Design, synthesis and evaluation of caffeic acid phenethyl ester-based inhibitors targeting a selectivity pocket in the active site of human aldo-keto reductase 1B10

Article abstract of DOI:10.1016/j.ejmech.2011.12.034

Inhibitors of a human aldo-keto reductase, AKR1B10, are regarded as promising therapeutics for the treatment of cancer, but those with both high potency and selectivity compared to the structurally similar aldose reductase (AKR1B1) have not been reported.

Full text of DOI:10.1016/j.ejmech.2011.12.034

European Journal of Medicinal Chemistry 48 (2012) 321e329
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Original article
Design, synthesis and evaluation of caffeic acid phenethyl ester-based inhibitors
targeting a selectivity pocket in the active site of human aldoeketo reductase 1B10
,
b
,
,
Midori Soda a 1, Dawei Hu c 1, Satoshi Endo a, Mayuko Takemura a, Jie Li c, Ryogo Wada c, Syohei Ifuku c,
Hai-Tao Zhao d, Ossama El-Kabbani d, Shozo Ohta e, Keiko Yamamura b, Naoki Toyooka c f, Akira Hara a,
,
Toshiyuki Matsunaga a
,
*
a Gifu Pharmaceutical University, 1-25-4 Daigaku-nishi, Gifu 501-1196, Japan
b School of Pharmacy, 1-100 Kusumoto-cho, Chikusa-ku, Aichi Gakuin University, Aichi 464-8650, Japan
c Graduate School of Science and Technology for Research, University of Toyama, 3190 Gohuku, Toyama 930-8555, Japan
d Monash Institute of Pharmaceutical Sciences, 381 Royal Parade, Parkville, Victoria 3052, Australia
e Nagaragawa Research Center, API Co., Ltd., 692-3 Yamasaki, Nagara, Gifu 502-0071, Japan
f GraduateSchool of Innovative Life Science, University of Toyama, 3190 Gohuku, Toyama 930-8555, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Inhibitors of a human aldoeketo reductase, AKR1B10, are regarded as promising therapeutics for the
treatment of cancer, but those with both high potency and selectivity compared to the structurally
similar aldose reductase (AKR1B1) have not been reported. In this study, we have found that, among
honeybee propolis products, caffeic acid phenethyl ester (CAPE) inhibited AKR1B10 (IC50 ¼ 80 nM) with
7-fold selectivity over AKR1B1. Based on a model of docked CAPE in AKR1B10, its derivatives were
designed, synthesized and evaluated for inhibitory potency. Among them, 3-(4-hydroxy-2-
methoxyphenyl)acrylic acid 3-(3-hydroxyphenyl)propyl ester (10c) was the most potent competitive
inhibitor (Ki ¼ 2.6 nM) with 790-fold selectivity for AKR1B10 over AKR1B1. Molecular docking of 10c and
site-directed mutagenesis of AKR1B10 residues suggested that the interactions between the 2-methoxy
and 3-hydroxy groups of 10c and the enzyme’s Val301 and Gln114, respectively, are important for the
Received 2 September 2011
Received in revised form
20 December 2011
Accepted 22 December 2011
Available online 29 December 2011
Keywords:
AKR1B10
Aldose reductase
Caffeic acid phenethyl ester
Inhibitor
inhibitor’s selectivity. Additionally, the sub-
metabolism and cellular proliferation in AKR1B10-overexpressing cells.
Ó 2011 Elsevier Masson SAS. All rights reserved.
mM concentration of 10c significantly suppressed the farnesal
Selectivity
Cancer
1. Introduction
hepatocellular carcinoma xenografts in mice [8]. Since AKR1B10
shows high catalytic efficiency towards aliphatic aldehydes, retinals
and isoprenyl aldehydes [1,6,7,9e11], its roles in cell carcinogenesis
and tumor development have been suggested to be detoxification
of cytotoxic carbonyls derived from lipid peroxidation [6,7,9],
decrease in retinoic acid synthesis [10], and regulation of cellular
fatty acid synthesis and lipid metabolism [7,11], which lead to cell
survival and proliferation. In addition, AKR1B10 is up-regulated in
cancer cell lines resistant to anticancer drugs, pre-activated cyclo-
phosphamide [12], and mitomycin C [13] of which the mitomycin C
resistance is suggested to be mediated by the enzyme’s ability to
detoxify cytotoxic aldehydes. AKR1B10 catalyzes the reduction of
anticancer drugs such as daunorubicin, oracin and idarubicin,
which is also thought to cause the chemoresistance of cancer cells
[14,15]. Thus, this enzyme has been recognized not only as
a potential diagnostic and/or prognostic marker, but also as a target
for the prevention and treatment of the above types of cancer [16].
Previously reported inhibitors of AKR1B10 included bile
acids [11,14], synthetic aldose reductase inhibitors [10,11,17],
A human member of the aldoeketo reductase (AKR) super-
family, AKR1B10, is a NADPH-dependent reductase, which was
originally identified as a human aldose reductase (AKR1B1)-like
protein that is up-regulated in hepatocellular carcinomas [1].
AKR1B10 is normally expressed in the intestine and colon with
lower levels in the liver, but overexpressed in human tumors, such
as smokers’ non-small cell lung carcinomas [2], uterine carcinomas
[3], cholangiocarcinomas [4], and early stage gastric cancer [5], as
well as hepatocellular carcinomas. The silencing of the AKR1B10
gene results in growth inhibition of cancer cells [6e8] and
Abbreviations: AKR, Aldoeketo reductase; AKR1B1, Human aldose reductase;
AKR1B10, A human member in the AKR1B subfamily of the AKR superfamily; CAPE,
Caffeic acid phenethyl ester.
* Corresponding author. Tel.: þ81 58 230 8100; fax: þ81 58 230 8105.
E-mail address: matsunagat@gifu-pu.ac.jp (T. Matsunaga).
These authors contributed equally to this work and share first authorship.
1
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.12.034

322
M. Soda et al. / European Journal of Medicinal Chemistry 48 (2012) 321e329
anti-inflammatory agents, flavonoids [18], additional plant poly-
phenols [19], 9-methyl-2,3,7-trihydroxy-6-fluorone [20], chro-
mene-3-carboxamide derivatives [21], triterpenoids [22], fibrates
[23], and prostaglandin A1 [24]. Among them, tolrestat, an aldose
reductase inhibitor, and the chromene-3-carboxamide derivatives
are potent inhibitors, but almost equally inhibit the structurally
similar AKR1B1. While AKR1B1 is involved in diabetic complica-
tions under hyperglycaemia conditions, it also plays important
roles in prostaglandin metabolism and detoxification of lipid per-
oxidation products and precursors of advanced glycation end
products [25e27]. In this respect, selective inhibition of AKR1B10
over AKR1B1 is required for the development of anticancer agents
mainly targeting AKR1B10. Although a triterpenoid, oleanolic acid,
was reported to be the most selective inhibitor to AKR1B10 over
AKR1B1, compared to other inhibitors its potency is not high [22].
Recent studies reported the crystal structure [28] and docked
models of AKR1B10-coenzyme-inhibitor complexes [11,18e22]. The
structural studies suggested several selectivity determinant resi-
dues (Gln114, Lys125, Val301, Gln303 and/or Ser304) that are not
conserved in AKR1B1, forming an inhibitor selectivity pocket in the
active site AKR1B10.
