives were synthesized and tested for antiproliferative activity towards HL-60 cells. In gallamide derivatives having a short alkyl chain, the additional galloyl group enhanced the antiproliferative activity. In contrast, in the gallate derivatives, the additional galloyl group had no effect on the antiproliferative activity.

Full text of DOI:10.1248/cpb.57.190

190
Notes
Chem. Pharm. Bull. 57(2) 190—194 (2009)
Vol. 57, No. 2
Structure–Activity Relationship of Bis-Galloyl Derivatives Related to (ꢀ)-
Epigallocatechin Gallate
a
Kosuke DODO, ,a,b Taro MINATO,a and Yuichi HASHIMOTO
*
a Institute of Molecular and Cellular Biosciences, University of Tokyo; 1–1–1 Yayoi, Bunkyo-ku, Tokyo 113–0032, Japan:
and b RIKEN (The Institute of Physical and Chemical Research); 2–1 Hirosawa, Wako, Saitama 351–0198, Japan.
Received July 22, 2008; accepted November 10, 2008
Green tea and (ꢀ)-epigallocatechin gallate (EGCG: one of main components of green tea) are well known to
have preventive activities against human cancers. Previously, using a galloyl group as a core structure derived
from EGCG, we developed alkyl gallate and gallamide derivatives, which showed strong antiproliferative activity
towards human leukemia HL-60 cells by inducing apoptosis. Here, as a further structural development study, we
planned to introduce an additional galloyl group into alkyl gallates and gallamides. According to this strategy,
various bisgallate and bisgallamide derivatives were synthesized and tested for antiproliferative activity towards
HL-60 cells. In gallamide derivatives having a short alkyl chain, the additional galloyl group enhanced the an-
tiproliferative activity. In contrast, in the gallate derivatives, the additional galloyl group had no effect on the an-
tiproliferative activity.
Key words (ꢀ)-epigallocatechin gallate; gallic acid; antiproliferative activity
Various epidemiological studies on the relationship be- the hydrophobicity of their alkyl chains was thought to be
tween intake of green tea and risk of cancer indicate that important for the antiproliferative activity. On the other hand,
green tea has preventive activities against human cancers.1,2) the hydroxyl groups of the galloyl moiety were also discov-
(ꢀ)-Epigallocatechin gallate (EGCG), one of the major com- ered to be essential for cell growth inhibition, indicating that
ponents of green tea,3) was reported to have a range of activi- the galloyl group greatly contributes to the activity. There-
ties, including cell growth inhibition, apoptosis induction, fore, we planned to introduce an additional galloyl group into
and cell cycle arrest.4,5) Therefore, EGCG and related gallate and gallamide derivatives in an attempt to increase the
polyphenolic compounds were thought to contribute to the activity (Fig. 2).
antitumor activity of green tea. To clarify the essential struc-
ture for the antitumor activity, EGCG and related com- Results and Discussion
pounds, such as epicatechin, epicatechin gallate (ECG), epi-
Initially, we synthesized bisgallate derivatives having vari-
gallocatechin (EGC), were tested for antiproliferative activity ous lengths of alkyl chain according to the previously re-
towards PC-9 human lung cancer cells (Fig. 1).6) It was con- ported method, as shown in Chart 1. To obtain bisgallate de-
cluded that a galloyl group, which is composed of one ben- rivatives, benzyl-protected gallic acid 67) was introduced at
zoyl and three phenoxy groups, contributes significantly to both ends of the alkyl chain via transformation of 6 to acyl
the antiproliferative activity. Based on this concept, we previ- chloride (7), followed by debenzylation using H2/Pd–C to af-
ously developed various alkyl gallate and gallamide deriva- ford bisgallate derivatives 9a—c. Considering the possibility
tives having a galloyl group and evaluated their cell growth- of intracellular hydrolysis of the ester group, bisgallamide
inhibitory activity towards HL-60 human leukemia cells.7) In derivatives 11a—c having amide bonds instead of ester
our previous studies, gallates 1 and gallamides 2 (Fig. 1) bonds were also synthesized. As shown in Chart 2, bisgal-
were found to have potent cell growth-inhibitory activity, and lamide derivatives having various lengths of alkyl chain were
Fig. 1. Structures of EGCG and Related Compounds
∗ To whom correspondence should be addressed. e-mail: dodo@iam.u-tokyo.ac.jp; dodo@riken.jp
© 2009 Pharmaceutical Society of Japan

February 2009
191
Fig. 2. Design of Bis-Galloyl Derivatives
Reagents and conditions: (a) H2N(CH2)2NH2, EDCl, HOBt, CH2Cl2; (b) 10% Pd/C,
H2, AcOEt, rt; (c) Boc2O, CH2Cl2/MeOH; (d) 6, EDCl, HOBt, DMF; (e) TFA, CH2Cl2;
(f) 6, EDCl, HOBt, CH2Cl2; (b) 10% Pd/C, H2, AcOEt, rt; (c) Boc2O, CH2Cl2/MeOH;
(d) 6, EDCl, HOBt, DMF; (e) TFA, CH2Cl2; (f) 6, EDCl, HOBt, DMF; (g) 10% Pd/C,
H2, AcOEt, rt.
