Synthesis and antitumor evaluation of arctigenin derivatives based on antiausterity strategy

Article abstract of DOI:10.1016/j.ejmech.2012.11.031

A series of new (-)-arctigenin derivatives with variably modified O-alkyl groups were synthesized and their preferential cytotoxicity was evaluated against human pancreatic cancer cell line PANC-1 under nutrient-deprived conditions. The results showed that monoethoxy derivative 4i (PC₅₀, 0.49 μM), diethoxy derivative 4h (PC₅₀, 0.66 μM), and triethoxy derivative 4m (PC₅₀, 0.78 μM) showed the preferential cytotoxicities under nutrient-deprived conditions, which were identical to or more potent than (-)-arctigenin (1) (PC₅₀, 0.80 μM). Among them, we selected the triethoxy derivative 4m and examined its in vivo antitumor activity using a mouse xenograft model. Triethoxy derivative 4m exhibited also in vivo antitumor activity with the potency identical to or slightly more than (-)-arctigenin (1). These results would suggest that a modification of (-)-arctigenin structure could lead to a new drug based on the antiausterity strategy.

Full text of DOI:10.1016/j.ejmech.2012.11.031

European Journal of Medicinal Chemistry 60 (2013) 76e88
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European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and antitumor evaluation of arctigenin derivatives based on
antiausterity strategy
Naoki Kudou a, Akira Taniguchi b, Kenji Sugimoto a, Yuji Matsuya a, Masashi Kawasaki c,
,
d
,
e
f
g
e
Naoki Toyooka b , Chika Miyoshi , Suresh Awale , Dya Fita Dibwe , Hiroyasu Esumi ,
*
Shigetoshi Kadota g, Yasuhiro Tezuka g
,
**
a Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama, 930-0194, Japan
b Graduate School of Science and Technology for Research, University of Toyama, 3190 Gofuku, Toyama 930-8555, Japan
c Department of Liberal Arts and Sciences, Faculty of Engineering, Toyama Prefectural University, 5180 Kurokawa, Kosugi-Machi, Toyama 939-0398, Japan
d Graduate School of Innovative Life Science, University of Toyama, 3190 Gofuku, Toyama 930-8555, Japan
e Cancer Physiology Project, Research Center for Innovative Oncology, National Cancer Center Hospital East, Chiba, Japan
f Frontier Research Core for Life Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan
g Institute of Natural Medicine, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of new (ꢀ)-arctigenin derivatives with variably modified O-alkyl groups were synthesized and
Received 24 July 2012
Received in revised form
14 September 2012
Accepted 21 November 2012
Available online 28 November 2012
their preferential cytotoxicity was evaluated against human pancreatic cancer cell line PANC-1 under
nutrient-deprived conditions. The results showed that monoethoxy derivative 4i (PC50, 0.49
diethoxy derivative 4h (PC50, 0.66 M), and triethoxy derivative 4m (PC50, 0.78 M) showed the pref-
erential cytotoxicities under nutrient-deprived conditions, which were identical to or more potent than
M). Among them, we selected the triethoxy derivative 4m and examined
mM),
m
m
(ꢀ)-arctigenin (1) (PC50, 0.80
m
its in vivo antitumor activity using a mouse xenograft model. Triethoxy derivative 4m exhibited also
in vivo antitumor activity with the potency identical to or slightly more than (ꢀ)-arctigenin (1). These
results would suggest that a modification of (ꢀ)-arctigenin structure could lead to a new drug based on
the antiausterity strategy.
Keywords:
(ꢀ)-Arctigenin derivatives
Antiausteric activity
Synthesis
Ó 2012 Elsevier Masson SAS. All rights reserved.
Pancreatic cancer
1. Introduction
with 5-fluorouracil and gemcitabine is also used for palliative
therapy of advanced pancreatic cancer. However pancreatic cancer
Pancreatic cancer is the most aggressive cancer of all and has an
exceptionally high global mortality rate, with an estimated 267,000
deaths worldwide in 2008. It ranks 8th or 9th as the most frequent
cause of cancer death worldwide and is the 4th or 5th most frequent
cause of cancer death in most developed countries, including the
United States, Europe, and Japan [1]. Moreover, it has been estimated
that the number of deaths from pancreatic cancer will reach
484,000 by 2030 [1]. Pancreatic cancer rapidly metastases and lead
the patients to die in a short period of the diagnosis. Thus, the 5-year
survival rate of the patients with the pancreatic cancer is the lowest
among several cancers [2,3]. Though surgery is the only treatment
method that offers any prospect of potential cure, chemotherapy
is largely resistant to most known chemotherapeutic agents
including 5-fluorouracil and gemcitabine [4]. Therefore effective
chemotherapeutic agents that target pancreatic cancer are urgently
needed.
Tumor cells, in general, proliferate very fast, and the demand for
essential nutrients, oxygen, etc. is always high. The immediate envi-
ronment of cancers increasing in size, however, often becomes
heterogeneous and some regions of large cancers often possess
microenvironmental niches, which exhibit a significant gradient of
critical metabolites including oxygen, glucose, other nutrients, and
growth factors [5]. Thus, manycancer cells get the critical metabolites
by randomly recruiting new blood vessels, a phenomenon commonly
known as angiogenesis, to survive under such severe conditions.
However, human pancreatic cancer survives with an extremely poor
blood supplyand becomes more malignant [6]. The method by which
pancreatic cancer survives is by getting a remarkable tolerance to
extreme nutrient starvation [7]. Therefore, it has been hypothesized
that eliminating the tolerance of cancer cells to nutrition starvation
* Corresponding author. Graduate School of Science and Technology for Research,
University of Toyama, 3190 Gofuku, Toyama 930-8555, Japan. Tel.: þ8176 445 6859.
** Corresponding author. Tel.: þ81 76 434 7627; fax: þ81 76 434 5059.
E-mail addresses: toyooka@eng.u-toyama.ac.jp (N. Toyooka), tezuka@inm.u-
toyama.ac.jp (Y. Tezuka).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.11.031

N. Kudou et al. / European Journal of Medicinal Chemistry 60 (2013) 76e88
77
may allow a novel biochemical approach known as “anti-austerity”
for cancer therapy [8].
3,4-Dihydroxybenzaldehyde was converted to the alcohol 7 via
known benzyl ether 5 [13] and aldehyde 6 [14]. Mono-alkylation of
diethyl malonate with the mesylate of 7 afforded the ester 8.
Reduction of 8 and lipase-mediated transesterification of the
resulting diol provided the mono-acetate (þ)-9. The enantiomeric
excess of (þ)-9 was determined to be 98% ee by the HPLC analysis
using the chiral column (Chiralcel OJ). The absolute stereochem-
istry of (þ)-9 was determined by the comparison of the optical
rotation with known lactone 13a, prepared from (þ)-9 via mesylate
10, benzyl ether 11, and lactone 12a as shown in Scheme 2. Other
lactones 13bef were also prepared from (þ)-9, and these lactones
In this regard, we screened 500 medicinal plants used in Kampo
medicine to identify agents that preferentially reduce the survival of
nutrient-deprived human pancreatic cancer PANC-1 cells. The
screen led to the isolation of (ꢀ)-arctigenin (1) as the active principle
of Arctium lappa [9]. In addition to pancreatic cancer, arctigenin has
been reported to inhibit lung, skin, and stomach cancers [10]. Thus,
we started the synthetic work of arctigenin derivatives to obtain
more effective drugs against pancreatic cancer. In A. lappa,
(ꢀ)-arctigenin is mainly contained as its glucoside, arctiin, and after
consumption arctiin was reported to be deglucosidated to (ꢀ)-arc-
tigenin (1), followed by demethylation and dehydroxylation by
intestinal bacteria to metabolites IeV [11]. As reported previously,
(ꢀ)-arctigenin showed potent preferential cytotoxicity, whereas its
glucoside, arctiin, showed no cytotoxicity [9]. In our preliminary
examination, moreover, metabolites I and V (Fig. 1) showed weaker
activity. These facts should suggest that the 40-hydroxyl group
should be important for the preferential cytotoxicity and that
(ꢀ)-arctigenin is deactivated through the demethylation/deme-
thoxylation. In addition, the enantiomer of (ꢀ)-arctigenin (1),
(þ)-arctigenin (Fig. 1), showed very weak preferential cytotoxicity,
indicating the importance of the 2R,3R absolute stereochemistry of
(ꢀ)-form. Thus, with an intention to improve the metabolism
stability, we have synthesized 15 arctigenin derivatives 4aeo with
different alkoxy substituent and the 2R,3R-configuration, and the
in vitro preferential cytotoxicity of them was characterized under
nutrient-deprived conditions. Then, the triethoxy derivative 4m,
exhibiting the in vitro activity identical to 1 and having no methoxy
group which may be metabolized, was selected and further evalu-
ated the effect against tumorcell growth invivo in a cancer xenograft
mouse model.
13bef were alkylated on the a-position with several alkyl halides to
afford the di-substituted lactones 14aei. Finally deprotection of the
benzyl group furnished the desired derivatives 4geo.
From the comparison of the in vitro activity of the synthesized
derivatives 4aeo against the human pancreatic cancer cell line
PANC-1, the triethoxy derivative 4m was chosen as the potent
candidate for the in vivo experiment. As the more effective
synthesis of 4m, we investigated the modified synthesis of the
lactone 13d. 3,4-Dihydroxybenzaldehyde was converted to the
ester 17 via known aldehyde 15 [15] and alcohol 16 [16] as the same
procedure for the synthesis of 8. After reduction of 17, lipase-
mediated transesterification of the resulting diol afforded the
mono-acetate 18, whose enantiomeric excess was determined to be
98% ee again by the Mosher method. The mono-acetate 18 was then
transformed into the lactone 13d via mesylate 19 (Scheme 3).
2.2. In vitro preferential cytotoxicity of arctigenin derivatives
All of the (ꢀ)-arctigenin derivatives 4aeo were evaluated for
their in vitro preferential cytotoxic activity against human pancre-
atic cancer PANC-1 cells in nutrient-deprived medium (NDM). The
PANC-1 cell line is highly resistant to nutrient starvation, and can
survive in NDM even after 48 h of starvation [6,7,8]. However, this
tolerance to nutrient starvation was remarkably eliminated by the
tested compounds in a concentration-dependent manner. The
tested compounds exhibited different potency of toxicity (Fig. 2)
and their preferential cytotoxicities are obtained as the 50% cyto-
toxic concentration in NDM (PC50 value) (Table 1). Among the
(ꢀ)-arctigenin derivatives 4aeo, monoethoxy derivative 4i showed
2. Results and discussion
2.1. Chemistry
First we planned the synthesis of derivatives on the 30 position
of (ꢀ)-arctigenin. For this purpose, (ꢀ)-arctigenin (1) was con-
verted to the diol 2 [12], which was transformed into 6 derivatives
4aef via selective protection of 2, alkylation of 3, followed by
deprotection of the benzyl group (Scheme 1).
the most potent preferential cytotoxicity (PC50, 0.49
by diethoxy derivative 4h (PC50, 0.66 M) and triethoxy derivative
4m (PC50, 0.78 M), which were identical to or more potent than
(ꢀ)-arctigenin (1) (PC50, 0.80 M).
mM), followed
m
m
Next we planned the efficient and flexible synthesis of a variety
m
of derivatives on the 30, 300, and 400 positions of (ꢀ)-arctigenin.
On the relationship between the substituents and the prefer-
ential activity, the 30 position seems to favor smaller substituent
since the PC50 values of 1 and 4aed increase in the order: 1
(MeO) < 4a (EtO) ¼ 4b (n-PrO) < 4c (i-PrO) < 4d (n-BuO). This
would suggest the importance of the 40-hydroxy group for the
preferential activity. On the other hand, there is not clear rela-
tionship on the substituents at the 300 and 400 positions, although
smaller substituents seems to be favor.
H
H
H
H
O
O
O
3'
4'
MeO
HO
MeO
HO
MeO
GlcO
R
S
O
O
O
S
R
H
H
3''
OMe
OMe
OMe
4''
OMe
OMe
OMe
The order of in vitro preferential cytotoxicity (PC50) was
4i > 4h > 4m. Whereas 4h and 4i have the methoxy groups which
was reported to be demethylated and then deoxygenated by
intestinal bacteria and/or hepatic enzyme [11]. Thus, we selected
the triethoxy derivative 4m to pursue a further examination, from
a viewpoint of metabolism stability.
arctiin
(–)-arctigenin (1)
(+)-arctigenin
R1
R2
R3
H
O
HO
R1
I
OH
Me
Me
H
OMe
OMe
OMe
OH
O
II
III
IV
V
H
H
H
H
H
2.3. In vivo antitumor activity of triethoxy derivative 4m
H
The triethoxy derivative 4m showed the in vitro preferential
cytotoxicity also against human pancreatic cancer cell line CAPAN-1
under glucose deficient conditions with a intensity similar to
(ꢀ)-arctigenin (1) (Fig. 3). We used PANC-1 cell line for in vitro
OR2
H
H
R3
Fig. 1. Structures of (ꢀ)-arctigenin (1) and its analogs.

