Synthesis and neuroprotective activity of bergenin derivatives with antioxidant activity

Article abstract of DOI:10.1016/S0968-0896(02)00666-1

Norbergenin, which is the O-demethyl derivative of bergenin, the main component of Mallotus japonicus, has been found to show moderate antioxidant activity (IC₅₀ 13 μM in DPPH radical scavenging; 32 μM in superoxide anion scavenging). Modification of sugar part on norbergenin by coupling with a variety of fatty acids was employed for increasing its antioxidant activity. Selective esterification of hydroxyl groups on the sugar part enhanced greatly antioxidant activity. The most potent one is norbergenin 11-caproate, which not only exhibits stronger antioxidant activity than that of catechin but also prevents neuronal death at 10 μM on the primary culture of rat cortical neurons in DMEM supplemented with N2.

Full text of DOI:10.1016/S0968-0896(02)00666-1

Bioorganic & Medicinal Chemistry 11 (2003) 1781–1788
Synthesis and Neuroprotective Activity of Bergenin
Derivatives with Antioxidant Activity
Hironobu Takahashi, Masamiti Kosaka, Yasutoshi Watanabe,
Kousuke Nakade and Yoshiyasu Fukuyama*
Institute of Pharmacognosy, Faculty of Pharmaceutical Sciences, Tokushima Bunri University,
Yamashiro-cho, Tokushima 770-8514, Japan
Received 29 October 2002; accepted 13 December 2002
Abstract—Norbergenin, which is the O-demethyl derivative of bergenin, the main component of Mallotus japonicus, has been found
to show moderate antioxidant activity (IC50 13 mM in DPPH radical scavenging; 32 mM in superoxide anion scavenging). Modifi-
cation of sugar part on norbergenin by coupling with a variety of fatty acids was employed for increasing its antioxidant activity.
Selective esterification of hydroxyl groups on the sugar part enhanced greatly antioxidant activity. The most potent one is norber-
genin 11-caproate, which not only exhibits stronger antioxidant activity than that of catechin but also prevents neuronal death at
10 mM on the primary culture of rat cortical neurons in DMEM supplemented with N2.
# 2003 Elsevier Science Ltd. All rights reserved.
Introduction
antioxidant properties. Bergenin (1) is the main compo-
nent of the bark of Mallotus japonicus, which has been
used as a traditional drug for treatment of hyperacid
and a stomach ulcer in Japan. In addition, it has been
reported that bergenin has anti-HIV activity,11 and
antiarrhythmic,12 hepatoprotective13 and antiin-
flammatory effects.14 Since norbergenin (2) can be
regarded as a C-glucoside of a potent antioxidant gallic
acid, we have envisaged that 2 is a natural source sui-
table for developing effective antioxidants. Thus, com-
pound 2 readily available from 1 is modified by
coupling with a variety of fatty acids on different posi-
tion of its sugar part. In this paper, we describe the
synthesis of bergenin derivatives and the relationship
between their structures and antioxidant activity, as well
as their neuroprotective activities on a primary culture
of fetal rat cortical neurons in DMEM supplemented
with N2 (Fig. 1).
Various reactive oxygen species (ROS), such as the
hydroxyl radical and superoxide anion radical, has been
known to induce lipid peroxidation as well as to damage
membranes, proteins and DNA. Enzymatic and non-
enzymatic antioxidative defense systems can remove
ROS under normal conditions. Oxidative stress, how-
ever, occurring when antioxidant systems are inade-
quate and/or active oxygen species are overproduced,
can damage the tissues and DNA, thus resulting in a
progression of a number of human diseases such as
atheriosclerosis,1 diabetes, inflammation,2 Alzheimer’s
disease3 and senescence. Hence, antioxidants are expec-
ted to be promising drugs for treatment of these diseases
by removing oxidative stress.