In order to synthesize more potent and selective inhibitors of
ARK1B10, we have searched lead compounds that can be struc-
turally modified to bind tightly and target the selectivity pocket of
the enzyme. Since natural substances, cinnamic acid and its
phenolic analogs, have been received attention as anticancer agents
[29], we compared the inhibitory effects of cinnamic acid deriva-
tives isolated from propolis on recombinant AKR1B10 and AKR1B1,
and found that caffeic acid phenethyl ester (CAPE) is the most
potent and selective inhibitor for AKR1B10. Based on the subse-
quent molecular docking of CAPE in AKR1B10, we designed and
synthesized CAPE-based derivatives that are expected to interact
more tightly with the residues in the selectivity pocket, and
evaluated them for enzyme inhibitory activity. We show that
(7-fold versus AKR1B1), contrast to selectivity of the other deriva-
tives to AKR1B1. In addition to its anticancer property [29],
CAPE exhibits inhibitory effects on the activities of matrix
metalloproteinase-2 and -9 [31], cyclooxygenase-1 and -2 [32], 5-
lipoxygenase [33], xanthine oxidase [34], 5
type 2 [35], catechol-O-methyltransferase [36], and HIV integrase
[37]. The IC50 values for these enzymes are 2e82 M, and are much
higher than that for AKR1B10 (0.08 M), suggesting that CAPE
a-reductase type 1 and
m
m
tightly binds to AKR1B10. CAPE exhibited a mixed-type inhibition
with respect to pyridine-3-aldehyde in the reduction reaction by
AKR1B10, and displayed a competitive inhibition with respect to
the alcohol substrate, geraniol, in the reverse reaction (the Ki value
was 46 ꢀ 2 nM). The different inhibition patterns in the forward
and reverse reactions were similar to other AKR1B10 inhibitors
[11,18e22], including tolrestat that binds to the substrate-binding
site in the crystal structure of the enzyme-NADPþ-inhibitor
ternary complex [28]. Thus, CAPE binds to the substrate-binding
site of AKR1B10, and its affinity for the enzyme-NADPþ binary
complex can be expressed only as the Ki value determined in the
geraniol oxidation, but not as the two Ki values obtained from the
mixed-type inhibition of the pyridine-3-aldehyde reductase
activity. The affinity (Ki value) for CAPE is higher than those for the
chromene-3-carboxamide derivatives (2.7e24 nM) [21], iso-
lithocholic acid (15 nM), tolrestat (46 nM) [11] and bisdemethox-
ycurcumin (22 nM) [19], but is lower than those for other known
AKR1B10 inhibitors [18e20,22].
2.2. Synthesis of CAPE derivatives based on its docked model
To design more potent and selective CAPE-based inhibitors, we
constructed a model of docked CAPE in the AKR1B10-NADPþ
complex (Fig. 1). In this model, CAPE was surrounded by 16 amino
acids, including the catalytic residues (Tyr49 and His111), which
form hydrogen bond interactions with the 4-hydroxy group of the
caffeic acid moiety. Van der Waals contacts were present between
a
derivative, 3-(4-hydroxy-2-methoxyphenyl)acrylic acid 3-(3-
hydroxyphenyl)propyl ester (10c) is a highly potent and selective
inhibitor for AKR1B10. Additionally, we analysed 10c for cellular
efficacy and proposed the binding mode of the inhibitor in the
active site of the enzyme.
2. Results and discussion
2.1. Inhibitory potency of constituents of propolis
The inhibitory effects of eight cinnamic acid derivatives isolated
from Brazilian propolis on the pyridine-3-aldehyde reductase
activities of AKR1B10 and AKR1B1 were compared (Table 1). The
compounds, except for 3,5-dicaffeoylquinic acid AKR1B10 [30],
were evaluated for the first time in this study. Among them, CAPE
exhibited the lowest IC50 value and highest selectivity to AKR1B10
Table 1
Inhibitory effects of cinnamic acid derivatives of propolis on activities of AKR1B10
and AKR1B1.
Compound
IC50 (mM)
AKR1B10
AKR1B1
CAPE
0.080 ꢀ 0.010
0.13 ꢀ 0.01
0.24 ꢀ 0.01
7.9 ꢀ 0.3
49 ꢀ 6
0.57 ꢀ 0.06
0.088 ꢀ 0.003
0.078 ꢀ 0.01
0.30 ꢀ 0.01
38 ꢀ 2
3,5-Dicaffeoylquinic acid
3,4-Dicaffeoylquinic acid
3-Caffeoylquinic acid
Drupanin
Artepillin C
p-Coumaric acid
Caffeic acid
Fig. 1. Model of docked CAPE in AKR1B10-NADPþ complex. The bound NADPþ and the
enzyme’s residues within 4.0 Å from CAPE are depicted. The distances between the
4-hydroxy group of CAPE and catalytic residues (Tyr49 and His111) are given in
angstroms.
53 ꢀ 4
23 ꢀ 1
63 ꢀ 6
76 ꢀ 3
90 ꢀ 8
32 ꢀ 1

M. Soda et al. / European Journal of Medicinal Chemistry 48 (2012) 321e329
323
Trp220 and the aromatic ring of the caffeic acid moiety (3.77 Å),
which was also stacked against Trp21. The phenethyl ester moiety
was positioned in the so-called selectivity pocket that is composed
of non-conserved residues Gln114, Val301, Q303 and Ser304 in
AKR1B10 (the respective residues in AKR1B1 are Thr, Leu, Ser and
Ala, respectively).
did not significantly improve the inhibitor potency, the
3-hydroxy derivative (10c) of 10a showed the lowest IC50 value
(6.2 nM) and highest selectivity (IC50 ratio of AKR1B1/
AKR1B10 ¼ 790) among the synthesized CAPE-based derivatives. In
addition, 30
mM 10c did not inhibit human aldehyde reductase
(AKR1A1) that is functionally similar to AKR1B1 and AKR1B10.
Compound 10c inhibited competitively with respect to the alcohol
substrate in the geraniol oxidation by AKR1B10, showing its Ki
value of 2.6 nM. The Ki value is comparable to those for (Z)-2-(4-
methoxyphenylimino)-7-hydroxy-N-(pyridin-2-yl)-2H-chromene-
3-carboxamide [21], which had been known as the most potent
AKR1B10 inhibitor. In addition, the inhibitory selectivity of 10c is
much superior to those of known potent inhibitors such as the
chromene-3-carboxamide derivative, isolithocholic acid, bisde-
methoxycurcumin and tolrestat, whose IC50 ratios of AKR1B1/
AKR1B10 are 1.8, 255, 85 and 0.3, respectively [11,18,19,21].
To confirm the interaction of the 4-hydroxy group of the caffeic
acid moiety suggested from the docked model, we synthesized
CAPE derivatives without both (1) [38] and either of the 3,4-
dihydroxy groups (2 and 3) [39]. Compound 3 with only the 4-
hydroxy group retained high inhibitory potency towards
AKR1B10 in contrast to marked declines in inhibition by 1 and 2,
and showed 4-fold higher selectivity (IC50 ratio of AKR1B1/
AKR1B10) than CAPE (Table 2). To evaluate the effect of the struc-
ture of the ester moiety on the inhibitory potency, the derivatives
with benzyl (4 [40] and 5 [41]), isopropyl (6) [42] and phenpropyl
esters (7 and 8) were synthesized. The derivatives (7e10d) were
synthesized using the approach outlined in Scheme 1. Compounds
4e6 with short ester moieties were less potent inhibitors compared
to CAPE and 3, whereas 8 with the 4-hydroxy group and phen-
propyl ester moiety showed greater inhibitory potency and selec-
tivity than 3.
The CAPE-docked model suggested that the introduction of
a small hydrophobic substituent at the 2-position of CAPE is ex-
pected to enhance the inhibitor potency through the hydrophobic
interaction with the side-chain of Phe123 or Val301 (Fig. 1). We
synthesized 2-methoxy derivatives of 3 and 8 (9a and 10a,
respectively). The introduction of the methoxy group slightly
improved the inhibitor potency towards AKR1B10, but significantly
decreased inhibition towards AKR1B1, resulting in a great increase
in selectivity.