78
N. Kudou et al. / European Journal of Medicinal Chemistry 60 (2013) 76e88
H
H
H
H
H
H
O
O
H
H
O
O
MeO
HO
HO
HO
HO
RO
HO
a
b
c, d
O
O
O
O
BnO
OMe
OMe
OMe
OMe
OMe
OMe
2
OMe
3
OMe
(–)-arctigenin (1)
4a: R = Et
4b: R = n-Pr
4c: R = i-Pr
4d: R = n-Bu
4e: R = n-Hex
4f: R = HO(CH2)2
Scheme 1. Reagents and conditions: a: AlCl3, pyridine, CH2Cl2, reflux (quant.); b: BnBr, K2CO3, KI, acetone, reflux (63%); c: RI or RBr, K2CO3, acetone, reflux for 4aee or 2-
benzyloxyethanol, Ph3P, DEAD, CH2Cl2, rt for 4f; d: H2, Pd(OH)2, MeOH, rt.
study because of its ready growth [17], while mouse xenograft
model can be prepared with CAPAN-1 cell line more easily than
with PANC-1 cell line [18]. Thus, we used mouse xenograft model
with CAPAN-1 cell line for comparing the in vivo effect of triethoxy
derivative 4m with (ꢀ)-arctigenin (1).
the tested compounds might have no toxicity at the dose used. The
treatment was initiated from the 15th day by i.p. injection of the
drug at the dose of 50 mg/mouse/d on 6 days of the week (or vehicle
in the control group) until the 28th day. The tumor size was
measured weekly. As is evident from the tumor growth curve
shown in Fig. 4B, the tumor volume increased steadily in the
control group, whereas the increase was significantly less prom-
inent in the groups treated by triethoxy derivative 4m or (ꢀ)-arc-
tigenin (1). There was a significant difference in the tumor size at
the day 21 between the groups treated by triethoxy derivative 4m
or (ꢀ)-arctigenin (1) and the control group (P < 0.05). Similarly, the
mean wet weight and the size of the tumor were higher in the
Mice were inoculated with 5 ꢁ106 CAPAN-1 cells s.c. on the back
and then administered triethoxy derivative 4m, (ꢀ)-arctigenin (1),
or vehicle, as described in Experimental. The body weight of the
animals was monitored weekly (Fig. 4A) and no significant body
weight loss was recognized in the treated group versus the vehicle
control group at any time during the experimental period. This fact,
together with the behavior of the treated animals, indicated that
HO
HO
HO
MOMO
BnO
MOMO
BnO
MOMO
BnO
a
b
c
d, e
CO2Et
CO2Et
OH
CHO
BnO
CHO
h
CHO
6
7
8
5
O
O
O
MOMO
BnO
OH
MOMO
BnO
OMs
OAc
HO
R1O
BnO
l
f, g
i, j, k
O
OAc
BnO
H
H
H
H
12a: R1 = Me
12b: R1 = Et
9
10
11
12c: R1 = n-Pr
O
O
O
H
H
H
H
R3O
BnO
R3O
HO
R1O
R2O
o
p
m, n
O
O
O
H
13a: R1 , R2 = Me
13b: R1 = Me, R2 = Et
13c: R1 = Et, R2 = Me
13d: R1, R2 = Et
OR2
OR2
OR1
OR1
14a: R1, R3 = Me, R2 = Et
14b: R1 = Me, R2, R3 = Et
14c: R1 = Et, R2, R3 = Me
14d: R1, R3 = Et, R2 = Me
14e: R1 = Me, R2 = Et, R3 = n-Pr
14f: R1, R2 = Et, R3 = Me
14g: R1, R2, R3 = Et
4g: R1, R3 = Me, R2 = Et
4h: R1 = Me, R2, R3 = Et
4i: R1 = Et, R2, R3 = Me
4j: R1, R3 = Et, R2 = Me
13e: R1 = Me, R2 = n-Pr
13f: R1 = Et, R2 = n-Pr
4k: R1 = Me, R2 = Et, R3 = n-Pr
4l: R1, R2 = Et, R3 = Me
4m: R1, R2, R3 = Et
14h: R1 = Me, R2, R3 = n-Pr
14i: R1 = Et, R2, R3 = n-Pr
4n: R1 = Me, R2, R3 = n-Pr
4o: R1 = Et, R2, R3 = n-Pr
Scheme 2. Reagents and conditions: a: BnBr, K2CO3, KI, acetone, reflux (64%); b: MOMCl, DIPEA, CH2Cl2, rt (quant.); c: NaBH4, MeOH, rt (95%); d: MsCl, Et3N, CH2Cl2, rt; e: diethyl
malonate NaH, DMF, rt (72% in 2 steps); f: LiAlH4, THF, reflux; g: lipase-PS (Amano), vinyl acetate, i-Pr2OeTHF rt (80% in 2 steps, 98% ee); h: MsCl, Et3N, CH2Cl2, rt; i: KCN, DMSO,
90 ꢂC; j: LiOH, THFeH2O, rt; k: 10% NaOH (aq), reflux, then 10% HCl (aq)eTHF, rt (73% in 4 steps); l: MeI or EtI or n-PrBr, K2CO3, acetone, reflux (88% for 12a, 86% for 12b, 87% for 12c);
m: H2, Pd(OH)2, MeOH; n: MeI or EtI, K2CO3, acetone, reflux (55% in 2 steps for 13a, 55% in 2 steps for 13b, 55% in 2 steps for 13c, 47% in 2 steps for 13d, 80% in 2 steps for 13e, 77% in
2 steps for 13f); o: LiHMDS, substituted BnBr, HMPA, THF, ꢀ78 ꢂC to rt (44% for 14a, 59% for 14b, 43% for 14c, 53% for 14d, 40% for 14e; 48% for 14f; 56% for 14g, 49% for 14h; 33% for
14i); p: H2, Pd(OH)2, MeOH (89% for 4g, 63% for 4h, 57% for 4i, 63% for 4j, 56% for 4k, 81% for 4l, 66% for 4m, 46% for 4n, 63% for 4o).

N. Kudou et al. / European Journal of Medicinal Chemistry 60 (2013) 76e88
79
HO
HO
EtO
EtO
EtO
EtO
EtO
EtO
EtO
EtO
OH
OAc
a
b
c, d
e, f
CO2Et
CO2Et
OH
CHO
CHO
H
15
16
17
18
O
EtO
EtO
OMs
OAc
EtO
EtO
g
h, i, j
O
H
H
13d
19
Scheme 3. Reagents and conditions: a: EtI, K2CO3, acetone, reflux (92%); b: NaBH4, MeOH, rt (74%); c: MsCl, Et3N, CH2Cl2, rt; d: diethyl malonate NaH, DMF, rt (87% in 2 steps); e:
LiAlH4, THF, reflux; f: lipase-PS (Amano), vinyl acetate, i-Pr2OeTHF rt (53% in 2 steps, 98% ee); g: MsCl, Et3N, CH2Cl2, rt (79%); h: KCN, DMSO, 90 ꢂC; i: LiOH, THFeH2O, rt; j: 10% NaOH
(aq), reflux, then 10% HCl (aq)-THF, rt (60% in 3 steps).
control group than the groups treated by triethoxy derivative 4m or
(ꢀ)-arctigenin (1) (Fig. 4CeF). These data indicate that triethoxy
derivative 4m also exerted antitumor activity in vivo with the
potency identical to or slightly more than (ꢀ)-arctigenin (1).
4.1.2. Synthesis of (ꢀ)-arctigenin derivatives 4ae4f
4.1.2.1. (3R,4R)-3-(4-Benzyloxy-3-hydroxybenzyl)-4-(3,4-dimethoxyb-
enzyl)dihydrofuran-2-one (3). To a stirred solution of (3R,4R)-3-(3,4-
dihydroxybenzyl)-4-(3,4-dimethoxybenzyl)dihydrofuran-2-one (2)
[12] (65.4 mg, 0.18 mmol) in acetone (2 mL) were added K2CO3
3. Conclusion
(37.3 mg, 0.27 mmol), KI (5.97 mg, 0.036 mmol), and BnBr (21.4 mL,
0.18 mmol), and the resulting mixture was refluex for 5 h. After
cooling, the reaction mixture was filtered, and the filtrate was
evaporated. The residue was chromatographed on silica gel (10 g,
hexane:acetone ¼ 4:1) to give 3 (51.2 mg, 63%) as a pale yellow oil:
In summary, a series of new (ꢀ)-arctigenin derivatives modified
on O-alkyl groups were synthesized and their preferential cytotox-
icity was evaluated against human pancreatic cancer cell line PANC-
1 under nutrient-deprived conditions. The results showed that
1H NMR (300 MHz, CDCl3)
d: 1.60 (1H, br), 2.47e2.63 (4H, m), 2.86e
monoethoxy derivative 4i (PC50, 0.49
m
M), diethoxy derivative 4h
2.98 (2H, m), 3.80 (3H, s), 3.85 (3H, s), 3.80e3.89 (1H, m), 4.09e4.14
(PC50, 0.66 M), and triethoxy derivative 4m (PC50, 0.78
m
mM) showed
(1H, m), 5.13 (2H, s), 6.47e6.80 (6H, m), 7.28e7.44 (5H, m); 13C NMR
the preferential cytotoxicities under nutrient-deprived conditions,
(75 MHz, CDCl3) d: 34.59, 38.19, 41.15, 46.53, 55.82, 55.98, 71.08,
which were identical to or more potent than (ꢀ)-arctigenin (1)
71.21, 111.22, 111.72, 112.79, 113.95, 120.43, 121.20, 127.12, 127.69,
128.39, 130.30, 130.73, 136.98, 146.91, 147.67, 148.84, 149.63, 178.46;
IR (neat): 1514 (C]C), 1769 (C]O) cmꢀ1; MS (EI) m/z 449 (Mþ);
HRMS (EI): calcd for C27H28O6: 448.1886 (Mþ), found: 448.2743;
(PC50, 0.80 mM). Among them, we selected the triethoxy derivative
4m and examined in vivo antitumor activity with mouse xenograft
model. Triethoxy derivative 4m exhibited also in vivo antitumor
activity with the potency identical to (ꢀ)-arctigenin (1). These
results would suggest that a modification of (ꢀ)-arctigenin structure
could lead to a new drug based on the antiausterity strategy.
½
a 2D6
ꢃ
ꢀ20.7 (c 0.85, CHCl3).
4.1.2.2. (3R,4R)-4-(3,4-Dimethoxybenzyl)-3-(3-ethoxy-4-hydroxyb-
enzyl)dihydrofuran-2-one (4a). To a stirred solution of 3 (44.7 mg,
0.10 mmol) in acetone (5 mL) were added K2CO3 (82.6 mg,
4. Experimental
0.60 mmol), EtI (26.5 mL, 0.33 mmol), and the reaction mixture was
4.1. Chemistry
refluxed for 48 h. After cooling, the reaction mixture was filtered, and
the filtrate was evaporated. The residue was dissolved in MeOH
(6 mL). To the solution was added 20% Pd(OH)2 (10 mg), and the
resulting suspension was stirred under a hydrogen atmosphere at
1 atm for 16 h. The catalyst was removed by filtration and the filtrate
was evaporated. The residue was chromatographed on silica gel (7 g,
hexane : acetone ¼ 3:1) to give 4a (13.4 mg, 35% in 2 steps) as
4.1.1. General conditions
Chemicals were purchased from SigmaeAldrich, Merck, Nakalai
Tesque, Wako Pure Chemicals, and Kanto Chemicals, and used
without further purification. Column chromatography was done on
Cica silica gel 60N (spherical, neutral; particle size, 40e50
mm,
Kanto Chemical Co., Inc., Tokyo, Japan), while thin-layer chroma-
tography (TLC) was performed on Merck silica gel 60F254 plates
(Merck KGaA, Darmstadt, Germany). Melting points were taken on
a Yanaco micromelting point apparatus and are uncorrected. The
nuclear magnetic resonance (NMR) spectra were acquired in the
specified solvent, in a Varian Gemini 300 spectrometer (300 and
75 MHz for 1H and 13C, respectively) or Varian UNITY plus 500
spectrometer (500 and 125 MHz for 1H and 13C, respectively)
(Varian Inc., Palo Alto, CA, USA), with tetramethylsilane (TMS) as
a colorless oil: 1H NMR (300 MHz, CDCl3)
d: 1.41 (3H, t, J ¼ 7.1 Hz),
2.42e2.64 (4H, m), 2.90 (2H, d, J ¼ 5.2 Hz), 3.80 (3H, s), 3.84 (3H, s),
3.80e3.88 (1H, m), 4.02 (2H, q, J ¼ 7.1 Hz), 4.08e4.13 (1H, m), 5.66
(1H, br), 6.46e6.75 (4H, m), 6.81 (1H, d, J ¼ 8.0 Hz); 13C NMR
(75 MHz, CDCl3) d: 14.85, 30.94, 34.48, 38.15, 40.90, 46.58, 55.85,
64.38, 71.24, 111.11, 111.61, 112.29, 113.94, 120.43, 121.83, 129.20,
130.30, 144.47, 145.80, 147.62, 148.81, 178.51; IR (neat): 1516 (C]C),
1766 (C]O), 3446 (OH) cmꢀ1; MS (EI) m/z 386 (Mþ); HRMS (EI):
calcd for C22H26O6: 386.1729 (Mþ), found: 386.1724; ½a 2D6
ꢀ20.5 (c
ꢃ
internal standard. The chemical shifts (
d
) are reported in ppm
0.98, CHCl3).
downfield from TMS and coupling constants (J) are expressed in
Hertz. Optical rotations were obtained in the specified solvent on
a JASCO DIP-1000 digital polarimeter (JASCO Corp., Tokyo, Japan). IR
spectra were measured with a JASCO FT/IR-460 Plus spectropho-
tometer (JASCO Corp.). The low-resolution mass spectra (MS) and
high-resolution mass spectra (HRMS) were obtained with a Shi-
madzu GCMS-QP 500 mass spectrometer (Shimadzu Corp., Kyoto,
Japan), JEOL D-200, or JEOL AX505 mass spectrometer (JEOL Ltd.,
Tokyo, Japan) in the electron impact mode at the ionization
potential of 70 eV.
4.1.2.3. (3R,4R)-4-(3,4-Dimethoxybenzyl)-3-(4-hydroxy-3-propoxyb-
enzyl)dihydrofuran-2-one (4b). By the procedure similar to synthesis
of 4a, (ꢀ)-arctigenin derivative 4b was prepared from 3 and n-PrBr
1
(18% in 2 steps) as a colorless oil: H NMR (300 MHz, CDCl3)
d: 1.04
(3H, t, J ¼ 1.9 Hz), 1.77e1.87 (2H, m), 2.42e2.67 (4H, m), 2.81e3.01
(2H, m), 3.78e3.86 (7H, m), 3.90e4.00 (2H, m), 4.09e4.14 (1H, m),
5.59e5.63 (1H, br), 6.47e6.85 (6H, m); 13C NMR (75 MHz, CDCl3)
d:
10.60, 22.60, 29.34, 31.81, 34.55, 38.22, 41.49, 46.65, 53.80, 55.82,
70.35, 71.29, 111.71, 113.94, 115.27, 120.47, 121.85, 112.34, 129.28,