Chart 2. Modification of Terminal Carbon of Alkylgallamide
Reagents and conditions: (a) MeOH, H2SO4, 100 °C, 2 h; (b) BnCl, K2CO3, DMF,
80 °C, 15 h; (c) NaOH, MeOH/H2O/dioxane, 120 °C, 6 h; (d) (COCl)2, DMF, toluene,
rt; (e) HO(CH2)nOH, K2CO3, DMF, rt, overnight; (f) 10% Pd/C, H2, AcOEt, rt.
Table 1. Antiproliferative Activity of Alkyl Mono- and Bis-Gallate and
Gallamide Derivatives
Chart 1. Modification of Terminal Carbon of Alkylgallate
Compound
X
n
IC50 (mM)
synthesized by condensation of alkyldiamine with protected
gallic acid 6 in the presence of 1-ethyl-3-(3-dimethylamino-
propyl) carbodiimide (EDCI) and HOBt, followed by the
debenzylation using H2/Pd–C to give 11a—c.
1a
1b
1c
2a
2b
2c
O
O
O
NH
NH
NH
2
42
6
12
2
6
12
4.4
1.1
16
24
3.0
Then, mono-gallates 1a—c, bis-gallates 9a—c, mono-gal-
lamides 2a—c, and bis-gallamides 11a—c were tested for
antiproliferative activity by calculating the IC50 values from
the viability of human leukemia HL-60 cells treated with
each compound, as shown in Table 1. In all cases, disappoint-
ingly, an additional galloyl group did not elicit an improve-
ment of antiproliferative activity. As for gallate derivatives
(1a—c and 9a—c), the relationship between antiproliferative
activity and the length of the alkyl chain was similar in
mono- and bis-gallates, i.e., the longer the alkyl chain length,
more potent the antiproliferative activity. But, interestingly,
in gallamide derivatives (2a—c and 11a—c), the relationship
9a
9b
9c
11a
11b
11c
O
O
O
NH
NH
NH
2
6
12
2
6
12
29
14
1.4
5.2
7.1
12
EGCG
9.4
was different for mono- and bis-gallamides. In the case of changes, the DNA fragmentation, and the activation of cas-
monogallamide derivatives (2a—c), the longer the alkyl pase-3. Therefore, to clarify the difference between gallate
chain, the greater the antiproliferative activity. On the con- and gallamide derivatives, we examined the apoptosis-induc-
trary, in the case of bisgallamide derivatives (11a—c), the ing activity of gallate and gallamide derivatives. As a result,
relationship was reversed, i.e., the shorter the alkyl chain a great difference between bisgallates and bisgallamides was
length, the greater the antiproliferative activity. The results found in the activation of caspase-3, a typical hallmark of
suggest that the effect of an additional galloyl moiety is dif- apoptosis. As shown in Fig. 3, potently antiproliferative gal-
ferent between gallate and gallamide derivatives. In the pre- lates, 1c (IC50ꢁ1.1 mM) and 9c (IC50ꢁ1.4 mM), efficiently in-
vious report,7) we confirmed the apoptosis-inducing activity duced the activation of caspase-3 to the extent of 350—
of galloyl derivatives based on the apoptotic morphological 510%. A moderately potent antiproliferative monogallamide,

192
Vol. 57, No. 2
2c (IC50ꢁ3.0 mM), also induced the activation of caspase-3 by hydrophobicity of the alkyl chain is important for the activ-
ca. 200%. However, no apparent activation of caspase-3 was ity.
found with bis-gallamide derivatives 11a—c (IC50 values of
In this study, as a further structural development of alkyl
5.2—12 mM) (Fig. 3). These results indicate that bisgal- gallate and gallamide derivatives derived from EGCG, we
lamides may elicit the antiproliferative activity without in- synthesized various bisgallate and bisgallamide derivatives
ducing apoptosis.
having an additional galloyl group and tested them for an-
Although the antiproliferative activity was not increased tiproliferative activity in HL-60 cells. The effects of the addi-
by introduction of an additional galloyl group, the additional tional galloyl group were different between gallate and gal-
galloyl group in bis-gallate derivatives might have some ef- lamide derivatives. In gallamide derivatives having a short
fect. To examine this possibility, we modified one of galloyl alkyl chain, the additional galloyl group enhanced the an-
groups in the bis-gallate derivative 9c. In the previous study, tiproliferative activity. In contrast, in the gallate derivatives,
the hydroxyl group(s) of mono-galloyl derivatives was found the additional galloyl group had no effect on the antiprolifer-
to be essential for the antiproliferative activity.7) Therefore, ative activity. Further structural development and biological
various 9c-related gallate derivatives were synthesized as studies of the molecular mechanisms involved are in
shown in Chart 3, and their antiproliferative activities were progress.
