Inhibitory effect of novel 5-O-acyl juglones on mammalian DNA polymerase activity, cancer cell growth and inflammatory response

Article abstract of DOI:10.1016/j.bmc.2011.08.023

We previously found that vitamin K₃ (menadione, 2-methyl-1,4-naphthoquinone) inhibits the activity of human mitochondrial DNA polymerase γ (pol γ). In this study, we focused on juglone (5-hydroxy-1,4-naphthoquinone), which is a 1,4-naphthoquinone derivative, and chemically synthesized novel juglones conjugated with C2:0 to C22:6 fatty acid (5-O-acyl juglones). The chemically modified juglones enhanced mammalian pol inhibition and their cytotoxic and anti-inflammatory activities. The juglone conjugated with oleic acid (C18:1-acyl juglone) showed the strongest inhibition of DNA replicative pol α activity and human colon carcinoma (HCT116) cell growth in 10 synthesized 5-O-acyl juglones. C12:0-Acyl juglone was the strongest inhibitor of DNA repair-related pol λ, as well as the strongest suppression of the production of tumor necrosis factor (TNF)-α production induced by lipopolysaccharide (LPS) in the compounds tested. Moreover, this compound caused the greatest reduction in 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced acute inflammation in mouse ears. C12:0- and C18:1-Acyl juglones selectively inhibited the activities of mammalian pol species, but did not influence the activities of other pols and DNA metabolic enzymes tested. These data indicate that the novel 5-O-acyl juglones target anti-cancer and/or anti-inflammatory agents based on mammalian pol inhibition. Moreover, the results suggest that acylation of juglone is an effective chemical modification to improve the anti-cancer and anti-inflammation of vitamin K₃ derivatives, such as juglone.

Full text of DOI:10.1016/j.bmc.2011.08.023

Bioorganic & Medicinal Chemistry 19 (2011) 5803–5812
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Inhibitory effect of novel 5-O-acyl juglones on mammalian DNA
polymerase activity, cancer cell growth and inflammatory response
Sayako Maruo a, Isoko Kuriyama b, Kouji Kuramochi a, , Kazunori Tsubaki a, Hiromi Yoshida b,c
,
⇑
Yoshiyuki Mizushina b,c,
⇑
a Graduate School of Life and Environmental Sciences, Kyoto Prefectural University, Kyoto 606-8522, Japan
b Laboratory of Food and Nutritional Sciences, Department of Nutritional Science, Kobe-Gakuin University, Nishi-ku, Kobe, Hyogo 651-2180, Japan
c Cooperative Research Center of Life Sciences, Kobe-Gakuin University, Chuo-ku, Kobe, Hyogo 650-8586, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
We previously found that vitamin K3 (menadione, 2-methyl-1,4-naphthoquinone) inhibits the activity of
human mitochondrial DNA polymerase (pol ). In this study, we focused on juglone (5-hydroxy-1,4-
naphthoquinone), which is a 1,4-naphthoquinone derivative, and chemically synthesized novel juglones
conjugated with C2:0 to C22:6 fatty acid (5-O-acyl juglones). The chemically modified juglones enhanced
mammalian pol inhibition and their cytotoxic and anti-inflammatory activities. The juglone conjugated
Received 25 July 2011
Revised 9 August 2011
Accepted 10 August 2011
Available online 18 August 2011
c
c
with oleic acid (C18:1-acyl juglone) showed the strongest inhibition of DNA replicative pol
and human colon carcinoma (HCT116) cell growth in 10 synthesized 5-O-acyl juglones. C12:0-Acyl jug-
lone was the strongest inhibitor of DNA repair-related pol k, as well as the strongest suppression of the
production of tumor necrosis factor (TNF)-a production induced by lipopolysaccharide (LPS) in the com-
pounds tested. Moreover, this compound caused the greatest reduction in 12-O-tetradecanoylphorbol-
13-acetate (TPA)-induced acute inflammation in mouse ears. C12:0- and C18:1-Acyl juglones selectively
inhibited the activities of mammalian pol species, but did not influence the activities of other pols and
DNA metabolic enzymes tested. These data indicate that the novel 5-O-acyl juglones target anti-cancer
and/or anti-inflammatory agents based on mammalian pol inhibition. Moreover, the results suggest that
acylation of juglone is an effective chemical modification to improve the anti-cancer and anti-inflamma-
tion of vitamin K3 derivatives, such as juglone.
a activity
Keywords:
5-O-Acyl juglone
DNA polymerase
Enzyme inhibitor
Anti-cancer
Anti-inflammation
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
pols g, i, and j, in addition to REV1. We have been studying selec-
tive inhibitors of each pol derived from natural products including
food materials and nutrients for more than 16 years.5,6 We have
found that vitamin K3, but not K1 or K2, is a potent inhibitor of
The human genome encodes at least 15 DNA polymerases (pols,
i.e., DNA-dependent DNA polymerase, E.C. 2.7.7.7) that participate
in cellular DNA synthesis.1,2 Eukaryotic cells contain 3 replicative
human pol c 7–11
.
pols (
replicative pols (b, f,
a
, d, and
e
), 1 mitochondrial pol (
c
), and at least 11 non-
Vitamin K3 (menadione, 2-methyl-1,4-naphthoquinone) is a fat-
soluble compound that contains quinone as its principle chemical
feature. Quinones are a class of organic compounds that are de-
rived from aromatic compounds via the exchange of an even num-
ber of –CH@ groups for –C(@O)– groups, and any necessary
rearrangement of double bonds, resulting in a fully conjugated cyc-
lic dione structure. The toxicological properties of quinones, which
act as alkylating agents, have also been examined. For example,
quinones are known to interact with flavoproteins to generate
reactive oxygen species (ROS) that can induce biological in-
jury.12–15 In this study, we focused on 5-hydroxy-1,4-naphthoqui-
none (juglone, 1 of Fig. 1), which has the common naphthoquinone
skeleton and a hydroxyl group at the C-5 position. Juglone occurs
naturally in the leaves, roots, husks, and bark of plants in the
Juglandaceae family, particularly the black walnut (Juglans nigra),
and is toxic or growth stunting in many types of plants. It is
g
, h, , k, , terminal deoxynucleotidyl
i
,
j
l, m
transferase (TdT), and REV1).3,4 Pols have a highly conserved struc-
ture, which means that their catalytic subunits show little variance
among species. Enzymes with conserved structures usually per-
form important cellular functions, the maintenance of which has
evolutionary advantages. On the basis of their sequence homology,
eukaryotic pols can be divided into 4 main families, termed A, B, X,
and Y.4 Family A includes mitochondrial pol
. Family B includes 3 replicative pols ( , d, and
, as well as TdT, and family Y includes
c, as well as pols h and
m
a
e
) and pol f. Family
X comprises pols b, k, and
l
⇑
Corresponding authors. Tel./fax: +81 75 703 5603 (K.K.), tel.: +81 78 974
1551x3232, fax: +81 78 974 5689 (Y.M.).
E-mail addresses:
kobegakuin.ac.jp (Y. Mizushina).
kuramoch@kpu.ac.jp (K. Kuramochi),
mizushin@nutr.