80
N. Kudou et al. / European Journal of Medicinal Chemistry 60 (2013) 76e88
Fig. 2. Effects of (ꢀ)-arctigenin derivatives on cell survival in the PANC-1 cell line under nutrient-deprived conditions. Cells were seeded at a density of 2 ꢁ 104 per well in 96-well
plates and incubated in fresh complete medium for 24 h. The cells were then washed with PBS and the medium was changed to nutrient-deprived medium (NDM, C) or normal
DMEM (B) together containing graded concentrations of (ꢀ)-arctigenin derivatives. Points, mean from triplicate experiments. The cell number at the start of the starvation was
considered to be 100%. The cell count was measured by the WST-8 cell counting kit method, as described in experimental. The numbers 1 and 4aeo mean the data of (ꢀ)-arctigenin
(1) and (ꢀ)-arctigenin derivatives 4aeo, respectively.
130.59, 144.52, 147.69, 178.54; IR (neat): 1456 (C]C), 1769 (C]O)
cmꢀ1; MS (EI) m/z 400 (Mþ); HRMS (EI): calcd for C23H28O6: 400.1886
m), 6.48e6.84 (6H, m); 13C NMR (75 MHz, CDCl3)
d: 22.02, 34.39,
3.12, 41.45, 46.65, 55.81, 71.19, 111.25, 111.68, 113.41, 114.18, 115.49,
120.61, 122.09, 129.26, 130.43, 144.70, 145.48, 146.59, 147.84,149.02,
178.72; IR (neat): 1716 (C]O), 3629 (OH) cmꢀ1; MS (EI) m/z 400
(Mþ); HRMS (EI): calcd for C23H28O6: 400.1886 (Mþ), found:
(Mþ), found: 400.1893; ½a 2D6
ꢀ15.7 (c 1.45, CHCl3).
ꢃ
4.1.2.4. (3R,4R)-4-(3,4-Dimethoxybenzyl)-3-(4-hydroxy-3-i-propox-
ybenzyl)dihydrofuran-2-one (4c). By the procedure similar to
synthesis of 4a, (ꢀ)-arctigenin derivative 4c was prepared from 3
and i-PrI (18% in 2 steps) as a pale yellow oil: 1H NMR (300 MHz,
400.1926; ½a 2D4
ꢀ37.7 (c 0.41, CHCl3).
ꢃ
4.1.2.5. (3R,4R)-4-(3,4-Dimethoxybenzyl)-3-(4-hydroxy-3-butylox-
ybenzyl)dihydrofuran-2-one (4d). By the procedure similar to
synthesis of 4a, (ꢀ)-arctigenin derivative 4d was prepared from 3
CDCl3)
d: 1.31e1.35 (6H, m), 1.59 (1H, br), 2.41e2.68 (4H, m), 2.80e
3.00 (2H, m), 3.80e3.88 (7H, m), 4.07e4.12 (1H, m), 4.49e4.57 (1H,

N. Kudou et al. / European Journal of Medicinal Chemistry 60 (2013) 76e88
81
Table 1
Preferential cytotoxicity of (ꢀ)-arctigenin (1) and series of new (ꢀ)-arctigenin derivatives 4ae4o against human pancreatic cancer PANC-1 cells in nutrient-deprived medium
(NDM).
Compound
R1
R2
R3
PC50
(m
M)
Compound
R1
R2
R3
PC50
(mM)
1 (arctigenin)
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Et
Me
Et
0.80
3.74
3.74
4.16
7.14
3.89
7.70
4.71
4h
4i
4j
4k
4l
4m
4n
4o
Me
Et
Et
Me
Et
Et
Et
Et
Me
Et
n-Pr
Me
Et
n-Pr
n-Pr
0.66
4a
4b
4c
4d
4e
4f
Me
Me
Et
Et
Et
0.49
4.77
3.54
4.85
0.78
n-Pr
i-Pr
n-Bu
n-Hex
HO(CH2)2
Me
Me
Et
n-Pr
n-Pr
13.6
28.6
4g
and n-BuBr (25% in 2 steps) as a colorless oil: 1H NMR (300 MHz,
CDCl3)
IR (neat): 1457 (C]C), 1764 (C]O), 3689 (OH) cmꢀ1; MS (EI) m/z
d
: 0.98 (3H, t, J ¼ 7.1 Hz), 1.48 (2H, dd, J ¼ 15.1, 7.1 Hz), 1.74e
442 (Mþ); HRMS (EI): calcd for C26H34O6: 442.2355 (Mþ), found:
a 26
1.83 (2H, m), 2.41e2.66 (4H, m), 2.80e3.02 (2H, m), 3.82 (3H, s),
3.83 (3H, s), 3.85 (1H, m), 3.94e4.03 (2H, m), 4.08e4.14 (1H, m),
442.2336; ½ ꢃD ꢀ10.1 (c 0.65, CHCl3).
5.59 (1H, m), 6.50e6.84 (6H, m); 13C NMR (75 MHz, CDCl3)
d
: 13.97,
4.1.2.7. (3R,4R)-4-(3,4-Dimethoxybenzyl)-3-[4-hydroxy-3-(2-
hydroxyethoxy)benzyl]dihydrofuran-2-one (4f). By the procedure
similar to synthesis of 4a, (ꢀ)-arctigenin derivative 4f was prepared
from 3 and 2-benzyloxyethanol (20% in 2 steps) as a colorless oil:
19.32, 31.31, 55.82, 55.92, 68.60, 68.65, 71.21, 71.27, 111.19, 111.67,
112.32, 113.92, 120.46, 129.29, 130.34, 130.46, 144.52, 144.71, 145.59,
145.96, 147.69, 148.92, 178.53; IR (neat): 1515 (C]C), 1769 (C]O),
3446 (OH) cmꢀ1; MS (EI) m/z 414 (Mþ); HRMS (EI): calcd for
1H NMR (300 MHz, CDCl3)
d: 2.42e2.59 (4H, m), 2.78e2.94 (2H, m),
C24H30O6: 414.2042 (Mþ), found: 414.2000; ½a 2D6
ꢃ
ꢀ20.2 (c 1.15,
3.76 (3H, s), 3.83 (3H, s), 3.73e3.80 (1H, m), 3.86e4.07 (6H, m),
CHCl3).
4.13e4.16 (1H, m), 6.40e6.75 (4H, m), 6.81 (1H, d, J ¼ 8.0 Hz); 13C
NMR (75 MHz, CDCl3) d: 28.24, 38.22, 40.69, 46.53, 55.72, 55.97,
4.1.2.6. (3R,4R)-4-(3,4-Dimethoxybenzyl)-3-(3-hexyloxy-4-hydrox-
ybenzyl)dihydrofuran-2-one (4e). By the procedure similar to
synthesis of 4a, (ꢀ)-arctigenin derivative 4e was prepared from 3
and 1-bromohexane (35% in 2 steps) as a pale yellow oil: 1H NMR
61.08, 69.82, 71.45, 111.30, 111.56, 113.00, 115.02, 120.67, 122.55,
129.00, 130.44, 145.02, 146.10, 147.38, 148.72, 178.83; IR (neat): 1517
(C]C), 1765 (C]O), 3420 (OH) cmꢀ1; MS (EI) m/z 402 (Mþ); HRMS
(EI): calcd for C23H28O6: 402.1679 (Mþ), found: 402.1671;
(300 MHz, CDCl3)
d
: 0.90 (3H, t, J ¼ 6.4 Hz), 1.25e1.27 (2H, m), 1.33e
½
a 2D6
ꢃ
ꢀ19.7 (c 1.10, CHCl3).
1.35 (4H, m), 1.45 (2H, m), 1.75e2.66 (4H, m), 2.81e3.01 (2H, m),
3.82 (3H, s), 3.85 (3H, s), 3.84e3.89 (1H, m), 3.94e4.02 (2H, m),
4.09e4.14 (1H, m), 5.56e5.61 (1H, m), 6.47e6.84 (6H, m); 13C NMR
4.1.3. Synthesis of (ꢀ)-arctigenin derivatives 4ge4o
4.1.3.1. (4-Benzyloxy-3-methoxymethoxyphenyl)methanol (7). To a stir-
red solution of 4-benzyloxy-3-methoxymethoxybenzaldehyde (6)
[14] (7.03 g, 25.8 mmol) in MeOH (50 mL) was added NaBH4 (3.88 g,
103 mmol) at 0 ꢂC, and the resulting mixture was stirred at room
temperature for 2 h. The reaction was quenched with H2O (50 mL),
and the aqueous mixture was extracted with CH2Cl2 (50 mL ꢁ 3).
The organic extracts were combined, dried over MgSO4. The solvent
was removed under reduced pressure, and the residue was chro-
matographed on silica gel (40 g, hexane:acetone ¼ 3:1) to give 7
(75 MHz, CDCl3) d: 14.11, 22.67, 25.78, 29.25, 31.62, 34.56, 38.22,
40.02, 46.65, 55.82, 68.92, 71.21,111.19,111.67,112.32,113.92, 115.25,
120.56, 121.83, 129.29, 130.43, 130.34, 144.52, 147.67, 148.92, 178.53;
120.0
100.0
80.0
60.0
40.0
20.0
0.0
(6.66 g, 95%) as a pale yellow oil: 1H NMR (300 MHz, CDCl3)
d: 1.26
(1H, br), 3.53 (3H, s), 5.01 (2H, s), 5.16 (2H, s), 5.24 (2H, s), 6.88e6.96
(2H, m), 7.16 (1H, d, J ¼ 1.9 Hz), 7.30e7.45 (5H, m); 13C NMR
(75 MHz, CDCl3) d: 56.13, 64.64, 70.88, 95.40, 114.25, 116.22, 121.06,
126.98, 127.61, 128.27, 134.16, 136.82, 146.60, 148.19; IR (neat): 1511
(C]C), 3419 (OH) cmꢀ1; MS (EI) m/z 274 (Mþ); HRMS (EI): calcd for
C16H18O4: 274.1205 (Mþ), found: 274.1188.
4.1.3.2. 2-(4-Benzyloxy-3-methoxymethoxybenzyl)malonic acid die-
thyl ester (8). To a stirred solution of 7 (711 mg, 2.59 mmol) in
CH2Cl2 (26 mL) were added NEt3 (0.43 mL, 3.11 mmol) and MsCl
(0.22 mL, 2.85 mmol) at 0 ꢂC, and the reaction mixture was stirred
at room temperature for 0.5 h. The reaction was quenched with sat.
NaHCO3 (aq) (20 mL), and the organic layer was separated. The
aqueous layer was extracted with CH2Cl2 (30 mL ꢁ 3), and the
organic layer and extracts were combined, dried over MgSO4. The
Concentration (µM)
Fig. 3. Effect of triethoxy derivative 4m and (ꢀ)-arctigenin (1) on cell survival in the
CAPAN-1 cell line under glucose-deprived conditions. C, (ꢀ)-arctigenin (1) in normal
DMEM; :, triethoxy derivative 4m in normal DMEM; B, (ꢀ)-arctigenin (1) in glucose-
deprived medium;
6, triethoxy derivative 4m in glucose-deprived medium.