tested (Table 2). However there was little change in the IC50
values of compounds 20 (IC50 values of 0.84—1.7 mM) and
9c (IC50ꢁ1.4 mM), indicating that the second galloyl group
had little effect on the antiproliferative activity. Moreover,
the non-hydroxyl derivative 20d showed the most potent ac-
tivity among this series of compounds, which implies that the
Experimental
Biology. Cell Culture HL-60 cells were maintained in RPMI 1640
medium supplemented with 100 U/ml penicillin, 100 mg/ml streptomycin,
and 10% heat-inactivated fetal bovine serum (FBS). Cells were grown in a
humidified incubator at 37 °C under 5% CO2/95% air.
Cell Viability Assay HL-60 cells (1ꢂ105 cells/well) suspended in fresh
medium in a 96-well plate (100 ml/well) were treated with test compounds
(dimethyl sulfoxide (DMSO) solution, 0.5 ml/well). After 3-d incubation,
10 ml of Cell Counting Kit (Dojindo) was added to each well. The cell via-
bility was determined based on the increase of absorbance (450 nm) during
4 h incubation.
Measurement of Caspase-3 Activation HL-60 cells (3ꢂ105 cells/ml)
suspended in fresh medium in a 96-well plate (100 ml/well) were treated
with test compounds (DMSO solution, 0.5 ml/well). After 4 h incubation,
100 ml of homogeneous caspase-3 assay kit (Roche) was added to each well.
The activity of caspase-3 was determined based on the fluorescence of
cleaved substrate (excitation 485 nm/emission 535 nm).
Chemistry. General 1H-NMR (500 MHz) spectra were recorded on a
JEOL JNM-a 500 spectrometer. The 1H-NMR chemical shifts were reported
in parts per million (ppm) relative to the singlet at 7.26 ppm for chloroform
in deuteriochloroform or 2.49 ppm for DMSO in DMSO-d6 and coupling
constants were given in hertz (Hz). The following abbreviations are used for
spin multiplicity: sꢁsinglet, dꢁdoublet, tꢁtriplet, qꢁquartet, quintꢁquin-
tet, septꢁseptet, mꢁmultiplet, brꢁbroad. Mass spectra were recorded on
JEOL JMA-HX110 spectrometer with m-nitrobenzyl alcohol or JMS T100-
LC spectrometer. Routine thin layer chromatography (TLC) was performed
on silica gel 60 F254 plates (Merck, Germany). Flash column chromatogra-
phy was performed on silica gel (spherical, particle size 40—100 mm,
Kanto).
2-(3,4,5-Trisbenzyloxybenzoyloxy)ethyl 3,4,5-Trisbenzyloxybenzoate
(8a) To a stirred solution of 7 (418 mg, 0.910 mmol), K2CO3 (254 mg,
1.84 mmol) in N,Nꢃ-dimethylformamide (DMF) (20 ml) was added ethylene
glycol (30 ml, 0.538 mmol) and stirring was continued overnight. The reac-
tion mixture was extracted with ethyl acetate three times and washed with
sat NaHCO3 aq. and brine. The organic layer was dried with MgSO4 and fil-
tered. The residue was purified by column chromatography (toluene : ethyl
acetateꢁ7 : 1) to afford 8a (195 mg, 0.215 mmol 47%). 1H-NMR (500 MHz,
Fig. 3. Caspase-3 Activation Induced by Galloyl Derivatives (10 mM, 4 h)
Table 2. Antiproliferative Activity of Alkyl Bis-Gallate and Related De-
rivatives
Compound
R1
R2
R3
IC50 (mM)
9c
20a
20b
20c
20d
OH
OH
OH
H
OH
OH
H
OH
H
OH
H
H
H
H
1.4
1.7
1.2
1.3
0.84
H
Reagents and conditions: (a) HO(CH2)nOH, K2CO3, DMF, rt; (b) CBr4, PPh3, rt, overnight; (c) 17, K2CO3, DMF, rt, overnight; (d) 10% Pd/C, H2, AcOEt, rt.
Chart 3. Modification of Hydroxyl Groups of 9a

February 2009
193
1H-NMR (500 MHz, DMSO-d6) d: 8.96 (s, 4H), 8.58 (s, 2H), 7.98 (t,
Jꢁ5.5 Hz, 2H), 6.78 (s, 4H), 3.14 (q, Jꢁ6.5 Hz, 4H), 1.44 (quint, Jꢁ6.5 Hz,
4H), 1.35 (br, 4H), 1.27—1.19 (m, 16H). HR-MS (ESI, [M+Na]+) Calcd for
C26H36N2Na1O8 527.2369, Found 527.2363.