In our antioxidative screening program of natural pro-
ducts using free radical scavenging activity of the a,a-
diphenyl-b-picrylhydrazyl radical (DPPH)4 and super-
oxide anion (OÀ2 ) scavenging activity,5 we have already
reported some antioxidant natural products.6À8 Further,
we have found that norbergenin (2)9 which is the
O-demethyl derivative of bergenin (1)10 show moderate
Results and Discussion
Synthesis of norbergenin derivatives
Bergenin (1), readily obtained in 22.5% yield from
MeOH extract of the bark of M. japonicus, was con-
verted to norbergenin (2) by acetylation with acetic
anhydride and deprotection of the O-methyl group with
*Corresponding author. Tel.: +81-88-622-9611; fax: +81-88-655-
3051; e-mail: fukuyama@ph.bunri-u.ac.jp
0968-0896/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0968-0896(02)00666-1

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H. Takahashi et al. / Bioorg. Med. Chem. 11 (2003) 1781–1788
BCl3 followed by hydrolysis with K2CO3 in 71% over
three steps. The phenolic hydroxyl groups in 2 were
selectively benzylated with benzyl bromide under the
basic conditions to give 3. In order to increase lipophilic
properties of 2, hydroxyl groups on the sugar part of 3
were esterified with various chain lengths of fatty acids.
The acids with the chain lengths of n=0and n=2 were
coupled with acetic anhydride and butyric anhydride in
pyridine to give triacetate and tributyrate, respectively.
The fatty acids with the chain lengths of n=4, 6, 14 and
16 were coupled with 1,3-dicyclohexylcarbodiimide
(DCC) and fatty acid with corresponding chain length
to give triacylate. Finally, hydrogenation of triacylate
led to a series of triacylated norbergenins (4a–4f)
(Scheme 1). Among triacylated derivatives 4a–4f, nor-
bergenin 11-caproate (4c) was found to significantly
enhance antioxidant activity.
Since compound 4c with caproyl ester (n=4) showed
the most potent antioxidant activity, regioselective
monoesterfication of
2 with hexanoic acid was
employed to know which hydroxyl group on the sugar
part is essential for increasement of antioxidant activity
as outlined in Scheme 2. Esterification of the hydroxyl
group at C-11 was accomplished by coupling 3 with
DCC and hexanoic acid (1.5 equiv) followed by hydro-
genation with Pd/C to gave 5. On the other hand, com-
pound 6 having hexanoic acid ester on the C-3 position
was obtained from acid hydrolysis of 4c. Compound 7
esterified on the C-4 position was obtained as follows:
both the hydroxyl groups at C-11 and C-3 in 3 were
protected as an acetal group with p-anisaldehyde-
dimethylacetal and pyridinium p-toluenesulfonate
(PPTS), and then the free hydroxyl group at C-4 was
Figure 1. Structure of bergenin derivatives.
Scheme 1. Reagents and conditions: (a) (CH3CO)2O, Py, 96%; (b) BCl3, CH2Cl2; (c) 10% K2CO3, two steps 74%; (d) K2CO3, BnBr, DMF/acetone,
66%; (e) (i) (CH3CO)2O, Py; or (ii) CH3(CH2)nCOOH, DCC, DMAP, Py; (f) H2/Pd/C, EtOH/CH2Cl2.

H. Takahashi et al. / Bioorg. Med. Chem. 11 (2003) 1781–1788
1783
Scheme 2. Reagents and conditions: (a) 1.5 equiv CH3(CH2)4COOH, DCC, DMAP, py; (b) H2/Pd/C, EtOH/CH2Cl2; (c) HCl, MeOH, 28%; (d)
p-anisaldehyde dimethylacetal, PPTS, CH2Cl2, 89%; (e) CH3(CH2)4COOH, EDCl, DMAP, py, 82%; (f) H2/Pd/C, EtOH/CH2Cl2, 72%.
coupled with 1-ethyl-3-(3-dimethylaminopropyl)-carbo-
diimide (EDCl) and hexanoic acid, finally, all of the
benzyl groups and the p-methoxy denzilidene were
removed by hydrogenation, to giving rise to 7.
scavenging activity (4a–4c) is tending to be enhanced in
parallel with the DPPH radical scavenging activity.
Compounds (4d–4f) with the alkyl chains higher than
n=6 failed to be tested due to poor solubility in water.