82
N. Kudou et al. / European Journal of Medicinal Chemistry 60 (2013) 76e88
B 1200
1000
800
C
A
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
30
29
28
27
26
25
24
23
22
21
20
600
400
*
200
*
0
0
7
14 21 28
day
0
7
14 21 28
control
1
4m
day
D
E
F
Fig. 4. Effect of triethoxy derivative 4m and (ꢀ)-arctigenin (1) on the growth of CAPAN-1 cells in nude mice. A, body weight of mice. B, control group (n ¼ 5); C, group treated
with triethoxy derivative 4m (n ¼ 6); :, group treated with (ꢀ)-arctigenin (1) (n ¼ 5). B, the tumor volume in the mice. B, control group; C, group treated with triethoxy
derivative 4m; :, group treated with (ꢀ)-arctigenin (1). C, wet weight of the tumor in the mice on the last day of the experiment. DeF, photographs of the tumor after sacrifice on
the last day of control group, of group treated with (ꢀ)-arctigenin (1), and of group treated with triethoxy derivative 4m, respectively.
solvent was removed under reduced pressure to give a pale yellow
oil, which was used directly in the next step. To a stirred solution of
diethyl malonate (0.79 mL, 5.18 mmol) in DMF (10 mL) was added
NaH (60%, 207 mg, 5.18 mmol) at 0 ꢂC, and the resulting mixture
was stirred at room temperature for 1 h. To the solution was added
a solution of the oil obtained above in DMF (2 mL) at 0 ꢂC, and the
reaction mixture was stirred at room temperature for 20 h. The
reaction was quenched with sat. NaHCO3 (aq) (10 mL), and the
aqueous mixture was extracted with Et2O (20 mL ꢁ 3). The organic
extracts were combined, dried over MgSO4, evaporated to give
a pale yellow oil which was chromatographed on silica gel (20 g,
hexane:acetone ¼ 15:1) to give 8 (776 mg, 72% in 2 steps) as a pale
5.67 mmol), and the reaction mixture was stirred at room
temperature for 2 h. The catalyst was filtered and the filtrate was
evaporated to give residue, which was chromatographed on silica
gel (30 g, hexane:acetone ¼ 15:1) to give (þ)-9 (1.20 g, 80% in 2
steps) as a pale yellow oil. The enantiomeric excess of (þ)-9 was
determined to be a 98% ee by the following HPLC analysis; chiralcel
OJ (0.46 cm ꢁ 25 cm), hexane/2-propanol ¼ 1/1, flow rate ¼ 0.5 mL/
min,
l
¼ 254 nm, (þ)-9; tR ¼ 29.7 min, (ꢀ)-9; 25.5 min 1H NMR
(300 MHz, CDCl3) d: 1.70 (1H, br), 2.09 (3H, s), 2.17 (1H, s), 2.55e
2.62 (2H, m), 3.47e3.62 (2H, m), 3.52 (3H, s), 4.03e4.20 (2H, m),
5.13 (2H, s), 5.21 (2H, s), 6.72e6.98 (3H, m), 7.30e7.44 (5H, m); 13C
NMR (75 MHz, CDCl3) d: 20.99, 33.75, 42.47, 56.27, 62.08, 63.94,
yellow oil: 1H NMR (300 MHz, CDCl3)
d
: 1.22 (6H, t, J ¼ 7.1 Hz), 3.13
71.14, 95.72, 114.534, 118.21, 122.92, 127.11, 127.71, 128.40, 132.46,
137.11, 146.82, 147.48, 171.47; IR (neat): 1739 (C]O), 3165 (OH)
cmꢀ1; MS (EI) m/z 374 (Mþ); HRMS (EI): calcd for C21H26O6:
(2H, d, J ¼ 7.6 Hz), 3.50 (3H, s), 3.60 (1H, t, J ¼ 7.6 Hz), 4.16 (4H, q,
J ¼ 7.1 Hz), 5.11 (2H, s), 5.19 (2H, s), 6.74e6.83 (2H, m), 7.00 (1H, d,
J ¼ 1.7 Hz), 7.28e7.42 (5H, m); 13C NMR (75 MHz, CDCl3)
d
: 14.05,
374.1729 (Mþ), found: 374.1723; ½a 2D6
þ13.5 (c 1.14, CHCl3).
ꢃ
34.09, 53.93, 56.16, 61.35, 70.92, 95.64, 114.31, 117.98, 122.74, 127.01,
127.63, 128.31, 130.09, 136.95, 146.64, 147.71, 168.56; IR (neat): 1510
(C]C), 1732 (C]O) cmꢀ1; MS (EI) m/z 416 (Mþ); HRMS (EI): calcd
for C23H28O7: 416.1835 (Mþ), found: 416.1832.
4.1.3.4. (R)-Acetic acid 3-(4-benzyloxy-3-methoxymethoxyphenyl)-
2-methanesulfonyloxymethylpropyl ester (10). To a stirred solution
of (þ)-9 (666 mg, 1.78 mmol) in CH2Cl2 (8 mL) were added MsCl
(0.15 mL, 1.95 mmol) and NEt3 (0.32 mL, 2.31 mmol) at 0 ꢂC, and the
reaction mixture was stirred at room temperature for 0.5 h. The
reaction was quenched with H2O (8 mL), and the aqueous mixture
was extracted with CH2Cl2 (10 mL ꢁ 3). The organic extracts were
combined dried over MgSO4, and evaporated. The residue was
chromatographed on silica gel (30 g, hexane:acetone ¼ 15:1) to give
4.1.3.3. (R)-Acetic acid 3-(4-benzyloxy-3-methoxymethoxyphenyl)-
2-hydroxymethylpropyl ester ((þ)-9). To a stirred solution of 8
(1.66 g, 3.98 mmol) in THF (40 mL) was added LiAlH4 (378 mg,
9.96 mmol) at 0 ꢂC, and the resulting suspension was refluxed for
12 h. The reaction was quenched with 10% NaOH (aq) (20 mL), and
the mixture was extracted with AcOEt (20 mL ꢁ 5). The organic
extracts were combined dried over MgSO4, and the solvent was
evaporated to give diol, which was used directly in the next step. To
a stirred solution of the diol obtained above in i-Pr2OeTHF (20 mL,
4:1) were added lipase-PS (397 mg) and vinyl acetate (0.52 mL,
10 (775 mg, 96%) as a pale yellow oil: 1H NMR (300 MHz, CDCl3)
d:
2.03 (3H, s), 2.27e2.34 (1H, m), 2.61 (2H, d, J ¼ 7.42 Hz), 2.93 (3H, s),
3.47 (3H, s), 3.96e4.19 (4H, m), 5.08 (2H, s), 5.18 (2H, s), 6.69e6.95
(3H, m), 7.24e7.41 (5H, m); 13C NMR (75 MHz, CDCl3)
d
: 20.91,
33.41, 37.26, 39.61, 56.29, 62.94, 68.34, 71.13, 95.67, 114.65, 118.05,