2.3. Molecular docking and site-directed mutagenesis of inhibitor
binding residues
The underlying structural reason for the high inhibitory potency
and selectivity of 10c was examined by constructing a model of
docked 10c in the AKR1B10-NADPþ complex (Fig. 2). In this model,
the 4-hydroxy group of 10c closely interacted with the catalytic
residues, Tyr49 (3.40 Å) and His111 (2.94 Å), and thus the modeled
10c was bound more deeply to the active site of the enzyme than
the case of CAPE. The 2-methoxy group of 10c was close (3.25 Å) to
the side-chain of Val301, forming a tight hydrophobic interaction.
The indole ring of Trp220 also formed hydrophobic/van der Waals
interactions with the cinnamic acid moiety of 10c. The 3-
hydroxyphenpropyl moiety of 10c occupied the selectivity pocket,
and its 3-hydroxy group formed a hydrogen bond with the side-chain
of Gln114 (3.19 Å). The other part of the 3-hydroxyphenpropyl moiety
was surrounded by the side-chains of Gln303 (3.29 Å), Phe123
(3.42 Å) and Phe116 (3.66 Å), which interacted with this moiety
through hydrophobic/van der Waals contacts.
Derivatives of 9a and 10a with a hydroxyl group at 2-, 3- or 4-
position on their ester phenyl rings were also synthesized (9bed
and 10bed), because an additional interaction between the
hydroxyl group and the non-conserved residue(s) in the selectivity
pocket of AKR1B10 was suggested by the CAPE-docked model.
Although the introduction of the hydroxyl group into 9a (9bed)
Table 2
Inhibition of AKR1B10 and AKR1B1 by the CAPE-based derivatives.
Compound
R1a
R2a
R3a
R4a
IC50 (nM)
AKR1B10
Ratiob
AKR1B1
CAPE
1
2
3
4
5
6
7
8
9a
9b
9c
9d
10a
10b
10c
10d
(CH2)2Ph
(CH2)2Ph
(CH2)2Ph
(CH2)2Ph
CH2Ph
CH2Ph
CH(CH3)2
(CH2)3Ph
H
H
H
H
H
H
H
H
OH
H
OH
H
OH
H
H
OH
H
H
H
H
OH
H
H
80 ꢀ 10
570 ꢀ 60
7
1
4
31
2
17
7
13
52
554
558
562
229
688
766
790
478
18000 ꢀ 2000
19000 ꢀ 2000
69 ꢀ 12
23000 ꢀ 2000
73000 ꢀ 4000
2200 ꢀ 300
400 ꢀ 30
OH
OH
OH
OH
H
OH
OH
OH
OH
OH
OH
OH
OH
OH
210 ꢀ 10
130 ꢀ 10
920 ꢀ 10
3100 ꢀ 400
21 ꢀ 3
2200 ꢀ 300
6100 ꢀ 70
41000 ꢀ 3000
1100 ꢀ 70
(CH2)3Ph
(CH2)2Ph
H
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
13 ꢀ 2
7200 ꢀ 500
9500 ꢀ 600
7300 ꢀ 500
3200 ꢀ 400
11000 ꢀ 1000
6900 ꢀ 100
4900 ꢀ 400
11000 ꢀ 600
(CH2)2Ph(2eOH)
(CH2)2Ph(3eOH)
(CH2)2Ph(4eOH)
(CH2)3Ph
(CH2)3Ph(2eOH)
(CH2)3Ph(3eOH)
(CH2)3Ph(4eOH)
17 ꢀ 3
13 ꢀ 2
H
H
H
H
14 ꢀ 1
16 ꢀ 2
9.0 ꢀ 0.2
6.2 ꢀ 0.1
23 ꢀ 1
H
a
R4
O
R3
R1
R2
O
b
Selectivity, IC50 ratio of AKR1B1/AKR1B10.

324
M. Soda et al. / European Journal of Medicinal Chemistry 48 (2012) 321e329
R2
R2
R1
p-TsOH
O
toluene, reflux
R1
COOH
O
OH
3-hydroxy or
7: R1 = OH, R2 = H
4-hydroxycinnamic acid
8: R1 = H, R2 = OH
R2
MOMO
MOMO
MOMO
R1
R3
(CO2H)2, pyridine, aniline
toluene, reflux
EDC, DMAP
CH2Cl2, rt
O
CHO
OMe
COOH
OMe
n
R2
OMe
O
R1
R3
A
B
Ca: R1, R2, R3 = H, n = 2
Cb: R1 = OMOM, R2, R3 = H, n = 2
Cc: R1, R3 = H, R2 = OMOM, n = 2
Cd: R1, R2 = H, R3 = OMOM, n = 2
Ce: R1, R2, R3 = H, n = 3
HO
n
R2
Cf: R1 = OMOM, R2, R3 = H, n = 3
Cg: R1, R3 = H, R2 = OMOM, n = 3
Ch: R1, R2 = H, R3 = OMOM, n = 3
HO
R1
R3
10% HCl
O
THF, reflux
n
OMe
O
9a: R1, R2, R3 = H, n = 2
9b: R1 = OH, R2, R3 = H, n = 2
9c: R1, R3 = H, R2 = OH, n = 2
9d: R1, R2 = H, R3 = OH, n = 2
10a: R1, R2, R3 = H, n = 3
10b: R1 = OH, R2, R3 = H, n = 3
10c: R1, R3 = H, R2 = OH, n = 3
10d: R1, R2 = H, R3 = OH, n = 3
Scheme 1. Synthesis of CAPE-based derivatives.
The above interactions suggested from the docked model were
the wild-type enzyme. While the effects caused by these single
mutations were low, that caused by the double Val301Leu and
Glu303Ser mutation was large and synergistic, suggesting that at
least Val301 and Gln303 have a combined role in inhibitor binding
and selectivity.
supported by the site-directed mutagenesis of the AKR1B10
residues (Gln114, Val301, and Gln303) into the corresponding
residues in AKR1B1, as well as the mutations of Phe123Ala and
Trp220Tyr (Table 3). All the mutations, except for Ser304Ala,
resulted in 2e5 fold increases in the Ki value for 10c compared to
2.4. Effect of 10c on cellular metabolism and proliferation of
AKR1B10-overexpressing cancer cells
The inhibitory effects of 10c and CAPE on AKR1B10 at a cellular
level were compared in the metabolism of an isoprenyl aldehyde,
farnesal, which is efficiently reduced into farnesol by AKR1B10
in vitro and in the enzyme-overexpressed HeLa cells [11].
Compound 10c inhibited the farnesal metabolism more potently
than CAPE, and was effective from 0.1
0.3 M (Fig. 3A). The IC50 value is lower than those of known
AKR1B10 inhibitors determined under the same assay conditions
(0.8e61 M) [11,19,21,22].
mM, showing an IC50 value of
m
m
As the silencing of AKR1B10 gene results in growth inhibition of
cancer cells [6e8], we found that the growth of human lymphoma
U937 cells was stimulated approximately 1.3-fold compared to the
control cells, at 3 days after the transient transfection of the
Table 3
Effects of mutations of AKR1B10 on Ki values for compound 10c.
Enzyme
Ki (nM)
Mu/Wt.a
Wild-type
Gln114Thr
Phe123Ala
Trp220Tyr
Val301Leu
Gln303Ser
Ser304Ala
Val301Leu/Gln303Ser
2.6 ꢀ 0.5
7.6 ꢀ 0.2
5.1 ꢀ 0.3
15 ꢀ 2
e
3
2
6
2
3
1
9
5.8 ꢀ 0.3
7.1 ꢀ 0.2
3.5 ꢀ 0.2
23 ꢀ 2
Fig. 2. Model of docked compound 10c in the AKR1B10-NADPþ complex. The bound
NADPþ and the enzyme’s residues within 3.5 Å from 10c are depicted. The possible
hydrogen bond interactions were illustrated as dotted lines with their distance given in
angstroms.
a
Ki ratio of mutant enzyme/wild-type enzyme.