On the other hand, caproates (5–7) showed high com-
parable activity regardless of the esterified positions of
hydroxyl groups. Judging from these results, the modi-
fication of the primary hydroxyl group on the C-11
Free radical scavenging activity
The DPPH radical scavenging activity of bergenin deri-
vatives (4–7) is summarized in Table 1. First of all, it
should be emphasized that the radical scavenging degree
for O-demethylated compound 2 is enhanced seventy
times more than that of bergenin (1). Compound 4c and
4d, which bear ester groups with C6 and C8 alkylating
chains, respectively, showed the highest activity among
a series of tri-ester derivatives (4a–4f) and their poten-
cies were comparable to that of catechin as a positive
standard compound. These results clearly indicate that
hexanoic acid is appropriate for a modification of the
sugar part in 2. Next, our attention has been directed to
which hydroxyl group on the sugar part is significance
for being esterified with hexanoic acid. Compound 5,
having caproyl group on the C-11 position was found to
show most potent activity, whereas 6 and 7 having the
same ester on the C-3 and C-4 position respectively
decreased the DPPH radical scavenging activities
slightly than 4c.
Table 1. Antioxidant activity of bergenin derivatives (1–7)
IC50 (mM)a
DPPH radicalb
O2À
c
1
2
4a
4b
4c
921
13
32
12
9
NTd
32
72
62
44
4d
4e
4f
9
NTd
NTd
NTd
3
25
25
8
5
6
15
16
15
3
2
2
7
Catechin
aInhibitory activity was expressed as the mean of 50% inhibitory con-
centration of triplicate determination, obtained by interpolation of
concentration-inhibition curves.
bChemically stable radical scavenging activity [a,a-diphenyl-b-picryl-
hydrazyl (DPPH) radical].
The superoxide anion radical scavenging activity also
were examined (Table 1). As the number of the methyl-
ene group is increasing, the superoxide anion radical
cSuperoxide anion scavenging activity.
dNot tested due to poor solubility.

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H. Takahashi et al. / Bioorg. Med. Chem. 11 (2003) 1781–1788
position in norbergenin (2) with appropriate groups
allows to provide a potential way for developing a pro-
mising antioxidant agent.
The three norbergenin derivatives 2, 4c and 5, the
stronger antioxidants than the others, were evaluated
for neuroprotective effect. The primary cultures per-
formed in DMEM medium23 supplemented with N2
was used for this purpose and all viability of rat cortical
neurons was measured by the WST-8 reduction assay.
We have already established an assay system24 to pri-
marily evaluate neuroprotective effect of compounds by
using the primary culture of rat cortical neurons plated
at cell density 2Â105 cells cmÀ2 in serum-free medium
supplemental with N2, the culture conditions of which
naturally can cause neuronal death within 5 days. As
shown in Figure 2, compounds 4c and 5 enhanced cell
viability at 1 and 10 mM, respectively. But, lower con-
centration did not show positive effect on neuronal sur-
vival. Morphological evaluations as shown in Figure 3,
the control culture (a) shows a small number of neu-
rons, but the cultures (b) and (c), in the presence of 4c
and 5, increased the number of survival neurons. On the
other hand, 2 showed no effect on neuronal survival at
concentration of 0.01–10 mM. However, neuroprotective
activity of 4c and 5 is not dose-dependent and seems to
be limited to a narrow range of concentration. We have
not figured out why their effective dose-range is so
Neuroprotective activity
The brain consumes about 20% of oxygen in body, and
the generation of ROS in brain is assumed to be so
much. Therefore, the ROS plays some risky roles in a
progression of many neurodegenerative pathologies of
the central nervous system such as Parkinson’s and
Alzheimer’s diseases. It should be noted that the search
and synthesis for efficient antioxidants can provide a
defensive system against a variety of diseases caused by
a ROS. In a previous paper,15 we reported that masti-
gophorenes A and B, which was isolated from Masti-
gophora diclados,16 showed not only potent antioxidant
activity but also neuroprotective effect on neuronal
death in the primary culture of fetal rat cortical neu-
rons. Additionally, some antioxidants such as a-toco-
pherol,17 1,4-benzoxazines,18À20 flavanoid glycosides21
and a triterpene ester22 were reported to also show neu-
roprotective activity. Thus, the antioxidants appear to
have close relation with neuroprotective effect.