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.08.023

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S. Maruo et al. / Bioorg. Med. Chem. 19 (2011) 5803–5812
under basic conditions. The instability of 1 prompted us to exam-
O
OH
O
O
ine the condensation of 1 with carboxylic acids. After a systematic
survey of condensing reagents, catalysts and solvents, we obtained
5-O-acyl juglones (3b–d) by condensation of 1 with carboxylic
acids (2b–d) using 2-methyl-6-nitrobenzoic anhydride (MNBA)
with DMAP (0.1 equiv) in CH2Cl2.23
However, this protocol is not effective for condensation of 1
with long fatty acids (2e–j). For example, coupling of 1 with eico-
sapentanoic acid (2i) gave only mixed anhydride of 2i and 2-
methyl-6-nitrobenzoic acid (date not shown). The combination of
long acyl moieties with 1 was achieved only when acyl chlorides
were used as acylation reagents (Scheme 2). Carboxylic acids
(2e–j) were converted to the corresponding acyl chlorides, which
were treated with 1 (3 equiv) and DMAP (0.1 equiv) in pyridine
to give 3e–j.
R
O
O
O
+
R
OH
O
2a
: Acetic acid (C2:0)
Juglone (1)
5-O-Acyl juglone (3a-j)
2b
: Propionic acid (C3:0)
: Caproic acid (C6:0)
2c
2d
: Lauric acid (C12:0)
: Stearic acid (C18:0)
2e
2f: Oleic acid (C18:1)
2g: Linoleic acid (C18:2)
2h: α-Linolenic acid (C18:3)
2i: Eicosapentaenoic acid (C20:5)
2j: Docosahexaenoic acid (C22:6)
Figure 1. Structure of juglone (1), fatty acid (2) and 5-O-acyl juglone (3).
2.2. Effect of the synthesized 5-O-acyl juglones on mammalian
pol activity
sometimes used as a herbicide, as a dye for cloth and inks, and as a
coloring agent for foods and cosmetics. Juglone is known for its
wide range of biological activities, such as induction of oxidative
stress in many animal cell systems,16 inhibition of germination of
various plant species,17 and inhibition of the peptidyl-prolyl isom-
erase Pin1.18
We previously found that the catechin and epicatechin deriva-
tives conjugated with fatty acids, such as 3-O-acylcatechins and
3-O-acylepicatechins, respectively, were stronger pol inhibitors
than catechin and epicatechin;19–21 therefore, the 5-O-acylated
derivatives of juglone (3a–j of Fig. 1) were chemically synthesized
from juglone (1 of Fig. 1) and fatty acids (2a–j of Fig. 1). In this
study, we investigated the inhibitory effects of 10 5-O-acyl jugl-
ones on mammalian pol activity, human cancer cell growth and
the inflammatory response comparing juglone and fatty acids.
The relationship between pol inhibitory and cytotoxic or anti-
inflammatory effects of vitamin K3-based acylated derivatives is
discussed.
Initially, we investigated the in vitro biochemical action of jug-
lone (1), 10 fatty acids (2a–j) and their 10 chemically synthesized
derivatives, 5-O-acyl juglones (3a–j). The inhibition of four mam-
malian pols, namely calf pol
man pol k, by 10 M of each compound was investigated. Pols
and k were used as representatives of the B, A, Y and X families of
pols, respectively.1–3 As shown in Figure 2, juglone, a derivative of
vitamin K3, inhibited the activity of mammalian pols and , with
IC50 values of less than 10 M. Although vitamin K3 selectively
inhibited pol
among the mammalian pols tested,7–11 juglone
was an inhibitor of all families of pols. No fatty acids tested (C2:0
to C22:6) at 10 M had any effect on pol activity. On the other
a, human pol c, human pol j and hu-
l
a, c,
j
a
c
l
c
l
hand, the synthesized 5-O-acyl juglones were more potent pol
inhibitors than the individual compounds, such as juglones and
fatty acids. In the 5-O-acyl juglones, the juglone conjugated with
lauric acid (C12:0-acyl juglone) (3d) was the strongest inhibitor
of pols
pol inhibitor of pols
jugated with saturated fatty acids (3a–e) on pols
j
and k, and C18:1-acyl juglone (3f) was the most potent
and . The inhibitory effect of juglone con-
and k ranked
a
c
2. Results and discussion
j
as follows: C12:0-acyl juglone (3d) > C18:0-acyl juglone
(3e) > C6:0-acyl juglone (3c) > C3:0-acyl juglone (3b) > C2:0-acyl
juglone (3a); and the inhibitory effect of juglone conjugated with
2.1. Synthesis of 5-O-acyl juglones
5-O-Acetoxy-1,4-naphthoquinone (3a)22 was prepared by treat-
ing juglone (1) with acetic anhydride in pyridine in 79% yield
(Scheme 1). In this reaction, we found that 1 readily decomposes
unsaturated fatty acids (3f–j) on pols
a and c ranked as follows:
C18:1-acyl juglone (3f) > C20:5-acyl juglone (3i) > C22:6-acyl jug-
lone (3j) > C18:2-acyl juglone (3g) > C18:3-acyl juglone (3h).
When activated DNA (i.e., bovine deoxyribonuclease I-treated
DNA) and dNTP were used as the DNA template–primer and
O
O
O
O
OH
O
O
Ac2O
O
O
Oxalyl chloride
Pyridine
79%
R
OH
R
Cl
CH2Cl2
2
3a
1
O
1
R
O
O
O
O
DMAP
R
O
O
2
RCO2H ( )
Pyridine
MNBA, DMAP
1
CH2Cl2
3e : R = n-C17H35
3f : R = cis-CH3(CH2)7CH=CH(CH2)7
3g :
O
R = cis,cis-CH3(CH2)4CH=CHCH2CH=CH(CH2)7
3b
: R = C2H5 (64%)
3h : R = all cis-CH3CH2(CH=CHCH2)3(CH2)6
3i : R = all cis-CH3CH2(CH=CHCH2)5(CH2)2
3j : R = all cis-CH3CH2(CH=CHCH2)6CH2
3c: R = n-C5H11 (76%)
3d
: R = n-C11H23 (62%)
Scheme 1. Preparation of 5-O-acyl juglones (3a–d).
Scheme 2. Preparation of 5-O-acyl juglones (3e–j).