CDCl3) d: 7.36—7.26 (m, 30H), 7.22 (s, 4H), 5.06 (s, 4H), 5.04 (s, 8H),
4.60 (s, 4H). MS (FAB, [MꢄH]ꢄ) m/z 908.
6-(3,4,5-Trisbenzyloxybenzoyloxy)hexyl 3,4,5-Trisbenzyloxybenzoate
(8b) According to the same procedure used for 8a, starting from 7
(418 mg, 0.910 mmol), 8b (133 mg, 0.138 mmol, 30%) was obtained. 1H-
NMR (500 MHz, CDCl3) d: 7.41—7.25 (m 30H), 7.21 (s, 4H), 5.10 (s, 8H),
5.09 (s, 4H), 4.28 (t, Jꢁ6.5 Hz, 4H), 1.77 (quint, Jꢁ6.5 Hz, 4H), 1.47 (br,
4H). MS (FAB, [MꢄH]ꢄ) m/z 964.
12-(3,4,5-Trisbenzyloxybenzoyloxy)dodecyl 3,4,5-Trisbenzyloxyben-
zoate (8c) According to the same procedure used for 8a, starting from 7
(646 mg, 1.41 mmol), 8c (327 mg, 0.312 mmol, 44%) was obtained. MS
(FAB, [MꢄH]ꢄ) m/z 1048.
2-(3,4,5-Trihydroxybenzoyloxy)ethyl 3,4,5-Trihydroxybenzoate (9a)
To a stirred solution of 8a (99 mg, 0.109 mmol) in THF was added Pd/C
(37 mg), and the reaction mixture was stirred at room temperature under a
H2 atmosphere for 1 h, then filtered and concentrated. The residue was re-
crystalized from ethanol and hexane. 9a (31 mg, 0.085 mmol, 78%) was ob-
tained. 1H-NMR (500 MHz, DMSO-d6) d: 9.26 (s, 4H), 8.96 (s, 2H), 6.94 (s,
4H), 4.45 (s, 4H). HR-MS (FAB, [MꢄH]ꢄ) Calcd for C16H14O10 367.0665,
Found 367.0670.
6-(3,4,5-Trihydroxybenzoyloxy)hexyl 3,4,5-Trihydroxybenzoate (9b)
According to the same procedure used for 9a, starting from 8b (279 mg,
0.290 mmol), 9b (97 mg, 0.230 mmol, 79%) was obtained. 1H-NMR
(500 MHz, DMSO-d6) d: 6.93 (s, 4H), 4.15 (t, Jꢁ6.5 Hz, 4H), 1.67 (quint,
Jꢁ6.5 Hz, 4H), 1.42 (br, 4H). HR-MS (FAB, [MꢄH]ꢄ) Calcd for C20H23O10
423.1291, Found 423.1300.
tert-Butyl 6-Aminohexylcarbamate (13b) Boc2O (1900 mg, 8.71
mmol) was dissolved in CH2Cl2 (20 ml) and methanol (30 ml) and cooled to
0 °C. This solution was dropped into a stirred solution of hexyldiamine 12b
(2187 mg, 10.9 mmol) in CH2Cl2 (200 ml) at 0 °C for 2 h, and stirring was
continued overnight at room temperature. The reaction mixture was concen-
trated in vacuo, water was added, and the whole was extracted with ethyl ac-
etate three times. The organic layer was washed with half-saturated NaCl
aq., dried with MgSO4, filtered and concentrated in vacuo. The residue was
purified by column chromatography on silica gel (CH2Cl2 : methanol : NH3
aq.ꢁ100 : 20 : 1) to afford 13b (1112 mg, 5.14 mmol, 59%). MS (FAB,
[MꢄH]ꢄ) m/z 217.
tert-Butyl 12-Aminododecylcarbamate (13c) According to the same
procedure used for 13b, starting from dodecyldiamine 12c (2690 mg,
13.4 mmol) and Boc2O (633 mg, 2.90 mmol), 13c (502 mg, 1.67 mmol, 58%)
was obtained. 1H-NMR (500 MHz, DMSO-d6) d: 6.75 (t, Jꢁ5.5 Hz, 1H),
2.87 (q, Jꢁ6.5 Hz, 2H), 2.52 (t, Jꢁ6.5 Hz, 2H), 1.35 (s, 9H), 1.24—1.18 (m,
20H). MS (FAB, [MꢄH]ꢄ) m/z 301.