M. Soda et al. / European Journal of Medicinal Chemistry 48 (2012) 321e329
325
4
A
B
100
80
1
2
3
2
1
0
*
60
40
20
0
*
*
0.1 0.2 0.5 1.0 2.0 5.0 10
Concentration ( M)
24
48
72
0
Culture (h)
Fig. 3. Inhibitory effects of compound 10c and CAPE on cellular AKR1B10. (A) effect on cellular farnesal reduction. The AKR1B10-expressed HeLa cells were pre-treated with the
indicated concentrations of 10c (closed bar) and CAPE (open bar) for 2 h, and then incubated with 20
M [1-14C] farnesol for 6 h. The inhibition percentages of the farnesal reduction
m
by the inhibitors are expressed as the mean of duplicate experiments. (B) Effect on proliferation of AKR1B10-expressed U937 cells. Together with the control cells (B—B) that were
transfected with the empty vector, the AKR1B10-expressed cells (5 ꢂ 104 cells/well) were incubated without (C) or with 1.0
m
M CAPE (,), 0.2
m
M 10c (:), and 1.0
) and that without inhibitor (C),
mM 10c (6), and
then the viable cell numbers were estimated at the indicated times. *Significant difference between the group with 0.2
m
M (:) or 1.0
mM 10c (6
p < 0.05 (by statistical evaluation using the unpaired Student’s t-test). The inset shows the Western blot analysis of AKR1B10 expression in the extracts (each 20 mg) of the control
(lane 1) and AKR1B10-expressed cells (lane 2).
AKR1B10 cDNA (Fig. 3B). The transfected cells expressed AKR1B10,
which was hardly detected in the control cells transfected with the
vector alone. The addition of 10c decreased the growth of the
its efficacy as a candidate of therapeutics for cancers, in which
AKR1B10 is overexpressed.
transfected cells, and its efficacy was evident at 0.2
mM and much
4. Experimental
superior to that of 1.0 M CAPE. It should be noted that CAPE and
m
10c were not cytotoxic at their low concentrations under the above
assay conditions using HeLa and U937 cells. As CAPE was reported
to be cytotoxic to several cancer cells [39], CAPE and 10c showed
4.1. Compounds and materials
Brazilian propolis components listed in Table 1 were gifted by
API Co. Ltd. (Gifu, Japan). [1-14C] Farnesol was purchased from
American Radiolabeled Chemicals (St. Louis, MO). Human HeLa,
and U937 cells were obtained from American Type Culture
Collection (Manassas, VA).
high LD50 values of 56 ꢀ 4 and 36 ꢀ 3
mM, respectively, when HeLa
cells were treated for 24 h. The results clearly indicate that the
involvement of AKR1B10 in U937 cell proliferation, and suggests
that usefulness of the selective inhibitor 10c for validating the role
of the up-regulated enzyme in other cancer cells.
4.2. Preparation of recombinant enzymes
3. Conclusion
Recombinant AKR1B1, AKR1A1 [43] wild-type AKR1B10 with
the N-terminal 6-His tag [11], and its mutant forms (Gln114Thr,
Trp220Tyr, Val301Leu, Gln303Ser, Ser304Ala, and double mutant
Val301Leu/Gln303Ser) [18,19] were expressed in Escherichia coli
BL21 (DE3) pLysS cells transformed with expression plasmids
harboring their cDNAs, and purified to homogeneity, as described
previously. The Phe123Ala mutant enzyme of AKR1B10 was
prepared by site-directed mutagenesis [19] using a sense primer
(50-AGTCTGGGGATGACCTTGCCCCCAAAGATGATAAAGG-30) and the
corresponding antisense primer, expressed in the E. coli cells, and
purified to homogeneity as described above.
The molecular docking of the new lead CAPE in the crystal
structure of AKR1B10 has allowed the design of a novel CAPE-based
inhibitor (10c) with improved potency (Ki ¼ 2.6 nM) and selectivity
(790-fold over AKR1B1). As the inhibitor was designed to target
a selectivity pocket in the active site of AKR1B10, site-directed
mutagenesis, together with the molecular docking of 10c in
AKR1B10, demonstrated that the high selectivity is attributed to the
interactions of 10c with Gln114, Val301 and Gln303 in the selec-
tivity pocket. Especially, the interaction of the 2-methoxy group of
10c with Val301 is a novel structural feature for selectivity, and may
contribute to future synthesis of selective AKR1B10 inhibitors, in
order to prevent possible side effects caused by inhibition of other
AKR enzymes including AKR1B1. Compound 10c significantly
decreased the metabolism of farnesal in the cells with an IC50 value
4.3. Assay of enzyme activity
The reductase and dehydrogenase activities of the enzymes
were determined at 25 ꢁC by measuring the rate of change in
NADPH absorbance (at 340 nm) and fluorescence (at 455 nm with
an excitation wavelength of 340 nm), respectively [11]. The IC50
values for inhibitors were determined in the reaction mixture that
consisted of 0.1 M potassium phosphate, pH 7.4, 0.1 mM NADPH,
of 0.3 mM, which is superior to the IC50 values of the previously
reported inhibitors of AKR1B10. Furthermore, the growth stimu-
lation of U937 cells by the overexpression of AKR1B10 supports the
involvement of the enzyme in proliferation of cancer cells [6e8],
and the inhibition of the cell growth by 0.2 mM 10c is indicative of

326
M. Soda et al. / European Journal of Medicinal Chemistry 48 (2012) 321e329
0.2 mM pyridine-3-aldehyde, and enzyme (AKR1B1 or AKR1B10),
in a total volume of 2.0 mL. Kinetic studies in the presence of
inhibitors were carried out in both pyridine-3-aldehyde reduction
and NADPþ-linked geraniol oxidation over a range of five substrate
concentrations (0.5e5 ꢂ Km) at a saturating concentration of
coenzyme. The IC50 and Ki values are expressed as the means of at
least three determinations.
13C NMR (100 MHz, CDCl3)
d 30.17, 32.16, 64.20, 114.64, 117.68,
118.05, 120.58, 126.00, 128.37, 128.43, 130.06, 135.70, 141.09, 145.03,
156.35, 167.62; IR (KBr): 3398, 1686, 1636 cmꢄ1; MS (EI): m/z 282
(Mþ); HRMS: Calcd for C18H18O3 282.1256, Found 282.1256.
4.6.2.2. 3-(4-Hydroxyphenyl)acrylic acid phenpropyl ester (8).
Yield: 76%; mp: 82e84 ꢁC; 1H NMR (400 MHz, CDCl3)
d 2.03 (2H,
quint, J ¼ 7.8 Hz), 2.74 (2H, t, J ¼ 7.8 Hz), 4.22 (2H, t, J ¼ 7.8 Hz), 5.28
(1H, br), 6.31 (1H, d, J ¼ 16.1 Hz), 6.85 (2H, d, J ¼ 8.6 Hz), 7.20e7.32
(5H, m), 7.44 (2H, d, J ¼ 8.6 Hz), 7.62 (1H, d, J ¼ 16.1 Hz); 13C NMR
4.4. Molecular docking
The coordinates for AKR1B10 (PDB code: 1ZUA) [28] were ob-
tained from the RCSB Protein Data Bank. The structure was
prepared using the Maestro (Schrödinger, LLC) software package
Version 8.5, as described previously [11]. In order to eliminate any
bond length and bond angle biases, the ligand (CAPE or 10c) was
subjected to a full minimization prior to the docking. The docking
calculations were performed using the program Glide 5.0 [44] on
a Linux workstation under the conditions described previously [11].