S. Maruo et al. / Bioorg. Med. Chem. 19 (2011) 5803–5812
5805
Juglone (1)
C2:0 (2a)
C3:0 (2b)
C6:0 (2c)
C12:0 (2d)
C18:0 (2e)
C18:1 (2f)
C18:2 (2g)
C18:3 (2h)
C20:5 (2i)
C22:6 (2j)
C2:0-Acyl juglone (3a)
C3:0-Acyl juglone (3b)
C6:0-Acyl juglone (3c)
C12:0-Acyl juglone (3d)
C18:0-Acyl juglone (3e)
C18:1-Acyl juglone (3f)
C18:2-Acyl juglone (3g)
C18:3-Acyl juglone (3h)
C20:5-Acyl juglone (3i)
C22:6-Acyl juglone (3j)
Pol α
Pol γ
Pol κ
Pol λ
0
20
40
60
80
100
Pol relative activity (%)
Figure 2. Inhibitory effects of juglone (1), fatty acids (2a–j) and 5-O-acyl juglones (3a–j) on the activity of mammalian pols. Each compound (10
pol (B-family pol), human pol (A-family pol), human pol
was taken as 100%, and the relative activity is shown. Data are shown as the mean SE (n = 3).
l
M) was incubated with calf
a
c
j (Y-family pol) and human pol k (X-family pol) (0.05 units each). Pol activity in the absence of the compound
nucleotide substrate instead of synthesized DNA [poly(dA)/oli-
go(dT)18 (A/T = 2/1)] and dTTP, respectively, the inhibitory effects
of these compounds did not change (data not shown).
the A, B, X and Y families of pols was observed at a dose of 0.72,
0.68–0.74, 10.2–11.9 and 8.1–8.7 M, respectively; therefore, the
inhibitory effect of this compound on the A- and B-families of pols
was more than 10-fold stronger than that on X- and Y-families of
pols.
l
2.3. Effect of the synthesized 5-O-acyl juglones on pols and
other DNA metabolic enzymes
By contrast, these compounds had no effect on plant pols, such
as cauliflower pol
a or rice pol k, or prokaryotic pols, such as Esch-
Among the 10 5-O-acyl juglones investigated, C12:0-acyl jug-
lone (3d) and C18:1-acyl juglone (3f) displayed the strongest
inhibitory effect on mammalian pols (Fig. 2) and was therefore
the focus of this section. As described briefly in the introduction,
we succeeded in obtaining eleven mammalian pol species, includ-
erichia coli pol I, Taq pol or T4 pol (Table 1). The three-dimensional
structures of eukaryotic pols are likely to differ greatly from those
of prokaryotic pols. These compounds did not inhibit the activity of
other DNA metabolic enzymes, such as calf primase pol
a, HIV-1
reverse transcriptase, T7 RNA polymerase, mouse IMP dehydroge-
nase (type II), T4 polynucleotide kinase, or bovine deoxyribonucle-
ase I. These results suggest that 5-O-acyl juglones may be selective
inhibitors of mammalian pols; in particular, juglone conjugated
with unsaturated fatty acids, such as C18:1-acyl juglone (3f),
potently inhibited the activities of A- and B-families of pols.
To test whether these 5-O-acyl juglones are intercalating agents
that distort DNA and subsequently inhibits enzyme activity, we
measured the thermal transition of DNA in the presence or absence
of these compounds. The thermal transition profile of DNA was the
same with or without the compound (data not shown). Therefore,
ing pols
a, b, c, d, e, g, i, j, k and l, and TdT; however, pols f, h and
m
, and REV1 are not yet available. Currently, eukaryotes are
thought to express at least 15 species of pols,1,2 and we are still
in an era when most pols are very difficult to obtain in their puri-
fied form in a laboratory. Table 1 shows the inhibitory effect (IC50
value) of these 5-O-acyl juglones against various pol species
including the eleven mammalian pols that could be obtained.
C12:0-Acyl juglone (3d) inhibited the activity of all of the pols from
mammals with IC50 values of 6.3–8.4
lM. C18:1-Acyl juglone (3f)
also inhibited all mammalian pols tested, and 50% inhibition of

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S. Maruo et al. / Bioorg. Med. Chem. 19 (2011) 5803–5812
Table 1
2.5. Effect of the synthesized 5-O-acyl juglones on the LPS-
induced inflammatory response in cultured macrophage cells
IC50 values of C12:0-acyl juglone (3d) and C18:1-acyl juglone (3f) for mammalian
pols, various pols and other DNA metabolic enzymes
Enzyme
IC50 values (lM)
Next, we investigated whether chemically synthesized 5-O-acyl
juglones can inhibit TNF-
ride (LPS) stimulation in cultured mouse macrophage RAW264.7
cells. The inflammatory cytokine TNF- activates the major inflam-
mation signaling pathway by binding to the TNF- receptor (TNFR),
resulting in various inflammatory diseases.24 In RAW264.7 cells, no
compound showed cytotoxicity at 5 M, because the LD50 values of
these compounds were >10 M. As shown in Figure 4, juglone (1) at
M moderately suppressed the LPS-stimulated production of
TNF- , although 10 fatty acids (2a-j) displayed no inhibitory effect
on TNF- production. The inhibitory effects of C12:0-acyl juglone
(3d) were the strongest among these 10 5-O-acyl juglones. The ef-
fect of the compounds on the suppression of LPS-evoked TNF- pro-
a production induced by lipopolysaccha-
C12:0-Acyl juglone
(3d)
C18:1-Acyl juglone
(3f)
a
Mammalian pols
[A-Family of pol]
a
Human pol
[B-Family of pols]
Calf pol
Human pol d
Human pol
c
6.5 0.40
0.72 0.04
l
a
6.3 0.38
6.9 0.41
6.6 0.40
0.68 0.04
0.74 0.05
0.70 0.04
l
5
l
e
[X-Family of pols]
Rat pol b
Human pol k
a
7.6 0.46
7.0 0.43
7.9 0.48
8.4 0.50
11.5 0.69
10.2 0.61
10.8 0.65
11.9 0.72
a
Human pol
Calf TdT
l
a
[Y-Family of pols]
duction showed almost the same tendency as their inhibitory effect
on mammalian pol k. These results suggest that 5-O-acyl juglones,
such as C12:0-acyl juglone (3d), inhibit the activities of mammalian
Human pol
Mouse pol
Human pol
g
i
j
6.9 0.42
7.1 0.43
6.8 0.41
8.4 0.51
8.7 0.53
8.1 0.49
pols, and then prevent TNF-
a production in LPS-induced macro-
Plant pol
phages, but do not affect cell growth.
Cauliflower pol
a
>100
>100
>200
>200
Rice pol k
2.6. Effect of the synthesized 5-O-acyl juglones on TPA-induced
anti-inflammatory activity
Prokaryotic pols
E. coli pol I
Taq pol
>100
>100
>100
>100
>100
>100
T4 pol
In a previous pol inhibitor study, we found a relationship
between pol
k inhibitors and 12-O-tetradecanoylphorbol-13-
Other DNA metabolic enzymes
Calf primase of pol
HIV-1 reverse transcriptase
T7 RNA polymerase
Mouse IMP dehydrogenase
(type II)
acetate (TPA)-induced acute anti-inflammatory activity.6,25,26 Thus,
using the mouse ear inflammatory test, we examined the anti-
inflammatory activity of juglone (1), fatty acids (2a–j) and 5-O-acyl
a
>100
>100
>100
>100
>100
>100
>100
>100
juglones (3a–j) at 500 lg/ear. Application of TPA (0.5 lg) to the
T4 polynucleotide kinase
Bovine deoxyribonuclease I
>100
>100
>100
>100
mouse ear induced edema, resulting in a 241% increase in the
weight of the ear disk 7 h after application. As shown in Figure 5,
pretreatment with juglone moderately suppressed inflammation,
but 10 fatty acids had a weak effect on the level of inflammation.