N-(6-(tert-Butyl-carbamoyloxy)hexyl)-3,4,5-trisbenzyloxybenzamide
(14b) To a stirred solution of 6 (616 mg, 1.40 mmol), EDCI (402 mg,
2,10 mmol), DMAP (41 mg, 0.336 mmol) in CH2Cl2 (30 ml) was added 13b
(412 mg, 1.96 mmol). The reaction mixture was stirred at room temperature
under Ar atmosphere for 2 h, acidified with HCl aq., washed with brine,
dried with MgSO4, and concentrated in vacuo. 14b (894 mg, 1.40 mmol
12-(3,4,5-Trihydroxybenzoyloxy)dodecyl
3,4,5-Trihydroxybenzoate
1
(9c) According to the same procedure used for 9a, starting from 8c
(275 mg, 0.263 mmol), 9c (74 mg, 0.146 mmol, 56%) was obtained. 1H-
NMR (500 MHz, DMSO-d6) d: 6.92 (s, 4H), 4.13 (t, Jꢁ6.5 Hz, 4H), 1.63
(quint, Jꢁ6.5 Hz, 4H), 1.35 (br, 4H), 1.32—1.18 (m, 12H). HR-MS (FAB,
[MꢄH]ꢄ) Calcd for C26H35O10 507.2230, Found 507.2231.
N-(2-(3,4,5-Trisbenzyloxybenzamido)ethyl)-3,4,5-trisbenzyloxybenz-
amide (10a) To a stirred solution of 4 (591 mg, 1.34 mmol), EDCI
(382 mg, 1.99 mmol), DMAP (28 mg, 0.229 mmol) in dichloromethane was
added ethylene diamine (90 ml, 0.896 mmol). The reaction mixture was
stirred at room temperature under a N2 atmosphere for 7 h, then filtered and
concentrated. The residue was purified with column chromatography
(dichloromethane : methanolꢁ10 : 3) to afford 10a (334 mg, 0.369 mmol,
55%). 1H-NMR (500 MHz, DMSO-d6) d: 8.66 (t, Jꢁ5.5 Hz, 2H), 7.45—
7.24 (m, 34H), 5.13 (s, 8H), 4.96 (s, 4H), 3.43 (br, 4H). MS (FAB, [MꢄH]ꢄ)
m/z 906.
99%) was obtained. H-NMR (500 MHz, CDCl3) d: 7.41—7.27 (m, 15H),
7.09 (s, 2H), 6.20 (br, 1H), 5.12 (s, 4H), 5.07 (s, 2H), 3.38 (q, Jꢁ7.0 Hz,
2H), 3.11 (br, 2H), 1.47—1.36 (m, 17H).
N-(12-(tert-Butyl-carbamoyloxy)-dodecyl)-3,4,5-trisbenzyloxybenz-
amide (14c) According to the same procedure used for 14b, starting from
6 (439 mg, 0.997 mmol), 14c (688 mg, 0.952 mmol, 95%) was obtained. 1H-
NMR (500 MHz, CDCl3) d: 7.41—7.34 (m, 15H), 7.02 (s, 2H), 5.12 (s, 4H),
5.07 (s, 2H), 3.38 (q, Jꢁ6.5 Hz, 2H), 3.14—3.02 (br, 2H) 1.46—1.41 (m,
12H), 1.34—1.23 (m, 17H). MS (FAB, [MꢄH]ꢄ) m/z 723.
N-(6-Aminohexyl)-3,4,5-trisbenzyloxybenzamide (15b) To a stirred
solution of 14b (940 mg, 1.47 mmol) in CH2Cl2 (20 ml) was added TFA
(5 ml). The reaction mixture was stirred at room temperature for 1 h, then
concentrated. The residue was taken up in ethyl acetate (50 ml) and washed
with NaHCO3 aq., water, and brine. The organic layer was dried with
MgSO4, filtered and concentrated in vacuo. The residue was purified by col-
umn chromatography on silica gel (CH2Cl2 : methanolꢁ10 : 2) to afford 15b
N-(6-(3,4,5-Trisbenzyloxybenzamido)hexyl)-3,4,5-trisbenzyloxybenz-
amide (10b) To a stirred solution of 15b (220 mg, 0.408 mmol), EDCI
(124 mg, 0.647 mmol), DMAP (8 mg, 0.065 mmol) in CH2Cl2 (10 ml) was
added 6 (218 mg, 0.495 mmol). The reaction mixture was stirred at room
temperature under a N2 atmosphere for 6 h, then filtered and concentrated.
The residue was purified with column chromatography (hexane : ethyl ac-
1
(487 mg, 0.904 mmol, 62%). H-NMR (500 MHz, CDCl3) d: 8.12 (br, 1H),
7.41—7.25 (m, 15H), 7.05 (s, 2H), 5.04 (s, 4H), 5.02 (s, 2H), 3.30 (br, 2H),
2.86 (br, 2H) 1.64 (br, 2H), 1.51 (br, 2H), 1.35 (br, 2H), 1.28 (br, 2H). MS
(FAB, [MꢄH]ꢄ) m/z 539.