N. Kudou et al. / European Journal of Medicinal Chemistry 60 (2013) 76e88
83
122.84, 127.12, 127.76, 128.42, 130.88, 136.98, 146.98, 147.72, 170.56;
IR (KBr): 1242 (S]O), 1736 (C]O) cmꢀ1; MS (EI) m/z 452 (Mþ);
HRMS (EI): calcd for C22H28O8S: 452.1505 (Mþ), found: 452.1512;
4.1.3.8. (R)-4-(4-Benzyloxy-3-propoxybenzyl)dihydrofuran-2-one
(12c). By the procedure similar to preparation of 12a, 12c was
prepared from 11 and n-PrBr (87%) as a colorless oil: 1H NMR
½
a 2D5
ꢃ
þ2.76 (c 1.40, CHCl3).
(600 MHz, CDCl3)
d
: 1.04 (3H, t, J ¼ 7.0 Hz), 1.84 (2H, sextet,
J ¼ 7.0 Hz), 2.26 (1H, dd, J ¼ 17.5, 7.0 Hz), 2.57 (1H, dd, J ¼ 17.5, 8.1 Hz),
2.64e2.71 (2H, m), 2.74e2.83 (1H, m), 3.96 (2H, t, J ¼ 7.0 Hz), 4.00
(1H, dd, J ¼ 9.2, 5.9 Hz), 4.30 (1H, dd, J ¼ 9.2, 7.0 Hz), 5.09 (2H, s), 6.60
(1H, d, J ¼ 8.1 Hz), 6.67 (1H, s), 6.82 (1H, d, J ¼ 8.1 Hz), 7.27e7.42 (5H,
4.1.3.5. (R)-4-(4-Benzyloxy-3-hydeorxybenzyl)dihydrofuran-2-one
(11). To a stirred solution of 10 (993 mg, 2.19 mmol) in DMSO
(20 mL) was added KCN (150 mg, 2.19 mmol), and the resulting
mixture was heated at 90 ꢂC for 3 h. After cooling, the reaction was
quenched with H2O (20 mL), and the aqueous mixture was
extracted with Et2O/AcOEt (1:1, 20 mL ꢁ 3). The organic extracts
were combined, dried over MgSO4, and evaporated to give cyanide,
which was used directly in the next step. To a stirred solution of
cyanide obtained above in THFeH2O (3:1, 8 mL) was added
LiOH$H2O (91.9 mg, 2.19 mmol), and the reaction mixture was
stirred at room temperature for 24 h. The reaction mixture was
diluted with H2O (10 mL), and the aqueous mixture was extracted
with Et2O (20 mL ꢁ 3). The organic extracts were combined, dried
over MgSO4, and evaporated to give alcohol, which was used
directly in the next step. The alcohol obtained above was dissolved
in 10% NaOH (aq) (10 mL), and the mixture was refluxed for 5 h.
After cooling, 10% HCl (aq) (20 mL) and THF (20 mL) were added to
the reaction mixture, and the resulting solution was stirred at room
temperature for 50 h. The aqueous reaction mixture was extracted
with Et2O (30 mL ꢁ 3), and the organic extracts were combined,
dried over MgSO4, and evaporated to give a residue, which was
chromatographed on silica gel (20 g, hexane:acetone ¼ 3:1) to give
11 (479 mg, 73% in 4 steps) as a colorless solid: 1H NMR (300 MHz,
m); 13C NMR (100 MHz, CDCl3)
d: 10.46, 22.55, 34.07, 37.15, 38.43,
70.65, 71.32, 72.55, 114.28, 115.38, 120.69, 127.10, 127.63, 128.34,
131.61, 137.31, 147.37, 149.52, 176.84; IR (neat): 1508 (C]C), 1773
(C]O) cmꢀ1; MS (EI) m/z 340 (Mþ); HRMS (EI): calcd for 340.1675
(Mþ), found: 340.1667; ½a 2D6
ꢀ1.0 (c 1.05, CHCl3).
ꢃ
4.1.3.9. (R)-4-(3,4-Dimethoxybenzyl)dihydrofuran-2-one
(13a).
To a stirred solution of 12a (302 mg, 0.97 mmol) in MeOH (5 mL)
was added 20% Pd(OH)2 (20 mg), and the resulting suspension
was stirred under a hydrogen atmosphere at 1 atm for 15 h. The
catalyst was removed by filtration and the filtrate was evapo-
rated to give phenol, which was used directly in the next step. To
a stirred solution of the phenol obtained above in acetone
(10 mL) were added K2CO3 (201.1 mg, 1.46 mmol) and MeI
(0.18 mL, 2.92 mmol), and the resulting mixture was refluxed for
19 h. After cooling, the insoluble materials were filtered, and the
filtrate was evaporated to give a residue, which was chromato-
graphed on silica gel (10 g, hexane:acetone ¼ 4:1) to give 13a
(130 mg, 55% in 2 steps) as a pale yellow oil: 1H NMR (300 MHz,
CDCl3)
d
: 2.33 (1H, dd, J ¼ 18.0, 9.3 Hz), 2.61 (1H, dd, J ¼ 17.4,
CDCl3)
3.91e4.05 (1H, m), 4.30e4.36 (1H, m), 5.09 (2H, s), 5.67 (1H, br),
6.59e6.89 (3H, m), 7.36e7.85 (5H, m); 13C NMR (75 MHz, CDCl3)
d: 2.17e2.32 (1H, m), 2.52e2.69 (3H, m), 2.74e2.86 (1H, m),
8.1 Hz), 2.70e2.87 (3H, m), 3.87 (3H, s), 3.88 (3H, s), 4.05 (1H, dd,
J ¼ 9.3, 6.3 Hz), 4.33 (1H, dd, J ¼ 9.3, 6.6 Hz), 6.66e6.72 (2H, m),
d:
6.82 (1H, d, J ¼ 8.1 Hz); ½a D24
þ22.2 (c 0.87, CHCl3) (ref. [19],
ꢃ
34.25, 37.22, 38.41, 71.22, 72.63, 112.26, 114.78, 120.04, 127.69,
128.32, 128.61, 131.72, 136.09, 144.50, 145.89, 176.68; IR (KBr): 1647
(C]O), 3445 (OH) cmꢀ1; MS (EI) m/z 298 (Mþ); HRMS (EI): calcd for
½
a 2D5
ꢃ
þ23.8).
4.1.3.10. (R)-4-(4-Ethoxy-3-methoxybenzyl)dihydrofuran-2-one
(13b). By the procedure similar to preparation of 13a, 13b was
prepared from 12a and EtI (55% in 2 steps) as a pale yellow oil: 1H
C18H18O4: 298.1205 (Mþ), found: 298.1204; ½a 2D6
þ5.6 (c 0.13,
ꢃ
CHCl3); mp: 137e139 ꢂC.
NMR (300 MHz, CDCl3)
d
: 1.46 (3H, t, J ¼ 7.1 Hz), 2.29 (1H, dd,
4.1.3.6. (R)-4-(4-Benzyloxy-3-methoxybenzyl)dihydrofuran-2-one
(12a). To a stirred solution of 11 (330 mg, 1.1 mmol) in acetone
(15 mL) were added K2CO3 (168 mg, 1.2 mmol) and MeI (0.41 mL,
6.6 mmol), and the reaction mixture was refluxed for 24 h. After
cooling, the insoluble materials were filtered, and the filtrate was
evaporated to give a residue, which was chromatographed on silica
gel (15 g, hexane:acetone ¼ 4:1) to give 12a (304 mg, 88%) as
J ¼ 17.6, 6.9 Hz), 2.63 (1H, dd, J ¼ 17.6, 8.0 Hz), 2.71e2.88 (3H, m),
3.86 (3H, s), 4.03 (1H, dd, J ¼ 9.1, 6.9 Hz), 4.06 (2H, q, J ¼ 7.1 Hz), 4.34
(1H, dd, J ¼ 9.1, 6.9 Hz), 6.65e6.68 (2H, m), 6.81 (1H, d, J ¼ 8.2 Hz);
13C NMR (75 MHz, CDCl3)
d: 14.92, 34.32, 37.36, 38.65, 55.98, 64.38,
72.63, 112.01, 112.84, 120.56, 130.59, 147.06, 149.26, 176.68; IR
(neat): 1514 (C]C),1778 (C]O) cmꢀ1; MS (EI) m/z 250 (Mþ); HRMS
(EI): calcd for C14H18O4: 250.1205 (Mþ), found: 250.1192; ½a 2D4
þ4.4
ꢃ
a colorless oil: 1H NMR (300 MHz, CDCl3)
d: 2.17 (2H, s), 2.24e2.30
(c 1.66, CHCl3).
(1H, m), 3.88 (3H, s), 4.03e4.05 (1H, m), 4.30e4.35 (1H, m), 5.13
(2H, s), 6.61e6.64 (2H, m), 6.81e6.83 (1H, m), 7.27e7.45 (5H, m);
4.1.3.11. (R)-4-(3-Ethoxy-4-methoxybenzyl)dihydrofuran-2-one
(13c). By the procedure similar to preparation of 13a, 13c was
prepared from 12b and MeI (55% in 2 steps) as a pale yellow oil: 1H
13C NMR (75 MHz, CDCl3)
d: 34.29, 37.30, 38.64, 56.06, 71.11, 72.60,
112.32, 114.23, 120.52, 127.12, 127.72, 128.40, 131.25, 136.96, 146.91,
149.66, 176.65; IR (neat): 1654 (C]C), 1774 (C]O) cmꢀ1; MS (EI) m/
z 312 (Mþ); HRMS (EI): calcd for C19H20O4: 312.1362 (Mþ), found:
NMR (300 MHz, CDCl3)
d
: 1.47 (3H, t, J ¼ 6.9 Hz), 2.29 (1H, dd
J ¼ 17.3, 6.6 Hz), 2.61 (1H, dd, J ¼ 17.3, 8.0 Hz), 2.67e2.87 (3H, m),
312.1380; ½a 2D5
þ4.9 (c 0.95, CHCl3).
ꢃ
3.86(3H, s), 4.01e4.12 (3H, m), 4.34 (1H, dd, J ¼ 9.1, 6.6 Hz), 6.62e
6.69 (2H, m), 6.81 (1H, d, J ¼ 8.0 Hz); 13C NMR (75 MHz, CDCl3)
d:
4.1.3.7. (R)-4-(4-Benzyloxy-3-ethoxybenzyl)dihydrofuran-2-one
(12b). By the procedure similar to preparation of 12a, 12b was
prepared from 11 and EtI (84%) as a pale yellow oil: 1H NMR
14.87, 34.24, 37.31, 38.54, 55.95, 64.35, 72.58, 111.61, 113.24, 120.54,
130.52, 148.01, 148.22, 176.63; IR (neat): 1541 (C]C), 1771 (C]O)
cmꢀ1; MS (EI) m/z 250 (Mþ); HRMS (EI): calcd for C14H18O4:
(300 MHz, CDCl3)
d
: 1.44 (3H, t, J ¼ 4.4 Hz), 2.28 (1H, dd, J ¼ 17.3,
250.1205 (Mþ), found: 250.1207; ½a 2D7
þ4.4 (c 1.94, CHCl3).
ꢃ
6.9 Hz), 2.60 (1H, dd, J ¼ 17.3, 8.0 Hz), 2.67e2.84 (3H, m), 4.02e4.13
(3H, m), 4.32 (1H, dd, J ¼ 9.1, 6.9 Hz), 5.12 (2H, s), 6.60e6.69 (2H, m),
6.84 (1H, d, J ¼ 8.2 Hz), 7.30e7.77 (5H, m); 13C NMR (75 MHz, CDCl3)
4.1.3.12. (R)-4-(3,4-Diethoxybenzyl)dihydrofuran-2-one (13d). By the
procedure similar to preparation of 13a, 13d was prepared from 12b
and EtI (47% in 2 steps) as a pale yellow oil: 1H NMR (300 MHz, CDCl3)
d: 15.03, 34.29, 37.31, 38.61, 64.74, 71.37, 72.62, 114.29, 115.22,
120.73, 127.08, 127.63, 128.34, 131.46, 137.20, 147.33, 149.18, 176.67;
IR (neat): 1507 (C]C), 1772 cmꢀ1 (C]O); MS (EI) m/z 326 (Mþ);
HRMS (EI): calcd for C20H22O4: 326.1518 (Mþ), found: 326.1523;
d
: 1.41e1.47 (6H, m), 2.28 (1H, dd, J ¼ 17.3, 6.6 Hz), 2.59 (1H, dd,
J ¼ 17.3, 8.0 Hz), 2.67e2.86 (3H, m), 4.00e4.11 (5H, m), 4.32 (1H, dd,
J ¼ 9.3, 6.6 Hz), 6.64e6.67 (2H, m), 6.81 (1H, d, J ¼ 8.5 Hz); 13C NMR
½
a 2D6
ꢃ
þ3.4 (c 1.78, CHCl3).
(75 MHz, CDCl3) d: 14.97, 34.30, 37.36, 38.61, 64.64, 64.69, 72.65,