The anti-inflammatory effect of the chemically synthesized 5-O-
acyl juglones was stronger than that of juglone and fatty acid,
and C12:0-acyl juglone (3d) was the strongest among these 10
5-O-acyl juglones. Therefore, the mouse ear anti-inflammatory ef-
fect of these compounds correlated with their inhibitory effects on
Each compound was incubated with each enzyme (0.05 units). Enzyme activity in
the absence of the compounds was taken as 100 %. Data are shown as the mean-
SE of three independent experiments.
s
the inhibition of pols by 5-O-acyl juglones is not due to DNA distor-
tion, but seems to be due to the direct effect of this compound on
the enzymes themselves.
both mammalian pol k (Fig. 2) and TNF-
a production in macro-
phages (Fig. 4), and C12:0-acyl juglone (3d) had the strongest ef-
fect among the synthesized 5-O-acyl juglones. These results
suggest that inhibition of pol k activity has a positive correlation
with the anti-inflammatory activity observed.
2.4. Effect of the synthesized 5-O-acyl juglones on cultured
human cancer cells
Pols have recently emerged as important cellular targets for
chemical intervention in the development of anti-cancer agents.5
The chemically synthesized 5-O-acyl juglones could therefore be
useful in chemotherapy, and we investigated the cytotoxic effect
of juglone (1), fatty acids (2a–j) and 5-O-acyl juglones (3a–j)
against HCT116 human colon carcinoma cultured cell line. As
2.7. Three-dimensional structures of juglone, fatty acids and 5-
O-acyl juglones
To obtain information about the molecular basis of the inhibi-
tory properties of 5-O-acyl juglones, computational analyses were
performed using molecular simulation. We focused on the calcu-
lated logP (C log P) value (partition coefficients for octanol/water)
of the synthesized 10 5-O-acyl juglones (3a–j) and their part com-
pounds, such as juglone (1) and fatty acids (2a–j), as chemical
properties (Table 2). The values of ClogP, which indicate hydro-
phobicity, in 5-O-acyl juglones had almost the same tendency
and numerical values as those of fatty acids, which are consisted
of 5-O-acyl juglones; therefore, the ClogP value of 5-O-acyl jugl-
ones depends on the acyl-chain length, and does not affect bioac-
tivity, such as pol inhibition, human cancer cell growth
suppression and anti-inflammatory response.
shown in Figure 3, 100
lM juglone suppressed cell growth with
an LD50 value of less than 100
lM, whereas the 10 fatty acids
had no effect on cell viability. In the synthesized 5-O-acyl juglones,
C18:1-acyl juglone (3f) had the strongest growth inhibitory effect
on HCT116 cells. The influence of 5-O-acyl juglones on HCT116 cell
growth showed the same tendency as that on the inhibition of pols
a
and
these compounds may be related to inhibition of the activities of
DNA replicative pols, such as pol ; in particular, the inhibition
c (Fig. 2), suggesting that cancer cell growth prevention by
a
of both A- and B-families of pols by these compounds must be
important for HCT116 cell proliferation. C18:1-Acyl juglone (3f)
more strongly suppressed the growth of HCT116 cells than the
same concentrations of aphidicolin, camptothecin and etoposide,
which are inhibitors of replicative pols, DNA topoisomerase I and
DNA topoisomerase II, respectively (data not shown).
The length and width of the three-dimensional structures, from
which the energy-minimized compounds were calculated, are
shown in Table 2. The molecular length of 5-O-acyl juglones was
approximately 5–6 Å longer than that of fatty acids consisting of

S. Maruo et al. / Bioorg. Med. Chem. 19 (2011) 5803–5812
5807
10 μM
100 μM
Juglone (1)
C2:0 (2a)
C3:0 (2b)
C6:0 (2c)
C12:0 (2d)
C18:0 (2e)
C18:1 (2f)
C18:2 (2g)
C18:3 (2h)
C20:5 (2i)
C22:6 (2j)
C2:0-Acyl juglone (3a)
C3:0-Acyl juglone (3b)
C6:0-Acyl juglone (3c)
C12:0-Acyl juglone (3d)
C18:0-Acyl juglone (3e)
C18:1-Acyl juglone (3f)
C18:2-Acyl juglone (3g)
C18:3-Acyl juglone (3h)
C20:5-Acyl juglone (3i)
C22:6-Acyl juglone (3j)
0
20
40
60
80
100
Rate of HCT116 cell growth (%)
Figure 3. Effect of juglone (1), fatty acids (2a–j) and 5-O-acyl juglones (3a–j) on the proliferation of HCT116 human colon carcinoma cultured cell growth. Each compound (10
and 100 M) was added to the culture of HCT116 cells. The cells were incubated for 24 h, and the rate of cultured cell growth inhibition was determined by WST-1 assay.44
Cell growth inhibition of the cancer cells in the absence of the compound was taken as 100%. Data are shown as the mean SE (n = 5).
l
the 5-O-acyl juglones. The 5-O-acyl juglones conjugated with satu-
rated fatty acid (i.e., 3a–e) were almost the same molecular width
(5.07–5.13 Å). The length and width difference between 5-O-acyl
juglones and fatty acids is caused by the juglone moiety
(5.15 ꢀ 5.23 Å) of 5-O-acyl juglones, and the molecule of both
length and width of >8.28 and >5.07 Å, respectively, might be nec-
essary to have the bioactivity. Moreover, the molecular length and
width of C12:0-acyl juglone (3d) and C18:1-acyl juglone (3f),
ring structure, the aliphatic side chains attached to the 3-positions
of vitamins K1 and K2 may reduce their pol inhibitory and anti-
inflammatory activities.10 5-Hydroxy-1,4-naphthoquinone (jug-
lone) conjugated with fatty acid (i.e., 5-O-acyl juglone) was found
to be a stronger inhibitor of mammalian pols, human cancer cell
growth and inflammation than other 1,4-naphthoquinone based
compounds, such as vitamin K3 and juglone; therefore, 5-O-acyl
juglones could be chemotherapy agents for anti-cancer and/or
anti-inflammation based on mammalian pol inhibition.
which are the strongest inhibitors of mammalian pols j, k and pols
a
,
c
, respectively (Fig. 2), are 20.54 ꢀ 5.13 and 25.49 ꢀ 7.04 Å,
respectively; therefore, the three-dimensional length and width
of these compounds might be important for the inhibition of mam-
malian pol species.
4. Experimental procedure
4.1. Materials
3. Conclusion
A chemically synthesized DNA template, such as poly(dA), was
purchased from GE Healthcare Bio-Sciences (Little Chalfont, UK).
The oligo(dT)18 DNA primer was customized by Sigma–Aldrich Ja-
pan K.K. (Hokkaido, Japan). The synthesized radioactive nucleo-
tides, such as [3H]-deoxythymidine 50-triphosphate (dTTP) (43 Ci/
mmol), were obtained from MP Biomedicals LLC (Solon, OH,
USA). All other reagents were of analytical grade and purchased
from Nacalai Tesque Inc. (Kyoto, Japan).