N-(12-Aminododecyl)-3,4,5-trisbenzyloxybenzamide (15c) According
to the same procedure used for 15b, starting from 14c (202 mg,
0.279 mmol), 15c (172 mg, 0.276 mmol, 99%) was obtained. 1H-NMR
(500 MHz, DMSO-d6) d: 8.40 (t, Jꢁ5.5 Hz, 2H), 7.47—7.25 (m, 17H), 5.15
(s, 4H), 4.98 (s, 2H), 3.23 (q, Jꢁ6.5 Hz, 2H), 2.73 (quint, Jꢁ6.5 Hz, 2H)
1.52—1.44 (m, 4H), 1.30—1.22 (m, 16H). MS (FAB, [MꢄH]ꢄ) m/z 623.
12-Hydroxydodecyl 3,4,5-Trisbenzyloxybenzoate (16) To a stirred so-
lution of 7 (646 mg, 1.41 mmol), K2CO3 (393 mg, 2.85 mmol) in DMF was
added 1,12-dodecanediol (168 mg, 0.833 mmol) at room temperature. The
reaction mixture stirred overnight. Water was added, and the whole was ex-
tracted with ethyl acetate three times and washed with NaHCO3 aq. and
brine. The organic layer was dried with MgSO4, filtered and concentrated.
The residue was purified by column chromatography on silica gel to afford
1
etateꢁ3 : 2) to afford 10b (151 mg, 0.157 mmol, 39%). H-NMR (500 MHz,
DMSO-d6) d: 7.46—7.24 (m, 34H), 5.97 (t, Jꢁ5.5 Hz, 2H), 5.09 (s, 8H),
5.06 (s, 4H), 3.25 (q, Jꢁ6.0 Hz, 4H), 1.53 (quint, Jꢁ6.0 Hz, 4H), 1.34 (m,
4H). MS (FAB, [MꢄH]ꢄ) m/z 962.
N-(12-(3,4,5-Trisbenzyloxybenzamido)dodecyl)-3,4,5-trisbenzyloxy-
benzamide (10c) According to the same procedure used for 10b, starting
from 6 (485 mg, 1.10 mmol), 10c (569 mg, 0.544 mmol, 49%) was obtained.
1H-NMR (500 MHz, CDCl3) d: 7.46—7.25 (m, 30H), 7.03 (s, 4H), 5.94 (t,
Jꢁ5.0 Hz, 2H), 5.09 (s, 4H), 5.06 (s, 4H), 3.36 (q, Jꢁ6.5 Hz, 4H), 1.42—
1.27 (m, 16H). MS (FAB, [MꢄH]ꢄ) m/z 1046.
N-(2-(3,4,5-Trihydroxybenzamido)ethyl)-3,4,5-trihydroxybenzamide
(11a) To a stirred solution of 10a (306 mg, 0.338 mmol) in THF (10 ml)
and methanol (3 ml) was added 10% Pd/C (54 mg). The reaction mixture
was stirred at room temperature under a H2 atmosphere for 3 h, then filtered
1
white solid 16 (429 mg, 0.687 mmol, 49%). H-NMR (500 MHz, CDCl3) d:
7.42—7.29 (m, 17H), 5.12 (s, 4H), 5.09 (s, 2H), 4.25 (t, Jꢁ6.7 Hz, 2H), 3.38
(t, Jꢁ6.7 Hz, 2H), 1.82 (quint, Jꢁ6.7 Hz, 2H), 1.72 (quint, Jꢁ6.7 Hz, 2H),
1.39—1.27 (m, 16H). MS (FAB, [MꢄH]ꢄ) m/z 625.
1
and concentrated. 11a (120 mg, 0.329 mmol, 97%) was obtained. H-NMR
(500 MHz, DMSO-d6) d: 6.84 (s 4H), 3.50 (s 4H). HR-MS (FAB, [MꢄH]ꢄ)
Calcd for C16H17N2O8 365.0985, Found 365.0984.
12-Bromododecyl 3,4,5-Trisbenzyloxybenzoate (17) To a stirred solu-
tion of 16 (409 mg, 0.655 mmol), triphenylphosphine (352 mg, 1.34 mmol)
in THF (10 ml) was added tetrabromomethane (441 mg, 1.33 mmol). The re-
action mixture was stirred overnight, then evaporated. The residue was taken
up in water and extracted with ethyl acetate three times. The organic layer
was washed with water and brine, dried over MgSO4, filtered and concen-
trated in vacuo. The residue was purified by silica gel chromatography
(hexane : ethyl acetateꢁ15 : 1) to afford white solid 17 (400 mg, 0.582 mmol,
N-(6-(3,4,5-Trihydroxybenzamido)hexyl)-3,4,5-trihydroxybenzamide
(11b) According to the same procedure used for 11a, starting from 10b
(149 mg, 0.155 mmol), 11b (62 mg, 0.147 mmol, 95%) was obtained. 1H-
NMR (500 MHz, DMSO-d6) d: 8.98 (br, 6H), 7.99 (t, Jꢁ6.0 Hz, 2H), 6.78
(s, 4H), 3.15 (br, 4H), 1.45 (quint, Jꢁ6.5 Hz, 4H), 1.29 (br, 4H). HR-MS
(FAB, [MꢄH]ꢄ) Calcd for C20H25N2O8 421.1611, Found 421.1609.