Figure 2. Cell viability of rat cortical neurons enhanced by compounds 2, 4c and 5 in primary clutures. Effects of survival were assessed by the WST-
8 reduction assay. The data are expressed as means SE (n=6); **p<0.01 versus control.
Figure 3. Enhancement of survival of rat cortical neurons by bergenin derivatives in primary cultures. After the neuronal cells (2Â105 cells/cm2)
cultured for 3 days in the presence of 0.5% EtOH and 5c and 6 were fixed by 4% paraformaldehyde-PBS, the immunohistochemical staining for the
microtuble associated protein-2 (MAP-2) were performed. Pictures taken with 100 magnifications. (a) 0.5% EtOH, (b) 10 mM 5c, and (c) 1 mM 6.

H. Takahashi et al. / Bioorg. Med. Chem. 11 (2003) 1781–1788
1785
narrow. These results suggest that increase of lipophilic
property of norbergenin (2) can enhance antioxidant
activity, which can be further related to protecting neu-
rons from damage by ROS.25
in vacuo to give pentaacetate bergenin (15.9 g, 96%).
The obtained residue (4.4 g, 8.4 mmol) was dissolved in
CH2Cl2 (100 mL) and then BCl3 (1.0M in CH 2Cl2,
30mL, 30mmol) was added at 0 ꢀC and stirred for 1 h.
After stirring at room temperature for 24 h, the reaction
mixture was poured into an ice-water. The organic sol-
vent was removed in vacuo, and then the water layer
added to 10% K2CO3 solution, and stirred for 1 h. The
reaction mixture was acidified with 2 M HCl, and then
water layer absorbed with HP-20(500mL). The fraction
eluted with MeOH was concentrated in vacuo, and then
residue purified by recrystallization to afford 2 (1.89 g,
74%) as a white powder. Mp 250 ꢀC (dec.); HR-EIMS
m/z 314.0635 (calcd 314.0638 for C13H14O9); IR 3385,
Conclusion
We have synthesized a series of bergenin derivatives
aimed at increase of antioxidant activity and lipophilic
properties. Norbergenin 11–caprorate (5) showed not
only the highest antioxidant activity but also could pre-
vent neuronal death on the primary culture of rat cor-
tical neurons. These results indicated that the
modification of the primary hydroxyl group at the C-11
position in norbergenin with fatty acid made a con-
tribution to increase antioxidant activity. As we have
found relationship between antioxidant activity and
neuroprotective activity in norbergenin derivatives,
synthetic studies of norbergenin derivatives, the C-11
hydroxyl group of which is modified with various fatty
acids and/or other functional groups, will continue to
exploit more efficient and potent neuroprotective agent.
Moreover, our studies are under way to clarify neuro-
protective profile of 4c and 5 against neuronal death
caused by reactive oxygen species.
1
1707, 1622, 1477, 1325 cmÀ1; H NMR (Py-d5) d 4.20
(3H, m), 4.46 (1H, dd, J=8.4, 9.5 Hz), 4.60(1H, dd,
J=9.5, 10.3 Hz), 4.64 (1H, dd, J=5.1, 10.3 Hz), 5.25
(1H, d, J=10.3 Hz), 7.86 (1H, s).
Tribenzyl norbergenin (3). To a solution of 2 (515 mg,
1.64 mmol) in DMF (6 mL) and acetone (6 mL) was
added K2CO3 (1.13 g, 8.2 mmol) and benzyl bromide
(0.98 mL, 8.2 mmol) at 0 ꢀC. After stirring at room tem-
perature for 24 h, the reaction mixture was diluted with
water and extracted with EtOAc. The combined organic
layers were washed with brine, dried over MgSO4 and
concentrated in vacuo. The residue was chromato-
graphed on silica gel to afford 3 (635 mg, 66%) as a white
powder. Mp 160 ꢀC; HR-FABMS m/z 585.2114 (calcd
585.2100 for C32H34O9Na); IR 3335, 1734, 1593, 1444,
Experimental
General
1
1332 cmÀ1; H NMR (Py-d5) d 4.07 (1H, m), 4.43 (2H,
m), 4.52 (2H, m), 4.62 (1H, dd, J=9.2, 9.7 Hz), 5.08 (1H,
d, J=9.7 Hz), 5.20(2H, s), 5.24 (1H, d, J=10.6 Hz), 5.29
(1H, d, J=10.6 Hz), 5.39 (1H, d, J=10.6 Hz), 5.44 (1H,
d, J=10.6 Hz), 7.30–7.43 (9H, m), 7.51–7.76 (7H, m).