Recently, we found that vitamin K3, but not K1 or K2, suppressed
inflammation in in vitro cell culture experiments and in vivo ani-
mal experiments.10 Since vitamin K3 (2-methyl-1,4-naphthoqui-
none) has a 1,4-naphthoquinone backbone, this structure must
be important for the abovementioned activities. Although the vita-
min K group of vitamins shares a methylated 1,4-naphthoquinone

5808
S. Maruo et al. / Bioorg. Med. Chem. 19 (2011) 5803–5812
1 μM
5 μM
Juglone (1)
C2:0 (2a)
C3:0 (2b)
C6:0 (2c)
C12:0 (2d)
C18:0 (2e)
C18:1 (2f)
C18:2 (2g)
C18:3 (2h)
C20:5 (2i)
C22:6 (2j)
C2:0-Acyl juglone (3a)
C3:0-Acyl juglone (3b)
C6:0-Acyl juglone (3c)
C12:0-Acyl juglone (3d)
C18:0-Acyl juglone (3e)
C18:1-Acyl juglone (3f)
C18:2-Acyl juglone (3g)
C18:3-Acyl juglone (3h)
C20:5-Acyl juglone (3i)
C22:6-Acyl juglone (3j)
0
20
40
60
80
100
Rate of TNF-α production (%)
Figure 4. Inhibitory effects of juglone (1), fatty acids (2a–j) and 5-O-acyl juglones (3a–j) on LPS-induced production of TNF-
These cells were pretreated with 1 and 5 M of each compound as a vehicle control (TNF- level, 43 pg/mL) for 30 min and then treated with 100 ng/mL LPS for 24 h (LPS-
evoked TNF- level, 538 pg/mL). The TNF- concentration in the cell medium was measured by ELISA. The relative effect in the absence of the compound was taken as 100%.
a in the mouse macrophage cell line RAW264.7.
l
a
a
a
Data are shown as the mean SE (n = 4).
4.2. Preparation of enzymes
purified according to a method described by Shimazaki et al.33 Re-
combinant human His-pol was overexpressed in E. coli BL21 and
l
Pol
a
was purified from the calf thymus by immuno-affinity col-
purified by Glutathione SepharoseTM 4B (GE Healthcare Bio-Sci-
ence Corp., Piscataway, NJ, USA) column chromatography (Maeza-
umn chromatography, as described by Tamai et al.27 Recombinant
rat pol b was purified from E. coli JMpb5, as described by Date
wa et al. in preparation). Pol a from a higher plant, cauliflower
et al.28 The human pol
c
catalytic gene was cloned into pFastBac
inflorescence, was purified according to the method outlined by
Sakaguchi et al.34 Recombinant rice (Oryza sativa L. cv. Nipponbare)
pol k tagged with His6 at the C-terminus was expressed in E. coli
and purified as described by Uchiyama et al.35 Calf thymus TdT
and bovine pancreas deoxyribonuclease I were obtained from
Stratagene Cloning Systems (La Jolla, CA, USA). The Klenow frag-
ment of pol I from E. coli and human immunodeficiency virus
type-1 (HIV-1) reverse transcriptase were purchased from Wor-
thington Biochemical Corp. (Freehold, NJ, USA). Taq pol, T4 pol,
T7 RNA polymerase, and T4 polynucleotide kinase were purchased
from Takara Bio (Tokyo, Japan).
(Invitrogen Japan K.K., Tokyo Japan). Histidine-tagged enzyme
was expressed using the BAC-TO-BAC HT Baculovirus Expression
System according to the supplier’s manual (Life Technologies,
MD, USA) and was purified with ProBoundresin (Invitrogen Japan
K.K.).29 Human pols d and
e
were purified by nuclear fractionation
of human peripheral blood cancer cells (Molt-4) using the second
subunit of pols d and -conjugated affinity column chromatogra-
phy, respectively.30 A truncated form of human pol
(residues
e
g
1–511), tagged with His6 at its C-terminus, was expressed in
E. coli cells and was purified as described by Kusumoto et al.31 A re-
combinant mouse pol i, tagged with His6 at its C-terminus, was ex-
pressed and purified by Ni-NTA column chromatography as
4.3. DNA polymerase assays
described elsewhere (Masutani et al. in preparation). A truncated
form of pol
j
(residues 1–560) tagged with His6 at its C-terminus
The reaction mixtures for pol
otic pols have been described previously.36,37 Those for pol
pols d and
were as described by Umeda et al.29 and Ogawa et
a
, pol b, plant pols and prokary-
was overproduced in E. coli and purified as described by Ohashi
c
, and
et al.32 Recombinant human His-pol k was overexpressed and
e

S. Maruo et al. / Bioorg. Med. Chem. 19 (2011) 5803–5812
5809
Juglone (1)
C2:0 (2a)
C3:0 (2b)
C6:0 (2c)
C12:0 (2d)
C18:0 (2e)
C18:1 (2f)
C18:2 (2g)
C18:3 (2h)
C20:5 (2i)
C22:6 (2j)
C2:0-Acyl juglone (3a)
C3:0-Acyl juglone (3b)
C6:0-Acyl juglone (3c)
C12:0-Acyl juglone (3d)
C18:0-Acyl juglone (3e)
C18:1-Acyl juglone (3f)
C18:2-Acyl juglone (3g)
C18:3-Acyl juglone (3h)
C20:5-Acyl juglone (3i)
C22:6-Acyl juglone (3j)
0
10
20
30
40
50
60
70
Anti-inflammatory effect (%)
Figure 5. Anti-inflammatory activity of juglone (1), fatty acids (2a–j) and 5-O-acyl juglones (3a–j) toward TPA-induced edema on mouse ear. Each compound (500
lg) was
applied individually to one ear of a mouse, and after 30 min TPA (0.5 lg) was applied to both ears. Edema was evaluated after 7 h. The inhibitory effect is expressed as the
percentage of edema. Data are shown as the means SE (n = 6).
al.38 respectively. The reaction mixtures for pols
the same as for pol , and the reaction mixture for pols k and
g
,
i
and
j
were
4.4. Other DNA metabolic enzymes assays
The activities of calf primase of pol , T7 RNA polymerase,
mouse IMP dehydrogenase (type II), T4 polynucleotide kinase
and bovine deoxyribonuclease I were measured in standard assays
according to the manufacturer’s specifications, as described by
Tamiya-Koizumi et al.39 Nakayama and Saneyoshi,40 Mizushina et
al.41 Soltis et al.42 and Lu and Sakaguchi,43 respectively.
a
l
was the same as for pol b. For pols, poly(dA)/oligo(dT)18 (A/T = 2/
1) and dTTP were used as the DNA template–primer and nucleotide
(i.e., dNTP) substrate, respectively. For TdT, oligo(dT)18 (30-OH) and
dTTP were used as the DNA primer and nucleotide substrate,
respectively. For HIV-1 reverse transcriptase, poly(rA)/oligo(dT)18
(A/T = 2/1) and dTTP were used as the template–primer and nucle-
otide substrate, respectively.