N-(12-(3,4,5-Trihydroxybenzamido)dodecyl)-3,4,5-trihydroxybenz-
amide (11c) According to the same procedure used for 11a, starting from
10c (301 mg, 0.288 mmol), 11c (144 mg, 0.285 mmol, 99%) was obtained.
1
89%). H-NMR (500 MHz, CDCl3) d: 7.42—7.29 (m, 17H), 5.12 (s, 4H),
5.09 (s, 2H), 4.25 (t, Jꢁ6.7 Hz, 2H), 3.38 (t, Jꢁ6.7 Hz, 2H), 1.82 (quint,

194
Vol. 57, No. 2
According to the same procedure used for 20a, starting from 19c (73 mg,
0.087 mmol), 20b (23 mg, 0.048 mmol, 56%) was obtained. 1H-NMR
(500 MHz, CDCl3) d: 9.34 (br, 3H), 7.45—7.35 (m, 3H), 7.07 (d, Jꢁ7.3 Hz,
1H), 7.00 (s, 1H), 4.29 (t, Jꢁ6.7 Hz, 2H), 4.20 (t, Jꢁ6.7 Hz, 2H), 1.73
(quint, Jꢁ6.7 Hz, 2H), 1.69 (quint, Jꢁ6.7 Hz, 2H), 1.43—1.31 (m, 16H).
MS (FAB, MHꢄ) m/z 475. HR-MS (FAB, [MꢄH]ꢄ) Calcd for C26H35O8
475.2332, Found 475.2344.
12-(4-Hydroxybenzoyloxy)dodecyl 3,4,5-Trihydroxybenzoate (20c)
According to the same procedure used for 20a, starting from 19d (66 mg,
0.079 mmol), 20c (35 mg, 0.074 mmol, 93%) was obtained. 1H-NMR
(500 MHz, CDCl3) d: 10.37 (s, 1H), 9.31 (s, 2H), 8.99 (s, 1H), 7.85 (d,
Jꢁ7.3 Hz, 1H), 6.99 (s, 1H), 6.90 (d, Jꢁ7.3 Hz, 1H), 4.24 (t, Jꢁ6.7 Hz, 2H),
4.20 (t, Jꢁ6.7 Hz, 2H), 1.74—1.67 (m, 4H), 1.41—1.31 (m, 16H). MS
(FAB, MHꢄ) m/z 475. HR-MS (FAB, [MꢄH]ꢄ) Calcd for C20H35O8
475.2332, Found 475.2361.
Jꢁ6.7 Hz, 2H), 1.72 (quint, Jꢁ6.7 Hz, 2H), 1.39—1.27 (m, 16H). MS (FAB,
[MꢄH]ꢄ) m/z 688.
12-(3,4-Trisbenzyloxybenzoyloxy)dodecyl 3,4,5-Trisbenzyloxyben-
zoate (19a) To a stirred solution of 17 (81 mg, 0.119 mmol), K2CO3
(30 mg, 0.217 mmol) in DMF (10 ml) was added 18a (59 mg, 0.134 mmol).
The reaction mixture was stirred at room temperature overnight under a N2
atmosphere. Water was added and the whole was extracted with ethyl acetate
three times. The organic layer was washed with water, sat NaHCO3 aq. and
brine, dried over MgSO4, and concentrated in vacuo. The residue was puri-
fied by silica gel chromatography (hexane : ethyl acetateꢁ10 : 1) to afford
19a (110 mg, 0.105 mmol, 84%). 1H-NMR (500 MHz, CDCl3) d: 7.62—
7.29 (m 28H), 6.90 (d, Jꢁ8.55 Hz, 2H), 5.20 (s, 2H), 5.17 (s, 2H), 5.12 (s,
4H), 5.09 (s, 2H), 4.25 (t, Jꢁ6.7 Hz, 2H), 4.23 (t, Jꢁ6.7 Hz, 2H), 1.71
(quint, Jꢁ6.7 Hz, 4H) 1.38—1.24 (m, 16H). MS (FAB, [MꢄH]ꢄ) m/z 941.