84
N. Kudou et al. / European Journal of Medicinal Chemistry 60 (2013) 76e88
113.70, 114.12, 120.78, 130.70, 147.53, 148.72, 176.68; IR (neat): 1507
MS (EI) m/z 490 (Mþ); HRMS (EI): calcd for C30H34O6: 490.2355
(C]C), 1771 (C]O) cmꢀ1; MS (EI) m/z 264 (Mþ); HRMS (EI): calcd for
(Mþ), found: 490.2383; ½a 2D6
ꢀ14.8 (c 1.46, CHCl3).
ꢃ
C15H20O4: 264.1362 (Mþ), found: 264.1369; ½a 2D6
þ5.4 (c 1.29, CHCl3).
ꢃ
4.1.3.17. (3R,4R)-3-(4-Benzyloxy-3-methoxybenzyl)-4-(4-ethoxy-3-
methoxybenzyl)dihydrofuran-2-one (14c). By the procedure similar
to preparation of 14a, 14c was prepared from 13c and 4-benzyloxy-
3-methoxybenzyl bromide [20] (43%) as a pale yellow oil: 1H NMR
4.1.3.13. (R)-4-(4-Methoxy-3-propoxybenzyl)dihydrofuran-2-one
(13e). By the procedure similar to preparation of 13a, 13e was
1
prepared from 12c and MeI (80% in 2 steps) as a pale yellow oil: H
NMR (400 MHz, CDCl3)
d
: 1.05 (3H, t, J ¼ 7.1 Hz), 1.87 (2H, sextet,
(300 MHz, CDCl3)
d
: 1.45 (3H, t, J ¼ 6.9 Hz), 2.47e2.63 (4H, m),
J ¼ 7.1 Hz), 2.29 (1H, dd, J ¼ 17.5, 6.8 Hz), 2.60 (1H, dd, J ¼ 17.5, 8.1 Hz),
2.65e2.73 (2H, m), 2.77e2.84 (1H, m), 3.85 (1H, s), 3.96 (2H, t,
J ¼ 7.1 Hz), 4.03 (1H, dd, J ¼ 9.3, 6.1 Hz), 4.33 (1H, dd, J ¼ 9.3, 7.0 Hz),
6.67 (1H, s), 6.68 (1H, d, J ¼ 7.8 Hz), 6.81 (1H, d, J ¼ 7.8 Hz); 13C NMR
2.91e2.95 (2H, m), 3.84 (3H, s), 3.91 (3H, s), 3.91e3.95 (1H, m), 4.09
(2H, q, J ¼ 6.9 Hz), 4.03e4.14 (1H, m), 5.16 (2H, s), 6.48e6.96 (6H,
m), 7.28e7.45 (5H, m); 13C NMR (75 MHz, CDCl3)
d: 14.90, 34.58,
38.19, 41.13, 46.55, 55.98, 64.35, 65.29, 71.06, 110.88, 112.03, 112.82,
113.89, 113.97, 119.21, 120.44, 121.22, 127.69, 128.40, 130.26, 130.73,
134.03, 136.98, 146.91, 149.61, 178.49; IR (neat): 1261 (C]C), 1770
(C]O) cmꢀ1; MS (EI) m/z 476 (Mþ); HRMS (EI): calcd for C29H32O6:
(100 MHz, CDCl3)d: 10.37, 22.44, 34.15, 37.23, 38.45, 56.00, 70.51, 72,57,
111.89, 113.52, 120.60, 130.67, 148.25, 148.62, 176.86; IR (neat): 1516
(C]C), 1778 (C]O) cmꢀ1; MS (EI) m/z 264 (Mþ); HRMS(EI): calcd for
C15H20O4: 264.1362 (Mþ), found: 264.1345; ½a 2D6
ꢃ
þ3.2 (c 1.05, CHCl3).
476.2199 (Mþ), found: 476.2209; ½a 2D6
ꢀ9.0 (c 1.75, CHCl3).
ꢃ
4.1.3.14. (R)-4-(4-Ethoxy-3-propoxybenzyl)dihydrofuran-2-one
(13f). By the procedure similar to preparation of 13a, 13f was
prepared from 12c and EtI (77% in 2 steps) as a pale yellow oil: 1H
4.1.3.18. (3R,4R)-3-(4-Benzyloxy-3-ethoxybenzyl)-4-(4-ethoxy-3-
methoxybenzyl)dihydrofuran-2-one (14d). By the procedure similar
to preparation of 14a, 14d was prepared from 13c and 4-benzyloxy-
3-ethoxybenzyl bromide [21] (53%) as a pale yellow oil: 1H NMR
NMR (400 MHz, CDCl3)
d
: 1.05 (3H, t, J ¼ 7.0 Hz), 1,42 (3H, t,
J ¼ 6.8 Hz),1.87 (2H, sextet, J ¼ 7.0 Hz), 2.28 (1H, dd, J ¼ 17.5, 7.0 Hz),
2.60 (1H, dd, J ¼ 17.5, 8.0 Hz), 2.64e2.72 (2H, m), 2.74e2.85 (1H, m),
3.94 (2H, t, J ¼ 7.0 Hz), 4.01e4.09 (3H, m), 4.32 (1H, dd, J ¼ 9.1,
6.9 Hz), 6.64 (1H, s), 6.65 (1H, d, J ¼ 8.0 Hz), 6.81 (1H, d, J ¼ 8.0 Hz);
(300 MHz, CDCl3) d: 1.41e1.48 (6H, m), 2.44e2.67 (4H, m), 2.88e
2.93 (2H, m), 3.79 (3H, s), 3.80e3.87 (1H, m), 4.02e4.14 (5H, m),
5.11 (2H, s), 6.45e6.96 (6H, m), 7.27e7.45 (5H, m); 13C NMR
(75 MHz, CDCl3) d: 14.62, 14.69, 30.69, 34.27 37.89, 40.87, 46.28,
13C NMR (100 MHz, CDCl3)
d: 10.35, 14.79, 22.50, 34.10, 37.16, 38.38,
55.66, 64.12, 64.35, 71.04, 71.12, 111.92, 112.61, 114.52, 114.84,
120.38, 121.31, 127.02, 127.52, 128.23, 130.29, 130.91, 137.17, 146.91,
147.20, 149.10, 178.55; IR (neat): 1515 (C]C), 1771 (C]O) cmꢀ1; MS
(EI) m/z 490 (Mþ); HRMS (EI): calcd for C30H34O6: 490.2355 (Mþ),
64.67, 70.72, 72.56, 114.05, 114.31, 120.78, 130.88, 147.64, 149.09,
176.85; IR (neat): 1510 (C]C), 1774 (C]O) cmꢀ1; MS (EI) m/z 278
(Mþ); HRMS (EI): calcd for C16H22O4: 278.1518 (Mþ), found:
278.1512; ½a 2D6
ꢃ
þ1.2 (c 1.05, CHCl3).
found: 490.2383; ½a D24
ꢀ17.9 (c 1.14, CHCl3).
ꢃ
4.1.3.15. (3R,4R)-3-(4-Benzyloxy-3-methoxybenzyl)-4-(3-ethoxy-
4-methoxybenzyl)dihydrofuran-2-one (14a). To a stirredsolution of
13b (29.6 mg, 0.12 mmol) inTHF (2 mL) were added LiHMDS (1.6 M in
4.1.3.19. (3R,4R)-3-(4-Benzyloxy-3-propoxybenzyl)-4-(3-ethoxy-4-
methoxybenzyl)dihydrofuran-2-one (14e). By the procedure similar
to preparation of 14a,14e was prepared from 13b and 4-benzyloxy-3-
THF, 0.12 mL, 0.18 mmol), HMPA (31
m
L, 0.18 mmol) at ꢀ78 ꢂC, and the
propoxybenzyl
propoxybenzaldehyde [22], (40%) as a pale yellow oil: 1H NMR
(400 MHz, CDCl3)
bromide,
prepared
from
4-benzyl-3-
resulting solution was stirred at the same temperature for 0.5 h. To
the reaction mixture was added a solution of 4-benzyloxy-3-
methoxybenzyl bromide [20] (52.3 mg, 0.19 mmol) in THF (2 mL),
and allowed to warm to room temperature over 1 h, and then stirred
at the same temperature for 20 h. The reaction was quenched with
H2O (4 mL), and the aqueous mixture was extracted with Et2O
(10 mL ꢁ 3). The organic extracts were combined, dried over MgSO4,
and evaporated to give residue, which waschromatographed on silica
gel (10 g, hexane:acetone ¼ 4:1) to give 14a (25 mg, 44%) as a pale
d
: 1.05 (3H, t, J ¼ 7.1 Hz),1.45 (3H, t, J ¼ 7.8 Hz),1.84
(2H, sextet, J ¼ 7.1 Hz), 2.46e2.64 (4H, m), 2.86e2.99 (2H, m), 3.80e
3.87 (4H, m), 3.94 (2H, t, J ¼ 7.1 Hz), 4.00 (2H, q, J ¼ 7.8 Hz), 4.06e4.11
(1H, m), 5.10 (2H, s), 6.48e6.82 (6H, m), 7.28e7.44 (5H, m); 13C NMR
(100 MHz, CDCl3) d: 10.47, 14.75, 22.56, 34.47, 38.00, 41.10, 46.40,
55.87, 64.25, 70.53, 71.35,111.55,113.29,114.71,115.25,120.52,121.37,
127.18, 127.57, 127.63, 128.33, 130.35, 131.126, 137.34, 147.40, 148.07,
148.26, 149.57, 178.64; IR (neat): 1514 (C]C), 1771 (C]O) cmꢀ1; MS
(EI) m/z 504 (Mþ); HRMS(EI): calcd for C31H36O6: 504.2512 (Mþ),
yellow oil: 1H NMR (300 MHz, CDCl3)
d
: 1.44 (3H, t, J ¼ 6.9 Hz), 2.46e
2.65 (4H, m), 2.91e2.95 (2H, m), 3.79e3.90 (1H, m), 3.84 (6H, s), 4.01
(2H, q, J ¼ 6.9 Hz), 4.08e4.20 (1H, m), 5.12 (2H, s), 6.50e6.80 (6H, m),
found: 504.2538; ½a D24
ꢀ10.7 (c 0.75, CHCl3).
ꢃ
7.28e7.43 (5H, m); 13C NMR (75 MHz, CDCl3)
d: 14.77, 34.48, 38.02,
4.1.3.20. (3R,4R)-3-(4-Benzyloxy-3-methoxybenzyl)-4-(3,4-
diethoxybenzyl)dihydrofuran-2-one (14f). By the procedure similar
to preparation of 14a, 14f was prepared from 13d and 4-benzyloxy-
3-methoxybenzyl bromide [20] (48%) as a pale yellow oil: 1H NMR
41.09, 46.42, 55.90, 64.28, 65.18, 71.03, 71.16, 111.57, 112.85, 113.35,
113.92, 114.02, 120.54, 121.28, 127.17, 127.20, 127.76, 128.46, 130.32,
130.82, 137.06, 147.01, 148.09, 148.26, 149.73, 178.65; IR (neat): 1515
(C]C),1770 (C]O) cmꢀ1; MS (EI) m/z 476 (Mþ); HRMS (EI): calcd for
(300 MHz, CDCl3) d: 1.41e1.59 (6H, m), 2.43e2.63 (4H, m), 2.91e
C29H32O6: 476.2199 (Mþ), found: 476.2197; ½a 2D5
ꢃ
ꢀ16.4 (c 0.77, CHCl3).
2.95 (2H, m), 3.82e3.90 (1H, m), 3.85 (3H, s), 3.82e3.89 (1H, m),
3.97e4.12 (5H, m), 5.12 (2H, s), 6.49e6.80 (6H, m), 7.26e7.44 (5H,
4.1.3.16. (3R,4R)-3-(4-Benzyloxy-3-ethoxybenzyl)-4-(3-ethoxy-4-
methoxybenzyl)dihydrofuran-2-one (14b). By the procedure similar
to preparation of 14a, 14b was prepared from 13b and 4-benzyloxy-
3-ethoxybenzyl bromide [21] (59%) as a pale yellow oil: 1H NMR
m); 13C NMR (75 MHz, CDCl3)
d: 14.85, 34.50, 38.07, 41.12, 46.50,
55.96, 64.59, 71.09, 71.21, 112.90, 113.63, 114.06, 114.20, 120.78,
121.33, 127.26, 127.81, 128.51, 130.50, 130.86, 137.12, 147.06, 147.60,
148.77,149.76,178.70; IR (neat): 1509 (C]C),1772 (C]O) cmꢀ1; MS
(EI) m/z 490 (Mþ); HRMS (EI): calcd for C30H34O6: 490.2355 (Mþ),
(300 MHz, CDCl3) d: 1.34e1.40 (6H, m), 2.36e2.51 (4H, m), 2.81e
2.85 (2H, m), 3.71e3.78 (1H, m), 3.75 (3H, s), 3.90e4.05 (5H, m),
found: 490.2388; ½a D25
ꢀ13.5 (c 0.98, CHCl3).
ꢃ
5.02 (2H, s), 6.40e6.80 (6H, m), 7.14e7.35 (5H, m); 13C NMR
(75 MHz, CDCl3)
d
: 14.75, 14.82, 34.43, 37.99, 41.05, 46.39, 55.86,
4.1.3.21. (3R,4R)-3-(4-Benzyloxy-3-ethoxybenzyl)-4-(3,4-diethoxyb-
enzyl)dihydrofuran-2-one (14g). By the procedure similar to prepa-
ration of 14a, 14g was prepared from 13d and 4-benzyloxy-3-
ethoxybenzyl bromide [21] (56%) as a pale yellow oil: 1H NMR
64.22, 64.47, 71.14, 71.26, 111.51, 113.24, 114.59, 114.97, 120.51,
121.39, 127.11, 127.64, 128.35, 130.31, 130.99, 137.27, 147.33, 148.03,
148.23, 149.24, 178.65; IR (neat): 1507 (C]C), 1771 (C]O) cmꢀ1
;