a
Each compound was dissolved in distilled dimethyl sulfoxide
(DMSO) at various concentrations and sonicated for 30 s. Aliquots
4.5. Cell culture and measurement of cell viability
of 4
l
L sonicated samples were mixed with 16
l
L of each enzyme
The cultured human cancer cell line, HCT116 (colon carci-
noma cells), was obtained from the American Type Culture Col-
lection (ATCC, Manassas, VA, USA). Human cancer cells were
cultured in McCoy’s 5A medium supplemented with 10% fetal
bovine serum, penicillin (100 units/mL) and streptomycin
(100 mg/mL). HCT116 cells were cultured at 37 °C in a humid
atmosphere of 5% CO2/95% air. For the cell growth assay, the
cells were plated at 1 ꢀ 104 cells into each well of 96-well
microplates with various concentrations of the isolated com-
pounds. These compounds were dissolved in DMSO at a concen-
tration of 10 mM as a stock solution. The stock solutions were
diluted to the appropriate final concentrations with growth med-
ium as 0.5% DMSO just before use. Cell viability was determined
by WST-1 assay.44
(final amount 0.05 units) in 50 mM Tris–HCl (pH 7.5) containing
1 mM dithiothreitol, 50% glycerol and 0.1 mM ethylenediamine
tetraacetic acid (EDTA), and kept at 0 °C for 10 min. These inhibi-
tor–enzyme mixtures (8 lL) were added to 16 lL of each of the en-
zyme standard reaction mixtures, and incubation was carried out
at 37 °C for 60 min, except for Taq pol, which was incubated at
74 °C for 60 min. Activity without the inhibitor was considered
100%, and the remaining activity at each concentration of the
inhibitor was determined relative to this value. One unit of pol
activity was defined as the amount of enzyme that catalyzed the
incorporation of 1 nmol dNTP (i.e., dTTP) into synthetic DNA tem-
plate–primers in 60 min at 37 °C under the normal reaction condi-
tions for each enzyme.33,34

5810
S. Maruo et al. / Bioorg. Med. Chem. 19 (2011) 5803–5812
Table 2
4.8. Molecular modeling
C log P values and molecular length and width of the three-dimensional structure of
juglone (1), fatty acids (2a–j) and 5-O-acyl juglones (3a–j)
Structures were minimized by using the molecular dynamics
and MM2 force field incorporated in Chem3D version 5.0 software
(Cambridge Soft, USA). To obtain theoretical molecular properties,
further semiempirical minimization with MOPAC (Chem3D) was
carried out. For semiempirical calculations, the PM3 method was
used for compounds 2a–h. The AM1 method was used for com-
pounds 1, 2i, 2j, and 3a–j.
Compound
C log Pa
Lengthb (Å)
5.15
Widthb (Å)
5.23
Juglone (1)
0.52
Fatty acid (2)
2a
2b
2c
2d
2e
2f
2g
2h
2i
ꢁ0.07
0.39
1.76
4.49
7.23
6.52
5.80
5.65
5.85
6.36
2.41
3.73
7.41
14.94
22.48
20.61
16.42
15.46
9.91
1.97
2.24
1.95
2.01
2.02
4.47
7.00
8.05
8.51
8.51
4.9. Instrumental analyses
1H and 13C NMR spectra were recorded on a JEOL 270 MHz
spectrometer (EX-270 W). Chemical shifts are expressed in d
(ppm) relative to Me4Si or the residual solvent resonance, and
coupling constants (J) are expressed in Hz. Melting point (Mp)
data were determined with a Yanaco MP-3S instrument and are
uncorrected. Infrared (IR) spectra were recorded on a Horiba
FT210 spectrometer, using NaCl (neat) or KBr pellet (solid). Mass
spectra (MS) for compound 3b–d, 3h, and 3i were obtained on an
Applied Biosystems mass spectrometer (API QSTAR pulsar i).
Mass spectra (MS) for compound 3e–3g and 3j were obtained
on JEOL’s high-resolution double-focusing mass spectrometer,
the JMS-700 (‘MStation’). Analytical thin-layer chromatography
was performed on Silica Gel 60 F254 plates (Merck, Germany).
Flash chromatography was carried out on PSQ 100B (Fuji Silysia
Co., Japan).
2j
9.19
5-O-Acyl juglone (3)
3a
3b
3c
3d
3e
3f
3g
3h
3i
0.50
1.15
2.40
4.91
7.41
7.09
6.77
6.45
6.65
7.16
8.25
9.50
5.11
5.07
5.12
5.13
5.13
7.04
8.08
8.43
7.17
8.65
13.17
20.54
28.11
25.49
21.58
20.78
16.51
14.53
3j
a
The C log P values were obtained using CS ChemDraw version 5.0 software.
The length and width were calculated using CS Chem3D version 5.0 software.
b
4.10. General procedure for the preparation of 5-O-acyl juglones
using MNBA
4.6. Measurement of TNF-
mouse macrophages
a level in the cell culture medium of
To a solution of juglone (1 equiv) and carboxylic acid (1.2 equiv)
in CH2Cl2, Et3N (2.2 equiv), MNBA (1.2 equiv) and DMAP
(0.1 equiv) were added at room temperature. After the mixture
had been stirred for 24 h, the reaction was quenched by the addi-
tion of H2O. The resulting mixture was extracted with CHCl3. The
combined extracts were washed with brine, dried (Na2SO4), and
concentrated in vacuo. The residue was purified by silica gel chro-
matography using hexanes/ethyl acetate as the eluent.
A mouse macrophage cell line, RAW264.7, was obtained from
the American Type Culture Collection (ATCC) (Manassas, VA,
U.S.). The cells were cultured in Eagle’s Minimum Essential Med-
ium (MEM) supplemented with 4.5 g glucose per liter plus 10% fe-
tal calf serum, 5 mM
L-glutamine, 50 units/mL penicillin and
50 units/mL streptomycin. The cells were cultured at 37 °C in stan-
dard medium in a humidified atmosphere of 5% CO2–95% air.
RAW264.7 cells were placed in a 12-well plate at 5 ꢀ 104 cells/
well and incubated for 24 h. The cells were pretreated with the
compounds (final concentrations of 1 and 5 lM) for 30 min before
the addition of 100 ng/mL of LPS. After stimulation with LPS for
24 h, the cell culture medium was collected to measure the amount
of TNF-a secreted. The concentration of TNF-a in the culture med-
ium was quantified by using a commercially available enzyme-
linked immunosorbent assay (ELISA) development system (Bay
Bioscience Co., Ltd, Kobe, Japan) in accordance with the manufac-
turer’s protocol.
4.10.1. 5-O-Propionyloxy-1,4-naphthoquinone (3b)
The title compound was prepared in 64% yield according to the
general procedure described above. Yellow solid; Mp = 113–
114 °C; IR (KBr) 2972, 1757, 1662, 1601, 1452, 1367, 1329,
1290, 1234, 1142, 1038, 1009, 926, 872, 781 cmꢁ1 1H NMR
;
(270 MHz, CDCl3) d 8.05 (dd, J = 7.6 Hz, 1.4 Hz, 1H), 7.76 (t,
J = 7.6 Hz, 1H), 7.39 (dd, J = 7.6 Hz, 1.4 Hz, 1H), 6.94 (d,
J = 10.3 Hz, 1H), 6.84 (d, J = 10.3 Hz, 1H), 2.78 (q, J = 7.6 Hz, 2H),
1.33 (t, J = 7.6 Hz, 3H); 13C NMR (68 MHz, CDCl3) d 184.0, 183.4,
172.6, 149.4, 139.7, 137.1, 134.7, 133.4, 129.6, 124.8, 123.2,
27.7, 8.8; HRMS, calcd for C13H10O4Na ([M+Na]+) 253.0471, found
253.0455.