Figures were generated using PyMOL (DeLano Scientific).
(100 MHz, CDCl3)
d 30.25, 32.20, 64.07, 115.04, 115.93, 125.99,
128.40, 128.43, 130.02, 130.05, 141.16, 145.01, 158.19, 168.12; IR
(KBr): 3395, 1690, 1601 cmꢄ1; MS (EI): m/z 282 (Mþ); HRMS: Calcd
for C18H18O3 282.1256, Found 282.1253.
4.6.2.3. 3-(2-Methoxy-4-methoxymethoxyphenyl)acrylic acid (B). To
a stirred solution of aldehyde (A) [46] (1.23 g, 6.28 mmol) in toluene
(15 mL) were added malonic acid (980 mg, 9.41 mmol), pyridine
(0.78 mL, 9.67 mmol), and aniline (0.07 mL, 0.75 mmol), and the
resulting mixture was refluxed for 24 h. After cooling, the reaction
was quenched by 10% HCl. The aqueous mixture was extracted with
EtOAc (15 mL ꢂ 4). The organic extracts were combined, washed
with brine, dried over Na2SO4, and evaporated. The residue was
chromatographed on silica gel (30 g, CH2Cl2:MeOH ¼ 50:1e30:1) to
give B as a colorless solid. Yield: 97%; mp: 115e117 ꢁC; 1H NMR
4.5. Cell culture experiments
The human cancer cells (HeLa and U937) were cultured in
Dulbecco’s modified Eagle’s medium supplemented with 10% fetal
bovine serum, 100 U/mL penicillin, and 100 mg/mL streptomycin at
37 ꢁC in a humidified incubator containing 5% CO2. The value of
LD50 (50% lethal dose) was calculated from the viabilities of HeLa
and A549 cells treated for 24 h with increasing concentrations of
the for the tested compound. The cell viability was measured by
a tetrazolium dye-based cytotoxicity assay using 2-(4-iodophenyl)-
(400 MHz, CDCl3) d 3.49 (3H, s), 3.88 (3H, s), 5.21 (2H, s), 6.46 (1H, d,
J ¼ 16.0 Hz), 6.60 (1H, s), 6.65 (1H, d, J ¼ 8.5 Hz), 7.45 (1H, d,
J ¼ 8.5 Hz), 8.00 (1H, d, J ¼ 16.0 Hz); 13C NMR (100 MHz, CDCl3)
d
55.51, 56.21, 94.23, 99.92, 107.86, 115.49, 117.10, 130.63, 142.19,
160.00, 160.64, 173.22; IR (KBr): 2723, 2602, 2519, 2299, 1697, 1610,
1576, 1504 cmꢄ1; MS (EI): m/z 194 (Mþ); HRMS: Calcd for C10H10O4
194.0579, Found 194.0560.
3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium
sodium salt [45].
mono-
The transfection of the pGW1 plasmids harboring the cDNA for
AKR1B10 into HeLa and U937 cells, Western blot analysis of the
expressed enzyme and analysis of the metabolism of [1-14C]far-
nesol in the HeLa cells were carried out as described previously
[11]. In the experiments of growth of U937 cells, the overexpressing
and control cells were suspended in fresh growth medium, seeded
at a density of 5 ꢂ 104 cells/ml into a 24-well multiplate, and then
treated for 0, 24, 48, 72 h with CAPE or 10c. The number of the
viable cells was estimated by the trypan blue dye-exclusion
method.
4.6.3. General procedure for condensation using EDC
To a stirred solution of carboxylic acid (1 mmol) in CH2Cl2
(10 mL) were added EDCꢃHCl (1.2 mmol), DMAP (0.1 mmol), and
alcohol (1.2 mmol), and the resulting mixture was stirred at room
temperature for 24 h. The solvent was removed under reduced
pressure, and the residue was chromatographed on silica gel (15 g,
hexane:acetone ¼ 30:1e10:1) to give the corresponding ester.
4.6.3.1. 3-(2-Methoxy-4-methoxymethoxyphenyl)acrylic acid phe-
nethyl ester (Ca). Yield: 49%; 1H NMR (400 MHz, CDCl3)
d 3.02 (2H,
4.6. Chemical synthesis
t, J ¼ 7.1 Hz), 3.49 (3H, s), 3.89 (3H, s), 4.40 (2H, t, J ¼ 7.1 Hz), 5.20
(2H, s), 6.42 (1H, d, J ¼ 16.1 Hz), 6.59 (1H, s), 6.64 (1H, d-like,
J ¼ 8.5 Hz), 7.24e7.34 (5H, m), 7.42 (1H, d, J ¼ 8.5 Hz), 7.91 (1H, d,
4.6.1. General
The known esters (1e6) were synthesized by literature proce-
dure [38e42]. The esters (7, 8, 9aed, and 10aed) were synthesized
from corresponding carboxylic acids by Fischer esterification or
condensation using EDC with appropriate alcohols, and then
deprotection of the MOM group for 9aed and 10aed.
J ¼ 16.1 Hz); 13C NMR (100 MHz, CDCl3)
d 35.26, 55.49, 56.19, 64.75,
94.24, 99.93, 107.75, 116.29, 117.40, 126.46, 128.45, 128.95, 130.17,
138.03, 140.03, 159.70, 160.24, 167.70; IR (neat): 2955, 1705,
1605 cmꢄ1; MS (EI): m/z 342 (Mþ); HRMS: Calcd for C20H22O5
342.1467, Found 342.1466.
4.6.2. General procedure for Fischer esterification
To a stirred solution of carboxylic acid (1 mmol) in toluene
(10 mL) were added alcohol (15 mmol), and p-TsOHꢃH2O
(0.04 mmol), and the resulting mixture was refluxed for 24 h.
After cooling, the solvent was removed under reduced pressure,
and the residue was chromatographed on silica gel (15 g,
hexane:acetone ¼ 20:1e15:1) to give corresponding ester.
4.6.3.2. 3-(2-Methoxy-4-methoxymethoxyphenyl)acrylic acid 2-(2-
methoxymethoxyphenyl)ethyl ester (Cb). Yield: 47%; 1H NMR
(400 MHz, CDCl3)
d
3.06 (2H, t, J ¼ 7.2 Hz), 3.48 (6H, s), 3.87 (3H, s),
4.40 (2H, t, J ¼ 7.2 Hz), 5.20 (2H, s), 5.23 (2H, s), 6.42 (1H, d,
J ¼ 16.1 Hz), 6.59 (1H, s), 6.64 (1H, dd, J ¼ 8.5 Hz), 6.95 (1H, t,
J ¼ 7.3 Hz), 7.09 (1H, d, J ¼ 7.3 Hz), 7.18e7.23 (2H, m), 7.41 (1H, d,
J ¼ 8.5 Hz), 7.89 (1H, d, J ¼ 16.1 Hz); 13C NMR (100 MHz, CDCl3)
4.6.2.1. 3-(3-Hydroxyphenyl)acrylic acid phenpropyl ester (7). Yield:
d 30.06, 55.45, 56.02, 56.14, 63.71, 94.24, 94.27, 99.94,107.76,113.75,
81%; mp: 77e78 ꢁC; 1H NMR (400 MHz, CDCl3)
d
2.04 (2H, quint,
116.47, 117.45, 121,58, 126.80, 127.83, 130.13, 130.84, 139.84, 155.35,
159.67, 160.19, 167.74; IR (neat): 1701, 1628, 1609,1155 cmꢄ1; MS
(EI): m/z 402 (Mþ); HRMS: Calcd for C22H6O7 402.1679, Found
402.1685.