N. Kudou et al. / European Journal of Medicinal Chemistry 60 (2013) 76e88
85
(300 MHz, CDCl3)
(1H, m), 4.02e4.13 (7H, m), 5.11 (2H, s), 6.47e6.81 (6H, m), 7.27e7.44
(5H, m); 13C NMR (75 MHz, CDCl3)
: 14.87, 34.50, 38.09, 41.11, 46.50,
d
: 1.41e1.44 (9H, m), 2.42e2.60 (4H, m), 3.82e3.86
(neat): 1516 (C]C), 1768 (C]O) cmꢀ1; MS (EI) m/z 400 (Mþ); HRMS
(EI): calcd for C23H28O6: 400.1886 (Mþ), found: 400.1868;
d
½
a 2D7
ꢃ
ꢀ16.9 (c 1.13, CHCl3).
64.60, 71.22, 71.37, 113.63, 114.17, 114.69, 115.08, 120.79, 121.48, 127.21,
127.71, 128.42, 130.53, 131.07, 137.35, 147.41, 147.95, 148.78, 149.32,
178.73; IR (neat): 1514 (C]C), 1770 (C]O) cmꢀ1; MS (EI) m/z 504
(Mþ); HRMS (EI): calcd for C31H36O6: 504.2512 (Mþ), found:
4.1.3.26. (3R,4R)-4-(4-Ethoxy-3-methoxybenzyl)-3-(4-hydroxy-3-
methoxybenzyl)dihydrofuran-2-one (4i). By the procedure similar
to preparation of 4g, 4i was prepared from 14c (57%) as a pale
504.6139; ½a 2D5
ꢃ
ꢀ12.0 (c 0.58, CHCl3).
yellow oil: 1H NMR (300 MHz, CDCl3)
d: 1.45 (3H, t, J ¼ 7.1 Hz),
2.44e2.67 (4H, m), 2.93 (2H, d, J ¼ 5.8 Hz), 3.81 (3H, s), 3.82 (3H, s),
3.84e3.99 (1H, m), 4.03e4.15 (1H, m), 4.08 (2H, q, J ¼ 7.1 Hz), 5.30
(1H, br), 6.47e6.66 (4H, m), 6.75 (1H, d, J ¼ 8.0 Hz), 6.82 (1H, d,
4.1.3.22. (3R,4R)-3-(4-Benzyloxy-3-propoxybenzyl)-4-(4-methoxy-
3-propoxybenzyl)dihydrofuran-2-one (14h). By the procedure
similar to preparation of 14a, 14h was prepared from 13e and 4-
benzyloxy-3-propoxybenzyl bromide (49%) as a pale yellow oil:
J ¼ 8.0 Hz); 13C NMR (75 MHz, CDCl3)
d: 14.90, 30.99, 34.53, 38.22,
40.97, 46.63, 55.89, 64.35, 71.31, 111.48, 112.00, 112.71, 114.04,
120.47, 122.01, 129.37, 130.30, 144.39, 146.54, 146.99, 149.18, 178.56;
IR (neat): 1749 (C]O), 3648 (OH) cmꢀ1; MS (EI) m/z 386 (Mþ);
HRMS (EI): calcd for C22H26O6: 386.1729 (Mþ), found: 386.1693;
1H NMR (400 MHz, CDCl3)
d: 1.02e1.08 (6H, m), 1.82e1.88 (4H, m),
2.45e2.63 (4H, m), 2.85e2.97 (2H, m), 3.83 (3H, s), 3.83e4.60 (6H,
m), 5.10 (2H, s), 6.51e6.96 (6H, m), 7.28e7.45 (5H, m); 13C NMR
(100 MHz, CDCl3)
d: 10.48, 14.84, 22.56, 34.46, 38.00, 41.07, 46.45,
½
a 2D6
ꢃ
ꢀ9.5 (c 0.71, CHCl3).
64.71, 65.15, 70.55, 70.74, 71.18, 71.35, 71.37, 112.76, 114.42, 115.12,
119.34, 120.77, 121.40, 127.13, 127.15, 127.64, 128.34, 128.36, 130.63,
131.13, 137.36, 147.98, 149.58, 178.73; IR (neat): 1514 (C]C), 1771
(C]O) cmꢀ1; MS (EI) m/z 518 (Mþ); HRMS(EI): calcd for C32H38O6:
4.1.3.27. (3R,4R)-4-(3-Ethoxy-4-hydroxybenzyl)-3-(4-ethoxy-3-
methoxybenzyl)dihydrofuran-2-one (4j). By the procedure similar
to preparation of 4g, 4j was prepared from 14d (63%) as a pale
a 25
þ
518.2668 (M ), found: 518.2669; ½ ꢃD ꢀ12.2 (c 0.75, CHCl3).
yellow oil: 1H NMR (300 MHz, CDCl3)
d: 1.39e1.46 (6H, m), 2.41e
2.66 (4H, m), 2.91 (2H, d, J ¼ 6.0 Hz), 3.80 (3H, s), 3.81e3.87 (1H,
4.1.3.23. (3R,4R)-3-(4-Benzyloxy-3-propoxybenzyl)-4-(4-ethoxy-3-
propoxybenzyl)dihydrofuran-2-one (14i). By the procedure similar
to preparation of 14a, 14i was prepared from 13f and 4-benzyloxy-
3-propoxybenzyl bromide (33%) as a pale yellow oil: 1H NMR
m), 4.00e4.10 (5H, m), 5.64 (1H, br), 6.47e6.65 (4H, m), 6.74 (1H, d,
J ¼ 8.2 Hz), 6.82 (1H, d, J ¼ 8.2 Hz); 13C NMR (75 MHz, CDCl3)
d:
14.75, 30.86, 34.35, 38.06, 40.84, 46.54, 55.78, 64.26, 64.37, 71.24,
111.98, 112.38, 112.69, 114.00, 120.52, 121.95, 129.32, 130.38, 144.58,
145.91, 147.07, 149.25; IR (neat): 1771 (C]O), 3548 (OH) cmꢀ1; MS
(EI) m/z 400 (Mþ); HRMS (EI): calcd for C23H28O6: 400.1886 (Mþ),
(400 MHz, CDCl3)
d
: 1.02e1.08 (6H, m), 1.41 (3H, t, J ¼ 7.1 Hz), 1.80e
1.91 (4H, m), 2.41e2.63 (4H, m), 2.87e2.94 (2H, m), 3.82e3.96 (5H,
m), 4.01 (2H, q, J ¼ 7.1 Hz), 4.05e4.10 (1H, m), 5.10 (2H, s), 6.49e
found: 400.1897; ½a D26
ꢀ12.4 (c 1.04, CHCl3).
ꢃ
6.96 (6H, m), 7.28e7.45 (5H, m); 13C NMR (100 MHz, CDCl3)
d:
10.50, 22.59, 34.51, 38.05, 41.12, 46.48, 56.00, 65.22, 70.57, 71.40,
111.86, 112.79, 113.63, 114.72, 115.30, 119.36, 120.55, 121.40, 127.16,
127.28, 127.65, 128.36, 130.41, 131.16, 137.37, 147.43, 148.25, 148.58,
149.62, 178.70; IR (neat): 1508 (C]C), 1767 (C]O) cmꢀ1; MS (EI) m/
z 532 (Mþ); HRMS (EI): calcd for C33H40O6: 532.2825 (Mþ), found:
4.1.3.28. (3R,4R)-4-(3-Ethoxy-4-methoxybenzyl)-3-(4-hydroxy-3-
propoxybenzyl)dihydrofuran-2-one (4k). By the procedure similar
to preparation of 4g, 4k was prepared from 14e (56%) as a pale
yellow oil: 1H NMR (400 MHz, CDCl3)
d: 1.04 (3H, t, J ¼ 7.4 Hz), 1.45
(3H, t, J ¼ 7.1 Hz), 1.82 (2H, sextet, J ¼ 7.4 Hz), 2.48e2.63 (4H, m),
2.91 (2H, d, J ¼ 5.9 Hz), 3.81e3.88 (4H, m), 3.93 (2H, t, J ¼ 7.4 Hz),
4.02 (2H, q, J ¼ 7.1 Hz), 4.06e4.12 (1H, m), 5.57 (1H, s), 6.48 (1H, s),
6.54 (1H, d, J ¼ 10.2 Hz), 6.60 (1H, d, J ¼ 10.2 Hz), 6.66 (1H,s), 6.75
(1H, d, J ¼ 8.2 Hz), 6.82 (1H, d, J ¼ 8.2 Hz); 13C NMR (100 MHz,
518.2817; ½a 2D5
ꢀ6.3 (c 0.80, CHCl3).
ꢃ
4.1.3.24. (3R,4R)-4-(3-Ethoxy-4-methoxybenzyl)-3-(4-hydroxy-3-
methoxybenzyl)dihydrofuran-2-one (4g). To a stirred solution of 14a
(47.5 mg, 0.10 mmol) in MeOH (5 mL) was added 20% Pd(OH)2
(20 mg), and the resulting suspension was stirred under a hydrogen
atmosphere at 1 atm for 20 h. The catalyst was removed by filtra-
tion and the filtrate was evaporated to give a residue, which was
chromatographed on silica gel (10 g, hexane:acetone ¼ 3:1) to give
CDCl3) d: 10.43,14.79, 22.49, 34.41, 38.05, 40.95, 46.54, 55.92, 64.29,
70.32, 71.24, 111.55, 112.43, 113.26, 114.01, 120.57, 121.93, 129.37,
130.37, 144.64, 146.05, 148.12, 148.33, 178.75; IR (neat): 1516 (C]C),
1769 (C]O), 3589 (OH) cmꢀ1; MS (EI) m/z 414 (Mþ); HRMS (EI):
calcd for C24H30O6: 414.2042 (Mþ), found: 414.2046; ½a 2D6
ꢀ10.6 (c
ꢃ
4g (34.1 mg, 89%) as a pale yellow oil: 1H NMR (300 MHz, CDCl3)
d:
1.10, CHCl3).
1.45 (3H, t, J ¼ 7.1 Hz), 2.43e2.65 (4H, m), 2.91e2.94 (2H, m), 3.81e
3.89 (1H, m), 3.83 (3H, s), 3.84 (3H, s), 4.01 (2H, q, J ¼ 7.1 Hz), 4.12
(1H, dd, J ¼ 9.1, 6.9 Hz), 5.53 (1H, s), 6.47e6.65 (4H, m), 6.69 (1H, d,
4.1.3.29. (3R,4R)-4-(3,4-Diethoxybenzyl)-3-(4-hydroxy-3-methoxyb-
enzyl)dihydrofuran-2-one (4l). By the procedure similar to prepara-
tion of 4g, 4l was prepared from 14f (81%) as a pale yellow oil: 1H
J ¼ 8.0 Hz), 6.82 (1H, d, J ¼ 8.0 Hz); 13C NMR (75 MHz, CDCl3)
d:
14.80, 30.91, 34.46, 38.09, 40.95, 46.55, 55.83, 55.94, 64.30, 71.27,
99.88, 111.54, 113.26, 114.10, 120.58, 122.08, 129.47, 130.35, 144.52,
146.67, 148.11, 148.33; IR (neat): 1513 (C]C), 1771 (C]O) cmꢀ1; MS
(EI) m/z 386 (Mþ); HRMS (EI): calcd for C22H26O6: 386.1729 (Mþ),
NMR (300 MHz, CDCl3) d: 1.25e1.45 (6H, m), 2.44e2.66 (4H, m), 2.92
(2H, d, J ¼ 6.0 Hz), 3.83 (3H, s), 3.85e3.89 (1H, m), 3.98e4.13 (5H, m),
5.55 (1H, br), 6.49e6.67 (4H, m), 6.76 (1H, d, J ¼ 7.8 Hz), 6.82 (1H, d,
J ¼ 7.8 Hz); 13C NMR (75 MHz, CDCl3)
d: 14.84, 30.91, 34.40, 38.04,
found: 386.1693; ½a D25
ꢃ
ꢀ17.2 (c 1.44, CHCl3).
40.94, 46.56, 55.84, 64.58, 71.25, 111.56, 113.60, 114.11, 120.77, 122.10,
129.47, 130.51, 144.51, 146.65, 147.57, 148.78, 178.75; IR (neat): 1766
(C]O), 2978 (OH) cmꢀ1; MS (EI) m/z 400 (Mþ); HRMS (EI): calcd for
4.1.3.25. (3R,4R)-4-(3-Ethoxy-4-hydroxybenzyl)-3-(3-ethoxy-4-
methoxybenzyl)dihydrofuran-2-one (4h). By the procedure similar
to preparation of 4g, 4h was prepared from 14b (63%) as a pale
C23H28O6: 400.1886 (Mþ), found: 400.1858; ½a 2D7
ꢀ16.0 (c 1.33,
ꢃ
CHCl3).
yellow oil: 1H NMR (300 MHz, CDCl3)
d: 1.42e1.47 (6H, m), 2.46e
2.63 (4H, m), 2.92 (2H, d, J ¼ 5.8 Hz), 3.81e3.89 (1H, m), 3.84
4.1.3.30. (3R,4R)-4-(3,4-Diethoxybenzyl)-3-(4-hydroxy-3-ethox-
ybenzyl)dihydrofuran-2-one (4m). By the procedure similar to
preparation of 4g, 4m was prepared from 14g (66%) as a pale yellow
(3H, s), 3.97e4.12 (5H, m), 5.60 (1H, br), 6.48e6.84 (6H, m); 13C
NMR (75 MHz, CDCl3) d: 14.78, 30.88, 34.39, 38.04, 40.90, 46.53,
55.90, 64.26, 64.39, 71.24, 111.51, 112.37, 113.21, 114.01, 120.55,
121.95, 129.34, 130.35, 144.59, 145.93, 148.08, 148.03, 178.74; IR
oil: 1H NMR (300 MHz, CDCl3)
d: 1.40e1.46 (9H, m), 2.42e2.67 (4H,
m), 2.91 (2H, d, J ¼ 5.7 Hz), 3.85 (1H, dd, J ¼ 9.1, 7.4 Hz), 3.97e4.12