4.7. TPA-induced anti-inflammatory assay in mouse
The mouse inflammatory test was performed according to
Gschwendt’s method.45 In brief, an acetone solution containing
the compounds (500 lg in 20 lL) or 20 lL acetone as a vehicle
control was applied to the inner part of the mouse ear. Thirty
minutes after the test compound had been applied, a TPA solution
4.10.2. 5-O-Hexanoyloxy-1,4-naphthoquinone (3c)
The title compound was prepared in 76% yield according to the
general procedure described above. Ocher solid; Mp = 44–46 °C; IR
(KBr) 2951, 2866, 1768, 1662, 1597, 1456, 1375, 1331, 1286,
(0.5 lg/20 lL of acetone) was applied to the same part of the ear.
1230, 1120, 1099, 1043, 939, 889, 849, 791 cmꢁ1
;
1H NMR
Acetone, followed by TPA solution, was applied to the other ear of
the same mouse as a control. After 7 h, a disk (6 mm diameter)
was obtained from the ear and weighed. The inhibitory effect
(IE) is presented as the ratio of the increase in weight of the
ear disks:
(270 MHz, CDCl3) d 8.04 (dd, J = 7.8 Hz, 1.4 Hz, 1H), 7.76 (t,
J = 7.8 Hz, 1H), 7.38 (dd, J = 7.8 Hz, 1.4 Hz, 1H), 6.94 (d,
J = 10.5 Hz, 1H), 6.84 (d, J = 10.5 Hz, 1H), 2.74 (t, J = 7.3 Hz, 2H),
1.83 (quin, J = 7.3 Hz, 2H), 1.51–1.34 (br m, 4H), 0.95 (t,
J = 7.3 Hz, 3H); 13C NMR (68 MHz, CDCl3) d 183.9, 183.4, 171.9,
149.4, 139.7, 137.1, 134.6, 133.3, 129.7, 124.7, 123.2, 34.2, 31.3,
IE ¼ ½ðTPA onlyÞ ꢁ ðtested compound þ TPAÞꢂ=½ðTPA onlyÞ
ꢁ ðvehicleÞꢂ ꢀ 100
24.1, 22.4, 14.0; HRMS, calcd for
295.0940, found 295.0937.
C
16H16O4Na ([M+Na]+)

S. Maruo et al. / Bioorg. Med. Chem. 19 (2011) 5803–5812
5811
4.10.3. 5-O-Dodecanoyloxy-1,4-naphthoquinone (3d)
1.82 (quin, J = 7.3 Hz, 2H), 1.48–1.26 (br m, 14H), 0.89 (t,
J = 7.0 Hz, 3H); 13C NMR (68 MHz, CDCl3) d 184.1, 183.5, 171.9,
149.5, 139.8, 137.2, 134.7, 133.4, 130.1, 130.0, 129.7, 127.9,
127.8, 124.8, 123.2, 34.2, 31.6, 29.7, 29.4, 29.3, 29.2 (3C), 27.3,
25.7, 24.5, 22.7, 14.2. HRMS, calcd for C28H36O4 (M+) 436.2614,
found 436.2632.
The title compound was prepared in 62% yield according to the
general procedure described above. Yellow solid; Mp = 63–65 °C;
IR (KBr) 3076, 2922, 2856, 1766, 1666, 1599, 1375, 1333, 1296,
1232, 1134, 1111, 993, 939, 895, 783 cmꢁ1 1H NMR (270 MHz,
;
CDCl3) d 8.04 (dd, J = 7.6 Hz, 1.4 Hz, 1H), 7.75 (t, J = 7.6 Hz, 1H),
7.37 (dd, J = 7.6 Hz, 1.4 Hz, 1H), 6.93 (d, J = 10.5, 1H), 6.84 (d,
J = 10.5, 1H), 2.74 (t, J = 7.6 Hz, 2H), 1.82 (quin, J = 7.6 Hz, 2H),
1.47–1.28 (br m, 16H), 0.88 (t, J = 6.8 Hz, 3H); 13C NMR (68 MHz,
CDCl3) d 184.0, 183.4, 171.9, 149.4, 139.7, 137.1, 134.6, 133.3,
129.7, 124.7, 123.2, 34.2, 31.9, 29.6 (2C), 29.5, 29.4, 29.3, 29.2,
4.11.4. 5-O-Linolenoyloxy-1,4-naphthoquinone (3h)
The title compound was prepared in 34% yield according to the
general procedure described above. Brown oil; IR (neat) 3012,
2927, 2858, 1766, 1670, 1601, 1454, 1327, 1284, 1114, 1037,
24.5, 22.7, 14.2; HRMS, calcd for
379.1879, found 379.1914.
C
22H28O4Na ([M+Na]+)
939, 852 cmꢁ1 1H NMR (270 MHz, CDCl3) d 8.04 (dd, J = 7.8 Hz,
;
1.4 Hz, 1H), 7.76 (t, J = 7.8 Hz, 1H), 7.38 (dd, J = 7.8 Hz, 1.4 Hz,
1H), 6.94 (d, J = 10.3 Hz, 1H), 6.84 (d, J = 10.3 Hz, 1H), 5.37 (m,
6H), 2.84–2.71 (br m, 4H), 2.74 (t, J = 7.8 Hz, 2H), 2.08 (br m, 4H),
1.82 (quin, J = 7.8 Hz, 2H), 1.36 (br m, 8H), 0.98 (t, J = 7.6 Hz, 3H);
13C NMR (68 MHz, CDCl3) d 184.0, 183.4, 171.9, 149.4, 139.8,
137.2, 134.7, 133.4, 131.8, 130.2, 129.7, 128.2, 128.1, 127.6,
127.0, 124.8, 123.2, 34.2, 29.7, 29.3, 29.2 (2C), 27.3, 25.7, 25.6,
24.6, 20.6, 14.4. HRMS, calcd for C28H34O4Na ([M+Na]+) 457.2349,
found 457.2335.
4.11. General procedure for the preparation of 5-O-acyl juglones
via acyl chlorides
To a solution of carboxylic acid (1 equiv) in CH2Cl2, oxalyl chlo-
ride (3.1 equiv) was added at 0 °C, and the mixture was stirred at
room temperature for 6 h. The solvent was removed to yield crude
acyl chloride.
A solution of the acyl chloride (1 equiv), juglone (3 equiv) and
DMAP (0.1 equiv) in pyridine was stirred at room temperature.
After the mixture had been stirred for 3 h, the reaction was
quenched by the addition of 1 M aqueous HCl solution. The result-
ing mixture was extracted with EtOAc. The extract was washed
with brine, dried (Na2SO4), and concentrated in vacuo. The residue
was purified by silica gel chromatography using hexanes/ethyl ace-
tate as the eluent.
4.11.5. 5-O-Eicosapentaenoyloxy-1,4-naphthoquinone (3i)
The title compound was prepared in 54% yield according to the
general procedure described above. Brown oil; IR (neat) 3012,
2962, 1768, 1668, 1599, 1450, 1327, 1284, 1203, 1117, 1038,
937, 891, 781, 715 cmꢁ1 1H NMR (270 MHz, CDCl3) d 8.05 (dd,
;
J = 8.0 Hz, 1.1 Hz, 1H), 7.76 (t, J = 8.0 Hz, 1H), 7.38 (dd, J = 8.0 Hz,
1.1 Hz, 1H), 6.94 (d, J = 10.3 Hz, 1H), 6.84 (d, J = 10.3 Hz, 1H),
5.50–5.26 (br m, 10H), 2.86–2.73 (br m, 10H), 2.26 (q, J = 7.0 Hz,
2H), 2.07 (quin, J = 7.0 Hz, 2H), 1.91 (quin, J = 7.6 Hz, 2H), 0.97 (t,
J = 7.6 Hz, 3H); 13C NMR (68 MHz, CDCl3) d 184.0, 183.4, 171.7,
149.4, 139.8, 137.2, 134.7, 133.4, 131.9, 129.7, 128.9, 128.8,
128.4, 128.1 (2C), 128.0, 127.9, 127.7, 126.9, 124.8, 33.6, 26.6,
25.7 (3C), 25.7, 25.6, 24.4, 20.6, 14.4; HRMS, calcd for C30H34O4Na
([M+Na]+) 481.2349, found 481.2366.