J ¼ 7.3 Hz), 2.75 (2H, t, J ¼ 7.3 Hz), 4.23 (2H, t, J ¼ 7.3 Hz), 4.19 (1H,
br), 15.9 (1H, d, J ¼ 15.9 Hz), 6.87 (1H, d-like, J ¼ 7.8 Hz), 7.0 (1H, s),
7.11 (1H, d, J ¼ 7.8 Hz), 7.20e7.32 (6H, m), 7.61 (1H, d, J ¼ 15.9 Hz);

M. Soda et al. / European Journal of Medicinal Chemistry 48 (2012) 321e329
327
4.6.3.3. 3-(2-Methoxy-4-methoxymethoxyphenyl)acrylic acid 2-(3-
J ¼ 6.9 Hz), 3.48 (3H, s), 3.49 (3H, s), 3.88 (3H, s), 4.20 (2H, t,
J ¼ 6.9 Hz), 5.15 (2H, s), 5.20 (2H, s), 6.45 (1H, d, J ¼ 16.1 Hz), 6.60
(1H, d, J ¼ 2.3 Hz), 6.65 (1H, dd, J ¼ 8.5, 2.2 Hz), 6.97 (2H, d,
J ¼ 8.5 Hz), 7.13 (2H, d, J ¼ 8.5 Hz), 7.44 (1H, d, J ¼ 8.5 Hz), 7.92 (1H,
methoxymethoxyphenyl)ethyl ester (Cc). Yield: 49%; 1H NMR
(400 MHz, CDCl3)
d
2.99 (1H, t, J ¼ 7.1 Hz), 3.47 (3H, s), 3.49 (3H, s),
3.87 (3H, s), 4.40 (2H, t, J ¼ 7.1 Hz), 5.17 (2H,s), 5.20 (2H, s), 6.42 (1H,
d, J ¼ 16.1 Hz), 6.59 (1H, s), 6.64 (1H, d-like, J ¼ 8.5 Hz), 6.90e6.95
(3H, m), 7.22 (1H, t, J ¼ 7.6 Hz), 7.42 (1H, d, J ¼ 8.5 Hz), 7.91 (1H, d,
d, J ¼ 16.1 Hz); 13C NMR (100 MHz, CDCl3)
d 30.52, 31.34, 55.49,
55.88, 56.17, 63.52, 94.26, 94.55, 107.77, 116.26, 116.42, 117.44,
129.34, 130.21, 134.75, 139.91, 155.47, 159.71, 160.23, 167.84; IR
(neat): 1703, 1628, 1607, 1150 cmꢄ1; MS (EI): m/z 416 (Mþ); HRMS:
Calcd for C23H28O7 416.1835, Found 416.1828.
J ¼ 16.1 Hz); 13C NMR (100 MHz, CDCl3)
d 35.21, 55.45, 55.93, 56.17,
64.61, 94.22, 94.38, 99.90, 107.73, 114.24, 116.29, 116.89, 117.38,
122.49, 129.42, 130.18, 139.67, 140.02, 157.33, 159.69, 160.23, 167.66;
IR (neat): 1713, 1606, 1573 cmꢄ1; MS (EI): m/z 402 (Mþ); HRMS:
Calcd for C22H26O7 402.1678, Found 402.1677.
4.6.4. General procedure for the deprotection of MOM group for
9aed and 10aed
4.6.3.4. 3-(2-Methoxy-4-methoxymethoxyphenyl)acrylic acid 2-(4-
To a stirred solution of ester (1 mmol) in THF (6 mL) was added
10% HCl (catalytic amounts), and the resulting mixture was heated
at 60 ꢁC for 24 h. After cooling, the solvent was removed under
reduced pressure, and the residue was chromatographed on silica
gel (15 g, hexane : acetone ¼ 5 : 1e2 : 1) to give corresponding
ester.
methoxymethoxyphenyl)ethyl ester (Cd). Yield: 41%; 1H NMR
(400 MHz, CDCl3)
d
2.96 (2H, t, J ¼ 7.2 Hz), 3.48 (3H, s), 3.50 (3H, s),
3.87 (3H, s), 4.36 (2H, t, J ¼ 7.1 Hz), 5.16 (2H, s), 5.20 (2H, s), 6.42
(1H, d, J ¼ 16.2 Hz), 6.59 (1H, d, J ¼ 2.2 Hz), 6.64 (1H, dd, J ¼ 8.5,
2.2 Hz), 6.99 (2H, d, J ¼ 8.5 Hz), 7.18 (2H, d, J ¼ 8.5 Hz), 7.43 (1H, d,
J ¼ 8.5 Hz), 7.91 (1H, d, J ¼ 16.2 Hz); 13C NMR (100 MHz, CDCl3)
d
34.38, 55.43, 55.84, 56.10, 64.86, 94.20, 94.44, 99.90, 107.74,
4.6.4.1. 3-(4-Hydroxy-2-methoxyphenyl)acrylic acid phenethyl ester
116.25, 117.35, 129.88, 130.09, 131.36, 139.94, 155.85, 159.65, 160.21,
167.23; IR (neat): 1703, 1628, 1609, 1151 cmꢄ1; MS (EI): m/z 402
(Mþ); HRMS: Calcd for C22H26O7 402.1679, Found 420.1688.
(9a). Yield: 70%; mp: 113e116 ꢁC; 1H NMR (400 MHz, CDCl3)
d 3.01
(2H, t, J ¼ 7.1 Hz), 3.86 (3H, s), 4.40 (2H, t, J ¼ 7.1 Hz), 5.01 (1H, br),
6.38e6.40 (2H, m), 6.41 (1H, d, J ¼ 16.3 Hz), 7.26e7.34 (5H, m), 7.38
(1H, d, J ¼ 8.8 Hz), 7.90 (1H, d, J ¼ 16.3 Hz); 13C NMR (100 MHz,
4.6.3.5. 3-(2-Methoxy-4-methoxymethoxyphenyl)acrylic acid phen-
CDCl3) d 35.17, 55.37, 65.12, 99.10, 108.01, 114.76, 126.52, 128.43,
propyl ester (Ce). Yield: 42%; 1H NMR (400 MHz, CDCl3)
d
2.03 (2H,
128.47, 128.92, 130.58, 137.81, 141.07, 159.93, 160.14, 168.90; IR
(KBr): 3348, 1676, 1599 cmꢄ1; MS (EI): m/z 298 (Mþ); HRMS: Calcd
for C18H18O4 298.1205, Found 298.1196; Anal. Calcd for C18H18O4 C,
72.47; H, 6.08. Found C, 72.29; H, 6.25.
quint, J ¼ 7.3 Hz), 2.75 (2H, t, J ¼ 7.3 Hz), 3.49 (3H, s), 3.88 (3H, s),
4.21 (2H, t, J ¼ 7.3 Hz), 5.20 (2H, s), 6.45 (1H, d, J ¼ 16.1), 6.59 (1H, s),
6.64 (1H, d, J ¼ 8.6 Hz), 7.19e7.31 (5H, m), 7.43 (1H, d, J ¼ 8.6 Hz),
7.91 (1H, d, J ¼ 16.1 Hz); 13C NMR (100 MHz, CDCl3)
d 30.39, 32.23,
55.51, 56.20, 63.57, 63.99, 94.28, 99.96,107.77,116.44,117.45,125.94,
128.40, 128.44, 130.25, 139.94, 141.37, 159.72, 160.23, 167.84; IR
(neat): 1713, 1607, 1573 cmꢄ1; MS (EI): m/z 356 (Mþ); HRMS: Calcd
for C21H24O5 356.1624, Found 356.1624.