1
IR spectra were recorded on Jasco 5300 FT-IR. H and
13C NMR spectra were taken on a JEOL ECP-400
spectrometer. Chemical shifts are expressed in d unit
(part per million downfield from tetramethylsilane.
Mass spectra (MS) were recorded on a JEOL AX-500.
Column chromatography was carried out on Kiselgel 60
(70–230, 230–400 mesh), Wakogel C-300, Sephadex LH-
20and DIAION HP-20. Analytical thin-layer chroma-
tographies were performed with Merck precoated TLC
plates, and spots were visualized with ultraviolet light
and CeSO4/H2SO4.
General method of preparation of triacyl norbergenin
Method A. To a solution of 3 (57 mg, 0.098 mmol) in
pyridine (2 mL) was added acetic anhydride (1 mL), and
the mixture was stirred for 12 h. The reaction mixture
was diluted with water, extracted EtOAc and washed
with Cu(NO3)2 solution and brine, dried over MgSO4
and concentrated in vacuo to give triacetate. A solution
of triacetate in EtOH (2 mL) and CH2Cl2 (1 mL) con-
taining palladium carbon (Pd/C, Pd: 10%, 15 mg) was
stirred under an atmospheric pressure of hydrogen for
12 h. The reaction mixture was filtrated, concentrated,
and the residue was purified by column chromato-
graphy (n-hexane/EtOAc=2:1) to afford 4a (43 mg,
100%). In the same manner, 4b was prepared.
Plant material. The bark of M. japonicus were pur-
chased from Nakai Koshindo (Kobe, Japan).
Bergenin (1). The dried powder bark of M. japonicus
(10kg) were extracted with MeOH at room temperature
to give 410g of extract. The extract (24 g) was chroma-
tographed on silica gel (C-300) eluted successively
CHCl3, CHCl3/MeOH (9:1), CHCl3/MeOH (4:1) and
CHCl3/MeOH (1:1) to yield five fractions. Fraction
third were subjected to column chromatography on
Sephadex LH-20and eluted with MeOH to give ber-
genin (5.35 g).
Method B. To a solution of 3 (101 mg, 0.17 mmol) in
pyridine
(3 mL)
was
added
1,3-dicyclohexyl-
carbodiimide (DCC) (206 mg, 1.0 mmol) and 4-di-
methyl- aminopyridine (DMAP) (122 mg, 1 mmol). To
this stirring mixture was added hexanoic acid (0.13 mL,
1.0mmol). The reaction mixture was added, stirring at
room temperature for 12 h. The reaction mixture was
diluted with water, washed with water, Cu(NO3)2 solu-
tion and brine, and dried over MgSO4 and concentrated
in vacuo. The residue was chromatographed on silica
gel to afford tricaproate. The crude product obtained
Norbergenin (2). To a solution of bergenin (10g,
30.8 mmol) in pyridine (20 mL), acetic anhydride
(20mL) was added, and the mixture was stirred for 24 h.
The reaction mixture was poured into water and extrac-
ted with EtOAc. The combined organic layers were
washed with brine, dried with MgSO4, and concentrated

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H. Takahashi et al. / Bioorg. Med. Chem. 11 (2003) 1781–1788
after evaporation of the solvent was subjected to the
same hydrogenation as Method A, and then the residue
was purified by column chromatography (n-hexane/
EtOAc=6:1) to afford 4c (144 mg, 100%). In the same
manner, 4d–4f were prepared.