12-(3-Benzyloxybenzoyloxy)dodecyl 3,4,5-Trisbenzyloxybenzoate
(19b) According to the same procedure used for 19a, starting from 17
(71 mg, 0.105 mmol), 19b (78 mg, 0.093 mmol, 89%) was obtained. 1H-
NMR (500 MHz, CDCl3) d: 7.64—7.62 (m, 2H), 7.43—7.31 (m, 20H),
7.15—7.13 (m, 2H), 5.12 (s, 4H), 5.09 (s, 2H), 5.08 (s, 2H), 4.28 (t,
Jꢁ6.7H z, 2H), 4.25 (t, Jꢁ6.7 Hz, 2H), 1.76—1.69 (m, 4H), 1.43—1.24 (m,
16H). MS (FAB, [MꢄH]ꢄ) m/z 835.
12-Benzoyloxydodecyl 3,4,5-Trihydroxybenzoate (20d) According to
the same procedure used for 20a, starting from 19e (62 mg, 0.085 mmol),
1
20d (37 mg, 0.081 mmol, 94%) was obtained. H-NMR (500 MHz, CDCl3)
d: 9.24 (s, 2H), 8.92(s, 1H), 7.94 (d, Jꢁ6.1 Hz, 2H), 7.64 (t, Jꢁ6.1 Hz, 1H),
7.52 (t, Jꢁ6.1 Hz, 2H), 6.93 (s, 2H), 4.25 (t, Jꢁ6.7 Hz, 2H), 4.13 (t,
Jꢁ6.7 Hz, 2H), 1.69 (quint, Jꢁ6.7 Hz, 2H), 1.61 (quint, Jꢁ6.7 Hz, 2H),
1.40—1.24 (m, 16H). MS (FAB, MHꢄ) m/z 459. HR-MS (FAB, [MꢄH]ꢄ)
Calcd for C26H35O7 459.2383, Found 459.2402.
12-(4-Benzyloxybenzoyloxy)dodecyl 3,4,5-Trisbenzyloxybenzoate
(19c) According to the same procedure used for 19a, starting from 17
(70 mg, 0.103 mmol), 19c (67 mg, 0.080 mmol, 78%) was obtained. 1H-
NMR (500 MHz, CDCl3) d: 7.97 (d, Jꢁ9.2 Hz, 2H), 7.42—7.31 (m, 22H),
6.96 (d, Jꢁ8.5Hz, 2H), 5.11 (s, 4H), 5.09 (s, 4H), 4.25 (t, Jꢁ6.7 Hz, 4H),
1.74—1.69 (m, 4H), 1.40—1.23 (m, 16H). MS (FAB, [MꢄH]ꢄ) m/z 835.
12-Benzoyloxydodecyl 3,4,5-Trisbenzyloxybenzoate (19d) According
to the same procedure used for 19a, starting from 17 (63 mg, 0.093 mmol),
Acknowledgment We thank K. Harata and Y. Hongo for recording
mass spectra at RIKEN. The work described in this paper was partially sup-
ported by Grants-in-Aid for Scientific Research from The Ministry of Edu-
cation, Culture, Sports, Science and Technology, Japan, and the Japan Soci-
ety for the Promotion of Science.
1
19d (64 mg, 0.088 mmol, 94%) was obtained. H-NMR (500 MHz, CDCl3)
References
d: 8.02 (d, Jꢁ7.9 Hz, 2H), 7.54—7.29 (m, 20H), 5.12 (s, 4H), 5.09 (s, 2H),
4.29 (t, Jꢁ6.7 Hz, 2H), 4.25 (t, Jꢁ6.7 Hz, 2H), 1.75—1.70 (m, 4H), 1.43—
1.24 (m, 16H). MS (FAB, [M]ꢄ) m/z 729.
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12-(3,4-Dihydroxybenzoyloxy)dodecyl 3,4,5-Trihydroxybenzoate (20a)
To a stirred solution of 19a (97 mg, 0.103 mmol), 10% Pd/C (43 mg) in
AcOEt (10 ml) was added at room temperature under a H2 atmosphere. Stir-
ring was continued for 6 h. The reaction mixture was filtered and concen-
trated. The residue was purified by silica gel chromatography (hexane : ethyl
acetateꢁ1 : 1) and recrystallized to afford white solid 20a (51 mg,
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1
0.102 mmol, 99%). H-NMR (500 MHz, DMSO-d6) d: 9.34 (br, 5H), 7.41
(s, 1H), 7.36 (d, Jꢁ8.5 Hz, 1H), 7.00 (s, 2H), 6.86 (d, Jꢁ8.5 Hz, 1H), 4.23
(t, Jꢁ6.7 Hz, 2H), 4.21 (t, Jꢁ6.7 Hz, 2H), 1.74—1.67 (m, 4H), 1.43—1.32
(m, 16H). MS (FAB, MHꢄ) m/z 491. HR-MS (FAB, [MꢄH]ꢄ) Calcd for
C26H35O9 491.2281, Found 491.2296.
12-(3-Hydroxybenzoyloxy)dodecyl 3,4,5-Trihydroxybenzoate (20b)