4.6.4.2. 3-(4-Hydroxy-2-methoxyphenyl)acrylic acid 2-(2-hydroxyphenyl)
ethyl ester (9b). Yield: 66%; mp: 155e157 ꢁC; 1H NMR (400 MHz,
CDCl3)
d
3.02 (2H, t, J ¼ 7.2 Hz), 3.86 (3H, s), 4.37 (2H, t, J ¼ 7.2 Hz),
6.39 (1H, br), 6.42 (1H, d, J ¼ 16.1 Hz), 6.43 (1H, s), 6.44 (1H, d,
J ¼ 9.0 Hz), 6.85e6.89 (2H, m), 7.12e7.17 (2H, m), 7.38 (1H, d,
J ¼ 9.0 Hz), 7.95 (1H, d, J ¼ 16.1 Hz); 13C NMR (100 Hz, CDCl3)
4.6.3.6. 3-(2-Methoxy-4-methoxymethoxyphenyl)acrylic acid 3-(2-
methoxymethoxyphenyl)propyl ester (Cf). Yield: 49%; 1H NMR
d 29.46, 55.46, 62.94, 99.03, 108.13, 113.64, 113.93, 114.92, 118.89,
(400 MHz, CDCl3)
d
2.02 (2H, quint, J ¼ 7.1 Hz), 2.77 (2H, t,
123.81, 127.54, 130.92, 130.55, 139.60, 155.39, 159.67, 161.48, 166.95;
IR (KBr): 3385, 1674, 1599 cmꢄ1; MS (EI): m/z 314 (Mþ); HRMS:
Calcd for C18H18O5 314.1156, Found 314.1161; Anal. Calcd for
C18H18O5 C, 68.78; H, 5.77. Found C, 68.84; H, 5.59.
J ¼ 7.1 Hz), 3.48 (3H, s), 3.49 (3H, s), 3.87 (3H, s), 4.22 (2H, t,
J ¼ 7.1 Hz), 5.20 (2H, s), 5.21 (2H, s), 6.44 (1H, d, J ¼ 16.1 Hz), 6.59
(1H, d, J ¼ 2.2 Hz), 6.65 (1H, dd, J ¼ 8.5, 2.3 Hz), 6.94 (1H, t,
J ¼ 8.5 Hz), 7.06 (1H, d, J ¼ 7.6 Hz), 7.16 (2H, t, J ¼ 7.6 Hz), 7.43 (1H, d,
J ¼ 8.5 Hz), 7.90 (1H, d, J ¼ 16.1 Hz); 13C NMR (100 MHz, CDCl3)
4.6.4.3. 3-(4-Hydroxy-2-methoxyphenyl)acrylic acid 2-(3-hydrox-
d
26.85, 29.00, 55.47, 55.99, 56.16, 63.54, 94.25, 94.31, 99.95, 107.76,
yphenyl)ethyl ester (9c). Yield: 80%; mp: 144e146 ꢁC; 1H NMR
113.80, 116.56, 117.46, 121.61, 127.22, 130.07, 130.16, 130.36, 139.77,
155.133, 159.67, 160.19, 167.84; IR (neat): 1705, 1628, 1606,
1153 cmꢄ1; MS (EI): m/z 416 (Mþ); HRMS: Calcd for C23H28O7
416.1835, Found 416.1852.
(400 MHz, CDCl3)
d
2.96 (2H, t, J ¼ 7.1 Hz), 3.83 (3H, s), 4.39 (2H, t,
J ¼ 7.1 Hz), 5.18 (1H, br), 5.80 (1H, br), 6.38 (1H, d, J ¼ 16.2 Hz),
6.41e6.43 (2H, m), 6.72 (1H, d-like, J ¼ 7.7 Hz), 6.76 (1H, s-like), 6.84
(1H, d, J ¼ 7.7 Hz), 7.18 (1H, t, J ¼ 7.7 Hz), 7.36 (1H, d, J ¼ 9.0 Hz), 7.91
(1H, d, J ¼ 16.2 Hz); 13C NMR (100 MHz, DMSO-d6)
d 34.51, 55.49,
4.6.3.7. 3-(2-Methoxy-4-methoxymethoxyphenyl)acrylic acid 3-(3-
64.19, 99.05, 108.16, 113.32, 113.63, 113.80, 115.71, 119.45, 129.30,
130.38, 139.44, 139.76, 157.35, 159.72, 161.53, 166.96; IR (KBr): 3398,
1676, 1587 cmꢄ1; MS (EI): m/z 314(Mþ); HRMS: Calcd for C18H18O5
314.1154, Found 314.1149; Anal. Calcd for C18H18O5 C, 68.78; H, 5.77.
Found C, 68.55; H, 5.81.
methoxymethoxyphenyl)propyl ester (Cg). Yield: 60%; 1H NMR
(400 MHz, CDCl3)
d
2.03 (2H, quint, J ¼ 7.8 Hz), 2.72 (2H, t,
J ¼ 7.8 Hz), 3.48 (3H, s), 3.49 (3H, s), 3.89 (3H, s), 4.21 (2H, t,
J ¼ 7.8 Hz), 5.17 (2H, s), 5.20 (2H, s), 6.45 (1H, d, J ¼ 16.1 Hz), 6.59
(1H, s), 6.64 (1H, d-like, J ¼ 8.5 Hz), 6.86e6.89 (3H, m), 7.20 (1H, t,
J ¼ 7.8 Hz), 7.43 (1H, d, J ¼ 8.5 Hz), 7.91 (1H, d, J ¼ 16.1 Hz); 13C NMR
4.6.4.4. 3-(4-Hydroxy-2-methoxyphenyl)acrylic acid 2-(4-hydrox-
(100 MHz, CDCl3)
d
30.24, 32.22, 55.49, 55.95, 56.18, 63.54, 94.25,
yphenyl)ethyl ester (9d). Yield: 56%; mp: 95e97 ꢁC; 1H NMR
94.39, 99.94, 107.75, 113.67, 116.42, 117.44, 122.02, 129.37, 130.25,
139.93, 143.06, 157.35, 159.72, 160.22, 167.82; IR (neat): 2955, 1705,
1689 cmꢄ1; MS (EI): m/z 416 (Mþ); HRMS: Calcd for C23H28O7
416.1835, Found 416.1830.
(400 MHz, CDCl3)
d
2.94 (2H, t, J ¼ 7.0 Hz), 3.86 (3H, s), 4.75 (1H, br),
6.38 (1H, d, J ¼ 16.1 Hz), 6.39e6.42 (2H, m), 6.78 (2H, d, J ¼ 8.1 Hz),
7.38 (1H, d, J ¼ 8.8 Hz), 7.90 (1H, d, J ¼ 16.1 Hz); 13C NMR (100 MHz,
DMSO-d6) d 30.70, 55.49, 64.58, 99.04, 108.16, 113.64, 113.84, 115.14,
128.01, 129.78, 130.31, 139.66, 155.83, 159.69, 161.51, 166.94; IR
(KBr): 3312, 1684, 1609, 1516 cmꢄ1; MS (EI): m/z 314 (Mþ); HRMS:
Calcd for C18H18O5 314.1154, Found 314.1157; Anal. Calcd for
C18H18O5 C, 68.78; H, 5.77. Found C, 68.69; H, 5.93.
4.6.3.8. 3-(2-Methoxy-4-methoxymethoxyphenyl)acrylic acid 3-(4-
methoxymethoxyphenyl)propyl ester (Ch). Yield: 67%; 1H NMR
(400 MHz, CDCl3)
d
2.00 (2H, quint, J ¼ 6.9 Hz), 2.69 (2H, t,

328
M. Soda et al. / European Journal of Medicinal Chemistry 48 (2012) 321e329
T. Kodama, Y. Araki, Aldo-keto reductase family 1, member B10 in uterine
carcinomas: a potential risk factor of recurrence after surgical therapy in
cervical cancer, Int. J. Gynecol. Cancer 17 (2007) 1300e1306.