4.11.1. 5-O-Octadecanoyloxy-1,4-naphthoquinone (3e)
The title compound was prepared in 24% yield according to the
general procedure described above. Yellow solid; Mp = 72–75 °C;
IR (KBr) 3074, 2924, 2852, 1763, 1666, 1597, 1460, 1371, 1331,
1292, 1144, 1107, 1036, 939, 897 cmꢁ1; 1H NMR (270 MHz, CDCl3)
d 8.04 (dd, J = 7.8 Hz, 1.4 Hz, 1H), 7.76 (t, J = 7.8 Hz, 1H), 7.38 (dd,
J = 7.8 Hz, 1.4 Hz, 1H), 6.94 (d, J = 10.3 Hz, 1H), 6.84 (d,
J = 10.3 Hz, 1H), 2.74 (t, J = 7.8 Hz, 2H), 1.82 (quin, J = 7.8 Hz, 2H),
1.33–1.25 (br m, 28H), 0.88 (t, J = 7.0 Hz, 3H); 13C NMR (68 MHz,
CDCl3) d 184.1, 183,5, 172.0, 149.5, 139.8, 137.2, 134.7, 133.4,
129.7, 124.8, 123.3, 34.3, 32.0, 29.8 (5C), 29.7 (2C), 29.7, 29.6,
29.4, 29.4, 29.2, 24.5, 22.8, 14.2. HRMS, calcd for C28H40O4 (M+)
440.2927, found 440.2941.
4.11.6. 5-O-Docosahexaenoyloxy-1,4-naphthoquinone (3j)
The title compound was prepared in 62% yield according to the
general procedure described above. Brown oil; IR (neat) 3012,
2964, 2924, 1768, 1670, 1601, 1450, 1363, 1327, 1284, 1225,
1119, 1038, 935, 850 cmꢁ1 1H NMR (270 MHz, CDCl3) d 8.05 (dd,
;
J = 8.0 Hz, 1.4 Hz, 1H), 7.76 (t, J = 8.0 Hz, 1H), 7.38 (dd, J = 8.0 Hz,
1.4 Hz, 1H), 6.94 (d, J = 10.3 Hz, 1H), 6.84 (d, J = 10.3 Hz, 1H), 5.41
(m, 12H), 2.92–2.76 (m, 12H), 2.59 (q, J = 7.0 Hz, 2H), 2.07 (quin,
J = 7.6 Hz, 2H), 0.97 (t, J = 7.8 Hz, 3H); 13C NMR (68 MHz, CDCl3) d
184.0, 183.4, 171.3, 149.4, 139.8, 137.2, 131.2, 134.7, 133.4,
131.9, 129.7, 129.4, 128.4, 128.2, 128.1, 128.1, 128.0, 127.9 (3C),
127.7, 127.7, 126.9, 34.2, 30.4, 25.7, 25.7, 25.7, 25.6, 22.4, 20.6,
14.4. HRMS, calcd for C32H36O4 (M+) 484.2613, found 484.2632.
4.11.2. 5-O-{(Z)-Octadecenoyloxy}-1,4-naphthoquinone (3f)
The title compound was prepared in 29% yield according to the
general procedure described above. Brown oil; IR (neat) 3005,
2924, 2856, 1766, 1668, 1599, 1456, 1327, 1284, 1113, 1036,
941, 850 cmꢁ1 1H NMR (270 MHz, CDCl3) d 8.05 (dd, J = 8.1 Hz,
;
1.4 Hz, 1H), 7.76 (t, J = 8.1 Hz, 1H), 7.38 (dd, J = 8.1 Hz, 1.4 Hz,
1H), 6.94 (d, J = 10.3 Hz, 1H), 6.84 (d, J = 10.3 Hz, 1H), 5.39–5.34
(m, 2H), 2.74 (t, J = 7.8 Hz, 2H), 2.03 (br m, 4H), 1.82 (quin,
J = 7.8 Hz, 2H), 1.45–1.26 (br m, 20H), 0.88 (t, J = 7.0 Hz, 3H); 13C
NMR (68 MHz, CDCl3) d 184.1, 183.5, 172.0, 149.5, 139.8, 137.2,
134.7, 133.4, 129.9, 129.7, 129.7, 124.8, 123.2, 34.3, 32.0, 29.8,
29.8, 29.6, 29.4 (2C), 29.3, 29.2 (2C), 27.3, 27.3, 24.5, 22.8, 14.2.
HRMS, calcd for C28H38O4 (M+) 438.2770, found 438.2770.
Acknowledgments
We are grateful for the donation of calf pol
of Tokyo University of Science (Tokyo, Japan); rat pol b, human pols
d and by Dr. K. Sakaguchi of Tokyo University of Science (Chiba,
Japan); human pol by Dr. M. Suzuki of Nagoya University School
of Medicine (Nagoya, Japan); mouse pol and human pol by
Dr. F. Hanaoka and Dr. C. Masutani of Osaka University (Osaka,
Japan); human pol by Dr. H. Ohmori of Kyoto University (Kyoto,
a by Dr. M. Takemura
e
c
g
i
4.11.3. 5-O-Linoleoyloxy-1,4-naphthoquinone (3g)
The title compound was prepared in 31% yield according to the
general procedure described above. Brown oil; IR (neat) 3008,
2925, 2858, 1768, 1670, 1599, 1456, 1329, 1286, 1113, 1038,
j
Japan); and human pols k and
l by Dr. O. Koiwai of Tokyo University
of Science (Chiba, Japan).
941, 852 cmꢁ1
;
1H NMR (270 MHz, CDCl3) d 8.04 (dd, J = 7.8 Hz,
I.K. acknowledges a Grant-in-Aid for Young Scientists (B) (No.
23710262) from MEXT (Ministry of Education, Culture, Sports, Sci-
ence and Technology). Y.M. acknowledges a Grant-in-Aid from the
Takeda Science Foundation (Japan).
1.4 Hz, 1H), 7.76 (t, J = 7.8 Hz, 1H), 7.38 (dd, J = 7.8 Hz, 1.4 Hz,
1H), 6.94 (d, J = 10.3 Hz, 1H), 6.84 (d, J = 10.3 Hz, 1H), 5.39 (m,
4H), 2.80–2.71 (m, 2H), 2.74 (t, J = 8.1 Hz, 2H), 2.06–2.04 (m, 4H),

5812
S. Maruo et al. / Bioorg. Med. Chem. 19 (2011) 5803–5812
24. Aggarwal, B. B. Nat. Rev. Immunol. 2003, 3, 745.
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