3418, 1745, 1620, 1467, 1315 cmÀ1; 1H NMR (CDCl3) d
0.88 (9H, t, J=7.0Hz), 1.24–1.26 (84H, m), 1.59–1.66
(6H, m), 2.30–2.40 (6H, m), 3.99 (1H, m, H-2), 4.11
(1H, dd, J=6.2, 12.5 Hz, H-11), 4.29 (1H, dd, J=10.3,
10.6 Hz, H-4a), 4.49 (1H, d, J=12.5 Hz, H-11), 4.96
(1H, d, J=10.6 Hz, H-10b), 5.09 (1H, dd, J=9.5,
9.9 Hz, H-3), 5.51 (1H, dd, J=9.5, 10.3 Hz, H-4), 7.31
(1H, s, H-7).
Norbergenin 3,4,11-triacetate (4a). HR-FABMS m/z
463.0878 (calcd 463.0877 for C19H21O12Na); IR 3409,
1
1730, 1622, 1528, 1478, 1318 cmÀ1; H NMR (CDCl3)
d 2.09 (3H, s, Ac), 2.10 (3H, s, Ac), 2.12 (3H, s, Ac),
4.03 (1H, dd, J=5.3, 9.7 Hz, H-2), 4.16 (1H, dd,
J=5.3, 12.6 Hz, H-11), 4.30(1H, dd, J=9.2, 10.2 Hz,
H-4a), 4.44 (1H, d, J=12.6 Hz, H-11), 4.98 (1H, d,
J=10.2 Hz, H-10b), 5.08 (1H, dd, J=9.7, 9.7 Hz,
H-3), 5.47 (1H, dd, J=9.2, 9.7 Hz, H-4), 7.28 (1H, s,
H-7).
Preparation of regioselective esterification of norbergenin
Norbergenin 11–caproate (5). To a solution of 3
(500 mg, 0.86 mmol) in pyridine (20 mL) was added
hexanoic acid (0.16 mL, 1.29 mmol), DCC (530 mg,
2.57 mmol) and DMAP (314 mg, 2.57 mmol). The mix-
ture was stirred at room temperature for 12 h. The
reaction mixture was diluted with water, washed with
water, Cu(NO3)2 solution and brine, and dried over
MgSO4. Evaporation of the solvent gave a crude pro-
duct, which was chromatographed on silica gel (CHCl3/
MeOH=24:1) to afford monoester (105 mg, 18%) and
starting material 3 (129 mg, 30%). A solution of
monoester (105 mg, 0.15 mmol) in ethanol (2 mL) and
CH2Cl2 (2 mL) containing palladium carbon (Pd/C, Pd:
10%, 15 mg) was stirred under an atmospheric pressure
of hydrogen for 24 h. The reaction mixture was filtrated,
concentrated, and the residue was purified by column
chromatography (CHCl3/MeOH=24:1) to afford 5
(27 mg, 42%). Mp 175 ꢀC; HR-FABMS m/z 413.1399
(calcd 413.1447 for C19H25O10); IR 3384, 1719, 1618,
Norbergenin 3,4,11-tributyrate (4b). HR-FABMS m/z
547.1810(calcd 547.1791 for C 25H32O12Na); IR 3420,
1
1738, 1622, 1524, 1478 cmÀ1; H NMR (CDCl3) d 0.94
(6H, t, J=7.3 Hz), 0.97 (3H, t, J=7.3 Hz), 1.61 (4H, q,
J=7.3 Hz), 1.69 (2H, q, J=7.3 Hz), 2.28–2.37 (6H, m),
4.03 (1H, dd, J=6.2, 9.5 Hz, H-2), 4.13 (1H, dd, J=6.2,
12.4 Hz, H-11), 4.29 (1H, dd, J=10.3, 10.6 Hz, H-4a),
4.47 (1H, d, J=12.4 Hz, H-11), 5.00 (1H, d, J=10.6 Hz,
H-10b), 5.12 (1H, dd, J=9.5, 9.5 Hz, H-3), 5.52 (1H,
dd, J=9.5, 10.3 Hz, H-4), 7.28 (1H, s, H-7).