86
N. Kudou et al. / European Journal of Medicinal Chemistry 60 (2013) 76e88
(7H, m), 5.59 (1H, br), 6.49e6.66 (4H, m), 6.75 (1H, d, J ¼ 8.0 Hz), 6.82
4.01e4.24 (8H, m), 6.68e6.78 (3H, m); 13C NMR (75 MHz, CDCl3)
d:
(1H, d, J ¼ 8.0 Hz); 13C NMR (75 MHz, CDCl3)
d: 14.81, 14.85, 34.39,
13.77, 13.83, 14.57, 14.60, 34.06, 41.37, 53.82, 61.11, 64.19, 113.33,
114.09, 120.83, 130.28, 147.28, 148.33,166.34, 168.64; IR (neat): 1516
(C]C), 1731 (C]O) cmꢀ1; MS (EI) m/z 338 (Mþ); HRMS (EI): calcd
for C23H28O6: 338.1729 (Mþ), found: 338.1766.
38.06, 40.92, 46.58, 64.42, 64.59, 71.27, 112.43, 113.62, 114.05, 114.10,
120.80, 122.00, 129.37, 130.53, 144.61, 145.94, 147.58, 148.80, 178.79;
IR (neat): 1516 (C]C), 1761 (C]O) cmꢀ1; MS (EI) m/z 414 (Mþ);
HRMS (EI): calcd for C24H30O6: 414.2042 (Mþ), found: 414.2024;
½
a 2D5
ꢃ
ꢀ14.0 (c 0.70, CHCl3).
4.1.4.2. (R)-Acetic acid 3-(3,4-diethoxyphenyl-2-hydroxymethylpropyl
ester (18). To a stirred solution of 17 (1.43 g, 4.23 mmol) in THF
(40 mL) was added LiAlH4 (401 mg, 10.6 mmol) at 0 ꢂC, and the
resulting suspension was refluxed for 12 h. The reaction was
quenched with 10% NaOH (aq) (20 mL), and the mixture was
extracted with AcOEt (20 mL ꢁ 5). The organic extracts were
combined dried over MgSO4, and the solvent was evaporated to give
diol, which was used directly in the next step. To a stirred solution of
the diol obtained above in i-Pr2OeTHF (15 mL, 4:1) were added
Lipase-PS (323 mg) and vinyl acetate (0.45 ml, 4.85 mmol), and the
reaction mixture was stirred at room temperature for 2 h. The
catalyst was filtered and the filtrate was evaporated to give residue,
4.1.3.31. (3R,4R)-4-(4-Methoxy-3-propoxybenzyl)-3-(4-hydroxy-3-
propoxybenzyl)dihydrofuran-2-one (4n). By the procedure similar
to preparation of 4g, 4n was prepared from 14h (46%) as a pale
yellow oil: 1H NMR (400 MHz, CDCl3)
d: 1.02e1.07 (6H, m), 1.78e
1.90 (4H, m), 2.47e2.65 (4H, m), 2.92 (2H, d, J ¼ 5.9 Hz), 3.81 (3H,
s), 3.81e3.95 (5H, m), 4.08e4.12 (1H, m), 5.56 (1H, br), 6.50 (1H, s),
6.53 (1H, d, J ¼ 7.9 Hz), 6.60 (1H, d, J ¼ 7.9 Hz), 6.66 (1H, s), 6.75 (1H,
d, J ¼ 8.2 Hz), 6.82 (1H, d, J ¼ 8.2 Hz); 13C NMR (100 MHz, CDCl3)
d:10.39, 22.45, 22.47, 34.37, 38.01, 40.91, 46.54, 56.00, 70.29, 70.48,
71.23, 77.21, 111.81, 112.43, 113.53, 114.00, 120.55, 121.90, 129.34,
130.41, 144.61, 146.03, 148.22, 148.58, 178.74; IR (neat): 1516 (C]C),
1767 (C]O), 3422 (OH) cmꢀ1; MS (EI) m/z 428 (Mþ); HRMS (EI):
which
was
chromatographed
on
silica
gel
(30
g,
hexane:acetone ¼ 4:1) to give 18 (669 mg, 53% in 2 steps) as a pale
calcd for C25H32O6: 428.2199 (Mþ), found: 428.2216; ½a 2D5
ꢀ13.7 (c
ꢃ
yellow oil. The enantiomeric excess of 18 was determined to be a 98%
0.70, CHCl3).
ee by the Moscher’s method [24]. 1H NMR (300 MHz, CDCl3)
d: 1.39e
1.44 (6H, m), 2.06 (3H, s), 2.23 (1H, br), 2.49e2.64 (2H, m), 3.45e3.59
(2H, m), 4.01e4.08 (6H, m), 4.15 (1H, dd, J ¼ 11.3, 4.7 Hz), 6.66e6.70
4.1.3.32. (3R,4R)-4-(4-Ethoxy-3-propoxybenzyl)-3-(4-hydroxy-3-
propoxybenzyl)dihydrofuran-2-one(4o). By the procedure similar to
preparation of 4g, 4o was prepared from 14i (63%) as a pale yellow
(2H, m), 6.78 (1H, d, J ¼ 8.0 Hz); 13C NMR (75 MHz, CDCl3)
d: 14.88,
20.91, 33.86, 42.53, 62.07, 64.03, 64.55, 64.63, 113.69, 114.52, 121.22,
131.91, 147.23, 148.70, 171.68; IR (neat): 1513 (C]C), 1721 (C]O)
cmꢀ1; MS (EI) m/z 296 (Mþ); HRMS (EI): calcd for C16H24O5:
oil: 1H NMR (400 MHz, CDCl3)
d: 1.02e1.07 (6H, m), 1.42 (3H, t,
J ¼ 7.1 Hz), 1.80e1.86 (4H, m), 2.41e2.63 (4H, m), 2.92 (2H, d,
J ¼ 5.9 Hz), 3.83e3.96 (5H, m), 4.01 (2H, q, J ¼ 7.1 Hz), 4.07e4.11
(1H, m), 5.57 (1H, s), 6.51e6.84 (6H, m); 13C NMR (100 MHz,
296.1624 (Mþ), found: 296.1594; ½a 2D8
þ18.8 (c 1.47, CHCl3); 98% ee.
ꢃ
CDCl3)
d
: 10.50, 22.59, 34.51, 38.05, 41.12, 46.48, 56.00, 65.22, 70.57,
4.1.4.3. (R)-Acetic acid 3-(3,4-Diethoxyphenyl-2-methanesulfony-
loxymethylpropyl ester (19). To a stirred solution of 18 (1.45 g,
4.96 mmol) in CH2Cl2 (25 mL) were added MsCl (0.42 ml, 5.45 mmol)
and NEt3 (0.89 ml, 6.45 mmol) at 0 ꢂC, and the reaction mixture was
stirred at room temperature for 0.5 h. The reaction was quenched
with H2O (20 mL), and the aqueous mixture was extracted with
CH2Cl2 (20 mL ꢁ 3). The organic extracts were combined dried over
MgSO4, and evaporated. The residue was chromatographed on silica
gel (40 g, hexane:acetone ¼ 4:1) to give 19 (1.48 g, 79%) as a pale
71.40, 111.86, 112.79, 113.63, 114.72, 115.30, 119.36, 120.55, 121.40,
127.16, 127.28, 127.65, 128.36, 130.41, 131.16, 137.37, 147.43, 148.25,
148.58, 149.62, 178.7010.45, 14.87, 22.50, 22.60, 34.37, 38.04, 40.92,
46.58, 64,76, 70.33, 70.76, 71.26, 100.36, 112.48, 114.02, 114.06,
114.38, 129.37, 130.67, 144.63, 146.04, 147.70, 149.14; IR (neat): 1508
(C]C), 1770 (C]O) cmꢀ1; MS (EI) m/z 442 (Mþ); HRMS (EI): calcd
for C26H34O6: 442.2355 (Mþ), found: 442.2350; ½a 2D5
ꢀ12.9 (c 0.50,
ꢃ
CHCl3).
yellow oil: 1H NMR (300 MHz, CDCl3)
d: 1.41e1.46 (6H, m), 2.08 (3H,
4.1.4. Effective synthesis of (3R,4R)-4-(3,4-diethoxybenzyl)-3-(4-
hydroxy-3-ethoxybenzyl)dihydrofuran-2-one (4m)
s), 2.32e2.36 (1H, m), 2.65 (2H, d, J ¼ 7.4 Hz), 2.99 (3H, s), 4.00e4.23
(8H, m), 6.65e6.70 (2H, m), 6.80 (1H, d, J ¼ 8.0 Hz); 13C NMR
4.1.4.1. 2-(3,4-Diehtoxybenzyl)malonic acid diethyl ester (17).
To a stirred solution of (3,4-diehtoxyphenyl)methanol (16) [16,23]
(733 mg, 3.74 mmol) in CH2Cl2 (20 mL) were added NEt3
(0.67 mL, 4.86 mmol) and MsCl (0.32 mL, 4.11 mmol) at 0 ꢂC, and
the reaction mixture was stirred at room temperature for 0.5 h. The
reaction was quenched with sat. NaHCO3 (aq) (10 mL), and the
organic layer were separated. The aqueous layer was extracted with
CH2Cl2 (20 mL ꢁ 3), and the organic layer and extracts were
combined, dried over MgSO4. The solvent was removed under
reduced pressure to give a pale yellow oil, which was used directly
in the next step. To a stirred solution of diethyl malonate (1.14 ml,
7.48 mmol) in DMF (20 mL) was added NaH (60%, 299 mg,
7.48 mmol) at 0 ꢂC, and the resulting mixture was stirred at room
temperature for 1 h. To the solution was added a solution of the oil
obtained above in DMF (2 mL) at 0 ꢂC, and the reaction mixture was
stirred at room temperature for 25 h. The reaction was quenched
with sat. NaHCO3 (aq) (10 mL), and the aqueous mixture was
extracted with Et2O (20 mL ꢁ 3). The organic extracts were
combined, dried over MgSO4, evaporated to give a pale yellow oil
(75 MHz, CDCl3) d: 14.85, 20.81, 30.90, 33.49, 37.21, 39.71, 63.02,
64.60, 68.48, 113.70, 114.40, 121.22, 130.36, 147.53, 148.82, 170.78; IR
(neat): 1512 (C]C), 1735 (C]O) cmꢀ1; MS (EI) m/z 374 (Mþ); HRMS
(EI): calcd for C17H26O7S: 374.1399 (Mþ), found: 374.1362; ½a 2D5
þ2.1
ꢃ
(c 0.68, CHCl3).
4.1.4.4. (R)-4-(3,4-Diethoxybenzyl)dihydrofuran-2-one (13d) from
19. To a stirred solution of 19 (1.12 g, 3.00 mmol) in DMSO (25 mL)
was added KCN (205 mg, 3.00 mmol), and the resulting mixture
was heated at 90 ꢂC for 3 h. After cooling, the reaction was
quenched with H2O (25 mL), and the aqueous mixture was
extracted with Et2O/AcOEt (1:1, 20 mL ꢁ 3). The organic extracts
were combined, dried over MgSO4, and evaporated to give cyanide,
which was used directly in the next step. To a stirred solution of
cyanide obtained above in THFeH2O (3:1, 12 mL) was added
LiOH$H2O (126 mg, 3.00 mmol), and the reaction mixture was
stirred at room temperature for 24 h. The reaction mixture was
diluted with H2O (10 mL), and the aqueous mixture was extracted
with Et2O (20 mL ꢁ 3). The organic extracts were combined, dried
over MgSO4, and evaporated to give alcohol, which was used
directly in the next step. The alcohol obtained above was dissolved
in 10% NaOH (aq) (15 mL), and the mixture was refluxed for 5 h.
After cooling, 10% HCl (aq) (30 mL) and THF (30 mL) were added to
which was chromatographed on
hexane:acetone ¼ 15:1) to give 17 (1.10 g, 87% in 2 steps) as a pale
yellow oil: 1H NMR (300 MHz, CDCl3)
: 1.15e1.30 (6H, m), 1.39e
1.46 (6H, m), 3.13 (2H, d, J ¼ 8.0 Hz), 3.59 (1H, t, J ¼ 8.0 Hz),
silica
gel
(20
g,
d

N. Kudou et al. / European Journal of Medicinal Chemistry 60 (2013) 76e88
87
the reaction mixture, and the resulting solution was stirred at room
temperature for 50 h. The aqueous reaction mixture was extracted
with Et2O (30 mL ꢁ 3), and the organic extracts were combined,
dried over MgSO4, and evaporated to give a residue, which was
chromatographed on silica gel (30 g, hexane:acetone ¼ 3:1) to give
13d (475 mg, 60% in 3 steps) as a pale yellow oil.
Results are expressed as means ꢄSD. Statistical comparisons were
conducted using Student’s t test after ANOVA. The results were
considered to be significant when P < 0.05.
Acknowledgments
This work was supported in part by grants from the Ministry of
Health and Welfare for the Third-Term Comprehensive 10-Year
Strategy for Cancer Control and by Grant-in-Aid for Scientific
Research (C) (No. 22590098) from Japan Society for the Promotion
of Science (JSPS).
4.2. In vitro preferential cytotoxicity
4.2.1. Cells and culture
Human pancreatic cancer cell lines, PANC-1 and CAPAN-1, were
maintained in Dulbecco’s modified Eagle’s medium (DMEM, Nissui
Pharmaceutical Co., Ltd., Tokyo, Japan) supplemented with 10%
fetal bovine serum (FBS, Gibco BRL Products, Gaithersburg, MD,
USA), 0.1% sodium bicarbonate (Nacalai Tesque Inc.), and 1%
antibiotic-antimycotic solution (SigmaeAldrich Inc., St. Louis, MO,
USA). Nutrient deprived medium (NDM) contained 265 mg/L
CaCl2$2H2O, 0.1 mg/L Fe(NO3)$9H2O, 400 mg/L KCl, 200 mg/L
MgSO4$7H2O, 6400 mg/L NaCl, 700 mg/L NaHCO3, 125 mg/L
NaH2PO4, 15 mg/L phenol red, 1 M HEPES buffer (pH 7.4, Wako Pure
Chemical Industries, Ltd., Osaka, Japan), and 10 mL MEM vitamin
solution (Life Technologies, Inc., Rockville, MD, USA). The final pH
was adjusted to 7.4 with 10% NaHCO3. For amino acid supplemen-
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi:10.1016/j.ejmech.2012.11.031H.
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After24 hofincubation, thecells werewashed withPBS, and 100 mL of
pamoate,
6-(dimethylamino)-2-[2-(2,5-dimethyl-1-phenyl-1H-pyrrol-3-yl)
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Kumamoto, Japan) was added to the wells. After 3 h of incubation, the
absorbance was measured at 450 nm. Cell viability was calculated
from the mean values for three wells using the following equation:
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¼
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