4.6.4.5. 3-(4-Hydroxy-2-methoxyphenyl)acrylic acid phenpropyl ester
(10a). Yield: 94%; mp: 89e93 ꢁC; 1H NMR (400 MHz, CDCl3)
2.03
d
(2H, quint, J ¼ 7.3 Hz), 2.75 (2H, t, J ¼ 7.3 Hz), 3.87 (3H, s), 4.21 (2H, t,
J ¼ 7.3 Hz), 5.05 (1H, br), 6.41 (1H, d, J ¼ 16.1 Hz), 6.45e6.43 (2H, m),
7.20e7.31 (4H, m), 7.39 (1H, d, J ¼ 9.0 Hz), 7.90 (1H, d, J ¼ 16.1 Hz);
[4] S. Heringlake, M. Hofdmann, A. Fiebeler, M.P. Manns, W. Schmiegel,
A. Tannapfel, Identification and expression analysis of the aldo-ketoreductase
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13C NMR(100 MHz, CDCl3)
d 30.27, 32.14, 55.37, 63.59, 99.11, 108.01,
114.90, 115.69, 125.95, 128.38, 128.40, 130.66, 140.97, 141.17, 159.90,
160.16, 169.05; IR (KBr): 3329, 1684, 1605 cmꢄ1; MS (EI): m/z 312
(Mþ); HRMS: Calcd for C19H20O4 312.1362, Found 312.1345; Anal.
Calcd for C19H20O4 C, 73.06; H, 6.45. Found C, 73.23; H, 6.22.
identifies CDH17 as
a prognostic marker in early stage gastric cancer,
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4.6.4.6. 3-(4-Hydroxy-2-methoxyphenyl)acrylic acid 3-(2-hydroxyphenyl)
propyl ester (10b). Yield: 62%; 1H NMR (400 MHz, CDCl3)
d 2.04 (2H,
quint, J ¼ 7.0 Hz), 2.75 (2H, t, J ¼ 7.0 Hz), 3.87(3H, s), 4.23 (2H, t,
J ¼ 7.0 Hz), 4.98 (1H, br), 5.09 (1H, br), 6.42e6.43 (2H, m), 6.44 (1H,
d, J ¼ 16.3 Hz), 6.77 (1H, d, J ¼ 7.8 Hz), 6.88 (1H, t, J ¼ 7.8 Hz), 7.10
(1H, t, J ¼ 7.8 Hz), 7.14 ( 1H, d, J ¼ 7.8 Hz), 7.40 (1H, d, J ¼ 9.3 Hz), 7.92
(1H, t, J ¼ 16.3 Hz); 13C NMR (100 MHz, DMSO-d6)
d 26.17, 28.40,
55.49, 63.38, 99.06, 108.16, 113.68, 114.01, 114.89, 118.89, 126.96,
127.27. 129.78, 130.39, 139.59, 155.13, 159.72, 161.47, 167.10; IR
(neat): 3421, 1684, 1609 cmꢄ1; MS (EI): m/z 328 (Mþ); HRMS: Calcd
for C19H20O5 328.1311, Found 328.1307.
4.6.4.7. 3-(4-Hydroxy-2-methoxyphenyl)acrylic acid 3-(3-hydrox-
yphenyl)propyl ester (10c). Yield: 60%; mp: 104e107 ꢁC; 1H NMR
(400 MHz, CDCl3)
d
2.01 (2H, quint, J ¼ 7.2 Hz), 2.69 (2H, t,
J ¼ 7.2 Hz), 3.83 (3H, s), 4.21 (2H, t, J ¼ 7.2 Hz), 6.41 (1H, d,
J ¼ 16.1 Hz), 6.43e6.45 (2H, m), 6.68 (1H, d, J ¼ 8.9 Hz), 6.70 (1H, s),
6.77(1H, d, J ¼ 7.2 Hz), 7.15 (1H, t, J ¼ 7.2 Hz), 7.36(1H, d, J ¼ 8.9 Hz),
7.91(1H, d, J ¼ 16.1 Hz); 13C NMR (100 MHz, DMSO-d6): 29.83, 31.44,
55.49, 63.03, 99.05, 108.16, 112.85, 113.64, 113.89, 115.17, 118.92,
129.27, 130.40, 139.67, 142.58, 157.35, 159.72, 161.53, 167.08; IR
(KBr): 3362, 1670, 1609 cmꢄ1; MS (EI): m/z 328 (Mþ); HRMS: Calcd
for C19H20O5 326.1311, Found 328.1306; Anal. Calcd for C19H20O5 C,
69.50; H, 6.14. Found C, 69.51; H, 6.01.
4.6.4.8. 3-(4-Hydroxy-2-methoxyphenyl)acrylic acid 3-(4-hydrox-
yphenyl)propyl ester (10d). Yield: 86%; 1H NMR (400 MHz, CDCl3)
d
1.99 (2H, quint, J ¼ 6.6 Hz), 2.67 (2H, t, J ¼ 6.6 Hz), 3.86 (3H, s), 4.20
(2H, t, J ¼ 6.6 Hz), 4.74 (1H, br), 5.30 (1H, br), 6.42 (2H, m), 6.43 (1H,
d, J ¼ 16.1 Hz), 6.77 (2H, d-like, J ¼ 8.5 Hz), 7.08 (2H, d-like,
J ¼ 8.5 Hz), 7.39 (1H, d-like, J ¼ 9.0 Hz), 7.90 (1H, d, J ¼ 16.1 Hz); 13C
NMR (100 MHz, DMSO-d6):
d 30.23, 30.58, 55.49, 63.01, 99.05,
108.15, 113.67, 113.93, 115.11, 129.13, 130.39, 131.15, 139. 63, 155.42,
159.72, 161.48, 167.09; IR (neat): 3383, 1684, 1607 cmꢄ1; MS (EI): m/
z 328 (Mþ); HRMS: Calcd for C19H20O5 328.1311, Found 328.1308.
Acknowledgments
[21] S. Endo, T. Matsunaga, K. Kuwata, H.T. Zhao, O. El-Kabbani, Y. Kitade, A. Hara,
Chromene-3-carboxamide derivatives discovered from virtual screening as
potent inhibitors of the tumour maker, AKR1B10, Bioorg. Med. Chem. 18
(2010) 2485e2490.
[22] M. Takemura, S. Endo, T. Matsunaga, M. Soda, H.T. Zhao, O. El-Kabbani,
K. Tajima, M. Iinuma, A. Hara, Selective inhibition of the tumor marker aldo-
keto reductase family member 1B10 by oleanolic acid, J. Nat. Prod. 74 (2011)
1201e1206.
This work was partly founded by grant-in-aid for Young Scien-
tists (B) and Scientific Research (C) from the Japan Society for the
Promotion of Science, Sasakawa Scientific Research Grant from
Japan Science Society, and scholarship from Monash University
Graduate Research School.
[23] G.K. Balendiran, H.J. Martin, Y. El-Hawari, E. Maser, Cancer biomarker
AKR1B10 and carbonyl metabolism, Chem. Biol. Interact. 178 (2009) 134e137.
[24] B. Díez-Dacal, J. Gayarre, S. Gharbi, J.F. Timms, C. Coderch, F. Gago, D. Pérez-
Sala, Identification of aldo-keto reductase AKR1B10 as a selective target for
modification and inhibition by prostaglandin A1: implications for antitumoral
activity, Cancer Res. 71 (2011) 4161e4171.
[25] O.A. Barski, S.M. Tipparaju, A. Bhatnagar, The aldoeketo reductase superfamily
and its role in drug metabolism and detoxification, Drug Metab. Rev. 40
(2008) 553e624.
[26] S.P. Baba, O.A. Barski, Y. Ahmed, T.E. O’Toole, D.J. Conklin, A. Bhatnagar,
S. Srivastava, Reductive metabolism of AGE precursors: a metabolic route for
preventing AGE accumulation in cardiovascular tissue, Diabetes 58 (2009)
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