Norbergenin 3,4,11-tricaproate (4c). Mp 190 ꢀC; HR-
FABMS m/z 631.2708 (calcd 631.2731 for
C31H44O12Na); IR 3406, 1743, 1620, 1469, 1311 cmÀ1
1H NMR (CDCl3) d 0.86 (3H, t, J=7.0Hz), 0.88 (3H, t,
J=7.7 Hz), 0.90 (3H, t, J=6.6 Hz), 1.21–1.30(12H, m),
;
1509, 1466, 1307 cmÀ1; H NMR (Py-d5) d 0.72 (3H, t,
1
J=6.6 Hz), 1.13 (4H, m), 1.57 (2H, m), 2.34 (2H, m),
4.09 (1H, dd, J=8.8, 9.2 Hz, H-3), 4.25 (1H, td, J=8.1,
9.2 Hz, H-2), 4.43 (1H, dd, J=8.8, 9.5 Hz, H-4), 4.57
(1H, dd, J=9.5, 10.3 Hz, H-4a), 4.61 (1H, dd, J=8.1,
11.7 Hz, H-11), 5.17 (1H, dd, J=8.1, 11.7 Hz, H-11),
5.25 (1H, d, J=10.3 Hz, H-10b), 7.86 (1H, s, H-7).
1.58–1.66 (6H, m), 2.29–2.43 (6H, m), 4.03 (1H, ddd,
J=2.0, 6.2, 9.9 Hz, H-2), 4.11 (1H, dd, J=6.2, 12.1 Hz,
H-11), 4.30(1H, dd, J=10.3, 10.6 Hz, H-4a), 4.47 (1H,
dd, J=2.0, 12.1 Hz, H-11), 5.01 (1H, d, J=10.6 Hz, H-
10b), 5.11 (1H, dd, J=9.5, 9.9 Hz, H-3), 5.52 (1H, dd,
J=9.5, 10.3 Hz, H-4), 7.29 (1H, s, H-7).
Norbergenin 3–caproate (6). To a solution of 4c (46 mg,
0.075 mmol) in MeOH (3 mL) was added 1 M HCl
(0.3 mL) and the mixture was refluxed for 4 h. The
reaction mixture was concentrated, and the residue was
purified by column chromatography (CHCl3/
MeOH=24:1) to afford 6 (11 mg, 28%). Mp 165 ꢀC;
HR-FABMS m/z 413.1419 (calcd 413.1447 for
Norbergenin 3,4,11-tricaprylate (4d). Mp 198 ꢀC; HR-
FABMS m/z 715.3643 (calcd 715.3670for
C37H56O12Na); IR 3256, 1732, 1597, 1467, 1358 cmÀ1
;
1H NMR (CDCl3) d 0.89 (9H, t, J=7.0Hz), 1.24–1.29
(24H, m), 1.59–1.66 (6H, m), 2.26–2.42 (6H, m), 4.01
(1H, ddd, J=1.8, 5.9, 9.9 Hz, H-2), 4.10(1H, dd,
J=5.9, 12.8 Hz, H-11), 4.30(1H, dd, J=9.5, 10.6 Hz,
H-4a), 4.50(1H, dd, J=1.8, 12.8 Hz, H-11), 4.97 (1H, d,
J=10.6 Hz, H-10b), 5.11 (1H, dd, J=9.5, 9.9 Hz, H-3),
5.52 (1H, dd, J=9.5, 9.5 Hz, H-4), 7.31 (1H, s, H-7).
1
C19H25O10); IR 3387, 1719, 1618, 1476, 1317 cmÀ1; H
NMR (Py-d5) d 0.75 (3H, t, J=6.6 Hz), 1.18 (4H, m),
1.60(2H, m), 2.38 (2H, m), 4.90 (1H, dd,
J=9.9,
12.1 Hz, H-11), 4.23 (1H, dd, J=2.2, 12.1 Hz, H-11),
4.30(1H, dd, J=8.7, 9.9 Hz, H-2), 4.58 (1H, dd, J=9.2,
9.5 Hz, H-4), 4.66 (1H, dd, J=9.5, 10.3 Hz, H-4a), 5.31
(1H, d, J=10.3 Hz, H-10b), 5.63 (1H, dd, J=8.8,
9.2 Hz, H-3), 7.86 (1H, s, H-7).
Norbergenin 3,4,11-tripalmitate (4e). HR-FABMS m/z
1051.7470 (calcd 1051.7426 for C61H104O12Na); IR 3408,
1
1745, 1620, 1467, 1313 cmÀ1; H NMR (CDCl3) d 0.88
(9H, t, J=7.0Hz), 1.26 (72H, m), 1.59–1.66 (6H, m),
2.30–2.40 (6H, m), 4.01 (1H, dd, J=5.9, 9.9 Hz, H-2),
4.11 (1H, dd, J=5.9, 12.4 Hz, H-11), 4.29 (1H, dd, J=9.9,
10.6 Hz, H-4a), 4.48 (1H, d, J=12.4 Hz, H-11), 4.96 (1H,
d, J=10.6 Hz, H-10b), 5.09 (1H, dd, J=9.5, 9.9 Hz, H-3),
5.51 (1H, dd, J=9.5, 9.9 Hz, H-4), 7.31 (1H, s, H-7).
Norbergenin 4–caproate (7). To a solution of 3 (103 mg,
0.18 mmol) in CH2Cl2 (3 mL) was added p-anisaldehyde
dimethyl acetal (0.30 mL, 1.76 mmol) and pyridinium p-
toluenesulfonate (PPTS, 89 mg, 0.35 mmol). The reaction
mixture was stirred for 24 h. The reaction mixture was
diluted with water, extracted with EtOAc, washed water
and brine, dried MgSO4, and concentrated in vacuo. The
residue was purified by column chromatography (CHCl3/
MeOH=24:1) to afford an acetal compound (110mg,
Norbergenin 3,4,11-tristearate (4f). HR-FABMS m/z
1135.8310(calcd 1135.8367 for C 67H116O12Na); IR

H. Takahashi et al. / Bioorg. Med. Chem. 11 (2003) 1781–1788
1787
89%). To a solution of the acetal compound (110mg,
0.16 mmol) in pyridine (2 mL) was added hexanoic acid
(0.12 mL, 0.93 mmol), EDC (180 mg, 0.94 mmol) and
DMAP (114 mg, 0.94 mmol). The mixture was stirred at
room temperature for 24 h. The reaction mixture diluted
with water, washed with water, Cu(NO3)2 solution and
brine, and dried over MgSO4. Evaporation of the solvent
gave a crude product, which was chromatographed on
silica gel (CHCl3/EtOAc=49:1) to afford monoester
(102 mg, 82%). A solution of the monoester (17 mg,
0.02 mmol) in ethanol (1 mL) and CH2Cl2 (1 mL) con-
taining palladium carbon (Pd/C, Pd: 10%, 5 mg) was
stirred under an atmospheric pressure of hydrogen for
24 h. The reaction mixture was filtrated, concentrated,
and the residue was purified by column chromatography
(EtOAc) to afford 7 (6 mg, 72%). Mp 168 ꢀC; HR-
FABMS m/z 413.1419 (calcd 413.1447 for C19H25O10);
counting kit-8 (Dojindo Co. Ltd.)29 according to the
manufacture’s procedure.
Acknowledgements
The authors thank Ms. Ikuko Okamoto for measuring
MS spectra. This work is supported in a part by a
Grant-in Aid for Scientific Research (No. 12480175)
from the Ministry of Education, Culture, Sports,
Science, and Technology of Japan, and the Open
Research Center Fund from the Promotion and Mutual
Aid Corporation for Private School of Japan.
References and Notes
1
IR 3393, 1718, 1617, 1522, 1477, 1319, 1089 cmÀ1; H
NMR (CD3OD) d 0.92 (3H, t, J=6.8 Hz), 1.37 (4H, m),
1.68 (2H, m), 2.43 (2H, t, J=7.7 Hz), 3.62 (1H, dd,
J=9.2, 9.2 Hz, H-3), 3.73 (2H, m, H-2 and 11), 4.02 (1H,
d, J=9.2 Hz, H-11), 4.23 (1H, dd, J=9.7, 10.2 Hz, H-4a),
5.05 (1H, d, J=10.2 Hz, H-10a), 5.37 (1H, dd, J=9.2,
9.7 Hz, H-4), 7.07 (1H, s, H-7).
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